Transcription Factors: Normal and Malignant Development of Blood Cells

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Transcription Factors: Normal and Malignant Development of Blood Cells. Edited by Katya Ravid, Jonathan D. Licht Copyright  2001 by Wiley-Liss, Inc. ISBNs: 0-471-35054-0 (Hardback); 0-471-22388-3 (Electronic)

TRANSCRIPTION FACTORS

TRANSCRIPTION FACTORS NORMAL AND MALIGNANT DEVELOPMENT OF BLOOD CELLS Edited By

KATYA RAVID Department of Biochemistry Boston University School of Medicine Boston, MA

JONATHAN D. LICHT Derald H. Ruttenberg Cancer Center and Department of Medicine Mount Sinai School of Medicine New York, NY

A JOHN WILEY & SONS, INC., PUBLICATION New York

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Chichester

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Weinheim

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Brisbane

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Singapore

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Toronto

Designations used by companies to distinguish their products are often claimed as trademarks. In all instances where John Wiley & Sons, Inc., is aware of a claim, the product names appear in initial capital or   . Readers, however, should contact the appropriate companies for more complete information regarding trademarks and registration. Copyright 2001 by Wiley-Liss, Inc. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic or mechanical, including uploading, downloading, printing, decompiling, recording, or otherwise, except as permitted under Sections 107 or 108 of the 1976 United States Copyright Act, without the prior written permission of the Publisher. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 605 Third Avenue, New York, NY 10158-0012, (212) 850-6011, fax (212) 850-6008, E-Mail: PERMREQWILEY.COM. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold with the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional person should be sought. ISBN 0-471-22388-3 This title is also available in print as ISBN 0-471-35054-0. For more information about Wiley products, visit our web site at www.Wiley.com.

CONTENTS

Introduction

ix

Katya Ravid and Jonathan D. Licht

Contributors

I

TRANSCRIPTION FACTORS AND THE MEGAKARYOCYTIC AND ERYTHROID LINEAGES 1 The Role of GATA-1 and FOG in Erythroid and Megakaryocytic Differentiation

xiii

0 1

Alice P. Tsang, John D. Crispino, and Stuart H. Orkin

2 Regulation of Megakaryocyte and Erythroid Differentiation by NF-E2

13

Sang-We Kim and Ramesh A. Shivdasani

3 Transcription Factors Involved in Lineage-Specific Gene Expression During Megakaryopoiesis

31

Yulia Kaluzhny, Mortimer Poncz, and Katya Ravid

4 Role of TAL1/SCL Transcription Factor in Normal and Leukemic Hematopoiesis

51

Stephen J. Brandt

5 EKLF and the Development of the Erythroid Lineage

71

James J. Bieker

II

TRANSCRIPTION FACTORS AND THE MYELOID LINEAGE

6 RUNX1(AML1) and CBFB: Genes Required for the Development of All Definitive Hematopoietic Lineages

85

87

Nancy A. Speck and Elaine Dzierzak

7 PU.1 and the Development of the Myeloid Lineage

103

Daniel G. Tenen

v

vi

CONTENTS

8 CCAAT/Enhancer-Binding Proteins in Myeloid Cells

117

Dong-Er Zhang

9 Homeobox Gene Networks and the Regulation of Hematopoiesis

133

Guy Sauvageau, R. Keith Humphries, H. Jeffrey Lawrence, and Corey Largman

10 The Role of Retinoic Acid Receptors in Myeloid Differentiation

149

Steven J. Collins, Barton S. Johnson, and Louise E. Purton

11 Transcriptional Targets of the Vitamin D3 Receptor During Myeloid Cell Differentiation

163

V. Carrie Bromleigh, Jeremy Ward, and Leonard P. Freedman

III

TRANSCRIPTION FACTORS AND THE LYMPHOID LINEAGE

12 The Role of Ikaros Family Genes in Lymphopcyte Differentiation and Proliferation

181

183

Nicole Avitahl, Susan Winandy and Katia Georgopoulos

13 The Role of PU.1 in B-Lymphocyte Development and Function

201

Sridhar Rao and M. Celeste Simon

14 The Role of Pax5 (BSAP) in Early and Late B-Cell Development

217

Markus Horcher, Dirk Eberhard, and Meinrad Busslinger

15 Janus Kinases and STAT Family Transcription Factors: Their Role in the Function and Development of Lymphoid Cells

229

Tammy P. Cheng, Jéro me Galon, Roberta Visconti, Massimo Gadina, and John J. O’Shea

16 E2A and the Development of B and T Lymphocytes

255

Barbara L. Kee and Cornelis Murre

17 The Role of BCL-6 in Normal Lymphoid System and non-Hodgkin’s Lymphomas

271

B. Hilda Ye

18 The Role of Octamer Factors and Their Coactivators in the Lymphoid System

291

Eric Bertolino, Ralph Tiedt, Patrick Matthias, and Harinder Singh

19 The Role of Early B-Cell Factor in B-Lymphocyte Development Mikael Sigvardsson

313

CONTENTS

IV.

TRANSCRIPTION FACTORS INVOLVED IN LEUKEMIAS DUE TO CHROMOSOMAL TRANSLOCATION

20 The Role of RAR and Its Fusion Partners in Acute Promyelocytic Leukemia

vii

325 327

Ari Melnick and Jonathan D. Licht

21 The Leukemogenic Function of the inv(16) Fusion Gene CBFB-MYH11

379

P. Paul Liu, Lucio H. Castilla, and Neeraj Adya

22 EVI1 Rearrangements in Malignant Hematopoiesis

393

Giuseppina Nucifora

23 t(8;21) AML and the AML1/ETO Fusion Gene: From Clinical Syndrome to Paradigm for the Molecular Basis of Acute Leukemia

409

Richard C. Frank and Stephen D. Nimer

24 TEL/ETV6 Gene Rearrangements in Human Leukemias

425

Ema Anastasiadou, Michael H. Tomasson, David W. Sternberg, Todd R. Golub, and Gary Gilliland

25 MLL in Normal and Malignant Hematopoiesis

447

Paul M. Ayton and Michael L. Cleary

26 Coactivators and Leukemia: The Acetylation Connection with Translocations Involving CBP, p300, TIF2, MOZ, and MLL

465

Vandana Chinwalla and Nancy J. Zeleznik-Le

27 The LMO2 Master Gene; Its Role as a Transcription Regulator Determining Cell Fate in Leukemogenesis and in Hematopoiesis

483

Yoshihiro Yamada and Terence H. Rabbitts

28 The Acetyltransferases CBP and p300: Molecular Integrators of Hematopoietic Transcription Involved in Chromosomal Translocations

497

Gerd A. Blobel

V.

ONCOGENESIS AND HEMATOPOIESIS

29 The Roles of the c-myc and c-myb Oncogenes in Hematopoiesis and Leukemogenesis

519

521

Marcello Arsura and Gail E. Sonenshein

30 NF- B in Cell Life and Death Winnie E. Tam, Jyoti Sen, and Ranjan Sen

551

viii

CONTENTS

VI.

SUMMARY OF TRANSCRIPTION FACTORS IMPLICATED IN HEMATOPOIESIS: IN VIVO STUDIES

31 Transcription Factors Implicated in Hematopoiesis: In Vivo Studies

571 573

Yulia Kaluzhny and Katya Ravid

32 Chromosomal Translocations Associated with Disruption of Transcriptional Regulators in Leukemia and Lymphoma

593

Jonathan D. Licht

Index

599

INTRODUCTION

During hematopoiesis, pluripotent hematopoietic stem cells (HSC) give rise to mature blood cells of different lineages. Hematopoiesis within mammalian embryos begins in the yolk sac blood island, progresses to the fetal liver, and then moves to the bone marrow where definitive hematopoiesis occurs. During embryonic development, the commitment to specialized organs and cell types is tightly regulated by extrinsic influences, including cell interactions and gradients of inducing factors within cellular microenvironments. Although not yet proven, it would be reasonable to assume that the production of stem cells in the embryo is similarly not occurring in a random fashion. The question remains as to whether pluripotent stem cells are randomly committed to different lineages or whether lineage commitment is a result of external stimuli directing a certain development pattern. We refer the reader to a recently published, lively debate on this issue (Enver et al., 1998; Metcalf, 1998). The debating parties acknowledge the reports that there is low-level transcription of several different lineage-associated transcription factors (nuclear proteins that bind to and alter gene expression) as well as different cytokine receptors in single, pluripotent bone marrow stem cells (e.g., Hu et al., 1997). The question at issue is whether bone marrow hematopoietic stem cells are induced to express high levels of a specific factor spontaneously or are directed to do so by extracellular signals (e.g., by elevated levels of specific cytokines or by specific cell contact events). Since a threshold level of a given transcription factor would appear to be associated with specific lineage outcomes (Kulessa et al., 1995), one would predict that both activation and transcriptional suppression

of regulatory nuclear factors take place during lineage determination. Thus, the processes of HSC commitment to distinct lineages and subsequent cellular maturation appear to be controlled by specific transcription factors as well as by growth factors. These may selectively induce higher-level expression of specific transcription factors and/or simply enhance cellular proliferation and survival during lineage commitment and development. This book focuses on the role of specific transcription factors in promoting the development of distinct hematopoietic lineages. The studies described involve the use of culture systems (primary cells and cell lines) to examine the consequences of forced expression of transcription factors as well as to identify the site and mode of action of specific nuclear factors. A strong emphasis is given to transgenic and knockout mouse models, which have greatly assisted in examining the importance of specific transcription factors for lineage selection and commitment (see Chapter 31). Several important conclusions arise from the studies outlined in this book: (1) No single cis-element within a gene is sufficient to promote gene expression in a hematopoietic lineage. (2) Transcription factors that play a major role in the development/maturation of a certain hematopoietic lineage (e.g., GATA-1 in erythroid and megakaryote development) may also be expressed in other lineages and cell types (e.g., GATA-1 is present in eosinophils, noncommitted hematopoietic progenitors, and Sertoli cells of the testis). In other lineages and cell types, these factors may play a developmental role in combination with other transcription factors. (3) The absence of a transcription factor may have a major impact on lineage development (e.g., ix

x

INTRODUCTION

Pax5 on B-cell precursors), indicating that the factor is essential, but not necessarily sufficient, for proper development. A combination of transcription factors and accessory proteins binding to a cis-acting element(s) of a gene may establish a high level of gene expression (e.g., Pax5 interaction with TFIID via coactivator and other proteins) and it influences final cellular identity. (4) Some transcription factors enhance the expression of lineage-specific genes (e.g., the positive effect of Ikaros in the lymphoid lineage) while also suppressing the expression of genes associated with other lineages (e.g., the negative effect of Ikaros in the myeloerythroid lineage). This dual effect of activation and suppression further promotes lineage selection. (5) The presence of chromatin remodeling proteins at regulatory regions in a gene ensures activation of a promoter by a transcription factor, thus introducing an additional level of selective gene expression. Hence, because of the complexities surrounding the sets of proteins associated with distinct transcription factors that promote lineage-specific gene expression, we refrain from presenting a simple scheme of the factors essential for the development of specific lineages. We address this complexity by referring the reader to the detailed picture presented in each chapter. While individual transcription factors promote normal development of specific blood cells, many abnormalities critical to the development of leukemias also involve these same nuclear factors. This book also updates the reader on the connection between chromosomal translocations involving transcription factors and cellular transformation leading to leukemia. Several general principles can be gleaned from a review of the role of transcription factors in leukemia: (1) Chromosomal translocations associated with the development of hematological malignancy consistently target transcription factors. The translocations may lead to aberrant overexpression of a factor normally tightly regulated in hematopoietic tissues (e.g., c-myc) or misexpression of factors normally absent or expressed at low to absent levels in blood cells (e.g., EVI1). However, even more frequently the translocation leads to the production of fusion proteins with novel transcriptional activities whose expression may be deregulated relative to the normal components of the fusion. (2) Translocations are rare in solid tumors and the

tropism for hematopoietic lineages may be due to the fact that at least in lymphoid tissues, gene rearrangement may be due to aberrant immunoglobulin gene rearrangement joining. In addition, the genes disrupted by the translocations may have tissue-specific functions and may not be able to transform epithelial and other cells (Rabbitts, 1999). (3) The fusion proteins of leukemia often affect chromatin remodeling. Many of the fusion proteins are aberrant transcriptional repressors that recruit corepressor factors and histone deacetylases to target promoters, shutting off genes normally activated in hematopoietic differentiation (Redner et al., 1999). This has led to the recent use of inhibitors of histone deacetylase in the therapy of leukemia. Other fusions affect presumed (MLL) or identified (p3000, CBP) transcriptional cofactors, suggesting that global changes in gene regulation may be found in these forms of leukemia. (4) Chromosomal translocations in leukemia may highlight general pathways critical for normal blood development. For example, a number of hox genes are rearranged in leukemia, and knockout and overexpression of such genes also has profound effects on hematopoiesis. (5) Specific transcriptional factors are repeatedly disrupted in different hematological malignancies. The universal rearrangement of the retinoic acid receptor (RAR) in acute promyelocytic leukemia indicates the importance of targets of RAR for the progression of myeloid differentiation. The frequent rearrangement of genes such as TEL and the genes of the AML1/CBF complex in a number of different forms of leukemia suggest a nodal role for these proteins in normal development. The case of MLL may stand apart. Its rearrangement in dozens of different chromosomal events may be due to a central role in gene control. However, the frequent association of the MLL fusions with genotoxic chemotherapy treatment indicates the particular fragility of this region of the genome and the propensity for chromosomal breakage. We end this overview leading to the following chapters with our expression of appreciation to the contributors for their thorough summary of the field and their interesting hypotheses. We also apologize to those whose studies were not quoted because of limited space. Important advances have been made in understanding

REFERENCES

the complex nature of transcription factors, associated proteins, and their potential roles in hematopoietic cell development and lineage restriction. We trust that the studies reviewed in this book, together with the intriguing questions posed, will inspire the reader to continue undertaking research challenges in the field.

REFERENCES Enver, T., Heyworth, C. M., and Dexter, T. M. (1998). Do stem cells play dice? Blood 92, 348—352. Hu, M., Krause, D., Greaves, M., Sharkia, S., Dexter, M., Heyworth, C., and Enver, T. (1997). Multilineage gene expression precedes commitment in the hematopoietic system. Genes Dev. 11, 774—779.

xi

Kulessa, H., Frampton, J., and Graf, T. (1995). GATA1 reprograms avian myelomonocytic cell lines into eosinophils, thromboblasts, and erythroblasts. Genes Dev. 9, 1250—1256. Metcalf, D. (1998). Lineage commitment and maturation in hematopoietic cells: the case for extrinsic regulation. Blood 92, 345—348. Rabbitts, T. H. (1999). Perspective: chromosomal translocations can affect genes controlling gene expression and differentiation — why are these functions targeted? J. Pathol. 187, 39—42. Redner, R. L., Wang, J., and Liu, J. M. (1999). Chromatin remodeling and leukemia: new therapeutic paradigms. Blood 94, 417—428.

CONTRIBUTORS

Dr. James J. Bieker Mount Sinai School of Medicine Department of Biochemistry and Molecular Biology One Gustave Lower Levy Place New York, NY 10029 Dr. Gerd A. Blobel Children’s Hospital of Philadelphia Research Hematology Abramson Research Center 316 34th & Civic Center Boulevard Philadelphia, PA 19104 Dr. Stephen J. Brandt Vanderbilt University Medical Center Division of Hematology-Oncology Room 547 MRB II Nashville, TN 37232 Dr. Meinrad Busslinger, Markus Horcher, and Dirk Eberhard Research Institute of Molecular Pathology Dr. Bohr-Gosse 7 Vienna A-1030 Austria Tammy P. Cheng Howard Hughes Medical Institute NIH Research Scholars Program Dr. Michael L. Cleary and Paul M. Ayton Stanford University Medical Center Department of Pathology Room L235 Stanford, CA 94305 Dr. Steven J. Collins, Barton S. Johnson, and Louise E. Purton Fred. Hutchinson Cancer Research Center C2-023 1100 Fairview Avenue North Seattle, WA 98109

Elaine Dzierzak Department of Cell Biology and Genetics Erasmus University The Netherlands Dr. Leonard P. Freedman, V. Carrie Bromleigh, and Jeremy Ward Memorial Sloan Kettering Cancer Center Cell Biology Program 1275 York Avenue New York, NY 10021 Dr. Katia Georgopoulos, Nicole Avitahl, and Susan Winandy MGH East CBRC Building 149 13th Street Charleston, MA 02129 Dr. D. Gary Gilliland, Ema Anastasiadou, Michael H. Tomasson, David W. Sternberg, and Todd R. Golub Harvard Institutes of Medicine Room 421 4 Blackfan Circle Boston, MA 02115 R. Keith Humphries Department of Medicine University of British Columbia Canada Dr. Barbara L. Kee and Cornelis Murre University of California, San Diego Department of Biology Pacific Hall-1st Floor 9500 Gilman Drive San Diego, CA 92093 H. Jeffrey Lawrence and Corey Largman Department of Medicine University of California California xiii

xiv

CONTRIBUTORS

Dr. Jonathan D. Licht and Ari Melnick Mount Sinai School of Medicine, Ruttenberg Cancer Center Departments of Medicine and Molecular Biology Box 1130 One Gustave Lower Levy Place New York, NY 10029 Dr. P. Paul Liu, Lucio H. Castilla, and Neeraj Adya NIH, NHGRI Building 49, Room 3A18 49 Convent Drive Bethesda, MD 20892 Patrick Matthias and Ralph Tiedt Friedrich Miescher Institute Basel, Switzerland Dr. Stephen Nimer and Richard C. Frank Memorial Sloan-Kettering Cancer Center Division of Hematologic Oncology 1275 York Avenue New York, NY 10021 Dr. Giuseppina Nucifora Loyola University Medical Center Cardinal Bernardin Cancer Center Building 112 2160 First Avenue Maywood, IL 60153 Dr. John J. O’Shea, Massimo Gadina, Jerome Galon, and Roberta Visconti NIH MSC-1820 Building 10, Room 9N252 10 Center Drive Bethesda, MD 20892 Dr. Stuart H. Orkin, John D. Crispino, and Alice P. Tsang Children’s Hospital Enders Research Building, Room 761 300 Longwood Avenue, Boston, MA 02115 Mortimer Poncz Department of Pediatrics University of Pennsylvania School of Medicine Philadelphia, PA 19104 Dr. Terence H. Rabbitts and Yoshihiro Yamada MRC Laboratory of Molecular Biology

Division of Protein and Nucleic Acid Chemistry Hills Road Cambridge CB2 2QH UK Sridhar Rao Department of Pathology The University of Chicago Chicago, IL Dr. Katya Ravid and Yulia Kaluzhny Boston University School of Medicine Department of Biochemistry Whitaker Cardiovascular Institute 115 Albany Street Boston, MA 02118 Dr. Guy Sauvageau Institute de Researches, Cliniques de Montreal 110 Pine Avenue West Montreal (Quebec) H2W 1R7 Canada Dr. Ranjan Sen, Winnie F. Tam, and Jyoti Sen Brandeis University Rosenstiel Basic Medical Sciences Research Center Mailstop 029 Waltham, MA 02454 Dr. Ramesh A. Shivdasani and Sang-We Kim Dana-Farber Cancer Institute 44 Binnay Street Boston, MA 02115 Dr. Mikael Sigvardsson Lund University The Department of Stem Cell Biology Lund S-221 07 Sweden Dr. Celeste Simon University of Chicago Howard Hughes Medical Institute MC 1028 5841 South Maryland Avenue Chicago, IL 60637 Dr. Harinder Singh and Eric Bertolino University of Chicago Howard Hughes Medical Institute MC 1028 5841 South Maryland Avenue Chicago, IL 60637

CONTRIBUTORS

Dr. Gail E. Sohenshein and Marcello Arsura and Dr. Marcello Arsura Boston University School of Medicine 715 Albany Street Boston, MA 02118 Dr. Nancy A. Speck Dartmouth Medical School Department of Biochemistry 7200 Vail Building, Room 315 Hanover, NH 03755 Dr. Daniel G. Tenen Harvard Institutes of Medicine Room 954 77 Avenue Louis Pasteur Boston, MA 02115 Dr. B. Hilda Ye Albert Einstein College of Medicine Department of Cell Biology

Jack & Pearl Resnick Campus 1300 Morris Park Avenue Bronx, NY 10461 Dr. Nancy J. Zeleznik-Le and Vandana Chinwalla Loyola University Medical Center Cardinal Bernardin Cancer Center 2160 South First Avenue Maywood, IL 60153 Dr. Dong-Er Zhang The Scripps Research Institute Molecular & Experimental Medicine MEM-L51 10550 North Torrey Pine Road. La Jolla, CA 92037

xv

Dedicated to our spouses and children, KR, JL.

INDEX

Abi1 gene, MLL fusion and leukemogenesis, 457 Acetylcholinesterase (AChE), Friend of GATA-1 (FOG) assay using, 3 Activation domains (ADs) E2A proteins, 258—259 retinoic acid receptor (RAR) transcription and, 328—329 Acute lymphoblastic leukemia (ALL) c-myb oncogene and, 537—538 Ikaros/Aiolos regulation of chromatin and, 196 TEL/ABL fusion protein, 430 TEL/AML1 fusion protein, pediatric patients clinical significance, 435 diagnostic applications, 439 favorable prognosis with, 436—438 minimal residual disease detection, 439 prevalence in, 435—436 prognosis and relapse predictions, 436—439 structural characteristics, 431—432 TEL/JAK2 fusion protein and, 430 Acute myelogenous leukemia (AML) CBFB-MYH11 fusion protein knock-in mouse model, 385—387 research background, 379—380 transgenic mouse models, 384—385 EVI1 gene involvement, 397—399 hox gene hematopoiesis regulation, 139—140 inv(16) fusion genes, research background, 379—380 MLL partial amino-terminal duplication mutants, 452, 455 MLL-p300 fusion protein, 472 MOZ-CBP fusion protein, 467—470 MOZ-TIF2 inversion, 472—474 myeloid differentiation and lineage, HoxA10 target gene, 168—169 t(8;21) AML transcript, 409—411 Acute promyelocytic leukemia (APL). See also PML gene

all-trans retinoic acid (ATRA), 149—150 therapy using, 330—331 fusion protein comparisons, 351—353 NPM-RAR fusion protein, 348—349 nucleophosmin (NPM) gene and, 347—348 NuMA-RAR fusion protein, 350 partner protein comparisons, 351, 354—355 PLZF gene and, 341—343 PML gene expression in, 331—332 PML-RAR fusion protein, 150 action model for, 341 APL model of action, 341—343 cellular models, 339—340 mouse models, 340—341 nuclear bodies, 338—339 nuclear body, 338—339 protein-protein interactions, 336—337 retinoid resistance, 338 structure, 336 transcriptional activity, 337—338 RAR403, hematopoiesis activation and, MPRO promyelocytes and, 151—152 retinoic acid receptor (RARa) fusion partners fusion protein comparisons, 351—353 myeloid differentiation, 329 N-protein/RARa fusion comparisons, 351, 354—356 nuclear matrix-mitotic apparatus protein (NuMa), 349—350 nucleophosmin (NPM) gene, 347—350 NPM-RAR fusion, 348—349 PLZF-RAR, 345—347 t(11;17)(q23;q21) APL model, 347 RAR-PLZF reciprocal transcript, 347 RAR-PML reciprocal fusion, 341 research background, 327—328 Stat5b-RARa, 350—351 target genes, 329—331 transcriptional function, 328—329

599

600

INDEX

Acute promyelocytic leukemia (Contd.) t(11;17)(q23;q21) model, 347 vitamin D transcriptional targets, monocytic differentiation, 171—175 AF6q21, MLL fusion and leukemogenesis, 457 AIDS-related NHL, BCL-6 mutations and, 282—283 Aiolos family proteins B-cell maturation and null mutations, 189 cell cycle regulation, nuclear localization, 192—194 dominant negative mutations and lymphoid defects, 189 gene expression, 185—186 lymphocyte development, 186—188 differentiation, 194—196 lymphocyte homeostasis, 190—194 nuclear localization during cell cycle, 192—194 null mice lymphoproliferation, 191—192 structure and function, 183—185 ALA-S, erythroid Krppel-like factor (EKLF) and, 80—81 ALL-1 related (ALR) protein, MLL gene and, 450 All-trans retinoic acid (ATRA) acute promyelocytic leukemia (APL) and, 327—328 CFC production in hematopoietic precursors, 155—156 future applications of, 159—160 hematopoiesis activation CFU-S production in hematopoietic precursors, 156 RAR403 construct, 151—152 myelopoiesis and, 158—159 NPM-RAR fusion protein, 348—349 nucleophosmin (NPM) gene and, 347—348 NuMA-RAR fusion protein, 350 PLZF gene and, 341—343 PML-RAR fusion protein, 150 cellular models, 339—340 mouse models, 340—341 nuclear bodies, 338—339 retinoid resistance, 338 transcriptional activity, 337—338 RAR-PML reciprocal fusion protein, 341 retinoic acid receptor (RAR) structure and, 327—328 myeloid differentiation and lineage, 329 target genes and, 329—331 RXR-RAR complexes, PML-RAR fusion degradation and, 330—331 short- and long-term marrow repopulation in stem cell assays, 156—158 structure and function, 149—150 t(11;17)(q23;q21) APL model and, 347 IIb gene, megakaryocyte transcription GATA-1 promoter studies, 35 gene targeting techniques in transgenic mice, 39—40 repressor elements, 38 Sp1 family, 37 Amino acid sequencing, EVI1 gene, 395—396

AML gene sensitivity, RXR-RAR heterodimer activation, in immature vs. mature myeloid cells, 154—155 AML1:CBF fusion protein characteristics of, 409 hematopoietic regulation, 412—413 inv16 abnormality and, 410—411 AML1/ETO fusion protein CBP/p300 coactivator regulation, 506 ETO members and function, 413—415 ETO transcriptional effects, 415—416 future research issues, 419 gain-of-function properties, 418—419 hematopoietic inhibition, 432—433 leukemogenesis, 416—418 cellular functions, 417—418 dominant transcriptional repression, 416—417 research background, 409 Runx1 and Cbf b genes, late hematopoiesis and, 94—95 t(8;21) AML translocation, 410—411 AML1 gene AML1A and AML1B forms, 412 AML1N, 412 413 CBP/p300 coactivator regulation, 505—506 EVI1 gene and, myeloid leukemias, 398—399 hematopoiesis and, 412—413 identification and related genes, 411—412 t(8;21) translocation, 409—411 AML1/MDS1/EVI1 fusion protein, transcriptional functions, 402 Amplification techniques, c-myc oncogenes, 527—528 Angiotensin II, Janus kinases and, 232 Antiapoptosis, NF-KB family and cell cycle progression, 558 gene regulation mechanisms, 559—560 Antigen-dependent B-cell differentiation, octamer factors, 299—301 germinal center formation, 301 Ig enhancer activation, 300—301 Ig transcription regulation by Oct-1/2 and OBF-1, 299—300 IL-6 and retinoic acid signaling, 301 terminal differentiation, 299 Anti-mouse antibody, Aiolos family proteins, lymphoproliferations in null mice and, 191—192 Antisense oligonucleotides, hox gene function in normal hematopoiesis, 139 Aorta/gonad/mesonephros (AGM) region CBP/p300 coactivator regulation, 507—508 Runx1 and Cbf b genes, definitive hematopoiesis and, 90—92 Apoptosis c-myb oncogene and, 536—537 c-myc oncogene and, 524—525 NF-KB family and cell viability, 558—559 gene regulation mechanisms, 559—560

INDEX

proapoptotic activity, 560—561 PML growth suppression and, 335—336 AP1 protein, PML-RAR fusion protein and, 338 ARNT transporter, TEL gene fusion and, 427 A20 zinc-finger protein, NF-KB family and cell cycle antiapoptosis, 560

Bach 1 and 2 proteins, NF-E2 function and, 18—19 BAZF protein, BCL-6 mutation signaling and, 280 B-box zinc fingers, PML gene structure, 331 nuclear bodies, 333 B cell Aiolos family proteins, null mutations and maturation of, 189 BCL-6 mutations in, 275—277 CBF genes and heavy chain switching in, 95 early B-cell factor (EBF) and development of development in absence of, 316—317 EBF structure and function, 313—315 future research issues, 320 genetic targets for, 317—320 E proteins and developmental function, 259—262 E2A protein transactivation, 258—259 future research applications, 265—266 HLH structure and function, 258 research background, 257—258 416B cells, Friend of GATA-1 (FOG) assay using, 3 function of, 255 hox gene expression in, 136—137 Ikaros family proteins gene expression, 186 nuclear localization, 192—194 null mutations and, 187—188 octamer factors and development of, 295—299 antigen-dependent differentiation, 299—301 germinal center formation, 301 Ig enhancer activation, 300—301 Ig transcription regulation by Oct-1/2 and OBF-1, 299—300 IL-6 and retinoic acid signaling, 301 terminal differentiation, 299 gene expression regulation, 298—299 germ-line V gene transcription and rearrangement, 297—298 OBF-1 and early development, 295—297 Oct-2-dependent genes, 299 Pax5 protein, structure and function, 217—220 future research issues, 224—225 gene repression and alternative fates, 220—222 late differentiation, 222—224 oncogenic activation in non-Hodgkin’s lymphoma, 224—225 PU.1 gene and development and function future research applications, 211—212

601

physicochemical characteristics, 201—202 protein-protein interactions, 205 PU.1:PIP interactions, 205—207 PU.1/SPi-1 gene expression, 202—203 PU.1/SPi-1 models, 208—211 Spi-B regulated protein, 207—208 structure-function relationships, 203—205 target gene characteristics, 207 stem cell genetics and, 255—257 B-cell receptor (BCR) Aiolos family proteins B-cell maturation and null mutation, 189 lymphoproliferations in null mice and, 191—192 PU.1/Spi-B genetic models and, 210—211 B-cell-specific activator protein (BSAP). See Pax5 protein early B-cell factor (EBF) and disruption by, 317 genetic targets, 319—320 B-cell tumors c-myb oncogene and, 537—538 c-myc oncogene and, 522 BCL-2 gene AML1/ETO fusion protein, 418—419 NF-KB family and cell cycle antiapoptosis, 559—560 BCL-3 gene, I-KB family interaction, 554 BCL-6 gene mutations gene expression patterns, 277—278 as histogenetic markers, 282—283 Non-Hodgkin’s lymphoma clinical relevance of translocation, 283—284 future research, 284—285 normal and NHL B cells, 275—277 regulators and effectors, 279—280 role in normal germinal center and NHL B cells, 280—281 as transcriptional repressor, 278—279 translocation mechanisms, 274—275 nonlymphoid cell types and, 282 structural characteristics, 276—277 T-cell differentiation and function, 281—282 BCR-ABL fusion kinase Jaks and Stats role in, 243 TEL/ABL fusion protein and, 430 Bernard-Soulier syndrome (BSS), megakaryopoiesis transcription factors GATA-1 promoter studies, 35 gene targeting techniques in transgenic mice, 40 B220 expression, E proteins in B-cell development, 259—260 B29 gene B-cell-specific gene expression, 298 early B-cell factor (EBF) interaction with, 317 B-Kru¨ppel-like factor (BKLF), DNA-binding activity, 80 BLR1 protein, germinal center formation, octamer involvement in, 301

602

INDEX

B-lymphoid kinase (Blk), early B-cell factor (EBF) interaction with, 317—319 Bmi1 gene, hox gene expression, upstream regulation with, 142 Breakpoint cluster regions BCL-6 structure, 274—275 PML-RAR fusion protein, 336 Bridge building models, CBP/p300 coactivator regulation, 508 Bromodomain, MOZ-CBP fusion protein, 469—470 BTB/POZ domain, BCL-6 mutation and, 278—279 Burkitt’s lymphoma, c-myc oncogene transformation, 526 527

Carboxy terminals, c-myc oncogenes, 523—524 Casein kinase II c-myb oncogene structure, 531 DNA-binding domain, 531 erythroid Kru¨ppel-like factor (EKLF) transcription activation, 75—78 I-KB family, 553—554 Catalysis, CBP/p300 coactivator actions and, 509—510 CBFB-MYH11 fusion protein acute myelogenous leukemias (AMLs) knock-in mouse model, 385—387 research background, 379—380 transgenic mouse models, 384—385 AML1 function and, 411 future research issues, 387—389 CBF protein, structural characteristics, 380—381 CBF-SMMHC fusion gene biochemical and cellular studies, 381—383 functional studies, 383—384 knock-in mouse models, 388—389 structural characteristics, 380—381 CBP coactivators CBP/p300 (See CBP/p300 coactivator) erythroid Krppel-like factor (EKLF) transcription activation, 76—78 MLL-CBP fusion protein, 470—472 MOZ-CBP fusion protein, leukemia translocations, 467—470 CBP/p300 coactivator AML1 gene and, 505—506 C/EBP and, 504—505 c-myb and, 500—501 E2A gene and, 501—502 EKLF and, 503—504 Ets family and, 505 functional mechanisms, 507—510 animal models, 507—508 bridge-building activities, 508—509 catalytic actions, 509—510 numerical strength, 508 future research issues, 510 GATA-1 and, 502—503

hematopoiesis and, 499—506 leukemia and, 465—466, 475—476, 506—507 NF-E2 and, 503 research background, 497 structure and function, 498—499 CD20/B1 gene promoter, B-cell-specific gene expression, 298 CD34 pathways HoxA10 target gene, myeloid differentiation and lineage, 168—169 human hematopoietic cells, hox genes, 136 NF-KB family and, 556 CD40 pathways Aiolos family proteins, B-cell maturation and null mutation, 189—190 Janus kinases and, 232 NF-KB family and cell cycle progression, 557—558 gene regulation mechanisms, 559—560 proapoptotic activity, 560—561 CD43 pathways, E proteins in B-cell development, 260 CD45 pathways, E proteins in B-cell development, 259—260 C/EBP family CBP/p300 coactivator regulation, 504—505 AML1 gene and, 506 C/EBP, 117—120 deletion in animal models, 123—125 gene regulation, 122—123 C/EBP, 120 c-myb interaction with, 534 deletion in animal models, 125 C/EBP, 120—121 deletion in animal models, 125 C/EBP, 121 deletion in animal models, 125 C/EBP, 121 deletion in animal models, 125 C/EBP, 121—122 deletion in animal models, 126 deficiency in animal models, 123—126 deficient animal models, 123—126 gene regulation by, 122—123 research background, 117 retinoic acid receptor (RAR) target genes and, 331 transportation factors in, 117—122 Cell cycle regulation AML1/ETO fusion protein and, 417—418 c-myb oncogene and, 534—537 c-myc oncogene and, 524—525 Ikaros family proteins and, 192 nuclear localization, 192—194 LMO2 gene family hematopoietic multiprotein complexes, 488—489 mouse embryonic vascular formation, 485—488 primitive and definitive hematopoiesis, 485 NF-KB family apoptotic genes, 559—560

INDEX

inhibition mechanisms, 556—557 proapoptotic genes, 560—561 progression mechanisms, 557—558 research background, 551—552 viability factors, 558—559 PML gene nuclear bodies and, 333 VDR target genes, 170—171 Cell proliferation c-myb oncogene and, 534—536 c-myc oncogene and, 524—525 Jaks and Stats role in, 242—243 c-erbB-2 gene, c-myb interaction with, 534 CGP57148, TEL/PDGFR fusion protein and, 429 Chemokines, megakaryocyte transcription, cisregulatory element binding, 34 Chicken myelomonocytic growth factor (cMGF), C/EBP regulation, 122 Chimeric proteins, CBFB-MYH11 fusion protein, knock-in mouse models, 386—389 Chromatin structure erythroid Kru¨ppel-like factor (EKLF)-protein interaction, 77—78 Ikaros/Aiolos regulation of, 194—196 malignancy development and, 196 NF-E2 function and, 17—18 Chronic lymphocytic leukemia (CLL), c-myb oncogene and, 537—538 Chronic myelogenous leukemia (CML) EVI1 gene involvement, 398—399 Ikaros/Aiolos regulation of chromatin and, 196 Chronic myelomonocytic leukemia (CMML), TEL/ PDGFR fusion protein and, 425—426, 428—429 Cis-regulatory elements, megakaryocyte transcription factors, 32—34 c-Jun proto-oncogene AML1/ETO fusion protein, 418—419 PU.1 gene and myeloid lineage development, 106—108 Ras mediation, 108—109 c-Maf transcription factor, c-myb interaction with, 534 c-myb oncogene alterations aberrant transcriptional activation, 533—534 DNA-binding domain and CKII phosphorylation sites, 531—532 Myb interaction with other factors, 534 negative regulatory domain, 532—533 transcriptional activation domain, 532 CBP/p300 coactivator regulation AML1 gene and, 506 hematopoiesis and, 500—501 future research issues, 538 hematopoietic cells apoptosis, 536—537 differentiation, 536 proliferation, 534—536

603

leukemia amplification, rearrangement and expression, 537—538 protein structure, 529—531 structural characteristics, 521—522, 529 c-myc oncogene future research issues, 529 megakaryopoiesis transcription, overexpression studies, 42 multiple complexes and, 525—526 myeloid differentiation and lineage, 1,25(OH) D /VDR direct and indirect targets, 170 neoplastic transformation, 526—529 amplification, 527—528 cancer mutations, 527 multistep process for, 528—529 translocations, 526—527 proliferation, differentiation and apoptosis, 524—525 protein products, 523—524 structure and transcripts, 523 CNC domain, NF-E2 biochemistry and expression, 15—16 Coactivators acute promyelocytic leukemia (APL), monocytic differentiation, 173—175 erythroid Kru¨ppel-like factor (EKLF) transcription activation, 76—78 leukemia and CBP/p300 coactivators, 465—466 functions of, 474—477 histone acetyltransferases (HATs), 466—467 MLL-CBP t(11;16) (q23;p13) translocation, 470—472 MLL-p300 (t11;22)(q23;q13) translocation, 472 MOZ-CBP (t8;16) (p11;p13) translocation, 467—470 MOZZ-TIF2 inversion (inv)(8) (p11q13) translocation, 472—474 structural properties, 465 TIF2/nuclear receptor coactivator, 466 octamer factors OCA-B/OBF-1/Bob-1, 293—295 octamer-binding proteins, 292—293 octamer motif, 292 PU.1 gene, myeloid development, 106—107 retinoic acid receptor (RAR) transcription and, 328—329 Coiled-coiled domain PML gene structure, 331 nuclear bodies, 333 Stat family structure and activation, 238—239 Collaborator oncogenes, hox-induced leukemias, 140 Colony-forming cell (CFC), retinoic acid (RA) receptor activation, ATRA-enhanced CFC production in hematopoietic precursors, 155—156 Colony-forming unit (CFU) PU.1 gene, growth factor regulation, 110

604

INDEX

Colony forming unit (Contd.) Runx1 and Cbf b genes definitive hematopoiesis and, 90—92 gene dosage effects, 93—94 Colony-forming unit-granulocyte macrophage (CFUGM), retinoic acid (RA) receptor activation, ATRA-enhanced CFC production in hematopoietic precursors, 155—156 Colony-forming unit-spleen (CFU-S) all-trans retinoic acid (ATRA)-enhanced hematopoiesis, 156 hox gene function in normal hematopoiesis, 139 Competitive repopulating cells, hox gene function in normal hematopoiesis, 139 Core-binding factor (CBF) genes. See also specific CBF fusions and transcripts hematopoiesis lineages dosage effects, 93—94 function in later stages, 94—95 future applications, 95 gene expression, 92—93 hematopoietic cell emergence, 90—92 research background, 87—90 TEL/AML1 fusion protein, hematopoietic inhibition, 432—433 CREB (cyclic AMP response element-binding protein), CBP/p300 coactivator, leukemia and, 465—466 c-Rel gene cell viability and, 559 NF-KB family and, 552—554 cell cycle inhibition and, 556—557 Rel gene knockout analysis, 555—556 Cre-loxP recombination system, Pax5 gene-mediated late B-cell differentiation, 223—224 C-termini c-myb oncogene structure, 531—533 PML gene structure, 331 Stat family transcription activation, 238 Cyclin-A Ikaros family proteins, cell cycle regulation and nuclear localization, 192—194 myeloid differentiation and lineage, 1,25(OH) D /VDR direct and indirect targets, 170 Cyclin-dependent kinases (CDKs) myeloid differentiation and lineage, p21waf1,cip1, 164—167 NF-KB family and, cell cycle inhibition and, 557 Cysteine-rich secreted protein (CRISP-3), oct-2 gene expression, 299 Cytokines. See also specific interferons and interleukins BCL-6 mutations and, 279—280 T cell differentiation and function, 281—282 C/EBP in myeloid cells and, 120 deletion in animal models, 125 gene regulation activities, 122—123

E proteins and, 266 Janus kinases and lymphoid cell development, 230—232 signal transduction initiation, 234 megakaryocyte development and, 31—32 octamer factors and signaling in, 301 Pax5 proteins, pro-B cell development and, 219—220 signaling receptors Janus kinase and Stats family identification, 229—230 Janus kinase structure and, 233 Stat function in malignant transformation and, 243 Stat1 function, 239 Stat2 function, 239 Stat3 function, 239—240 Stat4 function, 240 Stat5 function, 240—241 Stat6 function, 240

Definitive hematopoiesis LMO2 gene family and, 485 Runx1 and Cbfb genes and, 90—92 gene expression, 92—93 Differential screening, VDR target genes myeloid differentiation and lineage, 164—165 1,25(OH) D /VDR, U937 differential screening, 166—167 Diffuse large-cell lymphoma (DLCL) BCL-6 translocations and, 274, 283—284 oncogenesis in, 272—273 Dimer-dimer interaction, Stat family structure and activation, 236—237 dMax variant, c-myc oncogene structure, 524 DNA-binding proteins AML1 binding characteristics, 411—413 AML1/ETO transcriptional repression, 416—417 c-myb oncogene structure, 530—531 CKII phosphorylation and, 531—532 E proteins in B-cell development, 261—262 erythroid Kru¨ppel-like factor (EKLF), 72, 80—81 ETO family, 414—415 EVI1 gene, malignant hematopoiesis and, 394—396 hox gene expression and, 142—143 Ikaros family protein structure and, 183—185 dominant negative mutations and lymphoid defects, 188—189 NF-KB as, 552 octamer factor structure and function, 291—292 coactivators, 292—293 Pax5 protein and gene repression, 221—222 PU.1 binding sites, 203—205 Spi-B protein, 208—211 Stat family structure and activation, 235—238 TAL1/SCL transcription factor preferences, 59—60

INDEX

DNA methytransferase (DMT), MLL gene domains and, 449—450 Domain mapping. See also Transcription activation domain; specific domains, e.g. Kinase domain c-myb oncogene structural alteration, 532 aberrant activation, 533—534 c-myb oncogene structure, 531—533 MLL-CBP fusion protein, 471—472 MLL genes, 447—448 trx gene similarities, 448—450 MOZ-CBP fusion protein, 468—470 PLZF gene, 343 PML gene expression, PODs, 332—333 PU.1 gene structure-function relationships, 204—205 Rel/NF-KB family, 552—553 Stat family structure and activation, 236—239, 238 Dominant negative mutations, Ikaros family proteins, lymphoid defects and, 188—189 Drosophila trithorax (trx) gene, MLL genes and structural similarities, 448—449 unconserved domains, 449—450

Early B-cell factor (EBF) B cell development and development in absence of, 316—317 future research issues, 320 genetic targets for, 317—320 structure and function, 313—315 E proteins in B-cell development, 261 E-box sequences c-myc oncogene, 525—526 LMO2 gene family in T-cell acute lymphoblastic leukemia (T-ALL), 490—492 Ectopic gene expression c-myb oncogene and hematopoiesis, 534—537 early B-cell factor (EBF) and disruption mechanisms, 316—317 genetic targets, 319—320 megakaryocyte transcription factors, 38—39 myeloid differentiation and lineage, p21waf1,cip1, 165—167 NF-KB family and cell cycle inhibition, 556—557 regulatory mechanisms, 559—560 Pax5 oncogenic activation in Non-Hodgkin’s lymphoma, 224—225 E2F-1, megakaryopoiesis transcription, overexpression studies, 42—44 EF-1 protein, PML gene partnership with, 334 EGF-receptor (EGF-R), PML gene transcription and, 333 Embryogenesis erythroid Kru¨ppel-like factor (EKLF) gene expression, 73—74, 81 LMO2 gene family and vascular formation, 485— 488 Runx1 and Cbfb genes

605

definitive hematopoiesis and, 90—92 hematopoiesis lineages, 90—92 TAL1/SCL transcription factor function in, 57 Embryonic stem (ES) cell differentiation CBFB-MYH11 fusion protein, knock-in mouse model, 385—389 hox gene function in normal hematopoiesis, 139 EML cells RXR-RAR heterodimer activation, AML sensitivity in immature vs. mature myeloid cells, 154—155 RXR-RAR response element activation nuclear hormone receptor corepressor activity vs. MPRO cells, 153—154 SCF-dependent cells, 153 Enhancer regions c-myc oncogene and, 526—529 Ig gene, octamer activation of, 300—301 ENU chimera model, CBFB-MYH11 fusion protein, knock-in mouse models, 386—389 E proteins E1A protein CBP/p300 coactivator, 498—499 E2A protein encoding, 501—502 GATA-1 and, 502—503 hematopoiesis and, 499—500 c-myb interaction with, 534 E2A protein B-cell development, 259—262 CBP/p300 coactivator regulation, 501—502 early B-cell factor (EBF) disruption and, 316—317 T-cell development, 262—264 T-cell lymphoma, 264—265 transactivation in B and T cells, 258—259 E12 protein, CBP/p300 coactivator regulation, 501—502 E47 protein, CBP/p300 coactivator regulation, 501—502 future research issues, 265—266 lymphocyte development HLH domain structure and function, 258 research background, 257—258 Epstein-Barr virus (EBV), Janus kinases and, 232 E-RC1 CBP/p300 coactivator catalysis, 509—510 erythroid Kru¨ppel-like factor (EKLF)-protein interaction, 77—78 Erythroid development erythroid Kru¨ppel-like factor (EKLF) and future applications, 79—81 gene expression, role in, 72—74 identification of EKLF, 71—72 regulation of, 78—79 research background, 71 transcriptional activation mechanism, 74—78 Friend of GATA-1 (FOG) and, 4—5 GATA-1 and Friend of GATA-1 (FOG) and, 6—8

606

INDEX

Erythroid development (Contd.) gene expression, GATA-1:FOG complex, 9 structure and function, 1—2 in vivo cofactor, 2—4 NF-E2 function and, 19—20 Erythroid Kru¨ppel-like factor (EKLF) CBP/p300 coactivator regulation, 503—504 catalysis and, 509—510 erythroid lineage development and future applications, 79—81 gene expression, role in, 72—74 identification of EKLF, 71—72 regulation of, 78—79 research background, 71 transcriptional activation mechanism, 74—78 TAL1/SCL transcription factor modifications, 54 Estrogen receptor (ER) erythroid maturation and GATA-1 function, 8 Friend of GATA-1 (FOG) assay using, 3—4 ETO family members and function of, 413—415 t(8;21) AML translocation, 410—411 transcriptional effects, 415—416 Ets family. See also PU.1 gene; TEL gene CBP/p300 coactivator regulation, 505 c-myb interaction with, 534 megakaryopoiesis transcription factors GATA/Ets repeats, 36—37 in vivo promoter studies, 35—36 ETV6 gene family. See TEL fusion proteins EVI1 gene, malignant hematopoiesis EVI1 identification, 394 future research issues, 403—404 gene expression and function, 396—397 intro differentiation and cell growth, 402—403 MDS1/EVI1 fusion, 399—400 myeloid leukemia involvement, 397—399 research background, 393—394 structural characteristics, 394—396 transcriptional function, 400—401

F(ab ) fragments, Aiolos family proteins, lymphoproliferations in null mice and, 191—192 Fas gene promoters, NF-KB family and, proapoptotic activity, 560—561 FcRIIB1 receptor, Aiolos family proteins, lymphoproliferations in null mice and, 191—192 Fibroblast growth factor (FGF), Stat1 signaling, 239 F-Kru¨ppel-like factor (FKLF), DNA-binding activity, 81 Fli-1 promoter, megakaryopoiesis transcription factors, 36 Fluorescence-activated cell-sorting (FACS) C/EBP expression and, 119—120 retinoic acid (RA) receptor activation ATRA-enhanced CFC production in

hematopoietic precursors, 155—156 short- and long-term marrow repopulation in stem cell assays, 157—158 Follicular center lymphoma (FL), BCL-6 mutations and, 272—273 FPD/AML genetics, Runx1 and Cbfb genes, gene dosage effects, 93—94 Friend of GATA-1 (FOG) erythroid and megakaryotic differentiation, 2—4 erythroid development, implications for, 6—8 as essential GATA-1 cofactor, 9—10 gene expression, GATA-1:FOG complex and, 9 LMO2 gene family, multiprotein transcription, 489 megakaryocyte transcription factors cis-regulatory elements, 32—34 targeted disruption (knockout) via homologous recombination, 41 megakaryotic development requirement for, 5—6 PU.1 gene, myeloid lineage development and, 106—107 structural diagram of, 2 terminal erythroid maturation, 4—5 Fusion proteins. See specific fusion proteins, e.g. PML-RAR fusion protein

Gain-of-function structures, AML1/ETO fusion protein, 418—419 GAL family, erythroid Kru¨ppel-like factor (EKLF) transcription activation, 74—78 c cytokines, Janus kinases lymphoid cell development and, 230—232 structure and function, 233 T cells, E proteins and, 265 5 gene B cell development and, 313—315 early B-cell factor (EBF) interaction with, 319—320 GATA-1 CBP/p300 coactivator regulation, 502—503 catalysis and, 509—510 Ets family, 505 erythroid Kru¨ppel-like factor (EKLF) and gene expression regulation, 74, 79 transcription activation, 77—78 Friend of GATA-1 (FOG) as in vivo cofactor, 2—4 erythroid development, 4—8 megakaryotic GATA-1-independent development, 5—6 LMO2 gene family, multiprotein transcription, 488—489 megakaryopoiesis transcription factors, 34—35 GATA/Ets repeats, 36—37 targeted disruption (knockout) via homologous recombination, 40—41 in vitro ectopic expression, 38—39 N-finger and C-finger structures in, 2, 7—8 PU.1 gene and

INDEX

protein-protein interactions, 105—106 rescue assay development, 110—111 stem cell induction, 104—105 structure and function, 1—2 TAL1/SCL transcription factor modifications, 54, 60—62 GATA-1\ erythroid cell line (G1E) erythroid maturation and, 8 Friend of GATA-1 (FOG) assay using, 3—4 GATA-2 erythroid Kru¨ppel-like factor (EKLF) and, 79 PU.1 gene and, protein-protein interactions, 105— 106 structure and function, 1—2 GATA-3 structure and function, 1—2 TAL1/SCL transcription factor and, 60—62 GATA-4, structure and function, 1—2 GBX2 gene, c-myb oncogene structural alteration, 531—532 Gene dosage, Runx1 and Cbfb genes, hematopoiesis and, 93—94 Gene expression BCL-6 mutations, 277—278 histogenetic markers, 282—283 C/EBPs in myeloid cells, 120—122 erythroid Kru¨ppel-like factor (EKLF) regulation of, 78—79 role in erythroid expression, 72—74 EVI1 gene, 396—397 GATA-1:FOG complex in erythroid development and, 9 hox genes human hematopoietic cells, 136 murine hematopoietic cells, 137 noncluster genes, 137—138 upstream regulators, 140—142 Ikaros family genes, 185—186 megakaryocyte transcription factors cis-regulatory elements, 32—34 overexpression studies, 42—44 in vitro ectopic expression, 38—39 NF-E2 biochemistry and, 14—16 erythroid cell differentiation and, 19—20 octamer factors B-cell-specific expression, 298—299 cytokine promoters, 302—303 Pax5 proteins, B cell repression and, 220—222 PLZF gene, 344 PML gene structure, 332 nuclear bodies, 332—333 PU.1/Spi-1 identification, 202—203 Runx1 and Cbfb genes, definitive hematopoiesis and, 92—93 TAL1/SCL transcription factor regulation, 58—59 TEL/AML1 fusion protein, malignant transformation and, 433—434

607

Germinal centers (GCs) Aiolos family proteins, B-cell maturation and null mutation, 189 BCL-6 mutations and lymphoid cell development, 272—273 normal vs. NHL B cells, 276—277, 280—281 regulators and effectors, 279—280 octamer involvement in formation of, 301 Germ-line V gene transcription and rearrangement, octamer factors and, 297—298 Globin genes erythroid Kru¨ppel-like factor (EKLF) gene expression, 73—74, 80—81 protein interaction, 77—78 NF-E2 function and, 16—18 erythroid cell differentiation and, 20 3-Globin, erythroid Kru¨ppel-like factor (EKLF) transcription, 77—78, 80—81 Glycoprotein (GP)IIb megakaryocyte transcription factors, cis-regulatory elements, 32—34 platelet biogenesis and, 21—23 G1 progression genes, NF-KB family and, 561—563 Granulocyte-colony-stimulating factor (GM-CSF), MDS1/EVI1 fusion and, 403 Granulocyte-monocyte colony-stimulating factor (GM-CSF) hematopoiesis activation, RXR-RAR response elements in MPRO cells, 152—153 PU.1 gene and regulation of, 109—110 stem cell induction, 104—105 Grg4 protein, Pax5 protein interaction with, 221—222 Growth factors c-myc oncogene and, 524—525 PU.1 gene and, stem cell induction and, 104—105 PU.1 gene regulation of, 109—110

Heat-stable antigen (HSA), B cell development and, 313—315 HEB protein B-cell development, 262 T-cell development and, 263—264 Helios family proteins dominant negative mutations and lymphoid defects, 189 gene expression, 185—186 structure and function, 183—185 Helix-loop-helix (HLH) family early B-cell factor (EBF) development, 315 E protein structure, 257—258 development and, 259 myeloid differentiation and lineage, 1,25(OH)2 D3/VDR direct and indirect targets, 169—170 structure and function, 258 TAL1/SCL transcription factor and, 53—54

608

INDEX

Helix-turn-helix structure, homeobox genes, 133—135 Hematopoiesis AML1 transcriptional regulation, 412—413 CBFB/RUNX1 genes dosage effects, 93—94 function in later stages, 94—95 future applications, 95 gene expression, 92—93 hematopoietic cell emergence, 90—92 research background, 87—90 CBP/p300 coactivator and, 499—506 AML1 and, 505—506 C/EBP and, 504—505 c-myb regulation, 500—501 E2A protein encoding, 501—502 EKLF and, 503—504 Ets family and, 505 GATA-1 and, 502—503 NF-E2 and, 503 c-myb oncogene and apoptosis, 536—537 differentiation, 536 proliferation, 534—536 EVI1 gene rearrangements, malignancy and EVI1 identification, 394 future research issues, 403—404 gene expression and function, 396—397 intro differentiation and cell growth, 402—403 MDS1/EVI1 fusion, 399—400 myeloid leukemia involvement, 397—399 research background, 393—394 structural characteristics, 394—396 transcriptional function, 400—401 Friend of GATA-1 (FOG) absence and failure of, 4—5 GATA-1-FOG complex and, 7—8 hox gene regulation future research, 144 human gene expression, 135 leukemia hematopoiesis collaborator oncogenes, 140 human leukemias, 140 mouse acute leukemia, 139—140 loss-of-function studies, 137—138 lymphoid cell gene expression, 135—136 murine model gene expression, 136 noncluster gene expression, 136—137 overexpression studies, 138—139 protein-DNA interaction, 142—143 research background, 133—135 target genes, 143—144 upstream regulators, 140—142 in vitro EX differentiation model, 139 Ikaros null mutations and, 187—188 LMO2 gene family developmental regulation by, 483—484 multiprotein transcription, 488—489

primitive and definitive phases, 485 vascular formation and mouse embryogenesis, 485—488 MLL genes and acute leukemias, associated mutations, 452—455 AML-associated amino-terminal mutation, 452—455 fusion proteins from chromosomal translocations, 452 PHD finger 1 mutants with T-ALL, 455 ALL-1 related (ALR) protein, 450 alternatively spliced forms, 450 domain rupture and putative functions, 447—448 Drosophila trithorax (trx) gene similarities to, 448—449 unconserved domains, 449—450 fusion proteins and hematopoietic progenitors, 455—456 future research issues, 457—458 mammalian development, 450—451 partner proteins and leukemogenesis, 456—457 structural properties, 447 transformation mechanisms of fusion proteins, 455—456 NF-E2 biochemistry and expression, 15—16 retinoic acid (RA) receptor activation AML sensitivity nad RXR-RAR activation, 154—155 ATRA-enhanced CFC production in hematopoietic precursors, 155—156 ATRA-enhanced CFU-S production, 156 dominant negative RAR constructs, 151—152 nuclear hormone receptor corepressor activity in EML vs. MPRO cells, 153—154 RXR-RAR activation of GM-CSF-dependent MPRO cells, 152—153 RXR-RAR blunted activation in SCF-dependent EML cells, 153 TAL1/SCL transcription factor and developmental function, 57 DNA-binding preferences, 59—60 future research issues, 63 gene expression regulation, 58—59 leukemia involvement and discovery of, 51—53 oncogenic properties, 54—56 postnatal hematopoiesis, 58 posttranslational modifications, 54 protein products and interaction, 53—54 RNA and protein distribution, 56—57 target genes, 62—63 transcriptional properties, 60—62 TEL/AML1 fusion protein, core-binding factor (CBF) gene inhibition by, 432—433 TEL gene and malignant transformations, 426—427 normal development, 426 transcription factors and, in vivo studies, 574—584

INDEX

Hematopoietic stem cells (HSC) B and T lymphocytes and, 255—256 GATA-1 and development of, 1—2 HERF1 gene, AML1 targeting, 413 Hexapeptide group, homeobox genes, 134—135 Hex genes, expression in hematopoiesis, 137—138 HIS3 repression, GATA-1-FOG complex and, 7—8 Histogenetic markers, BCL-6 mutations as, 282—283, 284—285 Histone acetyltransferase (HAT) CBP/p300 coactivator and, 499 coactivators and leukemia CBP/p300 coactivator, 466 protein structure and classification, 466—467 research background, 465 E2A protein transactivation, 258—259 MOZ-CBP fusion protein, 469—470 MOZ-TIF2 inversion, 474 Histone deacetylase (HDAC) AML1/ETO transcriptional repression, 416—417 BCL-6 mutation and, 278—279 Ikaros/Aiolos regulation of chromatin, 194—196 malignancy development and, 196 PML-RAR fusion protein and, 337—338 RXR-RAR response element activation, EML vs. MPRO cells, 154 TAL1/SCL transcription factor and, 62 HL60 cells, PML-RAR fusion protein, 340 HL X gene, expression in hematopoiesis, 138 HMG1/Y complex, MLL gene domains and, 449—450 hMSH2 gene, TAL1/SCL transcription factor, oncogenesis and, 55 Hodgkin’s disease BCL-6 mutations and, 283 NF-KB family and cell cycle progression, 558 HOM-C cluster structure and function, 133—135 upstream regulation of gene expression, 141 Homeobox genes. See Hox genes Homologous recombination early B-cell factor (EBF) development, 314—315 Ikaros family protein structure and, 184—185 megakaryocyte transcription factors, targeted disruption (knockout) techniques, 40—42 HoxA10 target gene, myeloid differentiation and lineage, 167—169 Hox genes hematopoiesis regulation future research, 144 human gene expression, 135 loss-of-function studies, 137—138 lymphoid cell gene expression, 135—136 murine model gene expression, 136 noncluster gene expression, 136—137 overexpression studies, 138—139 protein-DNA interaction, 142—143 research background, 133—135

609

target genes, 143—144 upstream regulators, 140—142 in vitro EX differentiation model, 139 leukemia hematopoiesis regulation collaborator oncogenes, 140 human leukemias, 140 mouse acute leukemia, 139—140 structure and function, 135—136 Hypersensitive sites (HS) erythroid Kru¨ppel-like factor (EKLF)-protein interaction, 77—78 gene expression regulation, 78—79 NF-E2 function and, 16—18

IAP genes, NF-KB family and cell cycle antiapoptosis, 559—560 Id proteins, E proteins and future research issues, 266 T-cell development, 263—264 IEX-IL gene, NF-KB family and cell cycle antiapoptosis, 560 Ig gene enhancers, octamer activation of, 300—301 octamer regulation of, in activated B cells, 299—300 Ikaros family proteins future applications and research, 196—197 gene expression, 185—186 lymphocyte development, 186—189 dominant negative mutation and lymphoid defects, 188—189 lymphocyte differentiation, 194—196 chromatin regulation, 194—196 malignancy development mechanisms, 196 lymphocyte homeostasis, 190—194 cell cycle regulation, 192 lymphoproliferation in null mice, 191 nuclear localization changes, 192—194 T-cell malignancies in Ikaros DN]/- mice, 190—191 structure and function, 183—185 I-KB family cell cycle inhibition and, 556—557 cell cycle progression and, 557—558 cell viability and, 558—559 structural characteristics, 553—554 IL-3 oncogene, hox-induced leukemias, 140 Immunoglobulin A (IgA), CBF genes and heavy chain switching in, 95 Immunoglobulin heavy-chain (IgH) locus, B cell development and, 313—315 INK4A/INK4B loci, TAL1/SCL transcription factor, oncogenesis and, 55 Interferon/ malignant transformation and, 243 Stat2 activation, 239 Stat4 activation, 240

610

INDEX

Interferon regulatory factor (IRF) family, Pip coactivator, 205—206 Interferons. See also Cytokines; specific interferons PML gene and, 334—335 Interferon-stimulated gene factor-3. See STATs family Interleukin-2, octamer dependence and, 302 Interleukin-4 BCL-6 mutations and, 279—280 octamer dependence and, 302 Stat6 function and, 240 Interleukin-5, octamer-dependent gene regulation, 302—303 Interleukin-6, octamer factors and signaling, 301 Interleukin-7, early B-cell factor (EBF) disruption and, 316—317 Interleukin-10, Stat3 function and, 239—240 Interleukin-12, Stat4 function and, 240 Interleukin-13, Stat6 function and, 240 Interleukins. See Cytokines; specific interleukins Intra-aortic hematopoietic clusters, Runx1 and Cbf b genes, definitive hematopoiesis and, 92 Inv(16), acute myeloid leukemias (AML) and, 379— 380 In vitro ES cell differentiation, hox gene function in normal hematopoiesis, 139 In vivo promoter studies CBP/p300 coactivator regulation, 507—508 megakaryopoiesis transcription factors, 34—38 Ets family members, 35—36 GATA/Ets repeats, 36—37 GATA-1 promoters, 34—35 NF-E2 in megakaryocytes, 37 repressor elements, 38 Sp1 involvement, 37 transcription factors and hematopoiesis, 574—584 Inv(8)(p11q13), MOZ-TIF2 inversion, 473—474

Janus kinases (Jaks) associated molecules, 233—234 cellular proliferation and malignant transformation, 242—243 cytokine signaling and, 229—230 JAK1 kinase, lymphoid cell development, 231—232 JAK2 kinase erythroid Kru¨ppel-like factor (EKLF) gene expression, 79 lymphoid cell development, 230—232 TEL/JAK2 fusion protein, 429—430 JAK3 kinase, lymphoid cell development, 230—231 lymphoid cell development, 230—232 noncytokine receptors, 232 signal transduction initiation, 234 Stat5b-RAR fusion protein, 350—351 structure of, 232—233 Tyk2, lymphoid cell development, 232

Jun kinase 1(JNK1), PU.1 gene and myeloid lineage development, c-Jun proto-oncogene, 107—109 Jurkat cell line, E proteins and T-cell acute lymphoblastic leukemia (T-ALL), 265

Kinase domain, Janus kinases and, 230 autophosphorylation mechanisms, 233—234 Knock-in mouse genetics AML1/ETO fusion protein, 417—418 CBFB-MYH11 fusion protein, 385—389 Knockout mouse genetics CBP/p300 coactivator regulation, 507—508 megakaryopoiesis transcription factors, targeted disruption (knockout) via homologous recombination, 40—42 NF-KB family, Rel gene knockout analysis and, 555—556 Runx1 and Cbfb genes and, 95 Kru¨ppel zinc finger family. See also Erythroid Kru¨ppel-like factor (EKLF) and BCL-6 mutation and, 278—279 erythroid maturation and GATA-1 function, 8 EVI1 gene, malignant hematopoiesis and, 394—396

lacZ reporter, TAL1/SCL transcription factor, protein distribution and, 56—57 LAP domain, MOZ-CBP fusion protein, 468 LAP/LIP production, C/EBP in myeloid cells, 120 Leishmania infection, BCL-6 mutations and, 281—282 Leucine 115, c-myc oncogene mutations, 527 Leucine zipper domain, c-myb oncogene structure, 533 Leukemias AML1/ETO role in leukemogenesis, 416—418 CBF-SMMHC fusion gene and, 384 CBP/p300 coactivator regulation, chromosomal translocations, 506—507 c-myb oncogene, amplification, rearrangement and expression in, 537—538 c-myc oncogene future research issues, 529 multiple complexes and, 525—526 neoplastic transformation, 526—529 amplification, 527—528 cancer mutations, 527 multistep process for, 528—529 translocations, 526—527 proliferation, differentiation and apoptosis, 524— 525 protein products, 523—524 structure and transcripts, 523 coactivators and CBP/p300 coactivators, 465—466 functions of, 474—477 histone acetyltransferases (HATs), 466—467

INDEX

MLL-CBP t(11;16) (q23;p13) translocation, 470—472 MLL-p300 (t11;22)(q23;q13) translocation, 472 MOZ-CBP (t8;16) (p11;p13) translocation, 467— 470 MOZZ-TIF2 inversion (inv)(8) (p11q13) translocation, 472—474 structural properties, 465 TIF2/nuclear receptor coactivator, 466 hox gene hematopoiesis regulation collaborator oncogenes, 140 human leukemias, 140 mouse acute leukemia, 139—140 Jaks and Stats role in malignant transformation and, 242—243 MLL genes associated with, 452—455 AML-associated amino-terminal mutation, 452— 455 fusion proteins from chromosomal translocations, 452 partner proteins and leukemogenesis, 456—457 PHD finger 1 mutants with T-ALL, 455 Runx1 and Cbfb genes, role of, 94—95 TEL/ABL fusion protein, 430 TEL/AML1 fusion protein in core-binding factor complex inhibition, 432—433 gene expression and transformation, 433 malignant transformation, 433—434 pediatric acute lymphoblastic leukemia clinical significance, 435 favorable prognosis with, 436—438 minimal residual disease detection, 439 prevalence in, 435—436 structural characteristics, 431—432 TEL gene structure, 426—427 TEL/JAK2 fusion protein, 429—430 TEL/PDGFR fusion protein, 428—429 TEL/TRKC fusion protein, 430—431 tyrosine kinase fusions in, 427—428 LIF function, Stat3 activation and, 239 Ligand binding, Janus kinases and, signal transduction initiation, 234 LIM proteins homeobox genes, 134—135 LMO gene family and, 484—485 TAL1/SCL transcription factor, 53—54 oncogenesis functions, 55 Lineage-restricted transcription factor, NF-E2 as example of, 13—14 Lipopolysaccharide (LPS) cells, NF-KB family and cell cycle progression, 557—558 LMO3 gene, discovery of, 484 LMO4 gene, discovery of, 484 LMO1 gene family discovery of, 484 TAL1/SCL transcription factor and, 60—62 t(11;14) chromosomal translocation, 485

611

LMO2 gene family cell fate determination hematopoietic multiprotein complexes, 488—489 mouse embryonic vascular formation, 485—488 primitive and definitive hematopoiesis, 485 future research issues, 492 LIM-domain-only protein encoding, 484—485 molecular function, multiprotein complexes, 488 structural properties, 484 TAL1/SCL transcription factor and, 60—62 T-cell acute leukemia, 489—492 differentiation inhibition, 489—490 protein interaction models, 490—492 translocation, transcription and developmental regulation functions, 483—484 t(11;14) translocations with LMO1, 485 LMP1 protein, Janus kinases and, 232 Locus-control regions (LCRs) erythroid Kru¨ppel-like factor (EKLF) gene expression, 74 GATA-1 and, CBP/p300 coactivator regulation, 502—503 NF-E2 structure and, 13—14 erythroid development, 16—18 Long-term culture-initiating cells (LTC-IC), hox gene function in normal hematopoiesis, 138—139 Long-term repopulating hematopoietic stem cells (LTR-HSCs), Runx1 and Cbfb genes, definitive hematopoiesis and, 90—92 Loss-of-function studies hox gene function in normal hematopoiesis, 138—139 Ikaros family proteins, lymphoproliferations in null mice and, 191 MLL fusion proteins, 456 Loss of heterozygosity (LOH), Ikaros family proteins, malignancies in Ikaros DN]/- mice, 190—191 LRF transcription factor, BCL-6 mutation signaling and, 280 Ly1 cell line, BCL-6 gene mutations, 276—277 Lymphocyte development BCL-6 mutations and, 271—273 CBF genes and, 95 hox gene expression in, 136—137 Ikaros family proteins, 186—189 differentiation mechanisms, 194—196 chromatin regulation, 194—196 malignancy development mechanisms, 196 dominant negative mutation and lymphoid defects, 188—189 homeostasis mechanisms, 190—194 cell cycle regulation, 192 lymphoproliferation in null mice, 191 nuclear localization changes, 192—194 T-cell malignancies in Ikaros DN>\ mice, 190—191 Janus kinases and development of, 230—232

612

INDEX

Lymphocyte development (Contd.) STATs family and development of, 239—241 Lymphomas, E2A protein and, 264—265

Macrophage-colony-stimulating factor (M-CSF) B and T lymphocytes development, 255—256 PU.1 gene and regulation of, 109—110 Maf recognition element (MARE) megakaryopoiesis transcription factors, targeted disruption via homologous recombination, 42 NF-E2 biochemistry and expression, 15—16 Malignant transformation EVI1 gene and hematopoiesis EVI1 identification, 394 future research issues, 403—404 gene expression and function, 396—397 intro differentiation and cell growth, 402—403 MDS1/EVI1 fusion, 399—400 myeloid leukemia involvement, 397—399 research background, 393—394 structural characteristics, 394—396 transcriptional function, 400—401 Jaks and Stats role in, 242—243 TEL/AML1 fusion protein, gene expression mechanisms, 433—434 Mammalian development, MLL gene and, 450—451 MBD1 protein, MLL gene domains and, 449—450 mb-1 promoter, early B-cell factor (EBF) genetic targets, 317 MDS1/EVI1 fusion EVI1 and, 399—400 malignant hematopoiesis and, 393—394 myeloid leukemias and, 399 transcriptional functions, 400—402 in vitro hematopoietic differentiation and cell growth control, 402—403 Megakaryotic differentiation Friend of GATA-1 (FOG) functions in, 5—6, 10 GATA-1 and structure and function, 1—2 in vivo cofactor, 2—4 NF-E2 function and, 20—23 transcription factors and cis-regulatory element binding, 32—34 research background, 31—32 transgenic mice in vivo studies, 39—44 in vitro ectopic expression, 38—39 in vivo promoter studies, 34—38 Ets family members, 35—36 GATA/Ets repeats, 36—37 GATA-1 promoters, 34—35 NF-E2 in megakaryocytes, 37 repressor elements, 38 Sp1 involvement, 37 Meis1 oncogene hox gene-DNA binding, 143

hox-induced leukemias, 140 Mel-18 gene, hox gene expression, upstream regulation with, 142 Minimal residual disease detection, TEL/AML1 fusion protein, pediatric patients, 439 Misexpression studies, Pax5 oncogenic activation in Non-Hodgkin’s lymphoma, 224—225 Mitogen-activated protein kinase (MAPK), c-myb oncogene structure, 533 MLL-AF9 fusion protein, hematopoietic transformation, 455—456 MLL-AF10 fusion protein, leukemogenesis and, 457 MLL-CBP fusion protein, leukemia and translocation, 470—472 MLL-ENL fusion protein hematopoietic transformation, 455—456 leukemogenesis and, 457 MLL genes acute leukemias, associated mutations, 452—455 AML-associated amino-terminal mutation, 452—455 fusion proteins from chromosomal translocations, 452 PHD finger 1 mutants with T-ALL, 455 ALL-1 related (ALR) protein, 450 alternatively spliced forms, 450 domain rupture and putative functions, 447—448 Drosophila trithorax (trx) gene similarities to, 448—449 unconserved domains, 449—450 fusion proteins and hematopoietic progenitors, 455—456 future research issues, 457—458 hox gene expression, upstream regulation with, 141 mammalian development, 450—451 partner proteins and leukemogenesis, 456—457 structural properties, 447 MLL-p300 fusion protein, leukemia translocation, 472 Monocytic differentiation, acute promyelocytic leukemia, vitamin D3 transcriptional targets, 171—175 Mouse acute leukemia, hox gene hematopoiesis regulation, 139—140 Mouse models CBFB-MYH11 fusion protein, 385—389 LMO2 gene family and vascular formation in, 485—488 PML-RAR fusion protein, 340—341 Mouse myelomas, c-myc oncogene transformation, 526—527 MOZ-CBP fusion protein, leukemia translocations, 467—470, 477 MOZ-TIF2 inversion, leukemia translocations, 472—474 MPRO promyelocytes RAR403, hematopoiesis activation and, 151—152

INDEX

RXR-RAR heterodimer activation, AML sensitivity in immature vs. mature myeloid cells, 154—155 RXR-RAR response element activation in GM-CSF-dependent cells, 152—153 nuclear hormone receptor corepressor activity vs. EML cells, 153—154 SCF-dependent EML cells, 153 mRNA c-myb oncogene aberrant transcriptional activation, 533—534 c-myc oncogene transformation, 527 PML gene expression, 332 MRP8 gene, CBFB-MYH11 fusion protein, transgenic mice models, 385 MTG16 AML1 fusion with, 410 ETO gene family and, 415 MTG8 gene, ETO members and, 414—415 Multi-level gene regulation, BCL-6 mutations, 277— 278 Multiprotein complexes BCL-6 mutations, 277—278 CBP/p300 coactivator regulation, 508 c-myc oncogene, 525—526 ETO gene family, 413—416 LMO2 gene family bridging molecule function, 488 hematopoietic development and, 488—489 Multistep transformation, c-myc oncogene and, 528— 529 Murine erythroleukemia (MEL) cells, erythroid Kru¨ppel-like factor (EKLF) and, 72 Murine hematopoietic cells, hox gene expression, 137 Murine leukemia virus (MuLV), c-myc oncogene multistep transformation models, 528—529 Myb-binding sequence, c-myb oncogene development and, 530—531 Myb protein C/EBP regulation, 122 c-myb oncogene interaction with other factors, 534 Myelodysplastic syndrome (MDS), EVI1 gene involvement, 397—399 Myeloid leukemias, EVI1 gene involvement, 397—399 Myelopoiesis all-trans retinoic acid (ATRA) effects on, 158—159 C/EBP family and deficient animal models, 123—126 gene regulation by, 122—123 research background, 117 transportation factors in, 117—122 PU.1 gene c-Jun mediation of Ras, 108—109 future applications, 111 genetic model for, 208—211 growth factor receptor regulation, 109—110 lineage-specific coactivators, 105—108 protein-protein interactions, 104—105

613

rescue assay structure-function development, 110—111 research background, 103 stem cell induction, 103—105, 109 structure-function relationships, 204—205 target genes, 110 RA receptor regulation and, 155 retinoic acid receptor (RAR) and, 329 retinoic acid receptors AML sensitivity in immature vs. mature myeloid cells, 154—155 ATRA enhancement of CFC production, 155—159 future applications, 159—160 hematopoietic lineage-specific activation, 151—155 myelopoiesis regulation, 155 PML-RAR receptor fusion protein, 150 research background, 149—150 Spi-B genetic model, 208—211 Stat3 activation and, 239—240 vitamin D3 transcriptional targets acute promyelocytic leukemia, 171—175 1,25(OH)2 D3 induction, 172—173 p21 gene expression, 173—175 future applications, 175 research background, 163—164 VDR target genes, 164—171 cell cycle arrest and differentiation, 170—171 HoxA10, 167—169 1,25(OH) D /VDR, 169—170 p21waf1,cip1, 164—167 Myeloproliferative (Mpl) receptor, megakaryopoiesis transcription factors, 32 cis-regulatory element binding, 32—34 Ets family promoter studies, 36 MYH11 gene, acute myelogenous leukemias (AMLs), research background, 379—380 MYST domain, MOZ-CBP fusion protein, 468

NB4 cells, PML growth suppression and, 335 N-CoR AML1/ETO transcriptional repression, 416—417 ETO binding, 415—416 PML-RAR fusion protein and, 337—338 Negative regulatory domain, c-myb oncogene alteration, 531—533 Neoplastic transformation, c-myc oncogene involvement amplification, 527—528 multistep transformation, 528—529 mutations, 527 translocations, 526—527 Nervy homology regions (NHR), ETO gene family, 414—415 NF-E2-related factor 1 (Nrf1/LCR-F1/TCF11) erythroid cell differentiation and, 20 NF-E2 function and, 18—19

614

INDEX

NF-E2-related factor 2 (Nrf2) erythroid cell differentiation and, 20 NF-E2 function and, 18—19 N-finger structures BCL-6 mutation and, 278—279 GATA-1, 2, 7—8 Ikaros family proteins, 183—185 PML gene structure, 331 NF-KB family cancer and, 561 C/EBP gene regulation, 123 cell cycle regulation apoptotic genes, 559—560 inhibition mechanisms, 556—557 proapoptotic genes, 560—561 progression mechanisms, 557—558 research background, 551—552 viability factors, 558—559 function of, 554—555 future research issues, 561—563 octamer involvement in, 301 Rel gene knockout studies, 555—556 structural characteristics, 552—553 NF-M protein, C/EBP regulation, 122 NH2 terminal regions, NF-E2 biochemistry and expression, 15—16 NIH3T3 cells AML1/ETO fusion protein, 418 ETO gene family and, 415 NK cells hox gene expression in, 136—137 Ikaros family gene expression, 186 Noncluster homeobox genes, expression of, 137—138 Noncytokine receptors, Janus kinases and, 232 Non-Hodgkin’s lymphoma BCL-B expression, 277—278 BCL-6 mutations clinical relevance of translocation, 283—284 future research, 284—285 normal and NHL B cells, 275—277 regulators and effectors, 279—280 role in normal germinal center and NHL B cells, 280—281 as transcriptional repressor, 278—279 translocation mechanisms, 274—275 classification, 271—274 Pax5, oncogenic activation, 224—225 Nonlymphoid cells, BCL-6 mutations and, 282 Nonmammalian organisms, Stat function in, 241 Nov1 and 2 genes, oct-2 gene expression, 299 N-terminal dimer-dimer interaction domain c-myb oncogene structural alteration, 531—532 Stat family structure and activation, 236—237 Nuclear bodies PML gene expression, 332—333 growth suppression and apoptosis, 335—336 partner proteins, 334

PML-RAR fusion protein, 338—339 Nuclear factor-erythroid 2 (NF-E2) gene biochemistry aqnd expression, 14—16 CBP/p300 coactivator regulation, 503 erythroid cell differentiation, 19—20 Friend of GATA-1 (FOG) assay, 3, 10 functions of, 16—18 future applications of, 24 limits of research using, 23 megakaryocyte differentiation, 20—23 targeted disruption (knockout) via homologous recombination, 41—42 transcription factor promoters, 37 platelet biogenesis and, 20—23 related proteins, 18—19 structure and composition, 13—14 in vivo gene expression, 19—20 Nuclear factor of activated T cells (NF-AT), octamerdependent gene regulation, 302 Nuclear hormone receptor corepressor, RXR-RAR response element activation, EML vs. MPRO cells, 153—154 Nuclear localization Ikaros family proteins, 192—194 MOZ-CBP fusion protein, 468 PLZF gene, 343—344 PML gene structure, 331 Nuclear matrix-associated (NuMA) gene NuMA-RAR fusion protein, 350 RAR fusion with, research background, 327 structural characteristics, 349—350 Nuclear receptor coactivators leukemia translocations and function of, 474—476 TIF1/nuclear receptor coactivators, 466 Nuclear translocation, Stat family structure and activation, 238 Nucleophosmin (NPM) gene NPM-RAR fusion protein, 348—349 RAR fusion with, research background, 327 structural characteristics, 347—348 Null mutations Aiolos family proteins B-cell maturation and, 189 lymphoproliferations in null mice and, 191—192 Ikaros genes lymphocyte development and, 186—188 lymphoproliferations in null mice and, 191 PML growth suppression and apoptosis, 335—336 NuMA-RAR fusion protein, structural characteristics, 350 NUP98 gene, hox-induced leukemias, 140 NURD complex, Ikaros/Aiolos regulation of chromatin, 194—196

OBF-1 coactivator B-cell-specific gene expression, 298—299

INDEX

early B-cell development, 295—297 germinal center formation and, 301 Ig gene transcription and, 299—300 structure and function, 293—295 T-cell activation and, 301—302 OCA-B/OBF-1/Bob-1 coactivator, structure and function, 293—295 Octamer factors antigen-dependent B-cell differentiation, 299—301 germinal center formation, 301 Ig enhancer activation, 300—301 Ig transcription regulation by Oct-1/2 and OBF-1, 299—300 IL-6 and retinoic acid signaling, 301 terminal differentiation, 299 B-cell development, 295—299 gene expression regulation, 298—299 germ-line V gene transcription and rearrangement, 297—298 OBF-1 and early development, 295—297 Oct-2-dependent genes, 299 coactivators and OCA-B/OBF-1/Bob-1, 293—295 octamer-binding proteins, 292—293 octamer motif, 292 future research issues, 304—305 in lymphoid system, structure and function, 291— 292 T-cell binding sites, 301—304 IL-2 and IL-4 gene activation, 302 IL-5 gene regulation, 302—303 Oct-2 and OBF-1 activation, 301—302 Oct-1/2 POU domain, 304 TGF- and retinoic acid regulation, 303—304 Octapeptide sequences, Pax5-Grg4 protein interactions, 221—222 Oct-1 protein B-cell-specific gene expression, 298 Ig enhancer activation, 300—301 Ig gene transcription and, 299—300 structure and function, 292—293 T-cell transformation, 304 Oct-2 protein B-cell-specific gene expression, 298—299 early B-cell development and, 295—297 gene expression, 299 germinal center formation and, 301 Ig enhancer activation, 300—301 Ig gene transcription and, 299—300 structure and function, 292—293 T-cell activation and, 301—302 T-cell transformation, 304 1,25(OH) D /VDR monocytic differentiation, acute promyelocytic leukemia (APL), 171—175 myeloid differentiation and lineage direct and indirect targets, 169—170

615

HoxA10 target gene, 168—169 research background, 163—164 U937 differential screening, 166—167 Oncogenesis Jaks and Stats role in, 243 NF-KB family and, 561 non-Hodgkin’s lymphomas, 272—273 Pax5 gene in non-Hodgkin’s lymphoma, 224—225 TAL1/SCL transcription factor and, 54—56 Overexpression studies hox gene function in normal hematopoiesis, 139 Pax5 oncogenic activation in Non-Hodgkin’s lymphoma, 224—225

p100, c-myb interaction with, 534 Para-aortic splanchnopleure (PAS), Runx1 and Cbfb genes, definitive hematopoiesis and, 90—92 Partner proteins, MLL gene mutations, 452—454 future research issues, 457—458 leukemogenesis and, 456—457 PAS/bHLH domain, MOZ-TIF2 inversion, 474 Pax5 protein B-cell lineage commitment and, 218—220 future research issues, 224—225 gene repression and alternative fates, 220—222 late differentiation, 222—224 oncogenic activation in non-Hodgkin’s lymphoma, 224—225 E proteins in B-cell development, 261 structure and function, 217—218 PBX-EXD family, hox gene expression, DNA-binding proteins, 142—143 P/CAF transcriptional coactivator erythroid Kru¨ppel-like factor (EKLF) transcription activation, 76—78 MOZ-CBP fusion protein, 469—470 PEBP2/CBF complex, AML1 binding and, 411—412 Peripheral blood lymphocytes (PBLs), VDR target genes, U937 differential screening, 167 PEST domain c-myb oncogene structure, 533 I-KB family, 553—554 PU.1 gene gene expression, 202—203 structure-function relationship, 204—205 Spi-B protein, 207—208 p21 genes, acute promyelocytic leukemia (APL), monocytic differentiation, 173—175 PHD domain MLL-CBP fusion protein, 471—472 MLL finger 1 deletion mutants, 455 MOZ-CBP fusion protein, 468 trx gene function and, 448—449 Phosphorylation C/EBP in myeloid cells, 120

616

INDEX

Phosphorylation (Contd.) Stat function regulation, 241—242 TAL1/SCL transcription factor modifications, 54 Pim 1 and 2, c-myc oncogene multistep transformation models, 529 PIN*POINT assay, erythroid Kru¨ppel-like factor (EKLF)-protein interaction, 77—78 Pip coactivator, PU.1 interaction with, 205—207 p27Kip1 gene c-myc oncogene and, 525—526 TEL/AML1 fusion protein, malignant transformation and, 434 Platelet basic protein (PBP), megakaryopoiesis transcription factors cis-regulatory element binding, 34 Ets family promoter studies, 35—36 GATA-1 promoter studies, 34—35 Platelet biogenesis, NF-E2 function and, 20—23 Platelet factor 4 (PF4) megakaryopoiesis transcription cis-regulatory element binding, 34 GATA-1 promoter studies, 35 gene targeting techniques in transgenic mice, 39—40 overexpression studies, 43—44 megakaryopoiesis transcription factors, Ets family promoter studies, 36 platelet biogenesis and, 21—23 PLZF gene. See Promyelocytic leukemia zinc finger (PLZF) gene PLZF-RAR fusion protein CBP/p300 coactivator regulation, 507 structural characteristics, 345—347 t(11;17)(q23;q21) APL models, 347 PML gene expression mechanisms, 332 growth suppression and apoptosis, 335—336 interferon, viral infection and, 334—335 nuclear bodies and gene expression, 332—333 partner proteins, 334 structural characteristics, 331—332 transcription and, 333 PML-RAR fusion protein acute promyelocytic leukemia (APL) and, 150 monocytic differentiation, VDR target genes, 171—175 all-trans retinoic acid (ATRA), future applications of, 159—160 APL model of action, 341—343 CBP/p300 coactivator regulation, leukemia translocations, 507 cellular models, 339—340 mouse models, 340—341 nuclear body, 338—339 PML gene transcription and, 333 PML growth suppression and apoptosis, 335—336 protein-protein interactions, 336—337

RAR-PML reciprocal fusion, 341 retinoid resistance, 338 structure, 336 transcriptional activity, 337—338 viral infection and, 334—335 PNT domain, TEL/PDGFR fusion protein, 425—427 p45 nuclear factor-erythroid 2 (NF-E2) gene biochemistry and expression, 14—16 CBP/p300 coactivator regulation, 503 megakaryocyte transcription factors, targeted disruption (knockout) via homologous recombination, 41—42 platelet biogenesis and, 21—23 PODs (PML oncogenic domains), PML gene expression, 332—333 Point mutations, c-myc oncogene transformation, 527 Polycomb genes hox gene expression, upstream regulation with, 142 MLL genes and, 448—449 Polypeptide products, TAL1/SCL transcription factor, 53—54 Polyploid nuclei, megakaryocyte development and, 31 Porphobilinigen deaminase (PBGD), NF-E2 biochemistry and, 14—16 Postnatal hematopoiesis, TAL1/SCL transcription factor function in, 58 Posttranscriptional modification BCL-6 mutations, 277—278 C/EBP in myeloid cells, 120 Posttranslational modification erythroid Kru¨ppel-like factor (EKLF) transcription activation, 75—78 NF-KB family, 554 POU domain family homeobox genes, 134—135 octamer factors OBF-1 coactivator, 293—295 octamer-binding proteins, 292—293 Oct-1/2 T-cell transformation, 304 structure and function, 291—292 T-cell transcription inhibition, 304 POZ/BTB domain PLZF gene, 343—345 PLZF-RAR fusion protein, 345—347 p50 protein cell viability and, 559 I-KB family, 554 Rel/NF-KB family, 552—553 p52 protein I-KB family, 554 Rel/NF-KB family, 552—553 p65 protein NF-KB family and cell viability, 558—559 Rel/NF-KB family, 552—553 Pre-B cell receptor complex, Pax5 protein structure and, 218 Pre-T, T-cell development, 262—263

INDEX

Pro-B cells early B-cell factor (EBF) disruption and, 316—317 Pax5 protein and, 218—220 gene repression mechanisms, 220—222 Promyelocytic leukemia zinc finger (PLZF) gene acute promyelocytic leukemia (APL), monocytic differentiation, VDR target genes, 171—175 expression in, 344 growth suppression, 345 nuclear localization, 343—344 PML gene partnership with, 334 RAR fusion, research background, 327 structural characteristics, 341—343 transcriptional function, 344—345 Protein inhibitor of activated Stat (PIAS) family, Stat function regulation and, 242 Protein-protein interactions BCL-6 mutation, 279—280 erythroid Kru¨ppel-like factor (EKLF) transcription activation, 75—78 LMO2 gene family in T-cell acute lymphoblastic leukemia (T-ALL), 490—492 Pax5-Grg4 protein interactions, 221—222 PML-RAR fusion protein, 336—337 PU.1 gene B-cell development, 205 myeloid lineage development, 105—106 Protein structures c-myb oncogene, 529—531 c-myc oncogenes, 523—524 NF-E2 function and, 18—19 erythroid cell differentiation and, 20 Pseudokinase domain, Janus kinase structure and, 232—233 P/S/T (Proline/Serine/Threonine) domain, Spi-B protein, 208 p300 transcriptional coactivator CBP/p300 coactivator AML1 gene and, 505—506 C/EBP and, 504—505 c-myb and, 500—501 E2A gene and, 501—502 EKLF and, 503—504 Ets family and, 505 functional mechanisms, 507—508 future research issues, 510 GATA-1 and, 502—503 hematopoiesis and, 499—506 leukemia and, 465—466, 506—507 NF- and, 503 research background, 497 structure and function, 498—499 E2A protein transactivation, 259 erythroid Kru¨ppel-like factor (EKLF) transcription activation, 76—78 MLL-p300 fusion protein, 472

617

PU.1 gene B-lymphocyte development and function future research applications, 211—212 physicochemical characteristics, 201—202 protein-protein interactions, 205 PU.1:PIP interactions, 205—207 PU.1/SPI-1 gene expression, 202—203 PU.1/SPI-1 models, 208—211 Spi-B regulated protein, 207—208 structure-function relationships, 203—205 target gene characteristics, 207 megakaryopoiesis transcription factors, in vivo promoter studies, 35—36 myeloid lineage development c-Jun mediation of Ras, 108—109 future applications, 111 growth factor receptor regulation, 109—110 lineage-specific coactivators, 105—108 protein-protein interactions, 104—105 rescue assay structure-function development, 110—111 research background, 103 stem cell induction, 103—105, 109 target genes, 110 octamer factors, B-cell-specific gene expression and, 298—299 p21waf1,cip1, myeloid differentiation and lineage, 164— 167 RACE technique, MOZ-TIF2 inversion, 473—474 Rag proteins B cell development and, 313—315 E proteins and in B-cell development, 260—261 in T-cell development, 264 RAR403, hematopoiesis activation and, 151—152 RAR-PLZF reciprocal fusion protein, 347 structural characteristics, 347 RAR-PML reciprocal fusion protein, characteristics of, 341 Ras pathway, PU.1 gene and myeloid lineage development, c-Jun proto-oncogene, 107—109 Relapse predictions, TEL/AML1 fusion protein in ALL and, 436—439 Rel homology domain (RHD) AML1/ETO transcriptional repression, 416—417 Rel protein family and, 552—553 Rel proteins future research issues, 561—563 I-KB family, 553—554 NF-KB family and cancer and, 561 cell cycle progression and, 557—558 cell viability and, 558—559 functional analysis, 554—555 gene knockout analysis, 555—556 proapoptotic activity, 560—561

618

INDEX

Rel proteins (Contd.) research background, 551 structural characteristics, 552—553 Repressor promoters AML1/ETO role in leukemogenesis, 416—417 c-myc oncogenes, 524 megakaryopoiesis transcription factors, in vivo promoter studies, 38 Rescue assays, PU.1 gene structure-function, 110—111 Retinaldehyde dehydrogenase 2 (RALDH2) gene, TAL1/SCL transcription factor and, 60—62 Retinoblastoma protein (Rb), PU.1 gene proteinprotein interactions, 205 Retinoic acid, octamer factors signaling mechanisms, 301 transcription inhibition, 303—304 Retinoic acid receptor (RAR) acute promyelocytic leukemia fusion partners, research background, 327—328 fusion protein comparisons, 351—353 myeloid differentiation, 329 N-protein/RAR fusion comparisons, 351, 354—356 nuclear matrix-mitotic apparatus protein (NuMa), 349—350 nucleophosmin (NPM) gene, 347—350 NPM-RAR fusion, 348—349 PLZF-RAR, 345—347 t(11;17)(q23;q21) APL model, 347 PML-RAR fusion protein APL model of action, 341—343 cellular models, 339—340 mouse models, 340—341 nuclear body, 338—339 protein-protein interactions, 336—337 retinoid resistance, 338 structure, 336 transcriptional activity, 337—338 RAR-PLZF reciprocal transcript, 347 RAR-PML reciprocal fusion, 341 Stat5b-RAR, 350—351 target genes, 329—331 transcriptional function, 328—329 Retinoic acid receptor (RAR) acute promyelocytic leukemia (APL) and, monocytic differentiation, VDR target genes, 172—175 hox gene expression, upstream regulation, 140—141 myeloid differentiation and lineage ATRA enhancement of CFC production, 155—159 future applications, 159—160 hematopoietic lineage-specific activation, 151—155 myelopoiesis regulation, 155 PML-RAR receptor fusion protein, 150 research background, 149—150 RARA-Stat5b fusion, 243 structural characteristics, 327—328

Retinoic acid response elements (RAREs) hox gene expression, upstream regulation, 141 PLZF-RAR fusion protein, 345—347 PML-RAR fusion protein, protein-protein interactions, 336—337 retinoic acid receptor (RAR) target genes and, 330—331 retinoic acid receptor (RAR) transcription and, 328—329 Retinoic acid syndrome, all-trans retinoic acid (ATRA) treatment and, 330—331 Retinoid resistance, PML-RAR fusion protein and, 338 Retinoids, hox gene expression, upstream regulation, 140—141 Ribosomal P proteins, PML gene partnership with, 334 RING finger configuration c-myc oncogene multistep transformation models, 528—529 PML gene structure, 331 nuclear bodies, 333 RNA C/EBP expression in, 119—120 TAL1/SCL transcription factor, protein distribution and, 56—57 VDR target gene screening, myeloid differentiation and lineage, 164—165 Roaz protein, early B-cell factor (EBF) development, 315 Rubenstein-Taybi syndrome CBP/p300 coactivator regulation, 506—507 MOZ-CBP fusion protein, 467—470 Runt gene family, AML1 membership in hematopoietic function, 412—413 identification and related genes, 411—412 RUNX1 genes, hematopoiesis lineages dosage effects, 93—94 function in later stages, 94—95 future applications, 95 gene expression, 92—93 hematopoietic cell emergence, 90—92 research background, 87—90 RXR-RAR heterodimer acute promyelocytic leukemia (APL) and, monocytic differentiation, VDR target genes, 173—175 hematopoiesis activation and AML sensitivity in immature vs. mature myeloid cells, 154—155 GM-CSF-dependent MPRO cells, 152—153 nuclear hormone receptor corepressor activity in EML vs. MPRO cells, 153—154 SCF-dependent EML cells, 153 octamer factors, transcription inhibition, 304 PLZF-RAR fusion protein, 346—347 PML-RAR fusion protein, protein-protein interactions, 337

INDEX

retinoic acid receptor (RAR) transcription and, 329 myeloid differentiation and lineage, 329 PML-RAR fusion degradation, 330 331 structure and function, 149 150

SCL interrupting locus (SIL) gene, TAL1/SCL transcription factor and, 51—52 SET domain MDS1/EVI1 fusion and, 400 MLL-CBP fusion protein, 471—472 MLL genes, 456 trx gene function and, 448—449 Severe combined immunodeficiency (SCID), Janus kinases and lymphoid cell development, 230—232 SH2 molecules Janus kinases and, signal transduction initiation, 234 Stat family structure and activation, 237—238 Signal integration BCL-6 mutations and, 279—280 erythroid Kru¨ppel-like factor (EKLF)-protein interaction, 77—78 Signal transduction, Jaks activation and initiation of, 234 Small-MAF proteins megakaryopoiesis transcription factors, targeted disruption via homologous recombination, 42 NF-E2 biochemistry and expression, 15—16 platelet biogenesis and, 22—23 NF-E2 function and, 18—19 SMMHC gene MYH11 encoding of, 379—380 structural characteristics, 380—381 SMRT AML1/ETO transcriptional repression, 416—417 ETO binding, 415—416 PML-RAR fusion protein and, 337—338 SOCS family molecules, Janus kinases and, 233—234 Spi-B transcription factor B lymphocyte development and function protein regulation, 207—208 structural characteristics, 202 genetic model of, 208—211 Spi-1 transcription factor, B lymphocyte development and function gene expression patterns, 202—203 genetic models of, 208—211 Spleen focus forming virus (SFFV), PU.1/Spi-1 identification, 202—203 Sp-1 transcription factor, megakaryopoiesis, in vivo promoter studies, 37 Src homology, Janus kinase structure, 233 STAM molecule, Janus kinases and, 234

619

STAT family cellular proliferation and malignant transformation, 242—243 cloning and identification of, 235 functional regulation, 241—242 lymphoid cell development, 239—241 nonmammalian organism functions, 241 PML gene, viral infection and, 334—335 PML-RAR fusion protein and, 338 structure and activation, 235—239 coiled-coil domain, 238—239 DNA-binding domain, 235—236 N-terminal dimer-dimer interaction domain, 236—237 nuclear translocation, 238 SH2 domain and tyrosine phosphorylation site, 237—238 transcription activation domain, 238 TEL/JAK2 fusion protein, 430 STAT5 gene RAR fusion with, 327 Stat5-RAR fusion protein, 350—351 TEL/JAK2 fusion protein, 430 Stem cell assays, all-trans retinoic acid (ATRA), shortand long-term marrow repopulation in, 156—158 Stem cell factor (SCF), RXR-RAR response element activation, EML-dependent cells, 153 Stem cell induction, myeloid lineage development, PU.1 gene and, 103—104, 109 Stem cell leukemia, TAL1/SCL transcription factor and, 51 Subnuclear localization, MLL/trx gene function and, 449 SUMO-1 protein, PML gene and, 334 SWI/SNF complex, Ikaros/Aiolos regulation of chromatin, 194—196

TALE group, homeobox genes, 135 Tal-1 gene E proteins and, 265 LMO2 gene family, multiprotein transcription, 488—489 TALLA1 gene family, TAL1/SCL transcription factor and, 62—63 TAL1/SCL transcription factor, hematopoiesis and developmental function, 57 DNA-binding preferences, 59—60 future research issues, 63 gene expression regulation, 58—59 leukemia involvement and discovery of, 51—53 oncogenic properties, 54—56 postnatal hematopoiesis, 58 posttranslational modifications, 54 protein products and interaction, 53—54 RNA and protein distribution, 56—57

620

INDEX

TAL1/SCL transcription factor (Contd.) target genes, 62—63 transcriptional properties, 60—62 t(8;21)AML biologic and genetic features, 409—411 future research issues, 419 Target genes. See also specific genes, e.g. Vitamin D3 transcriptional targets BCL-6 mutation, 279—280 c-myb oncogene and hematopoiesis, 534—537 early B-cell factor (EBF) characterization of, 317—320 structural characterization, 314—315 targeted disruptions of, 316—317 E protein, future research issues, 266 hox gene regulation, 143—144 non-Hodgkin’s lymphoma and, 272—274 PU.1 gene as, 110 B-cell specificity, 207 structure-function relationships, 203—205 retinoic acid receptor (RAR) fusion and, 329—331 TAL1/SCL transcription factor and, 62—63 TATA-binding protein (TBP) C/EBP and, 118—120 c-myc oncogenes, 524 PU.1 gene protein-protein interactions, 205 B cell target specificity, 207 T cell Aiolos family proteins, lymphoproliferations in null mice and, 191—192 BCL-6 mutations and, role in differentiation and function, 281—282 E proteins and developmental function, 259, 262—264 E2A protein transactivation, 258—259 future research applications, 265—266 HLH structure and function, 258 lymphoma and E2A, 264—265 research background, 257—258 function of, 255 hox gene expression in, 136—137 Ikaros family proteins cell cycle regulation, 192 gene expression, 186 malignancies in Ikaros DN]/- mice, 190—191 nuclear localization, 192—194 null mutations and, 187—188 octamer factor binding sites, 301—304 IL-2 and IL-4 gene activation, 302 IL-5 gene regulation, 302—303 Oct-2 and OBF-1 activation, 301—302 Oct-1/2 POU domain, 304 Pax5 proteins and, 220 PU.1/Spi-B genetic models and, 209—211 Runx1 gene and, 95 Stat3 activation and, 239—240 Stat6 activation and, 240

stem cell genetics and, 255—257 T-cell acute lymphoblastic leukemia (T-ALL) c-myc oncogene transformation, 526—527 E2A protein and, 265 LMO2 gene family, 489—492 differentiation inhibition, 489—490 protein interaction models, 490—492 translocation mechanisms, 483—484 MLL PHD finger 1 deletion mutants and, 455 TAL1/SCL transcription factor and, 51 oncogenesis functions, 54—56 T-cell receptor (TCR) CBF genes and, 95 E proteins and development of, 263 transcription factor function and, 256—257 T-cell receptor (TCRb) chain ETO transcriptional effects, 416 LMO1 and LMO2 translocations, 485 T-cell receptor (TCR) locus c-myb interaction with, 534 LMO1 and LMO2 translocations, 485 TEL/ABL fusion protein diagnostic applications, 439 prognosis and relapse predictions with, 436—439 structural properties, 430 TEL/AML1 fusion protein core-binding factor complex inhibition, 432—433 gene expression and transformation, 433 malignant transformation, 433—434 pediatric acute lymphoblastic leukemia clinical significance, 435 favorable prognosis with, 436—438 minimal residual disease detection, 439 prevalence in, 435—436 structural characteristics, 431—432 TEL gene EVI1 gene and, 399 hematopoiesis and, 426 Jaks fusion with, 242—243 malignant hematologic transformation, 426—427 structural properties, 425—426 tyrosine kinase fusion, 427—428 TEL/JAK2 fusion, structural properties, 429—430 TEL/PDGFR fusion protein structural characteristics, 428—429 structural properties, 425—426 TEL/TRKC fusion protein hematologic malignancies and, 426—427 structural properties, 430 Ternary complex, PU.1:Pip interactions and formation of, 205—207 TFIIB, C/EBP and, 118—120 TFIID, c-myb interaction with, 534 TFIIH, TAL1/SCL transcription factor interactions, 54 t(16;16) gene rearrangement, acute myeloid leukemias (AML) and, research background, 379—380

INDEX

t(16;21) gene rearrangement, AML1/ETO fusion protein, 410 Threonine 58, c-myc oncogene mutations, 527 Thrombocytopenia, p45 NF-E2 absence and, 21—23 Thrombopoietin (Tpo) Friend of GATA-1 (FOG) and megakaryotic development, 5—6 megakaryopoiesis transcription factors, 32 Ets family promoter studies, 36 GATA-1 promoter studies, 35 NF-E2 biochemistry and expression, 15—16 Thromboxane synthase, NF-E2 biochemistry and expression, megakaryotic differentiation and, 23 Thymocyte development BCL-6 mutations and, 281—282 E proteins and, 263—264 E2A protein and T-cell lymphoma, 264—265 hox gene function in normal hematopoiesis, size parameters, 139 Ikaros family proteins lymphoproliferations in null mice and, 191 malignancies in Ikaros DN>\ mice, 190—191 null mutations and, 187—188 TIF2 coactivator, MOZ-TIF2 inversion, 472—474 TIF1/nuclear receptor coactivators, leukemia and, 466 t(11;17)(q23;q21) APL model, PLZF-RAR fusion protein and, 347 Trans-acting factors, megakaryocyte transcription, 34 Transactivation elements C/EBP and, 118—120 E2A proteins, 258—259 PU.1 gene protein-protein interactions, B-cell development, 205 Transcription activation domain. See Domain mapping Transcriptional functions AML1 gene, 413 erythroid Kru¨ppel-like factor (EKLF) activation mechanism, 74—78 ETO gene family, 415—416 LMO2 gene family, 483—484 MDS1/EVI1 fusion and EVI1, 400—402 PLZF gene, 344 PML gene and, 333 PML-RAR fusion protein, 337—338 retinoic acid receptor (RAR), 328—329 TAL1/SCL transcription factor, 60—62 Transcriptional repressor, BCL-6 mutation as, 278— 279 Transcription factors. See also specific transcription factors c-myc oncogene structure, 523—524 hematopoiesis and, in vivo studies, 574—584 megakaryotic differentiation and cis-regulatory element binding, 32—34 research background, 31—32 transgenic mice in vivo studies, 39—44

621

in vitro ectopic expression, 38—39 in vivo promoter studies, 34—38 Ets family members, 35—36 GATA/Ets repeats, 36—37 GATA-1 promoters, 34—35 NF-E2 in megakaryocytes, 37 repressor elements, 38 Sp1 involvement, 37 Stat family association with, 238—239 Transforming growth factor- MDS1/EVI1 fusion and, 403 octamer-dependent transcription, 303—304 Transgenic mice CBFB-MYH11 fusion protein model, 384—385 c-myc oncogene multistep transformation models, 528—529 megakaryocyte transcription factors gene targeting techniques, 39—44 overexpression studies, 42—44 regulatory regions of specific genes, 39—40 targeted disruption (knockout) via homologous recombination, 40—42 Translocations BCL-6 mutations and, 283—284 CBP/p300 coactivator regulation, leukemia chromosomes, 506—507 c-myc oncogene, neoplastic transformation and, 526—527 coactivators and leukemia CBP/p300 coactivators, 465—466 functions of, 474—477 histone acetyltransferases (HATs), 466—467 MLL-CBP t(11;16) (q23;p13) translocation, 470—472 MLL-p300 (t11;22)(q23;q13) translocation, 472 MOZ-CBP (t8;16) (p11;p13) translocation, 467—470 MOZZ-TIF2 inversion (inv)(8) (p11q13) translocation, 472—474 structural properties, 465 TIF2/nuclear receptor coactivator, 466 LMO2 gene family, 483—484 MLL fusion proteins, 452—454 Trithorax, hox gene expression, upstream regulation, 141 TRR gene, MLL gene and ALL-1 related (ALR) protein, 450 1-Tubulin, megakaryopoiesis transcription factors, targeted disruption (knockout) via homologous recombination, 41—42 Tumor suppression E proteins and, 264—265 MDS1/EVI1 fusion, 402—403 NHL genes and, 273 PLZF gene, 344 PML growth suppression and apoptosis, 335—336

622

INDEX

Tyk2 classification, 229—230 lymphoid cell development, 231—232 Tyrosine kinase TEL gene fusion in leukemia and, 427—428 TEL/PDGFR fusion protein and, 429 Tyrosine phosphorylation Stat family structure and activation, 237—238 Stat function regulation and, 241—242

U937 differential screening HoxA10 target gene, 168—169 1,25(OH) D /VDR, 166—167 PML-RAR fusion protein, 340 Upstream regulators BCL-6 mutation, 279—280 hox gene expression, 140—142

Vascular formation, LMO2 gene family and, 485—488 VDR target genes acute promyelocytic leukemia (APL), monocytic differentiation, 171—175 myeloid differentiation and lineage, 164—171 cell cycle arrest and differentiation, 170—171 HoxA10, 167—169 1,25(OH) D /VDR, 169—170 p21waf1,cip1, 164—167 V gene rearrangement, octamer factors and, 297—298 V205G mutant, erythroid maturation and GATA-1 function, 8 Viral infection, PML gene and, 334—335 Vitamin D response element (VDRE), acute promyelocytic leukemia (APL) and, monocytic differentiation, VDR target genes, 172—175

Vitamin D transcriptional targets myeloid differentiation and lineage acute promyelocytic leukemia, 171—175 1,25(OH) D induction, 172—173 p21 gene expression, 173—175 future applications, 175 research background, 163—164 VDR target genes, 164—171 cell cycle arrest and differentiation, 170—171 HoxA10, 167—169 1,25(OH) D /VDR, 169—170 p21waf1,cip1, 164—167 PML-RAR fusion protein and, 338 v-myb oncogene CBP/p300 coactivator regulation, hematopoiesis and, 500—501 c-myb oncogene development and, 529—531 structural alterations, 531—533 research background, 521 transcription factor interaction with, 534 v-myc oncogene c-myc research and, 522 research background, 521 von Willebrand factor (vWF), megakaryopoiesis transcription factors cis-regulatory element binding, 32—34 Ets family promoters, 36 VpreB genes B cell development and, 313—315 early B-cell factor (EBF) interaction with, 319

WEHI-231 cells, NF-KB family and cell cycle inhibition, 559—560

Xamli gene, definitive hematopoiesis and, 92

PART I


TRANSCRIPTION FACTORS AND THE MEGAKARYOCYTIC AND ERYTHROID LINEAGES

CHAPTER 1


THE ROLE OF GATA-1 AND FOG IN ERYTHROID AND MEGAKARYOCYTIC DIFFERENTIATION ALICE P. TSANG, JOHN D. CRISPINO, AND STUART H. ORKIN Division of Hematology-Oncology, Children’s Hospital and the Dana-Farber Cancer Institute, Department of Pediatrics, Harvard Medical School and the Howard Hughes Medical Institute

INTRODUCTION The development of mature blood cells of distinct lineages from pluripotent hematopoietic stem cells is controlled by cell-restricted transcription factors. Among these, the zinc finger protein GATA-1 is notable in several respects. First, its expression is highly restricted to hematopoietic cells, specifically to the erythroid, megakaryocytic, mast, and eosinophilic lineages (for review, see Orkin, 1998). Second, its cognate DNA-binding motif is present in the cis-regulatory elements of virtually all characterized genes specifically expressed in the erythroid and megakaryocytic lineages (Orkin, 1992; Shivdasani, 1997). Third, enforced expression of GATA-1 reprograms transformed myeloblasts into erythroid cells, megakaryocytes, or eosinophils (Kulessa et al., 1995; Visvader and Adams, 1993). Finally, loss of GATA-1 function in mice

leads to blocks in both erythroid and megakaryocytic cell maturation, with accompanying apoptosis or unrestrained proliferation of precursor cells, respectively (Pevny et al., 1991; Shivdasani et al., 1997; Weiss et al., 1994; Weiss and Orkin, 1995). Thus, GATA-1 is essential for the development of at least two hematopoietic lineages. Other GATA transcription factors, related to GATA-1 by virtue of their DNA-binding domains, have also been shown to serve essential but distinct roles in development. Within the hematopoietic system, GATA-2 is necessary for the proliferation and/or survival of early hematopoietic progenitors (Tsai et al., 1994), whereas GATA-3 is required specifically for the development of the T-cell lineage (Ting et al., 1996; Zheng and Flavell, 1997). The nonhematopoietic GATA factors, GATA-4, -5, and -6, are expressed at multiple sites, including the heart and

Transcription Factors: Normal and Malignant Development of Blood Cells, Edited by Katya Ravid and Jonathan D. Licht. ISBN 0-471-35054-0 Copyright 2001 by Wiley-Liss, Inc.

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GATA-1 AND FOG IN HEMATOPOIESIS

intestinal epithelium (Gao et al., 1998). Gene targeting has revealed a crucial requirement for GATA-4 in heart tube formation and ventral morphogenesis (Kuo et al., 1997; Molkentin et al., 1997). Fundamental to understanding how GATA factors such as GATA-1 function in differentiation and development is the elucidation of the mechanisms by which they act in transcription. The DNA-binding domain of GATA-1 is comprised of two homologous zinc fingers, designated N-finger and C-finger (Evans and Felsenfeld, 1989; Tsai et al., 1989). The C-finger is essential for sequence-specific binding to the consensus motif (A/T)GATA(A/G). In contrast, the N-finger is dispensable for binding to simple GATA motifs, but enhances the stability and specificity of binding to more complex motifs, termed palindromic GATA sites (Trainor et al., 1996). Although early studies implicated the amino-terminal activation domain of GATA-1 as critical for function based on its transactivation potential in fibroblasts (Martin and Orkin, 1990), later studies suggested that the biological activity of GATA-1 resides principally in its zinc finger DNA-binding domain, as assayed in 416B cells (Visvader et al., 1995) and GATA-1\ embryoid bodies (Blobel et al., 1995). In addition, a differentiation assay based on the rescue of GATA-1\ erythroid precursor cells revealed that the N-finger of GATA-1, which is largely dispensable for both DNA binding and transactivation in fibroblasts, is absolutely required for terminal erythroid differentiation (Weiss et al., 1997).

Taken together, these studies suggested that GATA-1 might regulate transcription of critical target genes during erythroid and megakaryocytic differentiation by associating with another hematopoietic-specific nuclear factor, possibly via its N-finger. By direct interaction with GATA-1, such a cofactor could link DNAbound GATA-1 to other transcription components, and, in so doing, provide a combinatorial signal for cell-specific gene expression and differentiation (Tsang et al., 1997; Weiss et al., 1997).

IDENTIFICATION OF FRIEND OF GATA-1: A COFACTOR FOR GATA-1 IN ERYTHROID AND MEGAKARYOCYTIC DIFFERENTIATION To address the possibility of an in vivo cofactor for GATA-1 action, yeast two-hybrid interaction screening was performed (Tsang et al., 1997). Based on the in vivo requirement for the N-finger in erythroid cells (Weiss et al., 1997), our bait consisted of the GAL4 DNA-binding domain fused to the N-finger of GATA-1. In a screen of 6 ; 10 primary transformants of a murine erythroleukemia (MEL) cell cDNA expression library, two independent partial cDNAs were isolated that encoded a novel zincfinger protein, designated Friend of GATA-1 (FOG). Sequence analysis revealed the presence of nine putative zinc fingers (F1 — F9) of two different types, C H and C2HC, distributed throughout the protein (Fig. 1.1). In yeast, FOG specifically recognized the N-, but not the C-

Figure 1.1. Schematic diagram of the structure of Friend of GATA-1 (FOG), a novel, multitype zinc-finger protein. The regions of FOG encoded by the partial cDNA clones M10 and M22 are also diagramed. Zinc fingers are symbolized by ovals. Note that fingers 1 and 6 are each sufficient to mediate interaction with GATA-1.

IDENTIFICATION OF FRIEND OF GATA-1

finger, of GATA-1. In addition, FOG associated with GATA-1 both in vitro and in vivo, and was also able to interact with DNA-bound GATA-1 to form a ternary complex, as shown by a yeast one-hybrid assay. Further characterization of FOG revealed an expression pattern strikingly similar to that of GATA-1 (Tsang et al., 1997). Among hematopoietic cell lines, FOG was shown to be strongly expressed in erythroid, megakaryocytic, and multipotential progenitor cell lines. Interestingly, in contrast to GATA-1, FOG was not detectable in mast cell lines. Immunofluorescence staining using -FOG antibody confirmed the presence of FOG protein in the nuclei of primary erythroblasts and cultured megakaryocytes. Furthermore, RNA in situ hybridization analysis of mouse embryos demonstrated high-level expression of FOG in yolk sac blood islands and fetal liver, both of which are sites of high-level GATA-1 expression and active erythropoiesis. This coexpression of FOG and GATA-1 during embryonic as well as hematopoietic development hinted at an important functional role for FOG in hematopoiesis. To begin to establish this role, three independent assays were developed to assess for potential synergism or cooperation between FOG and GATA-1. In the first assay, FOG’s ability to synergize with GATA-1 in activating transcription from a native hematopoietic-specific promoter was tested. The reporter plasmid contained the growth hormone gene fused to the entire 7 kb upstream regulatory region of the erythroid/ megakaryocytic-expressed p45 NF-E2 gene (Andrews et al., 1993). Significantly, NF-E2, a basic zipper heterodimeric transcription factor, has been implicated in erythroid cell globin gene expression and is also essential for megakaryocyte maturation and platelet production (see Shivdasani et al., 1995, and references therein). Transient transfection experiments in fibroblast cells demonstrated that while neither GATA1 nor FOG alone significantly stimulated transcription of the reporter gene, cotransfection of GATA-1 and FOG resulted in a greater than 10-fold activation of gene expression (Tsang et al., 1997). This synergism between FOG and GATA1 provided the first evidence of a functional role for FOG in GATA-1—mediated transcription. The ability of FOG to cooperate with GATA1 was next examined in a biological assay for

3

megakaryocytic differentiation. This assay was based upon the conversion of early myeloid 416B cells to megakaryocytes upon enforced expression of GATA-1 (Visvader et al., 1992). To determine whether FOG can enhance GATA-1’s ability to induce differentiation, expression vectors containing FOG or GATA-1 cDNAs were stably introduced into 416B cells. Transfectants expressing FOG alone resembled parental 416B cells and expressed only very low levels of acetylcholinesterase (AChE), a specific marker of mouse megakaryocytes. Consistent with previous studies, GATA-1 transfected cells exhibited morphological and histochemical evidence of megakaryocytic differentiation. However, transfectants expressing both FOG and GATA-1 displayed a significantly greater degree of differentiation (:3—4—fold), as determined by the degree of AChE staining and the level of AChE transcripts (Tsang et al., 1997). Thus, in cell culture FOG enhances the ability of GATA1 to induce megakaryocytic differentiation. In a third assay for cooperativity between FOG and GATA-1, the ability of FOG to cooperate with GATA-1 in rescuing terminal erythroid maturation of a GATA-1\ erythroid cell line (G1E) (Weiss et al., 1997) was examined. To this end, a conditionally active form of GATA-1 was created by fusing its coding region to the ligand-binding domain of the human estrogen receptor (ER). This GATA-1/ER fusion protein, when stably expressed in G1E cells, induced erythroid differentiation in an estrogen-dependent manner. G1E cells stably expressing GATA-1/ER, termed G1ER cells, were infected with retrovirus harboring FOG cDNA and were retested for estrogen-dependent differentiation. Upon induction of differentiation, infected G1ER cells expressing exogenous FOG displayed a 3 — 4 — fold increase in the percentage of benzidine-positive cells relative to control cells. Consistent with these results, - and globin mRNAs were induced to levels 3 — 4 — fold higher in the presence of FOG (Tsang et al., 1997). Thus, similar to megakaryocytic induction of 416B cells, FOG enhances GATA-1 — mediated rescue of terminal erythroid differentiation. This cooperative action of FOG and GATA-1 in erythroid as well as megakaryocytic differentiation, together with the proteins’ specific interaction and their coexpression during hematopoietic development, strongly supported

4


assignment of FOG as the postulated cofactor for GATA-1 in hematopoiesis.

FOG AND TERMINAL ERYTHROID MATURATION To assess FOG’s role as a GATA-1 cofactor in vivo, the FOG gene was disrupted by homologous recombination in mouse ES cells (Tsang et al., 1998). FOG>\ mice appeared normal and were interbred to generate homozygous mutants (FOG\\). Among the liveborn offspring of FOG>\ crosses, none were homozygous mutant, indicating that loss of FOG is embryonic lethal. Whereas at embryonic day 10.5 (E10.5) :26% of viable embryos were of the FOG\\ genotype, at E11.5 only :13% of viable embryos were FOG\\, and none survived to E12.5. Thus, homozygous null mutations of the FOG gene result in embryonic lethality between E10.5 and E12.5. At E11.5, viable FOG\\ embryos were comparable in overall development to wild-type and heterozygous littermates, but were readily distinguished by their smaller size and marked pallor (Fig. 1.2). In contrast to the normal, easily visualized pattern of yolk sac vasculature

in littermates, the large yolk sac vessels of FOG\\ embryos appeared very pale and thin. Upon closer inspection, the small capillaries of mutant yolk sacs appeared abnormally dilated, presumably as a secondary consequence of anemia (see below). In addition, the fetal livers of mutant embryos were smaller than those of normal embryos and very pale, suggesting a significant reduction in effective fetal liver erythropoiesis (Tsang et al., 1998). The absence of gross morphological or histological abnormalities in FOG\\ embryos pointed to a specific failure of primitive hematopoiesis in the absence of FOG. To address this, the peripheral blood of E10.5 and E11.5 embryos was examined (Fig. 1.2). At this stage of development, peripheral blood normally contains large primitive (or embryonic) red cells arising from the yolk sac. In contrast to the population of maturing primitive erythroblasts evident in normal blood, the erythroid cells of mutant embryos exhibited a marked, but partial, arrest in development at the proerythroblast stage, reminiscent of GATA-1\ erythroid precursors. Consistent with a maturational arrest, the levels of embryonic and adult globin RNAs were significantly decreased (:5 fold) in mutant compared to heterozygous blood

Figure 1.2. Phenotypic comparison of GATA-1- and FOG\\ Embryos. A, B, and C: Wild-type E11.5, FOG\\ E11.5, and GATA-1\ E9.5 embryos with intact visceral yolk sacs. D, E, and F: May-Grunwald-Giemsa-staining of blood cells from wild-type E11.5, FOG\\E11.5, and GATA-1\ E10.5 yolk sacs. Original magnification 1000;.

A GATA-1—INDEPENDENDENT REQUIREMENT FOR FOG IN MEGAKARYOCYTE DEVELOPMENT

samples. In contrast, transcripts for actin and, notably, GATA-1 were present at comparable levels. Thus, primitive hematopoiesis is severely impaired in mice lacking FOG (Tsang et al., 1998). To determine whether FOG is also required for definitive (fetal liver stage) hematopoiesis, yolk sac and fetal liver cells were obtained for in vitro hematopoietic colony formation assays (Tsang et al., 1998). Under conditions optimal for the differentiation of definitive hematopoietic progenitor cells, wild-type and heterozygous cells generated numerous hemoglobinized erythroid colonies. By contrast, FOG\\ progenitor cells yielded only pale colonies that contained developmentally arrested proerythroblast-like cells. FOG\\ colonies also contained cells with fragmented nuclei and clumped chromatin, suggestive of apoptosis. In accordance with these in vitro findings, FOG\\ ES cells failed to contribute to mature, circulating red blood cells of chimeric animals, demonstrating that FOG is indeed necessary for the production of mature erythroid cells in vivo. Comparison of the consequences of FOG and GATA-1 deficiencies for erythroid development reveals several remarkable similarities (Fig. 1.2). For instance, both FOG and GATA-1 are essential for the terminal maturation of erythroid precursors in the primitive and definitive lineages. In the absence of either protein, precursors undergo developmental arrest and apoptosis at the proerythroblast stage (Pevny et al., 1995; Weiss et al., 1994; Weiss and Orkin, 1995). Furthermore, the blockage in erythroid maturation induced by loss of FOG or GATA-1 results in severe anemia and consequently embryonic lethality during midgestation (Fujiwara et al., 1996). This striking similarity of phenotypes further strengthened the notion that FOG and GATA-1 act in concert, providing a combinatorial signal for erythroid-specific gene expression and differentiation (Tsang et al., 1998).

A GATA-1—INDEPENDENT REQUIREMENT FOR FOG IN MEGAKARYOCYTE DEVELOPMENT As FOG cooperates with GATA-1 during megakaryocytic differentiation (Tsang et al., 1997),

5

the phenotypic consequences for megakaryocyte development induced by loss of FOG are of particular interest. As shown in Fig. 1.3, when cultivated in the presence of thrombopoietin (Tpo), wild-type yolk sac and fetal liver cells generated numerous megakaryocyte colonies. However, under identical conditions, no mature megakaryocyte colonies or cells were obtained from either the yolk sacs or fetal livers of viable FOG\\ embryos. In addition, megakaryocytes failed to develop from FOG\\ ES cells differentiated in vitro. While the presence of rare AChE-positive cells and low levels of platelet factor 4 (PF4) transcripts suggested that megakaryocytic commitment took place in the absence of FOG, at least to a small extent, these findings clearly demonstrated a nonredundant function for FOG in early megakaryocyte development (Tsang et al., 1998). This profound consequence of FOG loss was unexpected given FOG’s proposed mechanism of action as a GATA-1 cofactor in erythroid and megakaryocytic cells (Tsang et al., 1997). Although recent studies have demonstrated a critical role for GATA-1 in the megakaryocytic lineage, GATA-1, in contrast to FOG, appears to be necessary at a somewhat later stage of development. Specifically, GATA\\ megakaryocytes express PF4 and glycoprotein IIb (gpIIb), two early markers of the megakaryocytic lineage, and exhibit deregulated proliferation and severely impaired cytoplasmic maturation (Shivdasani et al., 1997). While the FOG\\ erythroid phenotype strongly supports a role for FOG as a GATA-1 cofactor in vivo, the megakaryocyte defect points to a pivotal GATA-1 — independent requirement for FOG in megakaryocyte development. It should be noted that the early effect of FOG deficiency on megakaryocyte development does not preclude a role for FOG as a GATA-1 cofactor during later stages. Rather, it seems plausible that FOG exhibits dual functions in megakaryocytes: an early GATA-1 — independent role, perhaps at the very earliest stage of development from the bipotential erythroid/megakaryocytic progenitor, and a later GATA-1 — dependent role during terminal megakaryocyte maturation. Thus, in the megakaryocyte lineage, FOG may act through both GATA-1—dependent as well as GATA-1 — independent mechanisms (Tsang et al., 1998).

6


Figure 1.3. Absolute requirement for FOG in early megakaryopoiesis. Yolk sac and fetal liver cells from wild-type and homozygous mutant (—/—) embryos were assayed in semisolid medium for megakaryocyte colonies. A: Number of megakaryocytic (MK) and granulocyte-macrophage (GM) colonies observed after 6 days of cultivation. Numbers (N) at the top of each error bar indicate the number of embryos analyzed. Error bars represent standard error of the mean. YS, yolk sac; FL, fetal liver. B: May-Grunwald-Giemsa and AChE staining of cells from wild-type and mutant (—/—) colonies. Wild-type megakaryocytes were readily identified by their large size, multilobed nuclei, granular cytoplasm, and AChE positivity. Small AChE-positive cells were very rarely observed in mutant cultures. Original magnification, 630;.

IMPLICATIONS OF FOG FOR GATA-1 FUNCTION IN ERYTHROID DEVELOPMENT Although numerous findings using cell culture assays and knockout animals were compatible with FOG and GATA-1 acting in a partnership,

other observations suggested that these two factors might have independent roles in hematopoiesis. As discussed previously the requirement for FOG, but not GATA-1, in early megakaryocyte development strongly suggests that FOG may function independently of

IMPLICATIONS OF FOG FOR GATA-1 FUNCTION IN ERYTHROID DEVELOPMENT

7

Figure 1.4. FOG Noninteracting mutants of GATA-1. A: Summary of noninteracting GATA-1 mutants. The amino acids comprising the N-finger are depicted in one line, while the selected mutants are shown below. B: Structural model of the N-finger of GATA-1, shown facing away from DNA. The novel mutations are labeled.

GATA-1 at this stage of megakaryopoiesis. Furthermore, while mice deficient for expression of either gene exhibit similar blocks in erythroid maturation at the proerythroblast stage, FOG\\ erythroid precursors appear to survive longer than GATA-1\ precursors, raising the possibility that GATA-1 might exert FOG-independent functions. These observations highlight the difficulty in interpreting the results of forced expression and gene ablation studies. Though these studies may demonstrate cooperation and a requirement for each protein, they cannot be used to determine the extent to which GATA-1 function is dependent on FOG or to establish the in vivo relevance of the GATA-1:FOG complex (Crispino et al., 1999). To address the significance of the GATA1:FOG complex in hematopoiesis, altered specificity mutants selected in yeast were employed (Crispino et al., 1999). A split two-hybrid system was used to identify mutations in GATA-1 that disrupt association with FOG (Shih et al., 1996). In this screen, interaction of two proteins drives expression of the Tet repressor, which represses transcription of HIS3. Disruption of the interaction leads to derepression of HIS3 transcription and growth on selective media. Since the Nfinger of GATA-1 is necessary and sufficient for interaction with FOG (Tsang et al, 1997), altered specificity mutants of this domain were sought. Among 16,000 transformants screened, 139 candidate mutants were obtained, 9 of which harbored single substitutions at noncysteine residues. Using the standard two-hybrid

assay in yeast as well as coimmuno-precipitation studies in transfected COS cells, four of the GATA-1 mutants — E203V, V205G, G208V, and H222R — were found to have impaired protein protein interactions. Remarkably, based on the structure of the N-finger of GATA-1 bound to DNA, these four mutated residues lie on a single face of the finger, positioned away from the predicted DNA-binding surface (Fig. 1.4). Thus, these non-contiguous residues comprise a potential interaction surface for FOG. The goal in selecting GATA-1 mutants impaired for FOG interaction was to discriminate N-finger functions involved in DNA binding and those involved in protein-protein interactions. Since prior studies have shown that the N-finger makes important contacts with palindromic GATA-1 sites to stabilize binding (Martin and Orkin, 1990; Trainor et al., 1996), we reasoned that mutations that significantly alter the structure of the N-finger, such as alterations in any one of the critical cysteines, would bind to the palindromic element less tightly and hence dissociate rapidly. In contrast, substitutions that selectively alter a protein interaction surface would be expected to display normal dissociation kinetics. Of the four N-finger GATA-1 mutants selected, three — E203V, V205G, and H222R — were found to dissociate from DNA with similar kinetics as wild type. Thus, these three mutants retain normal DNAbinding properties despite their inability to associate normally with FOG (Crispino et al., 1999).

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To determine the extent to which the requirement for GATA-1 in erythroid development reflects concerted action of a GATA-1:FOG complex rather than independent action of GATA-1, the ability of these noninteracting GATA-1 mutants to rescue terminal erythroid maturation of GATA-1\ G1E cells was examined (Weiss et al., 1997). Whereas expression of wild-type GATA-1 induced the appearance of benzidine-positive hemoglobinized cells, no positive cells were observed in populations expressing any of the three GATA-1 mutants. In addition, while the wildtype GATA-1/ER fusion protein induced terminal erythroid maturation of G1E cells in an estrogen dependent manner (Tsang et al., 1997), six independent clonal lines expressing a conditionally active form of the V205G mutant (V205G/ER) failed to mature into benzidine-positive erythroid cells upon estrogen induction, despite stable protein expression. Thus, GATA-1 mutants that do not associate with FOG but retain ostensibly normal DNA-binding properties fail to promote terminal erythroid maturation. These results strongly suggest that GATA-1 requires direct association with FOG to exert its effects on erythroid development (Crispino et al., 1999). In addition to interacting with FOG, the DNA-binding domain of GATA-1 appears to associate with two proteins of the zinc-finger Kru¨ppel family (Sp1 and EKLF) (Merika and Orkin, 1995) as well as the LIM protein LMO2 (Osada et al., 1995) and the CREB-binding protein (CBP)/p300 (Blobel et al., 1998). Given the multifunctional nature of the GATA-DNAbinding domain and the possible involvement of these other cofactors, effects of the novel Nfinger mutations on aspects of GATA-1 function unrelated to the FOG interaction could not be excluded a priori. Hence, compensatory mutations within FOG were sought that would restore the interaction with the V205G GATA-1 mutant. Using a yeast two-hybrid screen, three suppressor alleles of FOG were isolated. One FOG mutant, S706R, was selected for further analysis because it contained a single amino acid substitution within finger 6 and was capable of interacting with both V205G GATA-1 and wild-type GATA-1 (Crispino et al., 1999). As shown in Figure 1.5, the S706R FOG compensatory mutant promoted extensive estrogen-dependent differentiation (i.e., rescue) of

Figure 1.5. A Compensatory FOG mutant rescues differentiation of G1E cells harboring V205G/ER GATA-1. Benzidine staining of V205G/ER stable cells infected with wild-type FOG, FOG compensatory mutant, or vector alone. The figures are representative of three independent experiments. Original magnification 400;.

G1E cells expressing the V205G/ER fusion protein (:10% of cells). In the absence of this FOG mutant, no erythroid differentiation was observed. Interestingly, introduction of wild-type FOG resulted in weak rescue of these G1E cells (:2% of cells), a finding readily attributable to low residual affinity between the GATA-1 mutant and overproduced wild-type FOG. The markedly enhanced rescue by S706R FOG likely reflects the greater affinity of this altered specificity mutant for V205G GATA-1. That differentiation is rescued by overexpression of either wild-type or S706R FOG provides compelling evidence that the failure of V205G GATA-1 to direct normal cellular maturation is due to its reduced affinity for FOG, rather than an impairment in some other, undefined property of GATA-1. FOG is therefore an essential cofactor for GATA-1 in the terminal differentiation of erythroid cells (Crispino et al., 1999).

CONCLUSIONS AND FUTURE DIRECTIONS

THE ROLE OF THE GATA-1:FOG COMPLEX IN IN ERYTHROID GENE EXPRESSION Having established that terminal erythroid maturation is strictly dependent on the association of GATA-1 and FOG, the extent to which this partnership is required for expression of specific target genes was assessed. The use of altered specificity mutants allows discrimination between two classes of targets. The majority of downstream targets of GATA-1, represented by the globins and band 3, are not activated in the absence of a GATA-1:FOG association. Repressed genes, such as GATA-2 and c-myc, are also not regulated normally. Nonetheless, a subset of targets, including EKLF, HRI, and FOG itself, are relatively FOG independent. At these targets, GATA-1 acts either alone or in concert with a different cofactor(s). In the vast majority of cis elements of erythroid-expressed genes, GATA motifs are not found in a consistent relationship to other sequence motifs. We would propose that the GATA-1:FOG complex is primarily employed at these sites. Rarely, GATA motifs are present in a specific orientation and distance from an E-box motif, consistent with the assembly of a pentameric complex containing GATA-1, LMO2, SCL/tal-1, E2A, and Ldb1 (Wadman et al., 1997). Recently, it has been shown that an upstream enhancer of the EKLF gene relies on a composite GATA-E-box element (Anderson et al., 1998). In this setting the E-box-binding protein SCL/tal-1 and associated proteins may provide cofactor activity to GATA-1, obviating the need for FOG interaction. It is tempting to speculate that GATA-1 functions in alternative complexes, one in which FOG is the cofactor, and another in which SCL/tal-1 (and associated components) fulfill this role (Crispino et al., 1999).

CONCLUSION AND FUTURE DIRECTIONS To summarize, several lines of evidence suggest that FOG is an essential cofactor for GATA-1 in hematopoiesis. First, FOG is coexpressed with GATA-1 during embryonic development and within erythroid cells and megakaryocytes. Second, FOG cooperates with GATA-1 in promoting both erythroid and megakaryocytic

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maturation in cellular assays (Tsang et al., 1997). Third, both FOG and GATA-1 are required for terminal erythroid differentiation in vivo (Tsang et al., 1998). Finally, the physical interaction between FOG and GATA-1 is necessary for the differentiation activity of GATA-1 in erythroid cells (Crispino et al., 1999). Superficially, the association of GATA-1 and FOG is reminiscent of other partnerships in hematopoietic development. In B-lymphoid cells, the coactivator OCA-B (Bob-1, OBF-1) interacts with POU-containing transcription factors Oct-1/Oct-2 (Gstaiger et al., 1995; Luo and Roeder, 1995; Strubin et al., 1995) at a subset of octamer sites (Cepek et al., 1996; Gstaiger et al., 1996). Although both Oct-2 and its coactivator are essential for proper B-cell function, the contribution of the interaction alone remains unclear (Corcoran et al., 1993; Feldhaus et al., 1993; Kim et al., 1996; Nielsen et al., 1996). As described previously in erythroid cells, a pentameric complex can assemble on a composite GATA-E box DNA element (Wadman et al., 1997). Although the leukemia oncoproteins SCL/tal-1 and LMO2 are individually essential for development of the entire hematopoietic system, the contribution of the association of LMO2 to overall SCL/tal-1 function is uncertain. The GATA:FOG complex, however, is distinctive in two respects. First, FOG does not appear to contribute a typical activation domain (Tsang et al., 1997). Indeed, the principal role of FOG may be to recruit additional, as yet unknown, nuclear proteins via interaction with one or more of its available zinc fingers. Second, FOG does not appear to modulate the DNAbinding specificity of GATA-1. CASTing experiments using MEL cell nuclear extracts fail to demonstrate that a FOG:GATA complex recognizes a subset of GATA consensus sequences (A.P.T. and S.H.O., unpublished data). If FOG’s principal function is to couple GATA-1 to the transcriptional machinery, why might two cellrestricted proteins be necessary, when a single DNA-binding protein would, in principle, suffice? While appearing unnecessarily complex, this strategy may afford an additional means of regulation. Transcription could be controlled at the level of the GATA:FOG protein-protein interaction rather than by the binding of GATA-1 to DNA. In maturing erythroid cells,

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GATA-1 might be poised at specific elements. Transcription would ensue only upon FOG interaction and subsequent recruitment of other components. This scenario is consistent with the observation that locus control regions of globin clusters exhibit DNase hypersensitivity prior to activation of globin gene transcription (Jimenez et al., 1992). With respect to megakaryopoiesis, the role of FOG may be significantly more complex, involving both GATA-dependent as well as GATA-independent mechanisms. The coexpression of FOG and GATA-1 in megakaryocytes, as well as their cooperativity in megakaryocytic cell differentiation, suggests that FOG serves as a cofactor for GATA-1 function in the megakaryocytic as well as the erythroid lineages. As enhancement of megakaryocytic induction by FOG is dependent on the presence of the Nfinger of GATA-1 (Tsang et al., 1997), we infer that direct interaction between FOG and GATA-1 is required for their cooperativity, and that, as in erythroid differentiation, the association of FOG and GATA-1 is essential for the latter’s transcriptional function in megakaryocyte development. Curiously, while loss of GATA-1 leads to a block in terminal megakaryocyte maturation (Shivdasani et al., 1997), the absence of FOG results in the specific ablation of the megakaryocytic lineage at a very early stage (Tsang et al., 1998). To date, the specific ablation of the megakaryocytic lineage has not been a phenotypic consequence of any transcription factor gene knockout. Indeed, previous studies have identified only one other transcription factor besides GATA-1, NF-E2 (Andrews et al., 1993), that is specifically required for the development of megakaryocytes. Like GATA-1, however, NF-E2 is only necessary for advanced stages of megakaryocyte maturation, as endomitosis and expression of lineage-specific markers such as AChE appear to be unaffected in the absence of NF-E2 (Shivdasani et al., 1995). The only other genes required for the formation of megakaryocytes are those that are essential for the earliest hematopoietic progenitors, for example SCL/tal-1 (Porcher et al., 1996) and CBF (Okuda et al., 1996; Wang et al., 1996), and as such are required for the development of all hematopoietic lineages. Thus, among the known hematopoietic nuclear regulatory

factors, FOG is unique in defining the earliest stage in megakaryocyte development. Defining how FOG functions in these early phases of megakaryopoiesis should suggest novel GATAindependent mechanisms by which FOG, and related finger proteins, regulate transcription and development.

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Orkin, S. H. (1992). GATA-binding transcription factors in hematopoietic cells. Blood 80, 575 — 581. Orkin, S. H. 1998. Transcription factors regulating early hematopoietic development and lineage commitment. In Molecular biology of B-cell and T-cell Development (J. G. Monroe and E. V. Rothenberg, eds.). Totawa, NJ, Humana Press, Totawa, NJ, pp. 41 — 54. Osada, H., Grutz, G., Axelson, H., Forster, A., and Rabbitts, T. H. (1995). Association of erythroid transcription factors: complexes involving the LIM protein RBTN2 and the zinc-finger protein GATA1. Proc. Natl. Acad. Sci. USA 92, 9585 — 9589. Pevny, L., Simon, M. C., Robertson, E., Klein, W. H., Tsai, S. F., D’Agati, V., Orkin, S. H., and Costantini, F. (1991). Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. Nature 349, 257 — 260. Pevny, L., Lin, C. S., D’Agati, V., Simon, M. C., Orkin, S. H., and Costantini, F. (1995). Development of hematopoietic cells lacking transcription factor GATA-1. Development 121, 163 — 172. Porcher, C., Swat, W., Rockwell, K., Fujiwara, Y., Alt, F. W., and Orkin, S. H. (1996). The T cell leukemia oncoprotein SCL/tal-1 is essential for development of all hematopoietic lineages. Cell 86, 47 — 57. Shih, H. M., Goldman, P. S., DeMaggio, A. J., Hollenberg, S. M., Goodman, R. H., and Hoekstra, M. F. (1996). A positive genetic selection for disrupting protein-protein interactions: identification of CREB mutations that prevent association with the coactivator CBP. Proc. Natl. Acad. Sci. USA 93, 13,896 — 13,901. Shivdasani, R. A. (1997). Transcription factors in megakaryocyte differentiation and gene expression. In Thromboiesis and Thrombopoietins, D. J. Kuter, P. Hunt, W. Sheridan, and D. ZuckerFranklin, eds. (Totawa, NJ: Humana Press), pp. 189 — 202. Shivdasani, R. A., Rosenblatt, M. F., Zucker-Franklin, D., Jackson, C. W., Hunt, P., Saris, C. J., and Orkin, S. H. (1995). Transcription factor NF-E2 is required for platelet formation independent of the actions of thrombopoietin/MGDF in megakaryocyte development. Cell 81, 695 — 704. Shivdasani, R. A., Fujiwara, Y., McDevitt, M. A., and Orkin, S. H. (1997). A lineage-selective knockout establishes the critical role of transcription factor GATA-1 in megakaryocyte growth and platelet development. EMBO J. 16, 3965 — 3973. Strubin, M., Newell, J. W., and Matthias, P. (1995). OBF-1, a novel B cell—specific coactivator that stimulates immunoglobulin promoter activity through association with octamer-binding proteins. Cell 80, 497 — 506. Ting, C. N., Olson, M. C., Barton, K. P., and Leiden, J. M. (1996). Transcription factor GATA-3 is required for development of the T-cell lineage. Nature 384, 474 — 478.

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Trainor, C. D., Omichinski, J. G., Vandergon, T. L., Gronenborn, A. M., Clore, G. M., and Felsenfeld, G. (1996). A palindromic regulatory site within vertebrate GATA-1 promoters requires both zinc fingers of the GATA-1 DNA-binding domain for high-affinity interaction. Mol. Cell. Biol. 16, 2238 — 2247. Tsai, F. Y., Keller, G., Kuo, F. C., Weiss, M., Chen, J., Rosenblatt, M., Alt, F. W., and Orkin, S. H. (1994). An early haematopoietic defect in mice lacking the transcription factor GATA-2. Nature 371, 221 — 226. Tsai, S. F., Martin, D. I., Zon, L. I., D’Andrea, A. D., Wong, G. G., and Orkin, S. H. (1989). Cloning of cDNA for the major DNA-binding protein of the erythroid lineage through expression in mammalian cells. Nature 339, 446 — 451. Tsang, A. P., Visvader, J. E., Turner, C. A., Fujiwara, Y., Yu, C., Weiss, M. J., Crossley, M., and Orkin, S. H. (1997). FOG, a multitype zinc finger protein, acts as a cofactor for transcription factor GATA-1 in erythroid and megakaryocytic differentiation. Cell 90, 109 — 119. Tsang, A. P., Fujiwara, Y., Hom, D. B., and Orkin, S. H. (1998). Failure of megakaryopoiesis and arrested erythropoiesis in mice lacking the GATA-1 transcriptional cofactor FOG. Genes Dev. 12, 1176 — 1188. Visvader, J., and Adams, J. M. (1993). Megakaryocytic differentiation induced in 416B myeloid cells by GATA-2 and GATA-3 transgenes or 5-azacytidine is tightly coupled to GATA-1 expression. Blood 82, 1493 — 1501. Visvader, J. E., Elefanty, A. G., Strasser, A., and Adams, J. M. (1992). GATA-1 but not SCL indu-

ces megakaryocytic differentiation in an early myeloid line. EMBO J. 11, 4557 — 4564. Visvader, J. E., Crossley, M., Hill, J., Orkin, S. H., and Adams, J. M. (1995). The C-terminal zinc finger of GATA-1 or GATA-2 is sufficient to induce megakaryocytic differentiation of an early myeloid cell line. Mol. Cell. Biol. 15, 634 — 641. Wadman, I. A., Osada, H., Grutz, G. G., Agulnick, A. D., Westphal, H., Forster, A., and Rabbitts, T. H. (1997). The LIM-only protein Lmo2 is a bridging molecule assembling an erythroid, DNA-binding complex which includes the TAL1, E47, GATA-1 and Ldb1/NLI proteins. EMBO J. 16, 3145 — 3157. Wang, Q., Stacy, T., Miller, J. D., Lewis, A. F., Gu, T.-L., Huang, X., Bushweller, J. H., Bories, J.-C., Alt, F. W., Ryan, G., Liu, P. P., Wynshaw-Boris, A., Binder, M., Marin-Padilla, M., Sharpe, A. H., and Speck, N. A. (1996). The CBFb subunit is essential for CBFa2(AML1) function in vivo. Cell 87, 697 — 708. Weiss, M. J., and Orkin, S. H. (1995). Transcription factor GATA-1 permits survival and maturation of erythroid precursors by preventing apoptosis. Proc. Natl. Acad. Sci. USA 92, 9623 — 9627. Weiss, M. J., Keller, G., and Orkin, S. H. (1994). Novel insights into erythroid development revealed through in vitro differentiation of GATA-1 embryonic stem cells. Genes Dev. 8, 1184 — 1197. Weiss, M. J., Yu, C., and Orkin, S. H. (1997). Erythroid-cell-specific properties of transcription factor GATA-1 revealed by phenotypic rescue of a genetargeted cell line. Mol. Cell. Biol. 17, 1642 — 1651. Zheng, W., and Flavell, R. A. (1997). The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89, 587 — 596.

CHAPTER 2


REGULATION OF MEGAKARYOCYTE AND ERYTHROID DIFFERENTIATION BY NF-E2 SANG-WE KIM AND RAMESH A. SHIVDASANI Departments of Adult Oncology and Cancer Biology, Dana-Farber Cancer Institute, and Departments of Medicine, Brigham & Women’s Hospital and Harvard Medical School

INTRODUCTION Lineage-specific gene expression in terminally differentiated blood cells is regulated through the combinatorial action of lineage-restricted and ubiquitous or widely expressed transcription factors. Understanding the transcriptional networks that coordinate such programs of gene expression is an important focus in the study of cell differentiation. The biochemistry and essential functions of transcriptional regulators that are expressed selectively in certain blood cell lineages have provided particularly useful insights (Orkin, 1995; Shivdasani and Orkin, 1996). Nuclear factor — erythroid 2 (NF-E2), the subject of this chapter, is a lineage-restricted transcription factor with an essential requirement in terminal differentiation of megakaryocytes and a likely important role in proper maturation of circulating erythrocytes. Important lineage-restricted transcription fac-

tors have frequently been identified through their proposed roles in regulating expression of tissue-specific gene products, and the globin proteins, the major products of developing erythroid cells, have provided a useful paradigm. Among the several regulatory properties ascribed to the locus-control regions (LCRs) of the mammalian and globin genes, the enhancer functions map to AP-1—like cis elements with the core DNA sequence TGAGTCA. NF-E2 is a heterodimeric transcription factor of the basic leucine zipper (bZip) superfamily that binds this cis element. Studies in cultured erythroid cells and transgenic mice have implicated NF-E2 in regulating globin gene expression through these sites, and targeted gene disruption has established the requirement for NF-E2 in terminal aspects of megakaryocyte differentiation and thrombocytopoiesis. Here we provide a synopsis of in vitro and in vivo studies pertaining to the composition and functions of NF-E2 in hematopoiesis.


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REGULATION OF MEGAKARYOCYTE AND ERYTHROID DIFFERENTIATION BY NF-E2

Figure 2.1. A: Schema of the subunit composition and transcriptional functions of NF-E2. B: Functional subdomains of p45 NF-E2. as revealed through a variety of assays. CNC, cap-’n’-collar; PY, XPPXY, where P : proline, Y : tyrosine, X : any amino acid.

BIOCHEMISTRY AND EXPRESSION OF THE NF-E2 PROTEIN COMPLEX NF-E2 was initially described as a novel nuclear protein that recognized an AP-1 — like DNA sequence and acted as a transcriptional regulator for erythroid-specific expression of the gene encoding human porphobilinigen deaminase (PBGD) (Mignotte et al., 1989). Subsequently, the same protein was found to bind AP-1—like motifs located within the -globin LCR (Ney et al., 1990b; Talbot and Grosveld, 1991). NF-E2 is readily distinguished from the AP-1 (Jun/Fos) and related protein complexes by its faster migration in electrophoretic mobility shift assays (EMSA) (Romeo et al., 1990), restricted expression in hematopoietic cells (Andrews et al., 1993a), absence of fos/jun-family antigenic determinants (Ney et al., 1990b; Talbot and Grosveld, 1991), and additional, specific sequence requirements at position 92 relative to the AP-1 core for DNA binding (Mignotte et al., 1989; Ney et al., 1990b; Talbot et al., 1990). The consensus sequence for NF-E2 binding is (T/C)(G/A)CTGA(G/C)TCA(T/C), and the NF-E2 protein complex is a heterodimer comprised of 45 and 18 kD subunits (Andrews et al., 1993b; Ney et al., 1993) (Fig. 2.1A).

The larger subunit, p45NF-E2, was cloned from murine erythroleukemia (MEL) cells and the human erythroid cell line K562, and is a member of the CNC family of bZIP proteins (Andrews et al., 1993b; Chan et al., 1993b; Ney et al., 1993). The CNC domain, named for the prototype cap’n’collar protein in Drosophila, is distinguished by a region of 43 amino acids immediately N-terminal to the bZIP domain and is conserved in species from Caenorhabditis elegans to humans (Andrews et al., 1993b; Blackwell et al., 1994; Mohler et al., 1991). Expression of p45 NF-E2 mRNA is restricted to developing erythroid cells, hematopoietic progenitors, megakaryocytes, and mast cells (Andrews et al., 1993a). The full-length cDNA predicts a polypeptide of 373 amino acids, and two major polypeptides of apparent molecular mass 43-45 kD are evident upon electrophoresis, apparently reflecting translation from separate initiation codons (Andrews et al., 1993a,b; Ney et al., 1993). The p45 NF-E2 gene, located on chromosome 12q13 in man and on the distal third of mouse chromosome 15, includes three exons, the first of which is noncoding (Ney et al., 1993; Peters et al., 1993). The human fetal liver harbors a splicing variant of p45 NF-E2 mRNA, transcribed from an alternative promoter

BIOCHEMISTRY AND EXPRESSION OF THE NF-E2 PROTEIN COMPLEX

located within the first intron; both RNA isoforms share the second and third exons (Pischedda et al., 1995). The functional significance of two transcripts that encode the identical protein is unknown. p45 NF-E2 mRNA and protein expression are evident at the earliest stage of hematopoiesis, in the murine yolk sac, and persist into adulthood without detectable variation during development. The determinants of tissue-restricted expression of p45 NF-E2 RNA have not been characterized extensively, although there is some evidence of a role for the lineage-restricted transcriptional regulator GATA-1 and its coactivator FOG (Tsang et al., 1997). In GATA-1deficient megakaryocytes (Shivdasani et al., 1997), p45 NF-E2 mRNA expression is reduced 35-fold (Vyas et al., 1999). Thrombopoietin (Tpo) induces expression of p45 NF-E2 in the hematopoietic cell line FDC-P2 (Nagata et al., 1995) and p45 mRNA in mouse megakaryocytes increases in response to administration of Tpo (Zimmet et al., 1998); however, it is still not clear if Tpo is absolutely required for p45 NF-E2 expression in vivo. In MEL cells chemically stimulated to differentiate in vitro, NF-E2 activity in EMSAs is increased (Mignotte et al., 1989; Ney et al., 1990a). Although transcriptional activation of the p45 gene may make some contribution to this effect, posttranslational modification also likely plays a role, as suggested by studies in which NF-E2 activity is modulated in vitro by protein kinase A (Garingo et al., 1995), vanadate (Lam and Bresnick, 1995), or the Ras-Raf-MAP kinase signaling pathway (Nagai et al., 1998; Versaw et al., 1998). The smaller (:18 kD) subunit of NF-E2 can be any of at least three bZIP proteins related to the avian oncogene v-maf (Andrews et al., 1993b). The known small-Maf proteins MafK (p18 NF-E2), MafG, and MafF are composed of 149-162 amino acids, have a molecular mass of 18—20 kD, and share 60 — 70% amino acid sequence identity (Blank et al., 1997; Fujiwara et al., 1993; Igarashi et al., 1995; Motohashi et al., 1997). Tissue expression of mRNAs encoding the small-Maf proteins is ubiquitous, including hematopoietic cells, although transcript levels vary. The small-Maf proteins appear to interact with the larger (5) portion of the asymmetric consensus NF-E2 site, (G/A)CTGA(C/G) (An-

15

drews et al., 1993b; Igarashi et al., 1994; Johnsen et al., 1996), thus imparting to the p45-p18 complex the specificity for the extended AP-1 — like DNA sequence. Therefore, the NF-E2 consensus site is sometimes referred to as the Maf recognition element or MARE. p45 NF-E2 itself interacts only very weakly with the NF-E2 consensus site as a monomer or homodimer; in contrast, small-Maf proteins can bind as homodimers to extended (palindromic) NFE2—like DNA sites, at least when overexpressed in cultured cells (Igarashi et al., 1994). The hypothesis that this binding results in negative regulation of transcriptional activity remains to be validated in vivo. Because small-Maf proteins lack an activation domain, they are presumed to function through interaction with various bZIPclass transcription factors (Fujiwara et al., 1993; Igarashi et al., 1994). Indeed, each of them is capable of associating with p45 NF-E2 in vivo, as well as with Fos and with several p45-related proteins (Blank et al., 1997; Johnsen et al., 1996; Johnsen et al., 1998; Kataoka et al., 1994; Toki et al., 1997). Characterization of the NF-E2 protein complex in primary murine megakaryocytes is discussed in greater detail below, with special emphasis on the functional implications. The known relationships between the structure and functions of p45 NF-E2 are illustrated schematically in Figure 2.1B. The NH2-terminal proline- and serine-rich region encompassing :200 amino acids of the p45 subunit includes a functional transcriptional activation domain (Kotkow and Orkin, 1995), and the first 80 amino acids are required for activity in transient transfection assays (Amrolia et al., 1997; Nagai et al., 1998). In MEL cells lacking endogenous p45 NF-E2 expression, multiple regions of exogenous p45 NF-E2 are required to restore -globin gene expression, as revealed through detailed functional dissection of the molecule (Bean and Ney, 1997). Within the transactivation domain, two discrete proline-rich regions — the first in the NH2 terminus and the second near the CNC domain — are required for the rescue of -globin expression. Other mutations within the conserved CNC domain also markedly diminish the extent of rescue without affecting DNA binding (Bean and Ney, 1997). Thus it is suggested that the function of the CNC domain extends beyond a role in DNA binding

16


and probably into mediating critical proteinprotein interactions. Sequence-specific DNA-binding proteins function in part through physical interactions with other components of the transcriptional regulatory machinery and thus harbor the potential for numerous protein-protein interactions. The PPPPY sequence located at amino acids 79 — 83 of human p45 NF-E2 (PPPSY in the mouse protein) fits the consensus of the so-called PY motif (XPPXY); this serves as the ligand for the WW domain that is present in a number of signaling and regulatory proteins, including ubiquitin ligases (Gavva et al., 1997; Macias et al., 1996). Interestingly, RNA pol II-CTD and p45 NF-E2 bind similar WW domains (Gavva et al., 1997). The activation domain of p45 NF-E2 interacts with the TATAbinding protein-associated factor TAF 130 in '' erythroid cell lines, and absence of this domain interferes with - and -globin gene expression (Amrolia et al., 1997). p45 NF-E2 was also isolated independently in a yeast two-hybrid screen for proteins that interact with the thyroid hormone receptor and in turn was shown to interact with the general coactivator CBP (Cheng et al., 1997). Although the functional significance of the presumed multi-molecular protein complex in vivo remains unknown, these results illustrate the potential for diverse extracellular signals to influence the biological activity of NF-E2. For example, studies in MEL cells have indicated that NF-E2 activity may be influenced by phosphorylation by protein kinase A and MAP kinase (Garingo et al., 1995; Nagai et al., 1998; Versaw et al., 1998).

PROPOSED FUNCTIONS OF THE NF-E2 PROTEIN Erythroid cell- and developmental stage-specific expression of the - and -globin gene families requires functional, and probably physical, interactions between individual globin gene promoters and regulatory cis elements located within LCRs, at considerable distance from the structural genes (Crossley and Orkin, 1993). Experimental evidence indicates that the mechanisms of - and -globin LCR function in humans and mice are substantially similar (Trimborn et al., 1999).

The human -globin gene cluster, distributed over about 70 kb on the short arm of chromosome 11, contain 5 functional genes (, %, , and ) whose expression is both tissue- and developmental stage-specific: -, -, and -globin mRNAs are transcribed predominantly in embryonic, fetal, and adult erythroid cells, respectively (Collins and Weissman, 1984; Karlsson and Nienhuis, 1985). The human -globin LCR, located 6—22 kb upstream of the -globin gene, is a regulatory unit believed to control the transcription, chromatin structure, and replication timing of the -globin domain (Forrester et al., 1990; Forrester et al., 1989; Tuan et al., 1985). The LCR includes five DNase I hypersensitive sites (HS), four of which are specific to erythroid cells and developmentally stable; together these confer copy number-dependent high levels of erythroid-specific expression of linked reporter genes in transgenic mice, largely independent of the site of integration (Fraser et al., 1993; Grosveld et al., 1987; Ryan et al., 1989; Talbot et al., 1989; van Assendelft et al., 1989). The mouse locus includes a sixth HS that is not apparent in man (Bender et al., 1998). A Hispanic() thalassemia, in which the 5HSs 2—4, are deleted, has further implicated the LCR in the establishment of an erythroid-specific chromatin domain that is sensitive to digestion by DNaseI, and in timing of replication of the locus in S phase (Forrester et al., 1990). Dissection of the -globin LCR shows that the major activity is associated with 5HS 2—4, and at least three groups of cis elements, GATA, NF-E2/AP1, and CACCC, are found repeatedly within these core regions (Collis et al., 1990; Ellis et al., 1996; Fraser et al., 1990; Philipsen et al., 1990; Ryan et al., 1989; Talbot et al., 1989; 1990; Tuan et al., 1989). Tandem NF-E2 binding sites found within HS2 are required for high levels of transcription in both MEL cells and transgenic mice (Caterina et al., 1994; Moi and Kan, 1990; Ney et al., 1990a; Talbot and Grosveld, 1991). In contrast, HS3 and HS4, which include NF-E2 sites, show weak or no enhancer activity in transient assays, but are active when stably integrated (Ellis et al., 1996; Pruzina et al., 1991; Talbot et al., 1990). In stably transfected K562 erythroleukemia cells, only a 46 bp enhancer element containing the tandem NF-E2 binding sites is necessary and sufficient for increased -globin gene expression (Sorrentino et al., 1990).

PROPOSED FUNCTIONS OF THE NF-E2 PROTEIN

The human -globin gene complex, located on the short arm of chromosome 16, includes one embryonic () and two fetal/adult (2 and 1) globin genes whose transcription is controlled in part by an erythroid-specific DNase I hypersensitive site (HS-40) located 40 kb upstream of the globin gene (Higgs et al., 1990; Sharpe et al., 1992). HS-40 behaves as a classical erythroid cell-specific enhancer for the and globin gene promoters in transiently transfected erythroid cells (Zhang et al., 1993). The functional domain of HS-40 lies within a 300 bp stretch of DNA that contains three GATA sites, two NF-E2/AP1 motifs (5 and 3) and a CACCC sequence (Huang et al., 1998; Jarman et al., 1991); among these, the 3 NF-E2/AP1 site accounts for 75% of the enhancer function in K562 cells (Huang et al., 1998; Zhang et al., 1995). Thus, NF-E2 sites located in the -and -globin LCRs appear to be important regulators of high-level erythroid gene expression. Although the LCR is believed to modulate chromatin structure, the veracity and mechanism of such a function are unclear. The NF-E2 protein can bind to HS2 in chromatin and disrupt nucleosomes in an energy-dependent manner with resultant increased binding of GATA-1 to downstream sites (Armstrong and Emerson, 1996; Gong et al., 1996). The distance between the NF-E2 and GATA sites in HS2 is conserved in a variety of -globin cis elements, including human HS1, HS3, and HS4, suggesting a conserved mechanism of interaction between the two factors (Armstrong and Emerson, 1996). Site-directed mutagenesis further suggests a need for common structural elements, including NFE2 and GATA sites, within the core of each 5HS in the organization of erythroid-specific chromatin structure in the -globin LCR (Pomerantz et al., 1998; Stamatoyannopoulos et al., 1995). Although both HS1 and HS2 are detectable in MEL cell lines lacking NF-E2 protein and are unchanged in response to restored NFE2 activity, it is noteworthy that at least the tandem NF-E2 sites in HS2 are occupied by some protein other than NF-E2 in these cells (Kotkow and Orkin, 1995). The extent to which NF-E2 might contribute to generation and maintenance of an ‘‘open’’ chromatin domain in the -globin locus thus remains uncertain. Moreover, some experimental evidence has challenged the proposed dominant function of

17

the -globin LCR in establishing or maintaining a chromatin domain with increased sensitivity to in vitro digestion by DNase I. Developmentally regulated expression of the human - and -globin genes can occur in transgenic mice in the absence of the entire LCR (Starck et al., 1994). A mouse chromosome 7 carrying a targeted 25 kb deletion including all six DNase I HSs preserves the DNase I hypersensitivity of the rest of the locus as well as developmentally regulated -globin gene expression in an erythroid cell environment (Epner et al., 1998; Reik et al., 1998). The only detectable consequence of this deletion is a quantitative decrease in the levels of all -globin mRNAs, ranging from 60 to 95%, depending on the assay. These important studies indicate that the principal function of the LCR is as a classical transcription enhancer and not as a regulator of chromatin structure or of developmental specificity of gene expression, and that these distinct properties are not coupled in the manner that has dominated models of LCR function for over a decade. Nevertheless, the importance of the LCR and its NF-E2 binding sites in enhancing globin gene transcription has been demonstrated in every experimental system tested. In MEL cells expression of both - and globin genes is highly dependent on NF-E2: CB3, a MEL cell line that does not express endogenous p45 NF-E2 due to integration of Friend viral sequences within the gene locus, fails to express - and -globin genes, and restoration of expression is dependent on the levels of exogenously provided NF-E2 protein (Bean and Ney, 1997; Kotkow and Orkin, 1995; Lu et al., 1994). Consensus NF-E2 binding sites are also present in the promoters of several nonglobin erythroid-expressed genes, including PBGD, delta amino levulinic acid synthetase, ferrochelatase, and ferritin H, although these sites are not always conserved across species and in some cases do not bind the NF-E2 protein (Beaumont et al., 1994; Magness et al., 1998; Mignotte et al., 1989; Taketani et al., 1992). Nevertheless, this finding is reminiscent of the presence of the GATA sequence motif within promoters of virtually all erythroid-expressed genes (Orkin, 1992), of the importance of E-box motifs and basic helix-loop-helix (bHLH) proteins in activating muscle gene promoters (Olson and Klein, 1994), and of the role of the PU.1

18


protein in regulating myeloid gene expression (Tenen et al., 1997). Whereas the latter examples support the notion that selected lineage-restricted nuclear proteins may regulate an entire transcriptional program in terminally differentiated cells, there is as yet little direct experimental evidence for such a role for NF-E2.

PROTEINS RELATED TO NF-E2 IN STRUCTURE AND/OR FUNCTION The apparent importance of NF-E2 sites within the -globin LCR led several groups to employ a variety of approaches to identify the protein(s) that might bind this cis element. Among these, only the NF-E2 complex comprised of p45 NFE2 and small-Maf proteins is restricted in expression to hematopoietic cells and detected in EMSAs using erythroid or megakaryocytic nuclear extracts (Andrews et al., 1993b; Lecine et al., 1998a; Ney et al., 1993; Shivdasani and Orkin, 1995). NF-E2 — related factor 1 (Nrf1/ LCR-F1/TCF11) was isolated independently from hemin-treated K562 cDNA libraries by virtue of binding to the NF-E2 consensus sequence (Caterina et al., 1994) and through expression cloning in yeast (Chan et al., 1993a). Nrf1/LCR-F1 mRNA is widely expressed and encodes a 742 amino acid bZIP protein of the CNC-domain subfamily that can activate transcription via NF-E2 sites in transient transfection assays (Caterina et al., 1994). Targeted disruption of this gene in mice has been reported by two groups, with different results. In one case, the gene targeting introduced a selection cassette just upstream of the bZIP domain. Homozygous null fetuses were anemic and died between embryonic days 14 and 18; anemia resulted from an abnormal fetal liver environment rather than from intrinsic red blood cell abnormalities, and a role for the targeted gene in regulation of globin gene expression was not evident (Chan et al., 1998). In the second case, the gene targeting replaced exons 3 — 5 and most of exon 6. The mutant mice died much earlier as a result of a global failure of mesodermal differentiation; although early lethality precluded examination of erythropoiesis in vivo, studies using mutant ES cells again suggested a noncell-autonomous defect without reduced globin gene expression (Farmer et al., 1997). The pre-

cise reason for discrepant results with two genetargeting strategies remains unclear and includes the possibility that the more severe phenotype results from inadvertent inactivation of additional genes besides Nrf1/LCR-F1. NF-E2 — related factor 2 (Nrf2) was isolated independently from cDNA libraries derived from hemin-induced K562 cells (Moi et al., 1994), murine yolk sacs (Chui et al., 1995), and chicken erythrocytes (Itoh et al., 1995); it is a 66 kD protein with a bZIP domain that is highly homologous to that of p45 NF-E2. Consistent with this similarity, the protein can heterodimerize with small-Maf proteins and activate transcription through NF-E2 sites. Like Nrf1/LCRF1, however, it appears to be expressed almost ubiquitously and has no demonstrated role in regulating globin gene expression. Targeted disruption of the Nrf2 gene indicates that it is not essential for murine growth, development, or erythropoiesis (Chan et al., 1996); however, the induction of a small repertoire of genes responsive to oxidative stress is impared in Nrf2-deficient macrophages (Itoh et al., 1997), and additional evidence suggests that the principal function of this gene is in hepatic metabolism (Venugopal and Jaiswal, 1996, 1998). Bach 1 and Bach 2 belong to a novel family of CNC-type bZIP proteins that were isolated in a yeast two-hybrid screen using MafK/p18 as the bait (Oyake et al., 1996). They are characterized by the presence of a conserved hydrophobic N-terminal protein-protein interaction domain called BTB (for Broad complex/Tramtrack/ Bric-a-brac) or POZ (for Pox and Zinc finger) (Motohashi et al., 1997), previously recognized only in selected actin-binding and zinc-finger— containing proteins, some of which are known to modulate chromatin structure during development (Albagli et al., 1995; Dorn et al., 1993; Farkas et al., 1994). Expression of Bach 1 mRNA is ubiquitous, and that of Bach 2 is restricted to monocytes and neuronal cells. The Bach 1 and Bach 2 proteins have 716 and 739 amino acids, and molecular masses of 79 and 81 kD, respectively, and share little resemblance outside of the CNC and BTB domains. Neither is expressed at appreciable levels in mature erythroid cells, and each functions as a transcriptional repressor rather than as an activator in transient transfection assays (Igarashi et al., 1998; Oyake et al., 1996). The functions of these

THE ROLE OF NF-E2 IN ERYTHROID CELL DIFFERENTIATION AND GENE EXPRESSION IN VIVO

proteins in erythroid and other cells in vivo remains to be determined. There are seven known members of the Maf family, of which MafK (p18), MafF, and MafG constitute the subfamily of small-Maf proteins; the other four are large proteins that contain canonical trans-activation domains. c-Maf is a proto-oncogene in chickens and regulates Tcell—specific gene expression in mammals (Ho et al., 1996, 1998); MafB/Kreisler is implicated in central nervous system development (Cordes and Barsh, 1994); and the human gene Nrl and avian gene MafA control gene expression in the retina (Benkhelifa et al., 1998; Kumar et al., 1996). In hematopoietic cells, expression of MafB is restricted to myelomonocytes, in which it appears to interact physically with the nuclear proteins Ets-1 and c-Myb to modulate expression of lineage-restricted genes (Hedge et al., 1998; Sieweke et al., 1996). Targeted disruption of the murine MafG gene results in thrombocytopenia (see below), slow postnatal growth, and a movement disorder that may reflect expression of MafG RNA in the cerebellum (Shavit et al., 1998). Hence, both subunits of the functional NF-E2 transcription factor complex

19

belong to protein families that are implicated in regulating tissue-specific gene expression.

THE ROLE OF NF-E2 IN ERYTHROID CELL DIFFERENTIATION AND GENE EXPRESSION IN VIVO Surprisingly, mice lacking p45 NF-E2 show only a mild erythroid phenotype characterized by anemia, reticulocytosis, wide distribution of red cell size, and abnormal red cell morphology, including hypochromia, target cells, and frequent dysmorphic red cells. Some of these features are illustrated in the peripheral blood smears shown in Figure 2.2. Furthermore, the mice exhibit remarkable splenomegaly with active erythropoiesis. Erythroid cells developing in NF-E2 null mice do not show decreased levels of mRNA transcripts whose expression is potentially regulated by NF-E2, including - and -globin, PBGD, and ferrochelatase (Shivdasani and Orkin, 1995). This discrepancy between findings in MEL cells and p45 NF-E2 knockout mice may reflect the possibility that NF-E2 function in develop-

Figure 2.2. Peripheral blood smears from wild-type (left) and p45 NF-E2 knockout mice (right). In addition to the complete absence of platelets, p45 NF-E2\\ mice have abnormal red cells, including hypochromia, anisocytosis, poikilocytosis, dysmorphic red cell forms, and polychromasia. Original magnification 600;.

20


ing red cells in vivo is provided by other cellspecific or ubiquitous factors that are either absent or incompetent in MEL cells. In any case, the consistent presence of red cell abnormalities suggests that any potential compensation in vivo is incomplete. Candidate compensatory proteins include the AP1 complex and p45 NF-E2—related factors, including Nrf1, Nrf2, Bach 1, and Bach 2. However, the combined absence of p45 NF-E2 and c-jun, or of p45 NF-E2 and Nrf2, in double homozygote mice obtained by interbreeding of individual knockout strains, does not result in erythroid maturation defects beyond those seen with loss of p45 NF-E2 alone (Kuroha et al., 1998; Martin et al., 1998; Shivdasani and Orkin, 1995), and even in CB3 cells overexpression of Nrf1/LCRF1 cannot replace the function of p45 NF-E2 in increasing globin gene expression (Kotkow and Orkin, 1995). Thus, if one or more proteins can replace p45 NF-E2 function in globin gene regulation and red blood cell differentiation, their identity is unknown. Alternatively, other cis-acting DNA elements might compensate for the absence of functional NF-E2 protein in vivo. Indeed, targeted deletion of either the 5 HS2 or 5 HS3 regions has only modest effects on transcriptional output of the -globin locus (Fiering et al., 1995; Hug et al., 1996), again refuting predictions based on results in MEL cells and transgenic mice. Furthermore, mice lacking 5 HS3 show an erythroid phenotype consistent with mild thalassemia (Hug et al., 1996), with erythroid cell parameters nearly identical to those seen in adult NF-E2 knockout mice (Shivdasani and Orkin, 1995). These cis-element knockouts suggest that synergistic interactions between the 5HSs might confer the effects seen in some transgenic studies. More importantly, together with studies showing surprisingly modest effects on the globin gene locus when the entire LCR is deleted (Epner et al., 1998; Reik et al., 1998), they demonstrate that mechanisms of transcriptional regulation may acquire considerable specificity from the native chromatin environment. As a group, these experiments suggest that the control of high-level lineage-specific gene expression is likely to be far more complex than previously appreciated. Despite the apparent absence of severe globin gene expression abnormalities in the absence of

NF-E2, other defects in erythroid cell morphology are consistently present and point to a regulatory function for NF-E2 in red blood cell differentiation (Shivdasani and Orkin, 1995). As discussed below, mice lacking NF-E2 are profoundly thrombocytopenic and manifest a bleeding diathesis. Hence, the interpretation of an anemic phenotype is clouded by the simultaneous contributions of hemorrhage and intrinsic erythroid cell abnormalities. Nevertheless, experimental results have defined the scope of the latter defects, which include the presence of numerous small red cell fragments, abnormally wide distribution of red cell size, and baseline reticulocytosis of 6—24% (normal 2% in adult mice). As noted above, the magnitude of some abnormalities, including mean cell volume, hemoglobin content, and reticulocytosis, is very similar to values seen in mice with targeted disruption of the -globin LCR 5 HS3 (Hug et al., 1996). Importantly, these defects are observed to the same extent in all surviving adults, among whom the degree of hemorrhage is variable (Shivdasani and Orkin, 1995), and they are fully reproduced in lethally irradiated wild-type mice upon hematopoietic transfer of p45 NFE2—deficient cells (J.-L. Villeval and R. A. Shivdasani, unpublished data). The molecular basis of defective erythropoiesis in the absence of NF-E2 remains unclear.

THE ROLE OF NF-E2 IN MEGAKARYOCYTE DIFFERENTIATION AND PLATELET BIOGENESIS Although p45 NF-E2 is expressed in selected nonerythroid blood cell lineages, it was expected to manifest its most important functional requirements within developing red blood cells. However, the most remarkable aspect of mice with targeted disruption of p45 NF-E2 is profound thrombocytopenia (Shivdasani et al., 1995). Whereas automated platelet counts are approximately 10% of normal values, few if any platelets are detected on peripheral blood smears, and the mice display a florid hemorrhagic diathesis that results in 90% neonatal lethality. It is now apparent that the majority of particles recognized as platelets by automated means in fact represent small red cell fragments (RAS, unpublished data), presumably attesting

THE ROLE OF NF-E2 IN MEGAKARYOCYTE DIFFERIENTIATION AND PLATELET BIOGENESIS

to an independent requirement for NF-E2 function in erythroid homeostasis. The small minority of particles with platelet features show marked ultrastructural abnormalities and fail to be activated by platelet agonists in vitro (J.-P. Peng, S. A. Burstein, and RAS, unpublished data). We infer that these dysfunctional ‘‘platelets’’ are not produced through normal mechanisms and probably represent megakaryocyte debris. Several observations indicate that the profound thrombocytopenia seen in the absence of p45 NF-E2 reflects a primary cell-autonomous disturbance in terminal megakaryocyte differentiation. First, surviving p45 NF-E2—deficient mice show megakaryocytosis in the bone marrow and spleen (Shivdasani et al., 1995). Second, serum from the knockout mice does not lead to thrombocytopenia in normal recipients and neither recombinant thrombopoietin nor serum from normal mice corrects the platelet defect (Shivdasani et al., 1995), Third, the thrombocytopenia is not corrected by splenectomy (J. Levin and RAS, unpublished data). Finally, reconstitution of lethally irradiated normal recipients by hematopoietic cells derived from p45 NF-E2—deficient mice reproduces the phenotype of anemia, thrombocytopenia, and megakaryocytosis (Lecine et al., 1998b). To appreciate the basis of thrombocytopenia in the absence of NF—E2, it is first necessary to understand the normal mechanism of thrombocytopoiesis. Following lineage commitment, normal megakaryocytes initially undergo sequential rounds of DNA replication without cytoplasmic division (endomitosis) (Hegyi et al., 1991; Hoffman, 1989). Cytoplasmic maturation commences after endomitosis and is characterized by the development of platelet-specific granules and other organelles within a demarcation membrane system (DMS) that is unique to megakaryocytes (Behnke, 1968) and may provide the lipid surface necessary for a single megakaryocyte to produce hundreds or thousands of individual blood platelets (Radley and Haller, 1982). Specific molecular markers are known to be expressed sequentially during megakaryocyte differentiation. Glycoprotein (GP) IIb is one of the earliest well-characterized markers, and is expressed both in bipotential erythroid-megakaryocyte progenitors and committed early megakaryocytes (Levene et al.,

21

1985; Prandini et al., 1992). Platelet factor 4 (PF4) is expressed later than GPIIb but considerably earlier than P-selectin (Ryo et al., 1992; Schick et al., 1993). The cellular and molecular basis of megakaryocyte differentiation is poorly understood, and the mechanism of platelet release remains uncertain. The major tenable models invoke (1) fragmentation of the entire megakaryocyte cytoplasm, including preassembled platelets, or (2) platelet biogenesis through the generation of intermediate structures known as proplatelets (Radley and Scurfield, 1980; Stenberg and Levin, 1989). Notably, the DMS does not contain the microtubule bundles known to be a part of the mature platelet structure or other cytoskeletal elements considered necessary for platelet biogenesis (Radley and Haller, 1982). Proplatelets are long cytoplasmic extensions that contain plateletspecific organelles and microtubules within an area defined by constriction points between platelet-size particles; they have been recognized in vivo and in vitro (Becker and de Bruyn, 1976; Choi et al., 1995; Radley and Scurfield, 1980). Megakaryocytes lacking p45 NF-E2 have an abnormally large cytoplasm with an expanded DMS but very few platelet-specific granules (Shivdasani et al., 1995). They are responsive to thrombopoietin, endomitosis is unimpaired, and mRNA levels of many lineage-specific markers, including GPIIb and PF4, are the same as in control cells (Shivdasani et al., 1995). Together, these findings point to a late arrest in megakaryocyte development in the absence of NFE2. When hematopoietic cells are cultured in recombinant thrombopoietin, mature megakaryocytes produce impressive arrays of proplatelets (Choi et al., 1995; Cramer et al., 1997; Lecine et al., 1998b); although p45 NF-E2—deficient megakaryocytes attain a large size and polyploidy under these conditions, they fail to produce proplatelets (Lecine et al., 1998b), as illustrated in Figure 2.3. Other features are similar to those seen in vivo, including dearth of granules and abundance of DMS. Supernatants from cultures of wild-type megakaryocyte fail to stimulate proplatelet formation in p45 NF—E2 deficient cells, and supernants from cultures of NF-E2 null megakaryocyte do not inhibit proplatelet formation by wild-type cells (Lecine et al., 1998b). Thus, the differentiation arrest in the absence of NF-E2 function is manifested around

22


Figure 2.3. Scanning electron micrographs of p45 NF-E2>\ (a), p45 NF-E2>> (b), and p45 NF-E2\\ (c) cultured megakaryocytes. Control megakaryocyte shows proplatelet extensions; p45 NF-E2 knockout megakaryocytes grow to a larger size but do not develop proplatelets. (Reprinted from Lecine et al., 1998b, with permission from Blood)

the stage at which normal megakaryocytes prepare to generate proplatelets, the immediate precursors of circulating platelets. Thus, genes required for terminal aspects of megakaryocyte differentiation, including cytoplasmic reorganization and proplatelet formation, may be critical targets of NF-E2 function. The cell-autonomous nature of the phenotype further suggests that the relevant trancriptional targets of NF-E2 are megakaryocyte-expressed genes. We have characterized the nature of the NFE2 DNA-protein complex in primary murine megakaryocyte using EMSA and immunoblot analysis (Lecine et al., 1998a). Notably, a MafG antiserum (probably cross-reactive with the closely related MafF protein) consistently displayed a greater effect on the EMSA complex than did an anti-p18/MafK—specific antibody (Fig. 2.4A). Immunoblot analysis further indicated that 18—20 kD proteins corresponding to MafF and/or MafG were more abundant than MafK in primary megakaryocytes, with the reverse pattern seen in erythroid cells (Fig. 2.4B).

Importantly, although the predominant NF-E2 complex in primary mouse megakaryocytes is between p45 and either MafF or MafG, rather than p18/MafK, the DNA binding-site preference is the same for either complex. In contrast to MafK-deficient mice, which show no hematologic abnormalities (Kotkow and Orkin, 1996), mice lacking MafG are thrombocytopenic (Shavit et al., 1998), with platelet counts about half of the normal value and an increase in megakaryocyte numbers. Thus, whereas MafF or MafG can apparently substitute for MafK function in developing erythroid cells in vivo, any substitution of MafG in megakaryocytes may be incomplete. Given the high sequence similarity between the known small-Maf proteins and identical DNA-binding specificity of the various complexes, this difference is likely to reflect the expression pattern rather than a functional distinction between closely related proteins. The essential transcriptional targets of established regulators of megakaryocyte differenti-

CONCLUSION AND FUTURE DIRECTIONS

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Figure 2.4. Composition of the NF-E2 complex in mouse primary megakaryocytes (MK) and MEL cells. A: EMSA of nuclear extracts from MEL cells and primary MKs, using antibodies directed against p45 NF-E2 (lanes 3, 4, 10, 11) and small-Maf proteins (lanes 5—7, 12—14) The antisera p45A and p45N were generously provided by N. C. Andrews and P. Ney, respectively. B: Immunoblot analysis of nuclear extracts from MEL cells or primary MKs with p18/MafK, MafG/MafF, and p45 NF-E2 antisera. (Reprinted from Lecine et al., 1998a, with permission from T he Journal of Biological Chemistry.)

ation, including GATA-1, FOG, and NF-E2, are not well understood (Shivdasani et al., 1997; Tsang et al., 1997, 1998; Vyas et al., 1999). NF-E2 may regulate transcription of a limited number of genes, one or more of which is essential for late megakarycocyte maturation. Alternatively, NF-E2 may coordinate an entire transcriptional program of terminal megakaryocyte differentiation via regulation of multiple essential genes. Investigation of this question is an active pursuit in our laboratory. To date, thromboxane synthase is the only megakaryocyte-expressed gene with known functional NF-E2—binding sites within characterized cis elements (Deveaux et al., 1997; Zhang et al., 1997), although it is unclear if and how loss of this gene might contribute to profound thrombocytopenia. Recent unpublished work

from our laboratory strongly suggests that NFE2 regulates transcription of genes encoding megakaryocyte-specific cytoskeletal proteins; this finding is consistent with our previous analysis indicating that the essential need for NF-E2 function is manifested at a differentiation stage when megakaryocytes undergo extensive cytoplasmic and cytoskeletal reorganization to generate large numbers of proplatelets.

CONCLUSION AND FUTURE DIRECTIONS The transcription factor NF-E2 is a heterodimer comprised of hematopoietic-specific (p45) and widely expressed (p18/small-Maf) subunits, both of which belong to the bZip family of nuclear proteins. Expression of p45 NF-E2 mRNA is

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restricted to selected hematopoietic cell lineages. NF—E2 binds to critical cis elements in the and -globin LCRs and affects cell-specific enhancement of globin gene expression through these sites in transgenic mice and erythroid cell lines. NF-E2 binding sites have also been identified in the promoters of several nonglobin erythroid-restricted genes. Mice lacking p45 NF-E2 are anemic and have multiple red blood cell abnormalities, although these do not appear to reflect alterations in mRNA levels of any of these putative transcriptional target genes. The knockout mice are profoundly thrombocytopenic and thus reveal an essential requirement for NF-E2 in the terminal phase of megakaryocyte differentiation. This differentiation arrest is manifested at a stage when megakaryocytes normally undergo extensive cytoplasmic reorganization and produce proplatelets, the precursors of circulating platelets. Inasmuch as NF-E2 serves as one useful model for approaching problems in the transcriptional control of cell differentiation, important questions remain unanswered on several fronts. First, it is unknown whether the lack of severe erythroid defects and of diminished globin gene expression in the absence of NF-E2 reflect redundancy at the level of transcription factors, LCR cis elements, both, or neither. The discrepancies between the predictions from transgenic mice and the observations in knockout mice may well indicate complexities of gene regulation that defy simple explanations. A better understanding of the repertoire and hierarchy of nuclear proteins capable of providing activation through NF-E2 sites would help elucidate the transcriptional basis of erythroid cell differentiation and gene expression. Second, the important transcriptional targets of NF-E2 remain largely unknown. The expression pattern of p45 NF-E2 and the cell-autonomous nature of the knockout phenotype together indicate that these target genes of NF-E2 are expressed in erythroid cells and megakaryocytes. Target genes identified in megakaryocytes are particularly likely to provide useful insights into the mysterious process of mammalian platelet biogenesis. Third, identifying the determinants of lineage-specific expression of p45 NF-E2 itself will help elucidate the transcriptional hierarchies underlying hematopoietic cell differentiation. Finally, there is only limited appreciation

of the combinatorial protein-protein interactions and extracellular signals that modulate NF-E2 function and determine lineage-specific outcomes; characterization of these influences will advance our understanding of the molecular control of hematopoiesis.

ACKNOWLEDGMENTS We are grateful to Patrick Lecine, Stuart Orkin, and Jack Levin for helpful discussions. SWK is supported by the University of Ulsan and by a grant from the Asan Life Science Institute (Seoul, Korea). RAS is supported in part by grants from the Harcourt General Charitable Foundation, the Dolphin Trust, and the Cancer Research Fund of the Damon Runyon—Walter Winchell Foundation.

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region is necessary for gene expression in the human beta-globin locus but not the maintenance of an open chromatin structure in erythroid cells. Mol. Cell. Biol. 18, 5992—6000. Romeo, P.-H., Prandini, M.-H., Joulin, V., Mignotte, V., Prenant, M., Vainchenker, W., Marguerie, G., and Uzan, G. (1990). Megakaryocytic and erythrocytic lineages share specific transcription factors. Nature 344, 447—449. Ryan, T. M., Behringer, R. R., Martin, N. C., Townes, T. M., Palmiter, R. D., and Brinster, R. L. (1989). A single erythroid-specific DNase I super-hypersensitive site activates high levels of human betaglobin gene expression in transgenic mice. Genes Dev. 3, 314—323. Ryo, R., Yasunaga, M., Saigo, K., and Yamaguchi, N. (1992). Megakaryocytic leukemia and platelet factor 4. Leuk. Lymphoma 8, 327—336. Schick, P. K., Konkle, B. A., He, X., and Thornton, R. D. (1993). P-selectin mRNA is expressed at a later phase of megakaryocyte maturation than mRNAs for von Willebrand factor and glycoprotein Ibalpha. J. Lab. Clin. Med. 121, 714—721. Sharpe, J. A., Chan-Thomas, P. S., Lida, J., Ayyub, H., Wood, W. G., and Higgs, D. R. (1992). Analysis of the human globin upstream regulatory element (HS-40) in transgenic mice. EMBO J. 11, 4565— 4573. Shavit, J. A., Motohashi, H., Onodera, K., Akasaka, J., Yamamoto, M., and Engel, J. D. (1998). Impaired megakaryopoiesis and behavioral defects in mafGnull mutant mice. Genes Dev. 12, 2164—2174. Shivdasani, R. A., and Orkin, S. H. (1995). Erythropoiesis and globin gene expression in mice lacking the transcription factor NF-E2. Proc. Natl. Acad. Sci. USA 92, 8690—8694. Shivdasani, R. A., and Orkin, S. H. (1996). The transcriptional control of hematopoiesis. Blood 87, 4025—4039. Shivdasani, R. A., Rosenblatt, M. F., Zucker-Franklin, D., Jackson, C. W., Hunt, P., Saris, C., and Orkin, S. H. (1995). Transcription factor NF-E2 is required for platelet formation independent of the actions of thrombopoietin/MGDF in megakaryocyte development. Cell 81, 695—704. Shivdasani, R. A., Fujiwara, Y., McDevitt, M. A., and Orkin, S. H. (1997). A lineage-selective knockout establishes the critical role of transcription factor GATA-1 in megakaryocyte growth and platelet development. EMBO J. 16, 3965—3973. Sieweke, M. H., Tekotte, H., Frampton, J., and Graf, T. (1996). MafB is an interaction partner and repressor of Ets-1 that inhibits erythroid differentiation. Cell 85, 49—60. Sorrentino, B. P., Ney, P. A., Bodine, D. M., and Nienhuis, A. W. (1990). A 46 base pair enhancer sequence within the locus activating region is required for induced expression of the -globin gene during erythroid differentiation. Nucl. Acids Res. 18, 2721—2731.

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Stamatoyannopoulos, J. A., Goodwin, A., Joyce, T., and Lowrey, C. H. (1995). NF-E2 and GATA binding motifs are required for the formation of DNase I hypersensitive site 4 of the human globin locus control region. EMBO J. 14, 106—115. Starck, J., Sarkar, R., Romana, M., Bhargava, A., Scarpa, A. L., Tanaka, M., Chamberlain, J. W., Weissman, S. M., and Forget, B. G. (1994). Developmental regulation of human gamma- and betaglobin genes in the absence of the locus control region. Blood 84, 1656—1665. Stenberg, P. E., and Levin, J. (1989). Mechanisms of platelet production. Blood Cells 15, 23—47. Taketani, S., Inazawa, J., Nakahashi, Y., Abe, T., and Tokunaga, R. (1992). Structure of the human ferrochelatase gene. Eur. J. Biochem. 205, 217—222. Talbot, D., and Grosveld, F. (1991). The 5’ HS 2 of the globin locus control region enhances transcription through the interaction of a multimeric complex binding at two functionally distinct NF-E2 binding sites. EMBO J. 10, 1391—1398. Talbot, D., Philipsen, S., Fraser, P., and Grosveld, F. (1990). Detailed analysis of the site 3 region of the human b-globin dominant control region. EMBO J. 9, 2169—2178. Talbot, D., Collis, P., Antoniou, M., Vidal, M., Grosveld, F., and Greaves, D. R. (1989). A dominant control region from the human beta-globin locus conferring integration site-independent gene expression. Nature 338, 352—355. Tenen, D. G., Hromas, R., Licht, J. D., and Zhang, D.-E. (1997). Transcription factors, normal myeloid development, and leukemia. Blood 90, 489—519. Toki, T., Itoh, J., Kitazawa, J., Arai, K., Hatakeyama, K., Akasaka, J., Igarashi, K., Nomura, N., Yokoyama, M., Yamamoto, M., and Ito, E. (1997). Human small Maf proteins form heterodimers with CNC family transcription factors and recognize the NF-E2 motif. Oncogene 14, 1901—1910. Trimborn, T., Gribnau, J., Grosveld, F., and Fraser, P. (1999). Mechanisms of developmental control of transcription in the murine alpha- and beta-globin loci. Genes Dev. 13, 112—124. Tsang, A. P., Visvader, J. E., Turner, C. A., Fujiwara, Y., Yu, C., Weiss, M. J., Crossley, M., and Orkin, S. H. (1997). FOG, a multitype zinc finger protein, acts as a cofactor for transcription factor GATA-1 in erythroid and megakaryocytic differentiation. Cell 90, 109—119. Tsang, A. P., Fujiwara, Y., Hom, D. B., and Orkin, S. H. (1998). Failure of megakaryopoiesis and arrested erythropoiesis in mice lacking the GATA-1 transcriptional cofactor FOG. Genes Dev. 12, 1176—1188.

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Tuan, D., Solomon, W., Li, Q., and London, I. M. (1985). The ‘‘beta-like globin’’ gene domain in human erythroid cells. Proc. Natl. Acad. Sci. USA 82, 6384—6388. Tuan, D. Y., Solomon, W. B., London, I. M., and Lee, D. P. (1989). An erythroid-specific, developmentalstage-independent enhancer far upstream of the human ‘beta-like globin‘ genes. Proc. Natl. Acad. Sci. USA 86, 2554—2558. van Assendelft, B., Hanscombe, O., Grosveld, F., and Greaves, D. R. (1989). The -globin dominant control region activates homologous and heterologous promoter in a tissue-specific manner. Cell 56, 969—977. Venugopal, R., and Jaiswal, A. K. (1996). Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the human antioxidant response element— mediated expression of NAD(P)H:quinone oxidoreductase1 gene. Proc. Natl. Acad. Sci. USA 93, 14,960—14,965. Venugopal, R., and Jaiswal, A. K. (1998). Nrf2 and Nrf1 in association with Jun proteins regulate antioxidant response element—mediated expression and coordinated induction of genes encoding detoxifying enzymes. Oncogene 17, 3145—3156. Versaw, W. K., Blank, V., Andrews, N. M., and Bresnick, E. H. (1998). Mitogen-activated protein kinases enhance long-range activation by the betaglobin locus control region. Proc. Natl. Acad. Sci. USA 95, 8756—8760. Vyas, P., Ault, K., Jackson, C. W., Orkin, S. H., and Shivdasani, R. A. (1999). Consequences of GATA1 deficiency in megakaryocytes and platelets. Blood 93 2867—2875. Zhang, L., Xiao, H., Schultz, R. A., and Shen, R. F. (1997). Genomic organization, chromosomal localization, and expression of the murine thromboxane synthase gene. Genomics 45, 519—528. Zhang, Q., Rombel, I., Reddy, G. N., Gang, J. B., and Shen, C. K. (1995). Functional roles of in vivo footprinted DNA motifs within an alpha- globin enhancer. Erythroid lineage and developmental stage specificities. J. Biol. Chem. 270, 8501—8505. Zhang, Q., Reddy, P. M., Yu, C. Y., Bastiani, C., Higgs, D., Stamatoyannopoulos, G., Papayannopoulou, T., and Shen, C. K. (1993). Transcriptional activation of human zeta 2 globin promoter by the alpha globin regulatory element (HS-40): functional role of specific nuclear factor—DNA complexes. Mol. Cell. Biol. 13, 2298—2308. Zimmet, J., Toselli, P., and Ravid, K. (1998). Cyclin D3 and megakaryocyte development: exploration of a transgenic phenotype. Stem Cells 16(2),: 97— 106.

CHAPTER 3


TRANSCRIPTION FACTORS INVOLVED IN LINEAGE-SPECIFIC GENE EXPRESSION DURING MEGAKARYOPOIESIS YULIA KALUZHNY AND KATYA RAVID Department of Biochemistry and the Whitaker Cardiovascular Institute, Boston University School of Medicine

MORTIMER PONCZ Department of Pediatrics, University of Pennsylvania School of Medicine

INTRODUCTION Megakaryocytes are hematopoietic precursors of platelets, which play an essential role in thrombosis and hemostasis (Review in Packham, 1994). Megakaryopoiesis starts with the commitment of stem cells in response to various homeostatic stimuli. These stimuli induce proliferation of diploid promegakaryoblasts, producing a pool of mature megakaryocytes. The hallmark of megakaryocyte development is the formation of a large cell (:50 M diameter) containing a single, large, lobulated, polyploid nucleus. Unlike other cells, megakaryocytes undergo an endomitotic cell cycle during which they replicate DNA but do not undergo cytokinesis, and as a result, acquire a DNA content of up to 128N (where 2N is the DNA content of a somatic cell) (Jackson et al., 1984; Odell and Jackson, 1968; Odell et al., 1970). An individual mature megakaryocyte produces on the order of

2000 to 3000 platelets (Review in Long, 1998). All degrees of polyploidy are present in cells of each stage of cytoplasmic maturation, showing that a cell can mature cytoplasmically at any ploidy level. However, few if any tetraploid and no diploid megakaryocytes are found in normal animals, indicating that megakaryocyte proliferation, membrane demarcation, and platelet formation are not strictly sequential events (Ebbe, 1976). Historically, the study of megakaryocyte differentiation and its transcriptional regulation had been restricted by the scarcity of these cells in the bone marrow (:0.05% of total mononuclear cells), the difficulties associated with isolating and managing a sufficient number of primary megakaryocytes, and the limited megakaryocytic potential of most established cell lines. Various growth factors regulate megakaryocyte differentiation at different levels. Certain cytokines such as interlukin-3 (IL-3), IL-6,


31

32

TRANSCRIPTION FACTORS AND MEGAKARYOPOIESIS

IL-11, IL-12, granulocyte-macrophage colony stimulating factor (GM-CSF), and erythropoietin (EPO) stimulate proliferation of megakaryocytic progenitors (Gordon and Hoffman, 1992). Others have been reported to modulate megakaryocyte maturation and platelet development including IL-1 and Leukemia Inhibitory Factor (LIF) (Gordon and Hoffman, 1992; Vainchenker et al., 1995a). However, all of the above-mentioned growth factors and cytokines have a very broad effect on all hematopoietic cell lines. The discovery and cloning of thrombopoietin (TPO), the ligand of the myeloproliferative (Mpl) receptor (Souyri et al., 1990), opened a new perspective in megakaryocyte differentiation studies (Bartley et al., 1994; Kaushansky et al., 1994; Lok et al., 1994). TPO was found to be a specific factor that controls megakaryocytic cell proliferation as well as maturation (Arnold et al., 1997; de Sauvage et al., 1994; Wendling et al., 1994; Zucker-Franklin and Kaushansky, 1996). Abrogated expression of Mpl receptor in transgenic mice results in moderate thrombocytopenia, with an 85% decrease in the number of megakaryocytes in the bone marrow (Gurney et al., 1994). Availability of recombinant TPO increased the feasibility of studying transcriptional regulation of megakaryopoiesis in immortalized multipotential cell lines containing the Mpl receptor. Moreover, the high specificity of Mpl ligand makes it possible to achieve selective expansion of mature megakaryocytes from primary bone marrow cells at the expense of other hematopoietic lineages (Drachman et al., 1997). This chapter will focuses on reviewing studies related to the role of specific transcription factors in promoting gene expression in the megakaryocytic lineage. Some of these factors may mediate the effects of TPO on this lineage.

TRANSCRIPTION FACTORS BINDING TO Cis-REGULATORY ELEMENTS IN GENES SPECIFICALLY EXPRESSED IN MEGAKARYOCYTES Comprehending the entire complement of lineage-restricted transcription factor—mediated gene activation events at any given stage of differentiation of a given cell type is one of the

ultimate goals of developmental biology. At present, it seems that rather than being controlled by a single specific master regulator, lineagespecific gene expression depends on a specific complement of cell-restricted and ubiquitously expressed transcription factors that mediate the net effect of the variety of external signals (cytokines, interleukins, colony-stimulating factors, extracellular matrix) (Review in Tenen et al., 1997). At the same time, it has been shown that particular patterns of transcription factor expression can overlap not only in a temporal pattern but also in a spatial pattern, which suggests its functional overlap during development of various hematopoietic lineages (Review in Sieweke and Graf, 1998). Resent observations, such as in the case of FOG (Friend of GATA -1 transcription factor coactivator), demonstrate that not only protein-DNA interactions but also protein-protein interactions of transcription factors and cofactors can both stabilize DNA contacts and modify the function of specific nuclear proteins in a particular cell type (Tsang et al., 1998). At the present time it is difficult to report precisely what transcription factor(s) is essential for megakaryocytic gene expression, and a ‘‘master switch type’’ megakaryocyte-specific transcription factor has not yet been described. However, analyses of regulatory regions of megakaryocyte-specific genes have defined a number of transcription factors that play significant roles in megakaryocyte development. Each phase in cellular development represents a specific step in a program with subsequent changes in the complement of cytoplasmic proteins and nuclear factors. During the process of megakaryopoiesis, cells turn on various genes, some of which are expressed exclusively in megakaryocytes and platelets, and therefore serve as the specific markers of this lineage. Genes, whose expression is restricted to megakaryocytes and whose products are restricted to platelets, include (1) platelet glycoprotein IIb (GPIIb or IIb) — part of the integrin fibrinogen receptor, which facilitates platelet interaction with injured blood vessels (Prandini et al., 1988); (2) Mpl receptor (Vainchenker et al., 1995b); (3) von Willebrand factor (vWF) receptor subunits: platelet glycoproteins, GPIb, GPIb, GPIX, GPV — the receptor that mediates platelet adhesion at the site of vascular

TRANSCRIPTION FACTORS BINDING TO CIS-REGULATORY ELEMENTS IN GENES

33

TABLE 3.1. Transcription Factors Implicated in Megakaryocytic Development

Transcription Factors

Protein Class

Methods of Study Indicating Involvement in Megakaryopoiesis

GATA-1 GATA-2

Zinc finger

Gene targeting; Northern analysis; forced expression; EMSA; transactivation studies (GPIIb, PF4, Mpl, p45NF-E2 promoters)

p45 NF-E2

bZIP

Gene targeting; Northern analysis

MafG FOG

bZIP Zinc finger

CREB PU.1

CREB/ATF Ets

Ets-1

Ets

Ets-2

Ets

Fli-1

Ets

c-Myc

bHLH-ZIP

Gene targeting Gene targeting; forced expression; transactivation studies (p45NF-E2 promoter) Differentiation assay Northern analysis; in situ; EMSA; gene targeting; transactivation studies (GPIIb, Mpl, PBP promoters) Northern analysis; in situ; EMSA; transactivation studies (GPIIb, PF4, Mpl promoters) Northern analysis; EMSA; transactivation studies (GPIIb promoter) Northern analysis; EMSA; transactivation studies (Mpl, GPIX promoters) Gene targeting; Northern analysis; in situ Overexpression

c-Myb

Sp1

Zinc finger

EKLF HoxA10

Kru¨ppel-like Homeobox

Transactivation studies; EMSA (GPIIb, cyclin D3 promoters) Overexpression Overexpression

Assay Systems Cell culture; PBM; ES; TG

TG

TG TG; cell culture

References (Martin et al., 1990; Pevny et al., 1995; Ravid et al., 1991b; Shivdasani et al., 1997; Tsai et al., 1994; Vyas et al., 1999; Yamaguchi et al., 1998) (Lecine et al., 1998b, Shivdasani et al., 1995) (Shavit et al., 1998) (Tsang et al., 1997, 1998)

Cell culture Cell culture; ES

(Zauli et al., 1998) (Doubeikovsky et al., 1997; Hromas et al., 1993; Uzan et al., 1991; Zhang et al., 1997)

Cell culture

(Deveaux et al., 1996; Minami et al., 1998)

Cell culture

(Lemarchandel et al., 1993)

Cell culture

(Bastian et al., 1999; Ben-David et al., 1991; Deveaux et al., 1996; Melet et al., 1996) (Dorn et al., 1994; Thompson et al., 1996) (Guy et al., 1996; Rosson and O’Brien, 1995) (Block et al., 1996; Wang et al., 1999)

Cell culture; TG Cell culture

Cell culture; PBM

TG PBM

(Tewari et al., 1998) (Thorsteinsdottir et al., 1997)

Abbreviations: PBM, primary bone marrow culture; TG, transgenic mice; EMSA, electrophoretic mobility shift assay; Meg, megakaryocytes; ES, embryonic stem cells.

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injury (Hickey and Roth, 1993; Lanza et al., 1993; Rabellino et al., 1981); (4) platelet factor 4 (PF4)—chemokine component of the -granule of potential immunomodulatory and antiangiogenic activity (Deuel et al., 1981); (5) platelet basic protein (PBP), which is cleaved to thromboglobulin (TG) and further cleaved to neutrophil-activating peptide 2 (NAP-2) — another chemokine component of -granules, potentially involved in processes of coagulation, inflammation, and wound repair (Majumdar et al., 1991; Varma et al., 1982). It is believed that lineage-restricted transcription factors modulate cell progression through differentiation steps by activating specific genes. These genes are necessary for the progress of differentiation or are essential for the proper function of the final product, the platelet. Cloning of the above genes restrictively expressed in the megakaryocytic lineage, identification of promoters and enhancer regions within those genes, and analysis of cis-regulatory sequences allowed identification of several trans-acting factors that play an important role in the tissuespecific expression of these genes. Northern blot analysis and in situ hybridization analysis further confirmed that these transcription factors are expressed in the megakaryocytic lineage. The list of transcription factors implicated in megakaryopoiesis is provided in Table 3.1. Notice that: (1) none of the above transcription factors are expressed only in the megakaryocytic lineage. Many of the mentioned transcription factors are coexpressed in megakaryocytes, mast cells and maturing erythroid cells, which may lead one to believe that these three lineages represent the progeny of a common precursor cell; (2) some of the listed transcription factors have no known target genes but have been implicated in megakaryopoiesis and/or thrombopoiesis via gene knockout studies; (3) the degree of importance for megakaryocytic development of some of these transcription factors is not clear; and (4) expression kinetics during different stages of megakaryopoiesis for most of the listed transcription factors have not been studied extensively. Involvement of various transcription factors in lineage-specific gene expression has been studied in addition to promoter analysis by various methods including gene-inactivation studies, forced expression and overexpression

approaches in cell culture models, primary bone marrow cells, and transgenic animals (Shivdasani and Orkin, 1996; Tenen et al., 1997).

IN VITRO PROMOTER STUDIES Information on the specific roles of various transcription factors during megakaryopoiesis comes from studies of the immediate 5-flanking region of genes specifically expressed in megakaryocytes. Over the past decade, the 5-regulatory regions of the majority of these genes have been cloned and sequenced (Bastian et al., 1996; Block et al., 1994; Deveaux et al., 1996; Prandini et al., 1992; Ravanat et al., 1997; Ravid et al., 1991b). In vitro transfection experiments of promoter/reporter constructs proved to be a useful model as a first step in exploring important transcriptional regulators of megakaryopoiesis. These studies using various cell lines with megakaryocytic features (HEL, K562, L8057, U7, Meg-01, DAMI, and others) and chemicals (e.g., phorbol esters, dimethyl sulfoxide, phorbol dibutyrate) to induce differentiation were far from ideal. Many of the cell lines expressed only a subset of megakaryocyte markers and often the response to induction was very limited. Fortunately, as described below, research in primary cells and transgenic animals supported many of these transient expression studies.

GATA-1 and Megakaryocytic Promoters As a general theme, a relatively short promoter region (usually 1 kb long) of most megakaryocytic genes is able to direct tissue-specific expression of a reporter gene. For several genes, this expression is near the level seen for the native gene. So far, many of the genes expressed in megakaryocytes were found to contain active GATA protein-binding sites in their regulatory regions, suggesting a major role for GATA-1 in megakaryocyte differentiation (Deveaux et al., 1996; Hashimoto and Ware, 1995; Hickey and Roth, 1993; Lanza et al., 1993; Minami et al., 1998; Prandini et al., 1992; Ravid et al., 1991b; Tunnacliffe et al., 1992; Uzan et al., 1991; Zutter et al., 1994). However, there are a number of exceptions to this rule. For example, the PBP gene does not have a GATA-1 consensus bind-

IN VITRO PROMOTER STUDIES

ing site in its 5-flanking region nor does GATA1 increase reporter gene expression in transient expression studies (Zhang et al., 1997; MP, personal observation). The first study to implicate the GATA protein in the regulation of a gene exclusively expressed in megakaryocytes was with the PF4 gene (Ravid et al., 1991b). In these studies, the mechanism by which GATA factors contribute to PF4 gene activation in megakaryocytes was not established, yet evidence was presented that GATA operates with factors binding upstream to the GATA site. Also, proteins interacting with the core promoter GATA motif had been shown to interact with basal transcription factors and exhibited inhibitory action on the rat PF4 and EPO promoters (Aird et al., 1994). Concomitant with these findings, in transient transfection assays, a point mutation in the rat PF4 core promoter of GATAAA to TATAAA (at 931 upstream to the transcriptional start) did not significantly alter activity in megakaryocytes, but led to increases in the level of expression of the PF4 gene in nonmegakaryocytic cells (Ravid et al., 1991b). The above observations suggested that GATA-binding proteins can interfere with formation of preinitiation complexes and may represent a mechanism for repressing transcription in cell types that do not express genes specific for megakaryocytic lineage. In this context, in a different study (Rahuel et al., 1992) the distal region of the erythroid-specific human glycophorin B, which contains overlapping sites for GATA and an unidentified ubiquitous protein, was shown to completely repress transcriptional activity of a promoter proximal region. This study showed that this negative regulation can be removed if GATA-1 binds to the sequence, possibly by a displacement of the ubiquitous protein(s). Studies by Martin and colleagues (Martin et al., 1993) have dissected the transcriptional activity of four different GATA sites present in the promoter region of the IIb gene, which is also uniquely expressed in megakaryocytes. It had been demonstrated that all GATA sites bind GATA-1 transcription factor in vitro, but do not equally contribute to the promoter activity, with the most upstream GATA motif being dominant upon the others. Investigations by Block and co-workers (Block et al., 1994), who carried out transient expression studies with

35

various segments of the 5-upstream region of the rat IIb gene in rat bone marrow cells, have confirmed the importance of GATA for highlevel expression of the IIb gene in a lineagespecific manner. The induction of GATA-1 mRNA in response to TPO has been observed both in cell lines (Nagata et al., 1995) and in primary tissue (Matsumura et al., 1996), suggesting that a high level of GATA-1 expression is an important characteristic of megakaryocytic differentiation. Furthermore, the importance of GATA-1 in megakaryocyte development in humans has clinical support (Ludlow et al., 1996). A naturally occurring mutation in the GATA sequence within the promoter element of a gene expressed in megakaryocytes, GPIb, has been described as one of the causes for Bernard—Soulier syndrome (BSS), a rare congenital bleeding disorder due to absent or decreased expression of von Willebrand factor receptor complex on the platelet surface. This study described a unique case of BSS involving loss of one of the GPIb genes due to chromosome 22 microdeletion and a mutation in the promoter region of the remaining GPIb allele that alters a GATA-binding consensus site shown to be functionally important in transient expression assays.

Ets Family Members A number of transcription factors, which belong to the Ets family, including Ets-1, Ets-2, Fli-1, and PU.1, were reported to be expressed in megakaryocytes (Bastian et al., 1999; Doubeikovski et al., 1997; Lemarchandel et al., 1993). PU.1 expression was shown in the two promegakaryoblastic cell lines, HEL and K562, and in mature megakaryocytes by Northern analysis and by in situ immunohistochemistry, respectively (Hromas et al., 1993). The biological importance of PU.1, whose significance has previously been implicated only in the development of the lymphoid and myeloid lineages, has been recently reported in the regulated expression of the megakaryocyte-specific gene expression of PBP (Zhang et al., 1997). Analysis of PU.1 null embryonic stem (ES) cells differentiated in vitro into megakaryocytes revealed that PBP gene expression was more than four times lower than in the wild-type ES cells.

36


PU.1/Spi-1 nuclear binding protein was identified as the endogenous Ets transcription factor that preferentially interacted with the enhancer Ets-binding site on the IIb promoter (Doubeikovski et al., 1997). Furthermore, this GATA/Ets-containing enhancer is important for TPO responsiveness, and deletion of this promoter fragment or mutation of the Ets site significantly reduced this promoter activation by TPO. In this study, PU.1 gene expression was increased in response to TPO treatment, and greater levels of PU.1 were found to bind to the Ets consensus site. Investigation by T. Minami and associates (Minami et al., 1998) implicated a strong role for Ets-1 in the positive regulation of expression of the PF4 gene. In addition, it was shown that Ets-1 mRNA levels increased 10-fold upon DMSO-induced differentiation of HEL cells. It has also been described that c-Ets-1 and human GATA-1 can transactivate the IIb promoter in HeLa cells in cotransfection assays and can act additively (Lemarchandel et al., 1993). Studies of the mechanism of Mpl receptor activation by TPO in megakaryocytes revealed very high dependence of gene transactivation on an intact Ets-1—binding site. Furthermore, it was shown that GATA-1 and two Ets proteins, Ets-1 and Fli-1, can transactivate the Mpl promoter in heterologous cells (Deveaux et al., 1996). The role of Ets transcription factors was also demonstrated for von Willebrand factor (vWF) gene expression (Schwachtgen et al., 1997). Cotransfection of Ets-1 and Erg (Ets-related gene) expression plasmids was sufficient to induce vWF core promoter activity in HeLa cells. vWF is expressed both in endothelial cells and in developing megakaryocytes (Zimmerman and Ruggeri, 1982). Expression of Ets-1, Ets-2, and Erg was shown in endothelium, but what Ets family members transactivate the vWF gene in megakaryocytic cells remains to be determined. There is growing evidence that the Fli-1 transcription factor is in the network of gene regulation in the megakaryocytic lineage. The human leukemia cell line K562, induced to differentiate along the megakaryocytic lineage by phorbol esters, starts to express higher levels of Fli-1/ERGB transcription factor. Furthermore, introduction of a retroviral construct expressing human Fli-1/ERGB into K562 cells induces changes similar to those seen following phorbol

ester treatment, including increased adherence to the surface of the culture vessel, changes in cell size and morphology, and increased level of expression of IIb/3 (Athanasiou et al., 1996). Work by Bastian and colleagues (Bastian et al., 1999) also suggested Fli-1 factor as one of the regulators of lineage-specific genes during megakaryopoiesis. It was demonstrated that Fli-1 could transactivate the GPIX promoter in the presence of an intact GPIX Ets site. In addition, expression of Fli-1 was identified by immunohistochemistry in megakaryocytes derived from CD34> cells treated with TPO. All of these observations strongly implicate Ets family members of transcription factors in expression of megakaryocyte markers.

GATA/Ets Repeats There is growing evidence in the literature that the 5-flanking regions of many megakaryocytespecific genes contain GATA-and Ets-binding sites as tandem repeats (Deveaux et al., 1996; Minami et al., 1998; Ravanat et al., 1997). Lemarchandel and colleagues (Lemarchandel et al., 1993) presented a detailed study of the minimal cis-acting sequences of the IIb promoter that contained a GATA/Ets tandem promoting specific megakaryocytic activity. It was shown that Ets-1, a member of Ets family of transcription factors, was important for tissuespecific expression of the minimal promoter. Mutation in the GATA site reduced basal transcriptional activity but did not affect tissue-specific expression. In addition, Ets-1 mRNA was found in cell lines that expressed megakaryocytic markers (HEL and MEG 01) but not in cells that did not (K562, KU812, and HeLa). Another GATA/Ets joint site is found in the IIb promoter around 500 bp upstream of the transcriptional start site and was shown to direct an enhancer function, as deletions or mutations of these sites, individually or together, resulted in a 70% decrease of the promoter activity (Prandini et al., 1992). Several different studies indicated that the same GATA/Ets repeat is found in the human, mouse, and rat IIb enhancers as well as in the rat PF4 promoter (Block and Poncz, 1995; Block et al., 1996; Hickey and Roth, 1993; Martin et al., 1993; Minami et al., 1998; Prandini et al., 1996; Ravid

IN VITRO PROMOTER STUDIES

et al., 1991b). Analysis of the murine c-mpl gene promoter also revealed the existence of the GATA/Ets motif. It was shown that the c-mpl promoter/reporter construct can be transactivated by GATA-1 and Ets proteins (including Ets-1, Fli-1, and Elf-1) in vitro in heterologous cells (Deveaux et al., 1996). Ets motif adjacent to the GATA site was found to be crucial, as its inactivation reduced c-mpl reporter gene expression to 15% in HEL cells. All together, the above observations suggest that GATA/Ets binding sites are a hallmark of megakaryocytic regulatory regions and they may be important for shared mechanisms involved in transcriptional regulation of megakaryocyte-specific genes. However, GATA and Ets motifs alone cannot explain cell specificity of gene expression as evidenced by their importance for expression of P-selectin in endothelial cells as well as in megakaryocytic expression (Pan and McEver, 1993).

Sp1 Involvement The Sp1 family of transcription factors is often involved in the regulated expression of TATAless genes, frequently enhancing gene transcription. Engagement of this ubiquitous transcription factor in the regulation of genes expressed in megakaryocytes was reported in several studies (Block et al., 1999; Jin et al., 1998; Shou et al., 1998; Wang et al., 1999). Studies by Block and colleagues identified a critical Sp1 site involved in transcription initiation of the IIb gene and showed that a mutation in this site significantly decreased gene expression in primary megakaryocytes. In addition, they showed a cooperation between proteins binding to a site beginning 14 bp upstream of the transcriptional start—site: Sp1 at 914 bp and to Ets at 935 bp — and suggested a model in which an Ets complex at 935 bp is necessary for binding of Sp1 to its site. The total complex plays an important role for normal level of IIb gene transcription. On the other hand, investigations by Shou and co-workers presented a controversial role of Sp1 during megakaryocyte development. Their data indicated the presence of a Sp1 silencer domain, which also played an important role in IIb gene regulation. They demonstrated that an Sp1-binding element at 9135 bp in-

37

hibited the expression of the IIb gene in all megakaryocytic cell lines tested, but this inhibitory effect was reversed by sequences upstream to 9135 bp of the promoter. The mechanism of inhibition by Sp1 is unclear but could involve interaction with other nuclear elements previously shown (Zhang et al., 1995). The model presented by the authors implicated the GATA site at 9 454 bp in forming a powerful megakaryocyte-specific complex, which is able to overcome the Sp-1 silencer complex at 9 135 bp to promote interactions with the transcription initiation machinery. In the context of all these studies, it is interesting to note that TPO was found to increase Sp1 levels and the DNAbinding activity of Sp1 in a megakaryocytic cell line (Wang et al., 1999; Wang et al., 1996c). In this case, the nonphosphorylated form of Sp1 had a higher DNA-binding ability, as indicated by a reduction in this activity by inhibition of protein phosphatase 1 (PP1) activity (Wang et al., 1999). This is of great interest, as PP1 is activated by MAP kinase, which is well known to be activated by TPO (Rouyez et al., 1997; Yamada et al., 1995).

NF-E2 in Megakaryocytes The study of megakaryocytic differentiation in cell lines implicated NF-E2 as a possible transcription factor during development of this lineage (Romeo et al., 1990). NF-E2 induction and enhanced DNA-binding activity in response to TPO were observed during differentiation of the human megakaryoblastic cell line Meg-J under conditions when even GATA proteins, as well as Tal-1/SCL level of expression, were not increased (Kobayashi et al., 1998). Megakaryocytic differentiation of the FDC-P2 cell line in response to TPO also resulted in elevated levels of NF-E2 mRNA (Nagata et al., 1995). Further confirmation of a possible role of NFE2 in megakaryopoiesis came from in vivo studies in mice injected with TPO, from which splenic megakaryocytes were subjected to in situ hybridization with a NF-E2 riboprobe (Zimmet et al., 1998). However, as indicated below, the role of NF-E2 in megakaryopoiesis was truly established only when NF-E2 knockout mice were produced.

38


Repressor Elements The question of how the megakaryocyte-specific pattern of gene expression might arise from a common precursor cell is intriguing, especially in the context of the many common features exhibited by megakaryocytes and erythrocytes. IIb was shown to be transcriptionally active in erythro/megakaryocytic stem cells and early erythroblastic progenitors but inactive in mature erythrocytic cells (Okumura et al., 1992; Tronik-Le Roux et al., 1995), suggesting that transcription of this gene might be regulated negatively during erythropoiesis. Prandini and colleagues undertook a survey of negative elements in the regulation of tissue specificity of the IIb gene expression by monitoring the expression of progressively deleted IIb constructs, as well as scanning mutations in the context of the intact promoter, all in the nonhematopoietic cell line HeLa (Prandini et al., 1996). This study delineated a region in the IIb promoter (9120/993) that was directly involved in the negative control of cell specificity. This was also in agreement with previous observations in primary cultured rat megakaryocytes (Block et al., 1994) in which the 9150/9101 region in the rat IIb promoter also behaved like a repressor. It is important to notice that this promoter region overlaps with the 9124/ 999 domain that was suggested to be involved in the temporal activation of the gene during megakaryopoiesis (Fong and Santoro, 1994). Furthermore, this repressor sequence, in which the common motif in all genes is A/G,G/ C,CATGA,NC,C/A,A/C, was found in a number of other megakaryocytic promoters, such as PF4, GPIb, GPV, and GPIX, and its functional importance was characterized for some of them (Ramachandran et al., 1995). Moreover, Prandini presented evidence that nonmegakaryocytic cell lines expressed positive transcription factors capable of driving IIb transcription, as well as that megakaryocytic cell lines expressed transcription factors that bind to the repressor element (Prandini et al., 1996). Binding of an enhancing factor in megakaryocytes and a repressing factor in non-megakaryocytes to the same DNA region was also demonstrated for the PF4 gene (Ravid et al., 1991b). A tempting explanation for this phenomenon is that in megakaryocytic cells the binding of the repres-

sor is suppressed by the positive acting factors, which bind to other areas in the gene, expressed in megakaryocytes and/or by other protein-protein interactions specific for megakaryocytes. In nonmegakaryocytic cells, the repressor is unrestricted in its function.

IN VITRO ECTOPIC EXPRESSION OF TRANSCRIPTION FACTORS Forced expression of GATA-1 transcription factor in various nonmegakaryocytic cell lines highlighted the importance of this factor during megakaryocytic development and showed its involvement in important aspects of cell differentiation. These studies also pointed out that its overexpression alone was not sufficient to drive all aspects of megakaryocytic differentiation. Transfection of GATA-1 expression vectors in the murine myeloid cell line 416B induced the appearance of megakaryocytes as assessed by morphology, elevated DNA content, and raised levels of megakaryocytic markers (Visvader et al., 1992). An important experimental advantage of this cell line was that it originally had some megakaryocytic potential but no longer responded to growth factors and chemical agents (such as phorbol esters) that previously induced megakaryocytic differentiation. When the related transcription factors, GATA-2 and GATA-3, were overexpressed in the same multipotential myeloid cell line, surprisingly, the same effect was reached, although this may be linked to the increased expression of the endogenous GATA-1 (Visvader and Adams, 1993). Notably, megakaryocyte differentiation was also observed when a truncated product, corresponding to the C-terminal zinc-finger of GATA-1 or GATA-2, was tested in this assay (Visvader et al., 1995). In fact, all GATA family members share a high degree of homology in their DNA-binding domain. Remarkably, only 69 residues spanning the C-terminal finger were required to induce limited megakaryocytic differentiation. Again, endogenous GATA-1 mRNA was induced by most mutants and may have contributed to the observed differentiation. It was proposed that because the GATA Cterminal finger could bind its target site but not transactivate a minimal reporter, it may direct megakaryocytic maturation by derepressing

IN VIVO STUDIES IN TRANSGENIC MICE AND A GENE TARGETING APPROACH

specific genes and/or by interacting with another protein that provides the transactivation function. Another study of GATA-1 gene overexpression, this time in the murine myeloid leukemic cell line M1, switched cell development from myeloid to erythroid and megakaryocytic pathways (Yamaguchi et al., 1998). This observation was remarkable since the M1 cell line has been known for its potential for granulocyte-macrophage differentiation on stimulation with leukemia inhibitory factor (LIF) or IL-6, but not for megakaryocytic differentiation. Results from this study indicated that GATA-1 is able to regulate the switching of the differentiation program from bilineage precursors to cells of either the myeloid or erythroid/megakaryocytic progenitors. It was also shown that GATA-1 overexpression increased the level of c-mpl mRNA, which was consistent with the increase in c-mpl promoter activity in transfected cells. All together, these studies indicated that GATA1 is one of the transcription factors associated with the expression of the gene encoding c-mpl. The effect of forced GATA-1 expression in Myb-Ets—transformed myeloblasts again highlighted the importance of this factor for megakaryocytic development (Kulessa et al., 1995). Normally, differentiation of Myb-Ets—transformed chicken hematopoietic progenitors into myeloblasts involves GATA-1 downregulation. But expression of GATA-1 suppressed the appearance of myelomonocytic markers and reprogrammed myeloblasts into cells resembling either transformed eosinophils or thromboblasts, the avian equivalent of megakaryocytes. In addition, the correlation between the level of GATA-1 expression and the phenotype of the cell was observed with intermediate levels of the factor being expressed by eosinophils and high levels by thromboblasts, suggesting an additional dosage effect of GATA-1 expression. Forced expression of genes, such as c-myb or c-myc (to which a separate chapter is dedicated in this book) has also been studied with regard to megakaryopoiesis. Constitutive expression of c-myb in K562 and of c-myc in megakaryocytes of transgenic mice does not inhibit megakaryocytic differentiation, as assessed by cytochemical staining and expression of differentiation markers (Rosson and O’Brien, 1995; Thompson et al., 1996). In the case of c-myc, however, the

39

extent of polyploidization of megakaryocytes is reduced.

IN VIVO STUDIES IN TRANSGENIC MICE AND A GENE TARGETING APPROACH Studies With Transgenic Mice Revealing Regulatory Regions Of Genes Specifically Expressed in Megakaryocyte The regulatory regions of several megakaryocytic genes such as the rat and human PF4, human IIb and GPIb, have been tested in transgenic mice and have demonstrated tissue-specific expression of a reporter gene. A region spanning a 1.1—kb of the 5-untranslated region of the rat PF4 gene coupled to the prokaryotic -galactosidase gene was used to generate lines of transgenic mice. Studies of blood, bone marrow, spleen, and thymus revealed that platelets were the only circulating blood cells and megakaryocytes were the only hematopoietic precursor cells that expressed the prokaryotic enzyme (Ravid et al., 1991a). Furthermore, when a conditional oncogene was placed under the control of the rat PF4 promoter in transgenic mice, it resulted in severe thrombocytopenia with morphologically abnormal megakaryocytes and the development of megakaryocytic malignancies (Robinson et al., 1994). As little as 245 bp of the human PF4 gene promoter was sufficient to drive megakaryocytespecific gene expression in another transgenic model, and the level of expression was comparable to that driven by the 1.1kb of the rat PF4 promoter in other transgenic mouse lines (Cui et al., 1998). In transgenic mice in which expression of the herpes simplex virus thymidine kinase gene was directed by the 5-untranslated region of the human IIb gene, reporter gene expression was restricted to megakaryocytes and multipotential erythroid-megakaryocytic progenitors, reflecting the expression pattern of human IIb gene. Treatment of transgenic animals with gancyclovir (GCV) resulted in anemia and reversible thrombocytopenia associated with a decreased number of megakaryocytes (Tronik-Le Roux et al., 1995). Prolonged GCV treatment induced erythropenia in the transgenic mice. Assays of hematopoietic progenitor cells in vitro demon-

40


strated that the transgene was expressed in early erythro-megakaryocytic progenitor cells. In addition, the entire coding region of the human GPIb gene, including 3 kb of the 5-flanking region, was used in a transgenic Bernard—Soulier mouse model (Ware et al., 1993). As a result, human GPIb was expressed and formed a functional cross-species GPIb-GPIb-GPIXGPV complex, alleviating the bleeding diathesis. This demonstrated the use of transgenic engineering of platelet adhesion molecules as a new approach to manipulate platelet states in hemostasis and thrombosis. Targeted Disruption (Knockout) Of Genes via Homologous Recombination Targeted disruption of many transcription factors proved to be a powerful approach in determining their role during development. Unfortunately, gene knockout studies do not allow one to see the complex network of genes being turned on and off during development. The observed phenotype may be the result of failure of activation of one crucial gene of interest controlled by the targeted transcription factor, or the accumulated effect of multiple dysregulated genes that together interfere with the finely tuned process of megakaryopoiesis. Up to now, the specific ablation of the megakaryocytic lineage has not been an outcome of any transcription factor gene knockout. The only genes found to be indispensable for the formation of megakaryocytes were those necessary for the earliest hematopoietic progenitors (such as SCL/tal-1, Rbtn2, AML1, GATA-2) (Okuda et al., 1996; Porcher et al., 1996; Tsai et al., 1994; Wang et al., 1996a, 1996b; Warren et al., 1994). The Generation of megakaryocytes was, however, severely affected by ablation of the GATA-associated factor FOG and moderately affected by the knockout of small Maf proteins (see below). Although generation of mice with specific ablation of transcription factors has not pointed to any of the known nuclear regulatory proteins as the one required for megakaryocytic lineage commitment, ablation of the following genes indicates their role in the development of megakaryocytes and suggests their requirement in the late stages of differentiation. The various mouse knockout models demonstrating megakaryocytic lineage

defects will only be briefly described in this chapter, as detailed chapters are devoted in this book to the description of the consequence of elimination of GATA-1, p45NF-E2, FOG, and EKLF proteins in a mouse model. GATA-1. The lethality of the GATA-1 knockout (Fujiwara et al., 1996) has created the need for alternative methods to assess gene function during differentiation of other lineages, including megakaryocytes. GATA\\ ES cells contributed to phenotypically normal macrophages, neutrophils, and megakaryocytes in in vitro colony assays, indicating that GATA-1 was not required for the in vitro differentiation of cells of these lineages (Pevny et al., 1995). On the other hand, GATA-1\\ megakaryocytes were abnormally abundant in chimeric fetal livers, suggesting an alteration in the kinetics of their formation or turnover. Recently, the importance of GATA-1 in megakaryocyte development was shown using transgenic mice in which the gene was selectively lost in megakaryocytes (McDevitt et al., 1997; Shivdasani et al., 1997). Targeted modification of upstream sequences of GATA-1 locus created a ‘‘knockdown mutation,’’ resulting in a generation of mice with sufficient erythroid cells to avoid lethal anemia but with selective loss of megakaryocyte GATA1 expression. This resulted in the mutant mice having marked thrombocytopenia, deregulated megakaryocytic proliferation, and severely impaired cytoplasmic maturation of the resultant megakaryocytes. In vitro colony formation studies showed that most of the GATA-1\\ megakaryocytic colonies were abnormally larger in size. In addition to mature megakaryocytes, the colonies contained numerous small, morphologically very poorly differentiated cells having few -granules. The influence of GATA-1 factor on megakaryocyte maturation and platelet production was recently examined in great detail in GATA1—deficient primary megakaryocytes (Vyas et al., 1999). This study indicated that a significant proportion of cells, cultured ex vivo from embryonic-day 12.5 fetal livers, did not undergo endomitosis. The retarded nuclear and cytoplasmic maturation was associated with low levels of expression of most megakaryocyte-associated genes. In addition, bleeding times were significantly prolonged in mutant animals and plate-


lets showed a modest selective defect in activation by thrombin or by a combination of ADP and epinephrine. Interestingly, the resultant platelets exhibited greater evidence of cytoplasmic maturity than the corresponding megakaryocytes. This discrepancy in maturation state led to the speculation that the platelets in the circulation could be produced by the most mature megakaryocytes, which make up a very low percentage of the total megakaryocyte population. Taken together, these data indicate that GATA-1 function is not necessary for megakaryocytic lineage commitment but is critical for the proper balance between proliferation, cell death, and differentiation during megakaryopoiesis as well as for proper completion of the maturation process. FOG. Rather striking results in relation to megakaryopoiesis came from FOG knockout studies (Tsang et al., 1998). The zinc-finger protein FOG is known to act as a cofactor of GATA-1 during erythroid and megakaryocytic differentiation (Tsang et al., 1997). FOG is coexpressed with GATA-1 at a high level in both of these lineages, and its expression is not detectable in mast cells. FOG\\ mice die between embryonic days 10.5 and 12.5 with severe anemia, which further supports its role as a GATA1 cofactor in erythroid development. To investigate the role of FOG in different hematopoietic lineages, yolk sac and fetal liver cells were cultivated in the presence of various hematopoietic growth factors. In relation to megakaryocytes, the absence of FOG led to ablation of the megakaryocytic lineage. The presence of scarce acetylcholinesterase positive cells and minute levels of PF4 transcripts suggested that the commitment to the megakaryocytic lineage took place in the absence of FOG. FOG, thus, is a pivotal factor for early megakaryocyte development (as also detailed in Chapter One in this book). p45 NF-E2. p45 NF-E2 heterodimerizes with p18 (either MafG or MafK) to form the hematopoietic transcription factor NF-E2. It appears that p45 NF-E2 is another transcription factor with an important function in megakaryocyte differentiation. This became clear only after generation of mice with targeted disruption of the gene. A number of the immediate 5-flanking regions of genes exclusively expressed in mega-

41

karyocytes, such as IIb, PF4, and GPIX, contain binding sites for NF-E2, suggesting a role for this transcription factor in lineage development (Hickey and Roth, 1993; Prandini et al., 1992). The difficulty of studying NF-E2 transcription factor resides in the fact that this factor recognizes extended AP-1—like DNA sequences and can dimerize with several different smallMaf proteins (see below), which appear to dictate the binding site preference on DNA (Andrews et al., 1993a, 1993b; Igarashi et al., 1994; Ney et al., 1993). Although p45 NF-E2 recognizes regulatory regions of several erythroid genes, mice deficient for this protein display only mild red blood cell abnormalities (Shivdasani and Orkin, 1995). The most notable feature of this knockout experiment is impaired megakaryopoiesis with complete absence of circulating platelets, and consequently, high mortality of homozygous animals from hemorrhage (Shivdasani et al., 1995). Megakaryocytes are present in the spleen and bone marrow of the mutant mice, but the majority of these cells appear larger than control megakaryocytes and display severely impaired cytoplasmic maturation. Electron microscopy of megakaryocytes revealed an abnormal demarcation membrane system, a lack of appropriate laminal organization, a striking deficit in the number of granules, and an absence of platelet territories. On the other hand, the mRNA level of genes expressed in mutant fetal liver megakaryocytes, examined by RT-PCR, was found to be similar to that in control megakaryocytes. Endomitosis was not impaired in the mutant cells, as the modal ploidy of p45 NF-E2\\ megakaryocytes was 32N, compared to 16N in controls (as also detailed in Chapter 2 in this book). Other studies (Lecine et al., 2000) began to characterize the specific steps at which NF-E2 is involved in terminal megakaryocyte differentiation and platelet formation. Hence, 1-tubulin, whose expression in mature megakaryocytes was shown to be regulated by NF-E2, was identified as a specific and probably essential component for platelet formation and release. This study showed that 1-tubulin mRNA and protein were absent from the spleen, blood, and megakaryocytes of p45 NF-E2 knockout mice. It is important to point out that 1-tubulin mRNA expression increases with normal megakaryocyte maturation and especially coincides

42


with proplatelet formation. Consequently, p45 NF-E2 is not required for lineage commitment and megakaryopoiesis proceeds normally at early phases. However, this transcription factor is needed at late stages of differentiation for proper final maturation and release of circulating platelets. Small-Maf proteins. p18 NF-E2 belongs to an extended family of small Maf proteins and serves as the p45 NF-E2 heterodimeric partner (Igarashi et al., 1994). A significant function of MafG in megakaryopoiesis was also discovered after generation of a null mouse model (Shavit et al., 1998). The product of the avian oncogene Maf and related family members share a common conserved basic region and a leucine zipper motif, which mediate DNA binding and dimer formation. The small-Maf proteins form homoor heterodimers with other b-ZIP proteins present in the cell and bind to a Maf recognition element (MARE) that has a striking similarity to the NF-E2—binding sequence (Motohashi et al., 1997). Since small-Maf proteins lack known transcriptional activation domains, they likely function either as obligatory heterodimeric partners of other transcriptional activators, or alternatively as homo- or heterodimeric transcriptional regulators (Fujiwara et al., 1993; Kataoka et al., 1995). Some understanding of the roles of small-Maf proteins in development was derived from analysis of animal models. MafK null mutant mice were viable and fertile, with blood cell counts and red cell parameters within the normal range (Kotkow and Orkin, 1996). In addition, NF-E2—binding activity in fetal livers of MafK null mutants was indistinguishable from that of the wild-type animals, suggesting that another small-Maf protein might provide a complementing activity that rescues MafK homozygous animals. In contrast to MafK null mice, MafG null animals, although viable and fertile, exhibited abnormal megakaryocyte proliferation with accompanying thrombocytopenia, as well as behavioral defects (Shavit et al., 1998). Histological analysis revealed a 3-fold increase in the number of megakaryocytes in both the bone marrow and in spleen sections of MafG\\ animals in comparison with heterozygous littermates. Moreover, MafG\\ megakaryocytes expressed IIb mRNA and demon-

strated acetylcholinesterase activity, indicating a late block in maturation, as is also the case for p45 NF-E2 null mice. Since MafG and MafK display some spatial and temporal overlap in expression, these two genes may provide certain compensatory activity. On the other hand, MafG and MafK expression are not completely overlapping, which may explain the observed megakaryocyte defect only in the MafG knockout animals. From these knockout animal models of hematopoietic transcription factors, it appears that the transcription factors GATA-1, NF-E2, MafG, and the factor FOG, all affect the balance of expression of various megakaryocytic genes without affecting the basic commitment of hematopoietic cells to the megakaryocyte lineage. It appears that FOG affects an early stage, GATA-1 affects an intermediate stage, and p45 NF-E2 and MafG affect a late stage of megakaryocyte development. Yet it is also possible that all of these genes affect gene expression at the same point of development; only the particular pattern of gene dysregulation differs in each and results in the different observed phenotypes. Consequences Of Overexpression of Transcription Factors: Studies In Transgenic Animals c-myc. Several studies indicated that c-myc is transiently upregulated during the initiation of the differentiation of megakaryocytic cell lines (Bakic et al., 1993; Dorn et al., 1994; Gewirtz and Shen, 1990). Analysis of transgenic mice overexpressing c-myc in early committed megakaryocytes (which are able to activate the PF4 promoter) revealed that the number of megakaryocytes of low ploidy level increased significantly but that these cells did not lose the ability to express differentiation markers. This change in ploidy distribution was not accompanied by any significant change in platelet number (Thompson et al., 1996). This indicated that c-myc expression might be important for the regulation of the proliferation phase of cells that are already committed to the lineage. E2F-1. E2F-1, being a ubiquitous transcription factor that plays a central role in cell cycle regulation, was overexpressed in megakaryo-


43

Figure 3.1. The platelet factor four (PF4) gene as a model for megakaryocyte-restricted gene expression. A: The PF4 1.1 kb promoter region drives expression of prokaryotic -galactosidase only to megakaryocytes, among bone marrow cells, and to platelets (Ravid et al., 1991a), as also confirmed by others (Cui et al., 1998; Guy et al., 1996; Robinson et al., 1994). Presented are a smear of bone marrow cells (top) in which the -galactosidase-positive cells stain blue (dark image), and platelets possessing -galactosidase activity (bottom), both from PF4LacZ transgenic mice. B: Transcription factors essential for activation of the PF4 promoter (Minami et al., 1998; Ravid et al., 1991b; Tsang et al., 1998). The importance of the Ets and GATA factors is also common to other genes with restricted expression in megakaryocytes, such as the IIb gene (Block et al., 1996).

cytes of transgenic mice using the PF4 promoter (Guy et al., 1996). E2F-1 overexpression increased megakaryocyte number but blocked megakaryocyte differentiation during maturation, resulting in severe thrombocytopenia. The impaired maturation was manifested by

abnormal development of demarcation membranes and reduced numbers of -granules. This study suggested that high levels of E2F-1 can prevent terminal differentiation. Alternatively, as in the p45 NF-E2 knockout model, the overexpression of E2F-1 may limit the express-

44


ion of a critical cytoskeletal component such as 1-tubulin and prevent proper demarcation and platelet release. In addition, a growing number of gene overexpression studies in transgenic models as well as in vitro, contributed to the discovery of new roles of various transcription factors in megakaryocyte development, for example, overexpression of EKLF transcription factor or HoxA10 in bone marrow cells in vitro or in transgenic mice implied a role for these factors in megakaryocyte development, among other lineages (Tewari et al., 1998; Thorsteinsdottir et al., 1997).

marker genes (Fig. 3.1). However, GATA and Ets motifs alone cannot explain cell specificity as evidenced by their importance for expression of nonmegakaryocyte-specific genes as well. Nevertheless, it is very likely that additional transcription factors that play a critical role in the establishment of the genetic program during megakaryopoiesis still remain to be elucidated. Identification of these factors as well as factors that mediate gene activation in response to various cytokines, and specifically to TPO signaling, remains as a major goal. A summary of the studies pointing to regulators of megakaryopoiesis, as derived from promoter studies or in vivo analysis, is in Table 3.1.

CONCLUSIONS The complete program for megakaryopoiesis begins with a pluripotent hematopoietic stem cell that proceeds through stages of proliferation associated with increased cellular commitment followed by a phase of cellular differentiation involving increased cell ploidy, cytoplasmic maturation, and ending with the release of platelets. This complex process is controlled by a wide array of external signals, where TPO plays the dominant role. Each step of this program is controlled by ordered gene regulation that culminates in the expression of a unique complement of both specific and widely expressed transcription factors. Although the critical targets of TPO and other cytokines that direct transcriptional regulation during megakaryopoiesis remain to be elucidated, extensive research in cell culture, and in whole animal studies identified several transcription factors implicated in the network. While knockout phenotypes of various nuclear proteins have limitations to their interpretation, these models point to several transcription factors with important and unique roles in megakaryopoiesis. Studies of promoters of megakaryocytic marker genes have suggested that cell specificity lies not only within the promoter sequence but also in the balance of transcription factors that are expressed by megakaryocytes, and that not only a qualitative but also a quantitative difference must play a significant role. For instance, the GATA and Ets families of transcriptional factors have been implicated in the lineageselective expression of most megakaryocytic

ACKNOWLEDGMENTS We thank Leah Cataldo-Calahan for carefully reading this chapter.

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Binder, M., Marin-Padilla, M., Sharpe, A. H., and Speck, N. A. (1996b). The CBF beta subunit is essential for CBF alpha2 (AML1) function in vivo. Cell 87, 697—708. Wang, Z., Sicinski, P., Weinberg, R. A., Zhang, Y., and Ravid, K. (1996c). Characterization of the mouse cyclin D3 gene: exon/intron organization and promoter activity. Genomics 35, 156—163. Wang, Z., Zhang, Y., Lu, J., Sun, S., and Ravid, K. (1999). Mpl ligand enhances the transcription of the cyclin D3 gene: a potential role for Sp1 transcription factor. Blood 93, 4208—4221. Ware, J., Russell, S. R., Marchese, P., and Ruggeri, Z. M. (1993). Expression of human platelet glycoprotein Ib alpha in transgenic mice. J. Biol. Chem. 268, 8376—8382. Warren, A. J., Colledge, W. H., Carlton, M. B., Evans, M. J., Smith, A. J., and Rabbitts, T. H. (1994). The oncogenic cysteine-rich LIM domain protein rbtn2 is essential for erythroid development. Cell 78, 45—57. Wendling, F., Maraskovsky, E., Debili, N., Florindo, C., Teepe, M., Titeux, M., Methia, N., BretonGorius, J., Cosman, D., and Vainchenker, W. (1994). cMpl ligand is a humoral regulator of megakaryocytopoiesis. Nature 369, 571—574. Yamada, M., Komatsu, N., Okada, K., Kato, T., Miyazaki, H., and Miura, Y. (1995). Thrombopoietin induces tyrosine phosphorylation and activation of mitogen-activated protein kinases in a human thrombopoietin-dependent cell line. Biochem. Biophys. Res. Commun. 217, 230—237. Yamaguchi, Y., Zon, L. I., Ackerman, S. J., Yamamoto, M., and Suda, T. (1998). Forced GATA-1 expression in the murine myeloid cell line

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M1: induction of c-Mpl expression and megakaryocytic/erythroid differentiation. Blood 91, 450—457. Zauli, G., Gibellini, D., Vitale, M., Secchiero, P., Celeghini, C., Bassini, A., Pierpaoli, S., Marchisio, M., Guidotti, L., and Capitani, S. (1998). The induction of megakaryocyte differentiation is accompanied by selective Ser133 phosphorylation of the transcription factor CREB in both HEL cell line and primary CD34> cells. Blood 92, 472—480. Zhang, C., Gadue, P., Scott, E., Atchison, M., and Poncz, M. (1997). Activation of the megakaryocyte-specific gene platelet basic protein (PBP) by the Ets family factor PU.1. J. Biol. Chem. 272, 26,236—26,246. Zhang, R., Min, W., and Sessa, W. C. (1995). Functional analysis of the human endothelial nitric oxide synthase promoter. Sp1 and GATA factors are necessary for basal transcription in endothelial cells. J. Biol. Chem. 270, 15,320—15,326. Zimmerman, T. S., and Ruggeri, Z. M. (1982). Von Willebrand’s disease. Prog. Hemostat. Thromb. 6, 203—236. Zimmet, J., Toselli, P., and Ravid, K. (1998). Cyclin D3 and megakaryocyte development: exploration of a transgenic phenotype. Stem Cells 16, 97—106. Zucker-Franklin, D., and Kaushansky, K. (1996). Effect of thrombopoietin on the development of megakaryocytes and platelets: an ultrastructural analysis. Blood 88, 1632—1638. Zutter, M. M., Santoro, S. A., Painter, A. S., Tsung, Y. L., and Gafford, A. (1994). The human alpha 2 integrin gene promoter. Identification of positive and negative regulatory elements important for cell-type and developmentally restricted gene expression. J. Biol. Chem. 269, 463—469.

CHAPTER 4


ROLE OF THE TAL1/SCL TRANSCRIPTION FACTOR IN NORMAL AND LEUKEMIC HEMATOPOIESIS STEPHEN J. BRANDT Departments of Medicine and Cell Biology, Vanderbilt University Medical Center; Vanderbilt— Ingram Cancer Center; and Nashville Veterans Affairs Medical Center

INTRODUCTION: DISCOVERY AND INVOLVEMENT IN LEUKEMIA Genes important in hematopoietic cell proliferation and differentiation have frequently been discovered from investigation of recurrent chromosomal rearrangements associated with specific types of leukemia. The gene encoding the TAL1 (or SCL) transcription factor is involved by such a translocation, the t(1;14)(p34;q11), observed in approximately 3% of patients with T-cell acute lymphoblastic leukemia (T-ALL) (Carroll et al., 1990). First cloned in 1989 using cells derived from patients with T-ALL (Bernard et al., 1990; Chen et al., 1990a; Finger et al., 1989) or stem cell leukemia (Begley et al., 1989), the majority of breakpoints involve sequences 5 to its coding region on chromosome 1 and the / T-cell receptor gene on chromosome 14. Translocations 3 to the TAL1 locus, in some cases a considerable distance, have also been

described (Bernard et al., 1990; Chen et al., 1990b; Xia et al., 1992). Other, rare translocations, including the t(1;7)(p32;q35) (Fitzgerald et al., 1991), t(1;3)(p34;p21) (Aplan et al., 1992c), and t(1;5)(p32;q31) (François et al., 1998), involve the TAL1 locus but different derivative chromosomes. Two genes that share considerable sequence homology with TAL1, LYL-1 (Mellentin et al., 1989) and TAL2 (Xia et al., 1991), are similarly misexpressed in T-ALL as a result of their translocation into T-cell receptor loci, although far less frequently. In an additional 12—26% of individuals with T-ALL, a series of site-specific rearrangements (denoted as talB) delete 90—100 kb of DNA extending from the coding sequences of an immediately upstream gene, SIL (for SCL interrupting locus), into 5 noncoding exons of the TAL1 locus (Aplan et al., 1990b; Bernard et al., 1991; Brown et al., 1990). As in the chromosomal translocations, these deletions result from


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TAL1/SCL TRANSCRIPTION FACTOR IN NORMAL AND LEUKEMIC HEMATOPOIESIS

Figure 4.1. Functional domains in TAL1. Defined sites of phosphorylation (Ser and Ser), location of basic, HLH, and transcriptional activation domains, and regions of protein interaction are depicted. pp242* and pp472* refer to two isoforms of TAL1 identified in cells.

aberrant recombinase activity and are associated with T-cell receptor (TCR) lineage commitment (Breit et al., 1993b; Macintyre et al., 1992). The SIL-TAL1 deletions generally delete the entire SIL coding region while leaving TAL1 coding exons intact. This effectively places the TAL1 gene under the control of a promoter that is constitutively active in T-cell development (Aplan et al., 1992a). In one deletion (tald3 (Bash et al., 1993), however, and in certain translocations in which an otherwise cryptic promoter in exon 4 of the TAL1 locus is activated (Aplan et al., 1990a; Bernard et al., 1992), translation initiates at an in-frame AUG codon in the second coding exon (exon 5). In either situation, an amino terminally truncated protein that still retains both basic and HLH regions, pp242*, is the only TAL1 polypeptide made (Fig. 4.1). The SIL-TAL1 deletions can be detected by PCR of genomic DNA and SIL-TAL1 fusion transcripts by reverse transcriptase-PCR (Aplan et al., 1992a; Delabesse et al., 1997). Both assays have been exploited for the detection of minimal residual disease (Borkhardt et al., 1992; Breit et

al., 1993a; Janssen et al., 1993; Neale et al., 1994). Finally, TAL1 mRNA has been detected in T-cell leukemias that lack either kind of TAL1 gene rearrangemnent (Bash et al., 1995). In some of these cases, transcription of TAL1 was found to be monoallelic, consistent with a subtle abnormality in cis-regulatory sequences of one allele or an occult rearrangement some distance away from the gene (Xia et al., 1992), while biallelic expression, suggested to result from trans-activation of both TAL1 alleles, was noted in others. Although another study that analyzed TAL1 expression by immunohistochemistry suggested that contaminating normal cells were the source of TAL1 RNA in this latter group of patients (Delabesse et al., 1998), TAL1 misexpression represents, in any case, the most frequent gain-of-function mutation in T-ALL (Bash et al., 1995). As discussed below, although the possibility that it is expressed physiologically at some stage of T-cell development has not been completely excluded, TAL1 expression in leukemic T cells likely reflects unscheduled or aberrant transcription.

TAL1 PROTEIN PRODUCTS AND INTERACTING PROTEINS

TAL1 gene rearrangement has also been detected in rare patients with cutaneous T-cell lymphoma (Neri et al., 1995), large granular lymphocytic leukemia (Feher et al., 1995), and, in one case, a B-cell malignancy (Bernard et al., 1993). Finally, TAL1 RNA or protein have been noted in vascular tumors (Chetty et al., 1997) and acute nonlymphocytic leukemias (Shimamoto et al., 1994, 1995). In these latter neoplasms, TAL1 expression likely reflects their lack of differentiation or hematopoietic lineage and does not contribute to their malignant phenotype per se.

TALI PROTEIN PRODUCTS AND INTERACTING PROTEINS TAL1 belongs to the helix-loop-helix (HLH) family of transcription factors, named for the conformation of its defining motif of two amphipathic helices with an intervening loop (Murre et al., 1989). This HLH domain promotes the formation of protein hetero- and homodimers, while an adjacent region enriched in basic amino acids and found in many members of this class of transcription factors mediates sequence-specific DNA binding (Davis et al., 1990; Voronova and Baltimore, 1990). As do a number of other tissue-restricted basic domain-HLH (bHLH) proteins, TAL1 binds DNA in combination with any of a number of more widely expressed, or class A, bHLH proteins that are referred to collectively as E proteins (Doyle et al., 1994; Hsu et al., 1991; Hsu et al., 1994b). These include the three proteins derived by alternative splicing from the E2A gene, E12, E47, and E2-5, the related protein E2-2, and the HEB/HTF4 gene products (see Chapter 16 in this book). All such bHLH heterodimers recognize a nucleotide motif, CANNTG, termed the E box and function in transcriptional regulation. Within the greater HLH gene family, subgroups can be distinguished on the basis of sequence relatedness, expression pattern, and function. Four bHLH genes expressed in overlapping fashion in developing skeletal muscle share the ability to induce myogenic differentiation when introduced into certain cell lines (Weintraub et al., 1991) and appear capable of substituting for each other’s functions in vivo

53

(Wang et al., 1996). In addition to their association with a specific human leukemia, the proteins encoded by the TAL1, TAL2, and LYL-1 genes demonstrate greater than 85% identity in their DNA-binding and dimerization domains and constitute a similarly distinct HLH subfamily. Three overlapping polypeptide products of the TAL1 gene have been characterized. The full-length protein, with a molecular mass of 47 kDa (pp472*), has been noted in essentially all cells that express the gene (Cheng et al., 1993b; Hsu et al., 1994b), while a protein initiating at the second in-frame AUG codon as the result of leaky ribosomal scanning (pp452*) has been demonstrated in programmed reticulocyte lysates, transfected COS cells, and certain cell lines (Aplan et al., 1990a; Bernard et al., 1991; Goldfarb et al., 1992). A further truncated protein, with a molecular mass of 22—24 kDa (pp242*), derives from a series of spliced RNAs that are translated from an in-frame AUG codon proximal to the bHLH domain (Fig. 4.1). This isoform has been detected in human and murine leukemia cell lines (Cheng et al., 1993b; Hsu et al., 1994b) and Friend virus—induced proerythroblasts (Prasad et al., 1995). As discussed above, pp242* is also the sole TAL1 protein synthesized in leukemias with the tal d3 rearrangement. In addition to its E protein DNA-binding partners, TAL1 was found to interact with the products of two genes that were identified themselves from their involvement by chromosomal translocations (Boehm et al., 1988; McGuire et al., 1989). They share considerable homology within their LIM domains, a cysteine-rich motif that takes its eponym from the three transcription factors, lin-11, Isl-1, and mec-3, in which it was initially recognized. These LIM domain proteins, LMO1 and LMO2 (for LIM-only, formerly TTG-1/2 and rhombotin-1/2), were found to coimmunoprecipitate with TAL1 in T-ALL cell lines (Valge-Archer et al., 1994) and associate with TAL1 in a mammalian twohybrid assay (Wadman et al., 1994a). Subsequently, it became apparent that these proteins contributed with TAL1 to ternary complexes that in erythroid cells also include GATA-1 (Osada et al., 1995) and the LIM domain—binding protein Ldb1/NLI (Wadman et al., 1997), in CD4- CD8- thymocytes of Lmo2 transgenic

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mice include Ldb1/NLI (Gru¨tz et al., 1998), and in T-ALL cells include Ldb1/NLI and in some cases GATA-3 (Valge-Archer et al., 1998). As discussed below, these multiprotein complexes have a DNA-binding preference distinct from that of TAL1/E2A heterodimers. In addition to LMO1 and LMO2, we have found that TAL1 also interacts with the transcriptional coactivators p300 (Huang et al., 1999) and p300/ CBP-associated factor (P/CAF) (unpublished) and with the transcriptional corepressors mSin3A (Huang and Brandt, 2000) and mSin3B (unpublished). Finally, TAL1 was reported to interact with a subunit of the basal transcription factor TFIIH (Zhao and Aplan, 1999) and the cytoplasmic GTP-binding protein DRG (Mahajan et al., 1996; Zhao and Aplan, 1998). The functional importance of these latter interactions have not yet been established.

POSTTRANSLATIONAL MODIFICATIONS The TAL1 gene products are present in cells as serine phosphoproteins (Cheng et al., 1993b; Goldfarb et al., 1992; Prasad et al., 1995), with two phosphorylation sites in the molecule having thus far been mapped. Serine 122 (Fig. 4.1) represents a major site of phosphorylation in leukemic cell lines, Friend virus—elicited splenic erythroblasts, and transfected COS cells and is phosphorylated in vitro and in erythropoietinstimulated proerythroblasts by the mitogenactivated protein kinase ERK1 (Cheng et al., 1993a,b; Tang et al., 1999). In studies using transfected cells, phosphorylation of Ser was found to be important for the activity of a transcriptional activation domain within which this residue is placed (Wadman et al., 1994b). Serine 172, situated in a region proximal to the DNA-binding domain that shows considerable homology to a similarly positioned sequence in LYL-1, serves as a substrate in vitro and in transfected cells for cAMP-dependent protein kinase (Prasad and Brandt, 1997). Phosphorylation of Ser has a target-dependent effect on DNA-binding activity, suggesting it regulates DNA-binding site selection or affinity (Prasad and Brandt, 1997). As discussed below, data from our laboratory have shown that TAL1 interacts with the two

histone acetyltransferases p300 (Huang et al., 1999) and P/CAF (unpublished). Two other hematopoietic transcription factors, erythroid Kru¨ppel-like factor (EKLF) and GATA-1, similarly interact with transcriptional coactivators and are, in addition, substrates for their acetyltransferase activity (Boyes et al., 1998; Zhang and Bieker, 1998). Although TAL1 also appears to be acetylated by p300 and P/CAF (unpublished), the specific lysines affected and the effect acetylation has on protein function are still under investigation.

ONCOGENIC PROPERTIES In distinction to most of the other erythroidexpressed transcription factors discussed in this section, TAL1 is also an oncoprotein. Although transduction with pp472* and particularly pp242* increased the clonogenicity of NIH 3T3 cells in soft agar (Hsu and Cheng, 1997), it is clear that TAL1 is not a classically transforming oncogene. Indeed, in initial transgenic studies employing the T-cell—specific CD2 promoter (Larson et al., 1996; Robb et al., 1995b) and gene transfer studies with murine bone marrow cells (Elwood and Begley, 1995), enforced expression of pp472* proved insufficient to elicit leukemia. More recently, expression of human (Condorelli et al., 1996) and murine (Kelliher et al., 1996) pp472* cDNAs targeted to the thymus with the proximal Lck promoter was shown to produce thymic lymphomas in transgenic mice. However, the tumors appeared in these animals after a prolonged latency and exhibited diverse immunophenotypes, indicating the need for additional abnormalities for development of high-grade disease resembling TALL in humans. In fact, when interbred with p53\> gene—targeted mice (Condorelli et al., 1996) or casein kinase II transgenic mice (Kelliher et al., 1996), the time to appearance of disease was significantly shortened and the lymphomas that developed more closely resembled in immunophenotype those observed in T-ALL. Although loss of p53 is infrequent (Hsiao et al., 1994) and overexpression of casein kinase is not at all characteristic of T-ALL, these results illustrate the ability of TAL1 misexpression to

ONCOGENIC PROPERTIES

cooperate with other genetic lesions in an experimental model of T-cell leukemogenesis. TAL1 is often expressed with, and as described above, capable of interacting physically (Valge-Archer et al., 1994; Wadman et al., 1994a) and functionally with, the LIM domain proteins LMO1 and LMO2. Although coexpression of TAL1 with LMO1 or LMO2 appears to be common in T-ALL cell lines, occuring in every one of 11 lines determined to express TAL1 (Ono et al., 1997), and in Lmo2 transgenic (Gru¨tz et al., 1998) and Msh2\\ mice (Lowsky et al., 1997), it has not been determined how often such concordance occurs in primary tumor samples. Transgenic expression of LMO1 (Fisch et al., 1992; McGuire et al., 1992) or LMO2 (Fisch et al., 1992; Larson et al., 1994, 1995; Neale et al., 1995) was sufficient to induce thymic T-cell tumors characterized, as in the lckNPM-TAL1 transgenics, by a relatively long latency. Mice doubly transgenic for LMO2 and pp472* (Larson et al., 1996), LMO1 and pp472* (Chervinsky et al., 1999), or LMO1 and pp242* (Aplan et al., 1997; Chervinsky et al., 1999) developed disease with a shorter latency, higher penetrance, and greater resemblance to human T-ALL. Another lesion potentially capable of cooperating with TAL1 misexpression in T-cell leukemogenesis is mutation of the DNA mismatch repair gene hMSH2. Although uncommon in most hematologic malignancies, microsatellite instability resulting from loss of mismatch repair function has been noted in a subset of pediatric patients with T-ALL (Baccichet et al., 1997), and inactivating mutations in the hMSH2 coding region were identified in tumor samples from 2 of 10 patients with T-cell lymphoblastic lymphoma (Lowsky et al., 1997). Importantly, Msh2\\ mice developed thymic lymphomas that, similar to their human counterparts, frequently coexpressed Tal1 and Lmo2 (Lowsky et al., 1997). A more frequent finding in T-ALL than hMSH2 mutation is genetic loss at the INK4A and/or INK4B loci (Cayuela et al., 1995; Fizzotti et al., 1995; Hebert et al., 1994; Iolascon et al., 1996; Ogawa et al., 1995; Ohnishi et al., 1995; Otsuki et al., 1995; Quesnel et al., 1995; Rasool et al., 1995; Takeuchi et al., 1995). As with TAL1 gene rearrangements, deletions involving these

55

genes are more common in pediatric than adult patients (Okuda et al., 1995) and appear to be mediated by illegitimate recombinase activity (Cayuela et al., 1997). In addition to gene deletion, promoter methylation leading to transcriptional silencing has also been observed in TALL, more frequently at the INK4B than the INK4A locus (Batova et al., 1997). Overall, loss of expression of one or more of the three tumor suppressors derived from these loci, p16',) , p15',), and particularly p140$(Gardie et al., 1998), constitutes the most frequent genetic lesion of any kind in this disorder, in some series approaching an incidence of 100%. On a statistical basis alone, then, it could be predicted that loss-of-function mutations in these genes and gain-of-function mutations at the TAL1 locus would be present in half or more of patients with T-ALL. At this time, however, no data are available on the actual frequency of their concordance or whether they can cooperate in leukemogenesis. The oncogenic properties of TAL1 have been studied primarily through its enforced expression in cell lines. Transduction of a pp472* cDNA into a human leukemic T-cell line not otherwise expressing TAL1 inhibited the apoptosis of these cells to certain chemotherapeutic agents and Fas/CD95 cross-linking but not to serum depletion (Bernard et al., 1998). In contrast, transfection of TAL1-expressing Jurkat cells with a carboxy-terminal TAL1 truncation mutant that acted as a dominant-negative inhibitor of TAL1 DNA binding promoted apoptosis in response to serum deprivation but had no effect on etoposide- or Fas-triggered apoptosis (Leroy-Viard et al., 1995). Enforced expression of TAL1 in the growth factor—dependent 32D cell line and the HL-60 human myeloid leukemia cell line (Condorelli et al., 1997) induced proliferation, particularly under suboptimal culture conditions, inhibited apoptosis, and blocked granulocytic differentiation of 32D cells and both granulocytic and monocytic differentiation of HL-60 cells. Finally, retroviral transduction with Tal1 and LMO1 cDNAs, but neither cDNA alone, inhibited apoptosis of confluent NIH 3T3 cells induced by serum withdrawal and enhanced the reentry of subconfluent cells arrested in G back into cycle (unpublished). While it could have other actions

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as well, TAL1 inhibition of programmed cell death, stimulation of cell cycle progression, and blockade of differentiation all likely contribute to its ability to act as an oncoprotein.

TAL1 RNA AND PROTEIN DISTRIBUTION The TAL1 gene is expressed in mammalian embryogenesis prior to the first appearance of hematopoietic elements. Its expression is detectable initially in extraembryonic mesoderm (Kallianpur et al., 1994; Silver and Palis, 1997) and then in the endothelial and hematopoietic cells of yolk sac blood islands and the definitive blood cells in the aorto-gonado-mesonephric region (Labastie et al.,1998) and fetal liver. TAL1 expression also characterizes angioblasts and endothelial cells of blood vessels that form by vasculogenesis (Drake et al., 1997; Kallianpur et al., 1994). In Xenopus (Mead et al., 1998) and zebrafish (Gering et al., 1998; Liao et al., 1998) embryos, TAL1 is also expressed initially in cells that have hematopoietic and vasculogenic potential and subsequently in cells that establish definitive hematopoiesis. In addition to these cell types, TAL1 protein and/or RNA have been detected in neurons in the developing brain and spinal cord (Gering et al., 1998; Green et al., 1992; Kallianpur et al., 1994), melanocytes (Kallianpur et al., 1994), cells of the developing thymus (Kallianpur et al., 1994), and smooth muscle cells (Kallianpur et al., 1994). Postnatally, TAL1 expression is largely restricted to the erythroid, megakaryocytic, and mast cell lineages, although it has also been detected by in situ hybridization (Hwang et al., 1993) or immunohistochemistry (Pulford et al., 1995) in endothelial cells, tissue macrophages, and smooth muscle. TAL1 expression has been studied in several leukemia cell lines used as experimental models of lineage-specific differentiation. An increase in TAL1 mRNA was noted in human and murine erythroleukemia (MEL) cell lines induced to differentiate with chemical compounds (Green et al., 1991, 1992; Visvader et al., 1991) and explanted murine (Kallianpur et al., 1994; Prasad et al., 1995) and human (Miller et al., 1994) erythroid progenitors treated with erythropoietin and stem cell factor (SCF), respec-

tively. In contrast, myeloid differentiation of multipotent hematopoietic cell lines (Cross et al., 1994) and interleukin-6 (IL-6)—, leukemia inhibitory factor (LIF)—, or oncostatin M (OSM)— induced differentiation of the M1 myeloid leukemic cell line was accompanied by downregulation of Tal1 expression. TAL1 expression has also been extensively characterized in purified populations of immature hematopoietic cells. Studies using sensitive PCR-based or immunofluorescent techniques (Hoang et al., 1996; Quesenberry et al., 1996; Valtieri et al., 1998), including two that carried out single-cell analyses of paired daughter cells (Cheng et al., 1996; Ziegler et al., 1999), found that TAL1 is expressed at low levels in pluripotent stem cells and multipotent progenitors committed to erythrocyte and megakaryocytic differentiation (Quesenberry et al., 1996), in increased amounts in early erythroid progenitors (Ziegler et al., 1999), and then in decreasing abundance with terminal erythroid differentiation (Cheng et al., 1996). In studies of human hematopoietic progenitors in liquid suspension cultures, TAL1 and E2A mRNAs were coordinately upregulated with the onset of erythroid differentiation, increased further at the erythroblast stage, and declined late in culture, while Id2 expression was extinguished with erythroid differentiation (Condorelli et al., 1995). Interestingly, both TAL1 and E2A mRNAs were only transiently induced and Id2 transcription was maintained when these cells were induced to differentiate to granulocytes. As discussed below, a decline in the expression of the Id proteins, which act as inhibitors to class A bHLH proteins, would serve to augment TAL1 function. As a surrogate for gene expression, the activity of the TAL1 promoter(s) has also been investigated in mice carrying a lacZ reporter gene ‘‘knocked-in’’ to the TAL1 coding region (Elefanty et al., 1998). This study found reporter expression in multipotent progenitors capable of forming spleen colonies in irradiated mice (CFU-S), in myeloid, B-, and T-cell progenitors, and in erythroid progenitor cells (Elefanty et al., 1998). In sum, TAL1 is likely expressed at low levels in stem and myeloid progenitor cells and then is rapidly extinguished with further differentiation in all but a subset of cells in the

TAL1 FUNCTION IN DEVELOPMENT

macrophage lineage. In contrast, expression persists and likely increases in the erythroid lineage, particularly from the stage of BFU-E to CFU-E, declining ultimately with terminal differentiation. TAL1 may be expressed in similar fashion in megakaryocytic as in erythroid differentiation, although this has not been studied in detail. Finally, while its significance is unclear, Tal1 protein was noted in a small number of cells in sections of thymus from adult mice (Kallianpur et al., 1994), and TAL1 transcripts were detected by PCR in pooled human thymic RNA (Mouthon et al., 1993), raising the possibility that the gene is expressed at some stage of T-cell differentiation. It may be significant in this regard that a recently discovered enhancer 3 to the Tal1 coding region (see below) directed expression of a lacZ reporter to thymic T-cells of both adult and newborn mice (Sańchez et al., 1999). However, -galactosidase activity in the Tal1 lacZ knock-in mice was detected only in T cells derived from multipotent or pluripotent cells and not in resident thymocytes (Elefanty et al., 1998).

TAL1 FUNCTION IN DEVELOPMENT As applied so profitably to analysis of the function of other genes in vivo, gene-targeting studies have been carried out on TAL1 in two laboratories. Inactivation of the Tal1 gene in mice resulted in the complete absence of yolk sac erythropoiesis and embryonic loss in midgestation (Robb et al., 1995a; Shivdasani et al., 1995) in association with a total deficiency of all assayable hematopoietic progenitors. Further, embryoid bodies derived from Tal1\\ embryonic stem (ES) cells were incapable of generating hematopoietic colonies of any type (Porcher et al., 1996) and lacked GATA-1, EKLF, globin, PU.1, and myeloperoxidase mRNA (Elefanty et al., 1997; Robb et al., 1996). Tal1\\ ES lines were also used to derive chimeric mice, on both a wild-type background (Porcher et al., 1996; Robb et al., 1996) and, to more sensitively test their contribution to the lymphoid compartment, on a RAG-2\\ background (Porcher et al., 1996). Analysis of adult animals showed that the Tal1 \\ ES cells failed to contribute to any blood cells in these chimeras, including B and T

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lymphocytes (Porcher et al., 1996; Robb et al., 1996). While these results are compatible with a role for TAL1 in either specification of ventral mesoderm to a hematopoietic fate or in the generation and/or maintenance of pluripotent hematopoietic stem cells, experiments in Xenopus place TAL1 downstream of the ventralizing signal of bone morphogenetic protein-4 and suggest it functions in inducing mesoderm to acquire a hematopoietic phenotype (Mead et al., 1998). Although homozygous loss of function led to early mortality, it was nonetheless surprising that no vascular abnormalities were evident in Tal1\\ embryos. Considerable evidence points to the existence in early embryonic development of a common progenitor, termed the hemangioblast, for both blood cells and the blood vascular system. Indeed, a cell with this developmental capability was purified recently from gastrulation-stage mesoderm of chick embryos (Eichmann et al., 1997) and murine embryonic stem cell—derived embryoid bodies (Choi et al., 1998). Thus, the finding that TAL1 was expressed simultaneously by endothelial and hematopoietic cells in yolk sac blood islands (Kallianpur et al., 1994) and that its expression in avian embryos specifically marked angioblasts (Drake et al., 1997) led to the prediction that vasculogenesis, the process by which blood vessels are assembled from individual progenitor cells, would be disrupted. However, in Tal1\\ embryos in which erythropoiesis was partially rescued by a Gata-1 promoter-driven Tal1 transgene, Stuart Orkin and colleagues uncovered a lethal phenotype resulting not from a failure of vasculogenesis but of vascular remodeling (Visvader et al., 1998). In contrast, enforced expression of TAL1 in zebrafish embryos led to overproduction of hematopoietic and endothelial precursors at the expense of other, nonaxial mesoderm (Gering et al., 1998), and in the zebrafish mutant cloche partially rescued both hematopoietic and endothelial defects (Liao et al., 1998), suggesting that TAL1 specifies hemangioblast formation. Although it is possible that TAL1 subserves a different function in lower vertebrates than in mice or that aberrant vascular remodeling is in some way a late manifestation of defective angioblast function, these results are not easily reconciled.

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TAL1 FUNCTION IN POSTNATAL HEMATOPOIESIS The pattern of TAL1 gene expression observed in hematopoietic cells has been largely predictive of its actions in hematopoietic differentiation as assessed through overexpression or antisense inhibition. Two studies have evaluated the consequences of retroviral transduction of TAL1 into CD34> and committed progenitor cells from human bone marrow, peripheral blood, and umbilical cord blood (Elwood et al., 1998; Valtieri et al., 1998). In one study, enforced TAL1 expression increased BFU-E number and size, increased CFU-Mk number, decreased CFU-GM and CFU-G numbers, but was without effect on numbers of CFU-M. Its effect on multipotent or pluripotent progenitors was more complex, however, with stimulation of high proliferative potential colony-forming cells but not the more primitive long-term cultureinitiating cells (Valtieri et al., 1998). In the second study, transduction of CD34> cells increased the number, size, and rate of hemoglobinization of erythroid colonies, increased the number of megakaryocytic colonies, and produced a slight increase in granulocyte-macrophage colonies (Elwood et al., 1998). The ability of TAL1 to promote erythroid differentiation has been investigated in particular detail, both in erythroleukemia cell lines and normal erythroid progenitors. Forced expression of TAL1 in MEL cells enhanced their dimethylsulfoxide or hexamethylene bisacetamide-induced differentiation, while expression of TAL1 antisense RNA or a DNA-binding-defective, dominant negative mutant inhibited their differentiation (Aplan et al., 1992b). Similarly, transduction of a TAL1 cDNA into the bipotent leukemic cell line TF-1 (Hoang et al., 1996) and human leukemic cell line K562 (Aplan et al., 1992b) enhanced their erythroid differentiation. Finally, incubation of purified human progenitor cells with phosphorothioate antisense oligodeoxynucleotides to TAL1 selectively inhibited erythroid differentiation, while anti-Id2 oligomers induced erythroid colony formation (Condorelli et al., 1995). These results mirror the ability of overexpressed Id proteins to inhibit TAL1 DNA-binding activity (Hsu et al., 1994c; Lister and Baron, 1998; Voronova and Lee, 1994) and block MEL cell differentiation (Lister

et al., 1995; Shoji et al., 1994), and indicate that reciprocal changes in expression of Id1 or Id2 could further augment TAL1 function in erythroid differentiation. The decline in TAL1 expression with myeloid differentiation was found, similarly, to correlate with its ability to inhibit the differentiation of cells of this lineage when overexpressed. A differentiation-incompetent subline of the WEHI3B myelomonocytic leukemic cell line was shown to constitutively express Tal1 as the result of an intracisternal A particle inserting into the 3 untranslated region of the gene (Tanigawa et al., 1994), while, more directly, TAL1 transfection of the differentiation-capable parental line decreased its ability to differentiate to retinoic acid (Li et al., 1998). Enforced expression of TAL1 similarly inhibited LIF- and OSM- but, interestingly, not IL-6-induced differentiation of M1 cells (Tanigawa et al., 1993, 1995).

REGULATION OF TAL1 GENE EXPRESSION The structures of both the human (Aplan et al., 1990a) and mouse (Begley et al., 1994) TAL1 genes have been characterized, and considerable information is available on how TAL1 gene expression is itself regulated. Two promoters have been identified, designated 1a and 1b, which lie within an unmethylated CpG island (Aplan et al., 1990a), and a series of alternately spliced RNAs originating in these promoters and translated to yield pp472* have been described. Transcripts have also been found that splice from 5 noncoding exons to exon 5 or juxtapose exons 3 and 5 to produce the amino-terminally truncated isoform pp242* when translated (Aplan et al., 1990a; Begley et al., 1994). Promoter 1a is active in erythroid cells (Aplan et al., 1992b; Lecointe et al., 1994) and mast cells (Bockamp et al., 1998) but not in T cells (Bockamp et al., 1995), and two GATA binding sites and one Sp1 binding site appear to be important for this activity (Aplan et al., 1992b; Bockamp et al., 1995, 1997; Lecointe et al., 1994). In contrast, while promoter 1b was weakly active, if at all, in erythroid cells, it exhibited considerable activity in transient

TAL1 DNA-BINDING PREFERENCES

59

TABLE 4.1. Preferred DNA-Binding Sequences of TAL1-Containing Complexes?

Preferred Sequences AACAGATGGT AACAGATGGT AACAGATGKT RACAGATGKT CAGGTG(N) MGATARSG \ AMCATCTGTT(N) \ AACAGRTGTT

Complexes

References

TAL1/E47 TAL1/E2-2 TAL1/HEB TAL1 ; Jurkat cellular extracts TAL1;E2A;GATA-1;Ldb1 ;LMO2* (MEL cells) Tal1;E2A;Ldb1;Lmo2* (Lmo2 transgenic thymocytes)

(Hsu et al., 1994a) (Hsu et al., 1994a) (Hsu et al., 1994a) (Hsu et al., 1994a) (Wadman et al., 1997) (Gru¨tz et al., 1998)

?Compilation of the DNA sequences for TAL1-containing complexes identified in binding site selection assays. Shown are the nucleotide sequences preferred, the composition of the complex used for binding, and the relevant reference. E box and GATA motifs are shown in bold. Asterisk denotes complexes immunoprecipitated with antiserum to Lmo2.

transfection assays in CD34> (Bockamp et al., 1997) and mast cells (Bockamp et al., 1998). The activity of this promoter in CD34> cells was dependent on binding sites for the myc-associated zinc-finger protein ZF87/MAZ (Bossone et al., 1992) and ETS proteins, while its activity in mast cells required ETS- and Sp1/3-binding elements (Bockamp et al., 1998). The relatively low activity of both of these promoters when stably introduced into TAL1expressing cells prompted a search for additional regulatory elements important for gene expression (Bockamp et al., 1997). Through mapping of DNase I—hypersensitive sites (Go¨ttgens et al., 1997; Leroy-Viard et al., 1994) and phylogenetic sequence comparisons, enhancers upstream (Sinclair et al., 1999) and downstream (Sańchez et al., 1999) of the TAL1 coding region have been identified. The 5 enhancers directed the expression of a lacZ reporter to spatially distinct regions of Tal1 expression in developing brain, spinal cord, and endothelium in transgenic mice (Sinclair et al., 1999), while the region encompassing the DNase I—hypersensitive sites 3 to the gene targeted reporter expression to hematopoietic and endothelial cells (Sańchez et al., 1999). Given the likelihood that TAL1 is first expressed in hemangioblastic precursors in embryonic development, delineation of the specific sequences responsible for these activities and identification of the proteins that interact with them are of considerable interest.

TAL1 DNA-BINDING PREFERENCES TAL1 resembles other tissue-restricted bHLH proteins in binding DNA as a heterodimer with one of the more widely expressed E proteins. Site-selection assays, first carried out with TAL1/E47, TAL1/E2-2, and TAL1/HEB heterodimers, identified a single preferred binding element containing the sequence CAGATG, with preference extending two bases to either side of the E box as well (Hsu et al., 1994a) (Table 4.1). Virtually identical sequences were selected by pp472* and pp242*, and gel mobility shift assays showed the CAGATG element could be recognized by endogenous TAL1/E2A complexes in leukemic T cells. Work from Terence Rabbitts’s laboratory subsequently showed that a multimeric complex containing TAL1, E2A, LMO2, GATA-1, and Ldb1/NLI recognized a bipartite DNA motif comprised of a different E box (CAGGTG) and a GATA site separated by 9—12 bp (Wadman et al., 1997) (Table 4.1). DNA-binding assays using MEL cellular extracts and an oligonucleotide corresponding to this site demonstrated the presence of two highly retarded complexes containing all five of these proteins. In addition, a DNA-binding complex containing LMO2, Ldb1, E2A, and, unexpectedly, TAL1 was identified in CD4\ CD8\ thymocytes from Lmo2 transgenic mice that demonstrated a different DNA-binding preference than the ternary complex found in erythroid cells (Gru¨tz et al., 1998). This site

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Figure 4.2. Models of TAL1 function: transcriptional activation. In erythroid cells (A), a ternary complex containing TAL1, an E2A protein, LMO2, Ldb1, and GATA-1 is shown binding to a bipartite element containing a specific E box (CANNTG) and GATA site spaced :9—12 bp apart. In leukemic T cells (B—D), either LMO1 or LMO2 can serve as a bridging molecule at bipartite elements containing two E boxes (B) or an E box and GATA site (C). Although TAL1-E2A heterodimers are shown binding to both E box sites in B, the two DNA-binding complexes may not necessarily be identical. GATA-3, which is expressed in T cells, presumably binds to the GATA site of the bipartite E box-GATA site in C. In addition, as suggested by recent studies on the RALDH2 gene, both TAL1 and LMO1 (or LMO2) may act as cofactors at sites to which only GATA-3 is bound (D). Other proteins such as the transcriptional coactivators p300 and CBP, to which TAL1 and GATA-1 can both bind, may also be present in these complexes. For the purpose of clarity, nucleotide sequences that flank E box and GATA sites and likely contribute to binding specificity are not shown.

contained two E box elements of different sequence (Table 4.1 and Fig. 4.1).

TRANSCRIPTIONAL PROPERTIES TAL1/E2A heterodimers have been shown to activate transcription, most convincingly when associated with a member of the zinc finger GATA transcription factor family and LMO1 or LMO2 (Fig. 4.2). Terence Rabbitts and colleagues reported that coexpressed TAL1, E47, GATA-1, Lmo2, and Ldb1 stimulated the transcription of a luciferase reporter gene linked to two copies of the bipartite E box—GATA se-

quence (Wadman et al., 1997). Maximal transactivation required expression of all five proteins and correlated with the presence of two highly retarded binding complexes in gel mobility shift analysis. Osamu Yoshie and colleagues observed T-cell—specific transactivation of a minimal tk promoter linked to four copies of the preferred CAGATG E box site by TAL1, either LMO1 or LMO2, and, despite the absence of a GATA-binding site in their reporter, GATA-3 (Ono et al., 1997). This group also showed that TAL1 could act as a cofactor with LMO1 and endogenously expressed GATA-3 in transactivating the retinaldehyde dehydrogenase 2 (RALDH2) gene in T-ALL cells (Ono et al., 1998). Their study identified a T-ALL—specific

TRANSCRIPTIONAL PROPERTIES

61

Figure 4.3. Models of TAL1 function: transcriptional repression. E-box sequences through which E2A proteins would normally activate transcription (A) may be targets of repression in TAL1-expressing leukemic cells. TAL1/E2A heterodimers could inhibit transcription by a mechanism involving competition for DNA binding, masking of the E2A activation domain, or incompatibility in the TAL1 and E2A activation domains (B). Although not depicted, repression may also involve the recruitment of histone deacetylases through the mediation of the corepressors mSin3A and mSin3B. Finally, similar to the Id proteins, TAL1 could effect repression by interacting with E proteins (or other transcriptional activators or coactivators) and preventing their interaction with DNA (C).

intronic promoter in the RALDH2 gene that contained a GATA site that was necessary and sufficient for its function, while an upstream E box and TAL1 DNA-binding activity were dispensable (Ono et al., 1998). Thus, TAL1 can contribute, in some cases without the need to interact with DNA itself, to multiprotein transcriptional complexes containing one of its Eprotein DNA-binding partners, either LMO1 or LMO2, the LIM domain—binding protein Ldb1, and GATA-1 or GATA-3. The striking similarity in the phenotype of the Lmo2 and Tal1 knockout mice, both in embryonic (Warren et al., 1994) and adult (Yamada et al., 1998)

hematopoiesis, provides support for the notion that the two proteins subserve similar functions as components of such a complex. Moreover, the association of TAL1 and LMO1/LMO2 misexpression in T-ALL suggests that an analogous complex could have a role in T-cell leukemogenesis. Considerable evidence indicates that TAL1 can also inhibit transcription (Fig. 4.3). It has been shown to decrease transcription from artificial promoters containing multiple copies of a number of E box elements, including the CAGATG site that binds TAL1/E2A complexes with high affinity (Hsu et al., 1994c), the E box

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sites in the E2 and E2/E5 immunoglobulin enhancers (Doyle et al., 1994; Hofmann and Cole, 1996; Voronova and Lee, 1994), a MyoDbinding site (Goldfarb and Lewandowska, 1995; Hofmann and Cole, 1996), and an E box from a retroviral enhancer (Nielsen et al., 1996). In one report, TAL1 did so without binding DNA, and, similar to the actions of the Id proteins, was proposed to titrate E proteins into nonproductive binding complexes (Goldfarb and Lewandowska, 1995). For most of these promoters, however, TAL1/E protein complexes were found to bind DNA, although with a reduced transactivation potential compared to E protein dimers (Hofmann and Cole, 1996; Hsu et al., 1994c; Nielsen et al., 1996; Voronova and Lee, 1994). This was ascribed both to a domain in the carboxy-terminus of TAL1 acting to mask the transactivation domain(s) of its DNA-binding partner (Hofmann and Cole, 1996) and to a functional incompatibility between the proteins’ respective activation domains (Park and Sun, 1998). For those TAL1-containing complexes that include LMO1 or LMO2, another potentialmechanism for TAL1-directed repression would involve the LIM domain proteins acting as corepressors. Indeed, LMO1 was reported to potentiate TAL1 inhibition of E2-5—stimulated transcription from the E5, E2, and E3 E boxes (Chervinsky et al., 1999), and LMO2 was found to contain multiple repression, in addition to activation, domains (Mao et al., 1997). Finally, we recently discovered that TAL1 interacts in vitro and in vivo with two members of a nuclear corepressor complex, mSin3A and histone deacetylase (HDAC) 1, and that these TAL1-containing complexes exhibit histone deacetylase enzymatic activity (Huang and Brandt, 1999). Further, TAL1-mediated inhibition of E47-stimulated transactivation was relieved by the specific HDAC inhibitor trichostatin A, suggesting this TAL1-corepressor complex functions in inhibiting gene expression. The findings that E2A transduction of E2A-deficient T lymphoblasts (Engel and Murre, 1999) or TAL1expressing T-ALL cells (Park et al., 1999) inhibited their growth and/or induced their apoptosis and the observation that E2A knockout mice show a high incidence of thymic lymphomas (Bain et al., 1997; Yan et al., 1997) provide strong support for TAL1 inhibition of E

protein—stimulated transcription having an important role in T-cell leukemogenesis.

TARGET GENES Since TAL1 appears capable of either stimulating or inhibiting transcription, it follows that expression of some target genes may be induced while others could be repressed by TAL1-containing complexes. This may apply not only to TAL1-expressing leukemic cells but also to cells that express the gene physiologically. Candidate targets in both categories have been proposed recently, although not all have been validated fully. A gene belonging to the four-membranespanning domain superfamily, TALLA1 (Takagi et al., 1995), was suggested to be a target of TAL1 in leukemic lymphoblasts (Ono et al., 1997). Compellingly, a TALLA1-negative TALL cell line was induced to express the gene by cotransduction of TAL1 and LMO1 cDNAs but not by either alone (Ono et al., 1997). It was not demonstrated, however, that TAL1 induction of TALLA1 expression was mediated at a transcriptional level or what elements in the TALLA1 gene were required for this induction. As discussed above, this same group has also suggested that the RALDH2 gene is transactivated in leukemic T cells by TAL1, LMO1, and GATA-3 (Ono et al.,1998). The gene for the SCF receptor, c-Kit, was suggested to be a TAL1 downstream target by Trang Hoang and colleagues from experiments demonstrating that c-Kit expression and SCF responsiveness of a CD34> human leukemic cell line were reduced with inhibition of TAL1 expression or DNA binding (Krosl et al., 1998). In addition, TAL1 transduction of Ba/F3 cells, which lack expression of both TAL1 and c-Kit, activated c-Kit expression and rendered the cells SCF-dependent. However, transfected TAL1 protein had only a modest effect on the activity of a c-Kit promoter-reporter construct (Krosl et al., 1998), and c-Kit expression in Tal1\\ ES cells induced to undergo hematopoietic differentiation was not affected, at least not grossly, by loss of TAL1 function (Elefanty et al., 1997). Using the technique of chromatin immunoprecipitation to isolate specific DNA sequences to which TAL1 protein would be bound, PaulHenri Romeó and colleagues (Cohen-Kaminsky

REFERENCES

et al., 1998) identified a novel gene whose expression pattern is consistent with a TAL1 target. They found an E box—E box—GATA element in an intronic promoter of this gene that both bound an erythroid-specific multiprotein complex containing TAL1 and functioned as an enhancer in transient transfection assays. Finally, a bipartite GATA-E box element was found in an upstream enhancer of the GATA-1 gene that bound a multimeric complex containing GATA-1, TAL1, E2A, Lmo2, and Ldb1 (Vyas et al., 1999). Mutagenesis of the E box had no effect on enhancer function in transgenic assays, however, although DNA binding of this complex depended on the integrity of both sites (Vyas et al., 1999). In addition to these candidates, the EKLF (Anderson et al., 1998), erythroid-specific porphobilinogen deaminase (Chretien et al., 1988), and protein 4.2 (Karacay and Chang, 1999) promoters also represent possible targets for TAL1 based on their nucleotide sequence and the expression pattern of the genes they control. As discussed previously, TAL1 has also been shown capable of inhibiting transactivation by the E2A-, HEB-, or E2-2-encoded proteins, so genes whose transcription is stimulated by these E proteins could be targets of TAL1-directed repression. Expression of two such genes, p21WAF1 (Prabhu et al., 1997) and CD4 (Sawada and Littman, 1993), were in fact reduced in TAL1-expressing cells (Chervinsky et al., 1999; Park and Sun, 1998).

CONCLUSION AND FUTURE DIRECTIONS Since its initial discovery at the site of a recurrent chromosomal translocation, efforts by laboratories in the United States, Australia, the United Kingdom, Canada, and France have demonstrated the oncogenic consequences of ectopic TAL1 expression and the importance of this transcription factor for normal hematopoiesis. Important questions remain in both areas of research, however. First, although the LIM domain proteins LMO1 and LMO2 are clearly capable of interacting with TAL1 physically and functionally, the frequency with which they are actually coexpressed with TAL1 in leukemic T cells is unknown, as is the requirement for other genetic abnormalities, including loss of

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the tumor suppressor products of the INK4A, INK4B, and MSH2 loci, for disease pathogenesis. Further, the specific contributions that TAL1 makes to the leukemic phenotype, the full range of proteins with which it interacts, and its downstream targets in leukemic cells need delineation. Finally, it must be determined whether transcriptional repression, activation, or both, are most important for the oncogenic actions of this bHLH transcription factor. In addition to its actions in leukemogenesis, TAL1 is required for the specification of hematopoietic fate and possibly vascular patterning in embryogenesis. Because of early embryonic lethality associated with homozygous loss of TAL1 function, analysis of the gene’s role in the differentiation of individual hematopoietic lineages and the nonhematopoietic cell types in which it is expressed awaits the application of conditional gene-targeting strategies. Further, while some progress has been made recently in identification of TAL1 target genes, validation of the ones proposed, discovery of additional targets, and determination of how specific nucleotide sequences in the regulatory elements of these genes potentially influence its actions as a transcriptional repressor or activator are still required. Characterization of the regulatory elements of the TAL1 gene itself and identification of the specific proteins that bind them, particularly in embryonic hemangioblasts, represent important objectives toward which rapid progress can be expected. Finally, the sites in TAL1 protein that undergo phosphorylation and acetylation and the specific effects these posttranslational modifications have on TAL1 function need more complete definition. Research into these and related questions should continue to provide important insights into normal and leukemic hematopoiesis.

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Aplan, P. D., Lombardi, D. P., Ginsberg, A. M., Cossman, J., Bertness, V. L., and Kirsch, I. R. (1990b). Disruption of the human SCL locus by ‘‘illegitimate’’ V-(D)-J recombinase activity. Science 250, 1426—1429. Aplan, P. D., Lombardi, D. P., Reaman, G. H., Sather, H. N., Hammond, G. D., and Kirsch, I. R. (1992a). Involvement of the putative hematopoietic transcription factor SCL in T-cell acute lymphoblastic leukemia. Blood 79, 1327—1333. Aplan, P. D., Nakahara, K., Orkin, S. H., and Kirsch, I. R. (1992b). The SCL gene product: a positive regulator of erythroid differentiation. EMBO J. 11, 4073—4081. Aplan, P. D., Raimondi, S. C., and Kirsch, I. R. (1992c). Disruption of the SCL gene by a t(1;3)translocation in a patient with T cell acute lymphoblastic leukemia. J. Exp. Med. 176, 1303—1310. Aplan, P. D., Jones, C. A., Chervinsky, D. S., Zhao, X., Ellsworth, M., Wu, C., McGuire, E. A., and Gross, K. W. (1997). An scl gene product lacking the transactivation domain induces bony abnormalities and cooperates with LMO1 to generate T-cell malignancies in transgenic mice. EMBO J. 16, 2408—2419. Baccichet, A., Benachenhou, N., Couture, F., Leclerc, J. M., and Sinnett, D. (1997). Microsatellite instability in childhood T cell acute lymphoblastic leukemia. Leukemia 11, 797—802. Bain, G., Engel, I., Robanus Maandag, E. C., de Riele, H. P., Voland, J. R., Sharp, L. L., Chun, J., Huey, B., Pinkel, D., and Murre, C. (1997). E2A deficiency leads to abnormalities in T-cell development and to rapid development of T-cell lymphomas. Mol. Cell. Biol. 17, 4782—4791. Bash, R. O., Crist, W. M., Shuster, J. J., Link, M. P., Amylon, M., Pullen, J., Carroll, A. J., Buchanan, G. R., Smith, R. G., and Baer, R. (1993). Clinical features and outcome of T-cell acute lymphoblastic leukemia in childhood with respect to alterations at the TAL1 locus: a Pediatric Oncology Group study. Blood 81, 2110—2117. Bash, R. O., Hall, S., Timmons, C. F., Crist, W. M., Amylon, M., Smith, R. G., and Baer, R. (1995). Does activation of the TAL1 gene occur in a majority of patients with T- cell acute lymphoblastic leukemia? A Pediatric Oncology Group study. Blood 86, 666—676. Batova, A., Diccianni, M. B., Yu, J. C., Nobori, T., Link, M. P., Pullen, J., and Yu, A. L. (1997). Frequent and selective methylation of p15 and deletion of both p15 and p16 in T-cell acute lymphoblastic leukemia. Cancer Res. 57, 832—836. Begley, C. G., Aplan, P. D., Denning, S. M., Haynes, B. F., Waldmann, T. A., and Kirsch, I. R. (1989). The gene SCL is expressed during early hematopoiesis and encodes a differentiation related DNA-binding motif. Proc. Natl. Acad. Sci. USA 86, 10,128—10,132. Begley, C. G., Robb, L., Rockman, S., Visvader, J., Bockamp, E. O., Chan, Y. S., and Green, A. R. (1994). Structure of the gene encoding the murine

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selective expansion of CD4-CD8- lymphocytes in the thymus and T-cell tumors intransgenic mice. Blood 86, 3060—3071. Neri, A., Fracchiolla, N. S., Roscetti, E., Garatti, S., Trecca, D., Boletini, A., Perletti, L., Baldini, L., Maiolo, A. T., and Berti, E. (1995). Molecular analysis of cutaneous B- and T-cell lymphomas. Blood 86, 3160—3172. Nielsen, A. L., Nørby, P. L., Pedersen, F. S., and Jørgensen, P. (1996). E-box sequence and contextdependent TAL1/SCL modulation of basic helixloop-helix protein-mediated transcriptional activation. J. Biol. Chem. 271, 31,463—31,469. Ogawa, S., Hangaishi, A., Miyawaki, S., Hirosawa, S., Miura, Y., Takeyama, K., Kamada, N., Ohtake, S., Uike, N., Shimazaki, C., Toyama, K., Hirano, M., Mizoguchi, H., Kobayashi, Y., Furusawa, S., Saito, M., Emi, N., Yazaki, Y., Ueda, R., and Hirai, H. (1995). Loss of the cyclin-dependent kinase 4-inhibitor (p16;MTS1) gene is frequent in and highly specific to lymphoid tumors in primary human hematopoietic malignancies. Blood 86, 1548—1556. Ohnishi, H., Kawamura, M., Ida, K., Sheng, X. M., Hanada, R., Noburi, T., Yamamori, S., and Hayashi, Y. (1995). Homozygous deletions of p16/ MTS1 gene are frequent but mutations are infrequent in childhood T-cell acute lymphoblastic leukemia. Blood 86, 1269—1275. Okuda, T., Shurtleff, S. A., Valentine, M. B., Raimondi, S. C., Head, D. R., Behm, F., Curcio-Brint, A. M., Liu, Q., Pui, C.-H., Sherr, C. J., Beach, D., Look, A. T., and Downing, J. R. (1995). Frequent deletion of p16INK4a/MTS1 and p15INK4b/MTS2 in pediatric acute lymphoblastic leukemia. Blood 85, 2321—2330. Ono, Y., Fukuhara, N., and Yoshie, O. (1997). Transcriptional activity of TAL1 in T cell acute lymphoblastic leukemia (T-ALL) requires RBTN1 or -2 and induces TALLA1, a highly specific tumor marker of T-ALL. J. Biol. Chem. 272, 4576—4581. Ono, Y., Fukuhara, N., and Yoshie, O. (1998). TAL1 and LIM-only proteins synergistically induce retinaldehyde dehydrogenase 2 expression in T-cell acute lymphoblastic leukemia by acting as cofactors for GATA3. Mol. Cell. Biol. 18, 6939—6950. Osada, H., Grutz, G., Axelson, H., Forster, A., and Rabbitts, T. H. (1995). Association of erythroid transcription factors: complexes involving the LIM protein RBTN2 and the zinc-finger protein GATA1. Proc. Natl. Acad. Sci. USA 92, 9585—9589. Otsuki, T., Clark, H. M., Wellmann, A., Jaffe, E. S., and Raffeld, M. (1995). Involvement of CDKN2 (p16INK4A/MTS1) and p15INK4B/MTS2 in human leukemias and lymphomas. Cancer Res. 55, 1436— 1440. Park, S. T., and Sun, X.-H. (1998). The Tal1 oncoprotein inhibits E47-mediated transcription. Mechanism of inhibition. J. Biol. Chem. 273, 7030— 7037. Park, S. T., Nolan, G. P., and Sun, X.-H. (1999). Growth inhibition and apoptosis due to restoration of E2A activity in T cell acute lymphoblastic

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Tanigawa, T., Robb, L., Green, A. R., and Begley, C. G. (1994). Constitutive expression of the putative transcription factor SCL associated with proviral insertion in the myeloid leukemic cell line WEHI-3BD\. Cell Growth Diff. 5, 557—561. Tanigawa, T., Nicola, N., McArthur, G. A., Strasser, A., and Begley, C. G. (1995). Differential regulation of macrophage differentiation in response to leukemia inhibitory factor/oncostatin-M/interleukin-6: the effect of enforced expression of the SCL transcription factor. Blood 85, 379—390. Valge-Archer, V. E., Osada, H., Warren, A. J., Forster, A., Li, J., Baer, R., and Rabbitts, T. H. (1994). The LIM protein RBTN2 and the basic helix-loophelix protein TAL1 are present in a complex in erythroid cells. Proc. Natl. Acad. Sci. USA 91, 8617—8621. Valge-Archer, V., Forster, A., and Rabbitts, T. H. (1998). The LMO1 and LDB1 proteins interact in human T cell acute leukaemia with the chromosomal translocation t(11;14)(p15;q11). Oncogene 17, 3199—3202. Valtieri, M., Tocci, A., Gabbianelli, M., Luchetti, L., Masella, B., Vitelli, L., Botta, R., Testa., Condorelli, G. L., and Peschle, C. (1998). Enforced TAL-1 expression stimulates primitive, erythroid and megakaryocytic progenitors but blocks the granulopoietic differentiation program. Cancer Res. 58, 562—569. Visvader, J., Begley, C. G., and Adams, J. M. (1991). Differential expression of the LYL, SCL, and E2A helix-loop-helix genes within the hemopoietic system. Oncogene 6, 187—194. Visvader, J. E., Fujiwara, Y., and Orkin, S. H. (1998). Unsuspected role for the T-cell leukemia protein SCL/tal-1 in vascular development. Genes Dev. 12, 473—479. Voronova, A., and Baltimore, D. (1990). Mutations that disrupt DNA binding and dimer formation in the E47 helix-loop-helix protein map to distinct domains. Proc. Natl. Acad. Sci. USA 87, 4722— 4726. Voronova, A. F., and Lee, F. (1994). The E2A and tal-1 helix-loop-helix proteins associate in vivo and are modulated by Id proteins during interleukin 6-induced myeloid differentiation. Proc. Natl. Acad. Sci. USA 91, 5952—5956. Vyas, P., McDevitt, M. A., Cantor, A. B., Katz, S. G., Fujiwara, Y., and Orkin, S. H. (1999). Different sequence requirements for expression in erythroid and megakaryocytic cells within a regulatory element upstream of the GATA-1 gene. Development 126, 2799—2811. Wadman, I., Li, J., Bash, R. O., Forster, A., Osada, H., Rabbitts, T. H., and Baer, R. (1994a). Specific in vivo association between the bHLH and LIM proteins implicated in human T cell leukemia. EMBO J. 13, 4831—4839. Wadman, I. A., Hsu, H. L., Cobb, M. H., and Baer, R. (1994b). The MAP kinase phosphorylation site of TAL1 occurs within a transcriptional activation domain. Oncogene 9, 3713—3716.

Wadman, I. A., Osada, H., Gru¨tz, G. G., Agulnick, A. D., Westphal, H., Forster, A., and Rabbitts, T. H. (1997). The LIM-only protein Lmo2 is a bridging molecule assembling an erythroid, DNAbinding complex which includes the TAL1, E47, GATA-1 and Ldb1/NLI proteins. EMBO J. 16, 3145—3157. Wang, Y., Schnegelsberg, P. N., Dausman, J., and Jaenisch, R. (1996). Functional redundancy of the muscle-specific transcription factors Myf5 and myogenin. Nature 379, 823—825. Warren, A. J., Colledge, W. H., Carlton, M. B. L., Evans, M. J., Smith, A. J. H., and Rabbitts, T. H. (1994). The oncogenic cysteine-rich LIM domain protein Rbtn2 is essential for erythroid development. Cell 78, 45—57. Weintraub, H., Davis, R., Tapscott, S., Thayer, M., Krause, M., Benezra, R., Blackwell, T. K., Turner, D., Rupp, R., Hollenberg, S., Zhuang, Y., and Lassar, A. (1991). The myoD gene family: nodal point during specification of the muscle cell lineage. Science 251, 761—766. Xia, Y., Brown, L., Yang, C. Y.-C., Tsan, J. T., Siciliano, M. J., Espinosa, R., LeBeau, M. M., and Baer, R. J. (1991). Tal-2, a helix-loop-helix gene activated by the (7;9)(q34;q32) translocation in human T-cell leukemia. Proc. Natl. Acad. Sci. USA 88, 11,416—11,420. Xia, Y., Brown, L., Tsan, J. T., Yang, C. Y., Siciliano, M. J., Crist, W. M., Carroll, A. J., and Baer, R. (1992). The translocation (1;14)(p34;q11) in human T-cell leukemia: chromosome breakage 25 kilobase pairs downstream of the TAL1 protooncogene. Genes Chrom. Cancer 4, 211—216. Yamada, Y., Warren, A. J., Dobson, C., Forster, A., Pannell, R., and Rabbitts, T. H. (1998). The T cell leukemia protein Lmo2 is necessary for adult mouse hematopoiesis. Proc. Natl. Acad. USA 95, 3890—3895. Yan, W., Young, A. Z., Soares, A. C., Kelley, R., Benezra, R., and Zhuang, Y. (1997). High incidence of T-cell tumors in E2A-null mice and E2A/Id1 double-knockout mice. Mol. Cell. Biol, 17, 7317— 7327. Zhang, W., and Bieker, J. J. (1998). Acetylation and modulation of erythroid Kru¨ppel-like factor (EKLF) activity by interaction with histone acetyltransferases. Proc. Natl. Acad. Sci. USA 95, 9855— 9860. Zhao, X.-F., and Aplan, P. D. (1998). SCL binds the human homologue of DRG in vivo. Biochim. Biophys. Acta 1448, 109—114. Zhao, X. F., and Aplan, P. D. (1999). The hematopoietic transcription factor SCL binds the p44 subunit of TFIIH. J. Biol. Chem. 274, 1388—1393. Ziegler, B. L., Mu¨ller, R., Valtieri, M., Lamping, C. P., Thomas, C. A., Gabbianelli, M., Giesert, C., Bu¨hring, H.-J., Kanz, L., and Peschle, C. (1999). Unicellular-unilineage erythropoietic cultures: molecular analysis of regulatory gene expression at sibling cell level. Blood 93, 3355—3368.

CHAPTER 5


EKLF AND THE DEVELOPMENT OF THE ERYTHROID LINEAGE JAMES J. BIEKER Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine

INTRODUCTION Studies directed at an analysis of hemoglobin expression have historically been at the cutting edge of research, whether it has been the first description of developmentally regulated changes in sites of hemoglobinogenesis (Jordan and Spiedel, 1923; McCutcheon, 1936), determination of sickle cell anemia as an aberration of a single genetic locus (Pauling et al., 1949), analysis of the quaternary, three-dimensional structure of hemoglobin (Perutz, 1987), the early successes of molecular cloning approaches to gene structure determination (Lawn et al., 1978), or the introduction of foreign genes into mice (Costantini and Lacy, 1981). This use of novel approaches to address compelling issues of globin gene regulation have continued in recent years, as novel insights on the phenomena of distal locus control elements and their role in erythroid-specific and developmentally appropriate expression (Behringer et al., 1990; Enver et al., 1990; Grosveld et al., 1987) has been

abetted by the ability to analyze large constructs that contain the complete genomic globin cluster in transgenic mice (Gaensler et al., 1993; Peterson et al., 1993; Strouboulis et al., 1992). However, deciphering the mechanism by which erythroid genes are regulated has also depended on the isolation of trans-acting factors and identification of the specific cis-acting elements through which they exert their effects on gene expression (reviewed in Orkin, 1995; Baron, 1997; Bieker, 1998).

IDENTIFICATION OF EKLF This chapter focuses on the transcriptional activator known as erythroid Kru¨ppel-like factor (EKLF). EKLF was originally isolated via a differential screening approach whereby mRNA that was specifically expressed in erythroid cells was identified after subtraction with common messages expressed in a myeloid cell line


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EKLF AND THE DEVELOPMENT OF THE ERYTHROID LINEAGE

(Miller and Bieker, 1993; Ira J. Miller and James J. Bieker, unpublished). Specifically, murine erythroleukemia (MEL) cells provided the erythroid mRNA sample, and the J774 monocyte-macrophage cell line provided the subtracting mRNA sample. This removed 99% of MEL mRNA from consideration. The remaining material was directly cloned and sorted by a combination of cross-hybridization analysis of arrays of individual clones and gradual elimination of those clones that were most abundantly expressed. This left only 17 clones that represented almost all of the 1500 primary clones in the subtracted library. Most of these were related to Friend virus genes, metabolic clones (e.g., carbonic anhydrase), and transcription factors (e.g., c-Myb and GATA1), but some clones were novel at the time. A Northern blot analysis of adult murine tissues directed the focus to one particular clone uniquely expressed in bone marrow and spleen. Further analysis of this clone (EKLF) indicated that it was expressed only in erythroid cell lines, but not in any lymphoid or myeloid lines, with the exception of a small amount of expression in mast cell lines. A significant clue as to the function of this clone resulted from the completion of its primary sequence, which indicated that it contained three C2H2-type zinc fingers at the extreme carboxyl end, raising the possibility that it was a DNA-binding protein (Miller and Bieker, 1993). More precisely, it was most closely homologous to the Kru¨ppel family of transcription factors, which are named after the segmentation gene involved in Drosophila body patterning (Schuh et al., 1986). This was fortuitous, as a variety of finger-swapping experiments had been performed with other members of this family (e.g., Zif 268, Sp1, Krox 20) with the aim of predicting its target DNA-binding site. The molecular analysis culminated in the crystallographic structure of Zif 268 bound to its target site (Pavletich and Pabo, 1991). Of critical importance for predictive purposes were the interactions of basic residues at specific amino acid locations of Zif 268 with guanines within the DNA-binding sequence (Klevit, 1991). In addition, each finger interacted with a triplet of nucleotides. This information enabled the prediction that EKLF would interact with 3 GGNGNGGGN5. A subset of this sequence, known as the

5CACCC3 element, was already recognized as one of a trio of elements that are repeated within erythroid enhancers and promoters. In particular, the CAC site within the -globin gene (5CCACACCCT3) precisely matched the predicted EKLF consensus-binding sequence. An oligonucleotide containing this sequence was directly tested and found to bind EKLF in vitro with high affinity. In addition, transfection studies demonstrated that EKLF functioned as a transcriptional activator (not repressor) when bound to this site. These studies provided the initial isolation and characterization of this transcription factor (Miller and Bieker, 1993).

FUNCTIONAL ROLE OF EKLF IN ERYTHROID EXPRESSION The -globin gene CACCC element is the site of thalassemia-causing point mutations in humans (Stamatoyannopoulos and Nienhuis, 1994). As a result, individual examples of these were directly tested for their ability to interact with EKLF (Feng et al., 1994). The results were dramatic: not only was EKLF not able to transactivate through these mutated sites but the binding affinity was decreased 40- to 100-fold. Molecular modeling of these interactions indicated that both specific and nonspecific hydrogen bond interactions were adversely affected by what appeared to be minor changes in sequence of the CACCC element. Methylation interference assays further demonstrated that each of the guanines on the G-rich strand in the nine-base pair -globin CACCC element (3GGTGTGGGA5) were critical for optimal binding by EKLF. Analysis of EKLF biological expression also provided critical information about its role (Southwood et al., 1996). EKLF is not expressed in embryonic stem (ES) cells but becomes apparent during embryoid body differentiation in culture. Analysis of developing murine embryos indicated that EKLF is first expressed at the neural plate stage (i.e., day 7.5) within the extraembryonic mesoderm of the visceral yolk sac, the earliest morphologically identifiable time of blood island formation, after which its level continues to expand. It is then expressed at day 9.0 within the hepatic primordia, such that by day 14.5 the fetal liver is the only site of EKLF

FUNCTIONAL ROLE OF EKLF IN ERYTHROID EXPRESSION

expression. In the adult mouse spleen, it is expressed solely within the red pulp. These results complemented the early cell line studies and strengthened the idea that EKLF plays a critical role in erythropoietic differentiation, not only with respect to globin expression but potentially for activation of the many erythroid genes that contain CACCC elements within their promoters. Such a straightforward explanation for EKLF function was not to be. The initial hints had already followed from the point mutation analysis described above. Specifically, the murine embryonic (and human fetal) CACCC elements differ in one of the important guanines within the interaction sequence (3GAGGTGGGT5) (Bieker, 1994). Given the deleterious effects of the thalassemia-causing point mutations, it was felt that this change would also adversely affect binding. This was directly tested, and competition assays demonstrated that the embryonic CACCC element had an 8-fold lower affinity for EKLF compared to the adult CACCC element (Donze et al., 1995). The effects of such a mutation were tested in vivo by using K562 cells, a human erythroleukemic cell line that expresses -globin but not -globin. In this line, EKLF had minimal effects on the activity of an intact -globin promoter, but could specifically activate the -globin promoter over 1000-fold when it was linked in cis to the -promoter. These molecular data raised the possibility that EKLF may be important for the switch from fetal to adult -like globin expression. Expression of the -like globin genes is regulated in a specific temporal sequence during mammalian development (Baron, 1997; Orkin, 1995; Stamatoyannopoulos and Nienhuis, 1994). In humans, the first blood cells formed within the yolk sac express the embryonic () globin variant. After the site of hematopoiesis switches to the fetal liver, the fetal () globin genes are expressed. This is followed by yet another switch in expression to the adult () globin genes after the bone marrow becomes the major site of hematopoiesis. In mice, there is a single switch in molecular expression from embryonic (y, bh0, bh1) globin genes in the yolk sac to the adult () globin variant in the fetal liver and the adult bone marrow and spleen. Regulation of this large -like globin cluster is thought to occur by competition of each globin promoter member

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(embryonic, fetal, and adult) for interaction with the LCR, thus enabling high-level activity of only one member at its appropriate time during development. In reality, this control is more complex, as an endogenous silencing mechanism also plays a vital role in this process (Baron, 1997; Fraser et al., 1998; Orkin, 1995; Wijgerde et al., 1995). The precise role of the LCR and its individual hypersensitive site components in the process of transcription, chromatin structure, and developmental control are still not completely understood (Higgs, 1998). As a result, identifying the mechanism by which transcription factors affect or alter the control of globin switching are still critically required for illuminating this process (Bieker, 1998). The molecular ability of EKLF to discriminate among CACCC elements led to the prediction that an EKLF null mutation would lead to deficiency in adult, and not embryonic or fetal, globin expression, a conjecture that was borne out by its genetic knockout in mice (Nuez et al., 1995; Perkins et al., 1995). This led to an embryonic-lethal phenotype by day 14 due to lack of onset of -globin transcription, resulting in extreme anemia and prenatal death, precisely at the time of the switch; embryonic globins were expressed at normal levels. These effects were unusually specific, as -globins and other red cell—specific genes were unaffected by the lack of EKLF expression. Generation of committed erythroid cells, as monitored by BFU-E and CFU-E assays, was normal. Additional studies that utilized EKLF null embryonic stem cells to generate chimeric adult mice indicated that these cells did not contribute to the mature erythrocyte population, and that they were defective by virtue of a globin chain imbalance, leading to Heinz body formation, a shortened life span, and apparent clearance from the bloodstream (Lim et al., 1997). The previous data revealed that EKLF is critical for the onset of adult -globin expression. To address the effects on the other linked globin-like genes in the cluster, EKLF null mice were crossed with a murine line that carries the complete human -like globin cluster (Perkins et al., 1996; Wijgerde et al., 1996). As seen with the murine locus, adult -globin expression was completely dependent on EKLF. However, globin levels were 5-fold higher, and although they were eventually silenced (as the mice still

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Figure 5.1. Role of EKLF in consolidating the switch to adult -globin expression during development. In the presence of EKLF (left), the -globin promoter preferentially associates with the LCR, both enabling -globin transcription, and, by competition, decreasing the probability of -globin transcription. In the absence of EKLF (right), the -promoter is not able to adequately compete, leading to higher and more extended levels of -globin expression prior to its shutoff by autonomous silencing. (Based on Perkins et al., 1996. Copyright 1996 National Academy of Sciences, U.S.A.).

died from lack of adult -globin expression), this shutoff occurred significantly later than that observed in the presence of EKLF. In addition, most EKLF null CFU-E colonies were reprogrammed to high -globin expression. The mode of EKLF action thus appears to be in consolidating the switch from fetal to adult globin, possibly by enabling the -globin promoter to form a structure that is able to compete for interaction with the LCR to a greater extent that the embryonic or fetal globin promoters (Fig. 5.1). Consistent with this idea, the converse experiment (overexpression of EKLF) expedites the switch from - to -globin (Tewari et al., 1998). However, it must also be kept in mind that an active EKLF is necessary but not sufficient for activation of the -globin promoter. For example, differentiation of MEL cells can be blocked without altering the levels of EKLF and GATA-1 by forced expression of Id1 (Lister et al., 1995), a negative regulator of basic helixloop-helix proteins. In addition, treatment of the SKT6 erythroleukemic cell line, which already expresses EKLF and GATA-1, with erythropoietin is required to efficiently induce globin expression (Reese et al., 1997). At the same time, the presence of EKLF is essential for erythropoietin-mediated induction of hemoglobin in J2E erythroleukemia cells (Spadaccini et al., 1998). The details of EKLF interaction with CACCC elements have proved to be more complex than their simple discrimination by affinity (Asano and Stamatoyannopoulos, 1998). The

promoter context is also thought to play a major role in this process, as simple substitution of the adult -CACCC element in place of the fetal -CACCC element does not impart EKLFdependent activation to the -promoter (judged by in vivo cotransfection assays). Surprisingly, the reverse change (i.e., substitution of the fetal -CACCC element into the adult -CACCC site) did not alter the ability of EKLF to activate transcription via the adult promoter. To be fair, the promoters are not architecturally similar. However, these data raise the important point that the affinity is only part of the process, and that promoter-specific, protein-based complex formation can serve to stabilize what may initially be a weaker protein-DNA interaction.

MECHANISM OF EKLF TRANSCRIPTIONAL ACTIVATION Although all the initial studies focused on the EKLF zinc fingers and how they interact with their DNA target site, the rest of the molecule remained to be analyzed. This nonfinger region of the molecule functions as a classical transactivation module, as its fusion to a heterologous DNA-binding motif (GAL4) results in strong activation of a target reporter that contains the GAL DNA-binding motif, yet itself does not bind DNA (Bieker and Southwood, 1995). As this region of EKLF is not homologous to anything in GenBank, initial studies focused on utilizing a -globin promoter/transient reporter system that itself was regulated and subject to

MECHANISM OF EKLF TRANSCRIPTIONAL ACTIVATION

induction by HMBA after its transfection into MEL cells (Bieker and Southwood, 1995). Mutation of the CACCC element (to a GAL4-type DNA-binding sequence) within this reporter abrogated its activity, even after induction. However, transfection of a GAL-EKLF chimera that contained the GAL4 DNA-binding module fused to the EKLF transactivation domain reconstituted inducibility to this promoter. Intriguingly, fusion to the Sp1 transactivation domain, which has no sequence homology to EKLF beyond the zinc fingers, was not able to reconstitute inducibility to the -globin promoter. This specificity of action was lost, however, when the same experiment was performed in nonerythroid cells, or when a totally artificial promoter/reporter construct was used. This clearly indicated that the EKLF transactivation domain was specifically required at the -globin promoter for its regulated expression in the erythroid cell. A deletional analysis (Chen and Bieker, 1996) of the GAL-EKLF chimera was performed with a GAL-reporter system in the 32DEpo1 erythroid cell line. These data revealed that this 270 amino acid region contained subdomains, one of which was the minimal region (aa 20—120 at the amino terminus) required for transactivation of the reporter via cotransfection assays, and an inhibitory region (aa 195—275) whose removal led to an increase in transactivational activity. This inhibitory domain is likely to act in cis, as its removal also increased EKLF protein-DNA interactions in vitro. Using a different approach, a deletional study of EKLF’s ability to activate the -promoter within the --linked reporter system in K562 cells has mapped a second essential transactivation domain located at aa 157—196 (Tim M. Townes, manuscript in preparation). In vivo competition assays were used to assess whether EKLF can associate with proteins that affect its transactivational ability (Chen and Bieker, 1996). The assay monitors whether excess native EKLF can alter the transcriptional activity of a GAL-EKLF chimera on a GALbinding site-containing reporter gene in the 32DEpo1 erythroid cell line. The results demonstrated that increasing the level of EKLF proportionately decreased reporter gene expression, indicating that EKLF must be interacting with a positive-acting factor to induce transcription.

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Deletional analysis of this effect localized the relevant region to a 40 amino acid sequence at the amino terminus, overlapping the previously identified minimal activation domain at aa 20— 120. This strongly implied that EKLF activity is dependent on interactions with a coactivator protein via its amino terminus. It is of interest that this subdomain of the murine EKLF transactivation region is the most highly conserved with the human homologue (Bieker, 1996) in both primary sequence and predicted secondary structure (Fig. 5.2). These studies led to consideration of two additional aspects of EKLF function. One was whether posttranslational modification plays a role in EKLF activity, as the minimal activation and inhibitory domains each contain conserved sites for serine/threonine phosphorylation (Ouyang et al., 1998). In fact, one of the double amino acid mutations (at aa 43—44) that disrupted the in vivo competitive ability of EKLF overlapped a casein kinase II site (threonine at aa 41). As expected, immunoprecipitation of EKLF from erythroid cells revealed that it is a phosphoprotein in vivo, exclusively at serine and threonine (no evidence was seen for modification of tyrosine). In addition, it is a suitable substrate for casein kinase II in vitro, and thr41 phosphorylation was disrupted when the double mutant was used as a substrate. Further, the double mutant, or a direct mutant of thr41, was not able to transactivate a reporter in vivo. This loss was not observed when the charge at aa 41 was retained (i.e., after conversion to aspartic acid). The location of this important phosphorylation site within the interaction domain of the minimal activation region raises the possibility that EKLF interactions and activity may be modulated by phosphorylation. The second aspect of EKLF function that was raised by the deletional studies was the importance of protein-protein interactions. Although searches for potential partners by yeast twohybrid approaches or by probing of an expression library were unsuccessful (Chen, Zhang, and Bieker, unpublished), the possibility that EKLF may also be involved in chromatin structure at the -globin locus led to directly testing whether it interacted with the class of molecules known as histone acetyltransferases (Zhang and Bieker, 1998). These are known to modify histones via

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Figure 5.2. Conserved structural properties of EKLF protein. A schematic diagram of EKLF is used to demonstrate its minimal activation, inhibitory, and DNA-binding subdomains. A portion of the minimal activation domain is also involved in interacting with a positive-acting factor. The extent of amino acid conservation (indicated by percentage) between the murine (m) and human (h) EKLF homologues is shown for each region, along with preserved isoelectric points (pI) and predicted structures. (Based on Chen and Bieker, 1996).

acetylation of the amino terminal tails, leading to a more open chromatin structure at that region, thus enabling the transcription machinery to be fully recruited and active (Pazin and Kadonaga, 1997; Wolffe and Pruss, 1996). The finding that some transcriptional coactivators (such as CBP, p300, and P/CAF) harbor intrinsic histone acetyltransferase activity provides a mechanism to directly link transcriptional activation and alteration of chromatin structure (Berger, 1999). In the present case (Zhang and Bieker, 1998), EKLF was found to associate with p300, CBP, and P/CAF in vivo. Interestingly, EKLF is itself acetylated by p300 and CBP in vitro, but not by P/CAF. This specificity is also manifested after in vivo activation of a -globin promoter by cotransfection in erythroid cells, whereby additional p300 or CBP can superactivate EKLF further, but P/CAF inhibits its activity. The histone acetyltransferase activity of the coactivator is required for this effect, and a lysine adjacent to the EKLF zinc fingers appears to be the primary target for generating optimal activity (Wenjun Zhang and James J. Bieker, unpublished).

Although many of these coactivators are themselves not tissue specific, their own regulation, and thus that of any downstream targets, may be combinatorially controlled in conjunction with other intracellular regulators as a result of an extracellular stimulus (e.g., that leads to kinase activation; Berger, 1999). The tissue-specific target, EKLF in this case, would determine specificity of effect by virtue of its unique transactivation domain that is required both at the -globin promoter (Bieker and Southwood, 1995) and at the LCR (Gillemans et al., 1998; see below) for optimal gene activation. As EKLF is acetylated in vivo, interesting questions arise as to the mechanism of how lysine modifications alter EKLF transcriptional activity and further protein interactions. ‘‘Pull-down’’ assays, where the ability of two proteins to associate with each other in vitro is monitored, have been used to map one of the interaction domains between these proteins to the amino terminus of EKLF and to the E1A-interaction region of CBP (Steven M. Jane and John M. Cunningham, manuscript submitted).

MECHANISM OF EKLF TRANSCRIPTIONAL ACTIVATION

CACCC and GATA elements appear together nonrandomly at a number of locations within erythroid promoters and enhancers. GATA1 is the founding member of a related family of zinc-binding transcription factors that binds to this element and is critical for primitive and definitive erythropoiesis (Weiss and Orkin, 1995). In addition, GATA1 and EKLF expression colocalize during all phases of erythroid development. Indeed, artificial placement of GATA and CACCC elements upstream of a minimal promoter leads to its synergistic activation in vivo after cotransfection of GATA1 and EKLF (Gregory et al., 1996; Merika and Orkin, 1995). In addition, these two proteins interact in vitro, likely via their zinc-finger structures. Together with the previous results that indicate the EKLF fingers are not required for its interaction with the CBP coactivator (Zhang and Bieker, 1998), these data suggest that EKLF can potentially use both its transactivation and DNAbinding regions for interactions with other proteins. An additional area in which EKLF-protein interactions may be playing an important role is in chromatin remodeling (Armstrong and Emerson, 1998; Kadonaga, 1998). Using an in vitro chromatin reconstitution/transcription system, EKLF and an MEL-derived cell extract (ERC1) were both required to provide an open chromatin structure and transcription at the -globin promoter (Armstrong et al., 1998). In the absence of either component, or by using a mutated EKLF-binding site, the chromatin structure remained closed and refractory to transcription. Use of an EKLF deletion that had removed its amino terminal interaction domain still bound DNA and yielded an open chromatin structure, but no transcription was observed, thus uncoupling the two events. Intriguingly, purification of E-RC1 identified mammalian components of the SWI-SNF chromatin remodeling proteins, including BRG1, BAF170, BAF155, BAF47, and BAF57. Immunoprecipitation or immunoneutralization of this complex from the MEL fraction again led to an inability to activate the promoter. Elegant application of the PIN*POINT assay (Lee et al., 1998) has provided additional evidence that BRG1 and BAF170 are recruited to the -globin promoter, and that BRG1 recruitment is de-

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pendent on the presence of at least two LCR hypersensitive sites and an intact promoter (Lee et al., 1999b). The PIN*POINT assay detects recruitment of a tagged protein to DNA in vivo. These studies, taken together, implicate EKLF as a central player in the integration of a variety of signals that lead to protein modification, protein-protein interactions, opening of chromatin, and activation of transcription (Fig. 5.3). There is ample precedent for each of the steps in this pathway from other systems, and a testable challenge for future studies will be to determine whether and how EKLF functions within this hypothetical cascade of events. In addition, the biological relevance of these studies has followed from studies that crossed the EKLF null mice with mice that are transgenic for the complete human -like globin locus (Wijgerde et al., 1996). These studies demonstrated that the hypersensitive site (HS) at the -globin promoter is absent when EKLF is not present. In addition, HS3 within the LCR was adversely affected. Although multiple Kru¨ppel family members are able to bind the HS3 CACCC element, the requirement for EKLF at this site was elegantly demonstrated by showing that only the altered DNA-binding specificity mutant of EKLF (but not Sp1) could function at the altered CACCC element at HS3 to reconstitute normal levels of transgenic -globin expression and -promoter hypersensitive site formation (Gillemans et al., 1998). Consistent with this, use of the aforementioned PIN*POINT assay demonstrates that, unlike the lack of Sp1 recruitment under any condition, EKLF recruitment to HS3 occurs only in the erythroid cell and is dependent on intact CACCC and TATA elements at the -promoter (Lee et al., 1999a). Finally, varying the levels of EKLF can positively modulate the position effect variegation on transcription seen in mice that contain an incomplete human -globin locus transgene (described in Grosveld, 1999). Thus, EKLF is not only required for transcription of -globin in vivo but it also plays a role in forming the correct higher-order structure at the globin locus. EKLF interactions with histone acetyltransferases and with chromatin remodeling proteins provide a mechanism to partially account for how this may occur.

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Figure 5.3. EKLF functions as an integrator of signals at the adult -globin locus: a speculative sequence of interactions. Posttranslational modifications and protein-protein interactions (as discussed in the text) are laid out in a proposed scheme, each step of which may provide a means to control EKLF activity, and thus globin transcription in the red cell. EKLF is a phosphoprotein; of particular interest is thr41, whose phosphorylation is critical for activity. This may enable association with histone acetyltransferases (e.g., p300 or CBP) in a fashion similar to CREB (Chrivia et al., 1993). The resulting acetylation of lysines near the zinc fingers (particularly lys 288) may further play a role in recruiting the large erythroid complex (ERC-1) that contains SWI/SNF chromatin remodeling proteins to the promoter (Bresnick et al., 1997). The result is that the closed chromatin structure is derepressed at the -globin promoter, leading to its transcription, all of which is dependent on EKLF binding to the CACCC element and activating via its minimal interaction domain.

REGULATION OF EKLF EXPRESSION As EKLF is itself highly erythroid cell specific, its own regulation has been investigated (Anderson et al., 1998; Chen et al., 1998; Crossley et al.,

1994). Both the murine and human genomic clones have been isolated, sequenced, and mapped (Jenkins et al., 1998; van Ree et al., 1997). A surprisingly short region of the promoter is sufficient to reconstitute erythroid specificity to

UNRESOLVED ISSUES AND FUTURE DIRECTIONS

an adjacent reporter (Crossley et al., 1994). Although there is no TATA box, the proximal promoter region contains GATA and CCAAT sites. The importance of these sites was verified by transfection assays in erythroid cells (MEL and 32DEpo1 lines), and in addition, cotransfection with GATA-1 was found to be required for promoter activity in a nonerythroid cell. As expected, GATA-1 binds this proximal element in vitro. More distal elements have been implicated by identifying erythroid-specific DNase hypersensitive sites at the EKLF genomic locus (Chen et al., 1998). Two sites within 1000 bp 5 of the transcription start site were identified by this approach. This segment was also able to direct tissue-specific expression in transgenic mice (Anderson et al., 1998). The sequence surrounding the more distal hypersensitive site not only is highly conserved with the human EKLF promoter but behaves as a strong enhancer element, even when placed adjacent to a nonerythroid promoter. However, erythroid specificity can be reestablished when this element is placed adjacent to the EKLF promoter, with the proximal GATA site proving critical for this enhancement (Chen et al., 1998). This same segment is important for reporter activity in both 32DEpo1 and MEL cells; still, the details have diverged in the two cell lines. Experiments in 32DEpo1 cells have directed attention to a 49 bp sequence within this conserved, hypersensitive site that contains the core sequence responsible for enhancement and contains three protein-binding sites in vitro (Chen et al., 1998). Experiments in MEL cells have concentrated on a GATA/Ebox/GATA motif (in an adjacent region that was removed in the above experiments) that is critical for expression and binds GATA-1 and another protein (Anderson et al., 1998). Identification of the cognate-binding proteins will be necessary to resolve this issue and help determine how the EKLF regulator is itself regulated. One way in which the requirement for potential regulators can be tested is to address whether EKLF is expressed in gene-targeted nullizygous embryos. By this criterion, one can argue that the SCL (stem cell leukemia) factor is likely to play a role, as EKLF levels are significantly lower in SCL null embryoid bodies (Elefanty et al., 1997). A more complicated case is whether GATA-1 is involved in EKLF regu-

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lation. GATA-1 null ES cells still give rise to EKLF expression after embryoid body differentiation (Weiss et al., 1994); however, any definitive conclusion is compromised by the fact that GATA-2 levels are upregulated 50-fold in these cells, raising a potential redundancy of effect, or (more likely) implicating GATA-2 rather than GATA-1 as an EKLF regulator. Use of the FDCW2 myeloid cell line to address this issue indicates that high-level expression of exogenous GATA-1 (via stable transfection) can activate the endogenous EKLF locus (Seshasayee et al., 1998). On the other hand, absence of FOG, a coactivator of GATA-1, has little effect on EKLF expression (Crispino et al., 1999). In the same context, it is also of interest to address how EKLF is initially induced during early development, and what are the signal transduction pathways that accomplish this. For example, EKLF is still expressed in erythropoietin receptor knockout embryos (Kieran et al., 1996; Lin et al., 1996). In any scenario, use of a pathway involving JAK2 kinase (by the erythropoietin or another receptor) is excluded by the fact that EKLF is expressed in the JAK2\\ embryo (Parganas et al., 1998). The linkage between an extracellular signal and the intracellular induction of EKLF transcription will be of significant interest to decipher.

UNRESOLVED ISSUES AND FUTURE DIRECTIONS A paradox that results from the expression and genetic studies follows from the observation that EKLF message is expressed in both primitive (yolk sac) and definitive (mouse fetal liver) erythroid cells, yet is functionally only required for definitive erythropoiesis. As EKLF protein is present in both populations, it has been proposed that posttranslational modification or changes in protein-protein interactions might yield functionally altered EKLF states at different stages in development. However, transgenic studies with EKLF null mice indicate that EKLF is transcriptionally active and essential for -globin transgene expression within primitive cells (Guy et al., 1998; Tewari et al., 1998), still leaving this issue unresolved. This conundrum extends even deeper, as the EKLF message is detected quite early in hematopoietic

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Figure 5.4. Summation of EKLF structure/function. Diagram of EKLF, demarcation of its zinc fingers and their relation to its predicted binding site, and location of its actual -globin CACCC element-binding site. The numbers below the protein scheme denote amino acids, while those above the -globin gene denote the base position upstream to the transcriptional start. A recapitulation of EKLF’s role in erythroid gene control as discussed in the text is outlined.

differentiation, since it is already transcribed both in multipotential cell lines (Hu et al., 1997; Reese et al., 1997) and in enriched primary progenitors (early differentiating CD34>LIN\cells) (Ziegler et al., 1999). Again, the reason why EKLF should be expressed so precociously when its requirement is considerably later in differentiation, and whether this is functionally important, remains to be resolved. Another issue of intense interest is whether there are any other downstream targets of EKLF besides -globin. This issue arises primarily because a large number of erythroid gene promoters contain CACCC elements that theoretically should interact with EKLF. The initial knockout studies demonstrated that, in addition to embryonic -like globins, there were normal

levels of GATA-1, erythropoietin receptor, and porphobilinogen deaminase (Perkins et al., 1995). A recent exhaustive analysis by nuclear run-on assays of 40 genes in an erythroid cell line in which EKLF had been removed by antisense strategies revealed that only coproporphyrinogen oxidase, 5-aminolevulinic acid synthase (ALA-S; erythroid), and ferrochetalase were significantly downregulated along with globin (Spadaccini et al., 1998). Consistent with this, EKLF can bind and transactivate the ALA-S promoter (Surinya et al., 1997). Although the DNA-binding activity of another Kru¨ppel-like factor named BKLF is diminished in the EKLF null red cell, it appears that the effect on BKLF is translational rather than at the transcriptional level (Crossley et al., 1996).

REFERENCES

On a related issue, the absence of effect of EKLF upon embryonic/fetal globin expression begs the question of the identity of the protein that binds to those CACCC elements, which are themselves important for promoter activity and are occupied in vivo. This is not a trivial question to answer, as there are now numerous highly EKLF-related proteins that have been identified, form their own separate subfamily, and bind to very similar sites (reviewed in Turner and Crossley, 1999). However, based on the assumption that the cognate protein must be similar in finger structure to EKLF, a novel candidate named FKLF has been identified that might fit the bill, as it is predominantly expressed in erythroid fetal liver cells and transcriptionally activates the embryonic and fetal -like globin promoters in preference to the adult -globin promoter (Asano et al., 1999) The importance of both the DNA-binding and transactivation modules of EKLF in regulating -globin transcription and chromatin structure has inspired approaches to take advantage of these structures to alter globin regulation. For example, changing the defective -globin CAC site to an optimal EKLF-binding CACCC element reactivates this gene to highlevel expression, presumably because it is now able to bind EKLF (Donze et al., 1996; Tang et al., 1997; Tang and Rodgers, 1998). This raises the possibility of performing the converse experiment; that is, modifying the EKLF fingers to bind the defective CAC site, thus providing a therapeutic agent that increases levels of globin expression in -thalassemic or sickle cell patients. Alternatively, changing the EKLF activation module to a transrepressor could alter the structure of the locus enough to silence -globin yet reactivate the -globin gene, thus achieving the same end (Deepa Manwani and James J. Bieker, unpublished). EKLF clearly plays a crucial role in the controlled events that lead to developmental regulation of the -like globin cluster (Fig. 5.4). As a result, structural and functional analysis of EKLF has enabled a molecular window to be opened on the process of -globin gene regulation. Each EKLF module plays an important part in its functional specificity. The zinc fingers discriminate among very similar CACCC elements, and the transactivation domain plays a critical role in transcriptional activation and in

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opening the surrounding chromatin. Proteinprotein interactions are pivotal for this process; in this context, it will be critical to determine whether posttranslational modification (phosphorylation and acetylation) of EKLF is regulated during hematopoiesis and/or during development and how this affects the efficiency of these interactions, and thus of the onset of -globin expression. Any tie-in of these changes to the action of extracellular effectors will be critical for understanding the complete transduction cascade involved in the onset of -like globin transcription during development.

ACKNOWLEDGMENTS Work in the author’s lab has been supported by grants from NIDDK, NHLBI, and the Cooley’s Anemia Foundation. JJB is a Scholar of the Leukemia Society of America.

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tein in erythroid cells and selected other cells. Mol. Cell. Biol. 16, 1695—1705. Donze, D., Townes, T. M., and Bieker, J. J. (1995). Role of erythroid Kru¨ppel-like factor (EKLF) in human - to -globin switching. J. Biol. Chem. 270, 1955—1959. Donze, D., Jeancake, P. H., and Townes, T. M. (1996). Activation of -globin gene expression by erythroid Kru¨ppel-like factor: a potential approach for gene therapy of sickle cell disease. Blood 88, 4051— 4057. Elefanty, A. G., Robb, L., Birner, R., and Begley, C. G. (1997). Hematopoietic-specific genes are not induced during in vitro differentiation of scl-null embryonic stem cells. Blood 90, 1435—1447. Enver, T., Raich, N., Ebens, A. J., Papayannopoulou, T., Costantini, F., and Stamatoyannopoulos, G. (1990). Developmental regulation of human fetalto-adult globin gene switching in transgenic mice. Nature 344, 309—313. Feng, W. C., Southwood, C. M., and Bieker, J. J. (1994). Analyses of -thalassemia mutant DNA interactions with erythroid Kru¨ppel-like factor (EKLF), an erythroid cell-specific transcription factor. J. Biol. Chem. 269, 1493—1500. Fraser, P., Gribnau, J., and Trimborn, T. (1998). Mechanisms of developmental regulation in globin loci. Curr. Opin. Hematol. 5, 139—144. Gaensler, K. M., Kitamura, M., and Kan, Y. W. (1993). Germ-line transmission and developmental regulation of a 150-kb yeast artificial chromosome containing the human beta-globin locus in transgenic mice. Proc. Natl. Acad. Sci USA 90, 11,381— 11,385. Gillemans, N., Tewari, R., Lindeboom, F., Rottier, R., de Wit, T., Wijgerde, M., Grosveld, F., and Philipsen, S. (1998). Altered DNA-binding specificity mutants of EKLF and Sp1 show that EKLF is an activator of the beta-globin locus control region in vivo. Genes Dev. 12, 2863—2873. Gregory, R. C., Taxman, D. J., Seshasayee, D., Kensinger, M. H., Bieker, J. J., and Wojchowski, D. M. (1996). Functional interaction of GATA-1 with erythroid Kru¨ppel-like factor and SP1 at defined erythroid promoters. Blood 87, 1793—1801. Grosveld, F. (1999). Activation by locus control regions? Curr. Opin. Genet. Develop. 9, 152—157. Grosveld, F., Assendelft, G. B. v., Greaves, D. R., and Kollias, B. (1987). Position-independent, high-level expression of the human -globin gene in transgenic mice. Cell 51, 975—985. Guy, L. G., Mei, Q., Perkins, A. C., Orkin, S. H., and Wall, L. (1998). Erythroid Kru¨ppel-like factor is essential for beta-globin gene expression even in

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Perutz, M. F. (1987). Molecular anatomy, physiology, and pathology of hemoglobin. In The Molecular Bases of Blood Diseases, G. Stamatoyannopoulos, A. W. Nienhuis, P. Leder, and P. W. Majerus, eds. (Philadelphia: W. B. Saunders Co.). Peterson, K. R., Clegg, C. H., Huxley, C., Josephson, B. M., Haugen, H. S., Furukawa, T., and Stamatoyannopoulos, G. (1993). Transgenic mice containing a 248-kb yeast artificial chromosome carrying the human beta-globin locus display proper developmental control of human globin genes. Proc. Natl. Acad. Sci. USA 90, 7593—7597. Reese, T. T., Gregory, R. C., Sharlow, E. R., Pacifici, R. E., Crouse, J. A., Todokoro, K., and Wojchowski, D. M. (1997). Epo-induced hemoglobinization of SKT6 cells is mediated by minimal cytoplasmic domains of the Epo or prolactin receptors without modulation of GATA-1 or EKLF. Growth Factors 14, 161—176. Schuh, R., Aicher, W., Gaul, U., Ct, S., Preiss, A., Maier, D., Seifert, E., Nauber, U., Schro¨der, C., Kemler, R., and Ja¨ckle, H. (1986). A conserved family of nuclear proteins containing structural elements of the finger protein encoded by Kru¨ppel, a Drosophila segmentation gene. Cell 47, 1025— 1032. Seshasayee, D., Gaines, P., and Wojchowski, D. M. (1998). GATA-1 dominantly activates a program of erythroid gene expression in factor-dependent myeloid FDCW2 cells. Mol. Cell. Biol 18, 3278— 3288. Southwood, C. M., Downs, K. M., and Bieker, J. J. (1996). Erythroid Kru¨ppel-like factor (EKLF) exhibits an early and sequentially localized pattern of expression during mammalian erythroid ontogeny. Dev. Dyn. 206, 248—259. Spadaccini, A., Tilbrook, P. A., Sarna, M. K., Crossley, M., Bieker, J. J., and Klinken, S. P. (1998). Transcription factor erythroid Kru¨ppel-like factor (EKLF) is essential for the erythropoietin-induced hemoglobin production but not for proliferation, viability, or morphological maturation. J. Biol. Chem. 273, 23,793—23,798. Stamatoyannopoulos, G., and Nienhuis, A. W. (1994). Hemoglobin switching. In The Molecular Bases of Blood Diseases, G. Stamatoyannopoulos, A. W. Nienhuis, P. W. Majerus, and H. Varmus, eds. (Philadelphia: W. B. Saunders Co.), pp. 107—155. Strouboulis, J., Dillon, N., and Grosveld, F. (1992). Developmental regulation of a complete 70-kb human beta-globin locus in transgenic mice. Genes Dev. 6, 1857—1864. Surinya, K. H., Cox, T. C., and May, B. K. (1997). Transcriptional regulation of the human erythroid 5-aminolevulinate synthase gene. Identification of

promoter elements and role of regulatory proteins. J. Biol. Chem. 272, 26,585—26,594. Tang, D. C., and Rodgers, G. P. (1998). Activation of the human delta-globin gene promoter in primary adult erythroid cells. Br. J. Haematol. 103, 835— 838. Tang, D. C., Ebb, D., Hardison, R. C., and Rodgers, G. P. (1997). Restoration of the CCAAT box or insertion of the CACCC motif activate -globin gene expression. Blood 90, 421—427. Tewari, R., Gillemans, N., Wijgerde, M., Nuez, B., von Lindern, M., Grosveld, F., and Philipsen, S. (1998). Erythroid Kru¨ppel-like factor (EKLF) is active in primitive and definitive erythroid cells and is required for the function of 5HS3 of the beta-globin locus control region. EMBO J. 17, 2334—2341. Turner, J., and Crossley, M. (1999). Mammalian Kru¨ppel-like transcription factors: more than just a pretty finger [In Process Citation]. Trends Biochem. Sci. 24, 236—240. van Ree, J. H., Roskrow, M. A., Becher, A. M., McNall, R., Valentine, V. A., Jane, S. M., and Cunningham, J. M. (1997). The human erythroidspecific transcription factor EKLF localizes to chromosome 19p13.12-p13.13. Genomics 39, 393— 395. Weiss, M. J., Keller, G., and Orkin, S. H. (1994). Novel insights into erythroid development revealed through in vitro differentiation of GATA-1\ embryonic stem cells. Genes Dev. 8, 1184—1197. Weiss, M. J., and Orkin, S. H. (1995). GATA transcription factors: key regulators of hematopoiesis. Exp. Hematol. 23, 99—107. Wijgerde, M., Grosveld, F., and Fraser, P. (1995). Transcription complex stability and chromatin dynamics in vivo. Nature 377, 209—213. Wijgerde, M., Gribnau, J., Trimborn, T., Nuez, B., Philipsen, S., Grosveld, F., and Fraser, P. (1996). The role of EKLF in human -globin gene competition. Genes Dev. 10, 2894—2902. Wolffe, A. P., and Pruss, D. (1996). Targeting chromatin disruption: transcription regulators that acetylate histones. Cell 84, 817—819. Zhang, W., and Bieker, J. J. (1998). Acetylation and modulation of erythroid Kru¨ppel-like factor (EKLF) activity by interaction with histone acetyltransferases. Proc. Natl. Acad. Sci. USA 95, 9855— 9860. Ziegler, B. L., Muller, R., Valtieri, M., Lamping, C. P., Thomas, C. A., Gabbianelli, M., Giesert, C., Buhring, H. J., Kanz, L., and Peschle, C. (1999). Unicellular-unilineage erythropoietic cultures: molecular analysis of regulatory gene expression at sibling cell level. Blood 93, 3355—3368.

PART II


TRANSCRIPTION FACTORS AND THE MYELOID LINEAGE

CHAPTER 6


RUNX1(AML1) AND CBFB: GENES REQUIRED FOR THE DEVELOPMENT OF ALL DEFINITIVE HEMATOPOIETIC LINEAGES NANCY A. SPECK Department of Biochemistry, Dartmouth Medical School

ELAINE DZIERZAK Department of Cell Biology and Genetics, Erasmus University, The Netherlands

INTRODUCTION The core-binding factors (CBFs), which are also known as the polyomavirus enhancer binding protein 2 (PEBP2), constitute a small family of transcription factors that participate in multiple developmental processes in flies and mammals. CBFs are heterodimers consisting of a DNAbinding subunit (CBF) and a non-DNA-binding CBF subunit (Fig. 6.1) (Kamachi et al., 1990; Ogawa et al., 1993a, 1993b, Wang et al., 1993). CBF subunits by themselves can bind DNA in vitro, but their affinity for DNA is enhanced by the CBF subunit (Ogawa et al., 1993a; Wang et al., 1993). Two genes encoding CBF subunits (Runt and Lozenge) and two genes encoding CBF subunits (Brother and Big Brother) have been found in Drosophila. Mice and humans have three genes for CBF subunits

and one for CBF. Homologues have also been isolated from Caenorhadbitis elegans, sea urchins, chicken, and amphibians, but not from yeast (Castagnola et al., 1996; Coffman et al., 1996; Tracey et al., 1998). The gene nomenclature in mammals is somewhat confusing, with two or three names for each gene currently in use. Figure 6.1 lists the names of the mammalian genes. We use the official HUGO names RUNX1, RUNX2, and RUNX3 for the human CBF genes, and CBFB for the gene encoding the CBF subunit in this review. The corresponding mouse genes are Runx1, Runx2, Runx3, and Cbf b. We refer to the proteins in both species as Runx1, Runx2, Runx3, and CBF (Fig.6.2). The mammalian CBFs were originally identified as proteins regulating transcription of the mouse polyomavirus and the pathogenesis of


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RUNX1(AML1) AND CBFB

Figure 6.1. Subunit composition and genes. The CBF subunits alone can bind DNA in vitro. The CBF subunit heterodimerizes with CBF subunits on and off DNA, but does not contact additional bases or phosphates on the DNA molecule (Ogawa et al., 1993a; Wang et al., 1993). It has been proposed that CBF increases the affinity of the CBF subunit for DNA by stabilizing a high-affinity DNA-binding conformation of the DNA-binding domain (Berardi et al., 1999; Nagata et al., 1999). Gene names approved by the Mouse and HUGO nomenclature committees are shown, as are other names that commonly appear in the literature. RUNX2 is required for bone formation (Komori et al., 1997; Otto et al., 1997), and RUNX1 and CBFB for definitive hematopoiesis (Niki et al., 1997; Okuda et al., 1996; Sasaki et al., 1996; Wang et al., 1996a, 1996b). The function of RUNX3 is unknown.

murine leukemia viruses (Boral et al., 1989; Hallberg et al., 1991; Piette and Yaniv, 1987; Satake et al., 1988; Speck et al., 1990; Thornell et al., 1988). CBF-binding sites were also found in a number of genes transcribed in hematopoietic cells (Cameron et al., 1994; Giese et al., 1995; Hallberg et al., 1992; Hsiang et al., 1993; Nuchprayoon et al., 1994; Prosser et al., 1992; Redondo et al., 1992; Thornell et al., 1991; Wang and Speck, 1992; Zhang et al., 1994). CBF and subunits copurified (Kamachi et al., 1990; Wang and Speck, 1992), and genes encoding both subunits were cloned based on partial amino acid sequences (Ogawa et al., 1993a, 1993b; Wang et al., 1993). The human RUNX1 and CBFB genes were independently identified as commonly rearranged genes in human leukemias. RUNX1 (more commonly known as the acute myeloid leukemia 1, or AML1 gene) is disrupted by at least nine different chromosomal translocations associated with specific human leukemias (see Chapters 22, 23, and 24 in this book). RUNX1/ AML 1 was first identified as the gene at the breakpoint of chromosome 21 in the t(8;21)(q22;q22) found in the leukemic cells of approximately 40% of patients with the M2 subtype of acute myeloid leukemia (AML) (Bitter, 1987; Miyoshi, 1991). Later it was found that the t(12;21)(p13;q22), which occurs in 25%

of patients with childhood pre-B acute lymphocytic leukemia (ALL), also disrupts the RUNX1 gene (Golub et al., 1995 McLean et al., 1996; Romana et al., 1995a, 1995b; Shurtleff et al., 1995). More rare cytogenetic lesions involving RUNX1, including the t(1; 21) (p36; q22), t(3; 21) (q26; q22), t(5; 21) (q13; q22), t(12; 21) (q24; q22), t(14; 21) (q22; q22), t(15; 21) (q22; q22), t(16; 21) (q24; q22), and t(17; 21) (q11.2; q22), are found in tumor cells from patients with therapy-related myelodysplastic syndrome, therapy-related acute myeloid leukemia, and chronic myeloid leukemia in blast crisis (Gamou et al., 1998; Nucifora et al., 1993, 1994; Roulston et al., 1998). Biallelic nonsense mutations in RUNX1 have been identified in the most immature AMLs of the M0 subtype (Osato et al., 1999). Mutations in one copy of the RUNX1 gene cause a rare inherited condition called familial platelet disorder with predisposition to acute myelogenous leukemia (FPD/AML) (Song et al., 1999). The CBFB gene was cloned by virtue of its rearrangement by the inv(16)(p13;q22), t(16;16), and del(16)(q22) in all cases of acute myeloid leukemias of the M4 subtype with eosinophilia (Liu et al., 1993; (see also Chapter 21 in this book). The functional consequences of these CBF mutations are described in more detail in other chapters in this

INTRODUCTION

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Figure 6.2. The CBF and subunits. A: Shown is a transcriptionally active protein isoform from each of the mammalian RUNX genes and the Drosophila runt and lozenge genes. All CBF subunits share a 128 amino acid region of homology named the ‘‘Runt domain’’ (pink) after the founding member of the CBF family, the Drosophila Runt protein (Kagoshima et al., 1993). The Runt domain is responsible both for binding DNA and for heterodimerizing with the CBF subunit (Kagoshima et al., 1993; Meyers et al., 1993; Ogawa et al., 1993b). The Runt domain assumes a classic immunoglobulin (s-type) fold, consisting of seven -strands organized as two antiparallel -sheets, with the connections between the -strands provided by loop regions. The fold is similar to that of the DNA binding domains of NFAT, p53, STAT, and NF-B (Berardi et al., 1999; Nagata et al., 1999). A conserved C-terminal VWRPY sequence is shown in black. Stretches of glutamines (Q) and alanines (A) in the Runt, Lozenge, and Runx2 proteins are indicated. B: Functional domains in the Runx1 protein. Shown are context-dependent transactivation domains (Hiebert et al., 1996; Kanno et al., 1998a; Kitabayashi et al., 1998b; Zeng et al., 1997, 1998), interaction sites for transcriptional adapters (p300, ALY, mSinA, TLE, Ear2, YAP) (Ahn et al., 1998; Bruhn et al., 1997; Kitabayashi et al., 1998b; Levanon et al., 1998; Lutterbach et al., 1998; Yagi et al., 1999), nuclear matrix attachment sequences (Kanno et al., 1998a; Zeng et al., 1997), and sequences required for stimulation of mouse polyomavirus DNA replication (Chen et al., 1998). Arrows show positions where chromosomal translocations disrupt the protein sequence. C: A functional isoform of the mammalian CBF subunit. The heterodimerization domain (green) is contained within the N-terminal 135 amino acids (Kagoshima et al., 1996) and consists of four -helices and two 3-stranded antiparallel -sheets that combine to form a -sandwich (Goger et al., 1999; Huang et al., 1999). The heterodimerization itself appears to be sufficient for CBF function in vivo (Kanno et al., 1998b; Miller et al., 2000).

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book. This chapter focuses on RUNX1 and CBFB function in normal hematopoiesis.

Runx1, Cbfb, AND THE EMERGENCE OF DEFINITIVE HEMATOPOIETIC CELLS Gene disruption experiments in mice point to a crucial role for Runx1 and Cbfb in definitive hematopoiesis (Niki et al., 1997; Okuda et al., 1996; Sasaki et al., 1996; Wang et al., 1996a, 1996b). Recent evidence suggests that the earliest requirement for Runx1 and Cbfb may be for the emergence of the first definitive hematopoietic progenitors and stem cells in the embryo (North et al., 1999). A brief review of embryonic hematopoiesis is included here, but for a more complete description we refer the reader to Chapter 00. Hematopoiesis in the mouse embryo initiates in the yolk sac, at about 7.0 days postcoitus (dpc), and produces a burst of primitive nucleated erythrocytes (Haar and Ackerman, 1971; Moore and Metcalf, 1970). Primitive erythrocytes are thought to differentiate from a bipotential precursor called the hemangioblast,

that gives rise both to hematopoietic and endothelial cells (Murray, 1932; Sabin, 1920). Some of the genes required for primitive hematopoiesis include Flk1, which encodes a tyrosine kinase receptor (Shalaby et al., 1995, 1997), and the transcription factors Gata1, Fog, Scl, and Rbtn2 (Pevny et al., 1991; Robb et al., 1995; Shivdasani et al., 1995; Tsang et al., 1997; Warren et al., 1994). Flk1 is also necessary for the formation of endothelial cells in the embryo, and thus is thought to be required at the level of the hemangioblast. The second wave of hematopoiesis, often referred to as definitive hematopoiesis, produces enucleated definitive erythrocytes, myeloid cells, and lymphocytes. Progenitors for these cells begin to accumulate in the fetal liver starting at about 10.0 dpc, and by 12.5 dpc enucleated definitive erythrocytes and myeloid elements can be found in peripheral blood. The progenitors and stem cells for the definitive lineages are thought to migrate to the fetal liver from several sites in the embryo, including the yolk sac, a caudal part of the embryo called the para-aortic splanchnopleure (PAS), the vitelline and umbilical arteries, and the aorta/gonad/mesonephros

Figure 6.3. Hematopoietic sites in the early mouse embryo. Shown is a diagram of a 10.5 dpc mouse embryo. Definitive hematopoietic progenitors can be found in the yolk sac (ys), vitelline (v) and umbilical (u) arteries, the AGM (agm) region, and the fetal liver (fl). The umbilical artery connects the dorsal aorta to the placenta (pl), and the vitelline artery connects the dorsal aorta to the yolk sac. Shown in blue are the hematopoietic sites where Runx1 is expressed (North et al., 1999). Expression in the AGM region is confined to the ventral endothelium and para-aortic mesenchyme.

RUNX1, CBFB, AND THE EMERGENCE OF DEFINITIVE HEMATOPOIETIC CELLS

(AGM) region (Fig 6.3) (Dieterlen-Lie`vre and Martin, 1981; Eren et al., 1987; Godin et al., 1993, 1995; Medvinsky and Dzierzak, 1996; Medvinsky et al., 1993; Moore and Metcalf, 1970; Mu¨ller et al., 1994; Ogawa et al., 1988; Tavian et al., 1996; Toles et al., 1989; Weissman et al., 1977; Yoder et al., 1997). The PAS, which in mouse embryos forms at approximately 7.5— 8.5 dpc, includes the splanchnic mesoderm, endothelial cells of the vitelline artery and paired dorsal aortae, and the gut endoderm. The AGM corresponds to approximately the same region at later developmental stages (9.0—12.5 dpc), and includes the urogenital system (genital ridge and mesonephros) and dorsal aorta. Definitive hematopoietic progenitors in the yolk sac and in the PAS at 7.5 dpc are able to undergo differentiation into enucleated erythroid and myeloid cells (yolk sac), or erythroid, myeloid, and lymphoid cells (PAS) in vitro (Cumano et al., 1996; Godin et al., 1993; Moore and Metcalf, 1970). These progenitors cannot, however, successfully replace all the definitive hematopoietic lineages when injected into lethally irradiated adult mice. At 9.0 dpc, colony-forming units— spleen (CFU-S) progenitors, able to form macroscopic spleen colonies containing erythroid and myeloid cells upon injection into irradiated adult recipient mice, appear in the yolk sac and AGM region (Medvinsky and Dzierzak, 1996; Medvinsky et al., 1993, 1996). Long-term repopulating hematopoietic stem cells (LTR-HSCs) capable of colonizing the adult bone marrow and providing long-term reconstitution of the entire adult hematopoietic system do not appear until 10.0—10.5 dpc in the vitelline and umbilical arteries, and in the AGM region (de Bruijn et al., 2000; Medvinsky and Dzierzak, 1996; Mu¨ ller et al., 1994). By 11 dpc, LTR-HSCs can be isolated from the AGM region, the umbilical and vitelline arteries, fetal liver, and yolk sac (de Bruijn et al., 2000; Mu¨ ller et al., 1994). Since hematopoietic cells can circulate freely between these sites and may also migrate interstitially, the issue of whether LTRHSCs originate independently in all these sites, or whether they emerge in some sites and seed others, is still under debate. It is clear, however, that LTR-HSCs can autonomously emerge from the AGM region (Medvinsky and Dzierzak, 1996). Homozygous disruption of Runx1 or Cbfb results in identical defects in developing mice,

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including midgestation embryonic lethality between 12.5 and 13.5 dpc, and a profound block in definitive hematopoiesis (Niki et al., 1997; Okuda et al., 1996; Sasaki et al., 1996; Wang et al., 1996a, 1996b). Histological analyses of fetal livers from Runx1- or Cbfb-deficient mice reveal no definitive hematopoietic elements such as erythroblasts or megakaryocytes. In vitro colony-forming units—culture (CFU-C) assays demonstrate a virtual absence of definitive hematopoietic progenitors in either fetal liver or yolk sacs of Runx1- or Cbf b-deficient embryos. Runx1- and Cbf b-deficient ES cells are also incapable of giving rise to definitive erythroid and myeloid cells when cultured in vitro (Miller et al., 2000; Okuda et al., 1996; Wang et al., 1996a). Chimeric animals made from Runx1- or Cbf b deficient ES cells and wild-type mouse blastocysts contain no ES-derived cells in adult hematopoietic tissues (Okuda et al., 1996; Wang et al., 1996b). In contrast, primitive erythropoiesis in the yolk sac appears to be normal. The data allow us to draw several conclusions. First, definitive hematopoiesis requires the Runx1 and Cbf b genes. Runx1 and Cbf b thus join a small group of genes (1-integrin, c-myb, c-kit, and T ie2) that upon mutation cause widespread defects in definitive hematopoiesis but do not affect primitive erythropoiesis (Geissler et al., 1988; Hirsch et al., 1996; Mucenski et al., 1991; Takakura et al., 1998). These genes are thought to be required at the level of definitive hematopoietic progenitors or stem cells, since all definitive hematopoietic lineages are affected by mutations in these genes. Second, Runx1 and Cbf b are required in a cell autonomous fashion, as cells deficient in these genes are incapable of undergoing hematopoiesis in the normal microenvironment of a chimeric mouse. Third, the identical developmental defects associated with mutation of Runx1 and Cbf b indicate that both the and subunits of this CBF complex are essential for its function in hematopoiesis. Recent studies suggest that Runx1 (and by extension probably Cbf b) is required for the differentiation of definitive hematopoietic stem and/or progenitor cells from endothelial cells in the midgestation embryo (North et al., 1999). Runx1 is expressed by 8.5 dpc in endothelial cells in all sites where definitive hematopoietic cells emerge, including endothelial cells in the

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yolk sac, in the vitelline and umbilical arteries, and in the ventral portion (floor) of the dorsal aorta within the AGM region (North et al., 1999). Runx1 is also expressed in the mesenchyme ventral to the dorsal aorta in the AGM region. Runx1 is not, however, expressed in endothelial cells in the roof of the dorsal aorta, or in endothelial cells elsewhere in the embryo. Shortly after endothelial cells expressing Runx1 appear, clusters of hematopoietic cells expressing Runx1 can be found closely associated with the endothelium. Intra-aortic hematopoietic clusters in the dorsal aorta, vitelline and umbilical arteries have been documented in many species, including chicks, mice, and humans (Dieterlen-Lie`vre and Martin, 1981; Garcia-Porrero et al., 1995; Tavian et al., 1996). In chick embryos, it was shown that cells in the clusters lining the floor of the dorsal aorta lose expression of the endothelial marker Flk1 and acquire the pan leukocyte marker CD45 (Jaffredo et al., 1998). The appearance of intra-aortic hematopoietic clusters coincides with the emergence of definitive hematopoietic activity in these regions. Therefore, these clusters are believed to represent the newly emerging definitive hematopoietic progenitors and stem cells. More recent lineage tracing experiments suggest a precursor/ progeny relationship between endothelial cells in the floor of the dorsal aorta and the intraaortic hematopoietic clusters. Endothelial cells in chick embryos marked by uptake of lowdensity lipoproteins coupled to the fluorescent label DiI (LDL-DiI) give rise 1 day later to LDL-DiI—labeled intra-aortic hematopoietic clusters (Jaffredo et al., 1998). In a different approach, Nishikawa and colleagues (Nishikawa et al., 1998) demonstrated that VE-cadherin positive endothelial cells isolated from the caudal region of 8.5—10.5 dpc mouse embryos can give rise to B- and T-lymphoid cells when appropriately cultured in vitro. Taken together, the data suggest that at least some definitive hematopoietic progenitors and/or stem cells differentiate from endothelial cells. Although disruption of the Runx1 gene does not appear to impair the formation or integrity of the arteries in which it is expressed, it does impair formation of the intra-aortic hematopoietic clusters (North et al., 1999). Further-

more, Runx1 expression is initiated but fails to be maintained in endothelial cells in the vitelline and umbilical arteries, the yolk sac or the dorsal aorta in Runx1-deficient embryos (North et al., 1999). Thus, Runx1 appears to be required for the formation of intra-aortic hematopoietic clusters and for maintaining its own expression in endothelial cells. Runx1 may act to specify endothelial cells committed to a hematopoietic cell fate in the developing embryo. A Xenopus Runx1 homologue called Xaml has also been cloned and its expression pattern has been analyzed (Tracey et al., 1998). Xaml is expressed in the ventral blood islands of Xenopus embryos starting at stage 14, in a pattern anticipating that of later -globin gene expression. Injection of mRNA encoding a transdominant negative form of Xaml into fourcell-stage embryos inhibits primitive erythropoiesis, a result not predicted from the mouse data. It is possible that the requirement for Runx1 in primitive hematopoiesis differs in frogs and mice. Alternatively, overexpression of a transdominant negative Xaml protein may inhibit primitive erythropoiesis by affecting the activity of another protein or proteins required for primitive erythropoiesis.

EXPRESSION OF Runx1 AND Cbf b IN THE DEFINITIVE HEMATOPOIETIC LINEAGES Runx1 is expressed in the putative endothelial cell precursors of definitive hematopoietic cells (North et al., 1999) and continues to be expressed in many hematopoietic lineages (Corsetti and Calabi, 1997). FACS analysis documented Runx1 expression in 90% of ckit>CD34> fetal liver cells and in 90% of ckit>CD34> bone marrow cells (North et al., 1999). Definitive hematopoietic progenitors and LTR-HSCs are greatly enriched in the ckit>CD34> population of fetal liver cells (Sanchez et al., 1996); therefore, it is likely that Runx1 is expressed in definitive progenitors and LTR-HSCs. In fact, preliminary evidence indicates that the CFU-S activity in the E12.5 fetal liver resides in the population of cells expressing Runx1 (Speck et al., 1999). Runx1 expression has been detected by in situ hybridization in fetal liver, thymus, and spleen (Satake et al.,

GENE DOSAGE EFFECTS

1995; Simeone et al., 1995). Electrophoretic mobility shift assays combined with antibodies specific for Runx1 detected activity in nuclear extracts prepared from T cells, B cells, myeloid cells, thymus, and spleen (Meyers et al., 1996). Reverse transcriptase—polymerase chain reaction (RT-PCR) analysis also detected Runx1 expression in purified platelets (Song et al., 1999). Thus far, the only hematopoietic cells in which Runx1 expression does not appear to be maintained are erythroid cells. Runx1 is expressed in primitive erythroid cells as they emerge in the extraembryonic yolk sac, but expression in primitive erythrocytes declines significantly by 8.5 dpc in mice, and disappears entirely by 10.5 dpc (North et al., 1999). Expression in definitive erythrocytes in adult bone marrow is reported to largely decline by the TER119> erythroblast stage (Corsetti and Calabi, 1997). No direct target genes for Runx1 have been identified that are specifically expressed in the erythroid lineage in either mice or humans. Runx1 is expressed in many other tissues in developing mouse embryos, including (but not limited to) chondrocytes, external genitalia, the Mu¨llerian duct, olfactory epithelia, spinal ganglia, maxillary processes, a subset of CNS neurons, and denervated muscle (Castagnola et al., 1996; North et al., 1999; Simeone et al., 1995; Zhu et al., 1994). There is no evidence that Runx1 is required for the development of any of these tissues. However, the fact that the Drosophila genes runt and lozenge each play critical roles in multiple developmental processes hints that additional roles may exist for Runx1 as well. For example, runt functions in sex determination, segmentation, and neurogenesis (Duffy and Gergen, 1991; Duffy et al., 1991; Ingham and Gergen, 1988; Sanchez and Nothiger, 1983). lozenge is involved in the development of the eye, antenna, tarsal claws, and crystal cells, a blood cell lineage (Daga et al., 1996; Rizki et al., 1985; Stocker and Gendre, 1988). Experiments designed to remove Runx1 function in specific cells at later developmental stages may also unveil new roles for Runx1 in vivo. The Cbf b gene appears to be more broadly expressed than Runx1. Northern analyses detected abundant levels of mRNA in all tissues tested (Ogawa et al., 1993a; Wang et al., 1993).

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In situ hybridization in mice indicates that Cbf b expression is not uniform, however, and some tissues express the gene at significantly higher levels than others (Wang et al., 1996b). The same is true in Drosophila; the Cbf b homologues Brother and Big Brother are expressed at high levels in many tissues, but the expression patterns are not uniform and they change during development (Golling et al., 1996).

GENE DOSAGE EFFECTS Studies in mammals and Drosophila have documented dominant effects associated with reduced Runx gene dosage. Drosophila embryos hemizygous for amorphic and strong hypomorphic runt alleles display partially penetrant segmentation defects (Gergen and Wieschaus, 1986). Hemizygosity for mutant RUNX2 alleles is associated with inherited skeletal abnormalities in both mice and humans called cleidocranial dysplasia (Mundlos et al., 1997; Otto et al., 1997). These dominant gene dosage effects indicate that the levels of CBF subunits are limiting in these developmental contexts. Haploinsufficiency of RUNX1 in humans causes a rare autosomal-dominant disorder characterized by qualitative and quantitative platelet defects, and by the propensity to develop acute myelogenous leukemia (FPD/AML) (Song et al., 1999). Six FPD/AML pedigrees were analyzed; one contained an intragenic deletion of the RUNX1 gene, and the other five contained either nonsense or missense mutations predicted to inactivate DNA binding by the Runx1 protein. CFU-C assays on adult bone marrow and peripheral blood uncovered a marked defect in megakaryocyte progenitor numbers (CFU-Meg), as well as a decrease in the size of colonies formed from those progenitors. Decreases in CFU-GM and BFU-E progenitor numbers were also noted (Song et al., 1999). Thus, the correct RUNX1 dosage is critical for normal hematopoiesis in humans. In the mouse, mutation of one copy of the Runx1 gene causes a 2-fold decrease in the number of CFU-C progenitors during embryonic development, in both fetal livers and yolk sacs (Wang et al., 1996a, 1996b). Mutation of one copy of the Cbf b gene, on the other hand,

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does not decrease the number of definitive hematopoietic progenitor cells in fetal liver or yolk sac, indicating that the concentration of CBF is saturating in definitive hematopoietic progenitor cells in the embryo (Wang et al., 1996b). In contrast, no effect of Runx1 hemizygosity was detected in adult mice (Gu et al., 1999). Similar numbers of CFU-C progenitors were found in bone marrows of wild-type and Runx1>\ mice, although the numbers of platelets and white blood cells in peripheral blood is slightly lower (Gu et al., 1999). The loss of one functional copy of Runx1 may cause more than a 2-fold decrease in the cellular concentration of Runx1 if transcription is autoregulated. Suggestive evidence for a positive feedback loop has been obtained both for Runx1 and Runx2. Maintenance of Runx1 expression in putative hemogenic endothelial cells appears to require a functional Runx1 gene product (North et al., 1999). Runx2 expression in bone marrow is downregulated in 2-week-old mice transgenic for a dominant negative Runx2 gene (Ducy et al., 1999). It is not known whether the Runx1 and Runx2 genes are directly or indirectly regulated by their own protein products or by other CBF family members. However, the presence of CBFbinding sites in the promoters of both genes suggests that direct regulation is a plausible mechanism (Ducy et al., 1999; Ghozi et al., 1996).

POTENTIAL ROLES FOR RUNX1 AND CBFB IN LATER STAGES OF HEMATOPOIESIS Conventional gene disruption experiments can pinpoint the first requirement for a gene during development but may not address that gene’s role at later developmental stages. However, numerous findings hint at roles for CBF in later stages of hematopoiesis. The thrombocytopenia associated with Runx1 haploinsufficiency clearly documents a requirement for Runx1 in megakaryocyte development (Song et al., 1999). Additional evidence is the extensive involvement of the RUNX1 and CBFB genes in human leukemias. Rearrangements of the RUNX1 and CBFB genes are associated with acute myelogenous and acute lymphocytic (pre-B) leukemias and with therapy-related myelodysplasias and leukemias. The translocations that disrupt

RUNX1 and CBFB in leukemias create chimeric proteins that appear to behave as transdominant negative inhibitors of CBF function. This was most convincingly demonstrated for the AML1-ETO and CBF-SMMHC proteins, which are produced as a result of the t(8;21) and inv(16), respectively. The AML1-ETO protein contains the N-terminus of AML1 (Runx1) fused to a protein called ETO, which is thought to function as a transcriptional repressor (Erickson et al., 1992; Lenny et al., 1995; Meyers et al., 1995; Miyoshi et al., 1993; Nisson et al., 1992) (see Chapter 23 in this book). CBF-SMMHC contains most of the CBF protein fused to the coiled-coil tail region of a smooth muscle myosin heavy chain (Liu et al., 1993) (see Chapter 21 in this book). Mouse embryos heterozygous for ‘‘knocked-in’’ genes encoding either the AML1-ETO or CBF-SMMHC proteins (with one remaining wild type Runx1 or Cbf b allele) exhibit severely impaired definitive hematopoiesis, similar to that seen upon homozygous disruption of Runx1 and Cbf b (Castilla et al., 1996; Okuda et al., 1998; Yergeau et al., 1997). Definitive hematopoiesis is not, however, completely blocked, and some definitive hematopoietic progenitors emerge that have abnormally high self-renewal capacity and impaired differentiation potential (Castilla et al., 1999; Miller et al., 2000; Okuda et al., 1998). The ability to escape the block in stem cell emergence but to then exhibit other hematopoietic defects suggests that RUNX1 and CBFB are required at later stages of hematopoiesis. Consistent with this belief is the observation that when AML1-ETO is introduced into bone marrow or established myeloid cell lines, it impairs the differentiation potential of these cells. Infection of adult bone marrow with retroviruses expressing AML1-ETO resulted in the generation of immature hematopoietic colonyforming progenitors with a high self-renewal capacity and the ability to readily establish cell lines in vitro (Okuda et al., 1998). Overexpression of the AML1-ETO protein in myeloid progenitor cell lines such as L-G or 32Dc13 blocked their differentiation into granulocytes or neutrophils in response to G-CSF (Ahn et al., 1998; Kitabayashi et al., 1998a). Together with the association of the t(8;21) and inv(16) with leukemias of the myeloid lineage, the data strongly suggest that RUNX1 and CBFB are


required for normal myeloid cell growth and/or differentiation. Several genes specifically expressed in myeloid cells, including the CSF-1 receptor, myeloperoxidase, and neutrophil elastase, contain binding sites for CBF that are important for their transcription (Nuchprayoon et al., 1994; Zhang et al., 1994). Several lines of evidence also point to a role for CBFs in lymphocyte development. CBFbinding sites have been found in a number of genes specifically expressed in lymphoid cells, including genes encoding all the T-cell receptor chains (, , , ), CD3, granzyme B serine protease, IL-3, and the B-cell immunoglobulin heavy chain, and in most cases mutation of these sites had severe effects on transcription in vivo (Cameron et al., 1994; Erman et al., 1998; Giese et al., 1995; Hallberg et al., 1992; Hsiang et al., 1993; Prosser et al., 1992; Redondo et al., 1992; Shi and Stavnezer, 1998; Xie et al., 1999). VD to J rearrangement of a TCR minilocus in transgenic mice was also shown to require an intact CBF-binding site in the minimal core enhancer region (Lauzurica et al., 1997). Since rearrangement of the immunoglobulin and TCR genes requires the chromatin to be accessible to the recombinase (Sleckman et al., 1996), CBF may participate in establishing an open chromatin state of these genes. CBF may also be involved in mediating heavy chain class switching in B cells. The switch to IgA synthesis can be induced by TGF-1, and is preceded by the production of germ-line transcripts from the unrearranged IgA gene (Lebman et al., 1990; Shockett and Stavnezer, 1991). The promoter for the germ-line IgA gene contains multiple CBF-binding sites, and mutation of these sites impairs transcriptional induction by TGF-1 (Shi and Stavnezer, 1998; Xie et al., 1999). Which CBF proteins are responsible for transcription of the lymphoid-specific genes has not been established. Expression studies suggest that Runx1 may be most important in T cells, while Runx3 may be the predominant player in B cells (Meyers et al., 1996; Satake et al., 1995; Shi and Stavnezer, 1998). Runx2 is less likely to be important in hematopoiesis. Runx2 is required for bone formation; homozygous disruption of the Runx2 gene results in perinatal lethality and a complete lack of bone ossification caused by a maturational arrest in osteoblasts (Komori et al., 1997; Otto et al., 1997).

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Mice deficient for Runx2 exhibit excessive extramedullary hematopoiesis late in fetal development, but this is thought to be secondary to the congenital lack of a bone marrow cavity (Deguchi et al., 1999).

CONCLUSION AND FUTURE DIRECTIONS RUNX1(AML1) and CBFB encode two subunits of a sequence-specific DNA-binding protein. Both subunits are essential for definitive hematopoiesis, and are thought to be required for the emergence of definitive hematopoietic stem and/or progenitor cells from an endothelial intermediate in the embryo. Mutations in both RUNX1 and CBFB are associated with human myeloid and pre-B-cell leukemias, suggesting that both genes are also required for the normal proliferation and/or differentiation of lymphoid and myeloid cells. Several important questions remain that are subjects of current investigations. A key issue is the role of RUNX1 and CBFB in later stages of normal hematopoietic development. Conditional knockout or rescue strategies that will allow HSCs to emerge before removing RUNX1 or CBFB function should address this question. The precise role of RUNX1 and CBFB in early stages of HSC emergence also needs further definition. For example, what signals in the embryo activate Runx1 expression in the hemogenic endothelium? What are the earliest RUNX1:CBFB targets? Do some HSCs differentiate directly from a hemangioblast precursor, or is differentiation through an endothelial cell intermediate the path followed by all HSCs? Although it is known that RUNX1 and CBFB are required in a cell-autonomous fashion, is there also a non-cell-autonomous requirement for these genes? Related questions regard the role of RUNX1 and CBFB in other developmental pathways. Given that the Drosophila homologues runt and lozenge are involved in multiple developmental pathways, is this also true for the RUNX1 and CBFB genes, which are expressed in many different cell types? Conditional knockout and rescue approaches can be used to address this issue. Assessing the contribution of marked Runx1- or Cbf b-deficient ES cells to all cell types in chimeric mice may also uncover new developmental roles for these genes.

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CHAPTER 7


PU.1 AND THE DEVELOPMENT OF THE MYELOID LINEAGE DANIEL G. TENEN Harvard Institutes of Medicine

INTRODUCTION Transcription factors play a major role in hematopoietic lineage determination and differentiation (Shivdasani and Orkin, 1996; Tenen et al., 1997). This has become very apparent in the myeloid lineage, in that the most common form of acute leukemia in human adults results from a block in normal myeloid differentiation, and many of the abnormalities critical to development of myeloid leukemias involve transcription factors. As there have been a number of recent reviews which have covered transcription factors and myelopoiesis, and in particular the role of PU.1, Clarke and Gordon, 1998; Lawrence et al., 1996; Nichols and Nimer, 1992; Robertson et al., 1995; Shapiro and Look, 1995; Shivdasani and Orkin, 1996; Sieweke and Graf, 1998; Tenen et al., 1997; ) this chapter does not attempt to be exhaustive but rather to update the reader on newer concepts regarding PU.1 that have emerged in the past few years. The reader is referred to these previous reviews for a more

thorough background on published data, particularly the Tenen et al., 1997 reference. This chapter does not discuss embryonic development. It focuses on myeloid lineage commitment and differentiation. In addition, recent studies have emphasized several themes in terms of myeloid differentiation. These include mechanisms of downregulation of alternative pathways, as well as the increasingly important role of transcription factor interactions in lineage commitment decisions. Examples of these two mechanisms are highlighted as regulation of PU.1 function is likely to be mediated through these mechanisms.

INDUCTION OF THE STEM CELL TO COMMIT TO THE MYELOID LINEAGE Previous models have described stochastic, deterministic, and combination models of hematopoietic lineage commitment. Our view of myeloid commitment combines the two, but in


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PU.1 AND THE DEVELOPMENT OF THE MYELOID LINEAGE

Figure 7.1. Model of induction of myeloid development by the PU.1 transcription factor. In this model, transcription factors such as PU. 1 and GATA are expressed at low levels in CD34> stem cells (Cheng et al., 1996), as are specific growth factor receptors. Under the direction of signals that are as yet not defined, such as the influence of stromal interactions or growth factor signaling, specific transcription factors, such as PU. 1 (Voso et al., 1994), are upregulated. Upregulation of specific transcription factors leads to their autoregulation and upregulation of specific growth factor receptors, such as the GM-CSF receptor (Hohaus et al., 1995; Iwama et al., 1998), resulting in increases in proliferation, differentiation, and suppression of apoptosis of myeloid progenitors. Downregulation of specific transcription factors (such as GATA-1 during myeloid development) may also play an important role, as is inhibition of GATA-1 function by PU. 1 through direct protein-protein interactions (Rekhtman et al. 1999; Zhang et al., 1999; Zhang et al., 2000).

a manner different from traditional views. Several recent studies have augmented previous work suggesting that growth factor signaling does not determine myeloid development (Lagasse and Weissman, 1997; Stoffel et al., 1999). There is also accumulating data supporting a deterministic model of differentiation in which the determination factor is a transcription factor, not an external growth factor (see, for example, Kulessa et al., 1995; Nerlov and Graf, 1998; Radomska et al., 1998; Tenen et al., 1997) (Fig. 7.1; Table 7.1). Recent models propose that stem cells or early multipotential progenitor cells may coexpress low levels of lineage-specific transcription factors, such as GATA-1 and PU.1 (Hu et al., 1997; Tenen et al., 1997; Voso et al., 1994). Some event (and this may represent the stochastic aspect of differentiation) leads to an

increase in expression and/or activity of one or more of these transcription factors. Examples of possible initiating (or destabilizing) events include interactions with stroma, or perhaps local differences in concentrations of a growth factor leading to activation of specific growth factor pathways (Roberts et al., 1988). An important concept is that not only the absolute amount of any given transcription factor is critical but the relative increase in expression of one of these factors over the other by an initiating event may trigger differentiation. For example, treatment of CD34> cells with granulocyte-monocyte colony-stimulating factor (GM-CSF) leads to increases in PU.1 and decreases in GATA-1 mRNA (Voso et al., 1994). PU.1 autoregulates its own promoter (Chen et al., 1995a) and activates the gene for the GM-CSF receptor

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TABLE 7.1. Hypotheses Regarding Myeloid Development

Hypothesis

Traditional

Stochastic

Chance

Deterministic

CSFs induce differentiation

Stochastic/deterministic

Combines both

Default

Erythroid/monocytic

(Hohaus et al., 1995), leading to viability and proliferation of early myeloid progenitors (Fig. 7.1). In contrast, increases in GATA-1 induced in the same cells by erythropoietin could potentially lead to autoregulation (Tsai et al., 1991), activation of the erythropoietin receptor (Zon et al., 1991), and downregulation of PU.1 expression (Voso et al., 1994). Importantly, abnormal expression of these factors in the ‘‘wrong’’ lineage can result in a block of differentiation. For example, overexpression of GATA-1 in early myeloid cells blocks myeloid development (Visvader et al., 1992), and overexpression of PU.1 can block erythroid differentiation and lead to erythroleukemia (Moreau-Gachelin et al., 1996). Therefore, inhibition of pathways is also an important concept in understanding myeloid development (Table 7.2).

Transcriptocentric Chance encounter of early progenitor with stroma or CSF, leading to activation Transcription factors determine differentiation, modulated by expression and interactions with other factors Stromal and/or CSF signals induce transcription factor expression and/or activation Transcription factors modulate default pathways (e.g., C/EBP directs myeloid precursor away from default monocytic pathway)

THE ROLE OF PU.1 PROTEIN-PROTEIN INTERACTIONS IN MYELOID LINEAGE COMMITMENT Another emerging concept is that not only is the relative expression of transcription factors important but interactions among and combinations of factors are critical (Sieweke and Graf, 1998). A number of such interactions have been described previously (Tenen et al., 1997). Most of these have resulted in additive or synergistic activation of myeloid promoter activity (Zhang et al., 1996; Oelgeschlager et al., 1996). However, very recent results have indicated that factors such as GATA-1 and GATA-2 may interact with PU.1 to inhibit its function. We performed a two-hybrid screen using PU.1 as a bait, and isolated the GATA-2 cDNA.

TABLE 7.2. Concepts of Myeloid Development

Concept Transcription factors direct differentiation Low-level expression of multiple lineage— specific transcription factors in stem cells Autoregulation of expression Activation of growth factor receptors

Inhibition of alternate pathways Protein-protein interactions modulate function of lineage-determinating factors

Example GATA-1, PU.1, C/EBP GATA, PU.1 GATA-1, PU.1, C/EBP GATA ; EpoR PU.1 ; GM-CSFr, G-CSFr, M-CSFr C/EBP ; G-CSFr, IL-6r GATA inhibits PU.1; PU.1 inhibits GATA PU.1 and GATA, c-Jun, AML1, C/EBP

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Figure 7.2. A small protein-protein interaction domain of PU. 1 mediates interactions with other important hematopoietic regulators. Shown is a schematic of the PU. 1 protein structure, including the amino terminal transactivation domain (TAD), the PEST domain, and the Ets DNA-binding domain (DBD), including the winged helix-turn-helix (wHTH) structure (Kodandapani et al., 1996; Wara-aswapati et al., 1999). Functional domains are designated according to amino acid residue. A relatively small region of the PU. 1 Ets domain (Wara-aswapati et al., 1999) mediates inhibitory interactions with GATA-2 and GATA-1 (Zhang et al., 1999), as well as cooperative and/or synergistic positive interactions with AML1 (Petrovick et al., 1998) and C/EBP factors (Lefrancois et al., 1999;Wara-aswapati et al., 1999). c-Jun acts as an important coactivator of PU. 1 function (Behre et al., 1999; Lefrancois et al., 2000).

Subsequent studies indicated that both GATA-1 and GATA-2 can interact with PU.1 through a relatively small region within the PU.1 Ets domain (Zhang et al., 1999). Interestingly, this same region of PU.1 can interact with AML1, c-Jun, and C/EBP factors, and therefore comprises an important interaction domain of PU.1 mediating both positive and negative regulation of PU.1 activity (Fig. 7.2) (Wara-aswapati et al., 1999). These studies indicated that the conserved carboxyl zinc finger of the GATA proteins, which is important for GATA-1 DNA binding, interacts with PU.1 both in vitro in GST ‘‘pulldown’’ assays, and in vivo in coimmunoprecipitation studies, both using transfected cells and in K562 cells, which coexpress GATA-1 and PU.1 endogenous proteins. Further studies indicated that GATA did not inhibit PU.1 DNA-binding activity or the amino terminal transactivation domain, but rather GATA competed for c-Jun, a coactivator of PU.1 function (Behre et al., 1999), for binding to the PU.1 interaction domain (Figs. 7.2 and 7.3) (Zhang et al., 1999). Recent data demonstrated that PU.1 can also inhibit GATA function in early erythroid cells (Rekhtman et al., 1999; Zhang et al., 2000). Such complementary inhibitory functions could play an important role in early decisions of stem cells to become

myeloid cells (Fig. 7.3). In nonmyeloid cells, such as stem cells and/or erythroid cells, GATA2 and GATA-1, if present in excess, could inhibit PU.1 function by competing for binding of cJun, a coactivator of PU.1 function (Behre et al., 1999). In myeloid cells, PU.1 is more highly expressed than the GATA proteins, leading to inhibition of GATA function.

LINEAGE-SPECIFIC COACTIVATORS, PU.1, AND MYELOID DEVELOPMENT It is likely that in the next few years a number of myeloid-specific coactivators will be discovered. A very important example of this type of protein is the GATA coactivator FOG, which is specific for erythroid cells and critical for GATA function. FOG itself does not bind to DNA but interacts with the amino terminal zinc finger of GATA-1 and is essential for GATA function in induction of erythropoiesis (Crispino et al., 1999; Tsang et al.). A recent example of a potentially important myeloid coactivator is c-Jun (Behre et al., 1999). While c-Jun is not as specifically expressed as FOG, there is evidence to suggest that it does demonstrate very specific patterns of expression. The c-Jun proto-oncogene forms a heterodimer

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Figure 7.3. GATA-1 and GATA-2 inhibit PU. 1 function at different stages during hematopoietic cell differentiation to specific lineages through protein-protein interactions. The model hypothesizes that in stem cells, GATA-2 blocks PU. 1 and c-Jun interaction, and therefore inhibits PU. 1 activation of its downstream target genes. In erythroblasts, upregulation of GATA-1 blocks coactivation of PU. 1 by c-Jun. But in developing myeloid progenitors, with decreased expression of GATA-1 and GATA-2, PU. 1 and c-Jun synergistically activate PU. 1 target genes such as the M-CSF receptor (Behre et al., 1999). G-1: GATA-1; G-2: GATA-2.

with c-Fos to form the AP-1 transcription factor, which is involved in cell proliferation and activation (Bohmann et al., 1987). c-Jun mRNA is upregulated upon macrophage differentiation of bipotential myeloid cell lines (Gaynor et al., 1991; Mollinedo et al., 1993), and stable transfection of c-Fos (Lord et al., 1993), JunB (Lord et al., 1993), or c-Jun (Li et al., 1994; Szabo et al., 1994) in myeloid cell lines results in monocytic differentiation. c-Jun can physically interact with PU.1 in vitro (Bassuk and Leiden, 1995), and these studies demonstrated that cJun could synergize with Ets factors in inducing expression of target genes. These studies all indicate a role for c-Jun in normal myeloid development (Fig. 7.4). c-Jun can be activated by the Ras pathway through phosphorylation by Jun kinase 1 (JNK1) (Derijard et al., 1994; Smeal et al., 1991). In addition, c-Jun mRNA increases with macrophage differentiation (Gaynor et al., 1991; (Hass et al., 1997; Lord et al., 1993;). c-Jun expression is autoregulatory (Angel et al., 1988; Rozek and Pfeifer, 1993), and recent reports have shown that phosphorylation of the transcription factor MEF2C by LPS activation of p38 MAP kinase in monocytes can increase c-Jun gene transcription (Han et al., 1997). A more recent report has also linked MEF2 to c-Jun expression by a

JNK-independent but G-protein dependent pathway (Coso et al., 1997). Finally, c-Jun has been shown to be differentially upregulated during monocytic but not granulocytic differentiation of bipotential myeloid cell lines, implicating a specific role for c-Jun expression in macrophage development (Mollinedo et al., 1993). These studies indicate that regulation of c-Jun expression, as well as its activation, may be important in its role in myeloid development. As already noted, Ras activation is likely to be important in signaling c-Jun expression in monocytic cells. Ras proteins are GTP-dependent molecular switches that are essential for cell growth and differentiation (Marshall, 1995). In particular, macrophage differentiation and macrophage colony-stimulating factor (M-CSF)—dependent survival are altered in transgenic mice that express dominant suppressors of Ras signaling (Jin et al., 1995). Ras also serves to augment c-Jun expression (Sistonen et al., 1989). A number of hematopoietic cell lines undergo spontaneous monocytic differentiation in response to activated Ras expression (Hibi et al., 1993; Maher et al., 1996), and M-CSF, GMCSF, or interleukin-3—induced monocytopoiesis of CD34> cells is inhibited by N-Ras antisense oligonucleotides (Skorski et al., 1992). Activating Ras mutations occur in 20—40% of AML

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Figure 7.4. PU. 1 and myeloid development. In this model, PU. 1 is important for inducing multipotential hematopoietic cells toward myeloid multipotential progenitors, and induces monocytic differentiation as the ‘‘default’’ pathway along with its coactivator, c-Jun. C/EB acts as a switch (Radomska et al., 1998) to induce granulocytic differentiation.

(Farr et al., 1988) and myelodysplastic syndromes (MDS) (Lyons et al., 1988), especially chronic myelomonocytic leukemia (CMML) (HirschGinsberg et al., 1990). Some patients with juvenile chronic myelogenous leukemia (JCML) show loss of the neurofibromatosis type I (NF1) gene (Side et al., 1997), a Ras GTPase-activating protein, and NF1 gene loss by itself is sufficient to produce the myeloproliferative symptoms associated with human JCML (Largaespada et al., 1996). In chronic myelogenous leukemia (CML), the bcr-abl protein constitutively activates Ras and requires Ras (Sawyers et al., 1995) and c-Jun (Raitano et al., 1995) for transformation. Thus, Ras plays a critical role in normal myeloid differentiation and leukemogenesis.

ROLE OF c-Jun IN MEDIATING THE EFFECTS OF Ras ON PU.1 One candidate mediator of Ras stimulation of PU.1 function is c-Jun, whose expression can be induced and whose function can be activated by Ras. In addition, as already noted, c-Jun has been implicated in monocytic differentiation. We observed no activation of PU.1 by Ras in cJun—deficient F9 cells, and activation was restored by addition of c-Jun (Behre et al., 1999). A dominant negative mutant of c-Jun without the basic domain blocks the activation of PU.1

by Ras in CV-1 cells (which contain c-Jun), and cotransfection of MEKK1, which induces the expression of c-Jun via the JNK pathway, enhances PU.1 transactivation function as much as Ras does. However, posttranslational modification of c-Jun by Ras activation is not critical for PU.1 activation, because c-Jun mutated in the JNK phosphorylation sites (serines 63 and 73) synergizes with PU.1 as well as wild-type c-Jun does; instead, it appears that Ras activation mediates upregulation of c-Jun expression. These findings are consistent with recent results indicating that a mouse with a ‘‘knock-in’’ of the Ser63Ala/Ser73Ala mutations into both c-Jun alleles develops normally, with apparently normal myeloid development (Behrens et al., 1999), and that disruption of JNK1 does not affect monocytic development (Dong et al., 1998). c-Jun physically interacted in vitro to the PU.1 DNA-binding domain, but c-Fos did not (Behre et al., 1999). Expression of c-Fos, which heterodimerizes with c-Jun to form the DNA binding protein AP-1, blocked the synergy between c-Jun and PU.1. It appears that c-Jun, whose basic domain can bind to the PU.1 DNA- binding domain directly, augments PU.1 function without binding to DNA itself, as c-Jun does not bind to the M-CSF receptor promoter or the minimal promoter containing only PU.1 sites. These data suggest that Ras enhances, via the induction of c-Jun expression, the ability of

ROLE OF PU.1 IN REGULATION OF THE MYELOID GROWTH FACTOR RECEPTORS

PU.1 to activate critical monocytic target genes such as the M-CSF receptor promoter (Behre et al., 1999). Similar results from another laboratory have implicated synergism through PU.1, C/EBP, and c-Jun, in which c-Jun again acts as a coactivator (Lefrancois et al., 2000). A major conclusion of these experiments is that c-Jun is critical for PU.1 function in transactivation of monocytic target promoters, and that c-Jun acts as a specific coactivator for PU.1. In addition, the effect of Ras activation is not mediated through pathways dependent of JNK phosphorylation of c-Jun, but rather through pathways that induce upregulation of c-Jun expression in monocytic differentiation. Therefore, understanding c-Jun expression in monocytic cells is likely to be important. It is not known if PU.1 itself can regulate c-Jun expression.

THE ROLE OF PU.1 IN STEM CELL AND MYELOID DEVELOPMENT A major controversy regarding the role of PU.1 in hematopoiesis arose when the first groups reported on studies involving its targeted disruption in mice Henkel et al., 1996; McKercher et al., 1996; Olson et al., 1995; Scott et al., 1994. One mouse model was embryonic lethal and the PU.1 defect initially characterized as being a stem cell defect involving all lineages other than erythrocytes and megakaryocytes (Scott et al., 1994). In a second model, the animals were viable at birth, although they died within days to weeks (Henkel et al., 1996; McKercher et al., 1996;). In these animals, the deficit was characterized as primarily involving monocytes and B cells, with delayed maturation of T cells and neutrophils. Several explanations were discussed for these differences. As the second model involved an insertional strategy rather than a disruption, the possibility existed that perhaps it was really a hypomorph. However, this is clearly not the case, as subsequent studies have revealed that there is no PU.1 protein detectable in the knockout cells (Anderson et al., 1998b), and there is no significant expression of PU.1 mRNA in the PU.1\\ fetal livers from these mice, even using a probe specific for the 5 portion of the murine PU.1 cDNA (Chen et al., 1995b) on long exposure of heavily loaded

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Northern blots, indicating that the mRNA, if transcribed, is not sufficiently stable to be detectable at significant levels (Iwama et al., 1998). However, more recent studies make it appear that the two models are fairly similar in terms of what they tell us about PU.1 function in myeloid development. Although the cause of the embryonic lethality in the initial model is still not understood, it is clear that in this model early myeloid cells are produced, but in the absence of PU.1 they cannot differentiate further (DeKoter et al., 1998; Olson et al., 1995). In one of these studies (DeKoter et al., 1998), introduction of PU.1 but not CSF receptors (which are deficient in PU.1\\ cells; see below) was capable of rescuing monocytic development, further demonstrating that it is the transcription factor PU.1 and not CSF receptors that induce differentiation. Similar studies are discussed below for C/EBP. Therefore, one interpretation of these data is that the differences in the mouse models may be due to strain differences or other factors that do not reflect PU.1 function. Both models support the idea that very early myeloid progenitors can develop in the absence of PU.1, but that PU.1 is important for further development, and absolutely important for monocyte and macrophage differentiation from these earlier precursors.

ROLE OF PU.1 IN REGULATION OF THE MYELOID GROWTH FACTOR RECEPTORS: GM-CSF RECEPTOR , G-CSF RECEPTOR, AND M-CSF RECEPTOR Another controversy regarding PU.1 function was its role in regulation of myeloid CSF receptors. Initial transient transfection studies had identified PU.1 sites in the promoters for all three myeloid CSF receptors: those for GM-, G-, and M-CSF (Hohaus et al., 1995; Smith et al., 1996; Zhang et al., 1994). However, initial reports regarding the expression of these receptors in PU.1\\ cells indicated that expression of mRNA encoding the ‘‘earlier’’ GM-CSF receptor and G-CSF receptor were not affected in PU.1\\ cells, but only expression of M-CSF receptor mRNA was decreased (Olson et al., 1995). However, several recent studies have revisited this question.

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It is clear that there are deficiencies of all three receptors in PU.1\\ cells in both PU.1 knockout models (Anderson et al., 1998b; DeKoter et al., 1998; Iwama et al., 1998), and these cells have undetectable levels of all three receptors on their surface by colony-forming unit (CFU) assay, which is likely the most sensitive assay of receptor expression (Anderson et al., 1998a; DeKoter et al., 1998). In addition, direct measurement by Northern blot analysis of mRNA from PU.1 knockout fetal livers shows a significant decrease in all three receptors (GM-, G-, and M-CSF) (Iwama et al., 1998). Therefore, it is likely that the in vivo studies confirm the in vitro studies indicating that PU.1 is critical for expression of multiple CSF receptors. These results are quite distinct from those involving C/EBP, in which C/EBP can regulate all three in vitro, but only G-CSF receptor in the C/ EBP\\ animals.

ADDITIONAL PU.1 TARGET GENES In addition to the CSF receptors, a number of studies have identified additional PU.1 target genes that are important during early myeloid maturation. One interesting target appears to be c-myb, in that expression of PU.1 in 32D cells can downregulate c-myb expression by downregulating the c-myb promoter (Bellon et al., 1997). The function of c-myb can also be inhibited by protein-protein interactions with cMaf, which provides a second mechanism of downregulation of c-Myb activity during myeloid maturation (Hedge et al., 1998). Another study identified a number of novel PU.1 (and C/EBP) target genes by using representational difference analysis (RDA) (Iwama et al., 1998). Some of these were clearly expressed only early and not late during myeloid development, and are likely candidate direct gene targets of PU.1 that play a role in directing differentiation. It will be interesting to perform such studies in other cell types. For example, recent studies have also indicated that PU.1 is critical for development of the monocyte-related osteoclast, and that PU.1\\ mice develop osteoporosis (Tondravi et al., 1997). It is anticipated that there may be an entire novel set of PU.1 targets in this lineage. Finally, it will be of interest to further identify the differential roles of PU.1 and Spi-B in B-cell

development. Lack of Spi-B leads to very specific and selective defects in B-cell response (Su et al., 1997), quite different from the complete lack of B-cell development in PU.1\\ animals. One reason for these differences is the very different pattern of expression of PU.1 and Spi-B (Chen et al., 1995b). Recent studies characterizing the genomic structure of Spi-B and identification of two promoters and upstream regions of Spi-B (Chen et al., 1998) will allow a comparison with the PU.1 promoter (Chen et al., 1995a) in order to understand these differences.

PU.1 STRUCTURE-FUNCTION: DEVELOPMENT OF RESCUE ASSAYS Recently, four different models to investigate the biologic function of PU.1 were developed. One model involves introduction of PU.1 into multipotential chicken cells using a conditional retrovirus (Nerlov and Graf, 1998), while the other three are ‘‘rescue’’ assays of PU.1\\ cells (Anderson et al., 1999; DeKoter et al., 1998; Fisher et al., 1998). These studies will undoubtedly provide important insights into the role of PU.1 in myeloid development and indeed have already demonstrated that PU.1 and not myeloid CSF receptors are critical for induction of myeloid differentiation (Anderson et al., 1999; DeKoter et al., 1998). Also, these approaches will allow biologic assays of PU.1 structure and function, in addition to those already performed using transient transfection assays (Klemsz and Maki, 1996). These studies are critical, since it is clear from studies using GATA-1 that transient assays of transcription factor function do not necessarily define the important biologically functional domains, and in particular the transactivation domains identified in transient assays are not necessarily critical for lineage development (Blobel et al., 1995; Crispino et al., 1999; Tsang et al., 1997). This may be in part due to the fact that these transient assays do not adequately measure the effect of protein-protein interaction domains. For example, it is clear that the ‘‘transactivation domain’’ of GATA-1 is not the critical functional domain for induction of erythroid differentiation; rather, the domains that interact with proteins such as FOG are more important. Since it is clear that PU.1, like GATA-1, has an important interaction domain outside its

REFERENCES

transactivation domain, it is likely that the biologic assays described above will be critical to define their importance. Like GATA-1, the PU.1 domain that interacts with GATA, AML1, cJun, and C/EBP is part of the DNA-binding domain (Fig. 7.2), making it relatively difficult to identify mutants that selectively abrogate such protein-protein interactions but do not disrupt PU.1 DNA binding. Recently, an elegant yeast ‘‘split-hybrid’’ technique was used to identify GATA-1 mutants that retained DNA binding but no longer could interact with FOG (Crispino et al., 1999), and such techniques will likely be useful to define the importance of interactions of PU.1 with its partners. There have already been some interesting (and differing) findings regarding the use of these assays to define PU.1 function. One study found that the PU.1 transactivation domain was important in directing myeloid differentiation of multipotential cells, and that induction of differentiation correlated with transactivation function (Nerlov and Graf, 1998). Other studies indicated that some parts of the PU.1 transactivation domain (the acidic domain) were dispensable for macrophage rescue of PU.1\\ ES cells (Fisher et al., 1998). Obviously, some of the differences may be due to differences in the systems used, or even the promoter context. For example, another study indicated that PU.1 was important for activation of B-cell enhancers, and this function did not require the transactivation domain, perhaps because of the binding of multiple other proteins in this enhancer that interact with PU.1, including NF-EM5/PIP (Pongubala and Atchison, 1997). Finally, it may be the case that both the transactivation and protein interaction domains may be important, and related. For example, in the studies of c-Jun coactivation of PU.1 function of the M-CSF receptor, c-Jun bound to the PU.1 interaction domain within the carboxyl Ets domain, but the amino terminal transactivation domain was also necessary for this function. Another example is found in a recent study of the cooperation of PU.1 and AML1 in activation of the M-CSF receptor promoter. In this case, PU.1 and AML1 interact through their DNA-binding domains, but another region of AML1 is required for cooperative activation of the promoter (Petrovick et al., 1998). These studies suggest the possibility that there may be additional proteins mediating activation be-

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tween these myeloid regulators, and their identification will be an important goal of future investigations.

CONCLUSION AND FUTURE DIRECTIONS What are the major areas and questions involving PU.1 and myeloid development? Clearly, the control of expression and function of regulators such as PU.1 is critical, and we know very little about that subject. We know very little about feedback mechanisms, although a passive model, in which receptors serve to control CSF levels, is likely operating in megakaryocytopoiesis (Shivdasani et al., 1997). If such a mechanism if operative in myelopoiesis, then in PU.1\\ mice, which have low levels of GM-CSF, MCSF, and G-CSF receptors, we would predict to find elevated levels of the corresponding CSFs. Another important area for investigation is further identification of protein-protein interaction partners, whose importance is discussed in the beginning of this chapter. And finally, a very important area of investigation, and perhaps most relevant to medicine, is to relate our knowledge of normal human myeloid development to the pathogenesis of AML.

ACKNOWLEDGMENTS I would like to thank the many comments and contributions from members of my laboratory and my colleagues, particularly those who have made major contributions that were not discussed in this chapter due to space limitations, as well as the patience and expert editing of Katya Ravid and Jon Licht. This work was supported by grants CA41456, HL56745, and CA72009 from the National Institutes of Health.

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Smeal, T., Binetruy, B., Mercola, D. A., Birrer, M., and Karin, M. (1991). Oncogenic and transcriptional cooperation with Ha-Ras requires phosphorylation of c-Jun on serines 63 and 73. Nature 354, 494—496. Smith, L. T., Hohaus, S., Gonzalez, D. A., Dziennis, S. E., and Tenen, D. G. (1996). PU.1 (Spi-1) and C/EBP regulate the granulocyte colony-stimulating factor receptor promoter in myeloid cells. Blood 88, 1234—1247. Stoffel, R., Ziegler, S., Ghilardi, N., Ledermann, B., Desauvage, F. J., and Skoda, R. C. (1999). Permissive role of thrombopoietin and granulocyte colony-stimulating factor receptors in hematopoietic cell fate decisions in vivo. Proc. Natl. Acad. Sci. USA 96, 698—702. Su, G. H., Chen, H. M., Muthusamy, N., GarrettSinha, L. A., Baunoch, D., Tenen, D. G., and Simon, M. C. (1997). Defective B cell receptormediated responses in mice lacking the Ets protein, Spi-B. EMBO J. 16, 7118—7129. Szabo, E., Preis, L. H., and Birrer, M. J. (1994). Constitutive c-Jun expression induces partial macrophage differentiation in U-937 cells. Cell Growth Differ. 5, 439—446. Tenen, D. G., Hromas, R., Licht, J. D., and Zhang, D.-E. (1997). Transcription factors, normal myeloid development, and leukemia. Blood 90, 489—519. Tondravi, M. M., McKercher, S. R., Anderson, K., Erdmann, J. M., Quiroz, M., Maki, R., and Teitelbaum, S. L. (1997). Osteopetrosis in mice lacking haematopoietic transcription factor PU.1. Nature 386, 81—84. Tsai, S. F., Strauss, E., and Orkin, S. H. (1991). Functional analysis and in vivo footprinting implicate the erythroid transcription factor GATA-1 as a positive regulator of its own promoter. Genes Dev. 5, 919—931. Tsang, A. P., Visvader, J. E., Turner, C. A., Fujiwara, Y., Yu, C., Weiss, M. J., Crossley, M., and Orkin, S. H. (1997). FOG, a multitype zinc finger protein, acts as a cofactor for transcription factor GATA-1 in erythroid and megakaryocytic differentiation. Cell 90, 109—119. Visvader, J. E., Elefanty, A. G., Strasser, A., and Adams, J. M. (1992). GATA-1 but not SCL induces megakaryocytic differentiation in an early myeloid line. EMBO J. 11, 4557—4564. Voso, M. T., Burn, T. C., Wulf, G., Lim, B., Leone, G., and Tenen, D. G. (1994). Inhibition of hematopoiesis by competitive binding of the transcription factor PU.1. Proc. Natl. Acad. Sci. USA 91, 7932— 7936. Wara-aswapati, N., Koyama, Y., Behre, G., Tetradis, S., Tsukada, J., Ro, Y.-T., Tenen, D. G., and Auron, P. E. (2000). The wHTH wing: an interaction motif mediating association with a variety of transcription factors. submitted.

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CHAPTER 8


CCAAT/ENHANCER-BINDING PROTEINS IN MYELOID CELLS DONG-ER ZHANG Department of Molecular and Experimental Medicine, The Scripps Research Institute

INTRODUCTION During the initial stage of eukaryotic gene regulation studies, an enhancer core sequence 5TGTGG G-3 was identified in enhancers 2 2 2 of murine sarcoma virus, polyoma virus, and SV40 (Johnson et al., 1987). In addition, a functional DNA sequence 5-CCAAT-3 was identified in the promoter region of the herpes simplex virus thymidine kinase gene and the murine sarcoma virus long terminal repeat (Graves et al., 1986). Using protein-DNA interaction analysis, a heat-stable protein from rat liver nuclear extract was partially purified, which interacts with both the promoter CCAAT sequence and the enhancer core sequence (Graves et al., 1986; Johnson et al., 1987). This protein was soon cloned and designated as CCAAT/enhancer-binding protein (C/EBP) (Landschulz et al., 1988a). Amino acid sequence analysis identified a highly positively charged region and a periodic repeat of leucines at the C-terminal end of C/EBP. From here, a new

class of proteins containing a basic DNA-binding domain and a leucine zipper for dimerization (bZIP) was characterized (Landschulz et al., 1988b, 1989). Further study has shown that C/EBP is a family of transcription factors. They play many important roles during cell proliferation, differentiation, and survival. In this review, I focus on the function of C/EBP proteins in myeloid cells.

MEMBERS OF C/EBP FAMILY TRANSPORTATION FACTORS IN MYELOID CELLS Since the discovery of the first C/EBP protein (now known as C/EBP), five other C/EBP proteins have been identified. All of these proteins contain bZIP domains at the C-terminal region. There are various names used for these factors, which are mentioned in this review. However, to simplify the description, C/EBP, -, -, -, -, and - are used in this review as


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CCAAT/ENHANCER-BINDING PROTEINS IN MYELOID CELLS

Figure 8.1. Domain structure of C/EBP proteins. C/EBP activation domains are marked with slashed boxes. C/EBP negative regulatory domains are marked with dotted boxes. DNA- binding basic regions are marked with filled boxes. Leucine zipper dimerization domains are marked with boxes containing vertical lines.

proposed by Cao and colleagues (Cao et al., 1991). The structures of these proteins are illustrated in Figure 8.1. The founding member of the C/EBP transcription factor family is C/EBP (Cao et al., 1991; Landschulz et al., 1988a). It is encoded by an intronless gene (Birkenmeier et al., 1989) with an open reading frame for 358 amino acids. As shown in Figure 8.1, C/EBP has three transactivation elements (TEI-TEIII), a basic

region for interacting with DNA, and a leucine zipper domain for dimerization (Birkenmeier et al., 1989; Friedman and McKnight, 1990; Nerlov and Ziff, 1994). TE-I and TE-II cooperatively interact with TBP and TFIIB, which are essential components of the RNA polymerase II basal transcriptional apparatus (Nerlov and Ziff, 1995). The amino acid sequences of C/ EBP required for the interaction with TBP and TFIIB are conserved among members of the

MEMBERS OF C/EBP FAMILY TRANSPORTATION FACTORS IN MYELOID CELLS

C/EBP family, indicating a possible common scheme utilized by C/EBP proteins in gene activation. Using antibodies against the C-terminal region of C/EBP, three C/EBP products are detected from liver nuclear extracts. These proteins are C/EBP42, C/EBP30, and C/EBP20. The formation of the two shorter forms of C/EBP is due to a ribosome-scanning mechanism (Ossipow et al., 1993). In transient transfection assays, only C/EBP42 strongly stimulates transcription (Lin et al., 1993; Ossipow et al., 1993). Analysis of RNA prepared from different tissues of human, mouse, and rat reveals that C/EBP is highly expressed in placenta, liver, both brown and white fat, lung, peripheral leukocytes, small intestine, skeletal muscle, and colon; and is undetectable in brain, kidney, spleen, thymus, testis, and ovary (Antonson and Xanthopoulos, 1995; Birkenmeier et al., 1989), indicating a tissue-restricted pattern of C/EBP expression. Further analysis in the hematopoiesis system demonstrates that C/EBP is specifically expressed in myeloid cells, including human and mouse neutrophils, mouse bone marrow—derived macrophages and peritoneal macrophages, but not in human peripheral blood monocytes (Hu et al., 1998; Radomska et al., 1998; Scott et al., 1992). In mice, the amount of C/EBP RNA in peritoneal macrophages (with thioglycolate stimulation) is similar to its amount in the liver (Fig. 8.2). Hematopoietic cells representing different developmental stages and lineages can be prepared from the in vitro culture of human multipotential hematopoietic stem cells using fluorescence-activated cell sorting (FACS) by antibodies against different lineage markers. When cells are separated using FACS, C/EBP RNA is not expressed in G arrested hematopoietic stem cells, the CD34>CD33\ uncommitted progenitor cell, CD3> T cells, and CD19> B cells. However, C/EBP RNA is moderately expressed in CD34>CD33> committed myeloid precursor cells and highly expressed in CD11b> mature myeloid cells (Radomska et al., 1998). C/EBP is also expressed in myeloid cell lines, including both bipotential lines (HL60 and U937) or cells belonging to monocytic or granulocytic lineages (THP-1, Mono Mac 6, and 32Dcl3) (Natsuka et al., 1992; Pan et al., 1999; Radomska et al., 1998; Scott et al., 1992; Zhang et al., 1996). Studies

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Figure 8.2. C/EBP expression in mouse peritoneal macrophages and liver. RNA was prepared from different tissues of mice 48 hours after intraperitoneal injection of thioglycolate (0.1 g/mL, 1.5 mL/mouse). 28S ribosomal RNA is presented to show the loading of the RNA samples.

with chicken cell lines representing different hematopoietic lineages show that C/EBP is highly expressed in myeloid and eosinophil cells, but not in lymphoid and erythroid cells (Nerlov et al., 1998). C/EBP expression is regulated during the differentiation of myeloid cell lines induced by various reagents. A 42 kDa C/EBP protein is present at high levels in the uninduced myeloblastic murine cell line 32Dc13. Upon G-CSF treatment to induce granulocytic cell differentiation, the level of C/EBP increased 2-fold during the first day of differentiation, was maintained at this level for several days, and finally diminished to low levels by day 6 of differentiation (Scott et al., 1992). C/EBP protein expression in the uninduced human bipotential myeloblast cell line HL60 is high. Upon phorbol ester (PMA or TPA) treatment to differentiate these cells toward the monocytic lineage and dimethylsulfoxide (DMSO) or alltrans retinoic acid (RA) treatment to differentiate these cells toward the granulocytic lineage, C/EBP expression decreases (Radomska et al., 1998; Scott et al., 1992). When the U937 bipotential myeloblast cell line was treated with

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TPA to induce monocytic differentiation, a rapid decrease of C/EBP expression was also detected, and an increase in C/EBP expression was shown after RA treatment for U937 cell granulocytic differentiation (Radomska et al., 1998). Recently, we treated U937 cells with the combination of 1,25-dihydroxyvitamin D3 and TGF to induce monocytic cell differentiation. Upon such treatment for 48 hours, the macrophage differentiation marker CD14 is highly increased (over 30-fold) and C/EBP in nuclear extracts is also clearly increased 13-fold (Pan et al., 1999). These data indicate that C/EBP is tissue specifically expressed in myeloid cells and its expression is regulated during lineage commitment and differentiation. C/EBP is also called NF-IL6, IL6-DBP, LAP, CRP2, AGP/EBP, NF-M, and ApC/EBP due to its discovery in different systems (Akira et al., 1998; Cao et al., 1991; Descombes et al., 1990; Lekstrom-Himes and Xanthopoulos, 1998; Poli et al., 1990). Chicken C/EBP, known as NF-M, was cloned during the analysis of the transcription factors critical for chicken myelomonocytic growth factor (cMGF) gene regulation (Katz et al., 1993). With the leaky ribosome scanning mechanism, rat C/EBP was also translated into three different proteins (39 kD, 36 kD, and 20 kD) (Descombes and Schibler, 1991). The shorter form of C/EBP has only the bZIP region for DNA binding and dimerization. Transfection analysis demonstrates that the long forms of C/EBP (39 kD and 36 kD) are transcription activators (LAP) and the short form of C/EBP (20 kD) is a transcriptional inhibitory protein (LIP) (Descombes and Schibler, 1991). LIP/LIP and LIP/LAP dimers interact with DNA more strongly compared to LAP/LAP. Since LIP lacks any activation function, LIP becomes a dominant inhibitor of LAP function (Descombes and Schibler, 1991). The ratio of LAP/LIP production is regulated during cell differentiation (Descombes and Schibler, 1991). This could be one way to control LAP activity. C/EBP also has two negative regulatory elements, RD1 (a.a. 136—170) and RD2 (a.a. 183 - 213) (Williams et al., 1995). RD1 does not affect cell type specificity but inhibits the transactivation of GAL4-C/EBP hybrid proteins by a factor of 50. Deletion of RD2 increases C/ EBP activity in fibroblastic L1 cells. However, the same deletion of RD2 does not affect C/

EBP activity in HepG2 hepatoma cells. This indicates that RD2 is a tissue-specific regulatory domain. The most interesting feature of C/EBP is its regulated biological function. C/EBP transcription is activated during acute phase response induced by IL-1 and LPS treatment (Akira et al., 1990; Alam et al., 1992). Its nuclear localization is promoted via posttranscriptional regulation (Chinery et al., 1997; Yin et al., 1996). C/EBP can also be phosphorylated with a MAP kinase at Thr and such phosphorylation has been demonstrated to be an essential step for its transcription activation function (Engelman et al., 1998; Nakajima et al., 1993;). Its DNA binding is also regulated by phosphorylation. It was shown that phosphorylation of rat C/EBP at Ser by PKC will inhibit its binding to DNA (Trautwein et al., 1994). Ford and co-workers reported that C/EBP is unphosphorylated and localized in the cytoplasm of multipotential hematopoietic cells and presents as a phosphorylated nuclear enhancerbinding protein in committed granulocytic cells (Ford et al., 1996). Among myeloid cells, C/EBP is highly expressed in human monocytes (Natsuka et al., 1992). When monocytes are treated with LPS, a significant increase of C/EBP expression can be detected. The expression of C/EBP mRNA is also increased markedly during induction of macrophage differentiation of the monoblast cell line M1 with either IL-6 or LIF treatment, and in bipotential cell lines U937 and HL60 with TPA treatment (Natsuka et al., 1992). However, C/EBP mRNA is not increased when HL60 cells are treated with DMSO to induce granulocytic differentiation. When antibodies specifically against C/EBP are used to analyze C/ EBP protein expression, an increase of C/ EBP expression has been reported in 32Dc13 cells treated with G-CSF for granulocytic differentiation and in U937 cells treated with vitamin D3 and TGF for monocytic differentiation (Pan et al., 1999; Scott et al., 1992). C/EBP is another C/EBP family member (Fig. 8.1), also called NF-IL6, CRP3, CELP, RcC/EBP2 (Cao et al., 1991; Kageyama et al., 1991; Kinoshita et al., 1992; Lekstrom-Himes and Xanthopoulos, 1998). C/EBP is highly expressed in intestine, lung, and fat of mice (Cao et al., 1991). Its expression is highly inducible by

MEMBERS OF C/EBP FAMILY TRANSPORTATION FACTORS IN MYELOID CELLS

LPS in other tissues (Kinoshita et al., 1992). Its DNA-binding activity and transactivation ability are similar to C/EBP and C/EBP (Cao et al., 1991). C/EBP expression in hematopoietic cells has been studied using a series of murine hematopoietic cell lines representing different lineages (Scott et al., 1992). C/EBP expression is only detectable in myelomonocytic cells and not in erythroid and lymphoid cells. C/EBP expression is increased during G-CSF-induced 32Dcl3 cell granulocytic differentiation (Scott et al., 1992). C/EBP, another member of the C/EBP family, was originally cloned as a C/EBP related protein (CRP1) from a rat genomic library (Williams et al., 1991). The human C/EBP gene was cloned by genomic DNA screening using C/ EBP and C/EBP cDNA probes and by lowstringency homologous RT-PCR (Antonson et al., 1996; Chumakov et al., 1997). Further analysis demonstrated that the C/EBP gene has two introns and two promoter regions, yielding four different forms of mRNA and three protein products (Yamanaka et al., 1997a). The expression of C/EBP can only be detected in lymphoid and myeloid tissues. However, its expression in myeloid cells is much higher than in lymphoid cells (Antonson et al., 1996; Chumakov et al., 1997) C/EBP expression increases during myeloid cell differentiation in vitro (Williams et al., 1998; Yamanaka et al., 1997a). Overexpression of C/EBP in a promyeloid cell line (NB4) enhances cell proliferation (Chumakov et al., 1997). Since C/EBP is specifically expressed in myeloid cells and its expression is regulated during myeloid cell differentiation, it has been studied as a critical C/EBP protein related to myeloid cell function (Park et al., 1999). C/EBP is also called Ig/EBP. It was originally cloned following analyses of the immunoglobin heavy chain enhancer activity and the fetal liver -fetoprotein upstream enhancer (Roman et al., 1990; Thomassin et al., 1992). This protein contains a basic region and a leucine zipper domain, which has high homology with other C/EBP family members. It can recognize the same DNA sequence as C/EBP and C/EBP. However, C/EBP protein contains only 151 amino acids and does not contain any activation domain. By itself, C/EBP is neither a transcription activator nor a transcription repressor. However, it can heterodimerize

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with other C/EBP family members, such as C/EBP and C/EBP, to block C/EBP factormediated gene activation (Cooper et al., 1995). Recently, C/EBP was reported to directly interact with another non-C/EBP family bZIP protein, JDP2 (Broder et al., 1998). Besides its negative regulatory function through dimerizing with other C/EBP family members, C/EBP also has a positive regulatory function in directing -globin gene expression through interaction with other transcription factors (Wall et al., 1996). C/EBP is ubiquitously expressed, but is more highly expressed in early B cells relative to mature B cells (Roman et al., 1990). C/EBP was originally cloned as a growth arrest and DNA damage gene (GADD153) and as a gene encoding a C/EBP homologous protein CHOP-10 (Fornace et al., 1989; Ron and Habener, 1992). This gene has three introns and encodes a protein with 168 amino acids. C/ EBP is highly homologous to other C/EBP members in the basic leucine zipper region. Therefore, it forms dimers with other C/EBPs. However, C/EBP contains two prolines substituting for two residues in the basic region, which are critical for binding to DNA (Ron and Habener, 1992). Heterodimers of C/EBP with other C/EBPs are unable to bind to a common class of C/EBP recognition sequence. Therefore, it is a negative modulator for the function of other C/EBP proteins (Ron and Habener, 1992). However, experiments with in vitro PCR-based site selection assay indicate that C/EBP-C/ EBP heterodimers can specifically interact with a novel DNA sequence (PuPuPuTGCAAT/ CCCC) (Ubeda et al., 1996). C/EBP is ubiqui! tously expressed and is induced during cell stress, especially if the endoplasmic reticulum is stressed (Ron and Habener, 1992). Besides the increase in its expression, stress also enhances C/EBP phosphorylation by p38 MAP kinase. Such phosphorylation increases its ability to function as a transcriptional activator and is also required for its full inhibitory effect on adipose cell differentiation (Wang and Ron, 1996). C/EBP is involved in a chromosomal translocation t(12;16)(q13;p11) associated with malignant liposarcoma (Aman et al., 1992; Crozat et al., 1993; Rabbitts et al., 1993). This translocation generates a fusion protein TLSCHOP. TLS (translocated in liposarcoma) is an RNA-binding protein. C/EBP can block cell

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proliferation at the G1/S checkpoint. In contrast, TLS-CHOP has an opposite effect. Its overexpression in NIH-3T3 cells caused cell transformation (Barone et al., 1994). C/EBP expression has been analyzed in the myeloid cell line 32Dcl3 (Friedman, 1996). C/ EBP expression is not detectable when 32Dcl3 cells are cultured in IL-3, during their differentiation in response to G-CSF, or during their apoptosis upon withdrawal of both IL-3 and G-CSF. An alkylating agent, methylmethane sulfonate (MMS), can induce C/EBP expression in 32Dcl3 cells. 32Dcl3 cells expressing a high level of exogenous C/EBP have a decrease of C/EBP transcriptional activity and undergo rapid apoptosis without differentiation (Friedman, 1996). Chromosomal localization of several human C/EBP genes has been reported. C/EBP gene is located on chromosome 19q13 (HendricksTaylor et al., 1992; Szpirer et al., 1992). C/EBP gene is located on chromosome 20q13 (Hendricks-Taylor et al., 1992; Szpirer et al., 1991). C/EBP gene is located on chromosome 8p11 (Cleutjens et al., 1993; Wood et al., 1995). C/ EBP gene is located on chromosome 14q11 (Antonson et al., 1996). C/EBP gene is located on chromosome 12q13 (Park et al., 1992). The properties of the C/EBP family members are summarized in Table 8.I.

GENES THAT ARE REGULATED BY C/EBP IN MYELOID CELLS As DNA-binding transcription factors, C/EBP proteins are important regulators for the expression of critical genes in various biological systems, especially in adipocytes and hepatocytes. Their regulated expression in myeloid cells indicates that they are also important regulators of myeloid-specific gene expression. Chicken myelomonocytic growth factor (cMGF) is distantly related to the mammalian hematopoietic growth factors G-CSF and IL-6. It is specifically expressed in monocytic cells within the hematopoietic system and is required for the outgrowth of bone marrow—derived macrophage, granulocyte, and mixed granulocyte/macrophage colonies (Leutz et al., 1989). The cMGF promoter contains two critical sites that interact with the same nuclear

protein—NF-M (Sterneck et al., 1992). These binding sites are required for the tissue specificity of the promoter. Molecular cloning analysis has demonstrated that NF-M is chicken C/ EBP. Further analysis has revealed that a Myb target gene mim-1 can be activated by C/EBP (Burkhardt et al., 1991). Most interestedly, C/ EBP and Myb can synergistically activate the mim-1 promoter, and the coexpression of C/ EBP and Myb protein in nonmyeloid cells, such as erythroid and fibroblast cells, can induce the endogenous markers of myeloid differentiation, such as the mim-1 and lysozyme genes (Ness et al., 1993). These data indicate that the combinatorial signal from C/EBP and Myb plays an important role for myeloid lineage commitment and differentiation. Receptors for three myeloid lineage growth factors, M-CSF, G-CSF, and GM-CSF, are very important for lineage-specific signaling related to cell proliferation, differentiation, and survival. The promoters of these receptor genes have C/EBP binding sites (Hohaus et al., 1995; Smith et al., 1996; Zhang et al., 1996). Site-specific mutagenesis of C/EBP-binding sites and transactivation analysis has demonstrated that C/EBP family members C/EBP, -, and - can activate the promoters of these three receptors. Furthermore, these C/EBP proteins and another critical factor of myeloid cell development—AML1/CBF (Chapter 6 in this book)— can synergistically activate the promoter of MCSF receptor through their adjacent binding sites (Petrovick et al., 1998; Zhang et al., 1996). These results indicate the important function of C/EBP proteins in myeloid-specific growth factor signaling. C/EBP binding sites have also been identified in the regulatory elements of genes related to the function of myeloid cells, including the proinflammation cytokines IL-1, IL-1, IL-6, IL-8, IL-12, macrophage inflammatory protein 1, and TNF- (Grove and Plumb, 1993; Hu et al., 1993; Natuska et al., 1992; Plevy et al., 1997; Shirakawa et al., 1993; Stein and Baldwin, 1993); growth factor G-CSF (Natsuka et al., 1992); the monocyte LPS receptor CD14 (Pan et al., 1999); and important enzymes expressed in myeloid cells, such as nitric oxide synthase (Lowenstein et al., 1993), myeloperoxidase (Ford et al., 1996), neutrophil elastase (Nuchprayoon et al., 1994; Oelgeschlager et al., 1996), lysozyme (Goethe

STUDIES WITH C/EBP-DEFICIENT ANIMAL MODELS

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TABLE 8.1. C/EBP Family Members

Name

Alternative Name

C/EBP C/EBP

C/EBP NF-IL6, IL6-DBP LAP, CRP2, AFP/EBP NF-M, ApC/EBP NF-IL6, CRP3, CELP RcC/EBP2 CRP1 Ig/EBP GADD153, CHOP-10

C/EBP C/EBP C/EBP C/EBP

and Loc, 1994), lactoferrin (Khanna-Gupta et al., 1999), and major basic protein (Yamaguchi et al., 1999). Using representational difference analysis, Iwama and associates have reported the identification of a set of genes, including both primary and secondary granule protein genes, differentially expressed in C/EBP knockout mice relative to wild-type mice (Iwama et al., 1998). Furthermore, studies have shown that C/EBP can synergize with NFB to activate IL-8 and IL-12 (Oettgen et al., 1996; Plevy et al., 1997; Stein and Baldwin, 1993), synergyze with GATA1 to activate the major basic protein promoter in eosinophil (Yamaguchi et al., 1999), and cooperate with PU.1, GABP, or c-myb to activate the neutrophil elastase promoter (Oelgeschlager et al., 1996; Nuchprayoon et al., 1997). In addition, ectopic expression of C/EBPs in the P388 lymphoblastic cell line can induce IL-6 and monocyte chemoattractant protein-1 expression after stimulation with LPS. Overexpression of C/ EBP in a monoblastic cell line can induce CD14, osteopontin and macrophage inflammatory protein-1 expression (Hu et al., 1998; Matsumoto M. et al., 1998; Williams et al., 1998), indicating the importance of C/EBP factors in the regulation of the expression of additional myeloid-specific genes. Table 8.2 shows a list of target genes of C/EBP proteins. The role of C/EBP in myeloid development was studied by overexpressing C/EBP in hematopoietic cell lines. Ectopic expression of C/ EBP in E26 virus—transformed chicken multi-

Number of Exons

Chromosomal Location (Human)

1 1

19q13 20q13

1

8p11

3 1 4

14q11 12q13

potent hematopoietic progenitors induced eosinophil differentiation (Nerlov et al., 1998). Upon zinc treatment for 17 days, myeloid bipotential U937 cells stably transfected with zincinducible C/EBP-expressing construct exhibited a neutrophilic differentiation (Radomska et al., 1998). More recently, another group reported that overexpression of C/EBP in a C/EBP-estrogen receptor ligand-binding domain fusion protein in 32D cl3 myeloblast cells induced granulocytic differentiation and reduced cell proliferation (Wang et al., 1999). Interestingly, another myeloid transcription factor PU.1 was highly upregulated when C/EBP expression was induced with estradiol in stable 32D cl3 lines (Wang et al., 1999).

STUDIES WITH C/EBP-DEFICIENT ANIMAL MODELS Mice homozygous for the targeted deletion of the c/ebpa gene died within 8 hours after birth due to hypoglycemia (Flodby et al., 1996; Wang et al., 1995). These mutant mice lack hepatic glycogen storage. C/ebpa null mice also showed defects in the control of hepatic growth and lung development (Flodby et al., 1996). Specifically, the amount of c-myc, c-jun, -actin mRNA and proliferating cell nuclear antigen/cyclin protein in the liver of mutant mice were increased, suggesting an active proliferative state of the liver. This demonstrates the importance of C/ EBP in control of cell proliferation, which was

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TABLE 8.2. Myeloid Target Genes of C/EBP Proteins

Type of Genes

Name of Genes

Growth factors

MGF G-CSF G-CSF receptor M-CSF receptor GM-CSF receptor IL-1 IL-1 IL-6 IL-8 IL-12 TNF Macrophage inflammatory protein 1 Nitric oxide synthase Myeloid peroxidase Neutrophil elastase Lysozyme Lactoferrin Major basic protein CD14 mim-1

Growth factor receptors

Proinflammation cytokines

Meyloid functional proteins

also suggested by the growth-suppressive properties of C/EBP in in vitro cell transfection analysis in various cell lines (Diehl et al., 1996; Hendricks-Taylor and Darlington, 1995; Ramos et al., 1996; Timchenko et al., 1996; Umek et al., 1991). The activation of the cell cycle inhibitor p21 gene expression and/or the stabilization of p21 protein by C/EBP have been suggested as mechanisms of C/EBP-mediated growth suppression (Cram et al., 1998; Timchenko et al., 1996;). Most recently, C/EBP has been reported as a competitor of E2F for the formation of the S-phase-specific complex E2F-p107. Since this complex is important for normal cell proliferation, its loss in the presence of C/EBP can also explain the antiproliferative function of C/EBP (Timchenko et al., 1999). The most interesting phenotype of c/ebpa null mice for myeloid lineage development is that the mutant newborn mice do not have mature neutrophils and eosinophils in their liver and circulating blood (Zhang et al., 1997). In normal newborn mice, 80—90% of peripheral white blood cells are mature neutrophils. In mutant

References Leutz et al., 1989 Natsuka et al., 1992 Smith et al., 1996 Zhang et al., 1996 Hohaus et al., 1995 Natsuka et al., 1992 Shirakawa et al., 1993 Hu et al., 1998 Stein and Baldwin, 1993 Plevy et al., 1997 Natsuka et al., 1992 Grove and Plumb, 1993 Lowenstein et al., 1993 Ford et al., 1996 Nuchprayoon et al., 1994 Goethe and Loc, 1994 Khanna-Gupta et al., 1999 Yamaguchi et al., 1999 Pan et al., 1999 Burkhardt et al., 1991

mice, over 90% of peripheral white blood cells are immature myeloid blasts. When RNA prepared from newborn liver was analyzed for the expression of different growth factor receptors, a loss of the C/EBP target gene encoding the G-CSF receptor was identified. As a result, multipotential myeloid progenitors from the mutant fetal liver were unable to respond to G-CSF signaling, although they were capable of forming granulocyte-macrophage colonies in response to other growth factors. Furthermore, transplantation of fetal liver cells from mutant mice to irradiated recipient mice reconstituted lymphoid but not neutrophilic cells, demonstrating the intrinsic defect of hematopoiesis in myeloid progenitor cells. Additional experiments indicated that the IL-6 receptor was expressed at a very low level in C/EBP knockout mice (Zhang et al., 1998). These studies suggest a model by which transcription factors can direct the maturation of early committed progenitors through activation of expression of a specific growth factor receptor, allowing proliferation and differentiation in response to a spe-

STUDIES WITH C/EBP-DEFICIENT ANIMAL MODELS

cific extracellular signal. Furthermore, the data demonstrate the critical role of C/EBP in myeloid cell differentiation, indicating the possible function of C/EBP in the development of acute myelogenous leukemia, in which a block of the differentiation of myeloid precursors is a key feature of the disease. Mice with targeted disruption of C/EBP gene had almost normal morphology of blood cells. However, they are highly susceptible to infection by L isteria monocytogenes (Tanaka et al., 1995). A large number of pathogens escape from the phagosome to the cytoplasm in activated macrophages of the mutant mice. The tumor cytotoxicity of macrophages from the mutant mice was severely impaired. In addition, G-CSF induction with LPS stimulation was impaired, although induction of other cytokines, such as IFN, TNF, GM-CSF, M-CSF, IL-1, IL-6, and IL-10, was comparable to that observed in wild-type mice (Tanaka et al., 1995). C/EBP gene—deleted mice also showed a lymphoproliferative disorder (Screpanti et al., 1995), which is similar to mice overexpressing IL-6 and to Castleman’s disease in human patients (Fattori et al., 1994; Frizzera et al., 1985; Suematsu et al., 1989). Further analysis showed a higher circulating IL-6 level in the C/EBP-deficient mice, indicating that C/EBP is a negative regulator of IL-6 expression. There is also an overexpansion of the B-cell compartment in the lymph nodes and spleens of mutant mice (Screpanti et al., 1995). However, the number of bone marrow B cells is decreased and the expansion of bone marrow B cells is impaired in long-term lymphoid cultures (Chen et al., 1997). Besides the defect of C/EBP deficiency mice on hematopoiesis, these mice also show major defects in the differentiation of ovary periovulatory granulosa cells in response to luteinizing hormone (Sterneck et al., 1997), in epithelial cell proliferation and differentiation in mammary gland (Robinson et al., 1998), and in adipocyte differentiation (Tanaka et al., 1997). C/EBP-deficient mice do not have any obvious abnormal phenotype. However, 85% of C/ EBP and C/EBP double knockout mice died at the early neonatal stage (Tanaka et al., 1997), indicating the synergistic or redundant function of these two C/EBP proteins in regulating cellular functions. C/EBP and C/EBP double knockout mice lack lipid drop accumulation in

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the brown fat tissue. Although previous work has shown that expression of C/EBP and C/ EBP activate C/EBP and PPAR transcription during 3T3-L1 adipocyte differentiation, C/EBP and PPAR expression in the double knockout mice is normal. However, embryonic fibroblasts from the double knockout mice do not differentiate into adipocytes under standard differentiation stimulation (Tanaka et al., 1997). There is no report of impaired hematopoiesis or macrophage function in the double knockout mice relative to C/EBP\\ mice. C/EBP is primarily expressed in myeloid and lymphoid cells. C/EBP knockout mice are born at the expected Mendelian ratio and appear normal at birth (Yamanaka et al., 1997b). Young mutant mice do not have any difference from their wild-type and heterozygous littermates in behavior and reproductive ability. However, C/EBP\\ mice die within 3 to 5 months. Further analyses show that there is a marked increase in granulocytic progenitors in the bone marrow of these mutant mice. They also have increased numbers of hyposegmented morphologically atypical neutrophils in peripheral blood and decreased numbers of eosinophils in both peripheral blood and bone marrow. No defect of erythropoiesis or lymphopoiesis is detected in the mutant mice. Necropsy examination of C/EBP\\ mice at age 8 to 20 weeks shows a variety of lesions associated with opportunistic bacterial infections. All mutant mice eventually develop signs of myelodysplasia and abnormal granulopoiesis, demonstrating the critical role of C/EBP in the development of granulocytic cells (Yamanaka et al., 1997b). These results indicate that C/EBP is a critical factor for late granulocytic cell differentiation. C/EBP deficiency mice have not been reported. C/EBP\\ mice have been recently generated (Zinszner et al., 1998). The knockout mice are born at the expected Mendelian frequency, appear phenotypically normal, and have normal fertility and reproductive behavior. In addition, there is no difference among mouse embryonic fibroblasts (MEF) from wild-type, heterozygous, and homozygous C/EBP mutant mice regarding the response of endoplasmic reticulum (ER) stress, although C/EBP is highly inducible with such stress. However, programmed cell death in response to ER stress is

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Figure 8.3. Function of C/EBP proteins during myeloid cell differentiation.

attenuated in C/EBP\\ MEFs. Such decreased programmed cell death due to C/EBP deficiency in response to ER stress has also been detected in an in vivo animal model. Furthermore, lack of C/EBP, the major C/EBP dimerization partner in ER stress, also showed the attenuation of programmed cell death in response to ER stress. These data indicate that C/EBP? is not a critical factor for the induction of ER stress response but plays an important role in the resulting programmed cell death following ER stress. There is no report on hematopoietic analysis in C/EBP\\ mice.

CONCLUSION C/EBPs are important transcription factors in regulating a variety of genes during different stages of cell proliferation, differentiation, and survival. Many studies have been done in different cell lineages, especially in myeloid cells, hepatocytes, and adipocytes. Although in vitro analyses have shown the redundancy of C/EBP proteins in regulating promoter activity, in vivo assays clearly demonstrate the critical role of certain C/EBP proteins in particular cellular events. C/EBP, -, and - are three C/EBP proteins that generate the most interest in the field of myeloid cell differentiation and function (Fig. 8.3). The further analyses of their expression, production, modification, cellular localization, and cooperation with other transcription factors will provide valuable information about

myeloid cell differentiation and about the mechanism of myeloid leukemogenesis, which will help us in designing strategies to take control of diseases associated with the myeloid lineage.

ACKNOWLEDGMENTS I would like to thank Drs. Daniel Tenen, Alan Friedman, Katya Ravid, Jonathan Licht, and Richard Schwartz for the valuable discussion or the critical reading of the manuscript.

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CHAPTER 9


HOMEOBOX GENE NETWORKS AND THE REGULATION OF HEMATOPOIESIS GUY SAUVAGEAU Laboratory of Molecular Genetics of hemopoietic stem cells, Institut de Recherches Cliniques de Montreal

R. KEITH HUMPHRIES Department of Medicine, University of British Columbia

H. JEFFREY LAWRENCE Department of Medicine, University of California

COREY LARGMAN Department of Medicine and Dermatology, University of California School of Medicine

INTRODUCTION The term homeobox takes its origin from the Drosophila homeotic mutations in which the identity of one body segment was transformed into that of another, such as the development of legs in place of antennae. Many of the genes involved in these mutations (such as Antennapedia, Bithorax, and others) share a conserved 180 nt region called the homeobox, which encodes a 60-amino-acid DNA-binding motif known as the homeodomain. Homeobox genes are expressed in precise temporal and spatial

patterns to guide morphogenesis of the Drosophila embryo. One cluster of homeobox genes, the HOM-C or homeotic cluster, is expressed in ordered domains along the anteriorposterior axis of the embryo. Remarkably, this parallels the linear 3 to 5 order of the genes in the HOM-C clusters. Homeobox genes are present in all animal genomes, including humans and mice, and play a conserved role in morphogenesis and tissue identity. X-ray crystallographic and NMR analyses indicate that the 60-amino-acid homeodomain region forms a helix-turn-helix structure, with


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HOMEOBOX GENE NETWORKS AND THE REGULATION OF HEMATOPOIESIS

Figure 9.1. Mammalian Hox genes are located in four different clusters (A to D) and grouped into 13 paralogous groups depending on their relative 3 to 5 location. Hox gene products from paralog 9 and 10 cooperatively bind DNA with PBX or with Meis.

three distinct -helical regions (Gehring et al., 1994). The third (or recognition) helix contacts DNA through the major groove and makes specific base pair contacts. Homeobox proteins bind to DNA with increased affinity in combination with partner proteins such as PBX (see below) and recently such structures were also solved (Piper et al., 1999). There are hundreds of homeobox-containing genes in mammalian genomes that can be subdivided into superclasses, classes, and families. The assignment of genes to the various groups depends on shared homology within the homeobox region and similarities in motifs outside the homeobox. Among the classes of homeobox genes expressed in hematopoietic cells are (1) the hexapeptide group, including the Hox family (see below), which all have an N-terminal 6amino-acid protein interaction domain; (2) the POU group, which includes the octomer family of factors (see Chapter 18 in this book), containing an N-terminal POU domain; (3) the LIM group, with an N-terminal LIM domain in addition to the homeodomain; (4) the TALE group, which includes the PBX and Meis proteins, with homeodomains containing three extra amino acids located at the c-terminal end of helix 1. In mammals, there are 39 genes is organized into four conserved clusters of 9—11 genes, each situated on a different chromosome (Fig. 9.1). The four clusters are lettered A, B, C, and D and individual genes are numbered according to their 3 to 5 order on the chromosome — for example, Hoxa1, Hoxb2, and so forth. Chromosomal order correlates with the spatial and temporal order of expression of these genes along body axes during development. Of note, differen-

tiation of teratocarcinoma cells with retinoic acid stimulates a wave of Hox gene expression that proceeds from the 3 to 5 ends of the Hox cluster. Genes in the same position on different clusters — for example, Hoxa4, Hoxb4, Hoxc4 and Hoxd4 — are called paralogs and have particularly high sequence identity among one another, indicating that they probably arose from a single ancestral gene. There are 13 such paralog groups; however, none of the four clusters contains members of all of the groups, indicating gene loss and/or incomplete duplication of the clusters during evolution. The clusters are quite compact, spanning only an average of :100 kb, and non-Hox homeobox genes, such as members of the evx (even-skipped) and dlx (distalles) groups, are located in the same chromosomal region. Mammalian Hox genes have significant sequence identity to those of the single HOM-C cluster found in insects. For example, the paralog 1 genes resemble the Drosophila labial gene, the genes in the center of the Hox cluster resemble Antennapedia, and the 5 Hox paralogs resemble the Abdominal-B gene in the fly. This suggests strong evolution pressure to maintain the specific, clustered genomic organization of homeobox genes. During murine embryogenesis, Hox genes are expressed in overlapping temporal and spatial patterns along the body axes, including the AP axis of the spine, the proximal-distal axis of the limbs, the gastrointestinal tract, and the genitourinary system. As in Drosophila, the anteriorposterior order of expression follows the 3 to 5 order of genes within a cluster. Hox knockout mice display multiple morphogenetic abnormalities involving the axial skeleton, limbs, great

Hox GENES IN NORMAL HEMATOPOIESIS

vessels, nervous system, and genitourinary tract. In general, (1) the abnormalities observed with Hox gene mutation are spatially restricted, generally to the most anterior or proximal region of the gene’s usual domain of expression, (2) the defects typically affect several different tissues in the same region; and 3) unlike the fly, Hoxdeficient mice rarely display true homeosis or transformation of one body part into another, and instead show loss of structures.

Hox GENES IN NORMAL HEMATOPOIESIS Gene Expression In Human Hematopoietic Cells Early studies of Hox gene expression centered on immortalized leukemic cell lines, in which lineage-restricted patterns of Hox gene activation were observed. Hox A genes tended to be expressed in myeloid cell lines, Hox B genes were active in erythroid cell lines, Hox C genes were active in lymphoid cells, and Hox D genes were generally silent. Hox gene expression was later found in normal marrow cells by sensitive RNase protection assays. Hox A and B cluster genes including Hoxa10 (Lowney et al., 1991), B6 (Shen et al., 1989), and other B cluster genes (Mathews et al., 1991) were detected. Even more sensitive RT-PCR assays of enriched subpopulations of bone marrow were required to demonstrate complex patterns of Hox gene expression in normal hematopoietic cells. In normal CD34> marrow cells, which contain hematopoietic stem cells and committed progenitors, at least 9 of the 11 Hox A genes, 8 of the 10 Hox B genes, and 4 of the 9 Hox C genes but no Hox D genes (Giampaolo et al., 1994; Moretti et al., 1994; Sauvageau et al., 1994) were expressed. CD34> cells can be sorted into three functionally distinct subpopulations including (1) those enriched for primitive hematopoietic cells detectable as long-term cultureinitiating cells (LTC-IC), (2) granulocyte/macrophage progenitors, and (3) erythroid progenitors. Hox gene expression was highest in the most primitive subpopulation (Sauvageau et al., 1994). Hox genes located at the 3 region of the clusters, such as Hoxb3 and Hoxb4, are downregulated at the committed progenitor stage, while genes at the 5 portion of the cluster (e.g.,

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Hoxa9, Hoxb9 and Hoxa10) continued to be expressed, until cells differentiated past the CD34> compartment. Thus, a large set of Hox genes are active in the most primitive hematopoietic cells, followed by a sequential repression of Hox genes that possibly follows their linear 3 to 5 chromosomal position. This suggests a role for Hox genes in early hematopoietic proliferation and differentiation. Giampaola and colleagues studied expression of the Hox B genes in CD34> peripheral blood cells in vitro under various culture growth conditions (Giampaolo et al., 1994). While only Hoxb3 was expressed when cells were initially purified, after as little as 24 hours in culture, Hoxb3 and several neighboring genes—Hoxb2, Hoxb4, Hoxb5 and Hoxb6 were upregulated. Hoxb6 activation was noted under conditions supporting granulocytic differentiation; Hoxb2 was prominently detected in late stages of erythropoiesis while Hoxb3—Hoxb5 were expressed under conditions favoring both granulocytic/ monocytic and erythroid differentiation. While differing in detail from the studies of fresh bone marrow, these studies also indicate that Hox genes may be dynamically regulated during early hematopoiesis. Hox Gene Expression in Lymphoid Cells Distinct patterns of Hox B and Hox C gene expression are apparent during lymphoid development. In quiescent B, T, and NK cells from normal peripheral blood, Hox B genes are not expressed (Care´ et al., 1994; Petrini et al., 1992; Quaranta et al., 1996). Activation of T cells with phytohemoglutinin or NK cells with IL-2 and IL-1 resulted in sequential activation of several Hox B genes. In NK cells, beginning as early as 6 hours. postactivation, Hoxb2 and Hoxb3 were detected with increased expression and subsequent induction of Hoxb4 andHoxb5 over the next few days (Quaranta et al., 1996). With PHA stimulation of T cells, expression of Hoxb1 and Hoxb2 peaked within 10 minutes; maximal induction of Hoxb3—Hoxb5 occurred up to 2 hours later while Hoxb6, Hoxb7 and Hoxb9 were expressed later with the onset of expression noted approximately 1 hour postinduction (Care´ et al., 1994). Again, sequential activation of 3 to 5 genes was observed, reminiscent of the pattern of activation of these genes in embryonic

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development or following retinoic acid treatment of embryonic cells (Simeone et al., 1990, 1991). Hoxc4 and Hoxc6 but not Hoxc5 were detected in mature B cells derived from peripheral blood and tonsillar tissue (Bijl et al., 1996). However, stimulation of B and T cells did not result in detectable Hoxc5 expression (Bijl et al., 1996). Others reported that Hoxc4 (Meazza et al., 1995) and Hoxc8 (Lawrence et al., 1993) were expressed in resting peripheral blood lymphocytes and increased with PHA activation (Meazza et al., 1995). Hoxb7 was also found in activated B and T lymphocytes, the thymic cortex, and germinal centers of the adult tonsil (Deguchi and Kehrl, 1991b). Hoxa9, but not Hoxa10, also is expressed in early T- and B-cell subpopulations of mice (Lawrence et al., 1997; Thorsteinsdottir et al., 1997). Within T-cell subsets, Hoxa9 was detectable (at very low levels) in CD4>CD8> and CD4> cells. Although these expression studies are still somewhat limited, it is apparent that, as for myeloid cells, multiple members of the A, B, and C Hox cluster are differentially expressed within lymphoid cells. Expression is low in quiescent mature cells but stimulated during mitogen-induced activation. Furthermore, expression of these genes changes during early lymphoid development. Hox Expression In The Murine Model Limited data from mice indicate that Hox genes are expressed in a stage-specific manner during hemopoiesis. Several A, B, and C cluster Hox genes are expressed in early mouse yolk sac coincident with the onset of hematopoiesis (McGrath and Palis, 1997). Hoxb6 expression is detected in active sites of erythropoiesis throughout embryogenesis. Specific expression of Hoxb6 was noted in erythroid progenitors but not in multipotent hematopoietic stem cell populations (Zimmermann and Rich, 1997). This is consistent with the preferential expression of Hox B cluster genes in erythroid leukemic cell lines and the absence of expression of Hoxb6 in various CD34> subpopulations (Sauvageau et al., 1994). However, these data contrast with those associating Hoxb6 expression with granulocytic/monocytic growth of human peripheral blood cells (Giampaolo et al., 1994). There is less information regarding expression

of Hox genes in adult murine hematopoietic cells. Expression of Hoxa7 and Hoxb7 was found in the marrow and spleen of adult mice (Kongsuwan et al., 1988). To test for conservation of patterns of Hox gene expression between human and murine hematopoietic cells, we examined expression of two 3 genes, Hoxa4 and Hoxb4, and two 5 genes, Hoxa9 and Hoxa10, in purified hematopoietic subpopulations. All four genes were expressed in adult murine bone marrow and fetal liver cells. In addition, all four Hox genes were detected in rare Sca-1>lineage\ cells that include hematopoietic stem cells. Significantly lower levels were found in progenitor depleted fractions (e.g., Sca-1-lin>) (Pineault et al., 1997; N. Pineault and R. K. Humphries, unpublished observations). Together these data indicate that Hox genes are differentially expressed in primitive murine hematopoietic cells in fetal and adult stages of development. Since these studies were performed with cell populations, there remains the possibility of significant heterogeneity of Hox gene expression at the single cell level. Furthermore, these studies have not established the role of Hox genes in lineage specification. In contrast to findings with leukemic cell lines, analysis of normal human marrow did not reveal lineagerestricted patterns of Hox gene expression. However, minor contamination of granulocytic versus erythroid subpopulations could blur lineage—related expression patterns. Use of singlecell PCR-based techniques (Brady et al., 1990; Voura et al., 1997; Ying et al., 1999) or in situ hybridization may allow further resolution of Hox gene expression in different cells reflecting distinct stages of the hematopoietic hierarchy.

Expression Of Noncluster Homeobox Genes Homeobox genes outside of the Hox cluster are also expressed in normal hematopoietic cells. Several novel homeobox genes were isolated by RT-PCR analysis of murine marrow or CD34>enriched human marrow cells utilizing degenerative oligonucleotide primers complimentary to conserved regions of the homeobox (Bedford et al., 1993; Moretti et al., 1994). One such homeobox gene, Hex, was isolated from mouse

Hox GENES IN NORMAL HEMATOPOIESIS

bone marrow and peripheral blood (Bedford et al., 1993). Hex expression is restricted to the hematopoietic system including myeloid and Blineage cells. By a similar approach, other divergent homeobox genes were isolated from RNA derived from CD34> human bone marrow cells (Bedford et al., 1993). Another variant homeobox gene, initially called HB24 and now designated HL X, is expressed in mitogen-stimulated human B lymphocytes (Deguchi and Kehrl, 1991a). Later HL X, was shown to be expressed in CD34> but not more differentiated CD34\ cells, in a pattern similar to that obtained for the clustered Hox genes (Deguchi and Kehrl, 1991b; Sauvageau et al., 1994). Like many Hox genes, HL X was found to be inducible in both B and T lymphocytes. Engineered expression of HL X in the lymphoid lineage of transgenic mice yielded marked perturbations in B- and T-cell development (Deguchi et al., 1993), and HL X was tumorigenic when overexpressed in a human T-cell line (Deguchi and Kehrl, 1993). While antisense inhibition of HL X blunted proliferation of CD34> cells, transient overexpression of HL X impaired differentiation into mature hematopoietic cells (Deguchi et al., 1992), suggesting that HL X has a direct physiologic role in hematopoiesis. HL X null mice died at day 15 postconception with anemia and severe hypoplasia of the liver and gut. The block to hematopoiesis, however, appeared to be extrinsic, since HL X\\ hematopoietic cells behaved normally in vitro and upon transplantation (Hentsch et al., 1996).

Dissecting Hox Gene Function In Normal Hematopoiesis: Loss-Of-Function Studies A major strategy to assess the role of homeobox genes in hematopoiesis has been to utilize knockout mice to examine the impact of gene deletion on blood cell development. The Hoxa9 knockout yielded the best-characterized hematopoeitic phenotype for a Hox cluster gene to date. These mice were viable and fertile and had only mild skeletal defects, but showed defects in T and B cells and granulocytes. As noted above, Hoxa9 is expressed in CD34>Lin\ human hematopoietic cells as well as in more mature CD34> committed progenitor cells. Hoxa9 knockout mice had normal hematocrit and

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platelet counts but showed consistent, modest reductions in peripheral blood granulocytes, T cells and B cells. In addition, these mice had a reduced spleen size and a 2- to 3-fold decrease in marrow myeloid and pre-B-cell progenitors. Although their basal peripheral blood granulocyte counts were only mildly reduced, these mutant mice exhibited a blunted response to G-CSF (Lawrence et al., 1997). Adult Hoxa9\\ mice also showed a 2-fold decrease in thymocytes, while day 15.5 fetal thymuses were reduced 5- to 10-fold. Fetal thymocytes from Hoxa9\\ mice showed a profound delay in progression to CD4>CD8> T cells in fetal thymic organ cultures (Izon et al., 1998). This defect was associated with decreased expression of the IL-7 receptor and E-cadherin, downregulation of bcl-2, and increased apoptosis. Study of more pluripotent hematopoietic precursors in Hoxa9 mutant mice, as assayed by day 12 CFU-S or in vitro assays for long-term culture-initiating cells (LTC-IC), suggested that there was no defect in earlier hematopoietic cells. However a number of in vivo assays did indicate that these mice had defects in their hematopoietic stem cells (Lawrence et al., manuscript in preparation). Hoxa9\\ mice showed prolonged suppression of hematopoiesis following sublethal irradiation. Furthermore, competitive repopulation assays demonstrated that the marrow derived from Hoxa9 null animals had a 10-fold or greater defect in ability, compared to wild-type marrow, to reconstitute the lymphomyeloid compartments of lethally irradiated syngeneic mice. Thus, loss of Hoxa9 function affects hematopoiesis at the level of the stem cell as well as the committed progenitor. Whether this defect is due to a qualitative or quantitative defect in the stem cell is not yet certain. Characterization of hematopoiesis in other Hox knockout mice has been less revealing. Initial reports indicated the presence of mild erythroid abnormalities in Hoxc8-deficient mice and mild myeloid abnormalities in Hoxa10\\ animals. No major defects were found in homozygous mutants for Hoxb3 or Hoxc4 (Lawrence et al., unpublished data). Hoxb4\\ mice die perinatally with multiple skeletal defects and develop a macrocytic anemia late in fetal development. However, fetal liver progenitors from these animals can restore long-term

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normal hematopoiesis after transplantation into irradiated syngeneic animals. Therefore, the anemia in the primary animals may be secondary to other developmental defects, or may represent a mild qualitative defect in erythroid development. The lack of major hematopoietic defects in mice mutant for a single Hox gene (except for Hoxa9, above) suggests functional redundancy between Hox genes. The redundancy could be among paralogs in different clusters or between adjacent genes on the same cluster. Often, skeletal defects of compound mutants are more severe than those observed in single mutants. If this situation holds for the hematopoietic system, then the examination of compound mutants would likely reveal more significant abnormalities in blood development. Use of antisense oligonucleotides to downregulate Hox gene expression also provided evidence for a role of these genes in hematopoietic cells. Exposure of T cells to antisense Hoxb2 orHoxb4 (Care´ et al., 1994) or NK cells to antisense Hoxb2 oligonucleotides (Quaranta et al., 1996) blunted mitogen-induced proliferation and expression of activation markers. Erythropoiesis and myelopoiesis were also affected in several similar experiments. Antisense oligonucleotide-mediated inhibition of Hoxb7 expression was associated with a reduction in numbers of murine CFU-GM but not of BFU-E and CFUMK (Wu et al., 1992). Inhibition of Hoxc6 expression in human bone marrow cultures suppressed the growth of CFU-E but not BFU-E or myeloid progenitors (Takeshita et al., 1993). Hox gene suppression can also yield heterogeneous effects. Giampaolo and colleagues (1994) examined colony formation from human peripheral blood cells following exposure to antisense oligonucleotides to various Hox B genes. While an anti-Hoxb3 or to a lesser extent anti-Hoxb4 and Hoxb5 oligonucleotide inhibited both erythroid and myeloid colony formation, antiHoxb6 more specifically inhibited myeloid colony formation, and anti-Hoxb2, Hoxb7, and Hoxb9 had no significant effect. Although the specificity and magnitude of Hox gene suppression achieved in these studies is uncertain, together they indicate diverse roles for Hox genes, potentially in a stage- and lineage-specific manner, throughout the hierarchy of hematopoietic development.

Dissecting Hox Gene Function In Normal Hematopoiesis: Overexpression Studies The complementary strategy to determine homeobox gene function in blood cell development has been a gain of function experiments. In most cases, retrovirus was used to engineer the overexpression of specific Hox genes in primary mouse bone marrow. The first such report involved the overexpression of Hoxb8 (Perkins et al., 1990; Perkins and Cory, 1993). Hoxb8-transduced marrow yielded a relatively normal spectrum of colony types in culture except for the appearance of an unusual type of colony characterized by a compact center, a diffused halo, and an increased ability of these cells to form secondary colonies. Progenitors from these transduced marrows remained growth factor dependent and did not yield cell lines in the absence of a very high dose of IL-3. Animals transplanted with Hoxb8-transduced bone marrow had elevated myeloid progenitors but otherwise were normal. Our own studies involved overexpression of Hoxb3, Hoxb4, Hoxa9, and Hoxa10. All four genes are normally expressed in CD34> human marrow cells with expression of Hoxb3 and Hoxb4 restricted to the most primitive subpopulations (Sauvageau et al., 1994). Overexpression of Hoxb4 did not significantly affect the cellularity of the bone marrow and spleen. The numbers and distribution of peripheral blood cells were normal, and only mild—less than 2-fold—elevations in the myeloid or B-cell progenitor compartments were seen (Sauvageau et al., 1995). Furthermore, mice transplanted with these cells did not develop leukemia or marrow failure, even after extended periods of observation. However, dramatic expansion of the stem cell compartment was evident as determined by in vivo—limiting dilution assays for competitive repopulating cells (CRU) (Szilvassy et al., 1990). Hoxb4-transduced marrow yielded 50-fold higher levels of hematopoietic stem cells compared to those of control transplant recipients. Studies for up to 12 months indicated that Hoxb4 was a positive regulator of the expansion of the HSC pool but did not affect the differentiation or lineage choice of the HSC or the controls that limit progenitor and mature cell numbers (Thorsteinsdottir et al., 1999b).

Hox GENES IN LEUKAEMIA

Transplantation experiments with Hoxb3transduced bone marrow yielded very different results (Sauvageau et al., 1997), including marked reduction in thymic size. This was due to a drastic reduction of CD4>CD8> thymocytes. In contrast, there were marked increases in CD4\CD8\ T-cell receptor -positive thymocytes. Hoxb3 overexpression was also associated with reduced numbers of pre-B-cell progenitors, a large increase in myeloid progenitors, and an enlarged spleen consistent with the development of a myeloproliferative syndrome (leading to AML; U. Thorsteinsdottir et al. MCB, accepted). Overexpression of Hoxa10 results in yet a different set of hematological disturbances (Thorsteinsdottir et al., 1997). While the T-cell lineage was unaffected, there was a marked reduction in pre-B-cell progenitors and an almost complete loss of committed macrophage progenitors. Hoxa10 overexpression was also associated with the appearance of large numbers of megakaryocyte/ blast progenitors, which after 20 weeks evolved into acute myeloid leukemia (see next section). In addition, most Hox genes described above greatly increased the recovery of day 12 CFU-S following short-term in vitro culture of infected bone marrow cells (Sauvageau et al., 1995; Thorsteinsdottir et al., 1997). Taken together, these studies suggest that Hox genes have overlapping effects on the proliferation of primitive hematopoietic cells but may also exert genespecific effects on lineage choice and/or progression. Functional Studies In The In Vitro ES Differentiation Model The in vitro model of blood development after embryonic stem cell (ES) differentiation was also used to study the biologic function of Hox genes. ES cells can be induced to form embryoid bodies which contain a number of differentiated tissues, including blood. ‘‘Replating’’ of these cells under specific conditions can recapitulate many aspects of early blood development and provide a ready means for studying the hematopoietic consequences of engineered alterations in gene expression (Kabrun et al., 1997). Overexpression of Hoxb4 in ES cells after retroviral transduction resulted in a 6 to 10 fold

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increase in mixed erythroid/myeloid colonies and definitive, but not primitive, erythroid colonies. There was no effect on the efficiency of embryoid body formation or growth rate nor on the generation of granulocytic or monocyte progenitors. Overexpression of Hoxb3 resulted in the enhancement of the number of multipotential progenitors up to 75 fold. In contrast, Hoxa10 overexpression had its greatest effect at later stages of differentiation resulting in an increase in granulocyte/macrophage progenitors (Helgason et al., 1996; Helgason et al., 1997). Multipotential hematopoietic cell lines were derived from Hoxa10-transduced ES cells by isolating colonies arising from day 10 or 12 embryoid bodies. Similarly, Keller et al. derived factor-dependent hematopoietic cell lines representing novel stages of embryonic hematopoiesis from ES cells transduced with a retrovirus harboring the HOX11/TCL3 gene (Keller et al., 1998). Multipotent hematopoietic precursor cell lines responsive to Steel/Stem cell factor were also obtained after retrovirus-mediated overexpression of the LIM homeobox gene LH2 (Pinto do O et al., 1998). These experiments highlight the potential of using the non-transformed ES cell system to dissect the Hox regulatory network in early blood cell development, and to determine how alterations of this network may lead to leukemia.

Hox GENES IN LEUKAEMIA Hox Genes Involved In Mouse Acute Leukemia The first link between aberrant Hox gene expression and leukemia was obtained from genetic analysis of the murine WEHI-3B leukemic cell line. This cell was found to contain activating retroviral integrations adjacent to the Hoxb8 and interleukin-3 (IL -3) genes. Mice transplanted with bone marrow transduced with a retrovirus to simultaneously overexpress Hoxb8 and IL -3 die from an aggressive, polyclonal acute myeloid leukemia (AML), whereas no acute disease was detected in recipients’ marrow transduced with either Hoxb8 or IL -3 alone (Perkins et al., 1990). However, more recently, it was shown that a high proportion of mice transplanted with marrow engineered to

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overexpress Hoxb8, Hoxa9, Hoxa10, or Hoxb3, but not Hoxb4, will eventually develop acute myeloid leukemia after several months of latency (Thorsteinsdottir et al., 1997, 1999; Sauvageau et al., 1995, 1997; Kroon et al., 1998). The long latent period suggests the requirement of at least a second genetic event in Hoxinduced leukemic transformation. The time required for clinical presentation of leukemia varies considerably from one Hox gene to another. For example, even after 1 year of follow-up, mice reconstituted with Hoxb4-transduced bone marrow do not develop AML. In contrast, recipients of Hoxa10-tranduced cells succumb to AML as early as 3 months posttransplant and most animals will die of the disease within 12 months. Other studies similarly indicated that 100% of Hoxa9 and Hoxb3 recipients will die of AML within a year after transplantation (U. Thorsteinsdottir et al., MCB, accepted). Collaborator Oncogenes In Hox-Induced Leukemias The monoclonal nature and long latent period of Hox-induced leukemias indicate that Hox gene products do not by themselves acutely transform primary bone marrow cells and thus must cooperate with other oncogenes. IL -3 and Meis1 are the only two oncogenes currently known to collaborate with Hox (b8 and a9, respectively) in transformation. Meis1 was initially identified as a gene adjacent to a common retroviral integration site in acute leukemias that developed in BXH-2 mice (Moskow et al., 1995). Meis1 is frequently co-overexpressed with Hoxa9 or Hoxa7 in these leukemic cells (Nakamura et al., 1996b). More recently, it was demonstrated by retroviral gene transfer that Meis1 and Hoxa9 collaborate in leukemic transformation of primary bone marrow (Kroon et al., 1998). Hoxa9 gene is of particular interest because until recently it was the only Hox gene to be disrupted by a recurrent chromosomal translocation in a subgroup of human leukemia (Borrow et al., 1996; Nakamura et al., 1996a). Hoxa9 transformation may occur in collaboration with one of its DNA-binding cofactors (Meis1), possibly due to the more effective regulation of a set of critical target genes by combined action of these two proteins (see below). Collaboration between Hoxa9 and Meis1 is specific, since the expression of Hoxa9 with another

DNA-binding partner, Pbx1, does not acutely transform bone marrow. This situation is intriguing since Pbx1 can collaborate with other Hox gene products such as Hoxb3 or Hoxb4 to transform Rat-1 fibroblasts (Krosl et al., 1998). This indicates a potential combinatorial code for Hox gene action, and, as shown in Figure 9.1, suggests that Meis1 and Pbx1 may induce transformation when coexpressed with distinct Hox partners. The Importance Of Hox Genes In Human leukemia Relatively recently it was recognized that Hox genes might be involved in human hematopoietic malignancies. First Hoxa9 was shown to be fused to the NUP98 gene in the t(7;11)(p15;p15) translocation found in some myeloid leukemia patients (Nakamura et al., 1996a; Borrow et al., 1996). More recently, Hoxd13 was also identified as an alternative fusion partner with NUP98 in a therapy-related leukemia (Raza-Egilmez et al., 1998). The presence of a NUP98-Hox fusion protein is therefore a recurring theme in a small percentage of cases of AML. In addition, leukemias that involve Hox DNA-binding partners such as t(1;19), yielding the E2a-PBX1 protein (see Chapter 00 in this book), could affect the expression or function of Hox genes (Chang et al., 1997). In addition to its involvement in human and mouse leukemia, Hoxa9 (overexpression?) was recently shown to be the single most highly correlated gene (out of 6817 genes tested) to poor prognosis in human AML (Golub et al., 1999), thus suggesting a role for Hox genes in human leukemia beyond their simple implication in chromosomal translocations. A brief summary of Hox gene involvement in leukemia is provided in Table 9.1.

MOLECULAR BIOLOGIC ASPECTS OF THE REGULATION OF Hox GENES Upstream Regulators of Hox Gene Expression Retinoids and Retinoic Acid Receptors. The observation 10 years ago that low doses of retinoic

MOLECULAR BIOLOGIC ASPECTS OF THE REGULATION OF Hox GENES

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TABLE 9.1. Hox Genes and Leukemia/Lymphoma

Genes Human diseases PBX1 TCL3 Hoxa9 Hoxd13 Mouse diseases Hoxb8 Hoxb3 Hoxa9 Hoxa10

Genetic Anomalies

Types of Disease

t(1;19)E2a-PBX1 t(10;14) TCR-TCL3 t(7;11) NUP98-HOXA9 t(2;11)NUP98-HOXD13

Pre-B ALL T ALL AML (M2 or M4) MDS-AML

Proviral activation Retroviral overexpression Retroviral overexpression Retroviral overexpression

AML (;IL-3) AML AML (;Meis or ;E2a-PBX1) AML

acid (RA) induce the ordered, sequential expression of Hox genes in embryonic carcinoma cells suggested that retinoids and retinoic acid receptors (RARs) (see Chapter 20 in this book) might affect development through their effects on Hox gene activation. So far, there are two classes of nuclear receptors identified which mediate retinoid signaling. They are the Retinoic Acid Receptors (RARs) and Retinoid X Receptors (RXRs). Both groups of nuclear receptors mediate the morophogenetic signals provided by retinoids by binding in various combinations to the regulatory regions of key developmental genes. The transcriptional effects of retinoic acid receptor—DNA binding are dependent on whether the retinoic acid ligand is bound, and the exact DNA target sequence. Retinoic acid response elements (RAREs) were identified at the 3 end of the Hox A and B clusters and adjacent to single Hox genes. These sites appear to mediate RA-dependent activation of the adjacent Hox genes. Analysis of knockout mice and knockout embryonic carcinoma cell lines indicates that specific RARs and RXR are required for activation of Hox A versus Hox B genes. Retinoids are believed to play a role in hematopoiesis, and particularly in granulopoiesis. It is possible that the effects of retinoids on myelopoiesis are mediated by the activation of Hox genes through specific retinoic acid receptors. However, there are no published data showing that RA activates Hox genes in hematopoietic cells. Furthermore, knockout mice deleted for single RARs did not display any significant hematopoietic defects. Even compound RAR

mutant mice showed only subtle deficits in myelopoiesis (Labrecque et al., 1998). These findings may simply reflect functional redundancy within the RAR/RXR family, and perhaps other complex, compound RAR/RXR mutants will show hematologic defects. Trithorax. In Drosophila, the spatially restricted pattern of expression of homeotic genes is initiated by combined action of the pair-rule (activators) and gap (repressors) classes of segmentation genes. The maintenance of HOMC gene expression during later stages of development depends on the products of the trithorax (trx-G) and Polycomb group (PcG) loci (reviewed in Simon, 1995). In the fly, trithorax is required to maintain HOM-C genes in an activated state. The mammalian homolog of this gene known as AL L, ML L, or HRX was identified as a gene on chromosome 11q23 frequently rearranged in a variety of human leukemias, including biphenotypic leukemias seen in infants and etoposide-induced leukemias. Over 20 different genes may be rearranged with ML L in human leukemia (see Chapter 26 in this book). ML L knockout mice die in utero with a variety of morphogenetic defects associated with alterations in the expression patterns of Hox genes along the vertebral axis. These mice are anemic and have a marked reduction in colony-forming cells, although they possess the full array of erythroid, myeloid, monocytic, and megakaryocytic cells. This suggested that MLL influenced the growth of early hematopoietic precursors but did not regulate specific lineage determination.

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Polycomb Genes. The Polycomb (PcG) genes of Drosophila generally act in opposition to the effects of Trx, to maintain inactive HOM-C genes in a repressed state. Drosophila PcG mutants display pleiotropic phenotypes including aberrant body patterning, neural development and hematopoiesis (Breen and Duncan, 1986; Girton and Jeon, 1994; Ingham, 1984; Santamaria and Randsholt, 1995; Smouse et al., 1988). Severe hypomorphic alleles of the multisex comb (mxc) gene result in premature hemocyte differentiation and tumorous overgrowth of the larval hematopoietic organs (Gateff, 1978; Gateff and Mechler, 1989). This is particularly intriguing, since loss of Polycomb function would be expected to lead to inappropriate activation of some HOM-C genes. Analogously, as described above, engineered overexpression of Hox gene in murine marrow can clearly cause leukemia. There are several different mammalian PcG genes, many of which were cloned in mammalian cells because of their role in hematopoiesis. Among the first murine PcG mouse genes to be described were bmi1, which was cloned as a common site for retroviral integration in lymphomas developing in mice transgenic for c-myc (Haupt et al., 1991; van Lohuizen et al., 1991) and enx-1, which was isolated as a protein that interacts with Vav (Hobert et al., 1996). Bmi1 knockout mice display a progressive aplastic anemia with replacement of marrow by adipocytes. Furthermore, these animals have defects in T- and B-cell development, and reduced numbers of myeloid progenitors, findings reminiscent but more severe than those of Hoxa9\\ mice. Mice mutant for a bmi-related protein called mel-18 also have hematopoietic defects, including severe abnormalities in IL -7— responsive B-cell progenitors. Two other PcG gene mutant mice were reported. M33 mutant mice have profound defects in proliferation of several cell types including mature B and T cells. Rae-28/Mph-1 mutant mice die in the perinatal period with multiple anomalies including thymic hypoplasia, again showing a potential role for PcG gene products in the regulation of hematopoiesis. PcG genes may also be involved in hematopoietic malignancies. For example, transgenic mice engineered to overexpress bmi1 in the T or B lineage (Haupt et al., 1993; Alkema et al., 1997) developed lymphomas.

A study of the expression of PcG genes in purified subpopulations of human marrow cells showed that of nine PcG genes analyzed, eight were preferentially expressed in differentiated (i.e., CD34\) bone marrow cells, and one, bmi1, was preferentially expressed in a primitive subpopulation of bone marrow cells (i.e., CD34>CD71\CD45RA\) (Lessard et al., 1998). The expression profile of the majority of these PcG genes is consistent with a role for these genes in repressing the Hox cluster as Hox genes are preferentially expressed in primitive hematopoietic stem cells and progenitors. Recent functional studies have shown lympho- and myeloproliferative diseases in eed mutant mice, thus suggesting that this gene product may perform tumor-suppressive function in bone marrow cells. Moreover, a genetic interaction was identified between eed (suppressor of proliferation) and bmi1 (enhancer of proliferation) in B-cell precursors, suggesting that proliferation of certain bone marrow precursors is controlled by the combined action of PcG genes present in different complexes (complex A: eed and enx versus complex B: bmi1, mel-18, mph1, etc.) (Lessard et al., 1999). Homeobox Proteins--DNA Interactions Hox proteins are DNA-binding transcriptional regulators. Their mode of interaction with DNA and the identification of their target genes is critical for the understanding of their role in hematopoiesis. Although the isolated 60 amino acid homeodomain can bind DNA (Muller et al., 1988), many full-length homeodomain proteins have low inherent affinity for DNA (Shen et al., 1996). Furthermore, the 13 paralog groups of Hox proteins show little specificity, and most bind to the TAAT consensus site (Mann, 1995; Pellerin et al., 1994). It was hard to explain how Hox proteins, with their inherent properties, could direct the expression of specific downstream genetic events until the observation, first made in Drosophila (Chan et al., 1994; van Dijk and Murre, 1994), that Hox proteins bind cooperatively to DNA with the PBX/EXD family of homeodomain proteins (Lu et al., 1995). PBX1 was discovered as the fusion partner with the E2a gene in the t(1,19) associated with pre-B-cell leukemia (Kamps et al., 1990; Nourse et al., 1990). Three PBX genes, encoding a

MOLECULAR BIOLOGIC ASPECTS OF THE REGULATION OF Hox GENES

number of alternatively spliced protein products, were subsequently identified (Monica et al., 1991). All contain a homeodomain and MEIS interaction motif (Chang et al., 1997b), and interact with Hox proteins through a conserved Hox interaction domain (Chang et al., 1997b). PBX proteins do not bind strongly to DNA alone, but instead bind cooperatively with Hox proteins from paralog groups 1 through 10 (Chang et al., 1996; Shen et al., 1997). Interactions with PBX provide distinct DNA-binding specificity to each Hox protein paralog (Chang et al., 1996; Shen et al., 1997). Hox paralog proteins 1 through 8 bind to PBX through a tryptophan within a conserved YPWM motif (Lu and Kamps, 1996; Neuteboom et al., 1995; Phelan et al., 1995; Shen et al., 1996), while paralog 9 and 10 proteins use a tryptophan in a conserved ANW interaction domain (Shen et al., 1997). In addition, the non-Hox TCL3 protein (former Hox11), which is present in T-cell leukemias containing the t(10;14) translocation, also contains a YPWM-like motif and can cooperatively bind to DNA with PBX (Shen et al., 1996). The Hox9 and Hox10 proteins fall within the Abd-B—like subgrouping that encompasses paralogs 9 through 13. Proteins from Hox paralogs 11—13 do not bind DNA in cooperation with PBX (Shen et al., 1997), but instead these proteins bind to the MEIS1 homeodomain proteins (Shen et al., 1997) described above (see also Fig. 9.1). Although the MEIS proteins were initially described as Hox protein partners, later studies showed that in nonhematopoietic cells MEIS1 forms heterodimeric DNA-binding complexes with PBX (Chang et al., 1997b; Knoepfler et al., 1997). Several other MEIS-related proteins were described (Steelman et al., 1997; Nakamura et al., 1996b; Berthelsen et al., 1998a), some of which cooperatively bind DNA in either dimeric complexes with PBX (Berthelsen et al., 1998a), or in trimeric complexes with Hox or Hox-related proteins (Berthelsen et al., 1998b; Swift et al., 1998). Finally, recent studies in myeloid leukemia cell lines indicated that Hoxa9 forms trimeric complexes with PBX2 and MEIS1 (Shen et al., 1999). Although all three genes are expressed in normal myeloid cell populations, the presence of similar complexes has not yet been demonstrated. Together, these studies suggest a complex combinatorial pattern

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of interaction and competition among Hox and non-Hox homeodomain proteins. Both Hox and Meis genes are expressed in lineage- and stagespecific patterns in hematopoietic cells, while PBX genes are expressed in all hematopoietic cell types. Since each of the heterodimeric PBXHox, MEIS-Hox, or PBX-MEIS complexes bind to unique DNA sequences, the proposed competition for dimeric- and trimeric-binding partners would yield opportunities for diverse patterns of cell-specific gene regulation that may be critical for the ability of homeodomain proteins to regulate hematopoietic cell growth and differentiation. Target Genes Although homeodomain proteins are presumed to act as transcription factors, little is known about direct downstream target genes of these proteins in any cell type. Several classes of candidate genes were identified in Drosophila, including genes involved in cell shape and adhesion such as tubulin, centrosomin, and connectin, a GPI-linked adhesion molecule. Also suggested as targets have been other transcription factors, for example, homeobox genes such as Distal-less and empty spiracles, and genes encoding other classes of transcriptional factors including forkhead, nervy, and teashirt. Lastly, genes encoding signaling molecules including decapentaplegic (dpp), the Drosophila homolog of the TGF/BMP family, and wingless (Wg), the homolog of the mammalian Wnt gene family, may be Hox targets. In Drosophila, genetic evidence has implicated the Hox genes as both activators and repressors; however, in the absence of biochemical data it is uncertain whether these are direct or indirect effects. Even less is known about the genetic targets of vertebrate homeodomain proteins. In vertebrates, the only unequivocal targets of the Hox proteins are the Hox genes themselves, which are controlled in a variety of auto- and crossregulatory circuits. The autoregulatory circuits described to date are stimulatory, whereas the cross-regulatory circuits may either stimulate or inhibit Hox gene expression. Other putative candidate targets include those encoding adhesion molecules, such as members of the cadherin family (Goomer et al., 1994), and genes for peptide growth factors, such as bFGF (Care´ et

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al., 1996). For example, Hoxd9 protein positively regulates the chicken E-cadherin gene through a regulatory element in its second intron. This observation is intriguing as primitive thymocytes from mice deleted for Hoxa9, a paralog of Hoxd9, express only low levels of E-cadherin. Hoxa7 may regulate bFGF, an interesting finding in view of the role of the latter molecule in hematopoiesis. A great deal of work will be required to delineate the direct targets of homeodomain proteins. Furthermore, it will be quite difficult to prove the authenticity of a gene as a Hox protein target because (1) it was proposed that a given homeobox gene can directly regulate hundreds if not thousands of individual genes and (2) homeodomain proteins may not function as conventional activators or repressors, and hence their function may not be measured by conventional transcriptional assays.

CONCLUSIONS AND DIRECTIONS Homeobox genes, and Hox genes in particular, are expressed in complex stage and potentially lineage-specific patterns in normal hematopoietic cells. Perturbation of Hox gene expression yields a wide array of hematopoietic abnormalities. Many critical questions remain. A more precise determination of the pattern of Hox gene expression at different stages of hematopoietic development, using highly purified subpopulations (preferably at the single-cell level), is required. Such analysis may reveal checkpoints and branch points in the hematopoietic tree that may be controlled by Hox genes. The redundancy of the Hox gene clusters suggests that the true biologic function of a particular paralog or colinear cluster will be revealed only when multiple genes are knocked out. In this vein, we are currently analyzing mice deleted for the entire Hox B cluster for hematopoietic defects (J. Bijl, R. Solis, and GS). We are also embarking on a project to knock out each member of a single paralog group with the eventual aim of mating these mice to yield compound null animals. One obvious mystery is the identity of the downstream target of the Hox proteins in hematopoietic cells. It is unclear whether such targets will be specific for hematopoietic cells or instead

serve general biologic functions related to cell proliferation, growth, and adhesion. And finally it is not yet known whether homeobox proteins are activators or repressors of transcription in vivo or whether they serve some alternative function related to higher-order chromatin structure. Moving beyond the role of homeobox genes in normal hematopoiesis, there is now strong evidence for their involvement in murine and human leukemia. As suggested in a recent review by T. Look (1997), deregulation of Hox gene expression and/or function may represent a common final pathway for the leukemic transformation by several oncogenes, including the chimeric and overexpressed transcription factors generated by chromosomal translocations in human leukemia. Examples include E2a-PBX, PML-RAR, and the numerous MLL fusion proteins. If this speculation is correct, then strategies to interrupt Hox protein-protein or Hox protein-DNA interactions could offer a novel therapeutic approach for human malignancies.

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Quaranta, M. T., Petrini, M., Tritarelli, E., Samoggia, P., Care´, A., Bottero, L., Testa, U., and Peschle, C. (1996). HOXB cluster genes in activated natural killer lymphocytes. J Immunol 157, 2462—2469. Raza-Egilmez, S. Z., Jani-Sait, S. N., Grossi, M., Higgins, M. J. , Shows, T. B., and Aplan, P. D. (1998). NUP98—HOXD13 gene fusion in therapy-related acute myelogenous leukemia. Cancer Res. 58, 4269—4273. Santamaria, P. and Randsholt, N. B. (1995). Characterization of a region of the X chromosome of Drosophila including multisex combs (mxc), a polycomb group gene which also functions as a tumour suppressor. Mol. Gen. Genet. 246, 282— 290. Sauvageau, G., Lansdorp, P. M., Eaves, C. J., Hogge, D. E., Dragowska, W. H., Reid, D. S., Largman, C., Lawrence, H. J., and Humphries, R. K. (1994). Differential expression of homeobox genes in functionally distinct CD34> subpopulations of human bone marrow cells. Proc. Natl. Acad. Sci. USA 91, 12,223—12,227. Sauvageau, G., Thorsteinsdottir, U., Eaves, C. J., Lawrence, H. J., Largman, C., Lansdorp, P.M., and Humphries, R.K. (1995). Overexpression of HOXB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo. Genes Dev. 9, 1753—1765. Sauvageau, G., Thorsteinsdottir, U., Hough, M. R., Hugo, P., Lawrence, H.J., Largman, C., and Humphries, R.K. (1997). Over-expression of HOXB3 in hematopoietic cells causes defective lymphoid development and progressive myeloproliferation. Immunity 6, 13—22. Shen, W.F., Largman, C., Lowney, P., Hauser, C., Simonitch, T.A., Hack, F.M., and Lawrence, H.J. (1989). Lineage-restricted expression of homeobox-containing genes in human hematopoietic cell lines. Proc. Natl. Acad. Sci. USA 86, 8536—8540. Shen, W.F., Chang, C.P., Rozenfeld, S., Sauvageau, G., Humphries, R.K., Lu, M., Lawrence, H.J., Cleary, M.L., and Largman, C. (1996). Hox homeodomain proteins exhibit selective complex stabilities with Pbx and DNA. Nucl. Acids Res 24, 898—906. Shen, W.F., Montgomery, J.C., Rozenfeld, S., Moskow, J.J., Lawrence, H.J., Buchberg, A.M., and Largman, C. (1997). The AbdB-Like proteins stabilize DNA binding by the Meis1 homeodomain proteins. Mol. Cell. Biol. 17, 6448—6458. Shen, W.F., Rozenfeld, S., Kwong, A., Kom, V. L., Lawrence, H.J., and Largman, C. (1999). HOXA9 forms triple complexes with PBX2 and MEIS1 in myeloid cells. Mol. Cell. Biol. 19, 3051—3061. Simeone, A., Acampora, D., Arcioni, L., Andrews, P.W., Boncinelli, E., and Mavilio, F. (1990). Sequential activation of HOX2 homeobox genes by retinoic acid in human embryonal carcinoma cells. Nature 346, 763—766. Simeone, A., Acampora, D., Nigro, V., Faiella, A., D’Esposito, M., Stornaiuolo, A., Mavilio, F., and

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Boncinelli, E. (1991). Differential regulation by retinoic acid of the homeobox genes of the four HOX loci in human embryonal carcinoma cells. Mech. Dev. 33, 215—227. Simon, J. (1995). Locking in stable states of gene expression: transcriptional control during Drosophila development. [Review]. Curr. Opin. Cell. Biol. 7, 376—385. Smouse, D., Goodman, C., Mahowald, A., and Perrimon, N. (1988). Polyhomeotic: a gene required for the embryonic development of axon pathways in the central nervous system of Drosophila. Genes Dev. 2, 830—842. Steelman, S., Moskow, J. J., Muzynski, K., North, C., Druck, T., Montgomery, J. C., Huebner, K., Daar, I. O., and Buchberg, A. M. (1997). Identification of a conserved family of Meis-1-related homeobox genes. Genome Res. 7, 142—156. Swift, G. H., Liu, Y., Rose, S. D., Bischof, L. J., Steelman, S., Buchberg, A. M., Wright, C. V. E., and MacDonald, R. J. (1998). An endocrine-exocrine switch in the activity of the pancreatic homeodomain protein PDX1 through formation of a trimeric complex with PBX1b and MRG1 (MEIS2). Mol. Cell. Biol. 18, 5109—5120. Szilvassy, S. J., Humphries, R. K., Lansdorp, P. M., Eaves, A. C., and Eaves, C. J. (1990). Quantitative assay for totipotent reconstituting hematopoietic stem cells by a competitive repopulations strategy. Proc. Natl. Acad. Sci. USA 87, 8736—8740. Takeshita, K., Bollekens, J. A., Hijiya, N., Ratajczak, M., Ruddle, F. H., and Gewirtz, A. M. (1993). A homeobox gene of the Antennapedia class is required for human adult erythropoiesis. Proc. Natl. Acad. Sci. USA 90, 3535—3538. Thorsteinsdottir, U., Sauvageau, G., Hough, M. R., Lawrence, H. J., Largman, C., and Humphries, R. K. (1997). Overexpression of HOXA10 in murine hematopoietic cells perturbs both myeloid and lymphoid differentiation and leads to acute myeloid leukemia. Mol. Cell. Biol. 17, 495—505.

Thorsteinsdottir, U., Krosl, J., Kroon, E., Hamann, A., Hoang, T., and Sauvageau, G. (1999a). The oncogene E2A-Pbx1a collaborates with Hoxa9 to acutely transform primary bone marow cells. Mol. Cell. Biol. 19, 6355—6366. Thorsteinsdottir, U., Sauvageau, G., and Humphries, R. K. (1999b). Enhanced polyclonal regeneration of hematopoietic stem cells overexpressing HOXB4 following bone marrow transplantation. Blood 94, 2606—2612. van Dijk, M. A., and Murre, C. (1994). Extradenticle raises the DNA binding specificity of homeotic selector gene products. Cell 78, 617—624. van Lohuizen, M., Verbeek, S., Scheijen, B., Wientjens, E., van der Gulden, H., and Berns, A. (1991). Identification of cooperating oncogenes in E mumyc transgenic mice by provirus tagging [see comments]. Cell 65, 737—752. Voura, E. B., Billia, F., Iscove, N. N., and Hawley, R. G. (1997). Expression mapping of adhesion receptor genes during differentiation of individual hematopoietic precursors. Exp. Hematol. 25 1172— , 1179. Wu, J., Zhu, J. Q., Zhu, D. X., Scharfman, A., Lamblin, G., and Han, K. K. (1992). Selective inhibition of normal murine myelopoiesis in vitro by a Hox 2.3 antisense oligodeoxynucleotide. Cell. Mol. Biol. 38, 367—376. Ying, S. Y., Lui, H. M., Lin, S. L., and Chuong C. M. (1999). Generation of full-length cDNA library from single human prostate cancer cells. BioTechniques 27, 410—414. (GENERIC) Ref Type: Generic Zimmermann, F., and Rich, I. N. (1997). Mammalian homeobox B6 expression can be correlated with erythropoietin production sites and erythropoiesis during development, but not with hematopoietic or nonhematopoietic stem cell populations. Blood 89, 2723—2735.

CHAPTER 10


THE ROLE OF RETINOIC ACID RECEPTORS IN MYELOID DIFFERENTIATION STEVEN J. COLLINS, BARTON S. JOHNSON, AND LOUISE E. PURTON Human Biology Division, Fred Hutchinson Cancer Research Center

INTRODUCTION Retinoic acid (RA), the natural acidic derivative of vitamin A (retinol), regulates the growth and differentiation of a wide variety of different cell types. The biologic effects of retinoic acid are generally mediated through specific ligand-activated nuclear transcription factors, the retinoic acid receptors. These receptors consist of two distinct families, the RARs and RXRs, with both receptor types exhibiting modular structures harboring distinct DNA-binding and ligandbinding domains. These receptors likely mediate their biologic effects by binding as RAR-RXR heterodimers to regulatory elements (RAREs) in specific target genes (for reviews, see Kastner et al., 1995; Mangelsdorf and Evans, 1995). The transcriptional activity of the RA receptors is mediated through specific corepressors and coactivators. In the absence of ligand, the RXR-RAR heterodimer likely interacts with large nuclear proteins including N-CoR (nuclear

receptor co-repressor) (Horlein et al., 1995) and SMRT (silencing mediator for retinoid and thyroid hormone receptors) (Chen et al., 1995). These proteins mediate transcriptional repression by complexing with other proteins including mSin3A and histone deacetylases (HDACs). The addition of ligand (retinoic acid) results in a distinct conformational change in the RARRXR complex, resulting in the release of such corepressors and recruitment of transcriptional coactivators including SRC-1, ACTR, and GRIP-1 (for review, see Torchia et al., 1998). The RARs primarily consist of three family members, RAR, RAR, and RAR. The RAR appears to be of particular interest in hematopoiesis, since hematopoietic cells preferentially express this particular RA receptor isoform. Moreover, there is considerable interest in RAR with respect to myeloid differentiation, since as a single agent all-trans retinoic acid (ATRA) induces granulocytic differentiation of leukemia cells from patients with acute


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promyelocytic leukemia (APL). These leukemia cells frequently harbor an aberrant RAR; that is, the PML-RAR fusion transcript generated by the t(15;17) that is present in most cases of APL. Indeed, the presence of this PML-RAR fusion protein in patient leukemia cells is perhaps the best predictor of initial patient response to retinoic acid. In this chapter we focus on the role that retinoic acid receptors play in regulating myeloid differentiation. We address the central paradox of why the human leukemia cells that are the most sensitive to retinoic acid (i.e., APL) are those that harbor an aberrant retinoic acid receptor. We discuss some experimental evidence indicating differences in activity of RAR in different hematopoietic lineages. We also present some recent experimental observations indicating that ATRA, while being a potent inducer of promyelocyte differentiation, may enhance the self-renewal and actually prevent or delay the differentiation of more primitive hematopoietic precursors.

THE PARADOX OF THE PML-RAR FUSION TRANSCRIPT IN APL ATRA as a single agent induces complete remission in patients with APL and appears to exert this therapeutic effect by inducing terminal granulocytic differentiation of the leukemia cells (Breitman et al., 1981; Castaigne et al., 1990; EHuang et al., 1988; Warrell et al., 1991). The t(15;17) chromosome translocation is present in most cases of APL and results in aberrant RAR transcripts in which a portion of the PML gene on chromosome 15 is fused to a truncated coding region of RAR on chromosome 17, resulting in the aberrant PML-RAR fusion transcript (Alcalay et al.,1991; deThe et al., 1991; Kakizuka et al., 1991). The normal function of the PML gene is still unclear, and both the PML gene and the PML-RAR translocation are described in detail in Chapter 2 in this book. In contrast with acute promyelocytic leukemia, other types of human myelogenous leukemias express normal full-length RA receptors that do not harbor DNA mutations (Morosetti et al., 1996), and yet ATRA has

little biologic effect on such leukemias. Such observations lead to a central paradox: the ATRA-sensitive human myelogenous leukemias (APL) harbor aberrant (i.e., truncated, translocated) RA receptors; in contrast, the ATRAresistant human leukemias (the non-APL types) harbor full-length nonmutated RA receptors. Compounding this paradox is the accumulating evidence that the PML-RAR fusion protein, rather than sensitizing cells to the biologic effects of ATRA, actually acts as a dominant negative and appears to inhibit normal RAR transcriptional activity (Grignani et al., 1998; He et al., 1998; Lin et al., 1998). One explanation for this paradox reflects the likely differences in the sensitivity of normal hematopoietic precursors to differentiation induction by ATRA. That is, during normal myeloid (granulocyte) differentiation, cells may become increasingly sensitized to the differentiative effects of ATRA. Thus, normal primitive myeloid cells may be relatively resistant to the differentiative effects of endogenous levels of ATRA while more mature myeloid cells (i.e., normal promyelocytes) may be relatively sensitive. The presence of the dominant negative PML-RAR fusion protein in early myeloid cells does not give rise to leukemogenic cells representative of this early myeloid stage if the normal RAR exerts little differentiative effect on such primitive myeloid precursors. However, if RAR is involved at a later stage of myeloid differentiation to help trigger normal promyelocyte differentiation to granulocytes, then the presence in these more mature myeloid cells of the dominant negative PML-RAR fusion protein may block this granulocyte differentiation and generate leukemogenic promyelocytes. That is, the leukemogenic t(15;17) might occur in an early myeloid stem cell but would become phenotypically evident only at a relatively late ATRA-sensitive (i.e., promyelocyte) stage of myeloid development. Below we describe experiments utilizing a truncated, dominant negative form of RAR (RAR403) that offer considerable insight into the normal role of RAR in mediating granulocytic differentiation and that also indicate significant differences in retinoid-mediated activation of RA receptors in hematopoietic cells of different lineages.

HEMATOPOIETIC LINEAGE-SPECIFIC ACTIVATION OF RA RECEPTORS

HEMATOPOIETIC LINEAGE-SPECIFIC ACTIVATION OF RA RECEPTORS Establishment of Growth Factor--Dependent Mouse Hematopoietic Cell Lines Utilizing Dominant Negative RA Receptor Constructs Experiments introducing a dominant negative retinoic acid receptor into normal mouse bone marrow have indicated an important role for RA receptors in regulating normal myeloid differentiation. Truncating the 59 amino acids at the COOH-terminal end of RAR results in an altered RA receptor (designated RAR403) that inhibits the function of normal RA receptors (Damm et al., 1993; Durand et al., 1994; Robertson et al., 1992; Tsai et al., 1992). Such truncated RAR constructs lack the COOHterminal activation domain (AF2) while retaining the DNA-binding domain as well as the ability to heterodimerize with RXRs (Damm et al., 1993; Durand et al., 1994). It is likely that this truncated RAR403 receptor acts as a dominant negative by competing with the normal RA receptors in the formation of biologically active RXR-RAR heterodimers (Fig. 10.1). Introducing this truncated dominant negative RAR403 construct into normal mouse bone marrow generates hematopoietic growth factor—dependent cells ‘‘frozen’’ at a distinct stage of myeloid differentiation (Tsai et al., 1993,

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1994). These include the GM-CSF—dependent MPRO cells trapped at the promyelocyte stage of granulocytic differentiation (Tsai et al., 1993) and the more primitive SCF-dependent EML cells, which are multipotent and exhibit erythroid, lymphoid, and myeloid potential (Tsai et al., 1994). Interestingly, the effect of the dominant negative RAR403 construct is not absolute but can be overcome with the addition of relatively high pharmacological (1—10 uM) concentrations of RA. Thus 1-10 uM ATRA induces terminal granulocytic differentiation of the GMCSF—dependent MPRO cells (Tsai et al., 1993), while in the pluripotent SCF-dependent EML cells, ATRA potentiates the IL-3—mediated commitment of these cells to the granulocyte/ monocyte lineage (Tsai et al., 1994). There are a number of important parallels between the mouse MPRO promyelocytes and human promyelocytic leukemia (APL). Both consist of hematopoietic cells that are ‘‘frozen’’ at the promyelocyte stage of myeloid differentiation. The generation of both involves the expression of aberrant retinoic acid receptors exhibiting dominant negative activity (i.e., the truncated RAR403 construct in the MPRO cells and the PML-RAR fusion protein in human APL). Both types of cells can be induced to terminally differentiate to granulocytes utilizing relatively high concentrations of ATRA. Thus, both the mouse MPRO cells and the PMLRAR human APL cells emphasize the critical

Figure 10.1. The truncated RAR403 inhibits the function of normal RAR. A: The normal RAR binds to its DNA target sequences as a heterodimer with RXR. B: The truncated RAR403 binds to RXR and to the same DNA target genes as the full-length RAR but lacks the final 59 amino acids within the COOH terminal activation domain. The RAR403 likely acts as a dominant negative by competitively binding to RXRs and inhibiting normal RXR-RAR heterodimer formation. LBD, ligand-binding domain; DBD, DNA-binding domain; RARE, retinoic acid response element.

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role that activated RA receptors play in regulating terminal granulocytic differentiation.

RXR-RAR Response Elements Readily Activated in the GM-CSF‒Dependent MPRO cells In order to gain insight into how relatively high concentrations of ATRA might induce terminal granulocytic differentiation of the MPRO promyelocytes, we utilized synthetic retinoids displaying specific RXR-RAR agonist activity. Such synthetic retinoid agonists selectively bind RAR or RXRs and transactivate either RXRRAR or RXR-RXR response elements. In pursuing these studies, we made the unexpected observation that RXR-selective agonists were

potent inducers of MPRO granulocyte differentiation while RAR agonists were not. Indeed, the RXR agonist (designated AGN 194204) was approximately 100- to 1000-fold more potent than ATRA in inducing MPRO granulocytic differentiation (Johnson et al., 1999). The selective RXR agonist likely induces MPRO differentiation by triggering activation of RXR-RAR403 heterodimers, which appear to predominate in the MPRO cells (Johnson et al, 1999). This was confirmed in transient transfection studies utilizing a reporter construct driven by a promoter harboring a DR5 RA response element that selectively responds to RXR-RAR heterodimer (rather than RXR-RXR homodimer) activation. This reporter was activated some 8—10—fold by the RXR agonist in the transfected MPRO cells (Fig. 10.2, lane 4).

Figure 10.2. DR5 (RXR-RAR) activation triggered by different retinoids in MPRO versus EML cells. Relative luciferase activity was determined in MPRO (lanes 1—4) and EML (lanes 5—8) cells transfected with a reporter construct harboring a DR5 (RXR-RAR) response element (Johnson et al., 1999) and treated for 24 hours with the indicated retinoids. Concentrations of the RAR and RXR agonists (Johnson et al., 1999) were each 1 M. The data represent the average of three independent experiments.

HEMATOPOIETIC LINEAGE-SPECIFIC ACTIVATION OF RA RECEPTORS

In contrast, the RAR agonist, which is ineffective in activating MPRO differentiation, mediated only a 2-fold, at best, activation of the DR5 (RXR-RAR) reporter in MPRO cells (Fig. 10.2, lane 3). As previously detailed (Johnson et al., 1999), the RXR agonist triggered no activation of a luciferase reporter driven by a DR1 response element, which is preferentially activated by RXR-RXR homodimers, indicating that this RXR-selective agonist triggers MPRO granulocyte differentiation by activating the RXRRAR403 heterodimer rather than RXR-RXR homodimers.

Blunted Activation of RXR-RAR Response Elements in the SCF-Dependent EML Cells. We performed similar studies on the stem cell factor (SCF)—dependent pluripotent EML cells derived by transducing the dominant negative RAR403 construct into normal mouse hematopoietic cells and then culturing these cells in SCF (Tsai et al., 1994). Unlike the GM-CSF— dependent MPRO cells, which are strictly committed to granulocyte differentiation, the SCF—dependent EML cells are multipotent, exhibiting erythroid, lymphoid, and myeloid potential. The addition of IL-3 to EML cultures will induce commitment of these cells to the monocyte/granulocyte lineage (as measured by CFU—GM generation), and this IL-3-induced commitment to the monocyte/granulocyte lineage is potentiated by relatively high concentrations of ATRA (Tsai et al., 1994). Similar to the above studies in MPRO cells, we utilized the synthetic retinoids specific for RAR and RXR to determine whether the CFUGM generation observed in EML cells induced by relatively high concentrations of ATRA was mediated through RXR or RAR. We observed that in marked contrast to MPRO cells, where the RXR agonist (AGN 194204) exhibited potent activity in inducing granulocyte differentiation, this same RXR agonist had little effect in potentiating the IL-3—mediated CFU-GM production in the EML cells (Fig. 10.3, lane 4). Moreover, the RAR agonist also exhibited no significant biological effect, and the combination of RAR and RXR agonist exhibited no signifi-

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cant increase in CFU-GM generation compared with either agonist alone (Fig. 10.3, lane 6). Thus, there was a marked contrast of the RXR agonist activity in the two hematopoietic lineages, with this compound being a potent inducer of granulocytic differentiation in MPRO cells while exhibiting virtually no biological activity in the more immature EML cells. To further explore the activity of the RXR agonist in EML cells, we assessed the ability of this compound to trigger DR5 (RXR-RAR) reporter activation in transfected EML cells. We observed that in the pluripotent EML cells the DR5 (RAR-RXR) reporter exhibited a markedly reduced retinoid-mediated activation compared with MPRO cells. For example, the RXR agonist, which readily stimulates the DR5 (RXRRAR) reporter in MPRO cells (Fig. 10.2, lane 4), stimulated little if any activation of the same DR5 reporter in the transfected EML cells (Fig. 10.2, lane 8). ATRA consistently induced activation of the DR5 construct in EML cells, but this activation (3—4—fold) was less than the approximately 10-fold activation of the same construct induced by ATRA or 9-cis RA in the transfected MPRO cells (Fig. 10.2, lanes 2 vs. 6). Thus, in comparison with the MPRO promyelocytes, in the more immature, multipotent EML cells the RXR-RAR403 heterodimers exhibit a blunted response to retinoid-induced activation, and in these cells the RXR agonist by itself exhibits virtually no activation of these heterodimers.

Differences in Nuclear Hormone Receptor Corepressor Activity in EML vs. MPRO Cells Our observations indicate that the RXRRAR403 heterodimer is readily activated in one hematopoietic cell lineage (MPRO promyelocytes) but not in another closely related though distinct lineage (multipotent EML cells). Since nuclear hormone receptors regulate transcription by interacting with a complex array of coactivator or corepressor proteins (for review, see Torchia et al., 1998), our observations suggest that there may be significant differences in such coactivators or corepressors in the EML versus MPRO cells. Transcriptional repression by RA receptors may in part be mediated by specific corepressors

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Figure 10.3. Generation of CFU-GM in EML cultures. Numbers of CFU-GM were determined in EML cultures following 2—3 days of incubation with IL-3 and the indicated retinoid. ATRA concentration was 10 M while the concentration of the RAR and RXR agonists (Johnson et al., 1999) was 1 M. The data represent the average of three independent experiments.

such as N-CoR and SMRT, which interact with nuclear hormone receptors as multicomponent complexes including mSin3A and histone deacetylases (HDACs) (Heinzel et al., 1997; Horlein et al., 1995; Nagy et al., 1997). To compare the activity of such HDAC-containing repressor complexes in EML versus MPRO cells, we assessed the ability of the HDAC inhibitor trichostatin A (TSA) (Yoshida et al., 1990) to activate the DR5 (RAR-RXR) reporter in these different cell types. We observed that TSA readily activated this reporter in EML cells but had no effect on activating this same reporter in the MPRO cells even at higher concentrations (Johnson et al., 1999). Thus, repression of the DR5 appears dependent on HDAC activity in the EML but not the MPRO cells, indicating that there are significant differences in transcriptional repressor complexes interacting with the RXR-RAR403 heterodimer in these different cell types.

Do the Differences in AML Sensitivity to Retinoic Acid Reflect Differential Activation of RXR-RAR Heterodimers in Immature Versus Mature Myeloid Cells? The EML and MPRO cell lines, which both express the dominant negative retinoic acid receptor, in many respects closely resemble normal pluripotent hematopoietic progenitors and committed promyelocytes respectively. Both cell types can be reproducibly derived from normal mouse bone marrow cells, both cell types respond to physiologic hematopoietic growth and differentiation factors, and the patterns of gene expression observed in these hematopoietic cell lines mimic patterns of lineage-specific gene expression noted in normal hematopoietic cells (Lawson and Berliner, 1998; Weiler et al., 1999). In this respect, the marked difference in activation of RAR-RXR heterodimers noted in the EML versus MPRO cell lines (Fig. 10.2) may

RA RECEPTORS REGULATE THE GENERATION OF EARLY MYELOID PRECURSORS

mimic differences in RA receptor activity in immature multipotent hematopoietic precursors versus committed granulocyte/monocyte progenitors and may help explain the selective, paradoxical presence of the PML-RAR fusion protein in human promyelocytic leukemia rather than other forms of myeloid leukemia. That is, the generation of the dominant negative PML-RAR would be a leukemogenic event in committed promyelocytes, where the RA receptor is readily activated, but would not be a leukemogenic event in more immature hematopoietic precursors, where RA activation is normally blunted. This blunted activation of RA receptors might help explain why the great majority of human myeloid leukemia cells display little response to retinoic acid even though they harbor apparently normal RA receptors. Our experimental observations with the EML and MPRO cells described above suggest that the molecular basis for this difference in RXR-RAR activation may be related to hematopoietic lineage-specific differences in histone deacetylase repressor complexes that normally interact in the absence of ligand with the RA receptors in these different cell types.

RA RECEPTORS REGULATE THE GENERATION OF EARLY MYELOID PRECURSORS A Broader Role for RA Receptors in Regulating Myelopoiesis The above studies indicate a role for activated RA receptors in regulating the terminal differentiation of committed myeloid progenitors to granulocytes. ATRA clearly plays a role in inducing the terminal differentiation of leukemic promyelocytes and may also be involved in regulating normal monocyte/granulocyte progenitor differentiation as well. However, we have obtained recent experimental evidence that ATRA and activated RA receptors may play a significantly larger role in regulating hematopoiesis and that these effects may be directly related to the maturational state of the cell. Indeed, the observations described below suggest that RA receptors may enhance the maintenance and/or self-renewal of primitive hematopoietic precursors including the long-term marrow repopulating stem cell.

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ATRA Enhances CFC Production from Liquid Suspension Cultures of Primitive Hematopoietic Precursors When highly enriched hematopoietic precursors are cultured in liquid suspension in the presence of specific hematopoietic growth factors, they undergo extensive proliferation that is associated with progressive lineage commitment and terminal differentiation. In studies assessing the behavior of such cultures, we found that the addition of ATRA (10\ M) significantly alters the growth and differentiation of these primitive hematopoietic precursors. Starting with normal mouse bone marrow we utilized the fluorescentactivated cell sorter (FACS) to enrich for lin\, c-kit>, Sca-1> cells, a population that is known to be highly enriched for primitive hematopoietic precursors and to harbor the longterm repopulating hematopoietic stem cell (Okada et al., 1992). We cultured these precursors in liquid suspension supplemented with hematopoietic growth factors including SCF, IL-6, flt-3 ligand, and IL-11 in the presence or absence of 10\M ATRA (Purton et al., 1999). We then periodically harvested aliquots of these cultured cells and performed standard colonyforming cell (CFC) assays for the relatively mature colony-forming unit—granulocyte macrophage (CFU-GM) as well as for the more immature mixed (granulocyte/monocyte, erythroid, megakaryocytic) progenitors (HPP-mix). These highly enriched primitive hematopoietic progenitors cultured in liquid suspension in the absence of exogenous ATRA exhibited significant colony-forming activity during the first 14 days of liquid culture, after which the production of CFU-GM and HPP-mix was negligible. In contrast, the same hematopoietic progenitors cultured in the presence of ATRA exhibited prolonged and enhanced CFU-GM and HPP-mix production that continued for up to 21—28 days in liquid suspension culture (Purton et al., 1999). There was at least a 20—100— fold increase in both CFU-GM and HPP-mix production in the ATRA-treated cultures. In contrast to this enhanced CFU-GM and HPPmix production, there was no effect of ATRA on the number or size of erythroid colonies generated from hematopoietic precursor cells cultured in liquid suspension. This effect of ATRA was specific for the relatively primitive lin\, c-kit>,

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Sca-1> hematopoietic precursors, because ATRA did not exhibit any enhanced colony production in cultures of more committed lin\, c-kit> Sca-1\ hematopoietic progenitors. This enhanced CFC production noted in ATRA-treated liquid suspension cultures of primitive hematopoietic precursors (lin\, c-kit>, Sca-1>) suggested that ATRA enhanced the generation and/or maintenance of these hematopoietic progenitors. This could be a direct effect of ATRA on these colony-forming cells or alternatively could represent an ATRA-mediated enhancement of the maintenance and/or self-renewal of hematopoietic precursors that are more primitive and less differentiated than such colony-forming cells. To distinguish these possibilities, we determined the effect of ATRA on the generation of more primitive hematopoietic precursors in these cultures. CFU-S Production in Cultured Hematopoietic Precursors Is Enhanced by ATRA Colony-forming unit-spleen (CFU-S) populations are more primitive than colony-forming cells and likely represent pluripotent myeloid progenitors. Their presence in hematopoietic populations is quantitated by measuring the number of colonies arising 8—12 days following injection of the hematopoietic cells into lethally irradiated mice. The CFU-S day 8 likely reflects a more mature cell than the CFU-S day 12, but

both are more primitive than the in vitro colony-forming cell (CFC). To determine whether ATRA (10\M) had any effect on the generation of the CFU-S population in liquid suspension cultures of our highly enriched hematopoietic precursors, we performed CFU-S assays on lin\, c-kit>, Sca-1> precursors cultured for 7 and 14 days in liquid suspension in the presence or absence of ATRA. The results are presented in Table 10.1. We observed a pronounced effect of ATRA in stimulating CFU-S generation in these cultures. After 7 days of liquid suspension culture, the ATRA-treated cells generated approximately 12-fold more CFU-S day 8, and 30-fold more CFU-S day 12, than cells cultured without ATRA. After 14 days of culture, the effect of ATRA was even more dramatic, with a 600-fold increase in CFU-S day 8 in the ATRA-treated cells (Purton et al., 1999). There also appeared to be an increase in CFU-S day 12, but a comparative analysis could not be performed because all of the mice injected with cells cultured without ATRA had died. This likely indicates that ATRA may also enhance the generation of radioprotective cells in these liquid suspension cultures.

Short- and Long-Term Marrow Repopulating Stem Cell Assays We also determined whether ATRA influenced the maintenance or the generation of hema-

TABLE 10.1. ATRA Enhances CFU-S Production from Cultured lin\, c-kit>, Sca-1> Hematopoietic Precursors?

Day of Culture

ATRA (1 M)

Number of cells injected

CFU-S D8

CFU-S D12

0 7 7 14 14

9 9 ; 9 ;

500 4.80;10 3.08;10 3.92;10 4.28;10

4.25 < 0.85 21.0 < 2.65 242 < 51 2.0 < 1.15 1250 < 160

30.0 < 10.0 7.0 < 0.00 217 < 88 N/A@ 300

?CFU-S were counted per spleen at day 8 (D8) or day 12 (D12) following transplantation of the indicated number of cells into irradiated recipient mice following previously detailed procedures (Purton et al., 1999). CFU-S from day 0 are from 500 freshly sorted uncultured hematopoietic precursors (lin\ c-kit> Sca-1> cells). CFU-S after day 7 and 14 are from all cells that grew in liquid suspension from 500 or 1000 of these sorted hematopoietic precursors respectively. @N/A : not available due to deaths of all mice before day 12.

RA RECEPTORS REGULATE THE GENERATION OF EARLY MYELOID PRECURSORS

157

Figure 10.4. ATRA enhances generation of short- and long- term repopulating hematopoietic stem cells in liquid suspension culture. Murine FACS-enriched Ly5.2 hematopoietic precursors (lin\, c-kit>, Sca-1>) were cultured in liquid suspension in the presence or absence of ATRA (10\ M) as previously detailed (Purton et al., 1999). After 7 and 14 days of culture, aliquots of these cells were injected into irradiated Ly5.1 recipients. At the indicated monthly intervals, peripheral blood cells from these recipients were harvested and the percentage of donor cells (Ly5.2) was determined by FACS analysis.

topoietic precursors with the capability of repopulating the bone marrow of lethally irradiated mice. For these studies, we utilized mice congenic at the Ly5 locus such that cells from donor mice (Ly5.2) could be distinguished from cells of recipient mice (Ly5.1) utilizing FACS analysis with epitope-specific monoclonal antibodies. Cultured donor cells (Ly5.2) were injected into lethally irradiated recipients (Ly5.1), and then at periodic intervals the Ly5 phenotype of the peripheral blood cells in the recipient animals was determined. This permits a semiquantitative estimate of the number of short- and long-term repopulating cells in the cultured cell population. We established liquid suspension cultures of FACS-enriched lin\, c-kit>, Sca-1> precursors (Ly5.2), cultured them for 7—14 days in the presence or absence of ATRA (10\ M), and

then injected these cells into lethally irradiated recipient mice (Ly5.1) together with 10 competing normal bone marrow cells (Ly 5.1). Mice injected with hematopoietic precursors cultured for 7 days with or without ATRA displayed similar levels of long-term donor cell reconstitution (Fig. 10.4). In contrast, after 14 days of culture there was a significant difference in repopulating ability of cells from the ATRAtreated and untreated cultures. None of the 10 mice receiving cells cultured for 14 days without ATRA showed any donor cell reconstitution, whereas all mice receiving hematopoietic precursors cultured with ATRA for 14 days showed significant short- and long-term repopulating ability (Fig. 10.4). (Purton et al., 2000). We also assessed the effect of the addition of specific RA receptor antagonists on these cultured hematopoietic precursors. The RAR an-

158


Figure 10.5. The RAR antagonist AGN 193109 inhibits short- and long-term repopulating hematopoietic stem cells in liquid suspension. Murine FACS-enriched Ly5.2 hematopoietic precursors (lin\, c-kit> Sca-1>) were cultured for 7 days in liquid suspension in the presence or absence of the RAR antagonist AGN193109 (10\ M) and then injected into irradiated Ly5.1 recipients. At the indicated times, recipient peripheral blood cells were harvested and the percentage of donor cells (Ly5.2) was determined by FACS analysis.

tagonist AGN 193109 binds with high affinity to the ligand-binding domain of RAR but does not transactivate this receptor and thus acts as a competitive inhibitor of RA receptor activation by endogenous retinoids. We found that the addition of this RAR antagonist to cultures of the lin\, c-kit>, Sca-1> precursors markedly abrogated their short- and long-term repopulating ability (Fig. 10.5). Hematopoietic precursors cultured for 7 days in the cytokine cocktail clearly exhibited repopulating ability, but the addition of AGN 193109 to these cultures resulted in virtually undetectable donor cells in the recipient animals over both the short and long term (Fig. 10.5). Thus, in these liquid suspension cultures of primitive hematopoietic precursors, the retinoic acid receptor antagonist exhibited the opposite effect as the RA receptor agonist (ATRA) in generating marrow repopulating cells. This provides further evidence that activation of RA receptors is somehow involved in the maintenance and/or generation of primitive hematopoietic stem cells. (Purton et al., 2000).

Diverse Effects of ATRA on Myelopoiesis These observations indicating that ATRA enhances the maintenance and/or generation of primitive hematopoietic stem cells in liquid suspension cultures is surprising and counterintuitive given the well-documented effects of ATRA in enhancing promyelocytic differentiation. In fact, when we initially set up these experiments we hypothesized that the opposite would occur: that is, that the RAR agonist (ATRA) would inhibit hematopoietic precursor generation and that the RAR antagonist would enhance the production of such cells. These observations suggest that RA receptors are not only involved in regulating the differentiation of promyelocytes to granulocytes but also may be involved in regulating the maintenance and/or self-renewal of much earlier myeloid precursors. Our observation that the RA receptor antagonist AGN193109 blocks the generation of short- and long-term marrow repopulating cells suggests that endogenous retinoids may be involved in regulating the generation of early hematopoietic


stem cells. Such diverse effects of retinoic acid have been previously noted in other developmental systems, particularly embryonic limb development in the mouse. Here the application of pharmacological (i.e., micromolar) concentrations of ATRA 5 days postcoitum (dpc) induces limb duplications (Niederreither et al., 1996), but when the embryo is exposed to similar ATRA concentrations later in development (10— 12 dpc) the opposite effect (i.e., stunted limb development) occurs (Kochar, 1980). Similarly, activation of RA receptors has different effects on hematopoietic cells depending upon their maturational state, underscoring the complexity of RA receptor regulation of myelopoiesis.

CONCLUSION AND FUTURE DIRECTIONS The effect of RA receptors on myelopoiesis is clearly complex. The well-known ability of ATRA to induce the differentiation of PMLRAR—positive human promyelocytic leukemias to mature granulocytes suggests that RA receptor activation may play an important role in regulating normal granulocytic differentiation. This would likely occur in synergy with other hematopoietic growth factors such as GM-CSF or G-CSF. Since RA receptors are transcription factors that likely mediate most of their biological effects by regulating the expression of specific target genes, it would be important to identify the specific genes regulated by ATRA in mediating this terminal granulocytic differentiation. However, the identification of the direct targets for the activated RA receptor that trigger granulocytic differentiation has been surprisingly elusive. For instance, expression of the c-myc oncogene, an important regulator of cell cycle progression, is downregulated during ATRA-mediated differentiation of APL cells and likely represents a critical event in such differentiation, but no RA response element that binds directly to the activated RA receptor has been identified in the c-myc promoter/enhancer regulatory sequences. One possibly important direct target for the RA receptor is the p215! gene, encoding a cyclin-dependent kinase inhibitor that causes growth arrest, is upregulated during myeloid differentiation, and harbors an RA response

159

element in its promoter (Liu et al., 1996). We have identified other genes harboring RA response elements that are upregulated during granulocytic differentiation, but their role in directly triggering granulocytic differentiation is unclear (Scott et al., 1996.) Likely, RA receptor— triggered granulocytic differentiation represents a temporally regulated cascade of molecular events involving multiple transcription factors and target genes. The use of cDNA microarray technology, which can simultaneously quantify the expression of literally thousands of different genes at specific time points, holds significant promise in dissecting this complex series of molecular events that characterize ATRA-triggered granulocyte differentiation. Another unresolved question is why non-APL myeloid leukemias display little if any response to retinoic acid even though virtually all of these myeloid leukemias express apparently normal RA receptors. Perhaps some of this unresponsiveness relates to the specific RA receptor target genes. These genes may be ’’accessible’’ to RA receptor—mediated transcriptional activation in promyelocytic leukemias but not in more immature leukemias, perhaps because of hematopoietic lineage-specific differences in chromatin structure in such target genes. Alternatively, our studies with the EML and MPRO cells suggest that more immature hematopoietic cells may harbor transcriptional repressor complexes that blunt ATRA-mediated RA receptor activation. The characterization of such repressor complexes, which we postulate to be hematopoietic lineage-specific, may provide significant insight into the molecular basis for this ATRA resistance that characterizes nonAPL human myeloid leukemia cells. Further complicating the issue is our observation that ATRA, in pharmacological doses (10\M), appears to enhance the maintenance and/or self-renewal of normal mouse hematopoietic precursors grown in liquid suspension culture. The specific hematopoietic cell type that is the target of this activity remains unclear as well as the specific target genes regulating this activity. This observation raises the interesting possibility that ATRA may prove useful for the ex vivo expansion of hematopoietic stem cells as well as for improving the efficiency of retroviral vector-mediated transduction of hematopoietic stem cells in gene therapy trials.

160


Finally, there are other important transcription factors that are critical in regulating myeloid lineage development and that are described in other chapters in this book. Determining how the RA receptor interacts with other gene products that are critical to the development and differentiation of the myeloid lineage, such as PU.1, C-EBP-, c-EBP-, AML1, MZF-1, and the homeobox gene products, will undoubtedly provide significant insight into the molecular basis for myeloid lineage commitment and differentiation.

REFERENCES Alcalay, M., Zangrilli, D., Pandolfi, P., Longo L., Mencarelli, A., Giacomucci, A., Rocchi, M., Biondi, A., Rambaldi, A., LoCoco, F., Diverioi, D., Donti, E., Grignani, F., and Pelicci, P. (1991). Translocation breakpoint of acute promyelocytic leukemia lies within the retinoic acid receptor locus. Proc. Natl. Acad. Sci. USA 88, 1977—1981. Breitman, T., Collins, S. J., and Keene, B. (1981). Terminal differentiation of human promyelocytic leukemia cells in primary culture in response to retinoic acid. Blood 57, 1000—1004. Castaigne, S., Chomienne, C., Daniel, M., Berger, N., Fenaux, P., and Degos, L. (1990). All-trans retinoic acid as a differentiation therapy for acute promyelocytic leukemia. I. Clinical results. Blood 76, 1704—1712. Chen, J., and Evans, R., (1995). A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature 377, 454—457. Damm, K., Heyman, R. A., Umesono, K., and Evans, R. M. (1993). Functional inhibition of retinoic acid response by dominant negative retinoic acid receptor mutants. Proc. Natl. Acad. Sci. USA 90, 2989— 2993. de The, H., Lavfau, C., Marchio, A., Chomienne, C., Degos, L., and Dejean, A. (1991). The PML-RAR fusion mRNA generated by the t(15;17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR. Cell 66, 675—684. Durand, B., Saunders, M., Gaudon, C., Roy, B., Losson, R., and Chambon, P. (1994). Activation function 2 (AF-2) of retinoic acid receptor and 9-cis retinoic acid receptor: presence of a conserved autonomous constitutive activating domain and influence of the nature of the response element on AF-2 activity. EMBO J. 13, 5370—5382. Grignani, F., De Matteis, S., Nervi, C., Tomassoni, L., Gelmetti, V., Cioce, M., Fanelli, M., Ruthardt, M., Ferrara, F., Zamir, I., Seiser, C., Grignani, F., Lazar, M., Minucci, S., and Pelicci, P. G. (1998). Fusion proteins of the retinoic acid receptor-re-

cruit histone deacetylase in promyelocytic leukemia. Nature 319, 815—818. He, L-Z, Guidez, F., Tribioli, C., Peruzzi, D., Ruthardt, M., Zelent, A., and Pandolfi, P. P. (1998). Distinct interactions of PML-RAR and PLZF- RAR with co-repressors determine differential responses to RA in APL. Nat. Genet. 18, 126—135. Heinzel, T., Lavinsky, R., Mullen, T. M., Soderstrom, M., Laherty, C., Torchia, J., Yang, W-M., Brard, G., Ngo, S., Davie, J., Seto, E., Eisenman, R., Rose, D., Glass, C., and Rosenfeld, M. (1997). A complex containing N-CoR, mSin3 and histone deacetylase mediates transcriptional repression. Nature 387, 43—48. Horlein, A., Naar, A., Heinzel, T., Torchia, J., Gloss, B., Kurokawa, R., Ryan, A., Kamei, Y., Soderstrom, M., Glass, C., and Rosenfeld, M. (1995). Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor co-repressor. Nature 377, 397—404. Huang, M., Ye, Y. C., Chen, S. R., Chai, J. R., Lu, J. X., Zhoa, L., Gu, L. J., and Wang, Z. Y. (1988). Use of all trans retinoic acid in the treatment of acute promyelocytic leukemia. Blood 72, 567—574. Johnson, B., Chandraratna. R., Heyman, R., Mueller, L., and Collins, S. (1999). RXR agonist-induced activation of dominant negative RXR-RAR heterodimers is developmentally regulated during myeloid differentiation. Mol. Cell. Biol. 19, 3372— 3382. Kakizuka, A., Miller, W., Umesono, K., Warrell, R., Frankel, S., Murty, V., Dmitrovsky, E., and Evans, R. (1991). Chromosomal translocation t(15;17) in human acute promyelocytic leukemia fuses RAR with a novel putative transcription factor, PML. Cell 66, 663—674. Kastner, P., Mark, M., and Chambon, P. (1995). Nonsteroid nuclear receptors: what are genetic studies telling us about their role in real life? Cell 83, 859—869. Kochhar, D. M. (1980). In vitro testing of teratogenic agents using mammalian embryos. Teratogen. Carcinogen. Mutagen. 1, 63—69. Lawson, N., and Berliner, N. (1998). Representational difference analysis of a committed myeloid progenitor cell line reveals evidence for bilineage potential. Proc. Natl. Acad. Sci. USA 95, 10,129— 10,133. Lin, R., Nagy, L., Inoue, S., Shao, W., Miller, W., and Evans, R. (1998). Role of the histone deacetylase complex in acute promyelocytic leukemia. Nature 391, 811—814. Liu, M., Iavarone, A., and Freedman, L. (1996). Transcriptional activation of the human p215! gene by retinoic acid receptor. J. Biol. Chem. 271, 31,723—31,728. Mangelsdorf, D., and Evans, R. (1995). The RXR and orphan receptors. Cell 83, 841—850. Morosetti, R., Grignani, F., Liberatore, C., Pelicci, P.,

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Schiller, G., Kizaki, M., Bartram, C., Miller C., and Koeffler, H. P. (1996). Infrequent alterations of the RAR alpha gene in acute myelogenous leukemias, retinoic acid-resistant acute promyelocytic leukemias, myelodysplastic syndromes and cell lines. Blood 87, 4399—4403. Nagy, L., Kao, H-Y., Chakravarti, D., Lin, R., Hassig, C., Ayer, D., Schreiber, S., and Evans, R.(1997). Nuclear receptor repression mediated by a complex containing SMRT, mSin3A, and histone deacetylase. Cell 89, 373—380. Niederreither, K., Ward, S. J., Dolle, P., and Chambon, P. (1996). Morphological and molecular characterization of retinoic acid-induced limb duplications in mice. Dev. Biol. 176, 185—195. Okada, S., Nakauchi, H., Nagayoshi, K., Nishikawa, S., Miura, Y., and Suda, T. (1992). In vivo and in vitro stem cell function of c-kit> and Sca-1—positive murine hematopoietic stem cells. Blood 80, 3044—3052. Purton, L., Bernstein, I., and Collins, S. J. (1999). All-trans retinoic acid delays the differentiation of primitive hematopoietic precursors (lin- c-kit> Sca-1>) while enhancing the terminal maturation of committed granulocyte/monocyte progenitors. Blood 94, 483—495. Purton, L., Bernstein, I., and Collins, S. (2000). Alltrans retinoic acid enhances the long-term repopulating activity of cultured hematopoietic stem cells. Blood 95, 470—477. Robertson, K., Emami, B., and Collins, S. J. (1992). Retinoic acid-resistant HL-60R cells harbor a point mutation in the RA receptor ligand binding domain that confers dominant negative activity. Blood 80, 1885—1889. Scott, L., Mueller, L., and Collins, S. (1996). E3, a hematopoietic-specific transcript directly regulated

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by the retinoic acid receptor alpha. Blood 88, 2517—2530. Torchia, J., Glass, C., and Rosenfeld, G. (1998). Coactivators and co-repressors in the integration of transcriptional responses. Curr. Opin. Cell. Biol. 10(3), 373—383. Tsai, S., and Collins, S. (1993). A dominant negative retinoic acid receptor blocks neutrophil differentiation at the promyelocyte stage. Proc. Natl. Acad. Sci. USA 90, 7153—7157. Tsai, S., Bartelmez, S., Heyman, R., Damm, K., Evans, R., and Collins, S. (1992). A mutated retinoic acid receptor alpha exhibiting dominant negative activity alters the lineage development of a multipotent hematopoietic cell line. Genes Dev. 6, 2258—2269. Tsai, S., Bartelmez, S., Sitnicka, E., and Collins, S. (1994). Lymphohematopoietic progenitors immortalized by a retroviral vector harboring a dominant negative retinoic acid receptor can recapitulate lymphoid, myeloid and erythroid development. Genes Dev. 8, 2831—2842. Warrell, R., Frankel, S., Miller, W., Itri, L., Andreef, M., Jabukowski, A., Gabrilove, J., Gordon, M., and Dmitrovsky, E. (1991). Differentiation therapy of acute promyelocytic leukemia with tretinoin (all-trans retinoic acid). New Engl. J. Med. 324, 1385—1390. Weiler, S., Gooya, J., Ortiz, M., Tsai, S., Collins, S., and Keller, J. (1999). (D3): A gene induced during myeloid differentiation of linlo c-Kit>, Sca-1> progenitor cells. Blood 93, 527—536. Yoshida, M., Kijima, M., Akita, M., and Beppu, T. (1990). Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A. J. Biol. Chem. 265, 17,174— 17,179.

CHAPTER 11


TRANSCRIPTIONAL TARGETS OF THE VITAMIN D3 RECEPTOR DURING MYELOID CELL DIFFERENTIATION V. CARRIE BROMLEIGH, JEREMY WARD, AND LEONARD P. FREEDMAN Cell Biology Program, Memorial Sloan-Kettering Cancer Center, and Sloan-Kettering Division, Joan and Sanford I. Weill Graduate School of Medical Sciences of Cornell University

INTRODUCTION The fat-soluble vitamin D metabolite, 1,25 dihydroxyvitamin D [1,25(OH) D ], is a major regulator of mineral homeostasis and bone formation/remodeling (Feldman et al., 1997). Perhaps surprisingly, this ligand can also elicit potent growth inhibitory and differentiation effects on a variety of cell types, including myeloid cells. 1,25(OH) D transduces its signal directly through a regulable, DNA-binding transcription factor, the vitamin D receptor (VDR), which is a member of a large superfamily collectively known as nuclear hormone receptors. Thus, vitamin D -inducible effects on cell growth and differentiation are initiated through the direct activation or repression of target genes by VDR. The identities of such genes, however, have remained elusive until recently. 1,25(OH) D can induce normal and leuke mic hematopoietic cells to differentiate into cells displaying characteristics consistent with a more mature monocyte/macrophage phenotype, including a decrease or cessation in their prolifer-

ation (Abe et al., 1981; Bar-Shavit et al., 1983; Mangelsdorf et al., 1984; Munker et al., 1986). An examination of the role of 1,25(OH) D during hematopoiesis was prompted by two observations: first, that hematopoietic cells contain VDR (Mangelsdorf et al., 1984); and second, that osteoclasts arise from the fusion of circulating mononuclear precursor cells and therefore represent a terminal stage of mononuclear phagocyte differentiation (Kahn and Simmons, 1975; Moore, 1987). Indeed, Abe and colleagues (Abe et al., 1983) showed that 1,25(OH) D at concentrations in the nanomolar range were sufficient to induce fusion of mouse alveolar macrophages. This same group first demonstrated that a myeloid leukemic cell line, mouse M1, could be induced to differentiate along the macrophage lineage by 1,25(OH) D (Abe et al., 1981). Subsequently, they and others showed that the human promyelocytic leukemia cell line HL60 and the human myelomonocytic cell line U937 can also be induced to terminally differentiate by 1,25(OH) D (Ollson et al., 1983). A variety of other compounds, such as phorbol


163

164

VITAMIN D RECEPTOR AND MYELOID DIFFERENTIATION

esters, dimethyl sulfoxide (DMSO), and retinoic acid, also induce the differentiation of these cell lines (reviewed in Collins, 1987); however, HL60 cells differentiate into granulocytes upon exposure to DMSO or retinoic acid (Breitman, 1981; Collins, 1978), whereas they differentiate into cells exhibiting distinct monocyte/macrophage characteristics when treated with 1,25(OH) D (McCarthy, 1983). The role of hormone-induced transcription factors, such as VDR, in myeloid differentiation requires both an understanding of the molecular mechanism of transcription by nuclear receptors and identification and characterization of the target genes they regulate. Generally, ligand binding leads to the dissociation of corepressors and the recruitment of coactivators. Many of these factors, acting in large complexes, have emerged as chromatin remodelers through intrinsic histone modifying activities or through other novel functions. In addition, other ligandrecruited complexes appear to act more directly on the transcriptional appartus, suggesting that transcriptional regulation by nuclear receptors may involve a process of both chromatin alterations and direct recruitment of key initiation components at regulated promoters (Freedman, 1999).The identification of target genes regulated by VDR and 1,25(OH) D has recently been accelerated by improved techniques for isolating inducible genes. In this chapter, we first focus on a subset of genes isolated from a differential screen of myeloid cells induced to differentiate to macrophages upon addition of 1,25(OH) D . These genes encode cell cycle regulators and transcription factors. In the second part of the chapter, we discuss the relationship between 1,25(OH) D and chromosomal translocations that block 1,25(OH) D -induced differentiation, possibly leading to the misregulation of some of the key VDR target genes in myeloid cells. VDR TARGET GENES INVOLVED IN MYELOID DIFFERENTIATION A Differential Screen for Induced Target Genes Since the discovery that 1,25(OH) D could elicit a differentiative response from myeloid cells in culture, much work has been done to examine the expression of a variety of genes in

these cells in response to this hormone. A great deal of data has been produced in this area, but until recently none of the direct VDR target genes involved in this process had been identified. In 1995, a differential screen was initiated in order to identify the genes that are both directly regulated by VDR and responsible for the differentiation resulting from 1,25(OH) D treatment (Liu et al., 1996; see also Rots et al., 1998). The screen was modified from a method by Beadling and Smith (Beadling et al., 1993; Beadling and Smith, 1993) and was designed specifically to select for direct targets (Rots et al., 1998). As outlined in Figure 11.1, a cDNA library was generated from the polyA> RNA of the myelomonocytic cell line U937 cultured in the presence of 1,25(OH) D . The protein synthesis inhibitor cycloheximide was included to avoid the cloning of genes representing indirect VDR targets. 4-thiouridine and [H]-5,6-uridine were also included for the purposes of purification on a mercury column and for the tracing of nascent RNAs, respectively. This library was then probed with cDNA from the nascent RNA of U937 cells grown for 4 hours in the presence or absence of 1,25(OH) D . By design, this screen was used to single out mRNAs induced as a result of 1,25(OH) D treatment, due to the fact that the library was generated from nascent mRNA after hormone treatment. It was not used to detect genes that are targets of 1,25(OH) D -mediated repression. After several rounds of screening, candidates were verified by Northern blot analysis with RNA from cells treated or untreated, in the presence of cycloheximide (as detailed in Rots et al., 1998). The screen yielded a number of potential clones (see Table 11.1 and Rots et al., 1998), many of which proved to be repetitive elements. Some of the clones represent unidentified genes or genes of unknown function, and a small number proved to be previously identified genes. Some of these have been studied further to demonstrate the direct nature of their regulation and to understand how they might be important for the differentiation of myeloid cells.

p21waf1, cip1 One of the genes identified in this screen was the well-studied cyclin dependent kinase inhibitor

VDR TARGET GENES INVOLVED IN MYELOID DIFFERENTIATION

165

Figure 11.1. A schematic representation of the differential screen performed by Liu et al. (Liu et al., 1996) in order to identify direct 1,25(OH) D receptor (VDR) target genes involved in myeloid differentiation. Nascent RNA was purified as shown on a phenyl/mercury affinity columns from [1,25(OH) D ;-treated U937 cells and used to make a cDNA library. This library was probed with cDNA from [1,25(OH) D ;-treated or untreated cells in order to identify nascent clones specifically expressed in response to 1,25(OH) D .

(CKI) known alternatively as p21, cip1, or waf1. This gene was originally identified in two ways. One group cloned it as waf-1 by subtractive hybridization, in a screen designed to search for direct targets of the tumor suppresser protein p53 (el-Deiry et al., 1993). Another group reported p21 as an inhibitor of G1 cyclin-dependent kinases (Harper et al., 1993), after an earlier report where it was described as an unidentified protein in a complex with cyclin D1 and the proliferating cell nuclear antigen (PCNA) (Xiong et al., 1992). Since these initial reports, p21 has been well established as an inhibitor of many cyclin-dependent kinases (CDKs) and a potent effector of cell cycle arrest (Xiong et al., 1993). p21 was subsequently established as an inhibitor of DNA synthesis via its interaction

with PCNA (Waga et al., 1994), involving a domain separate from that involved in its activity as a CDK inhibitor (Chen et al., 1995) (Fig. 11.2A). In addition to its identification as a transcriptional target of 1,25(OH) D /VDR, the transi ent, ectopic expression of the p21 gene product was shown to be sufficient to double the percent of U937 cells expressing the monocyte/macrophage terminal differentiation markers CD11b and CD14, (as compared to untreated controls) (Rots et al., 1998). These results indicate that the p21 gene product may play a central role in directing the response of the cells to this hormone. In fact, they demonstrate that p21 expression alone can send a significant portion of myeloid cells down the differentiation pathway

166


TABLE 11.1. 1.25(OH)2D3-Induced Clones from U937 Differential Screen

Class

Clone

Extent of Induction?

I

42 116 124 316 322 344 346 347 19 26 174 303 309 310 340 63 168 31 304 308

;; ;;;; ;; ; ;; ;; ;;;; ;; ; ;; ; ; ;; ; ;;;;; ; ;;; ; ; ;;

II

III IV

Description from BLAST Search@ Gene encoding ribosomal protein L21 p21Cip1/Waf1 Gene encoding robisomal protein S4 CD14 HOXA10 Gene encoding cyclin A Gene encoding Ki antigen Mad1 Low similarity to dopamine receptor Similarity to NF-kB Similarity to microtubule-associated protein and triacylglyceride lipase Similarity to respiratory syncytial virus gag Similarity to human immunodeficiency virus env Low similarity to Fos-related antigen Low similarity to TAN-1 (Drosophila Notch homologue) EST EST Unknown Unknown Unknown

?Extent of induction was determined by Northern blot analysis. A single ; indicates a less than 5-fold induction; ;; indicates 5- to 10-fold, ;;;, 10 to 20-fold; ;;;;, 20- to 40-fold; and ;;;;; represents induction over 40-fold. @Clones are divided into four classes. Class I represents clones with 100% identity to genes that have been cloned (but not previously characterized as 1.25 (OH) D targets). Class II represents clones that have varying degrees of identity to known genes, or regions of known genes. Class III clones match expressed sequence tags (ESTs), while class IV clones are those with no match or significant homology in the GenBank or the EST database.

all the way to the monocytic phenotype (Asada et al., 1998; Liu et al., 1996). This is not a unique finding, in the sense that initial stages of differentiation in a number of hematopoietic lines (Jiang et al., 1994) as well as other cell types (Halevy et al., 1995; Macleod et al., 1995; Matsumoto et al., 1998; Missero et al., 1995; Steinman et al., 1994; Zhang et al., 1995) have been correlated with p21 induction. This has been seen in response to a variety of differentiating agents. Experiments in some cell types have shown that ectopic p21 expression can not only initiate cell cycle arrest but can result in a differentiated phenotype as well (Erhardt and Pittman, 1998). More recent work has implied that the p21 activity must initially increase, and subsequently be followed by a decrease, in order for cells to reach terminal differentiation (Di Cunto et al., 1998).

During the initial response to 1,25(OH) D treatment of the myeloid cell line U937, there is a proliferative burst that precedes cell cycle arrest (Rots et al., 1999). p21 induction was shown to occur during this phase as well as during the subsequent cell cycle arrest. Indeed, there is some evidence that low levels of p21 expression may be important for cyclin-CDK assembly (LaBaer et al., 1997). This function may contribute to, or even account for, the proliferative burst seen in U937 cells. In addition, some tumor-associated mutants that lack the kinase inhibitory function of p21 selectively stimulate cyclin/CDK activity, thereby facilitating entry into and passage through the cell cycle (Welcker et al., 1998). It appears, then, that p21 may work through multiple mechanisms, regulating the cell cycle in a complex way, according to its concentration and stoichiometry in the cell.


Recently, U937 cell lines harboring an inducible form of p21 have shed more light on the role of this protein in differentiation (Asada et al., 1998). Controlled induction of the p21 gene again showed that p21 expression is sufficient to elicit a differentiation response in this cell line. Interestingly, induction of the antisense version of this gene, which caused a significant reduction in p21 protein expression by 1,25(OH) D , resulted in an inhibition of the normal 1,25(OH) D -mediated differentiation (Asada et al., 1998). Thus, p21 is clearly not only capable of initiating differentiation during this hormonemediated response but is apparently necessary as well. Further findings from this study show that p21 antisense expression results in an increase of apoptotic activity. This result is very much in agreement with more recent findings regarding the role of p21 in apoptosis. While nuclear localization is required for the CDK and PCNA inhibitory effects of the p21 gene product, there also appears to be an important function for a cytoplasmic population of this protein (Asada et al., 1999). The differentiation of myeloid progenitors is associated with a relocalization of nuclear p21 to the cytoplasm. Both in U937 cells and peripheral blood lymphocytes (PBLs), cytoplasmic localization seems to act as an antiapoptotic signal. In experiments performed to address this possibility, U937 cells were stably transfected with a zinc-inducible p21 deletion mutant lacking the nuclear localization signal. When this version of p21 was expressed, the cells did not differentiate, but did remain resistant to apoptotic signals. Cytoplasmically localized p21 forms a complex with the apoptotic signal-regulating kinase-1 (ASK1), preventing it from initiating a kinase cascade that would normally lead to apoptosis. p21 was also reported to interact with caspase 3, a cysteine protease known to be an important regulator in cell death signaling (Suzuki et al., 1999). In one report, caspase 3 was shown to be cleaved by p21, activating its enzymatic activity, and thereby allowing the conversion of the human lung cancer cell line A549 from a growtharrested state to an apoptotic one (Zhang et al., 1999). In the other case, the binding of procaspase 3 to p21 inhibited caspase 3 activity (Suzuki et al., 1999). A binding domain for this procaspase was identified at the N terminus of

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p21 (Fig. 11.2A), and this domain proved sufficient in HepG2 cells to prevent Fas-mediated apoptosis. It is becoming increasingly clear that the p21 gene product plays multiple and critical roles in cell cycle arrest, differentiation, and apoptosis in a variety of cell types in response to a variety of signals. This is especially true for the 1,25 (OH) D -induced differentiation of myelo monocytic cells, though there is much left to learn in this area. Interestingly, it was shown several years ago that the inducible expression of antisense VDR in U937 cells, which effectively knocks out expression of the gene, resulted in an increase in apoptosis in these cells (Hewison et al., 1996).

HOXA10 A second direct transcriptional target of VDR is HOXA10. Northern blot analysis of RNA derived from U937 cells demonstrated a vitamin D —mediated induction of this gene that was resistant to the presence of cycloheximide (Liu et al., 1996; Rots et al., 1998). As in the case of p21, ectopic overexpression of the gene was sufficient to induce the differentiation of a significant percentage of U937 cells, implicating the involvement of HOXA10 in the 1,25(OH) D induced differentiation of these cells (Rots et al., 1998). HOXA10 is a member of a large gene family consisting of clustered genes that share a characteristic DNA-binding motif known as a homeobox. These genes are known to be transcription factors and are involved in a wide variety of developmental processes (Gehring et al., 1994). Genes of this family were originally discovered for their involvement in Drosophila pattern formation, but it is now clear that homeobox genes exist in all animal species (Magli et al., 1997, and references therein). A homozygous HOXA10 knockout mouse (Satokata et al., 1995) showed that HoxA10-deficient males exhibit bilateral cryptochordism (Kolon et al., 1999; Satokata et al., 1995). In females, the HoxA10 deficiency leads to infertility as revealed by test matings. On further examination, it was determined that absence of the normal expression of HoxA10 in the oviduct and uterus was responsible for this phenotype. In addition, both males and females displayed an anterior

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Figure 11.2. Schematic representations of the p21 promoter and the domain structure of the p21 protein. A: p21 protein functional domains, as described in the text. B: The two regions of the p21 promoter responsive to HoxA10, the region known to be responsive to 1,25(OH) D and several other well-known elements. DNaseI footprinting has demonstrated that there are as many as six 1,25(OH) D -binding sites in this promoter (VCB and LIF, unpublished data). The mechanism underlying this arrangement has yet to be deciphered.

homeotic transformation of the L1 into a T13 vertabrae, as well as other vertebral abnormalities (Satokata et al., 1995). As has been shown for expression during pattern formation in Drosophila (McGinnis and Krumlauf, 1992), it turns out that HOX genes are coordinately regulated in blocks during myeloid differentiation (Magli et al., 1991). Later work has shown that different types of leukemias can be characterized by their pattern of HOX gene expression, and it was suggested that this pattern of expression could be used to define the differentiation state of a particular leukemia (Celetti et al., 1993). HOXA10 has a fairly restricted expression pattern with respect to hematopoietic cell types. A detailed study of

the expression of HOX genes in well-defined subpopulations of both CD34\ and CD34> bone marrow cells revealed an expression pattern that is restricted to all subtypes of the CD34> population (the primitive progenitors) (Sauvageau et al., 1994). This expression is markedly down regulated in the CD34\ population. The CD34> population is considered to contain the most primitive blood cells, including erythroid and myeloid progenitors and the putative stem cells (Lawrence et al., 1995, and references therein). Transcripts of HOXA10 were absent in normal peripheral blood lymphocytes, monocytes, and granulocytes. Expression of HOXA10 was also detected in all acute myelogenous


leukemias (AMLs), with the exception of the acute promyelocytic leukemia (AML-M3), and expression was strongest in the least differentiated subtypes. HOXA10 message was also present in cases of chronic myelogenous leukemia (CML) but reduced in accelerated phase and blast crisis, particularly lymphoid blast crisis, and HOXA10 expression was very rarely observed in any lymphoid leukemias (Lawrence et al., 1995). The overall conclusion from these studies is that HOXA10 expression is restricted to early stages of myeloid differentiation. Experiments were carried out to look at the effect of retroviral-driven overexpression of HoxA10 in mouse bone marrow cells. This overexpression dramatically affected myeloid and B-lymphoid differentiation, leading to a cell population devoid of both pre-B-lymphoid cells and macrophages. (Thorsteinsdottir et al., 1997). The bone marrow of these mice contained, at high frequency, a unique progenitor that had the ability to form megakaryocyte colonies. Interestingly, a large proportion of mice receiving these cells developed a transplantable acute myeloid leukemia. Although much research has been presented concerning the regulation of a variety of HOX genes, very few target genes have been identified for these transcription factors, and no targets have yet been identified for HOXA10. A consensus DNA response element has been determined for this protein by the method of random-binding sight selection (Benson et al., 1995). This element is a 12-base-pair sequence consisting of a TTAT core typical of many HOX-binding sites, flanked by nucleotides to make up the full consensus sequence AA(A/T)TTTTATTAC. This protein can form heterodimers with the nonHox homeobox proteins PBX1 and MEIS1, in a way that increases the specificity of binding (Chang et al., 1996; Shen et al., 1997a, 1997b). Though the importance of the proper expression of HOXA10 during myeloid differentiation is clear, it can only be understood when the target genes are identified. We have recently shown by transient transfection that the p21 gene is a direct target of HOXA10. Overexpression of this HOX gene in U937 cells in the presence of a reporter gene construct containing the p21 enhancer gives a 50-fold induction of luciferase activity. This regulation could be seen,

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though to a lesser extent, in the nonmyeloid cell line 10-1 (Bromleigh and Freedman, 2000). Two responsive regions have been identified in the p21 promoter that may account for this effect (Fig. 11.2B). Each of these regions contains three potential and overlapping 12-base-pair elements, bearing striking resemblance to the consensus site. The more distal of the two is over 3 kilobases from the transcriptional start site and on its own is capable of providing a more than 7-fold induction of luciferase activity in the presence of HOXA10 protein. The more proximal region is located just downstream of the 1,25(OH) D -responsive elements previously identified (Fig 11.2B), (Liu et al., 1996; V. Carrie Bromleigh and Leonard Freedman, unpublished data). This region of 40 base pairs can, on its own, modulate a 15- to 20-fold increase in luciferase activity in the presence of HOXA10 protein, and this induction is further elevated when PBX1a is coexpressed. Site-directed mutagenesis and binding studies will be instrumental in narrowing down the necessary sequences involved in this regulation. The picture that begins to emerge from these studies is that 1,25(OH) D initiates a complex and precise regulation required for the differentiation, of myeloid cells. To verify the necessity of HOXA10 expression during this differentiation, it will be necessary to generate an inducible, stable U937 cell line that can express the antisense version of this gene, effectively knocking out its activity. Several questions arrise from this work, such as why is the direct regulation of p21 by VDR potentially insufficient, and why should HOXA10 expression be so restricted to early stages of myeloid differentiation? The identification of additional target genes for this transcription factor will be a critical part of understanding how 1,25(OH) D specifically instructs the myeloid cell type to become a monocyte/macrophage-like cell.

Additional Direct and Indirect Targets of 1,25 (OH)2 D3 /VDR The basic helix-loop-helix (bHLH) E-box protein Mad1 was also identified as a target of VDR by differential screening. Mad-1 is a known heterodimeric partner of the bHLH pro-

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tein Max (Ayer et al., 1993), which can also partner with c-Myc. c-Myc is promitogenic, a proto-oncogene, and can also act as an apoptotic signal, depending on the cellular environment. It was previously shown that during the phorbol ester—induced differentiation of U937 cells, Mad-1 protein levels are rapidly elevated, even prior to the loss of Myc protein, and are accompanied by a switch from Myc-Max to Mad-Max heterodimers (Ayer and Eisenman, 1993; Ayer et al., 1993). This switch is proposed to be an anti-proliferative signal, and the Mad1 partner may act as an antagonist of cMyc activity (Cultraro et al., 1997a, 1997b). It is tempting to speculate that this arrest signal serves as a nice complement to the antiproliferative signal provided by p21 in response to 1,25(OH) D , but unlike p21, its expression has not been established as an essential part of differentiation for myeloid cell models (Ryan and Birnie, 1997). c-Myc was previously found to be indirectly downregulated during 1,25(OH) D -induced differentiation of the myeloid cell line HL60 (Pan et al., 1996; Taoka et al., 1993). It has been established that during 1,25(OH) D -induced differentiation of these cells, c-Myc transcription is downregulated through the binding of proteins to three regions of the first intron of this gene (Pan et al., 1996). Binding of these proteins appears to require the activation of protein kinase C (Pan and Simpson, 1999), a process known to occur as a result of 1,25(OH) D treatment (Marcinkowska et al., 1997; SimboliCampbell et al., 1992). Cyclin A was also identified as a VDR target in the differential screen, though upregulation of this gene proved to be sensitive to the presence of cycloheximide, implicating it as an indirect target. Cyclin A is a cell cycle regulatory protein known to function in both the S phase and the G2-M transition in cycling cells (Girard et al., 1991; Pagano et al., 1992). It was not immediately clear why a protein involved in cell cycle progression would be the target of an antiproliferative hormonal signal. As mentioned, U937 cells undergo a brief burst of proliferation prior to withdrawal from the cell cycle in response to 1,25(OH) D . This proliferative burst was shown to be accompanied by a transient increase in cyclins A, D1 and E protein levels, followed by a decrease at about 48 hours after 1,25(OH) D treatment (Rots et al., 1999).

Cdk2 activity, which is considered the ratelimiting step for progression through the cell cycle (Sherr, 1993) was also shown to increase transiently in U937 cells after 1,25(OH) D treatment. This transient activity peaked at around 8 hours and fell off at about 12 hours after treatment with 1,25(OH) D . Interestingly, this phenomenon of a transient, accelerated burst of proliferation seems to be a common aspect of differentiating cells. In contrast, the breast tumor cell line MCF7, which is already differentiated, will undergo cell cycle arrest in response to 1,25(OH) D , but does not exhibit the same accelerated proliferative phase prior to that cell cycle arrest (Rots et al., 1999). Just exactly why this burst of proliferation may be required prior to withdrawal from the cell cycle, and subsequent differentiation, is unclear. This may be better understood as more information becomes available about the physiological role of 1,25(OH) D in the innate immune response of whole organisms.

Cell Cycle Arrest and Differentiation The VDR targets described here are all directly or indirectly involved in regulation of the cell cycle. This fact underscores the importance of controlling cell division as a critical aspect of achieving a more differentiated phenotype. Our current understanding of the regulation of 1,25(OH) D target genes during myeloid differ entiation is shown schematically in Figure 11.3. A number of genes are transcriptionally upregulated by VDR in the presence of the hormone. Seemingly key among them is the CDK inhibitor p21. Remarkably, p21 is not only directly regulated through VDR but is also induced by HOXA10, another target of VDR (Fig. 11.3). This network of regulation is complicated by the fact the p21 is also upregulated by the transcription factor Sp1 (Biggs et al., 1996), which is known to be active during myeloid differentiation (Chen et al., 1993; Clarke and Gordon, 1998). The identification of additional primary targets, along with a more in depth understanding of the multiple roles p21 plays during cell fate decisions, will undoubtedly further elucidate the mechanisms by which 1,25(OH) D mediates myeloid cell differenti ation.

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Figure 11.3. A model for 1,25(OH) D -induced myeloid cell differentiation. When myeloid cells are exposed to 1,25(OH) D , VDR forms a heterodimer with the retinoid X receptor (RXR), and this complex regulates the expression of many diverse target genes, resulting in differentiation. Several target genes have been identified (see Table 11.1). The p21 and HOXA10 genes are two such targets. p21 is also an indirect 1,25(OH) D target in that the transcription factor HOXA10 appears to up regulate the transcription of this gene. The net effect of the ligand to coordinate regulation on cell cycle arrest and perhaps direct influences on differentiation. Alternatively, differentiation may be a result of the arresting of the cell cycle.

VITAMIN D3-INDUCED MONOCYTIC DIFFERENTIATION AND ACUTE PROMYELOCYTIC LEUKEMIA As described above, 1,25(OH) D has been im plicated in the differentiation pathways of several leukemic cell lines. Acute promyelocytic leukemia (APL) has become the paradigm for studies of the molecular basis of retinoic acid (RA) and 1,25(OH) D -induced differentiation of leukemic cells. APL is a member of the M3 subtype of human acute leukemias characterized by a block in intermediate promyelocytic differentiation (Bennett et al., 1976; Grignani et al., 1993; Groopman and Ellman, 1979; Huang et al., 1988; Stone and Mayer, 1990). All patients with APL harbor a chromosomal translocation fusing any of several genes with the retinoic acid receptor alpha isoform (RAR). These genes include PML-t(15;17)(q22;q21) (de The et al., 1990; Goddard et al., 1991; Kakizuka et al., 1991; Pandolfi et al., 1991), PLZFt(11;17)(11q23;q21) (H. M. Chen et al., 1993; S.

J. Chen et al., 1993; Licht et al., 1995), nucleophosmin-t(5;17)(q35;q21) (Redner et al., 1996), and NuMA (nuclear matrix—associated antigen)-t(11;17)(q13;q21) (Wells et al., 1997). The most well studied of the APL translocations are the t(15;17) and the t(11;17) joining the PML and PLZF genes respectively with that of RAR to create the primary fusion proteins PML-RAR and PLZF-RAR. This section focuses on these two APL-related translocations and their resulting effects with respect to monocytic differentiation induced by 1,25(OH) D . Although a large majority of patients with APL contain the t(15;17) translocation, a small but significant number have the t(11;17)(q23;q21) translocation fusing the PLZF gene to the RAR gene. PML and PLZF have similar nuclear localizations and have been shown to interact with one another (Koken et al., 1997). Further, the respective fusion proteins have been shown to localize in similar microspeckled nuclear bodies, heterodimerize with their respective full-length N-terminal moieties, and interact

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with RXR (Dong et al., 1996). While neither PML nor PLZF have been assigned normal functions, significant work has been done on both proteins. An important assumption is that the PLZF and PML proteins have distinct functions that may independently affect the molecular biology of APL. The PML and PLZF proteins as well as the PML-RAR and PLZFRAR translocations are described in detail in Chapter 20 in this book.

1,25(OH)2 D3-Induced Monocytic Differentiation and APL In 1993, several groups began to investigate the effects of PML-RAR on 1,25(OH) D -induced differentiation and transcription. Treatment with 1,25(OH) D , or 1,25(OH) D ;TGF (but not TGF alone) induced terminal monocytic differentiation of human promyelocytic and monocytic cell lines such as HL-60 and U937 (Grignani et al., 1993; Testa et al., 1993). In addition to measuring cell surface markers of monocytic differentiation by flow cytometry, functional monocyte differentiation was also measured by assaying the amount of lysozyme, an enzyme involved in the phagocytic response, that was produced in response to the hormone treatment. Using this assay, two separate studies assessed the ability of PML-RAR or PLZFRAR to block differentiation when expressed in promyelo- or monocytic cell lines (Grignani et al., 1993; Ruthardt et al., 1997). Using retroviral insertion, U937 cell lines constitutively expressing PML-RAR were established. In addition, U937 or HL-60 cell lines containing PMLRAR and PLZF-RAR under the control of an inducible metallothionine promoter were constructed. The expression of PML-RAR, in U937 cells, whether constitutive or inducible, blocked monocytic differentiation as a result of treatment with 1,25(OH) D or TGF; 1,25(OH) D as measured by expression of the above-mentioned markers (Grignani et al., 1993). All of the markers were strongly reduced, with the exception of CD11b, possibly because it is expressed well by undifferentiated U937 cells (Grignani et al., 1993; Testa et al., 1993, 1994). The differentiation block was more ap-

parent in the inducible cell lines that produce higher levels of PML-RAR. Therefore, the exogenous expression of PML-RAR was able to abrogate U937 monocytic differentiation specifically initiated by treatment with either 1,25(OH) D alone or 1,25(OH) D ;TGF. In contrast to the results of 1,25(OH) D treat ment, the myelocytic differentiation response to RA was elevated in PML-RAR-expressing cells, a result similar to the RA hypersensitivity seen in APL blasts. In comparison, PLZFRAR expression was sufficient to block monocytic differentiation by 1,25(OH) D alone, 1,25(OH) D ;TGF, or RA in both U937 and HL-60 cells to levels equal or greater than that seen by PML-RAR. Coincident with the above differentiation studies, a molecular basis for the differentiation block in response to 1,25(OH) D was emerg ing. Ligand association with VDR induces dimerization with the retinoid X receptor (RXR) (Cheskis and Freedman, 1994). Dimerization with RXR is an obligate step in 1,25(OH) D mediated transcriptional activation (Lemon et al., 1997). Using an electrophoretic mobility shift assay (EMSA), in vitro translated PMLRAR was shown to inhibit the formation of a VDR/RXR heterodimer on the osteopontinin vitamin D response element (VDRE), a direct hexameric repeat with a spacing of three nucleotides between the repeats (Freedman and Towers, 1991; Perez et al., 1993). Perez and colleagues (Perez et al., 1993) showed further that addition of RXR rescued the loss of a heterodimeric shift. In transfections of Cos-1 cells, PML-RAR was shown to significantly reduce the activity of a VDRE-containing chloramphenicol acetyl transferase (CAT) reporter construct in the presence of VDR and 1,25(OH) D . However, when RXR was co transfected, normal VDR signaling was recovered. This observation in conjunction with the EMSA data led the authors to conclude that inhibition of 1,25(OH) D -mediated differenti ation was caused by the PML-RAR fusion protein heterodimerizing with and sequestering RXR, an obligate VDR partner. In contrast to this conclusion, the authors also reported that PML-RAR did not inhibit gene activation by the thyroid receptor, which also uses an RXR partner. A second report (Weis et al., 1994) also

VITAMIN D -INDUCED MONOCYTIC DIFFERENTIATION

suggested that RXR sequestration was the mechanism of leukemogenesis in APL. As described previously, PML-RAR is a complex protein with many potentially important domains. Grignani et al. (Grignani et al., 1996) defined regions necessary for the differentiation block in U937 cells exhibited above. The block of differentiation is dependent in part on the RAR DBD and/or the PML hydrophobic clusters in the context of the entire fusion protein. The two regions, when not in the context of the fusion protein, could not effect a block. A fusion protein missing the RAR DBD was still able to elicit a block, albeit weakly (Grignani et al., 1996). A mutant in which the first of the four hydrophobic amino acid clusters is deleted conferred a complete loss of the ability to elicit a differentiation block. Therefore, the regions responsible for mediating the differentiation block are the first of four cysteine-rich heptad repeats and the RAR DNA-binding domain, but only when combined in the context of the entire fusion protein (Fig. 11.4A). As a result of the initial experiments, PMLRAR was proposed to block 1,25(OH) D -de pendent transcription by sequestering RXR, an obligate partner of VDR. The mutant series generated in PML-RAR allowed the mechanism of RXR sequestration to be questioned. Several results obtained were inconsistent with sequestration of RXR as a mechanism for the actions of PML-RAR. Specifically, overexpression of RXR was unable to restore differentiation to U937 cells expressing PML-RAR. Second, PML-RAR mutants were found with intact RAR domains that were unable to block differentiation. In fact, colocalization or immunoprecipitation of RXR and PML-RAR could not be clearly demonstrated (Grignani et al., 1996). Another study reached similar conclusions from experiments with retinoic acid— resistant APL blasts (NB4). In this study, cell lines were created that do not express PMLRAR but are still resistent to the effects of 1,25(OH) D (Dermime et al., 1993, 1995). Taken together, these observations and those mentioned above indicate that simple sequestration of RXR is not enough to account for the block of differentiation in response to 1,25(OH) D seen in the presence of PML RAR.

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PML/PML-RAR, PLZF/PLZF-RAR, and the Expression of p21 Our laboratory has conducted several new studies that may contribute to the overall understanding of the mechanisms of APL. Specifically, we determined the transcriptional effects of PML and PML-RAR or PLZF and PLZFRAR on 1,25(OH) D -induced transcriptional activation in transient transfection assays. PML-RAR expressed in CV-1 cells effectively blocks ligand-dependent transcription of a VDRE-containing reporter gene. PLZF-RAR also blocks transcription of this reporter but to a much lesser extent. PML alone or RAR alone was not sufficient to account for the loss in transcription, supporting earlier work suggesting that the entire fusion protein was necessary for abrogated differentiation. In contrast to previously published work, overexpression of exogenous RXR could not rescue the loss of 1,25(OH) D -induced transcriptional activation in the presence of PML-RAR. Interestingly, squelching of 1,25(OH) D -activated transcrip tion caused by expression of RAR alone could be rescued by expression of RXR. These results would argue that RXR sequestration by PMLRAR may not be the sole reason for the loss of VDR-mediated transcription. PLZF alone exhibited significant repression of reporter activation (Ward and Freedman, unpublished data). There has been much recent work on the role of coactivators and nuclear receptor signaling (reviewed in Freedman, 1999). Several classes of coactivators, acting as single peptides or large complexes, have been isolated and characterized. To test whether the loss in VDR transcriptional activity caused by PML-RAR was a result of competition for coactivator proteins, several coactivators, including CBP, TIF1, SRC-1, and members of the DRIP complex (Rachez et al., 1998), were overexpressed in epithelial and monocytic cell lines in an attempt to rescue the effects of PML-RAR. None of the proteins expressed had the desired effect, leading to the conclusion that PML-RAR, although presumably able to associate with coactivators via its RAR moiety, was not simply interupting known VDR-signaling pathways. The possibility remains that expression of any single member of a larger complex may not be sufficient to

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Figure 11.4. A: A general schematic depicting PML, RAR and the short and long isoforms of PML-RAR. Shaded or stippled areas indicate generally distinct domains (not drawn precisely to scale). Several papers (Fagioli et al., 1992; Goddard et al., 1991; Pandolfi et al., 1991, 1992) referenced break points as slightly different; as such, the amino acid designations given are representative. B: General organization of PLZF, RAR, and PLZF-RAR proteins. As in A, the indicated domains are not drawn precisely to scale. The break point is referenced from Chen (Chen et al., 1993).

render the complex functional again. If PMLRAR were to severly disrupt such a complex, expression of one protein may not be enough to repair it. As mentioned earlier in this chapter, the p21 gene was identified as a direct transcriptional

target of VDR. A loss of VDR’s ability to transcriptionally regulate the p21 gene could conceivably contribute to the phenotype of APL. In U937 cells we assayed the transcriptional effects of APL-associated protein expression on the p21 promoter. PML expression,

ACKNOWLEDGMENTS

which did not affect the synthetic VDRE-containing reporter, enhanced both basal and 1,25(OH) D -activated transcription of the p21 promoter. PML has also been shown to upregulate RA induction of the p21 promoter (Wang et al., 1998). PLZF, on the other hand, abrogated 1,25(OH) D -dependent activation of the p21 reporter as well as on a VDRE-containing artificial promoter. These opposing effects suggest differences in the regulatory roles of PML and PLZF on cell cycle progression. The effects of the APL fusion proteins PMLRAR and PLZF-RAR on p21 were most striking and paradoxical. On a reporter gene construct containing a synthetic VDRE in the core promoter region, PML-RAR completely blocked 1,25(OH) D -dependent activation. However, on a reporter gene construct containing the entire p21 promoter, PML-RAR caused a dramatic ligand-independent constitutive activation of the reporter. PLZF-RAR also elicited the same response. What pathogenic mechanism this activation might elicit has yet to be determined. However, several recent studies suggested that aberrant regulation of the p21 promoter by PML-RAR may be deleterious to accurate cell cycle regulation and differentiation(Casini and Pelicci, 1999; Yang et al., 1999). In fact, another report implicates p21 expression as a mode of transformation of primary cells (Di Cunto et al., 1998). These studies suggest that a simple interpretation of p21 expression affecting G1 arrest and differentiation may not be accurate and that the actual mechanism may be far more complicated than originally thought. The transcriptional properties of PML-RAR and PLZF-RAR with respect to p21 (and other genes) are only beginning to be clarified and any conclusions about their effects on differentiation at this point would be premature. Both fusion proteins, when present in patient blasts or cell lines, block monocytic differentiation in response to 1,25(OH) D . Expression of monocytic surface antigens is lowered and there is a loss of phagocytic activity and production of the monocyte enzymatic machinery. At the molecular level, PML-RAR has been shown to antagonize VDR/RXR DNA binding and to block transactivation of a VDRE-containing reporter gene. A mechanism for this block has not yet been clearly defined. Although the block of 1,25(OH) D —mediated differenti

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ation is clearly significant, the role of PMLRAR and PLZF-RAR in inhibiting this and other differentiation pathways remains largely unexamined. Further, we have shown differential regulation of VDR-mediated transcription on the p21 promoter by PML and PLZF. However, PML-RAR and PLZF-RAR similarly confer significant constitutive expression of p21, suggesting the possibility of converging modes of leukemogenesis in APL by disrupting normal cell cycle progression.

CONCLUSIONS The emergence of 1,25(OH) D as a general inhibitor of cell proliferation and inducer of myeloid differentiation has renewed interest in this ligand as a potentially efficacious chemotherapeutic for several types of cancers, including leukemias, and solid tumors such as breast and prostate. This relatively new biology of vitamin D has also stimulated interest in the development of synthetic vitamin D compounds for a variety of clinical applications, especially cancer. Although several classes of analogues with high antiproliferative but low calcemic activities have been identified, the mechanisms by which these analogues possess potent effects in regulating cell proliferation and differentiation are poorly understood. In particular, whether these compounds work indirectly via pharmacokinetic phenomena or through direct conformational changes on VDR is still a matter for debate. A clearer understanding of the molecular mechanisms underlying 1,25(OH) D —me diated differentiation in the myeloid system, such as those described in this chapter, will undoubtedly lead to more potent and more specific drugs that act through this pathway of regulated transcription.

ACKNOWLEDGMENTS The authors are indebted to past and present members of the Freedman lab who have contributed to the work of the past few years, described in this review, especially M. Liu, N. Rots, E. Anderson, J. Soto, M. RodriquezCalvo, Z. Suldan, L. Spivak, M. Cohen, and M. Bommakanti. Funding for much of this work

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was provided by the NIH and the Leukemia Society of America. The work of V. C. Bromleigh was supported in part by the the Jack and Susan Rudin Scholarship and the Bruce Forbes Fellowship. The work of J. Ward was supported in part by the Jack and Susan Rudin Scholarship.

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PART III


TRANSCRIPTION FACTORS AND THE LYMPHOID LINEAGE

CHAPTER 12


THE ROLE OF IKAROS FAMILY GENES IN LYMPHOCYTE DIFFERENTIATION AND PROLIFERATION NICOLE AVITAHL, SUSAN WINANDY, AND KATIA GEORGOPOULOS Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School

DESCRIPTION OF IKAROS FAMILY PROTEINS AND GENE EXPRESSION Structure and Function of Ikaros Family Proteins Ikaros is the founding member of a family of homologous genes that include Aiolos and Helios (Georgopoulos et al., 1992; Hahm et al., 1998; Kelley et al., 1998; Morgan et al., 1997). The protein products of these three genes are thought to work in concert to promote the proper specification, differentiation, and function of lymphocytes (Georgopoulos et al., 1994; Wang et al., 1996, 1998). The Ikaros gene encodes, by means of alternative splicing, a family of eight zinc-finger proteins that are expressed in hemopoietic cells (Hahm et al., 1994; Klug et al., 1998; Molna´r and Georgopoulos, 1994; Molna´r et al., 1996) (Fig. 12.1). The zinc-finger modules

of Ikaros are spatially arranged in two domains that are critical for its ability to function in multiple capacities. Ikaros isoforms differ in the number of Nterminal zinc fingers they contain. A minimum of two N-terminal zinc fingers are required for binding to a core consensus DNA-binding site, 5-GGGA-3. Isoforms that bind DNA can function as transcriptional activators. Ikaros isoforms with less than two N-terminal zinc fingers do not bind DNA and, therefore, cannot activate transcription (Molna´r and Georgopoulos, 1994). Interactions between isoforms with a DNA-binding domain (i.e., Ik-1 and Ik-2) and those which lack a DNA-binding domain (i.e., Ik-5 and Ik-6) can modulate the DNA-binding properties of the resulting complex (Sun et al., 1996). That is, when Ikaros proteins without a DNA-binding domain are present in excess, they can interfere with the DNA-binding activity of


183

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THE ROLE OF IKAROS FAMILY GENES IN LYMPHOCYTE DIFFERENTIATION AND PROLIFERATION

Figure 12.1. Schematic representation of Ikaros, Aiolos, and Helios proteins including the different isoforms of Ikaros and Helios. The locations within exons 1—7 (Ex1/2—7) of the zinc finger domains (F1—F6) involved in DNA binding and dimerization as well as a conserved activation domain are indicated by arrows. Stars on Ikaros isoforms indicate their predominant production from the mutant dominant negative (DN) locus.

the Ikaros complex. All Ikaros isoforms share the C-terminal zinc-finger domain that mediates interaction of Ikaros with itself as well as with the Ikaros family members Aiolos and Helios (Hahm et al., 1998; Kelley et al., 1998; Morgan et al., 1997). Like the largest Ikaros isoform, Ik-1, the Aiolos gene contains seven exons that encode highly conserved N- and C-terminal fingers that mediate its DNA binding and dimerization, respectively (Fig. 12.1). The Aiolos mRNA is not alternatively spliced and gives rise to only one protein. The Helios gene encodes two isoforms

(Hel-1 and Hel-2) similar in structure to Ik-1 and Ik-2 (Fig. 12.1). Ikaros, Aiolos, and Helios share the highest degree of homology within their two functionally distinct zinc-finger regions. These Ikaros family proteins interact via their C-terminal zinc-finger domains and can function as transcriptional activators (Hahm et al., 1998; Kelley et al., 1998; Koipally et al., 1999; Morgan et al., 1997; Sun et al., 1996). Immunofluorescence microscopy has revealed that Ikaros, Aiolos, and Helios colocalize within higher-order chromatin structures in the nuclei of primary T cells, suggesting that these proteins

DESCRIPTION OF IKAROS FAMILY PROTEINS AND GENE EXPRESSION

exist as a multimeric complex in vivo (Kelley et al., 1998). When expressed ectopically, DNA-binding forms of Ikaros, Aiolos, and Helios can activate transcription of reporter genes driven by the Ikaros consensus DNA-binding site. For Ikaros and Aiolos, a transcriptional activation domain has been mapped within the protein sequences encoded by exon 7 (amino acid 283 to 364) (Molna´r and Georgopoulos, 1994; Sun et al., 1996). Ikaros proteins bind DNA very poorly as monomers; thus, protein interactions are pivotal for the ability of Ikaros family proteins to bind DNA and function as transcriptional activators. Mutation of one or both of the C-terminal zinc fingers abrogates both DNA-binding and transcriptional activity. Ikaros and Aiolos can also function as repressors when tethered to a heterologous DNAbinding domain (Koipally et al., 1999). Unlike activation, repression by Ikaros does not require either the DNA-binding or multimerization domains and is dependent on promoter context and cell type. Chromatin immunoprecipitation assays demonstrate that reporter constructs that are repressed by Ikaros display localized histone deacetylation around the promoter region, suggesting that histone modification is the underlying mechanism. Consistent with this, repression by Ikaros is relieved by the histone deacetylase inhibitor trichostatin A (Koipally et al., 1999). More details of the mechanisms employed by Ikaros in transcriptional regulation are presented in a later section.

Expression of Ikaros family genes Of the Ikaros family genes, Ikaros is the most widely expressed throughout the hemopoietic compartment, while the expression patterns of Aiolos and Helios, though in part overlapping with that of Ikaros, are more restricted. Ikaros is first detected in the developing embryo at sites populated with early hemopoietic progenitors. Its expression pattern varies to coincide with the major sites of hemo/lymphopoiesis as these change during embryogenesis, and in the adult animal, Ikaros is expressed in hemopoietic cells of the bone marrow, thymus, and secondary lymphoid organs. Expression in the mouse can be detected first by in situ hybridization at

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embryonic day 7.5—8 (E7.5—E8) in distinct mesodermal progenitors in the splanchnopleura region (Tohru Ikeda and Katia Georgopoulos, unpublished) and at E8 in the blood islands of the yolk sac (Kelley et al., 1998). By E9.5, Ikaros expression shifts to the fetal liver primordium at the time that this organ becomes the major site of hemopoiesis in the embryo proper (Georgopoulos et al., 1992). By E16, expression in the fetal liver has declined, correlating with a shift from primitive to more committed erythroid and myeloid precursors. In both the fetal liver and adult bone marrow hemopoietic progenitors, Ikaros message is detected in the Lin\ckit>Sca-1> population, which is enriched in hemopoietic stem cells (HSC), and in the Lin\ckit>Sca-1\ population, which is enriched for erythroid and myeloid precursors (Kelley et al., 1998; Morgan et al., 1997). Analysis performed with samples of 5—10 cells confirms that it is expressed in pluri- as well as multipotent HSCs (Klug et al., 1998). Although Ikaros is expressed at sites of early erythro/myelopoiesis, it becomes downregulated during differentiation along the erythroid lineage, while being expressed at relatively high levels in neutrophils (Klug et al., 1998). Like Ikaros, Helios is also expressed in sites of primitive hemopoiesis, that is, in yolk sac blood islands at E8 and in the E11 fetal liver. However, unlike Ikaros, which is expressed at high levels in the fetal liver, Helios is only expressed at low levels. While Helios is also expressed at early stages of erythro/myelopoiesis — that is, in the bone marrow Lin\c-kit>Sca-1\population — it is not expressed at later stages (Hahm et al., 1998; Kelley et al., 1998). In contrast to Ikaros and Helios, Aiolos expression cannot be detected by in situ hybridization in sites of primitive hemopoiesis, nor is its expression detected at any stage of erythroid or myeloid development. The earliest stage of embryonic hemopoiesis at which Aiolos can be detected by in situ hybridization is in the thymus at E16, when this organ is populated largely by CD4>8> thymocytes (Morgan et al., 1997). In contrast, Ikaros is detected in the thymus at E12, when this organ is first being seeded by the earliest thymocyte precursors (Georgopoulos et al., 1992), and low levels of Helios are detected at E13 (Kelley et al., 1998), indicating that Aiolos expression is induced at a

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later stage of lymphocyte development. By using the sensitive technique of reverse transcriptionpolymerase chain reaction (RT-PCR), Aiolos mRNA can be detected in a subpopulation of Lin\c-kit>Sca-1>cells which express the surface marker Sca-2> and have the potential for lymphoid differentiation (Morgan et al., 1997), as well as in a population of early lymphoid progenitors, which express low levels of CD4 and are negative for B220 (Nicole Avitahl and Katia Georgopoulos, unpublished). Thus, Aiolos expression coincides with the earliest step in lymphoid commitment. While Ikaros is expressed in all lymphoid lineages in the adult, including B, T, T, NK (natural killer) and dendritic antigenpresenting cells (APCs) of the thymus and spleen, Aiolos and Helios are expressed in a more restricted fashion. In contrast to Ikaros, which is expressed at comparably high levels in all lymphoid cells, Aiolos is expressed at very low levels in pro-B (B220>CD43>sIgM\) and pro-T (CD4\CD8\) cells, but undergoes a dramatic upregulation as these progress to the pre-B (B220>CD43\sIgM\) and pre-T (CD4>CD8>) cell stages, respectively, and it continues to be expressed at later developmental stages. High expression levels are specific to B and T cells, as Aiolos is not expressed in fetally derived T populations and only at very low levels in NK and dendritic APCs. Whereas Helios is expressed in early-stage pro-B cells (fraction A1, B220>CD43>sIgM\CD4 ), it is not expressed at later stages of B-cell development or in APCs (Kelley et al., 1998). Helios is expressed in T, T, and NK cells, with levels being higher in CD4>8> thymic cells than in mature splenic T cells, where it is only expressed in a small subset. In summary, expression of Ikaros and Helios is highest in the thymus, while Aiolos expression is highest in mature B cells. While Ikaros and Helios are expressed at the earliest stages of hemopoiesis, only Ikaros continues to be expressed in B and T lymphoid and in myeloid cells, whereas Helios becomes restricted to the T lineage. Aiolos expression is induced upon lymphoid lineage commitment and its expression is specific for B, T, and NK cells (summarized in Fig. 12.2). While Aiolos and Helios have only 1 and 2 splice forms, respectively, Ikaros has 8 different splicing variants (Fig. 12.1), some of which lack

a DNA-binding domain and can interfere with the DNA-binding ability of other Ikaros isoforms, as well as Aiolos and Helios. RT-PCR experiments performed on highly purified populations indicate that, at the RNA level, the relative levels of the different isoforms remain comparable from the HSC stage throughout hemopoiesis, with Ikaros isoforms 1 and 2 being expressed at much higher levels than isoforms 2A-6 (Kelley et al., 1998; Morgan et al., 1997). However, Ik-4 is expressed at relatively high levels (comparable to Ik-1 and Ik-2) in E14-E16 fetal liver hemopoietic populations and in E14 fetal thymocytes, which are predominantly CD4\CD8\ (Molna´r and Georgopoulos, 1994). It may, therefore, be that Ik-4 plays a specific role in fetal hemo/lymphopoiesis. Since the expression levels of the different Ikaros isoforms do not appear to vary significantly during development, mutations in Ikaros that deregulate splicing and alter the relative isoform levels would be expected to interfere with normal lymphocyte development and function. Indeed, this has been shown to be the case, as overexpression in the hemo/lymphoid system of Ikaros isoforms that lack a DNA-binding domain causes defects in lymphopoiesis and a deregulation in the homeostasis of mature thymocytes (Georgopoulos et al., 1994; Winandy et al., 1995, Susan Winandy and Katia Georgopoulos, unpublished).

THE ROLE OF IKAROS AND AIOLOS IN LYMPHOCYTE DEVELOPMENT Effects of an Ikaros Null Mutation on Lymphocyte Development In order to study the role of Ikaros in development of hemopoietic cells, two different functional domains of the Ikaros protein were targeted for deletion in mice (see Fig. 12.3). To determine the effect of loss of Ikaros activity, a mutation was targeted that resulted in deletion of most of the coding region of exon 7 (containing the activation domain and C-terminal zinc fingers) including its 5 splice donor site (Wang et al., 1996). This mutant Ikaros locus produces an unstable protein and mice homozygous for this mutation are functionally null for Ikaros

THE ROLE OF IKAROS AND AIOLOS IN LYMPHOCYTE DEVELOPMENT

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Figure 12.2. Summary of expression patterns of Ikaros (Ik), Aiolos (Aio), and Helios (Hel). The differentiation pathways of the erythroid, myeloid, and lymphoid lineages from the hemopoietic stem cell (HSC) are represented schematically, with each circle corresponding to a stage of development as defined by cell surface marker expression. Relative expression levels of each Ikaros family gene are indicated by 9 (not expressed), ;/9 (expressed at very low levels or only in a small subset of cells), and ; to ;;;; (moderate to very high levels).

activity (Ikaros null mice) (Wang et al., 1996). Ikaros null mice have multiple defects in hemopoiesis and lymphopoiesis (Wang et al., 1996). They display a complete block in both fetal and adult B—cell development (Fig. 12.3). NK and thymic dendritic cells, which arise from the earliest described T—cell progenitor (Ardavin et al., 1993; Shortman and Wu, 1996; Wu et al., 1997), are absent and significantly reduced, respectively. T-cell development is differentially affected by the lack of Ikaros activity. All fetal waves of T cells are missing, and therefore the thymuses of Ikaros null mice are devoid of identifiable lymphoid cells until the first few days after birth. Lack of fetal T-cell development results in the absence of dendritic

epidermal V3 T-cells, a TCR lineage subset whose development is restricted to fetal stages (Ikuta et al., 1990; Ikuta and Weissman, 1991). Other TCR lineages are differentially affected (Wang et al., 1996). Postnatally, reduced numbers of T-cell precursors appear in the thymus. This reduction in the number of pro-T, or CD4\CD8\TCR\, precursors is observed throughout the life of the animal. These precursors can give rise to some lineages of T cells and to T cells that show evidence of deregulated maturation. Ikaros null thymocytes are skewed toward CD4 single positive T cells and intermediates in transition to this lineage (Wang et al., 1996). This phenotype may, in part, be due to maturation of CD4> T cells in

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Figure 12.3. Summary of lymphoid phenotypes observed in mice heterozygous or homozygous for the Ikaros dominant negative (DN) mutation and homozygous for the Ikaros and Aiolos null mutations.

the absence of positive selection. Thymocytes that are positively selected upregulate the CD69 activation marker as a consequence of productive ligation of the TCR complex (Bendelac et al., 1992). Although all of the Ikaros null CD4> thymocytes express high levels of TCR, the majority do not express CD69, suggesting that they have not been positively selected. This hypothesis is further strengthened by the greatly reduced numbers of peripheral T cells observed in these animals, suggesting that they are being retained in the thymus due to incomplete differentiation. Finally, Ikaros null thymocytes and peripheral T cells display augmented proliferative responses when triggered via their TCR ex vivo and thymocytes undergo clonal expansions in vivo (Wang et al., 1996). These T-cell phenotypes in Ikaros null mice establish a role for Ikaros as an essential regulator of maturation and proliferation in the T-cell lineage.

More Severe Lymphoid Defects Caused by a Dominant Negative Mutation in Ikaros A dominant negative mutation was generated by targeting exons 3 and 4 in the Ikaros locus, thus deleting the high-affinity DNA-binding domain (Georgopoulos et al., 1994). Proteins generated from the mutant Ikaros locus cannot bind DNA, yet they retain the ability to interact with full-length Ikaros, Aiolos, and Helios proteins via the C-terminal zinc fingers and can thus interfere in a dominant fashion with the DNA-binding activity of full-length Ikaros family proteins (Sun et al., 1996). Indeed, in lymphocytes heterozygous for the dominant negative (DN) mutation, mutant Ikaros proteins colocalize in the nucleus with the DNA-binding isoforms made by the intact wild-type allele. Proteins generated predominantly by the mu-

THE ROLE OF IKAROS AND AIOLOS IN LYMPHOCYTE DEVELOPMENT

tant locus are Ik-6, Ik-7, and Ik-8 (see Figs. 12.1 and 12.3), which are also made by the wild-type locus but at much lower levels. Mice homozygous for the DN mutation (referred to as DN\\) display a more severe phenotype than mice homozygous for the null mutation. In DN\\ mice all lymphoid development is blocked, so even the postnatal wave of T-cell development is abolished (Fig. 12.3). Moreover, there is a dramatic increase in extramedullary hemopoiesis in the spleen (Georgopoulos et al., 1994). The severity of the DN\\ phenotype relative to the null \\ phenotype indicates that the dominant negative proteins target the activity of proteins other than Ikaros at early stages of hemopoiesis. A likely target is Helios activity, since the gene is expressed in both the HSC-enriched Lin\ckit>Sca-1>population and the c-kit>Sca-1\ population, which is enriched for erythroid- and myeloid-committed progenitors. Since Aiolos is not expressed in these populations, it is ruled out as a target at these early stages of differentiation. However, it is likely that the severe lymphoid defect is due to a dominant negative effect against the activities of both Aiolos and Helios at early stages of lymphoid development, while earlier hemopoietic effects might be due to dominant negative action against Helios in HSCs and at early stages of erythroid/myeloid specification. Future experiments with engineered mice lacking Helios activity will address this question.

Effects on B-Cell Maturation Caused by a Null Mutation in Aiolos As with the Ikaros gene, a null mutation was introduced into the Aiolos locus by targeting exon 7 (Wang et al., 1998). Mice homozygous for the mutation (Aiolos\\) display selective effects in the B lineage. In the bone marrow, there is an increase in the pre-B-cell population (B220>CD43\sIgM\), but a marked decrease in the long-lived, recirculating B-cell population (B220 CD43\sIgM ) that expresses the highest levels of Aiolos. In Aiolos null animals, a high percentage of splenic B cells display an activated phenotype based on expression of cell surface markers. Moreover, germinal centers

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(GCs) are seen in the spleen in the absence of antigenic challenge. GCs, the hallmark of a T-cell—dependent immune response, constitute sites of isotype switching, affinity maturation, and clonal expansion of B cells. Their formation in the absence of antigen challenge suggests that Aiolos null animals mount immune responses to inappropriate antigens, such as self-antigens or low levels of foreign antigen not sufficient to elicit a response in wild-type animals. Aiolos-deficient B cells are hyperresponsive to signaling through the B-cell receptor (BCR) and, to a lesser extent, CD40. It is likely that lower thresholds in both BCR and CD40 signaling pathways cooperate to trigger a GC reaction under conditions that fail to do so in a wild-type animal. Consistent with this, Aiolos null animals have increased levels of serum IgG2a, IgE, and IgG1, and autoantibodies are present in the sera of aging mutant animals. The selective overproduction of only certain isotypes suggests that Aiolos may play a role in specific signaling pathways directing isotype switching. The presence of high levels of sterile transcripts in the spleens of 50% of Aiolos null animals suggests that this role may be at the level of transcription. We propose that in Aiolos null splenic B cells, transcription from the I promoter can occur in response C to inappropriate signaling cues. The events triggering such signaling pathways may vary from animal to animal, thus explaining why this phenomenon is not observed in all mutant animals. Other populations affected in Aiolos null animals are the B-1a population of the peritoneum and the IgM marginal zone B cells that surround the follicle and separate the white from the red pulp in the spleen. Both of these populations, which are thought to arise from a common progenitor, are severely reduced in Aiolos null animals. In summary, whereas Ikaros is essential for the specification of B, NK, and certain T cells, Aiolos is not. However, Aiolos plays an essential role at later stages of B-cell differentiation (summarized in Fig. 12.3). In the absence of Aiolos activity, B-1a, marginal zone, and recirculating B-cell populations are severely reduced. In contrast, the number of conventional B cells is increased, possibly as a result of their hyperresponsiveness to antigenic stimuli.

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THE ROLE OF IKAROS AND AIOLOS IN LYMPHOCYTE HOMEOSTASIS Development of T-Cell Malignancies in Ikaros DN+/− Mice Studies with mice heterozygous for the dominant negative Ikaros mutation (DN>\) implicate Ikaros as a tumor suppressor gene for the T-cell lineage (Winandy et al., 1995). Initially, Ikaros DN>\ mice have grossly normal erythroid, myeloid, and lymphoid populations, as determined by flow cytometry. However, ‘‘phenotypically normal’’ Ikaros DN>\ thymocytes and splenocytes display augmented proliferative responses when triggered via the T-cell receptor ex vivo. This augmentation can be further amplified within the mutant T-cell population by addition of exogenous interleukin-2 (IL-2) to cultures of activated mutant T cells, suggesting deregulation of both TCR and IL-2R signaling pathways (Avitahl et al., 1999). Phenotypic changes in the thymocyte compartment of Ikaros DN>\ mice are detected between 2 and 3 months of age (Winandy et al., 1995). First, expansion of the intermediate double positive (CD4>8>) and single positive (CD4> or CD8>) thymocyte populations is observed. The intermediate double positive thymocytes correspond to cells in the process of downregulating either the CD4 or CD8 coreceptor to become mature single positive T cells ready for export into the periphery. This change in composition in the thymocyte compartment is quickly followed by the development of aggressive T-cell leukemias and lymphomas with 100% penetrance (Winandy et al., 1995). The malignant cells arise in the thymus, as determined by first evidence of clonal outgrowth. However, they rapidly spread to the periphery, resulting in complete takeover of the hemopoietic organs with clonal lymphoblastic T cells and extensive infiltration by these cells of nonlymphoid organs such as the lung, liver, and kidney. In all cases, the malignant cells express the CD3/TCR complex. However, the coreceptor phenotype differs between mice. CD8> populations arise with the highest frequency, but CD4>, CD4>CD8>, and CD4\CD8\ populations are also detected. Within a given animal, a percentage of these lymphoblastic T cells express the CD25 (interleukin-2 receptor

chain) activation marker. Most of the cell lines established from these mice express CD25, strongly suggesting that expression of CD25 correlates with a high degree of malignancy. Interestingly, however, we have found no evidence of production of interleukin-2 (IL-2) by these transformed cells (Susan Winandy and Katia Georgopoulos, unpublished). Genetic analysis of the malignant T cells in the Ikaros DN>\ mice revealed loss of Ikaros heterozygosity (LOH) at the DNA level. However, loss of wild-type Ikaros expression is not a prerequisite for transformation to occur. Mice transgenic for high copy numbers of a dominant negative Ikaros isoform (Ik-7, Fig. 12.1) develop leukemias and lymphomas with 100% penetrance indistinguishable from those that develop in the Ikaros DN>\ mice (Susan Winandy and Katia Georgopoulos, unpublished). The malignant T cells in these mice invariably retain normal levels of Ikaros expression from the wild-type Ikaros alleles. Mice that express low levels of the transgene never develop neoplasias, whereas mice with intermediate levels do so with delayed kinetics. Taken together, these observations prove that expression of the nonDNA-binding isoform in the transgenic or Ikaros DN>\ T-cell acts to titrate out Ikaros activity provided by the wild-type allele(s). If, however, low levels of the transgene are expressed that are insufficient to counteract the endogenous Ikaros activity, the transformation process does not occur. Ikaros-deficient thymocytes may be particularly vulnerable to transformation due to the receptor-mediated proliferative expansions coupled to differentiation events that they undergo in an ordered fashion. We have shown that immature thymocytes can be targets for transformation only in the presence of intact TCR or pre-TCR signaling pathways. Ikaros DN>\ mice that cannot recombine their TCR genes due to an additional mutation in the recombinase activating gene 1 (Rag 1\\) do not develop leukemias and lymphomas (Winandy et al, 1999). The transformation phenotype can be restored, however, when a TCR transgene is bred onto the Ikaros DN>\ x Rag 1\\ background (Winandy et al, 1999). Therefore, Ikaros controls proliferation events mediated by pre-TCR or TCR signaling. Decreasing the levels of Ikaros activity leads to

THE ROLE OF IKAROS AND AIOLOS IN LYMPHOCYTE HOMEOSTASIS

deregulation in pre-TCR and TCR-mediated proliferation, and causes T-cell transformation.

Lymphoproliferations in Ikaros Null Mice The transformation phenotype observed in the Ikaros DN>\ mice could be due to a combination of the functional inactivation of wild-type Ikaros proteins and a dominant-negative effect of the mutant Ikaros proteins toward the function of other proteins with which Ikaros interacts. However, the phenotype of Ikaros null mice demonstrates that Ikaros activity alone plays a central role in regulating T-cell proliferation. Lack of all Ikaros activity at the earliest stages of T-cell development in the Ikaros null mice results in deregulated maturation, hyperproliferation in response to activation signals and, eventually, transformation of thymocytes. Clonal expansions are detected within the Ikaros null postnatal thymus as early as 10 days after the appearance of T-cell precursors, and as soon as 1 month of age, single T-cell clones predominate in the thymus. This phenotype proves that loss of Ikaros activity alone, in the absence of dominant negative interfering isoforms, can lead to transformation of immature thymocytes. However, the Ikaros null malignancies are less aggressive than those that arise in the Ikaros DN>\ mice. After loss of heterozygosity, Ikaros DN\\ clones grow indefinitely ex vivo in the absence of added cytokines, whereas very few of the Ikaros-null clones display this property. Furthermore, whereas the DN\\ clones, derived from Ikaros DN>\ mice, quickly form solid tumors upon adoptive transfer into nude mice, very few of the Ikaros null clones, derived from mice homozygous for the Ikaros null mutation, do so, and the kinetics of tumor outgrowth is significantly slower. The more aggressive growth phenotype of the DN\\ malignancies suggests a dual molecular event. We hypothesize that a decrease of Ikaros activity, either by the absence of the protein (Ikaros null mice) or by the incorporation of dominant negative Ikaros proteins in Ikaros complexes (Ikaros DN>/\ mice), destabilizes homeostasis within the immature thymocyte population. However, for the development of highly aggressive malig-

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nancies, functional loss of another protein is required. Loss of function would be brought about by interaction of this protein with the dominant negative Ikaros protein. One candidate target of this dominant negative effect is Aiolos, which is also expressed in the developing thymocyte (Morgan et al., 1997). Aiolos, as well as wild-type Ikaros protein, can be coimmunoprecipitated with the dominant negative Ikaros protein, confirming that Aiolos is a target of dominant negative interference by the mutant Ikaros proteins (Morgan et al., 1997). In support of this hypothesis, we observe that, although Ikaros null>/\ and Aiolos null\\ mice rarely develop T-cell malignancies, these arise rapidly in mice in which the two mutations have been combined (Ikaros-null>/\ Aiolos null\\) (Marta Cortes and Katia Georgopoulos, unpublished).

Lymphoproliferations in Aiolos Null Mice Ikaros plays a central role in maintenance of T-cell growth control and Aiolos contributes to this role. In a manner parallel to that of Ikaros in the T-cell lineage, Aiolos is important for regulating B-cell responsiveness and proliferation. Aiolos null B cells hyperrespond relative to wild-type cells when triggered via the B-cell receptor (BCR). Treatment with whole antimouse antibody, which ligates both the BCR and FcRIIB1 receptor (the low-affinity receptor for IgG), causes little proliferation of wildtype B cells. This is due to negative signaling from the FcRIIB1R, which interferes with Ca> mobilization induced by BCR engagement, thus attenuating the proliferative response (D’Ambrosio et al., 1995, 1996; Hippen et al., 1997). Treatment of Aiolos-deficient B cells with anti- causes an increased proliferative response relative to wild type, which could be due to either an increase in positive signaling or a decrease in negative signaling. Using anti- F(ab) fragments as a stimulant eliminates negative signaling via the FcRIIB1R and addresses this question. Treatment with anti- F(ab) fragments brings about a greater prolif erative response in Aiolos null B cells relative to wild type, though the difference is less than stimulation with whole anti-. Therefore, in the absence of Aiolos, B cells are hyperresponsive to

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BCR signaling due to enhanced positive as well as diminished negative signaling. Moreover, Aiolos null B cells respond to concentrations of F(ab) fragment that are insufficient to elicit a proliferative response in wild-type B cells, indicating a lower activation threshold in the absence of Aiolos. Finally, the degree of hyperresponsiveness correlates inversely with the level of Aiolos activity. Therefore, Aiolos sets thresholds for signaling responses in B cells in a manner parallel to the thresholds set by Ikaros activity in T cells (described in the next section). Consistent with a role in regulating B-cell proliferative responses, the lack of Aiolos activity gives rise to lymphomas, the majority of which are derived from clonal B-cell expansions that cause enlargement of the spleen and lymph nodes (Wang et al., 1998). However, unlike the relatively rapid kinetics and 100% penetrance of T-cell lymphomagensis in Ikaros-mutant mice (3—6 months), B-cell lymphomas develop at a slower rate (8—10 months) in the Aiolos null background and the degree of penetrance is lower. In summary, Ikaros and Aiolos cooperate to regulate T- and B-cell proliferation (summarized in Fig. 12.3). However, gene disruption experiments indicate that Ikaros and Aiolos play predominant roles in regulating T- and B-cell proliferation, respectively. Possibly, these differences reflect the expression levels of these genes, as Ikaros expression peaks in thymic T cells, while Aiolos expression peaks in mature B cells.

The Role of Ikaros in Regulation of the Cell Cycle T-cell activation encompasses the molecular processes that permit a quiescent T cell (G0) to enter and progress through the cell cycle. T cells with reduced levels of Ikaros activity require fewer T-cell receptor engagement events to become activated (Avitahl et al., 1999). The amount of signal required for activation directly correlates with intracellular levels of Ikaros DNA-binding activity (Ikaros DN>\ Ikaros null>\ Ikaros wild type). In addition, Ikarosdeficient T cells progress faster from G1 into S phase, suggesting that Ikaros controls this transition (Avitahl et al., 1999). Intracellular path-

ways that lead to T-cell activation and proliferation do so by controlling expression of cell cycle regulators such as cyclin D, cyclin E, p21, p16, and p27, which act in concert to control progression through the cell cycle. Deregulated expression of these important proteins can lead to aberrant proliferation and transformation. However, there is no evidence for deregulated expression of these factors in Ikaros-mutant T cells (Avitahl et al., 1999), suggesting that Ikaros functions downstream of these proteins. The activity of Ikaros is epistatic to multiple signaling pathways triggered by extracellular signals received through TCR and/or IL-2 receptor complexes. Inhibitors of these signaling pathways, which mediate the G0 to G1 and G1 to S transitions in mature T cells (i.e., protein kinase C, phosphatidylinositol 3-kinase, ras), are less effective in preventing proliferation of Ikaros-deficient T cells as compared to wildtype T cells (Avitahl et al., 1999). Taken together, these observations suggest that Ikaros works as a negative regulator of T-cell proliferation and that its activity is modulated by pathways that lie downstream of TCR and IL-2 receptor signaling.

Changes in Nuclear Localization of Ikaros/Aiolos During the Cell Cycle Consistent with a role in regulating cell cycle progression, the nuclear staining patterns of both Ikaros and Aiolos change dramatically in primary murine B and T cells as these become activated and enter the cell cycle. In resting cells, both proteins are localized in the nucleus, with a portion being in a diffuse pattern distributed throughout the nucleus and a portion concentrated in speckles. Ikaros and Aiolos colocalize within these ‘‘speckles,’’ which are mostly excluded from heterochromatin, as revealed by Hoechst staining. However, in activated cells Ikaros and Aiolos display a striking toroid pattern of nuclear staining (see Fig. 12.4). These toroids form in mid-G1, persist to the end of S phase, and are associated with centromeric heterochromatin (Avitahl et al., 1999; Brown et al., 1997; Wang et al., 1998). Helios displays the same nuclear staining patterns in resting and activated T cells (Hahm et al., 1998; Kelley et al., 1998). Although Ikaros and Aiolos (and likely

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Figure 12.4. During an immune response, the antigen receptors on a given T cell are engaged by antigen presented in the correct context on the surface of an APC. T-cell activation requires a sufficient number of TCR engagement and signaling events to overcome response thresholds. In mature T cells, the level of Ikaros activity sets activation thresholds and regulates G0-G1-S transitions. In Ikaros-mutant T cells, where Ikaros activity is reduced, fewer TCR engagements are required to drive a quiescent T cell into the cell cycle, and the mutant cells progress more quickly through G1 to S. Similarly, B cells with reduced levels of Aiolos activity require fewer BCR engagements to become activated. The top bar represents the cell cycle progression of wild-type murine T cells while the bottom bar depicts the shortened G1 of Ikaros-mutant cells. Below the top bar are immunofluorescence confocal microscopy images showing the nuclear staining patterns of Ikaros in wild-type T cells at the corresponding points in the cell cycle. The cells have been pulsed with the nucleotide analog bromodeoxyuridine (BrdU), which becomes incorporated into nascent strands of replicating DNA. Staining with antibodies specific for BrdU reveals nuclear sites of active DNA replication. Ikaros is shown in red and replicated DNA is shown as green. In late S phase, Ikaros colocalizes with BrdU-labeled replication foci. Ikaros is not detected on condensed chromosomes from late prophase through early anaphase of mitosis (M). Ikaros and Aiolos display the same changes in nuclear staining patterns during the cell cycle in both B and T cells.

also Helios) are readily detectable in G0-S phase of the cell cycle, they are not detectable during prophase, metaphase, and early anaphase of mitosis (Avitahl et al., 1999; Brown et al., 1997) (see Fig. 12.4). However, these proteins can be seen concentrated at the centromeres at late anaphase/telophase. Recruitment of Ikaros and Aiolos into these heterochromatin-associated structures is blocked by inhibitors of TCR or IL-2R signaling, which target events in early to mid-G1, such as PD98059 and staurosporine, but it is unaffected by the inhibitors wortmannin and rapamycin, which arrest cells in late G1, prior to the transition into S phase (Avitahl et al., 1999). In addition, Ikaros toroids form less efficiently

in DN>\ and null>\ T cells, correlating with a decreased sensitivity of these cells to inhibitors of cell cycle progression. Thus, the recruitment of Ikaros into toroid structures in mid-G1 may be important in regulating G1-S transitions. The toroidal shape of the structures to which Ikaros and Aiolos become recruited upon Tand B-cell activation are reminiscent of the staining patterns of late-replicating heterochromatin (revealed by BrdU labeling) and of proteins associated with replicating DNA, such as methyltransferase and cyclin A. Indeed, both Ikaros and Aiolos colocalize with the maintenance methyltransferase 1 (Dnmt1) in all cells examined and with BrdU-labeled replication foci in a subset of cells in late S phase (Avitahl

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et al., 1999; Wang et al., 1998) (Fig. 12.4). Ikaros was also seen to colocalize with cyclin A in a subset of cells. The localization of Ikaros and Aiolos in replication factories suggests that these proteins participate in molecular events that control DNA replication. Consistent with this, in activated primary DN>\ or null\\ T cells, chromosomal aberrations, such as differences in Giemsa stain banding patterns and the presence of extrachromosomal fragments (double minutes), can be detected after 4—5 days in culture. Specifically, double minutes are thought to be the by-products of amplification or premature chromosome condensation events, both of which are consistent with a deregulation in DNA replication. Therefore, a reduction in Ikaros activity not only lowers signaling thresholds in T cells and causes an accelerated transition to S phase (summarized in Fig. 12.4), it also brings about illegitimate propagation of genetic material. How aberrant genetic propagation occurs is a subject of great interest. One possibility is that the accelerated transition to S phase — that is, shortened G1 — could cause premature entry into S phase, thus causing some replication origins to fire at the inappropriate time and possibly more than once per cell cycle. However, we favor the model (discussed in a later section) that Ikaros and Aiolos play a more direct role in regulating the activity of replication origins.

MECHANISMS OF IKAROS/AIOLOS ACTION IN LYMPHOCYTE DIFFERENTIATION Ikaros/Aiolos as a Regulator of Chromatin During Lymphocyte Activation How might Ikaros function as a regulator of events in transcription and DNA replication? An increasing body of work suggests that chromatin structure is a key factor in both these processes of DNA metabolism. It is, therefore, significant that Ikaros and Aiolos exist in what appear to be two separate 2 megadalton complexes that contain Mi-2/histone deacetylases (HDACs) and Brg-1/Swi-3/BAF-60, components of the NURD and SWI/SNF complexes, respectively (Kim et al., 1999). (For more information on these chromatin remodeling proteins,

please refer to Chapter 28 in this book). In addition, Ikaros and Aiolos exist in a separate HDAC-containing complex with mSin3A/B (Koipally et al., 1999). While Mi-2 and HDACs colocalize with Ikaros/Aiolos in heterochromatin-associated nuclear structures in activated cells, SWI/SNF complex components do not (Kim et al., 1999). The differential localization of these Ikaros complexes in heterochromatin versus euchromatin suggests that they play different roles in organizing chromatin structure in distinct nuclear subcompartments. Ikaros and Aiolos proteins may play a specific role in targeting chromatin remodeling activities in heterochromatin to propagate its inaccessible state, possibly by deacetylation of newly deposited histones. Significantly, Mi-2 fails to localize in heterochromatin-associated toroids in Ikaros-mutant T cells, indicating that Mi-2 is recruited to these structures by Ikaros. The biological relevance of these associations and heterochromatic targeting is provided by the chromosomal aberrations observed in Ikarosmutant T cells, which include deletions of centromeric regions (Avitahl et al., 1999). In yeast it has been shown that HDAC activity is important in maintaining the structure and function of centromeric heterochromatin (De Rubertis et al., 1996; Ekwall et al., 1997). Our findings, together with previous reports in the field, suggest that appropriate targeting of chromatin remodeling factors in centromeric regions is mediated by Ikaros family proteins in lymphocytes and is critical for the stable propagation of genetic material during an immune response. The distinct nuclear localization patterns of mSin3A/B versus Mi-2 in activated T cells, where only Mi-2 associates with heterochromatic structures, suggest that Ikaros/AiolosmSin3A/B complexes recruit HDAC activity to different sites than Ikaros/Aiolos-Mi-2. We propose that in the context of accessible chromatin, Ikaros and Aiolos may function as repressors by recruiting mSin3A/B and HDACs to bring about histone deacetylation at promoter regions (see Fig. 12.5). However, in the context of an inaccessible chromatin configuration, Ikaros and Aiolos might require the chromatin remodeling activity of complexes such as NURD in order to gain access to target genes and promote histone deacetylation. Similarly for ac-

MECHANISMS OF IKAROS/AIOLOS ACTION IN LYMPHOCYTE DIFFERENTIATION

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Figure 12.5. In the context of inaccessible chromatin (represented by upright rectangles as nucleosomes), Ikaros (Ik) and Aiolos (Aio) may function as activators by targeting the chromatin remodeling activity of complexes such as SWI/SNF to lymphocyte differentiation genes, thus making them accessible to the conventional transcriptional machinery. In the context of partially accessible chromatin (represented by slanted rectangles as nucleosomes), these proteins may function as repressors by recruiting the remodeler Mi-2 and HDACs in order to gain access to target genes, promote histone deacetylation, and establish an inaccessible chromatin configuration. Ikaros/ Aiolos-Mi-2—HDAC would then maintain this configuration after subsequent cell divisions, thus functioning in cell memory. In the context of accessible chromatin, Ikaros and Aiolos may function as repressors by recruiting mSin3A/B and HDACs to bring about histone deacetylation at promoter regions and establish inaccessibility.

tivation, Ikaros proteins may target the remodeling activity of complexes such as SWI/ SNF, which is implicated in gene activation (Cosma et al., 1999; Hirschhorn et al., 1992; Kwon et al., 1994), to make lymphocyte differentiation genes accessible to the conventional

transcriptional machinery and enable their activation during development. Establishing formally which genes are targets of either repression or activation by Ikaros family proteins and how this activity is regulated during differentiation will provide important insight

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into the mechanisms by which these proteins control gene expression.

Mechanisms of Ikaros Action in the Development of Lymphoid Malignancy: Implications for Human Disease The regulation of chromatin structure via ATPdependent remodelers, histone deacetylases, and possibly DNA methylases are important mechanisms for differentiation, maintenance of cellular memory and the stable propagation of genetic material (Chen et al., 1998; Jeppesen, 1997; Kingston et al., 1996; Ng and Bird, 1999). Consistent with this, the deregulated recruitment of histone deacetylases and altered methylation patterns have been correlated with the development of cancer in humans (Counts and Goodman, 1995; Gelmetti et al., 1998; Jones, 1996; Lengauer et al., 1998; Lin et al., 1998). An increasing body of work indicates that maintenance of old or induction of new gene programs in daughter cells is coupled to processes that occur either during replication or immediately afterward (Almouzni and Wolffe, 1993; Araujo et al., 1998; Brandeis et al., 1993; Chuang et al., 1997; Jeppesen, 1997; Shibahara and Stillman, 1999). It is particularly significant that Ikaros and Aiolos colocalize with sites of DNA replication and that they recruit HDAC complexes to these sites. Therefore, Ikaros and Aiolos proteins may regulate the induction of new gene programs and maintenance of cellular memory by modulating the structure of chromatin at specific sites during lymphocyte differentiation and activation. Modulation of chromatin structure is possibly the mechanism by which Ikaros proteins might set signaling thresholds in T cells to control cell cycle transitions (G0-G1-S). Diminishing or altering Ikaros/Aiolos activity through mutations in these genes would, therefore, perturb regulated changes in chromatin accessibility and have important consequences for both gene expression and DNA replication. This is becoming increasingly relevant to human malignancy in light of the first few reports linking mutations in Ikaros to leukemia in humans. Two studies demonstrate the overexpression of dominant negative Ikaros isoforms in leukemic cells in several cases of infant and childhood acute lym-

phoblastic leukemia (ALL) (Sun et al., 1999a; 1999b). Aberrantly high levels of Ik-7 and Ik-8 were observed suggesting that, like in the mouse model, overexpression of dominant negative proteins causes lymphoid malignancies that arise with rapid kinetics. Another report correlates the development of blast crisis in patients with chronic myelogenous leukemia (CML) with a reduction in Ikaros activity (Nakayama et al., 1999). In several bone marrow samples of patients who were in blast crisis, Ikaros expression was severely reduced at the RNA level, while in other samples, activity was reduced through overexpression of dominant negative Ik-6. In contrast, Ikaros expression was normal in bone marrow samples of CML patients who were in chronic phase of the disease (Nakayama et al., 1999). The report of Nakayama and co-workers suggests that, while a primary mutation involving a reciprocal translocation resulting in a BCR-ABL fusion protein initiates the disease, secondary mutations in the Ikaros locus might be a mechanism that produces the acute phase of CML.

CONCLUSION AND FUTURE DIRECTIONS Ikaros, aided by Aiolos, appears to play a central role in both B and T cells to integrate signals received from the cell surface to events regulating transcription, cell cycle progression, and DNA replication. We propose that Ikaros family proteins might nucleate the formation of different complexes in various cell types corresponding either to different lineages or developmental stages. These complexes would be tailored to respond to signaling cues relevant for the given cell. Identifying further proteins that interact with Ikaros family proteins is likely to aid in distinguishing these complexes and the nature of their action. The presence of Ikaros and Aiolos in complexes that modulate chromatin structure suggests that Ikaros family proteins target chromatin remodeling activity to specific sites. Studies on the chromatin-based role of Ikaros in the stable propagation of genetic material and its potential link to gene expression may provide the molecular basis for the control of lymphocyte differentiation and proliferation. Studies correlating mutations in Ikaros with human malignancy demonstrate

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ACKNOWLEDGMENTS The work reported in this chapter has been supported by funds from NIH grants AI-33062, AI-38342, and AI-42254. Nicole Avitahl is supported by a fellowship from the American Cancer Society, Massachusetts Division. Susan Winandy is a recipient of a King Trust Research Award from the Medical Foundation. Katia Georgopoulos is a scholar of the Leukemia Society of America.

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Kwon, H., Imbalzano, A. N., Khavari, P. A., Kingston, R. E., and Green, M. R. (1994). Nucleosome disruption and enhancement of activator binding by a human SWI/SNF complex for transcription. Nature 370, 477—481. Lengauer, C., Kinzler, K. W., and Vogelstein, B. (1998). Genetic instabilities in human cancers. Nature 396, 643—469. Lin, R. J., Nagy, L., Inoue, S., Shao, W., Miller, W. H. J., and Evans, R. M. (1998). Role of the histone deacetylase complex in acute promyelocytic leukemia. Nature 391, 811—818. Molna´r, AM., and Georgopoulos, K. (1994). The Ikaros gene encodes a family of functionally diverse zinc finger DNA binding proteins. Mol. Cell. Biol. 14, 785—794. Molna´r, AM., Wu , P., Largespada, D., Vortkamp, A., Scherer, S., Copeland, N., Jenkins, N., Bruns, G., and Georgopoulos, K. (1996). The Ikaros gene encodes a family of lymphocyte restricted zinc finger DNA binding proteins, highly conserved in human and mouse. J. Immunol. 156, 585—592. Morgan, B., Sun, L., Avitahl, N., Andrikopoulos, K., Gonzales, E., Nichogiannopoulou, A., Wu, P., Neben, S., and Georgopoulos, K. (1997). Aiolos, a lymphoid restricted transcription factor that interacts with Ikaros to regulate lymphocyte differentiation. EMBO J. 16, 2004—2013. Nakayama, H., Ishimaru, F., Avitahl, N., Sezaki, N., Fujii, N., Nakase, K., Ninomiya, Y., Harashima, A., Minowada, J., Tsuchiyama, J., Imajoh, K., Tsubota, T., Fukuda, S., Sezaki, T., Kojima, K., Takimoto, H., Yorimitsu, S., Takahashi, I., Miyata, A., Taniguchi, S., Tokunaga, Y., Gondo, H., Niho, Y., Nakao, S., Kyo, T., Dohy, H., Kamada, N., and Harada, M. (1999). Decreases in Ikaros activity correlate with blast crisis in patients with chronic myelogenous leukemia. Cancer Res. 59, 3931— 3934. Ng, H. H., and Bird, A. (1999). DNA methylation and chromatin modification. Curr. Opin. Genet. Dev. 9, 158—163. Shibahara, K., and Stillman, B. (1999). Replicationdependent marking of DNA by PCNA facilitates CAF-1-coupled inheritance of chromatin. Cell 96, 575—585. Shortman, K., and Wu, L. (1996). Early T lymphocyte progenitors. Annu. Rev. Immunol. 14, 29—47. Sun, L., Liu, A., and Georgopoulos, K. (1996). Zinc finger—mediated protein interactions modulate Ikaros activity, a molecular control of lymphocyte development. EMBO J. 15, 5358—5369. Sun, L., Crotty, M. L., Sensel, M., Sather, H., Navara, C., Nachman, J., Steinherz, P. G., Gaynon, P. S., Seibel, N., Mao, C., Vassilev, A., Reaman, G. H.,

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CHAPTER 13


THE ROLE OF PU.1 IN B-LYMPHOCYTE DEVELOPMENT AND FUNCTION SRIDHAR RAO Department of Pathology, The University of Chicago

M. CELESTE SIMON Howard Hughes Medical Institute, Departments of Medicine and Molecular Genetics and Cell Biology, The University of Chicago

INTRODUCTION Hematopoiesis represents the development of all five blood lineages (erythrocyte, lymphocyte, monocyte/myelocyte, granulocyte, and thrombocyte) from a self-renewing, pluripotent stem cell (reviewed in Shivdasani and Orkin, 1996; see the Introduction in this book). This process is regulated by the action of cytokines, growth factors, cell-cell interactions, and appropriate changes in gene expression modulated by transcription factors. By altering the pattern of gene expression in a given cell type, transcription factors play a pivotal role in not only lineage specification but also cell function. B lymphocytes provide an excellent model system to explore the role of transcription factors in both lineage development and function (reviewed in Desiderio, 1995; Reya and Grosschedl, 1998). The role(s) of B-cell transcription factors can be studied by the generation of gene-targeted mice (Dahl et al., 1999). For

example, mutations in E2A (Bain et al., 1994; Zhuang et al., 1994; see Chapter 16), Ikaros (Wang et al., 1996; see Chapter 12), EBF (Lin and R, 1995), Pax-5 (Urbanek et al., 1994; see Chapter 14), and OCA-B (Schubart et al., 1996; see Chapter 18) have all revealed critical functions for these transcription factors in lineage development, immunoglobulin (Ig) gene rearrangement, and mature B-cell function. Mutant mice are a powerful tool to understand many aspects of transcription factor biology. However, such mice do not answer all questions concerning how transcription factors coordinately regulate gene expression. Gene regulation in vivo involves the combinatorial interaction of different transcription factors through protein:DNA and protein:protein interactions for proper temporal and spatial gene expression. Therefore, to truly understand the role of a transcription factor in B-cell gene expression, both genetic and biochemical approaches to explore protein:protein interactions, DNA binding, and structure/ function relationships are required.


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THE ROLE OF PU.1 IN B-LYMPHOCYTE DEVELOPMENT AND FUNCTION

The Ets family transcription factor PU.1/Spi-1 is a critical player in B-cell transcription at multiple levels. Spi-B, an Ets transcription factor similar to PU.1, is also expressed in B lymphocytes. Both PU.1 and Spi-B have been shown to interact with the lymphoid co-activator Pip/IRF4/NF-EM5 to activate Ig lightchain transcription. PU.1 provides an interesting example of how genetic and biochemical studies can be used to understand the biology of a transcription factor. This review will focus on PU.1 function in B-cells and studies used to elucidate its role in transcriptional regulation. In addition, Spi-B and Pip will each be discussed to illustrate 1) how members of a gene family and 2) protein:protein interactions can influence transcription.

IDENTIFICATION OF PU.1/SPI-1 AND EXPRESSION PATTERN The PU.1/Spi-1 locus was first identified in retrovirally induced murine erythroleukemia by the spleen focus forming virus (SFFV) and

named Spi-1 for SFFV proviral integration (Moreau-Gachelin et al., 1988, 1989; Paul et al., 1991). PU.1 (for purine-rich sequence bindingprotein) was cloned by screening a gt11 library (Klemsz et al., 1990) and was later found to be the gene product of the Spi-1 locus (Goebl, 1990). The murine cDNA encodes a 272 amino acid protein with a C-terminal DNA-binding motif (Ets domain) exhibiting homology to the Ets-1 transcription factor (Boulukos et al., 1989). PU.1 also contains a PEST (proline, glutamic acid, serine, threonine-rich) domain that is involved in protein:protein interactions (Rogers et al., 1986) and an N-terminal transcriptional activation domain (Fig. 13.1). The overall homology between PU.1 and Ets-1 is only 40%, although they are more similar in their DNA-binding domains (60%). The expression pattern of PU.1/Spi-1 was initially thought to be limited to macrophages and B lymphocytes (Klemsz et al., 1990). In fact, PU.1 mRNA is expressed in bone marrow, spleen, testis, fetal liver, and at low levels in the thymus (Galson et al., 1993; Klemsz et al., 1990). PU.1 mRNA and/or protein is detected in

Figure 13.1. The domain structure of PU.1, Spi-B, Ets-1, and Pip. The Ets domains of PU.1, Spi-B, and Ets-1 are involved in DNA binding and protein:protein interactions; DBD indicates DNA-binding domain; AD indicates transcriptional activation domain; *indicates these domains are weakly acidic; hatched areas indicate an inhibitory domain that blocks DNA binding. The glutamine-rich, P/S/T, and acidic regions are transcriptional activation domains. A number on top of a circled S or T indicates aa number for a serine or threonine residue, respectively.

PU.1 STRUCTURE-FUNCTION RELATIONSHIPS

CD34> stem cells, B lymphocytes, immature T cells, immature erythrocytes, granulocytes, mast cells, macrophages/monocytes, and megakaryocytes, but not in mature T lymphocytes or mature erythrocytes (Chen et al., 1995; Galson et al., 1993; Hromas et al., 1993; Klemsz et al., 1990; Su et al., 1996). In addition, there is evidence that PU.1 is expressed in some hematopoietic progenitor populations (DeKoter et al., 1998; Henkel et al., 1999; Olson et al., 1995; Simon et al., 1996). During murine B-cell development, PU.1 mRNA appears to be expressed at a constant level (Klemsz et al., 1990; Rao et al., 1999a).

PU.1 STRUCTURE-FUNCTION RELATIONSHIPS PU.1, like most other Ets proteins, binds as a monomer preferentially to the purine-rich core GGAA. GAGGAA is the optimized PU.1 corebinding site identified in vitro; the SV40 enhan-

203

cer site is the classic in vivo example of this motif (Table 13.1). As shown in Table 13.1, most PU.1 B-cell target genes contain a GGAA core, and many have the complete GAGGAA sequence. However, one important exception to this rule is the Ig J-chain promoter, which binds PU.1 via a noncanonical AGAA site (Shin and Koshland, 1993). A similar DNA site is found in a PU.1 myeloid target gene, c-fms (Zhang et al., 1994). The AGAA J-chain site appears to bind PU.1 with comparable affinity to GGAA sites (Shin and Koshland, 1993; Su et al., 1996). These studies demonstrate that while PU.1 binds to typical Ets DNA sites, it also binds to a distinct DNA element (AGAA) that may provide an expanded range of target genes in vivo. A cocrystal structure of PU.1’s Ets domain with a 16 bp double-stranded DNA oligonucleotide (5 AAAAAGGGGAAGTGGG 3) was resolved to 2.3 Å (Kodandapani et al., 1996). PU.1’s Ets domain assumes a compact structure made up of three -helices and a 4-stranded antiparallel -sheet that resembles an ; he-

TABLE 13.1. PU.1 Binding Sites in Different Genes

Gene

DNA-Binding Site

Other Transcription Factor Sites

In vitro? J chain LSP1 Blk (5)

AAAAAGAGGAAGTAG GAAAGCAGAAGC GGAAGGAGGAAG TATAAGAGGAAGTCACT

Blk (3)

CTCGTGAGGAAGGACC

PU.1

TACAGGAAGTCTCT

CD72 SV40 Enh@ HC Enh@

AAAAGAGGAAGAAGG GAAAGAGGAACTTGG TTTGGGGAAGGGAAAA

mb-1 B29 Btk

CTCAAGGGAATTGTGC CATGGCAGGAAGGGGCC AAAGGGAACTGA

Oct, Ets-1, Sp1, LyF1 C/EBP, E-box, Sp1

CD20 Enh@

TTTCAAGAAGTGAAACCTGG TAAAAGGAAGTGAAACCAAG

Pip, USF1 Pip

Enh@

CTTGAGGAACTGAAAACAGAACCT

Pip, AP-1

Sp1, C/EBP BSAP, PEA3, Sp1, E-box BSAP, PEA3, Sp1, E-box Oct, Sp1 BSAP Ets-1, TFEC, AML1

References Ray-Gallet et al., 1995) Shin and Koshland, 1993 Omori et al., 1997 Lin et al., 1995 Lin et al., 1995 Chen et al., 1996; Kistler et al., 1995 Ying et al., 1998 Pettersson and Schaffner, 1987 Erman and Sen, 1996; Rao et al., 1997 Feldhaus et al., 1992 Omori and Wall, 1993 Himmelmann et al., 1996; Muller et al., 1996 Himmelmann et al., 1997 Eisenbeis et al., 1995; Eisenbeis et al., 1993 Pongubala et al., 1992

?In vitro indicates the sequence is an optimized binding site based on a PCR based site-selection experiment. @Enh indicates enhancer elements. If not stated, binding sites are in a promoter element.

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lix-turn-helix motif. Direct contact with the GGAA core contained within the major groove of DNA is accomplished through two arginine residues (Arg 232 and Arg 235) in helix 3. Two other critical residues make DNA contact: Lys 245 contacts the phosphate backbone of DNA 5 to the GGAA core in the minor groove, while Lys 219 contacts the backbone 3 of the core sequence on the opposite strand. These two residues, as well as other positively charged side chains in PU.1’s Ets domain are thought to play a critical role in phosphate neutralization, which induces an 8° bend in DNA that facilitates contact between the recognition helix and the major groove (Kodandapani et al., 1996; Pio et al., 1996; Strauss-Soukup and Maher, 1997). Since Lys 219 and 245 are both contained in long loop structures, the DNA recognition motif of PU.1, and most likely other Ets proteins, is better described as a loop-helixloop structure. PU.1 contains other domains involved in both transcriptional activation and protein:protein interactions (Fig. 13.1). Studies from several labs have explored how different PU.1 domains are involved in transcription. The most complete analysis of domains required for transcriptional activation in transient assays has been performed by Klemsz and Maki (Klemsz and Maki, 1996). Here, PU.1 deletion and point mutants were tested for their ability to activate transcription from a multimerized SV40 enhancer site using transient transfection assays in HeLa cells. The N-terminal 100 amino acids of PU.1 contains three subdomains that demonstrate transactivation potential. Amino acids (aa) 1—32 are weakly acidic, while aa 33—74 contain two separable domains that are strongly acidic in character, resembling the activation domain of the potent viral activator of transcription VP16 (Cress and Triezenberg, 1991). In marked contrast to the classic acidic domain contained in virtually all Ets proteins (Bassuk and Leiden, 1997), aa 74—100 activates transcription through a series of five glutamine residues. Importantly, the PEST domain of PU.1 is dispensable for reporter gene expression in this system, implying that it contains no intrinsic transcriptional activity. This mapping of the domains of PU.1 required for transcriptional activation was done using a reporter gene containing a viral DNA

sequence, not a native eukaryotic element. The glutamine-rich domain is uniquely important for transcriptional activation of a J-chain reporter in S194 plasmacytoma cells (Shin and Koshland, 1993). However, aa 33—100 of PU.1’s transactivation domain (containing one acidic domain and the glutamine-rich domain) are required for maximal transactivation of the monocyte-specific IL-1 promoter in HeLa cells (Kominato et al., 1995). These studies reveal an important principle related to PU.1’s structure: it contains multiple domains important for transcriptional activation at certain promoter elements. Though the mapping of different transcriptional activation domains of PU.1 is important to understand its role in target gene transcription, a more interesting experiment is to examine how distinct domains of PU.1 are utilized in lineage specification and/or function. Unfortunately, these experiments have not yet been performed in B lymphocytes. However, such experiments using myeloid cell development to test the role of PU.1 functional domains have been performed. One important study on the role of various PU.1 domains in myeloid development utilized PU.1\\ ES cells, which cannot form myeloid cells upon in vitro differentiation (discussed in detail below). Fisher and colleagues directly assesed the role of different domains to rescue macrophage formation by transfecting deletion constructs into PU.1\\ ES cells (Fisher et al., 1998). As expected, fulllength PU.1 cDNA rescued macrophage formation, while the Ets domain of PU.1 by itself could not. In contrast to the results in transient assays, aa 133—140 in the PU.1 PEST domain are strictly required for macrophage development, although no example of a Pip-like cofactor for PU.1 has been identified in this lineage (Pip is discussed in detail below). Lastly, the acidic transactivation domains of PU.1 are dispensable for myeloid development, while the glutamine rich domain is essential. This implies that PU.1 interacts with a currently unknown cofactor(s) in macrophages through its PEST and glutamine domains to activate a critical target gene(s) required for myeloid cell lineage development. This study demonstrates how different protein domains are important for a biologically relevant process such as lineage commitment. It also illustrates the power of

PU.1:PIP INTERACTIONS AS A MODEL OF TERNARY COMPLEX FORMATION AND GENE REGULATION

using gene-targeted ES cells as an in vitro reagent for exploring transcriptional regulation. A second study used chicken blastoderm cells transformed with a Myb-Ets-Gag E26 leukemia virus to create a progenitor cell line capable of differentiating in vitro into thrombocytes, erythrocytes, myeloblasts, and eosinophils. When these progenitor cells are transfected with PU.1, they irreversibly commit to the myeloid lineage ADDIN ENRfu (Nerlov and Graf, 1998). Myeloid cell commitment requires the N-terminal 100 aa of PU.1, proving that the process requires the ability of PU.1 to transcriptionally activate downstream target genes, agreeing with the work of Fisher and co-workers (Fisher et al., 1998).

PU.1 PROTEIN:PROTEIN INTERACTIONS The ability of PU.1 to function as a transcriptional activator at different promoters is aided by the modular nature of its transactivation (TA) domains but also by multiple protein:protein interactions. One feature of PU.1’s Ets and TA domains is their ability to interact with other transcriptional activators to allow the formation of an activation complex at a promoter. Perhaps the classic example is the interaction of PU.1 with AP-1 family members to form protein:protein complexes at the Ig 3 enhancer (Pongubala and Atchison, 1997) as well as macrophage-specific promoters such as CD11b (Li et al., 1998; Moulton et al., 1994) to activate transcription. In fact, direct interactions between the basic domain of c-Jun and Ets domains were demonstrated by affinity chromatography and coimmunoprecipitation studies (Bassuk and Leiden, 1995; Basuyaux et al., 1997; Rao et al., 1999b). The basic domain of C/EBP proteins, specifically C/EBP ( (NF-IL6(), also directly interacts with aa 245—272 of PU.1, which includes its Ets domain and C-terminal tail (Nagulapalli et al., 1995; Rao et al., 1999b). PU.1’s Ets domain can also mediate interactions with other Ets transcription factors (Erman and Sen, 1996; Rao et al., 1997, 1999b), and these interactions are important for Ig heavychain enhancer function. Thus, while Ets proteins are thought to bind DNA as monomers (Bassuk and Leiden, 1997), they may interact with other Ets factors bound to additional DNA

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elements. This would allow Ets proteins to act as a tether between enhancers and promoters to specifically regulate transcription. PU.1 was also shown to interact with two other important transcription factors, Sp1 (Block et al., 1996; Chen et al., 1993) and AML1/ core-binding factor (Petrovick et al., 1998), although these interactions were not shown to be important in the function of a B-cell promoter/enhancer. A combined Ets-AML1 motif was shown to be present in both the Ig ( heavy chain enhancer and a TCR enhancer (Erman et al., 1998), implying that this motif may be important for antigen receptor gene expression in both B and T lymphocytes. The N-terminus of PU.1, (aa 1—75), also interacts with TATA-binding protein (TBP) and the retionoblastoma protein (Rb) by affinity chromatography (Hagemeier et al., 1993). The direct protein:protein interaction between PU.1 and TBP appears to be critical for the activation of transcription from TATA-less genes, which are often included among PU.1 target genes (see below). The role of the PU.1 and Rb interaction is less well understood. While it was shown that Rb can directly suppress the transcriptional activity of PU.1, most likely by blocking interaction with the basal transcription machinery (Weintraub et al., 1995), this effect was never shown to be either lineage restricted or cell cycle regulated. Therefore, the biological relevance of the PU.1:Rb interaction remains to be elucidated.

PU.1:PIP INTERACTIONS AS A MODEL OF TERNARY COMPLEX FORMATION AND GENE REGULATION The ability of PU.1 to form protein:protein interactions important for transcriptional activation was elegantly demonstrated by the discovery of a ternary complex between PU.1, DNA, and the lymphoid-specific coactivator Pip (PU.1 interaction partner). Pip is a member of the IRF family (IRF4, interferon regulatory factor) of transcription factors. The ability of Pip to associate with PU.1 and activate transcription of the and light-chain genes has provided an excellent example of how two transcription factors can coordinately interact to mediate gene-specific transcription.

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Pip was originally identified as a DNA-binding activity at the E3 and E enhancer H\ elements that regulate light-chain gene expression and was named NF-EM5 (Eisenbeis et al., 1993; Pongubala et al., 1992). NF-EM5 binds adjacent to PU.1 in both enhancer elements only after PU.1 is bound to DNA, implying that PU.1 recruits NF-EM5 to DNA through a protein:protein interaction that relieves the natural autoinhibition of Pip to bind DNA, although Pip can bind DNA weakly by itself (Brass et al., 1996). This interaction is critically dependent upon phosphorylation of PU.1 at Ser 148 in the PEST domain (Pongubala et al., 1993). Phosphorylation of this site by casein kinase II is critical not only for proper PU.1:NF-EM5/Pip interaction but also to allow the two factors to synergize and activate transcription from their enhancer elements. The cDNA encoding the DNA-binding activity contained in NF-EM5 was cloned by screening a gt11 expression library using as a probe, the E enhancer, and renamed ‘‘Pip’’ (Brass H\ et al., 1996; Eisenbeis et al., 1995). Pip is a 52 kD lymphoid-restricted protein. Pip levels increase as B lymphocytes develop and during T-cell activation (Brass et al., 1996; Matsuyama et al., 1995). Based upon strong homology in its DNA-binding domain, Pip is a member of the IRF family (Eisenbeis et al., 1995). Several labs have undertaken detailed experiments to understand how PU.1 and Pip interact with DNA to form a ternary complex (Brass et al., 1996; Brass et al., 1999, 1998; Ortiz et al., 1999; Perkel and Atchison, 1998; Yee et al., 1998). To briefly summarize these experiments, the N-terminus (aa 1—140) of Pip contains the DNA-binding domain (Fig. 13.1). Pip possesses at least one independent transactivation domain (aa 140—207) and possibly a second domain located at aa 300—400. Interestingly, Pip contains a region (aa 207—300) that masks its transcriptional activation domain(s), allowing it to act as both a repressor and activator. A specific domain of Pip (aa 245—422) allows it to bind to PU.1 and then undergo a conformational change unmasking its DNA-binding domain. A specific -helix near the C-terminus of Pip (aa 389—413) appears to play a critical role in both ternary complex formation with PU.1 as well as autoinhibition of DNA binding. Surprisingly, these two distinct functions can be sepa-

rated by generating alanine-scanning mutations throughout this region (Brass et al., 1999). However, the identity of the residue of Pip that interacts with the phosphorylated Ser 148 in PU.1 is still controversial; one group argues it is Lys 399 in Pip (Brass et al., 1999) whereas a different group claims it is Arg 328 (Ortiz et al., 1999). A second discrepancy regarding PU.1:Pip interactions is whether the two proteins can interact weakly in solution through their DNAbinding domains. Using affinity chromatography in the absence of DNA, Perkel and colleagues (Perkel and Atchison, 1998) demonstrated that PU.1 and Pip could interact specifically through their DNA binding domains, implying that PU.1 recruits Pip to DNA through a two-step mechanism: (1) PU.1 and Pip interact in solution through their DNA-binding domains, then PU.1 specifically binds to DNA and (2) undergoes a conformational change that allows it to unmask Pip’s DNA-binding domain and subsequently recruit Pip to the site (Perkel and Atchison, 1998). However, using glutaraldehyde cross-linking, Brass and co-workers could only detect PU.1:Pip interaction in the presence of DNA template, implying that they do not interact directly while in solution (Brass et al., 1999). The distinction between the two groups may reflect differences in the techniques used and their respective sensitivities. However, there is no evidence that these two different mechanisms of interaction may affect gene transcription. Although some controversy related to the PU.1/Pip interaction remains, these proteins provide important insights into how two proteins can cooperate in DNA binding and transcriptional activation. The role of the PU.1:Pip complex in vivo is more difficult to determine. One important step toward understanding an in vivo role of PU.1 and Pip was the generation of Pip\\ mice (Mittrucker et al., 1997). These mice exhibit lymphadenopathy due to increased numbers of lymphocytes. Since light-chaindeficient mice fail to produce normal numbers of mature B cells (Zou et al., 1993) , Pip must not be absolutely required for and gene transcription. However, Pip\\ B cells produce very little serum Ig even after antigen stimulation. In addition, B and T lymphocytes from the mutant mice were not activated in response to multiple stimuli both in vivo and in vitro.

THE PU.1-RELATED PROTEIN, SPI-B

One possible explanation for why Pip\\ mice still produce light-chain molecules is that PU.1 is able to allow transcription of the and loci through their respective enhancers, even in the absence of Pip. A second explanation is that PU.1 can interact with another IRF family member to activate transcription of the and loci . Thus, the role of the PU.1:Pip dimer in light-chain transcription was addressed by Brass and co-workers by transducing cells with a synthetic protein harboring a dimer of the DNA-binding domains of PU.1 and Pip (Brass et al., 1999). Although this dimer has a lower affinity for DNA as compared to native PU.1 and Pip, it almost eliminated light-chain mRNA in a stably transfected J558L plasmacytoma cell line, and it lowered message levels for the J chain, a target gene of PU.1 alone. Under these conditions, without any transactivation domains, the PU.1:Pip dimer could actually block the transcription of the locus, proving that the binding of native PU.1 and Pip or another endogneous IRF family member plays an absolutely critical role in lightchain transcription.

B-CELL SPECIFIC PU.1 TARGET GENES: COMMON THEMES? As shown in Table 13.1, PU.1 targets a large number of B-cell—specific genes. The majority of these target genes fit into two categories: (1) B-cell receptor proteins ( heavy chain, and light chains, Ig , and Ig ) or (2) signaling molecules (Blk, Btk, CD20). Interestingly, all PU.1-regulated B-cell promoters do not contain a canonical TATA box, a common feature among myeloid-specific PU.1 target genes as well (Eichbaum et al., 1997; Heydemann et al., 1997; Heydemann et al., 1996; Li et al., 1998; Moulton et al., 1994; Ross et al., 1998). Some of these promoters contain initiator elements (Inr) that can be used in place of a TATA box to accurately initiate transcription; Inr elements also typically occur in combination with Sp1 sites (Javahery et al., 1994; Pugh and Tjian, 1990; Weis and Reinberg, 1992). Promoters that lack identifiable Inr elements may require PU.1 to recruit TBP to the promoter for transcription initiation (Fisher and Scott, 1998; Hagemeier et al., 1993). Therefore, PU.1 acts like a myeloid

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and B-lymphoid—restricted TBP, much like TRF and TRF2 (Hansen et al., 1997; Rabenstein et al., 1999). The ability of PU.1 to interact with multiple transcription factors was postulated to promote lineage—specific gene expression by combining a cell-type specific protein (PU.1) with other more ubiquitous proteins (AP1 or C/EBP family members). Using the E3 enhancer element as a model system, transcription levels were shown to depend on the coordinate action of PU.1, Pip, E2A, and ATF/CREB or AP1 proteins (Pongubala and Atchison, 1997). Remarkably, two deletion mutants of PU.1, which lack portions of PU.1’s transactivation domain, efficiently activate transcription as long as the other factors are present. Under these conditions, PU.1 nucleates the formation of an enhancer complex through multiple protein:protein interactions, which then allows transcription via this enhancer.

THE PU.1-RELATED PROTEIN, SPI-B Spi-B was originally identified by screening a human B-cell library under low-stringency conditions to identify PU.1-related genes (Ray et al., 1992). PU.1, Spi-B, and the recently cloned Spi-C (Bemark et al., 1999) form a distinct subgroup of Ets proteins. While PU.1 and Spi-B share 70% identity with each other in their Ets domain, they are less than 30% identical to Ets-1. PU.1 and Spi-B are very similar in their PEST domains as well (60% identity), but are approximately 20% identical in their N-termini, implying that their transactivation domains are actually quite different. Spi-B was originally believed to have an identical expression pattern to PU.1 (Ray et al., 1992). Subsequent investigation revealed that Spi-B is predominantly expressed in B cells (Chen et al., 1995; Su et al., 1996), with lower level expression in double negative T-cells (Su et al., 1996). However, one distinction between PU.1 and Spi-B expression in B cells is that Spi-B levels increase during B-cell development while PU.1 levels remain essentially constant (Rao et al., 1999a). In addition, while plasma cells express PU.1, they fail to produce Spi-B (Abraham Brass, personal communication).

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Lastly, in purified splenic B cells, PU.1 is the predominant DNA-binding activity on the Ets/ PU.1-binding site, demonstrating that Spi-B protein may be expressed at lower levels than PU.1 (Rao et al., 1999). Thus, while Spi-B and PU.1 are both present in B cells, their expression patterns differ during B-cell ontogeny. The high degree of similarity between the Ets domains of PU.1 and Spi-B allows them to bind to similar DNA sites (Rao et al., 1999b; Ray et al., 1992; Ray-Gallet et al., 1995; Su et al., 1996). One interesting difference was found with the myeloid-specific c-fes promoter, a known target gene of PU.1 (Heydemann et al., 1996, 1997). Originally, it was reported that both PU.1 and Spi-B could bind to a DNA site in the c-fes promoter and activate transcription (Ray-Gallet et al., 1995; Su et al., 1996). However, further work revealed that Spi-B bound to this DNA site with one-tenth the affinity of PU.1 and transactivated the c-fes promoter very poorly in NIH 3T3 cells (Rao et al., 1999b). Thus, the PU.1 site in the c-fes promoter appears to be specific for PU.1 due to differences in its sequence 3 to the core (5 TCAGGAACTG 3), suggesting that there are subtle differences in the 1 helix of the Spi-B Ets domain from PU.1 that change the positioning of a critical arginine (Arg-170) involved in neutralization of the phosphate backbone. The PEST domain of PU.1 is functionally well conserved with that of Spi-B (Rao et al., 1999b; Su et al., 1996). Like PU.1, Spi-B contains a serine residue (Ser 144) that is important in its interactions with Pip. Also, Spi-B can cooperate with Pip for transcriptional activation from a site in the E enhancer element by recruiting Pip. Thus, the ability to interact with Pip is conserved in the PU.1 and Spi-B PEST domain. It is notable that the PEST domains of PU.1 and Spi-B do not target these proteins for degradation, since deletion of the domain does not affect protein stability (Perkel and Atchison, 1998). Whereas the Ets domains of PU.1 and Spi-B are very similar, their N-terminal transactivation domains are only 20% identical, implying that they may contain very different transcriptional activation motifs and may interact with different cofactors through these domains. Our laboratory undertook a structure/function analysis of Spi-B to explore any similarities

and/or differences between it and PU.1 (Rao et al., 1999b). As summarized in Figure 13.1, Spi-B contains an acidic transactivation domain that is common among Ets proteins (Bassuk and Leiden, 1997). However, it also contains a Proline/Serine/Threonine (P/S/T) domain which is unique among Ets transcription factors, with other examples being found in other types of transcription factors such as Pax6 and GATA-4 (Morrisey et al., 1997; Tang et al., 1998). Much like the case for PU.1 at the J-chain promoter (Shin and Koshland, 1993), Spi-B in the presence of Pip only requires its P/S/T domain for efficient transactivation from the B site of the E enhancer element. Therefore, Spi-B, like H\ PU.1, uses a specific transactivation domain at a transcriptional regulatory element. PU.1 was implicated as an architectural transcription factor capable of making multiple protein:protein interactions with other transcription factors. Therefore, we used affinity chromatography to explore the interactions of Spi-B with proteins known to bind PU.1 (Rao et al., 1999b). Proteins that interact with the Ets domain of PU.1 such as c-Jun and other Ets proteins interact equivalently with the Ets domain of Spi-B. NF-IL6 (C/EBP), which interacts with the C-terminus of PU.1, also interacts with Spi-B, although less efficiently. This is most likely because the 12 aa region tail of PU.1 is not conserved in Spi-B. Two proteins that interact with the transactivation domains of PU.1 (Rb and TBP) also interact with Spi-B, implying that a conserved motif between the two proteins is involved in this interaction, most likely the acidic domain. Thus, Spi-B can form multiple protein:protein interactions through its transcriptional activation, PEST, and Ets domains.

GENETIC MODELS FOR PU.1 AND SPI-B FUNCTION IN B-CELLS Scott and co-workers generated PU.1\\ animals deleted for the DNA-binding domain (exon 5) of PU.1 by homologous recombination in ES cells (Scott et al., 1994). This allele of PU.1 produces no mRNA or protein (Scott et al., 1994, 1997). PU.1\\ mice die at embryonic day 17, which was originally attributed to a highly variable anemia. However, the anemia disappears when PU.1\\ mice are bred back onto

GENETIC MODELS FOR PU.1 AND SPI-B FUNCTION

C57/BL6 mice, although the mutant mice still die at the same stage of gestation (Fisher and Scott, 1998). These mutant mice completely lack B cells, T cells, and myeloid cells as assessed by FACS analysis of fetal organs. The block in myeloid development occurs at the stage of myeloid commitment, since no myeloid progenitors can be detected in colony-forming assays. However, the PU.1\\ mice contain normal numbers of megakaryocytes and platelets, showing that PU.1 is not required for the formation or function of this lineage. Since no markers for early B- and T-cell lineages (V preB and RAG1/ 2) or antigen receptor rearrangements can be detected in the mice, PU.1 could play a role in the development of multiple lineages from a recently described lymphoid-myeloid progenitor (Lacaud et al., 1998). To determine if the PU.1\\ progenitor defect is cell autonomous, PU.1\\ ES cells were generated (Olson et al., 1995; Scott et al., 1997) to assess their ability to commit to the hematopoietic lineage in chimeric mice (Nagy et al., 1990). The PU.1\\ ES cells did not contribute to the B, T, or myeloid compartments in either fetal or adult animals, proving that the defect is cell intrinsic. To further define the role of PU.1 in myeloid cell development, PU.1\\ ES cells were differentiated in vitro , a faithful recapitulation of yolk sac hematopoiesis (Keller et al., 1993). The PU.1\\-differentiated ES do not cells form myeloid cells as assessed by the myeloid-specific markers F4/80 and Mac-1. Early myeloid markers such as GM-CSFR, G-CSFR, and myeloperoxidase are expressed in differentiated PU.1\\ ES cells (as well as PU.1\\ embryos), but markers associated with terminal differentiation such as CD11b, CD64, and M-CSFR, are not (Olson et al., 1995; Simon et al., 1996). Importantly, introduction of a PU.1 cDNA driven by its own promoter can rescue macrophage formation in vitro, proving that the myeloid defect is due to the loss of PU.1 and not the disruption of a closely linked locus. Similar results were obtained when fetal liver hematopoietic progenitors were purified from PU.1\\ mice using a lineage-depletion protocol to enrich for progenitor cells, using the stem cell-restricted marker AA4.1 (DeKoter et al., 1998). PU.1\\ mice exhibit approximately 5fold fewer AA4.1> progenitors and virtually no myeloid progenitors. Importantly, these myeloid

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progenitors completely lack c-fms, the receptor for M-CSF and a known target gene of PU.1 (Reddy et al., 1994; Zhang et al., 1994), and therefore cannot proliferate properly. Transduction of PU.1\\ cells with a retrovirus expressing c-fms bypasses this block, allowing the myeloid progenitors to proliferate. However, these progenitors still fail to differentiate properly. PU.1 was not required for the formation of granulocyte precursors but was required for their subsequent differentiation. A second targeted disruption of the PU.1 locus was generated by insertion of a neomycin resistance cassette into exon 5 of the locus without any deletion (McKercher et al., 1996). These mice die 48 hours after birth from septicemia, although they can live for up to 2 weeks if they are maintained on antibiotics. These mice lack T cells at birth; however, normal levels of immature T cells can be detected in the thymus 1—2 weeks later. Importantly, these mice fail to generate cells of the monocyte/myeloid and granulocyte lineages. They also fail to form mature B cells, although there is an outgrowth of B220> CD43\ CD24> cells, which may represent an aberrant B-cell population. These mice clearly have phenotypic differences from mice generated by Scott and colleagues (for a complete discussion, see Fisher and Scott, 1998; Simon, 1998), possibly due to differences in their respective targeting strategies of ES cells. Briefly, two explanations for the differences in phenotypes of the two alleles of PU.1 have been put forth. First, one of the mutations may not be a null and may in fact produce a truncated protein. Of note, mice produced by Scott and colleagues have no PU.1 mRNA or protein (Scott et al., 1994). However, a direct comparison of protein levels in the two mouse strains using the same antibody reagents has not yet been done. The second explanation is that one or both mutations may affect a tightly linked locus, thereby causing unique phenotypes. Both of these explanations need to be thoroughly investigated before the exact nature of the phenotypic differences can be understood. These differences notwithstanding, both strains of PU.1\\ mice define a critical role for PU.1 in commitment to the myeloid and B-lymphoid lineages. In contrast to the embryonic lethality observed with PU.1\\ mice, Spi-B\\ mice are viable, fertile, and born in normal Mendelian

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ratios (Su et al., 1997). Spi-B\\ mice exhibit no Spi-B mRNA and no defect in the number of B or T cells in the bone marrow, spleen, or thymus. In addition, unimmunized Spi-B\\ mice and those challenged with a T-independent antigen (DNP-LPS) exhibit no defects in antibody secretion. When challenged with a T-dependent antigen (DNP-KLH), Spi-B\\ mice exhibit elevated IgM and decreased IgG2a in their serum during the primary response and a more dramatic decrease in both IgG2a and IgG2b during the secondary response. SPI-B\\ mice show normal germinal center formation after immunization, but these centers are not properly maintained. The decrease in antibody production and poor maintenance of germinal centers is due to increased apoptosis of B cells in the spleens of immunized Spi-B\\ mice. Purified lymphocytes from Spi-B\\ mice were examined in vitro for their response to different stimuli. Spi-B\\ T cells respond normally to stimulation by anti-CD3, ConA, or PMA; ionomycin treatment and proliferate. However, Spi-B\\ B-cells respond poorly to stimulation by cross-linking their surface IgM. Spi-B\\ B-cells respond normally to LPS and PMA; ionomycin, implying that the proliferation defect observed in Spi-B\\ B cells is specific to B-cell receptor (BCR) stimulation. Mutant B cells are activated during in vitro stimulation and enter the cell cycle, however they undergo increased apoptosis. Thus, the poor overall proliferation of Spi-B\\ B cells in vitro upon BCR cross-linking is most likely due to increased apoptosis. Since Spi-B and PU.1 transactivate a number of genes involved in BCR signaling, mRNA levels for btk, blk, lyn, and syk were examined and found to be normal. In addition, PU.1 mRNA levels are unchanged in Spi-B\\ B cells, proving that PU.1 is not completely functionally redundant for Spi-B. The study of PU.1\\ and Spi-B\\ animals demonstrates some of the strengths as well as problems encountered when examining genetargeted mice. The precise requirements of these proteins in vivo were identified by these mice: PU.1 is uniquely important for the production and/or proliferation of a common myeloid-lymphoid progenitor, whereas Spi-B is required for appropriate BCR-mediated signaling. However, it is impossible to study a role of PU.1 later in mature B cells, since PU.1\\ mice do not

produce any lymphoid cells. The Spi-B\\ mice may have a mild B-cell phenotype due to compensation by PU.1 (Garrett-Sinha et al., 1999). A similar situation occurred with the bHLH proteins Myf5 and MyoD: skeletal muscle development is normal in Myf5\\ (Braun et al., 1992) and MyoD\\ (Rudnicki et al., 1992) mice but is blocked in Myf5\\ and MyoD\\ mice (Rudnicki et al., 1993). To directly address the redundant function of PU.1 and Spi-B in hematopoiesis and especially in B-cell development and function, we crossed the PU.1>\ and Spi-B>\ mice to generate all possible genotypes (Garrett-Sinha et al., 1999). PU.1\\ Spi-B\\ die in utero with similar defects observed in PU.1\\ Spi-B>> animals. In contrast, PU.1>\ Spi-B\\ mice are viable, fertile, and born in the expected Mendelian ratios. Myeloid development and adult T-cell development are normal in PU.1>\ Spi-B\\ mice . However, these mice exhibit a 50—80% reduction of mature (IgM;) B cells in their peripheral lymphoid organs whereas PU.1>\ Spi-B>> mice exhibit no defect in lymphocyte numbers. The PU.1>\ Spi-B\\ mice produce normal numbers of pre—B cells in their bone marrow (B220 S7- HSA& ) but an approximately 50% reduction in immature B cells (B220& S7\ HSA ) due to increased apoptosis. This defect is not due to changes in light-chain transcription. Thus, PU.1 and Spi-B are required for immature B-cell survival. The PU.1>\ Spi-B\\ mice also exhibit a more severe reduction in serum Ig production in response to T-dependent antigens as compared to PU.1>> Spi-B\\ mice. While PU.1>> SpiB\\ mice form germinal centers in response to antigen challenge, PU.1>\ Spi-B\\ mice are incapable of forming germinal centers and display higher levels of apoptotic B cells in their spleens upon immunization. In vitro responses to BCR cross-linking of PU.1>\ Spi-B\\ B cells are reduced greater than 10-fold compared to wild type, while the response to PMA;ionomycin and LPS are normal. Thus, the PU.1>\ Spi-B\\ mice exhibit a more exaggerated defect in BCR mediated responses than PU.1>> Spi-B\\ mice. Purified B-cells from PU.1>\ Spi-B\\ were tested for tyrosine phosphorylation of various substrates after BCR cross-linking and showed a dramatic global decrease in phosphorylation compared to

CONCLUSIONS

wild-type cells. Surprisingly, membrane proximal signaling events such as tyrosine phosphorylation of Ig ; and syk recruitment to the BCR are all normal in PU.1>\ Spi-B\\ B cells. The activities of various kinases important for antigen receptor signaling such as lyn, fyn, and syk (reviewed in Pleiman et al., 1994) are all normal. In addition, all known members of the BCR complex and its signaling machinery appear to be expressed at normal levels. However, the phosphorylation of two important substrates of syk, BLNK/Slp-65 (Fu et al., 1998; Wienands et al., 1998; Zhang et al., 1998) and PLC- (Law et al., 1996), are dramatically reduced in PU.1>\ Spi-B\\ B-cells. Lastly, PU.1>\ Spi-B\\ B-cells exhibit a blunted calcium response in vitro as compared to wild-type controls. Taken together, these results indicate that PU.1 and Spi-B cooperate in the transcription of component(s) of the BCR signaling cascade that are required for coupling syk to its downstream effectors, such as BLNK and PLC- . In addition, the reduced phosphorylation of downstream substrates can explain the increased apoptosis observed both in vitro and in vivo when PU.1>\ Spi-B\\ B cells encounter antigen, since appropriate signaling by the BCR is required for B cells to survive (Lam et al., 1997). The decreased calcium response is most likely due to decreased activity of BLNK and PLC- , which are important for creating calcium fluxes. Perhaps more interesting is that a single allele of PU.1 is unable to allow for normal B-cell function. This indicates that PU.1 and Spi-B exhibit a gene dosage effect, similar to results reported for bHLH proteins of the E2A family where B-cell development is affected (Zhuang et al., 1996). In this situation, combinations of mutations in E2A, E2-2, and HEB revealed that a critical dosage of all three genes is required for efficient B-cell formation and development. Therefore, the ability of a B cell to mature and function normally requires a specific dosage of PU.1 and Spi-B.

CONCLUSIONS The role of PU.1 in B-lymphoid function has been difficult to directly assess using PU.1\\ mice. However, traditional studies to identify

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PU.1 target genes have given valuable insights into their role in B-lymphocyte transcription. The fact that all target genes containing PU.1dependent promoters lack TATA boxes coupled with the ability of PU.1 to interact with TBP, implies that PU.1 could function much like a lineage-restricted TBP. Many PU.1 target genes play important roles in BCR signaling. As discussed previously, both heavy and light chains, Ig and , blk, and btk, contain PU.1-binding sites either in promoter or enhancer elements. Based upon these facts, it is not surprising that PU.1>\ Spi-B\\ mice exhibit a dramatic decrease in BCR signaling. It is interesting to note that all known members of antigen receptor signaling pathways, including those genes that are direct target genes of PU.1 and Spi-B, are unaffected at the mRNA level. This implies that a single allele of PU.1 is sufficient for proper transcription of all these target genes. However, it is also implies that the dosage of both PU.1 and Spi-B is critical for the transcription of a currently unidentified component(s) of BCR signaling. Since PU.1>\ Spi-B\\ B cells are phenotypically normal when at rest but exhibit a strong phenotype when activated, they provide an excellent model system to identify target genes of both PU.1 and Spi-B in vivo using subtractive hybridization. Using unstimulated B cells prepared from PU.1>> SpiB>> and PU.1>\ Spi-B\\ mice, we have identified a lymphoid-restricted heptahelical receptor (P2Y10) whose transcription critically depends upon the dosage of PU.1 and Spi-B acting through at least one site in its proximal promoter (Rao et al., 1999a), and we are currently searching for other target genes. The gene dosage model for explaining the phenotype in PU.1>\ Spi-B\\ B cells is appealing, since both PU.1 and Spi-B appear to bind similar DNA sites. However, it is conceivable that PU.1 and Spi-B in fact have different in vivo target genes. The fact that Spi-B is unable to bind efficiently to a site in the c-fes promoter, a known target gene of PU.1, suggests that PU.1 and Spi-B may in fact have overlapping but not identical DNA-binding specificity (Rao et al., 1999b) . In addition, PU.1 and Spi-B have very different transactivation domains, with each protein containing a transcriptional activation motif unique among all Ets proteins. One hypothesis is that PU.1 and

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Spi-B transcriptional activation domains (Fig. 13.1) interact with a distinct subset of cofactors or components of the basal transcription machinery to activate different target genes. For example, PU.1 could use its glutamine-rich domain to bind a specific cofactor allowing it to transactivate certain target genes, whereas Spi-B could use its P/S/T domain to interact with a B-cell—specific TAF such as hTAF 105 (Dik'' stein et al., 1996). Given that PU.1 and Spi-B appear to interact with other transcription factors such as Pip, TBP, and C/EBP, differences in the ability of their N-termini to interact with other unknown proteins provide an appealing model for how they could each specifically transactivate certain target genes. In all cases, PU.1 and Spi-B have provided a unique model to study a variety of biological processes ranging from antigen-mediated responses by the immune system to ternary complex formation between two transcription factors with DNA. It is also clear that both traditional experimental approaches to address in vitro issues of transcriptional regulation and in vivo experiments with gene-targeted animals are needed to truly understand the biology of any transcription factor.

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Urbanek, P., Wang, Z. Q., Fetka, I., Wagner, E. F., and Busslinger, M. (1994). Complete block of early B cell differentiation and altered patterning of the posterior midbrain in mice lacking Pax-5/BSAP. Cell 79, 901—912. Wang, J., Nichogiannopoulou, A., Wu, L., Sun, L., Sharpe, A. H., Bigby, M., and Georgopoulos, K. (1996). Selective defect in the development of the fetal and adult lymphoid system in mice with an Ikaros null mutation. Immunity 5, 537—549. Weintraub, S. J., Chow, K. N. B., Luo, R. X., Zhang, S. H., He, S., and Dean, D. C. (1995). Mechanism of active transcriptional repression by the retinoblastoma protein. Nature 375, 812—815. Weis, L., and Reinberg, D. (1992). Transcription by RNA polymerase II: initiator-directed formation of transcription-competent complexes. Faseb J. 6, 3300—3309. Wienands, J., Schweikert, J., Wollscheid, B., Jumaa, H., Nielsen, P. J., and Reth, M. (1998). SLP-65: a new signaling component in B lymphocytes which requires expression of the antigen receptor for phosphorylation. J. Exp. Med. 188, 791—795. Yee, A. A., Yin, P., Siderovski, D. P., Mak, T. W., Litchfield, D. W., and Arrowsmith, C. H. (1998). Cooperative interaction between the DNA-binding domains of PU.1 and IRF4. J. Mol. Biol. 279, 1075—1083. Ying, H., Chang, J. F., and Parnes, J. R. (1998). PU.1/Spi-1 is essential for the B cell-specific activity of the mouse CD72 promoter. J. Immunol. 160, 2287—2296. Zhang, D. E., Hetherington, C. J., Chen, H. M., and Tenen, D. G. (1994). The macrophage transcription factor PU.1 directs tissue-specific expression of the macrophage colony-stimulating factor receptor. Mol. Cell. Biol. 14, 373—381. Zhang, Y., Wienands, J., Zurn, C., and Reth, M. (1998). Induction of the antigen receptor expression on B lymphocytes results in rapid competence for signaling of SLP-65 and Syk. EMBO J. 17, 7304—7310. Zhuang, Y., Soriano, P., and Weintraub, H. (1994). The helix-loop-helix gene E2A is required for B cell formation. Cell 79, 875—884. Zhuang, Y., Cheng, P., and Weintraub, H. (1996). B-lymphocyte development is regulated by the combined dosage of three basic helix-loop-helix genes, E2A, E2-2, and HEB. Mol. Cell. Biol. 16, 2898—2905. Zou, Y., Zou, S. T., and, Zou, K. R. (1993). Gene targeting in the Ig kappa locus: efficient generation of lambda expressing B-cells independent of gene rearrangements in Ig kappa. EMBO J. 12, 811— 820.

CHAPTER 14


THE ROLE OF Pax5 (BSAP) IN EARLY AND LATE B-CELL DEVELOPMENT MARKUS HORCHER, DIRK EBERHARD, AND MEINRAD BUSSLINGER Research Institute of Molecular Pathology, Vienna, Austria

INTRODUCTION The paired box-containing (Pax) genes constitute an evolutionarily ancient family of transcription factors characterized by a highly conserved DNA-binding motif, the so-called paired domain (Noll, 1993). Naturally occurring mutations as well as targeted mutagenesis demonstrated that all nine mammalian Pax genes play essential roles in early development and organogenesis (reviewed by Mansouri et al., 1996). Pax5 is, however, the only member of this gene family that is also expressed in the hematopoietic system. The Pax5 protein was initially identified as a B-lymphoid transcription factor known as B-cell-specific activator protein (BSAP) (Barberis et al., 1990). Biochemical purification and cDNA cloning subsequently revealed that BSAP is encoded by the Pax5 gene (Adams et al., 1992). Within the hematopoietic system, Pax5 is exclusively expressed in the B-lymphoid lineage from the earliest detectable precursor to the mature B-cell stage, but is then

repressed in terminally differentiated plasma cells (Adams et al., 1992; Li et al., 1996). Pax5 (BSAP) is one of several transcriptional regulators that are essential for early B-cell lymphopoiesis (Figure 14.1). Gene targeting revealed a differential dependency of fetal and adult B-lymphopoiesis on this transcription factor (Nutt et al., 1997; Urbańek et al., 1994). In the absence of Pax5, no B-cell precursors could be detected in the fetal liver. B-cell development is also aborted at the earliest stage in mice lacking the early B-cell factor (EBF) or the basic helix-loop-helix proteins encoded by the E2A gene (Bain et al., 1994; Lin and Grosschedl, 1995; Zhuang et al., 1994; see also Chapters 16 and 19 in this book). Hence, the three transcription factors EBF, E2A, and Pax5 seem to act at a similarly early stage in fetal B-cell lymphopoiesis (Fig. 14.1). In contrast, B-cell development proceeds in the bone marrow of Pax5 mutant mice to the stage of early progenitor (pro)-B cells, which are also known as pre-BI cells (Nutt et al., 1997; Urba´ nek et al., 1994). These Pax5-


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Pax5 AND B-CELL DEVELOPMENT

Figure 14.1. Transcriptional control of early B-cell development. A schematic diagram of early B-lymphopoiesis is shown together with the developmental stages (A—D) as defined by Hardy et al. (1991) and Li et al. (1996). The two distinct developmental blocks observed in fetal and adult B-lymphopoiesis of Pax5\\ mice are indicated together with the Pax5 expression pattern, the appearance of the pre-B-cell receptor (pre-BCR), and the approximate stage of the developmental arrest in mice lacking PU.1 (Scott et al., 1994), Ikaros (Wang et al., 1996), E2A (Bain et al., 1994; Zhuang et al., 1994), EBF (Lin and Grosschedl, 1995), and Sox4 (Schilham et al., 1996). pro, progenitor; pre, precursor.

deficient pro-B cells undergo normal D -J & & rearrangements at the immunogloblin heavychain (IgH) locus. The frequency of V -DJ & & recombination is, however, reduced :50-fold, thus preventing efficient synthesis of the Ig protein, which is an essential component of the pre-B cell receptor (pre-BCR) complex. Consequently, signaling through the pre-BCR, which corresponds to an important checkpoint in Bcell development (Rajewsky, 1996), cannot occur in Pax5\\ pro-B cells. Complementation with a functionally rearranged heavy-chain transgene failed, however, to advance B-cell development in Pax5\\ mice (The´venin et al., 1998). Hence, the absence of Pax5 arrests adult B-lymphopoiesis at an early pro-B cell stage that is not yet responsive to pre-BCR signaling. Here we summarize recent experimental data that have provided novel insight into the role of Pax5 in early B-cell development. In addition, we discuss the function of Pax5 in late B-lymphopoiesis based on the analysis of conditional and gain-of-function mutations. A more comprehensive overview of the literature dealing with different aspects of the transcription factor Pax5 (BSAP) has recently been published (Busslinger and Nutt, 1998; Morrison et al., 1998a).

Pax5 (BSAP) IS ESSENTIAL FOR B-LINEAGE COMMITMENT Homozygous Pax5 mutant mice are born at a normal Mendelian frequency, then become growth retarded and usually die within 3 weeks for unknown reasons (Urbańek et al., 1994). These mice show alterations in the morphology of the posterior midbrain and in the foliation pattern of the cerebellum, indicating a role of Pax5 in the development of the midbrain-hindbrain boundary region where this gene is expressed during embryogenesis (Urbańek et al., 1994). In agreement with the B-lymphoid—restricted expression of Pax5 within the hematopoietic system (Adams et al., 1992), B-cell development is arrested at an early pro-B cell stage in the bone marrow of Pax5\\ mice, whereas differentiation along all other hematopoietic lineages is normal both with regard to the presence and cell number of the different developmental stages (Urbańek et al., 1994). The pro-B cells from Pax5\\ bone marrow can be cultured in vitro on stromal cells in the presence of IL-7 with similar efficiency as wildtype pro-B cells (Nutt et al., 1997). The availability of large quantities of Pax5\\ pro-B cells provided a unique oppor-

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Figure 14.2. Developmental potential of the Pax5-deficient pro-B cell. Mature cells of different hematopoietic lineages can be derived from Pax5\\ pro-B cells by differentiation in the presence of the indicated cytokines (replacing interleukin [IL]-7). T-cell development (in vivo in the thymus) was observed upon injection of Pax5\\ pro-B cells into RAG2-deficient mice. B-cell differentiation (in vitro) required restoration of Pax5 expression by retroviral transduction. DC, dendritic cell; NK, natural killer cell; M, macrophage; ST2, stromal feeder cell line. For experimental details, see Nutt et al. (1999b) and Rolink et al. (1999).

tunity to characterize in detail the early developmental block of the Pax5 mutation. Flow cytometric analyses as well as gene expression studies indicated that wild-type and Pax5\\ pro-B cells did not differ significantly in the expression of the 50 B-cell—associated genes that were analyzed (Nutt et al., 1997; 1998). These data and the normal frequency of D-J & rearrangements in Pax5\\ pro-B cells suggested that the mutant pro-B cells may have undergone B-lineage commitment. Withdrawal of IL7 from the culture medium normally arrests proliferation of wild-type pro-B cells and simultaneously induces their differentiation to mature B cells in vitro (Rolink et al., 1996). Under the same conditions, the Pax5\\ pro-B cells are unable to differentiate along the B-lymphoid lineage, demonstrating that the Pax5 mutation arrests B-cell development at the same early stage in vitro as in vivo (Nutt et al., 1999b). This

differentation arrest could, however, be overcome by restoring Pax5 expression in Pax5\\ pro-B cells by infection with a BSAP (Pax5)expressing retrovirus (Figure 14.2). Based on these observations, we concluded at the time that Pax5 is not required for B-lineage commitment, but rather for the developmental progression beyond an early pro-B-cell stage in the bone marrow of adult mice (Nutt et al., 1997). However, recently we made the surprising discovery that the Pax5\\ pro-B cells radically differ from wild-type pro-B cells with regard to their developmental potential (Nutt et al., 1999b; Rolink et al., 1999). Initially we observed that Pax5\\ pro-B cells survive for up to 3 weeks in IL-7—depleted medium in marked contrast to wild-type pro-B cells. In the absence of IL-7, Pax5\\ pro-B cells change their morphology and then continue to proliferate, suggesting that they are able to differentiate along

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other hematopoietic lineages (Nutt et al., 1999b). Further investigation of this phenomenon revealed that Pax5\\ pro-B cells are in fact uncommitted hematopoietic progenitors with a broad lymphomyeloid developmental potential (Nutt et al., 1999b). Substitution of IL-7 with lineage-appropriate cytokines allowed Pax5\\ pro-B cells to differentiate in vitro to functional macrophages (;M-CSF), osteoclasts (;TRANCE), dendritic cells (;GM-CSF), granulocytes (;G-CSF) and natural killer cells (;IL-2; Fig. 14.2). This multilineage potential is a clonal property of the Pax5\\ pro-B cells, as it is also observed with clones derived by singlecell sorting, thus ruling out the possibility that these cells correspond to a heterogeneous mixture of lineage-committed precursor cells. Importantly, the pro-B cells from wild-type and RAG2\\ mice lack the same broad developmental potential, as they are only able to differentiate along the B-cell pathway. Hence, Pax5-expressing pro-B cells are committed to the B-lymphoid lineage, whereas Pax5\\ pro-B cells retain a broad multilineage differentiation potential. The in vivo developmental capacity of Pax5deficient pro-B cells was studied in mice that are genetically deficient in a particular hematopoietic lineage. The T-lymphoid potential was investigated by injecting Pax5\\ pro-B cells into RAG2\\ mice that have an early T-cell developmental defect due to the absence of V (D)J recombination (Shinkai et al., 1992). Within 3 weeks of injection, the thymus of the recipient mice is fully reconstituted by donor-derived lymphocytes, which give rise to all developmental stages and to the expected ratio of TCR > and > T lymphocytes seen in wild-type mice (Rolink et al., 1999). At 4 weeks, normal levels of CD4> and CD8> T cells are also observed in peripheral lymphoid organs. Interestingly, the injected Pax5\\ pro-B cells are able to home to the bone marrow in the recipient mouse where they undergo self-renewal (Rolink et al., 1999). As a consequence, these Pax5\\ pro-B cells could be reisolated from the bone marrow of RAG2\\ mice and were shown to restore T-cell development in injected secondary RAG2\\ recipients (Rolink et al., 1999). The correct homing of Pax5\\ pro-B cells suggested that these cells may also differentiate along the myeloid lineages

in vivo. The formation of myeloid cell types was, however, not detected in reconstituted RAG2\\ mice (Rolink et al., 1999), indicating that the Pax5\\ pro-B cells cannot efficiently compete with endogenous myeloid progenitors in the bone marrow. In contrast, the Pax5\\ pro-B cells could differentiate to osteoclasts upon injection into c- fos\\ mice (Nutt et al., 1999b), which normally fail to generate cells of the osteoclast lineage (Grigoriadis et al., 1994). Hence, the Pax5\\ pro-B cell behaves as a lymphomyeloid progenitor not only in vitro but also in vivo.

ALTERNATIVE FATES ARE SUPPRESSED AT B-LINEAGE COMMITMENT BY PAX5-MEDIATED GENE REPRESSION The multilineage potential of Pax5\\ pro-B cells clearly demonstrates that B-cell development in the bone marrow can progress relatively far down the B-cell pathway even in the absence of B-lineage commitment (Figure 14.1). Important insight into the function of Pax5 (BSAP) at the molecular level was obtained by comparative expression analysis of uncommitted Pax5 mutant and committed wild-type pro-B cells. Analysis of :50 B-lymphoid genes initially identified seven differentially expressed genes (Table 14.1). Five of these genes are under the direct control of Pax5 (BSAP), as their expression was rapidly regulated in Pax5\\ pro-B cells by a hormone-inducible BSAP-estrogen receptor fusion protein (Nutt et al., 1998). The genes coding for the cell surface proteins CD19 and Ig (mb-1) and the transcription factors LEF-1 and N-myc were thus shown to be positively regulated by Pax5, while the gene coding for the cell surface protein PD-1 was efficiently repressed (Table 14.1). These data therefore suggested that Pax5 functions not only as a transcriptional activator but also as a repressor at B-lineage commitment. Consistent with this idea, Pax5 was previously implicated in the repression of J-chain gene expression (Rinkenberger et al., 1996) and in the downregulation of immunoglobulin 3 enhancers (Neurath et al., 1994; Roque et al., 1996; Singh and Birshtein, 1993). Multipotent hematopoietic progenitor cells are thought to promiscuously activate genes

ALTERNATIVE FATES ARE SUPPRESSED AT B-LINEAGE COMMITMENT

TABLE 14.1. Pax5-dependent Gene Regulation in Pro-B Cellsa Activated Gene CD19* mb-1 (lg)* LEF-1* N-myc* bcl-xL

Repressed

Lineage

Gene

B-lymphoid B-lymphoid lymphoid broad broad

PD-1* c-myc M-CSF-R MPO perforinC pT C GATA-1C SCL/Tal-1C

Lineage lymphoid ubiquitous myeloid granulocytes NK cells T-lymphoid erythroid progenitors

?The expression of the indicated genes was compared between wild-type and Pax5\\ pro-B cells by RNase protection (boldface) (Nutt et al., 1998) or RT-PCR analysis (Nutt et al., 1999b). The activated genes are more highly expressed in wild-type pro-B cells, while the repressed genes are preferentially expressed in Pax5\\ pro-B cells. Genes that were shown to be directly regulated by Pax5 (Nutt et al., 1998) are indicated by an asterisk. The symbol C denotes genes that are expressed at a relatively low level in Pax5\\ pro-B cells (Nutt et al., 1999b).

from different lineage-affiliated programs in a process known as multilineage priming of gene expression (Enver and Greaves, 1998; Hu et al., 1997). This priming model predicts that the multipotent Pax5\\ pro-B cell should also express genes from different lineage programs. Indeed, RT-PCR analyses revealed that several genes (M-CSF-R, MPO, perforin, pTa, GATA1, and SCL/Tal-1) are exclusively transcribed in uncommitted Pax5\\ pro-B cells (Table 14.1). Importantly, the lineage-promiscuous expression of these genes is efficiently repressed in Pax5\\ pro-B cells upon restoration of Pax5 activity by retroviral transduction (Nutt et al., 1999b). Hence, Pax5 plays an important role in B-lineage commitment by repressing the transcription of lineage-inappropriate genes, which ultimately results in the suppression of alternative lineage choices. How the developmental options of the Pax5\\ pro-B cell are restricted at lineage commitment is best illustrated by the Pax5-dependent repression of the M-CSF-R gene, which renders B-cell precursors unrespon-

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sive to the myeloid cytokine M-CSF (Nutt et al., 1999b). The transcription factor Pax5 is known to recognize its target genes as a monomer protein via the bipartite DNA-binding motif encoded by the N-terminal paired domain (Czerny et al., 1993; Czerny and Busslinger, 1995; Xu et al., 1995). Pax5 also contains a potent C-terminal transactivation domain and a conserved octapeptide that were implicated in Pax5-dependent gene activation and repression, respectively (Do¨rfler and Busslinger, 1996; Lechner and Dressler, 1996) (Figure 14.3A). The question therefore arises of what determines Pax5 to act as either a transcriptional activator or repressor. Such a decision is frequently brought about by the selective recruitment of coactivators or corepressors by DNA-binding transcription factors (reviewed by Ayer, 1999). In a yeast two-hybrid screen, we have recently identified Grg4, a member of the mammalian Groucho family, as a Pax5-interacting protein (Eberhard et al., 2000). The Groucho protein was first described in the Drosophila system and has recently been demonstrated to be a potent corepressor of a specific subset of transcription factors (reviewed by Fisher and Caudy, 1998). The mammalian genome codes for four broadly expressed Groucho homologues that are known as Grg or TLE proteins (Fisher and Caudy, 1998; Stifani et al., 1992). All Grg proteins were shown to interact with Pax5 and to repress its transactivation function in transient cell transfection experiments, using a reporter gene under the control of multiple Pax-binding sites (Eberhard et al., 2000). The Pax5 and Grg4 proteins each contain two separate interaction domains, as revealed by mutagenesis and protein-binding assays (Figure 14.3A). The octapeptide of Pax5 is required for binding to a serine/proline (S/P)— rich region of Grg4, while the transactivation domain of Pax5 is masked by interaction with the conserved glutamine (Q)—rich region of Grg4 (Eberhard et al., 2000). Moreover, a mutant Pax5 protein lacking the octapeptide (OP) sequence is severely impaired in its repression function, whereas it is still able to activate gene transcription. Retroviral expression of this mutant protein in Pax5\\ pro-B cells was thus used to test the functional significance of the Pax5-Groucho interaction. The Pax5-OP pro-

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Figure 14.3. Interaction of Pax5 (BSAP) with corepressors of the Groucho family. A: Structure of Pax5 and the mammalian Groucho homologue Grg4. The different domains of the two proteins are shown together with the corresponding amino acid positions (Adams et al., 1992; Koop et al., 1996). The interaction of the two proteins is schematically indicated by arrows linking the domains involved in protein binding (Eberhard et al., 2000). PD, paired domain; OP, octapeptide; HD, partial homeodomain; TA, transactivation domain; ID, inhibitory domain; Q, glutamine-rich region; G/P, glycine/proline—rich domain; CcN, domain containing casein kinase II and cdc2 kinase sites as well as a nuclear localization sequence; S/P, serine/proline—rich domain; WD40, region consisting of WD40 repeat motifs. B: Model of Groucho-mediated gene repression by Pax5. As all mammalian Groucho proteins are expressed throughout B-cell development (Eberhard et al., 2000), it is assumed that Pax5 can stably recruit Groucho proteins to a specific promoter only in collaboration with a second Groucho-binding transcription factor (X). The partial homeodomain (HD) of Pax5 was previously shown to interact with the TATA-binding protein (TBP) of TFIID (Eberhard and Busslinger, 1999). CoA, unidentified coactivator protein(s) interacting with Pax5; BTM, basal transcription machinery.

tein was able to fully activate the endogenous CD19 gene, but failed to repress the transcription of non-B-lymphoid genes with a similar efficiency as the wild-type Pax5 protein. Hence, the interaction with Groucho appears to be essential for Pax5-mediated gene repression at B-lineage commitment (Eberhard et al., 1999). These data furthermore indicate that stable recruitment of Groucho by Pax5 is promoter context dependent and may require collaboration with a second Groucho-interacting transcription factor (Figure 14.3B) in analogy to the situation described for the Drosophila Dorsal protein (Dubnicoff et al., 1997).

THE ROLE OF Pax5 (BSAP) IN LATE B-CELL DIFFERENTIATION The continuous expression of Pax5 from early progenitors up to the mature B-cell stage suggests a critical role of this transcription factor also in late B-cell lymphopoiesis (Adams et al., 1992). Such a late function could, however, not be studied in Pax5\\ mice due to the complete absence of all B lymphocytes beyond the early pro-B cell stage (Figure 14.1) (Urbańek et al., 1994). The late function of Pax5 was instead investigated in established B-cell lines and splenic B cells by Pax5 overexpression or anti-

THE ROLE OF Pax5 (BSAP) IN LATE B-CELL DIFFERENTIATION

223

Figure 14.4. Conditional inactivation of Pax5 by Cre-mediated deletion. A: Schematic diagram of the floxed Pax5 allele. The different targeted Pax5 alleles are shown together with a schematic representation of the Pax5 locus (Busslinger et al., 1996). The Pax5 knock-out allele was previously described (Urbańek et al., 1994). B: Cre transgenic lines used for inactivation of the floxed Pax5 allele at late stages of B-cell development. The CD19-Cre line was generated by insertion of the Cre gene into the B-cell—specific CD19 locus (Rickert et al., 1995). The interferon-inducible Mx-Cre transgene is activated by intraperitoneal injection of the inducer pIpC (Ku¨hn et al., 1995).

sense oligonucleotide interference experiments (reviewed by Busslinger and Nutt, 1998). These analyses have implicated Pax5 in the control of B-cell proliferation (Wakatsuki et al., 1994), class switching of the IgH gene (Liao et al., 1994; Qiu and Stavnezer, 1998), negative regulation of immunoglobulin 3 enhancers (Neurath et al., 1994; Roque et al., 1996; Singh and Birshtein, 1993) and suppression of plasma cell differentiation (Usui et al., 1997). To study the late function of Pax5 in vivo, we have taken advantage of the site-specific CreloxP recombination system (Ku¨hn et al., 1995) to conditionally inactivate Pax5 at late stages of B-cell development. For this purpose, we generated a mouse strain containing loxP sites on either side of Pax5 exon 2 that codes for the

essential N-terminal region of the paired domain (Figure 14.4A) (Horcher and Busslinger, 2000). The floxed (F) Pax5 allele was shown to be fully functional, as Pax5$$ mice are normal with regard to survival, behavior, and B-cell development (Horcher and Busslinger, 1999). In contrast, Cre-mediated deletion of exon 2 in the germ line of Pax5 mice generated the previously described Pax5 mutant phenotype (Urbańek et al., 1994). Inactivation of the floxed Pax5 allele during B-lymphopoiesis was achieved with two different Cre transgenic lines. The interferon-inducible Mx-Cre transgene (Ku¨hn et al., 1995) was used for conditional inactivation of Pax5, whereas the B-cell—specific CD19-Cre gene (Rickert et al., 1997) resulted in continuous Pax5 deletion throughout B-cell de-

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velopment (Fig. 14.4B). Importantly, the results obtained with both approaches led to similar conclusions concerning the role of Pax5 in late B-cell differentiation (Horcher and Busslinger, 2000). Upon Pax5 inactivation, mature B cells were consistently lost in the spleen and lymph nodes, the two peripheral lymphoid organs analyzed (Horcher and Busslinger, 2000). Moreover, the recirculating B cells were absent in the bone marrow, further demonstrating that Pax5 is essential for the survival of mature B cells. Analysis of serum immunoglobulins revealed that the levels of all IgG isotypes were reduced up to 10-fold in CD19-Cre, Pax5$\ mice compared to control mice. In contrast, the IgM and IgA levels remained unchanged upon Pax5 deletion, indicating that the reduced IgG levels are unlikely to be a consequence of the lower number of B cells in CD19-Cre Pax5$\ mice. Mature Pax5-deficient B cells were furthermore unable to undergo efficient lymphoblast formation in response to lipopolysaccharide treatment. The observed reduction in proliferation potential could explain the decrease in serum IgG levels in CD19-Cre, Pax5$\ mice, as cell division is known to be a prerequisite for IgH class switching to occur in the B-cell follicles of the spleen. Surprisingly, the mature B cells radically changed their cell surface phenotype upon Pax5 inactivation. The expression of most B cell antigens (CD19, CD21, CD22, CD23, CD40, CD72, IgD, MHC II) was down-regulated, whereas two plasma cell markers (CD138, 493) were induced in mature B cells lacking Pax5 function. These experiments therefore indicate that Pax5 is essential for maintaining the phenotype of mature B cells, as its loss appears to initiate early plasma cell differentiation (Horcher and Busslinger, 2000).

ONCOGENIC ACTIVATION OF PAX5 IN NON-HODGKIN’S LYMPHOMA The human PAX5 gene is located on chromosome 9p13 (Stapleton et al., 1993), which is involved in a recurring translocation with the IgH locus (on 14q32) in a subset of non-Hodgkin’s lymphomas (Busslinger et al., 1996; Hamada et al., 1998; Iida et al., 1996; Morrison et al., 1998b). This specific t(9;14)(p13;q32) transloca-

tion is therefore thought to activate PAX5 as an oncogene that contributes to the formation of B-cell tumors. Molecular characterization of the t(9;14) translocation revealed that the coding region of the PAX5 gene remained intact in all tumors analyzed (Fig. 14.5). Hence, the t(9;14) translocation must be regarded as a regulatory mutation that brought the PAX5 gene under the control of the IgH locus. Analyses of the translocation breakpoints identified two different types of regulatory mutations. The breakpoint of some translocations occurred upstream of the two PAX5 promoters and thus led to insertion of the potent E enhancer of the IgH locus upstream of the PAX5 gene (Fig. 14.5) (Busslinger et al., 1996; Hamada et al., 1998). Alternatively, the endogenous PAX5 promoters were replaced by an antisense promoter located within the switch S region of the IgH locus (Iida et al., 1996; Morrison et al., 1998b). The enhancer insertion and promoter replacement mutations both led to a 5—10—fold increase in PAX5 mRNA levels in malignant B lymphocytes carrying the t(9;14) translocation (Iida et al., 1996; Morrison et al., 1998b). Two mutually nonexclusive explanations have been put forward to account for the oncogenic role of PAX5 in non-Hodgkin’s lymphoma (Busslinger et al., 1996; Iida et al., 1996). The first model predicts that the oncogenic effect is caused by PAX5 overexpression. The function of several different Pax proteins is indeed known to be highly dosage-sensitive (reviewed by Nutt and Busslinger, 1999; Strachan and Read, 1994). In analogy, moderate overexpression of Pax5 may also alter the normal function of this transcription factor in B lymphocytes. For instance, the proliferation rate of B cells could be increased based on results obtained with B-cell lines overexpressing Pax5 (Wakatsuki et al., 1994). Consequently, deregulation of PAX5 may perturb the cell cycle regulation of mature B cells and could thus contribute to lymphomagenesis. According to the second model, the oncogenic effect is caused by misexpression of PAX5 during the terminal phase of B-cell differentiation. The expression of PAX5 is known to be downregulated during plasma cell differentiation (Andersson et al., 1996; Barberis et al., 1990; Stu¨ber et al., 1995), whereas the IgH locus is transcriptionally most active in immunoglobulin-secret-

CONCLUSION

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Figure 14.5. Oncogenic activation of PAX5 by chromosomal translocations in non-Hodgkin’s lymphoma. A schematic diagram depicts the 5 region of the PAX5 gene and the arrangement of the IgH and PAX5 loci at the breakpoint of the t(9;14)(p13;q32) translocation on derivative chromosome 14. The enhancer insertion configuration was observed with patients KIS-1 (Busslinger et al., 1996) and 895 (Hamada et al., 1998), while the indicated promoter replacement was described for patients 1025 (Iida et al., 1996) and MZL-1 (Morrison et al., 1998b). The alternative exons 1A and 1B as well as exon 10 of the PAX5 gene are shown. Different elements of the IgH locus are indicated: E, intronic enhancer; 3E, downstream 3 enhancers; C, constant region gene; S, switch region.

ing plasma cells (Arulampalam et al., 1997). Hence, the juxtaposition of IgH control elements may force the translocated PAX5 allele to remain active when expression of the endogenous PAX5 gene is switched off. Consistent with this hypothesis, non-Hodgkin’s lymphomas carrying the t(9;14) translocation are thought to be derived from activated B cells and frequently exhibit features of plasmacytoid differentiation (Iida et al., 1996; Morrison et al., 1998b; Offit et al., 1992). Moreover, ectopic overexpression of PAX5 appears to interfere with immunoglobulin secretion in established cell lines representing late stages of B-cell differentiation (Usui et al., 1997). Hence, the sustained expression of the translocated PAX5 gene may prevent completion of the plasma cell differentiation program and could thus contribute to lymphoma formation. These hypotheses should now be verified in transgenic mice containing a Pax5 gene under the control of immunoglobulin regulatory elements.

CONCLUSION The identification of Pax5 as a key regulator of B-lineage commitment provided important insights into the molecular mechanisms controlling early B-cell specification. The lack of B-lineage cells in E2A- and EBF-deficient mice had previously implicated these two regulators in the commitment process (Bain et al., 1994; Lin and Grosschedl, 1995; Zhuang et al., 1994). However, the E2A and EBF genes are equally expressed in uncommitted Pax5-deficient and committed wild-type pro-B cells (Nutt et al., 1997). As a consequence, the Pax5 deletion has allowed for dissociation of two important processes occurring at the onset of B-cell development. The transcription factors E2A and EBF, which act upstream of Pax5 in the genetic hierarchy of adult B-lymphopoiesis (Fig. 14.1), are responsible for the initial activation of the Bcell—specific gene expression program. Pax5 subsequently functions to commit the early B-

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lymphoid progenitor to the B-cell pathway by suppressing alternative lineage options. Recently, we made the intriguing observation that the transcription of the Pax5 gene is randomly initiated from only one of its two alleles in pro-B cells (Nutt et al., 1999a). Hence, Pax5 appears to be activated in an inefficient, stochastic manner in the progenitor cell, which supports the notion that B-lineage commitment may be a stochastic rather than a deterministic process. Furthermore, the allele-specific regulation of Pax5 leads in heterozygous mutant mice to the deletion of those B lymphocytes which express only the mutant Pax5 allele (Nutt et al., 1999a). During B-lineage commitment, Pax5 fulfills a dual role by further activating the B-cell— specific program and by repressing the lineagepromiscuous transcription of other hematopoietic genes. The specific recruitment of corepressors of the Groucho family offers a molecular explanation of how Pax5 can exert its function as a transcriptional repressor of non-Blymphoid genes during the commitment process. To date, less is known about the role of Pax5 in late B-lymphopoiesis. In vitro experiments with established B-cell lines as well as conditional gene inactivation in the mouse have implicated Pax5 in several processes of late B-cell differentiation including the control of cell viability and IgH class switching. Moreover, the characterization of a specific t(9;14) translocation indicated that PAX5 can be activated as an oncogene in a subset of non-Hodgkin’s lymphomas. However, the molecular mechanism by which the activated PAX5 gene contributes to oncogenesis still remains to be elucidated.

ACKNOWLEDGMENTS We thank P. Pfeffer for critical reading of the manuscript. This work was supported by the I. M. P. Vienna and by a grant from the Austrian Industrial Research Promotion Fund.

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CHAPTER 15


JANUS KINASES AND STAT FAMILY TRANSCRIPTION FACTORS: THEIR ROLE IN THE FUNCTION AND DEVELOPMENT OF LYMPHOID CELLS TAMMY P. CHENG Howard Hughes Medical Institute—NIH Research Scholars Program

JE RO ME GALON, ROBERTA VISCONTI, MASSIMO GADINA, AND JOHN J. O’SHEA Lymphocyte Cell Biology Section, Arthritis and Rheumatism Branch, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health

INTRODUCTION Cytokines are a diverse group of secreted factors regulating cellular growth and differentiation. Knowledge of cytokine signaling has exponentially increased over the past few years. This chapter focuses on only two structurally related families of cytokine receptors, Type I and Type II receptor families, since their receptor phosphorylation leads to the activation of the Stat family of transcription factors. Members of these two receptor families have no intrinsic tyrosine kinase activity and utilize Janus kinases (Jaks) to initiate intracellular signaling. Type I receptors bind hormones such as erythropoietin, thrombopoietin, prolactin, growth hormone, and specific interleukins, while Type II receptors bind interferons (IFN). The structure and function of various Jaks and Stats are the focus of

this review; the latter in particular are emphasized in accordance with the transcription factor theme of this book. Since many interleukins are important in lymphoid development and immunoregulation, the critical roles that Jaks and Stats play in cytokine signaling may best be illustrated in the context of lessons from human disorders resulting from Jak mutations and the study of Jak/Stat deficient mice. The space constraint in this review makes citation of all of the studies contributing to the understanding of Jaks and Stats an impossible task. Hence, readers interested in more information are directed to some excellent reviews on this subject: Aringer et al., 1999; Bach et al., 1997; Baird et al., 1999; Carter-Su and Smit, 1998; Darnell et al., 1994; Hoey and Grusby, 1999; Ihle, 1995; Ihle et al., 1998; Leonard and O’Shea, 1998; O’Shea, 1997.


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JAKS AND STATS IN LYMPHOGENESIS

An important caveat should be made first, however. The fact that much work has focused on the role of Jaks and Stats in cytokine signaling has led to the notion of a ‘‘Jak-Stat’’ pathway. While it has been useful to think of signaling in these terms and genetic data on Drosophila can be interpreted as a linear pathway, such a simplistic view of mammalian Jak-Stat systems can be misleading, as is elaborated below.

that Jaks physically associate with cytokine receptors. Indeed, for IFN receptors, specific Jaks are required for efficient receptor expression at the cell surface. A few points are clear from these studies. That is, particular Jaks such as Jak1 and Jak2 are widely used by a variety of cytokine receptors. Jak3, in contrast, is used only by the so-called c cytokines, whose receptors comprise the common chain, c.

JANUS KINASES

Jak Function in Lymphoid and Other Cell Development

To date, four mammalian Jaks have been identified: Jak1, Jak2, Jak3, and Tyk2. The kinase domain, a highly conserved sequence across different kinase families, initially allowed the identification of this family via PCR strategies based on sequence homology or by low-stringency hybidization of cDNA libraries (Harpur et al., 1992; Kawamura et al., 1994; Krolewski et al., 1990; Wilks et al., 1991; Witthuhn et al., 1994; Pellegrini and Dusanter-Fourt, 1997). Since then, Jaks have been mapped to the following chromosomal loci: Jak1-1p31.3 (mouse chromosome 4), Jak2-9p24 (mouse chromosome 19), Jak3-19p13.1 (mouse chromosome 8), and Tyk2-19p13.2. Jaks have also been identified and cloned in teleosts and birds. Drosophila Jak, termed Hopscotch, is critical for embryonic segmentation by regulating pair-rule and segmentpolarity genes (Binari, 1994). No Jak was identified in the completed Caenorhadbitis elegans genome sequence, and it may be possible that C. elegans Stats are phosphorylated by evolutionarily more ancient kinases other than Jaks. Shortly after Jaks had been cloned, their essential functions in interferon and cytokine signaling were established. Using a panel of cell lines mutagenized to acquire resistance to interferons (Type II cytokines), it was first shown that Jaks are essential for interferon signal transduction (Silvennoinen et al., 1993; Velazquez et al., 1992; Watling et al., 1993). Subsequently, Type I cytokines were analyzed for their ability to activate Jaks, and it is now known that all the Types I and II cytokines activate various Jaks in some combination (Argetsinger et al., 1993; Ihle et al., 1995; Johnston et al., 1994; Leonard and O’Shea, 1998; Stahl et al., 1994; Witthuhn et al., 1993; Witthuhn et al., 1994) (Table 15.1). Moreover, we now know

The pivotal function of the Jaks is best illustrated in mice or humans deficient in these kinases. The first deficiency of a mammalian Jak was identified in a human disease (Macchi, 1995; Russell, 1995). The disease, severe combined immunodeficiency (SCID), is a heterogeneous group of disorders manifested by abnormalities in T- and B-lymphocyte function and severe infections early in life. The most common form of SCID is the X-linked form, so-called X-SCID, and is due to mutations of c (Noguchi, 1993). These patients have impaired signaling via all the cytokines that utilize c (IL-2, IL-4, IL-7, IL-9, and IL-15), resulting in impaired development of natural killer cells (NK) and T cells and abnormal function of B cells. This combination of lack of T-cell and NK-cell development and relatively normal Bcell development is referred to as the T\B>NK\ subtype of SCID. As c mutations were identified in SCID patients and shown to be the cause of this specific subtype of SCID, there were also female patients with the same T\B>NK\ phenotype, albeit with a presumed autosomal inheritance pattern. Because of previous work indicating that Jak3 bound c (Miyazaki, 1994; Russell, 1994), Jak3 mutations were sought and indeed identified in these patients. Thus, it became clear that mutation of either c or Jak3 leads to precisely the same defects. This established that Jak3 was essential for signaling by c cytokines. Equally important, these patients indicated that Jak3 does not have irreplaceable functions outside of the immune system. Subsequently, Jak3 knockout mice were generated, and they too have defects of T, B, and NK cells (Nosaka, et al., 1995; Park, 1995; Thomis, 1995; Thomis, 1997). No other defects have been reported.

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TABLE 15.1. c cytokines: IL-2, IL-4, IL-7, IL-9, IL-15; gp130 cytokines: IL-6, IL-11, OSM, CNTF, LIF, CT-1, IL-12, leptin; c cytokines: IL-3, IL-5, GM-CSF? Jak/Stat Cytokines That Activate Specific Jak or Stat Jak1 Jak2 Jak3 Tyk2 Stat1 Stat2 Stat3 Stat4 Stat5a Stat5b

c cytokines, gp130 cytokines, IL-10, IFN-, IFN-, IFN-, IL-13 EPO, c cytokines, gp130 cytokines, IL-13, IL-12, GH, prolactin, TPO, IFN- IL-2, IL-7, IL-15, c cytokines gp130-R cytokines, IL-13, IL-12, IFN-, IFN-, IL-10 IFN-, IFN-, IFN-, FGF, IL-2, IL-6, IL-10 IFN-, IFN- LIF, IL-10, IL-6, gp130 cytokines, c cytokines, GH IL-12 Prolactin, IL-2, EPO, c cytokines, c cytokines, GH, TPO GH, GM-CSF, IL-2, EPO, c cytokines, c cytokines, prolactin, TPO

Stat5a/b GH, IL-2, EPO Stat6 Stat4/6

IL-4, IL-13

Phenotype of Deficient Mice Neurologic defect, mice die perinatally Embryonically lethal, failed to develop red blood cells SCID ND Susceptibility to viral and some bacterial infections; defective signaling in response to IFNs Defective signaling in response to IFNs Embryonically lethal; conditional KO showed defective IL-6 and IL-10 signaling Defective Th1 development Defective lobuloalveolar development in breast Required for sexual dimorphic growth Defective GM-CSF signaling in macrophages Defective IL-2 signaling in T cells Infertile, mammary gland development defects, smaller body size, growth deficiency (GH signaling) Defective Th2 development No Th2 cells in vitro

?Cytokines in which the specific Jak or Stat plays an essential signaling role have been highlighted in bold letters.

T- and B-lymphocyte abnormalities in c- and Jak3-deficient mice, as well as humans, appear to be due to defective IL-7 signaling. IL-7 is important for T-cell proliferation and maturation in the thymus, and mice and humans with IL-7R mutations both develop SCID (Puel et al., 1998; von Freeden-Jeffry et al., 1995). The defective lymphoid development in IL-7R\\ mice can be reversed by overexpression of bcl-2, suggesting that a major function of IL-7 is to inhibit programmed cell death (Akashi et al., 1997; Kondo, 1997; Maravsky, 1997). Furthermore, Jak3 may also be important in mediating signals for TCR rearrangement during T-cell development, especially in the linage. The lack of NK development in c or Jak3 deficiency, on the other hand, is likely the result of defective IL-15 signaling (Lodolce et al., 1998). Occasionally in Jak3-SCID patients and more consistently in Jak3\\ mice, the few T cells that are produced have an activated phenotype. This may be attributed to impaired IL-2 signaling as IL-2-, IL-2R-, and IL-2R-deficient mice develop a lymphoproliferative disease thought to be

the result of inefficient apoptosis (Willerford et al., 1995). It is notable that not all cell lineages normally expressing Jak3 have impaired function in Jak3-SCID patients. Jak3 is inducible in monocytes, but data are conflicting as to whether there are defects in this lineage in the absence of Jak3 (Grossman et al., 1999; Musso et al., 1995; Villa et al., 1996). In contrast to Jak3, gene targeting of Jak1 and Jak2 resulted in more diverse abnormalities. Jak1\\ mice die perinatally, apparently due to an ill-characterized neurologic defect (Rodig et al., 1998). These mice also have SCID like Jak3\\ mice, a finding that may be explained by the fact that Jak1 binds to the ligand-specific receptor subunit of c-using cytokines. Other than the c cytokines, Jak1 is also activated by IL-6, leukemia inhibitory factor (LIF), IL-11, IFN/, IFN, IL-10, IL-13, and G-CSF. The physiologic relevance of Jak1 activation in these pathways remains to be elucidated with lineagespecific Jak1 knockout mice. Jak2 is activated by a myriad of cytokines, including erythropoietin, prolactin, growth hor-

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mone, thrombopoietin, IL-3, IL-5, IL-6, IL-11, IL-12, GM-CSF, LIF, G-CSF, and IFN. Gene targeting of Jak2 produced an embryonically lethal phenotype because Jak2-deficient mice failed to develop red blood cells (Neubauer et al., 1998). The fact that the Jak2\\ phenotype entirely coincides with that of EPO and EPOR\\ mice suggests an essential role for Jak2 in erythropoietin signal transduction. As with Jak1, whether Jak2 simply plays a redundant role in other pathways remains to be evaluated in cell-type-specific Jak2 knockouts. Tyk2, the Jak that associates with IFN-/ receptor and other cytokine receptors, has not yet been knocked out in mice. The signaling pathways for which Tyk2 are uniquely responsible remain to be defined.

Noncytokine Receptors and Jaks A number of receptors other than Type I and II cytokine receptors have been reported to activate Jaks, but whether Jaks play an essential role in the signaling pathways of these receptors is still controversial. For instance, Jak3 has been found to associate with CD40 (Hanissian and Geha, 1997), though normal and Jak3-deficient human B cells respond equally well to antiCD40 stimulation as measured by ICAM-1, CD23, CD80, and lymphotoxin- regulation (Jabara et al., 1998). Angiotensin II has also been found to activate Jak2 via the AT1 receptor, a G-protein coupled receptor (Marrero et al., 1995). Jak2 appears to directly associate with AT1, and angiotensin II stimulation increases Jak2 kinase activity. Nonetheless, whether Jak2 is essential for angiotensin II action has not yet been determined. In summary, at present the only receptors established to be absolutely dependent upon Jaks for signaling are cytokine receptors. The use of Jak-deficient animal models should help clarify whether Jaks play critical functions in the signaling pathways of other than Type I and II cytokines or are a dedicated class of kinases whose essential function is signaling by Type I and II cytokine receptors. At this point, the data favor the latter contention. Since the Janus kinases play such critical functions in effecting immune responses and modulating cell growth, it is not surprising,

then, that micro-organisms can subvert Jaks in order to activate STATs. The Epstein Barr viral protein LMP1, for example, binds and activates Jak3 (Gires et al. 1999); however, it should be noted that EBV transforms Jak3-deficient B cells, indicating that Jak3 is dispensable for transformation.

Jak Structure Three-dimensional structural data for any of the Jaks are still lacking. Based on primary sequence comparison, overall conservation of Janus kinases was noted, and seven homologous regions were termed Janus homology domains 1—7 (JH1—7), respectively (Fig. 15.1). Jaks have a C-terminal tyrosine kinase, or JH1 domain, which shares extensive homology with other tyrosine kinases. As expected, mutation of either the ATP-binding site or a conserved tyrosine residue in activation loop abolishes catalytic activity. A feature unique to the Janus kinase family members is the pseudokinase domain. Aminoterminal to the JH1 kinase domain, the pseudokinase domain (JH2) shares extensive homology with tyrosine kinase domains but lacks residues critical for kinase activity. Though lacking catalytic activity, the pseudokinase domain appears to have a regulatory role, since JH2 mutations have significant effects on the function of the Jaks as kinases. A number of patients with Jak3-SCID have mutations in the pseudokinase domain. A singlepoint mutation in the JH2 domain results in a constitutively phosphorylated Jak3, which, counterintuitively, demonstrated poor kinase activity in vitro and was unable to transmit cytokine-dependent signals (Candotti et al., 1997). A number of artificial constructs have been generated, and they too support the critical function of the JH2 domain (Chen et al., 2000). In Drosophila, an interesting gain-of-function mutation in the JH2 domain results in malignant transformation (Luo et al., 1997). Mutant Drosophila Jak showed increased kinase activity, and the corresponding mutation in murine Jak2, when transfected into COS cells, led to increased Stat5 phosphorylation. We have hypothesized, based on the mutant phenotypes and in vitro biochemical studies, that JH2 and

JANUS KINASES

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Figure 15.1. Schematic representations of Jaks and Stats. Regions of homology shared by Jaks have been termed Jak homology (JH) domains. JH1 is a kinase domain and JH2 is a pseudokinase domain. The N-terminus of the Jaks appears to be important for association with cytokine receptor subunits. Stats have a conserved tyrosine whose phosphorylation allows Stat dimerization, an Src homology (SH2) domain that mediates the dimerization, and an N-terminal region known to play a role in the dimerization of Stats dimer. The N-terminal, C-terminal, and coil-coiled regions of Stats interact with transcription factors.

JH1 domains closely associate (Chen et al., 2000). Presumably, the solution of the threedimensional structure of the Jaks will give clues as to precisely how this domain regulates the catalytic activity. Another key feature of the Jaks is their direct association with cytokine receptors. The receptor-kinase interactions have been characterized through mutagenesis and chimera constructions of both the receptors and the Jaks. For Jak3, the N-terminal JH6 and JH7 domains confer binding specificity for the receptor subunit c (Chen et al., 1997). A conserved segment within the JH7 domain is particularly critical for receptor binding, and a SCID patient with a mutation in a conserved residue in JH7 resulting in failure of Jak3 to associate with c was identified (Cacalano et al., 1999). Other Jak-receptor interactions also are thought to involve the Jak amino terminus as well, albeit the segments that mediate this interaction may be more extended than that of Jak3. Recently, it has been suggested that the N termini of Jaks share distant homology to band 4.1 and related proteins (Girault et al., 1999). The functional significance of this is still to be determined, however.

Many tyrosine kinases have src homology (SH) 2 and SH3 domains. While Jaks evidently lack the latter, JH3 domains do share homology with SH2 domains. Definitive data on the ability of JH3 domains to bind phosphotyrosine have not been reported. Jak-Associated molecules Kinase autophosphorylation typically serves not only to regulate catalytic activity but also to recruit other components in the signaling pathway. In the case of Jaks, a family of SH2containing molecules (alternatively termed Jab, SOCS, SSI, and CIS) are induced upon cytokine stimulation and can serve as classic feedback inhibitors of signaling (Endo et al., 1997; Naka et al., 1997; Starr et al., 1997). One such example is SOCS-1 (a.k.a. Jab or SSI-1). Via its SH2 domain, SOCS-1 binds the phosphorylated activation loop tyrosine residue in Jak2, thereby inhibiting Jak2 kinase activity (Sasaki et al., 1999). SOCS-1—deficient mice perish within 3 weeks after birth (Naka et al., 1998), and it has been shown recently that the primary cause of lethality in SOCS-1—deficient mice is exag-

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gerated IFN- responses (Alexander et al., 1999; Marine et al., 1999a). Similarly, SOCS-3 is transcriptionally upregulated and becomes phosphorylated after several cytokine stimuli (Adams et al., 1998; Cohney et al., 1999). Phosphorylated SOCS-3 directly associates with Jak1 and downregulates IL-2—dependent Stat5b phosphorylation (Adams et al., 1998). SOCS-3 knockout mice have demonstrated the critical role SOCS-3 plays in fetal liver hematopoiesis; deficiency of SOCS-3 results in lethal erythrocytosis on embryonic day 12.5 (Marine et al., 1999b). The erythropoietin signaling pathway does not appear to contribute to the erythrocytosis in SOCS-3 deficiency, however, and the signaling events regulating the temporal- and cell type—specific expression of SOCS-3 remain elusive. It is worth mentioning here that other SOCS family members such as CIS-1 may downregulate cytokine signaling by binding receptors, rather than Jaks. Furthermore, while several SOCS family members are clearly important for terminating cytokine signaling, the functional significance of other proteins with a putative ‘‘SOCS box’’remains to be determined. Whereas the SOCS family members downregulate Jak kinase activity, SH2B- associates with Jak2 and increases the catalytic activity of Jak2 (Rui and Carter-Su, 1999; Rui et al., 1997). SH2B- has an SH2 domain, a plextrin homology domain, and a proline-rich motif, which is phosphorylated in response to growth hormone stimulation. SH2B- binds to Jak2 via the former’s SH2 domain and becomes a substrate for Jak2. Three splice variants of SH2B exist (, , and ). STAM is a 70 kD adaptor molecule phosphorylated in response to IL-2, IL-3, IL-4, GMCSF, epidermal growth factor, and platelet-derived growth factor (Takeshita, 1997). Notable primary sequence motifs in STAM include an SH3 domain and an ITAM (immunoreceptor tyrosine-based activation motif). After cytokine stimulation, STAM becomes inducibly associated with Jak3 and Jak2 via the ITAM region as mutation of the ITAM blocks the interaction between Jaks and STAM. STAM may couple cytokine stimulation to DNA synthesis induction, since an SH3 deletion mutant acts in a dominant negative manner on cytokine-induced DNA synthesis. Two other STAM-associated

molecules were identified — Hrs and AMSH — and both have been hypothesized to act downstream of Jaks in cytokine signaling pathways (Asao et al., 1997; Tanaka et al., 1999).

Activation of Jaks and the Initiation of Signal Transduction Unlike receptor tyrosine kinases with intrinsic catalytic activity, Type I and II cytokine receptors have no such activity. Cytokine receptor signaling is initiated, however, by Jaks, which physically associate with receptor subunits. It was proposed that ligand binding effects aggregation of receptor subunits, bringing Jaks in close proximity, allowing Jaks to phosphorylate one another on residues in the activation loop and enhancing catalytic activity. An alternative hypothesis is an allosteric mechanism (Livnah et al., 1999; Remy et al., 1999). For the erythropoietin receptor, evidence suggests that Epo receptors exist as preformed dimers, although the Jaks associated with the receptor subunits are not in proximity to activate each other. Ligand binding induces a conformational change of receptor subunits, leading to Jak activation. Which mechanism is more important for activating various receptors has not been determined. After activation, the Jaks phosphorylate receptor subunits on tyrosine residues to recruit proteins with SH2 or phosphotyrosine-binding (PTB) domains. These proteins, in turn, are phosphorylated by Jaks. Phosphorylation by Jaks couples cytokine stimulation to a number of pathways including the Ras/Raf/MAPK pathway and the PI3 kinase pathway. These pathways, though important, have been extensively reviewed and are not discussed in detail here. In general, genetic studies of cytokine receptors have demonstrated that redundancy exists downstream of Jaks. That is, in many cases only the membrane proximal portion of cytokine receptors (i.e., the Jak-binding site) is absolutely required for mitogenesis. Mutations that disrupt Jak activation, by contrast, inevitably eliminate mitogenic responses and the induction of immediate early genes. Phosphorylation of cytokine receptors also generates docking sites for a class of SH-2 containing transcription factors termed the Stats.

STATS

STATs By identifying common elements in the promoters of interferon-inducible genes and purifying factors bound to these elements, members of James Darnell’s laboratory cloned the first members of the STAT (Signal Transducer and Activator of Transcription) family (Fu et al., 1992). The IFN complex was termed interferon-stimulated gene factor-3 (ISGF-3) (Fu et al., 1992; Schindler et al., 1992a). It contained a 91 kDa polypeptide, which later became known as Stat1. Also present in the ISGF-3 complex were an 84 kDa alternatively spliced Stat1 variant, a 113 kDa protein, later known as Stat2, and p48, a member of the interferon regulatory factor (IRF) family. Similarly, an IFN-induced DNA-binding complex, -activated factor (GAF), was demonstrated to be composed of Stat1 only (Shuai et al., 1992). The cloning of Stat1 and Stat2 was rapidly followed by the identification of the other family members: Stat3, Stat4, Stat5a, Stat5b, and Stat6 (Akira et al., 1994; Hou et al., 1994, 1995; Liu et al., 1995; Quelle et al., 1995; Wakao et al., 1994; Yamamoto et al., 1994; Zhang, 1994). These seven members may represent the entire Stat family in mammals, since no other mammalian Stats have been reported since the mid-1990s. Murine Stat genes map to three chromosomal clusters: Stat1 and Stat4 on chromosome 1; Stat2 and Stat6 on chromosome 10; Stat3, Stat5a, and Stat5b on chromosome 11 (Copeland et al., 1995). Most Stats are about 750 amino acids long, although Stat2 and Stat6 are somewhat larger (850 amino acids). As with Jaks, Stat-like transcription factors have also been found in nonvertebrates. A single Drosophila Stat, termed D-Stat or Marelle, has been identified (Sweitzer et al., 1995), as has a Dictyostelium Stat, Dd-Stat. After the completion of the C. elegans genome project, two putative nematode Stats have been identified in the data bank. Interestingly, another fly Stat, termed Ag-Stat has recently been identified in A. gambiae, and it is translocated to the nucleus after bacterial challenge (Barillas-Mury et al., 1999). Since Drosophilia Stat and the C. elegans Stats are most homologous with mammalian Stat5, it is possible that Stat5 represents the ancestral Stat in mammals and has given rise to Stat3 via duplication. Sequence analysis sug-

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gests that the remainder of the Stats were generated by subsequent duplications. After the initial cloning of the Stats, it was immediately clear that this novel family of transcription factors was very different from previously recognized DNA-binding proteins by virtue of their SH2 domains. In addition, the fact that Stats were themselves tyrosine phosphorylated was also recognized, and the following paradigm emerged (Fig. 15.2): latent Stats are cytosolic transcription factors that are recruited to phosphotyrosine residues on activated cytokine receptors and specifically bind to phosphorylated cytokine receptors via SH2 domains. Stats, in turn, become phosphorylated, dimerize via reciprocal SH2-phosphotyrosine interactions, and translocate to the nucleus to regulate gene transcription (Greenlund et al., 1994; Schindler et al., 1995; Shuai et al., 1993; Shual et al., 1993). The critical functions of Stat1 and Stat2 in transmitting cytokine-dependent signals were established through the use of mutagenized cell lines defective in IFN responses (Darnell, 1994) Reconstitution of these cell lines with the wild-type Stats restored interferon signaling.

Stat Structure and Activation Unlike the Janus kinases, we have considerable information on the structure of Stats (Becker et al., 1998; Chen et al., 1998; Vinkemeier et al., 1998) (Fig. 1). Stat molecules consist of a central DNA-binding domain flanked by N-terminal protein-protein interaction domains and a Cterminal linker domain followed by an SH2 domain and a conserved site of tyrosine phosphorylation. The very C-terminal ends in Stats are variable and contain a transcriptional activation domain. As we shall see below, these structural features provide important clues for Stat function. DNA-Binding Domain. The DNA-binding region of the Stats resides within the central 171 amino acids (amino residues 318—488 in Stat3) (Becker et al., 1998; Chen et al., 1998). While this sequence is not related to other DNAbinding proteins, the overall structure of this portion of the molecule is similar to other transcription factors such as NF-B and p53. The

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Figure 15.2. Model for cytokine signal transduction. Cytokines associate with cytokine receptor subunits and activate Jak kinases. The Jaks in turn phosphorylate tyrosine-based docking sites on the receptor. Stats then bind via their SH2 domains. The Stats are then phosphorylated by the Jaks, form homo-heterodimers and then translocate into the nucleus, where they bind target sequences such as -activated sequence motif (GAS). Transcriptional activation of genes may also require accessory transcription factors.

dimeric molecule forms a C-clamp structure surrounding the DNA, but, unlike NF-B and p53, there are fewer direct contact sites with the DNA backbone. Stats bind two types of DNA motifs: ISREs (IFN-stimulated response elements) with a consensus sequence of AGTTTNCNTTTCC and GAS (gamma-activated sequence) with a consensus sequence of TTCNNNGAA. ISREs bind Stat1, Stat2, and p48. In contrast, GAS sites bind Stat1, Stat3,

Stat4, Stat5a, and Stat5b. Stat6 binds a similar site: TTCNNNNGAA. N-Terminal Dimer-Dimer Interaction Domain. In view of the fact that, with the exception of Stat6, Stats bind rather indiscriminately to the same consensus sequences, it is notable that clustered imperfect Stat-binding sites are present in a number of relevant cytokine-inducible promoters. While these sites individually

STATS

bind Stats poorly, cooperative dimer-dimer interaction can occur at these sites (Xu et al., 1996). The conserved N-terminus of the Stats consists of eight helices to form a hooklike structure facilitating dimer-dimer interactions. Whereas this domain may not be important for the binding of Stats to a single consenus-binding site (Mikita et al., 1996), it does appear to be critical for binding to imperfect sites (John et al., 1999). SH2 Domain and Tyrosine Phosphorylation Site. The Stat SH2 domain (AA 600—700) serves two critical functions: (1) to allow Stats to bind phosphorylated receptor subunits in order to become phosphorylated on a conserved tyrosine residue by Jaks and (2) to enable phosphorylated Stats to dimerize. The SH2 domains of Stat1, Stat3, Stat4, Stat5a, and Stat5b share approximately 75% identity, whereas the homology between Stat1 and Stat6 is only about 35% identity. The SH2 domain of the STATs is pivotal in recruiting specific Stats to distinct receptor subunits. This was demonstrated by several approaches, including ex-

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changing SH2 domains of different Stats (Heim et al., 1995) and insertion of heterologous Statbinding sites in receptors (Stahl et al., 1995). Receptor-derived phosphopeptides were also shown to bind Stats and inhibit Stat dimer formation (Greenlund et al., 1994; Schindler et al., 1995). Experiments in which chimeric receptors were used to recruit different Jaks showed that while Jaks phosphorylate Stats, the specificity resides in the receptor, not the Jak. Some receptors have only one Stat-binding site whereas others have more than one. It has also been noted that Stats may associate with nonreceptor proteins such as the Jaks (Fujitani et al., 1997). The crystal structure of a Stat-DNA complex vividly demonstrates the importance of the SH2 domain (Fig. 15.3). The SH2-phosphotyrosine interaction forms the hinge of the C clamp, which binds DNA. The formation of the Stat dimer and dissociation from the phosphorylated receptor is thought to be due to the greater affinity of Stats for each other than for the receptor. The reciprocal nature of the dimer formation means that the overall affinity is the square of the individual affinity (i.e., between the

Figure 15.3. A schematic representation of a Stat-Stat dimer bound to DNA. After being phosphorylated on tyrosine residues, Stats dimerize with each other via reciprocal phosphotyrosine-SH2 interactions, which form the hinge of a C-clamp—like structure. The mount of the C-clamp binds DNA with minimal contact while the exterior of the C-clamp provides ample surface for protein-protein interaction between Stat and other transcription factors.

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SH2 domain on one Stat and the phosphotyrosine on the partner Stat). Despite the importance of SH2-phosphotyrosine interactions, there may be Stat functions independent of dimerization. For instance, a Stat1 mutant that had lost the conserved tyrosine residue (Y701) essential for dimerization was shown to restore TNF-induced apoptosis in a Stat1-deficient cell line (Kumar et al., 1997). Nuclear Translocation. Stats lack a classical nuclear localization signal. Stat dimerization is generally believed to be essential for the activation of a cryptic nuclear localization signal, and the nuclear transport may be an energy-dependent process since the nuclear import of Stat1 requires a small GTPase, Ran (Sekimoto et al., 1996; Sekimoto and Yoneda, 1998). A recent study has shown that Stat dimerization may not necessarily be sufficient for nuclear translocation. Strehlow and Schindler have presented data indicating that alternations of Stat N termini may interfere with nuclear translocation without affecting dimerization (Strehlow and Schindler, 1998). From the observation that receptors and cytokines are also found in the nucleus, one group has hypothesized that Stats may translocate to the nucleus via the nuclear localization signal of the ligands or receptors (Johnson et al., 1998; Subramaniam et al., 1999). The importance of this finding for Stat trafficking remains to be determined. Transcription Activation Domain. The Ctermini of Stat1, Stat2, and Stat5 are responsible for the ability of Stats to activate transcription (Bromberg et al., 1996; Mikita et al., 1996; Moriggl et al., 1996; Wen and Darnell, 1997; Wen et al., 1995). Furthermore, serine phosphorylation in response to cytokine stimulation at a conserved proline-directed kinase site (PXSP) in the transcriptional activation domain has been shown to increase transcription activation by Stat1, Stat3, Stat4 and Stat5, and mutations of the phosphorylation site abolish transcriptional activation (Visconti et al., 2000; Wen et al., 1995; Yamashita, 1998; Zhang et al., 1995; Zhu, 1997). Kinase or kinases that phosphorylate serine residues on Stat C-termini have not been characterized with certainty. Some

data point toward ERKs (Beadling, C. 1996; Chung et al., 1997b; Ng and Cantrell, 1997). Nonetheless, more recently a number of papers point to p38 and JNK (Goh et al., 1999; Lim and Cao, 1999; Turkson et al., 1999; Zauberman et al. 1999). At present, there is also much ongoing debate on the functional significance of serine phosphorylation in Stats (Ceresa et al., 1997; Jain et al., 1998). The Stat2 activation domain is distinct from other Stats. It lacks the putative proline-directed kinase phosphorylation site but is rich in acidic amino acids. Alternatively spliced forms exist for many of the Stats. Interestingly, in most of these cases the C-terminal transcriptional activation domain is deleted, engendering dominant negative inhibitors of the full-length forms. Coiled-Coil Domain and Association with Other Transcription Factors. One striking feature of the Stat crystal structures was the presence of coiled-coil domains (AA 136—317). Consisting of four -helices projecting away from the center of the C-clamp-like dimer, the sterically unhindered and hydrophilic coiled-coil domain provides a structure suitable for protein-protein interaction. Even prior to the solution of the crystal structure of Stat1, mutation studies have helped determine the importance of this region (AA 150—250) for binding p48. Stat1, Stat2, Stat3, and Stat5 have also been shown to associate with the coactivator p300/CBP (Bhattacharya et al., 1996; Horvai et al., 1997; Korzus et al., 1998; Zhang et al., 1996). Moreover, p300 can act as a bridge between Stat3 and Smad1 to provide a mechanism for cooperative signaling between two cytokine pathways, namely, the activation of Stat3 by LIF and the activation of Smad1 by bone morphogenetic protein-2 (Nakashima et al., 1999). Another transcription factor, Nmi, binds the Stat coiled-coil domain and enhances Stat transcriptional activation via Nmi’s association with CBP/p300 (Zhu et al., 1999). A recent paper suggests that adaptors other than CBP/p300 may link Stat to the basal transcriptional machinery since a mutant adenoviral E1A protein defective in CBP/p300 binding can still interact directly with Stat1 and inhibit Stat1-dependent transcription (Look et al., 1998). A number of other transcription factors also associate with Stats, although the domains re-

STATS

sponsible for these interactions have not necessarily been mapped. For example, Stat3 and Stat5 may associate with the glucocorticoid receptor, an interaction that can augment transcription activation by Stat (Cella et al., 1998; Stocklin et al., 1996, 1997). In addition, Stats have been reported to associate with Sp1, c-Jun, NF-B (Look et al., 1995; Schaefer et al., 1995; Shen and Stavnezer, 1998). Stat Function in Lymphoid and Other Cell Development The discovery of Stats was met with great excitement. Soon afterward, most cytokines and many noncytokine stimuli were evaluated for the ability to activate various Stats, and accordingly, both cytokines and noncytokine stimuli were found to activate Stats (e.g., Sadowski et al., 1993). Indeed, as antibody reagents improved, it became less clear which Stats were essential for specific cytokine signaling pathways, since often more than one Jak and more than one Stat were activated in each signaling pathway. Although to date no human disease as a result of Stat mutations has been identified, extensive data on Stat function in Stat-deficient mice have clearly demonstrated the nonredundant functions of particular Stat proteins. Stat1. As mentioned before, Stat1 is essential for IFN actions. In addition to IFN, Stat1 is also activated by epidermal growth factor and cytokines such as IL-2, IL-6, and IL-10. It was, therefore, striking that Stat1-deficient mice were found to develop normally but exhibit high susceptibility to viral and certain bacterial infections, a phenotype reminiscent of the defects seen in IFN receptor— and IFN receptor— deficient mice and IFN receptor—deficient humans (Durbin et al., 1996; Meraz et al., 1996). Indeed, cells from these mice were completely unresponsive to IFN/ or IFN, and IFNinducible genes such as IRF-1, MHC Class I, and MHC Class II were poorly expressed. Stat1 also evidently regulates apoptosis, and its absence is associated with tumorigenesis (see below). One function of Stat1 that appears to be unrelated to IFN signaling is its possible role in fibroblast growth factor (FGF) signaling (Li et al., 1999; Su et al., 1997). Analysis of Stat1\\

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mice suggests that Stat1 function is required for the FGF-mediated growth inhibition of chondrocytes (Sahni et al., 1999). Stat2. Similar to Stat1, Stat2 is mainly activated by IFN/. Indeed, only IFN/ has been reported to activate Stat2. Unlike the other Stats, Stat2 requires Stat1 and p48 for interaction with DNA (Bluyssen and Levy, 1997). Stat2\\ mice have not yet been formally reported, but, as might have been anticipated, they have severely impaired responses to IFN/ (Chris Schindler, personal communication). Stat3. Stat3 was first identified as a factor activated by cytokines signaling through gp130 (i.e., IL-6, leukemia inhibitory factor, and ciliary neurotrophic factor). Gene targeting of Stat3 leads to early embryonic lethality at days 6.5 to 7.5 of gestation (Takeda et al., 1997). Though unclear as to how the embryonic lethal phenotype develops, it is notable that the lethality seen in Stat3 null embryos is earlier than that associated with deficiency of gp130 itself. One hypothesis is that the lethality is related to impaired LIF function, since LIF\\ mice also perish prematurely due to the embryo’s failure to implant (Escary et al., 1993). Furthermore, Stat3 activation is important for mediating the effects of LIF on maintaining the undifferentiated state of embryonic stem cells (Raz et al., 1999). In order to evaluate Stat3 function in T cells and myeloid cells, Akira and colleagues have generated conditional knockouts (Takeda, 1999). Absence of Stat3 in T cells has no effect on lymphoid development and only a modest effect on lymphocyte proliferation, a finding consistent with work from Marrack’s group showing that IL-6 is a survival, rather than mitogenic, factor for T cells (Teague et al., 1997). Targeting of Stat3 in myeloid cells produced an interesting effect. Given the role of Stat3 in transmitting IL-6 signals, the expectation was that the lack of Stat3 in myeloid cells would result in a blunted inflammatory response. Curiously the opposite was found: Stat3-deficient mice developed an exaggerated inflammatory response and died prematurely, apparently due to defective IL-10 signaling (Takeda et al., 1999). Thus, Stat3 is essential for the antiinflammatory actions of IL-10, but its function

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for other cytokines remains to be clarified due to the lethality seen in Stat3\\ embryos. Stat4. In contrast to other Stats, Stat4 is activated by a limited number of cytokines. In mice, IL-12 is the only known Stat4 activator, whereas in humans IFN/ and IL-12 may both activate Stat4 (Bacon et al., 1995; Cho et al., 1996; Jacobson et al., 1995; Rogge et al., 1998). More recently, Stat4 has been reported to be activated by IL-2 in human NK cells (Wang et al., 1999), but the physiologic importance of Stat4 activation by IL-2 in NK cells remains to be defined. Stat4\\ mice develop normally and demonstrate only defects due to impaired IL-12 responses (i.e., IFN production, NK cytotoxicity and proliferation) (Kaplan et al., 1996b). These mice have defective cell-mediated immune response and T-helper 1 (Th1) differentiation and augmented Th2 development, a phenotype entirely consistent with the abnormalities seen in IL-12, IL-12R\\ mice, and IL-12R deficient humans (Magram et al., 1996; Wu et al., 1997; de Jong et al., 1998). Interestingly, in contrast to other Stats, which have broad tissue expression, Stat4 expression is found only in thymus, spleen, activated T cells, and NK cells. It is also expressed at high levels in testes, but Stat4-deficient mice have no defects in spermatogenesis or fertility. Recently, Stat4 has been found to be induced in activated macrophages, and the functional significance of activated Stat4 in macrophages is being characterized (Frucht et al., 2000). In summary, Stat4 appears to have very restricted immunologic functions and may play a key role in promoting cell-mediated immune responses. Stat6. Stat6 was originally identified as an IL4-inducible transcription factor (Hou et al., 1994). Because of the important role IL-4 plays in the differentiation of naive T cells toward a T-helper 2 (Th2) phenotype, Stat6-deficient mice, not surprisingly, fail to develop Th2 immune responses (Kaplan et al., 1996a; Takeda et al., 1996b). Furthermore, they demonstrate impaired regulation of IL-4—inducible genes such as CD23, MHC Class II, and IL-4R, mount defective IgE responses following parasitic infections; and have impaired clearance of parasites (Kaplan et al., 1998c). On the other hand, the crucial role IL-4 plays in orchestrating

maladaptive Th2 responses is established by findings that a lack of Stat6 dramatically attenuates allergic and asthmatic disease in animal models (Akimoto et al., 1998; Kuperman et al., 1998; Miller et al., 1998; Miyata et al., 1999). Interestingly, Stat6 deficiency does not result in complete abrogation of IL-4 responses. The antiapoptotic effects of IL-4 are preserved in Stat6-deficient cells (Vella et al., 1997). Lastly, the cytokine IL-13 shares a receptor subunit with IL-4 and also activates Stat6. As expected, IL-13 responses are abrogated in Stat6\\ mice (Takeda et al., 1996a). The transcriptional repressor Bcl-6 binds to the consensus Stat6 binding site (Dent et al., 1997). The observation that Bcl-6\\ mice develop exaggerated allergic responses suggests that in vivo Bcl-6 functions to suppress signaling by this element (Ye et al., 1997). Despite the findings in Stat4-deficient mice, naive T cells from Stat4/Stat6 double knockouts can be differentiated into Th1, but not Th2 cells, in vitro (Kaplan et al., 1998d, 1998e). Interestingly, these doubly deficient mice, albeit demonstrating less IL-12—induced IFN than wild-type mice, were capable of mounting the Th1-mediated delayed type hypersensitivity response, an observation suggesting that Stat4 is dispensable for Th1 differentiation in the absence of Stat6. The regulation of T helper differentiation to Th1 versus Th2 is a complex and exciting arena in immunology today. While Stat4 and Stat6 clearly play important roles in promoting the Th1 vs. Th2 dichotomy, an intriguing challenge lies in identifying and evaluating the significance of various interactions among Stat4, Stat6 and other transcription factors such as GATA-3, c-Maf, IRF-1, and new players to be discovered on the horizon (reviewed in Kuo and Leiden, 1999; Szabo et al., 1997).

Stat5. Encoded by two distinct genes, Stat5a and Stat5b share 93% identity at the protein level (Liu et al., 1995; Lin et al., 1996). Moreover, both are activated by a myriad of cytokines including prolactin, growth hormone, erythropoietin, thrombopoietin, and IL-2. The generation of Stat5a and Stat5b knockout mice, therefore, provided striking examples of the specific functions these two transcription factors serve. Stat5a knockout mice have impaired mammary gland development and failure of

STATS

lactation (Liu et al., 1997). In contrast, Stat5bdeficient mice have defective sexually dimorphic growth and growth hormone—dependent regulation of liver gene expression (Udy et al., 1997). Thus, despite the remarkable homology between these two transcription factors, they obviously serve very distinct biological functions. To evaluate whether Stat5a and Stat5b serve redundant functions for other cytokines, Stat5a/ 5b double knockout mice were generated (Teglund et al., 1998). A third of these mice died within 48 hours after birth. The surviving mice developed a smaller body size than wild-type littermates, apparently due to impaired growth hormone signaling. Stat5a/5b doubly deficient females are infertile with higher numbers of atrisial follicles in the corpus luteum than wild type. Unlike Jak2 knockout mice, which have severely defective hematopoiesis, Stat5a/5b doubly deficient mice have been reported to have only moderate anemia (Socolovsky et al., 1999). Although IL-7 also activates Stat5, Tand B-cell development is grossly normal in Stat5a/5b null mice. However, the T cells that do develop in Stat5a/5b\\ mice fail to proliferate in vitro presumably due to an inability to respond to IL-2 (Moriggl et al., 1999). Furthermore, like IL-2— and IL-2R—deficient mice, these animals develop a lymphoproliferative disease. The individual roles of Stat5a and Stat5b in lymphocyte function are controversial, with two groups reaching slightly different conclusions (Moriggl et al., 1999; Nakajima et al., 1997). Nonetheless, it is very clear that Stat5 is essential for the intracellular signaling of IL-2, but not IL-7, even though these two cytokines share a common receptor subunit and activate the same Jaks. In conclusion, Stats are a family of transcription factors conserved through evolution and diversified in higher organisms. Despite the high degree of sequence homology, mammalian Stats have remarkably distinct functions in diverse signaling pathways. In general, Stat1, Stat4, Stat6, and probably Stat2 can be viewed as transcription factors dedicated to host defense. Stat3 has more diverse functions loosely correlated with its ability to transmit gp130-dependent signals. Stat5a and Stat5b play essential roles for certain cytokines (i.e., prolactin and growth hormone) but overlapping functions for others (c cytokine).

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Stat Function in Nonmammalian Organisms Stats in lower organisms are less diversified, but Jaks and Stats still serve important physiological functions. Loss of function in Drosophilia Stat, D-Stat, has effects similar to the absence of Drosophila Jak, including disruption of larval development due to maternal and zygotic effects on expression of pair rule genes (Hou et al., 1996; Yan et al., 1996a; 1996b). Dictyostelium Stat, DdStat, is upregulated in a temporally and spatially specific manner during the culmination response, one of the initial steps leading to Dictyostelium reproduction (Kawata et al., 1997). As opposed to peptide cytokine stimuli, Dictyostelium Stat is activated by a chlorinated hexaphenone via a yet undetermined kinase. Furthermore, Dictyostelium Stat may also act as a transcriptional repressor, rather than an activator (Mohanty et al., 1999). One idea worth repeating is that Dictyostelium Stat is activated by a serpentine receptor, rather than Type I and II cytokine receptors, which are not known to exist in this organism. Moreover, mammalian Stats may be activated by tyrosine kinases other than Jaks (Chin et al., 1996; Sadowski et al., 1993; Sahni et al., 1999). Thus, whereas it may be argued that Jaks have a dedicated role in cytokine signaling, this may be less true for some Stats.

Regulation of Stat Function Stat functions are regulated at multiple levels. First, Stat phosphorylation is often transient, so presumably Stats are dephosphorylated or degraded. The putative Stat tyrosine phosphatase could either be nuclear or, if Stats are exported from the nucleus, cytoplasmic. The tyrosine phosphatase SHP-1 has been suggested as one regulator of receptor/Stat phosphorylation, but it is likely that others exist (Haque et al., 1998; Klingmuller et al., 1995; Migone et al., 1998). Since several Stats are phosphorylated on a C-terminal serine residue, one might also hypothesize that a serine phosphatase could regulate Stat activities as well. SOCS/Jab/SSI family members (Starr, 1998), discussed earlier as regulators of cytokine signaling, may also indirectly regulate Stat phosphorylation by inhibiting Jak activity. Moreover, one paper has reported that

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Stats are ubiquitinated after ligand-dependent activation, providing yet another mechanism by which signaling is terminated (Kim and Maniatis, 1996). Finally, a family of proteins termed the protein inhibitor of activated Stat (PIAS) family has been identified (Chung et al., 1997a; Liu et al., 1998). PIAS1 and PIAS3 bind to Stat1 and Stat3 respectively. They inhibit transcriptional activity of the Stats without affecting phosphorylation. Some of these family members were isolated in association with nonStat proteins, so it remains to be determined whether they are truly dedicated inhibitors or if they have broader functions. The Role of Jaks and Stats in Physiologic Cellular Proliferation and Malignant Transformation Given the role of Jaks and Stats in mediating cell growth and differentiation signals, it is not surprising, then, that Jaks and Stats have been implicated in malignant transformation. The most vivid example of the role of Jak in tumor formation comes from Drosophilia (Hou and Perrimon, 1996; Mathey-Prevot and Perrimon, 1998). A gain-of-function mutation of Drosophila Jak, or Hopscotch, results in the so-called tumorous-lethal (Tuml) phenotype (Hanratty

and Dearolf, 1993; Harrison et al., 1995; Luo et al., 1995; Mathey-Prevot and Perrimon, 1998). The hyperactive Drosophila Jak causes melanotic tumors and lymph gland hypertrophy, resulting in dysregulated hematopoiesis in fly. As mentioned in this chapter’s introduction, evidence that Jak and Stat have a linear relationship at the genetic level is supported by the discovery that a loss-of-function mutation of Drosophila Stat, Marelle, inhibits leukomogenesis and rescues the Tuml phenotype (Hou et al., 1996). Data from mammalian systems, nevertheless, depict a more complex picture of Jak and Stat regulation, since Jak deficiency rarely results in the same cluster of phenotypes as a Stat deficiency. In rare cases of human leukemia, the kinase domain of a Jak is fused to the Tel transcription factor (Lacronique et al., 1997; Peeters et al., 1997) (Fig. 15.4). Tel-Jak fusion proteins are transfoming in cell culture, as well as transgenic mice (Ho et al., 1999; Schwaller et al., 1998). In these leukemias, transformation appears to be caused by the constitutive kinase activity of the Tel-Jak fusion proteins and the concomitant constitutive phosphorylation of downstream Stats. Mammalian Stats may be targets of kinases other than Jaks and act as oncogene effectors (Bromberg, 1999). The BCR-ABL fusion

Figure 15.4. A schematic representation of TEL/Jak2 fusion protein variants found in three leukemic patients. TEL protein is represented in white, Jak2 protein in gray. The amino acid positions, where the fusion occurs, are indicated. T/B-ALL, T/B-cell acute lymphoblastic leukemia; CML, chronic myelomonocytic leukemia; bHLH, basic helix-loop-helix domain; JH1, Janus homology domain 1; JH2, Janus homology domain 2.

CONCLUSION

kinases, identified in human leukemic patients with Philadelphia chromosomal translocations, constitutively activate Stats 1, 3 and 5, suggesting that Stats may contribute to the transforming activity of BCR-ABL (Danial, 1995; Frank and Varticovski, 1996; Hilbert, 1996; Ilaria and van Etten, 1996). Lastly, Stat3 has been implicated as a phosphorylation target of v-src (Bromberg et al., 1998). The finding that dominant negative Stat3 mutants inhibited transformation by v-src suggests a critical role for Stat3 in v-src transformation. Recently, a retinoid acid- receptor (RARA)Stat5b fusion has been identified in a case of acute promyelocytic leukemia (Arnould et al., 1999). How the Stat5b portion may contribute to transformation remains to be evaluated. However, the fact that the RARA portion is common to most of the fusion proteins causing acute promyelocytic leukemia would argue against a critical function for Stat in transformation in APL. Additionally, there are a number of other circumstances in which constitutive activation of Jaks and Stats has been found in the context of transformation. These include HTLV-1, Sezary’s syndrome, and in cell lines from patients with leukemias and lymphomas (Chai, et al., 1997; Migone et al., 1995; Migone et al., 1998; Takemoto, et al., 1997; Zhang et al., 1996). Nevertheless, whether Jaks and Stats play a primary causative role in transformation remains to be defined in these cases. The mechanism by which constitutive activation occurs has also not been clarified. The roles that Stats play in cellular proliferation have been further elucidated in Stat-deficient cells. While no Stat-deficient human has been identified to date, Stat deficient mice and cell lines have suggested that specific Stats may have important functions in cell cycle regulation. For instance, Stat5a and Stat5b appear to regulate proliferation by transcriptionally modulating genes involved in the cell cycle, since peripheral T cells from Stat5a/b double knockout mice fail to proliferate in response to the mitogenic cytokine IL-2 (Moriggl et al., 1999). Similarly, Stat6 mediates the proliferative effects of IL-4 by regulating expression of the cyclin-dependent kinase inhibitor, p27kip1 (Kaplan et al., 1998a). It should be noted that in addition to acting as transcription factors, Stats

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may also function in proliferative signaling as adaptor molecules. For example, phosphatidylinositol 3 kinase (PI3K) binds to phosphorylated Stat3 docked at IFN receptor, and PI3K, in turn, becomes tyrosine phosphorylated and subsequently activates downstream serine kinases (Pfeffer et al., 1997). On the other hand, Stat1 is associated with downregulating cellular growth. The antiproliferative effects of IFN/ have been attributed to Stat1-dependent upregulation of a cyclin-dependent kinase inhibitor, p21waf1 (Chin et al., 1996). This function of Stat1 may also be responsible for type II thanatophoric dysplasia. In this type of human dwarfism, gainof-function mutations of the fibroblast growth factor receptor-3 (FGFR-3) result in constitutive activation of Stat1 and, consequently, increased p21waf1 expression in cartilage (Li et al., 1999; Su et al, 1997). Stat1-deficient mice also display increased incidence of tumor formation compared to wildtype mice when challenged with the carcinogen methylcholanthrene or when bred onto a p53deficient background (Kaplan et al., 1998b). The increased tumor incidence may be a result of a failure of immune surveillance presumably related to impaired IFN signaling, since IFN receptor\\ mice also have an increased incidence of methylcholanthrene-induced tumors. Another potential mechanism for tumorigenesis in Stat1 null mice is defective TNF-induced apoptosis and constitutively reduced expression of caspases (Kumar et al., 1997). Additionally, a third mechanism may be via the antiangiogenic effect of IFN on tumors (Coughlin et al., 1998).

CONCLUSION In conclusion, Jaks are a small family of tyrosine kinases with rather specific functions as illustrated by humans with mutations and genetargeted mice. At this point, the existing data indicate that they have dedicated and irreplaceable functions in transmitting cytokine-dependent signals. Whether they are essential in the signaling apparatus of other receptors remains to be determined. Although much has been learned about the function of Jaks, more detailed structural information is eagerly awaited. The Stats too appear to be a relatively small but

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evolutionarily conserved family of transcription factors with highly specific functions. Remarkably, it appears that at least four of the six mammalian Stats have major functions in regulating host defense and immune responses. There is much detailed structural information on Stat molecules to help elucidate Stat functions. Nonetheless, identification of cytokineinducible genes and evaluation of integrated transcriptional regulation by Stats and associated transcription factors represent exciting future directions in studying Stat function.

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USA., 94, 13897—13902. Takeshita, T., Arita, T., Higuchi, M., Asao, H., Endo, K., Kuroda, H., Tanaka, N., Murata, K., Ishii, N., and Sugamura, K. (1997). STAM, signal transducing adaptor molecule, is associated with Janus kinases and involved in signaling for cell growth and c-myc induction. Immunity 6, 449—457. Tanaka, N., Kaneko, K., Asao, H., Kasai, H., Endo, Y., Fujita, T., Takeshita, T., and Sugamura, K. (1999). Possible involvement of a novel STAMassociated molecule ‘‘AMSH’’ in intracellular signal transduction mediated by cytokines. J. Biol. Chem. 274, 19,129—19,135. Teague, T. K., Marrack, P., Kappler, J. W., and Vella, A. T. (1997). IL-6 rescues resting mouse T cells from apoptosis. J. Immunol. 158, 5791—5796. Teglund, S., McKay, C., Schuetz, E., van Deursen, J. M., Stravopodis, D., Topham, D. J., Wang, D., Brown, M., Bodner, S., Grosveld, G., and Ihle, J. N. (1998). STAT5a and STAT5b proteins have essential and nonessential, or redundant, roles in cytokine responses. Cell 93, 841—850. Thomis, D. C., and Berg, L. J. (1997). The role of Jak3 in lymphoid development, activation, and signaling. Curr. Opin. Immunol. 9, 541—547. Thomis, D. C., Gurniak, C. B., Tivol, E., Sharpe, A. H., and Berg, L. J. (1995). Defects in B lymphocyte maturation and T lymphocyte activation in mice lacking Jak3. Science 270, 794—797. Turkson, J., Bowman, T., Adnane, J., Zhang, Y., Djeu, J. Y., Sekharam, M., Frank, D. A., Holzman, L. B., Wu, J., Sebti, S., and Jove, R. (1999). Requirement for Ras/Rac1—Mediated p38 and c-Jun N-terminal kinase signaling in Stat3 transcriptional activity induced by the Src oncoprotein. Mol. Cell. Biol. 19, 7519—7528. Udy, G. B., Towers, R. P., Snell, R. G., Wilkins, R. J., Park, S. H., Ram, P. A., Waxman, D. J., and Davey, H. W. (1997). Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression. Proc. Natl. Acad. Sci. USA. 94, 7239—7244. Velazquez, L., Fellous, M., Stark, G. R., and Pellegrini, S. (1992). A protein tyrosine kinase in the interferon alpha/beta signaling pathway. Cell 70, 313— 322. Vella, A., Teague, T. K., Ihle, J., Kappler, J., and Marrack, P. (1997). Interleukin 4 (IL-4) or IL-7 prevents the death of resting T cells: stat6 is probably not required for the effect of IL-4. J. Exp. Med. 186, 325—330. Villa, A., Sironi, M., Macchi, P., Matteucci, C., Notarangelo, L. D., Vezzoni, P., and Mantovani, A. (1996). Monocyte function in a severe combined

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CHAPTER 16


E2A AND THE DEVELOPMENT OF B AND T LYMPHOCYTES BARBARA L. KEE AND CORNELIS MURRE Department of Biology, University of California

INTRODUCTION B and T lymphocytes constitute the major effector cells of the adaptive immune system. B lymphocytes produce immunoglobulin (Ig), or antibodies, the humoral mediators of the immune response. T lymphocytes assist in the production of appropriate antibody responses and are required for cell-mediated cytotoxicity, which is essential for the clearance of viral infections. Both cell types are necessary for appropriate antigen-specific responses and the formation of memory to a particular pathogen. The absence of functional mature B and T cells leads to severe immunodeficiencies, resulting in susceptibility to viral and bacterial infections. Moreover, inappropriate regulation of the development of these cells can lead to autoimmunity, leukemia, and lymphoma. Mature B and T lymphocytes develop from multipotent hematopoietic stem cells (HSC) through the progressive restriction in lineage differentiation options (Abramson et al., 1977; Wu et al., 1967). The mechanisms leading to this restriction in differentiation potential, while con-

troversial, are beginning to be identified. For many years, it was considered likely that the cytokines, which affect the development of each lineage specifically, might in fact induce commitment to that lineage (reviewed in Dexter and Spooncer, 1987). For example, a HSC that exits the self-renewing pool and finds itself in a high concentration of macrophage colony-stimulating factor (M-CSF) would be induced to differentiate down the macrophage lineage. However, experiments designed to demonstrate this point were difficult to interpret given the requirement for lineage-associated growth factors for the survival of committed cells. Recent studies have indicated that most cytokines promote the survival and proliferation of hematopoietic cells but do not significantly alter the decisions of a cell to differentiate down a particular lineage (Cross and Enver, 1997; Fairbairn et al., 1993; Kee and Paige, 1996; Wu et al., 1995). A hematopoietic cell is considered to be committed to differentiate through a particular lineage when it is unable to give rise to alternative cell types under conditions in which more primitive cells are able to develop into multiple


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Figure 16.1. Schematic representation of B- and T-cell development. The approximate timing of expression of Bor T-lineage—associated genes and Ig or TCR receptor gene rearrangements during B- and T-cell development is depicted. The stages of development affected by the deletion of specific transcription factors, including the E2A proteins, is indicated by an X.

lineages. During B- and T-cell development, the committed state has been associated with the expression of a number of ‘‘lineage-associated’’ genes (see Fig. 16.1). Generally, the patterns of expression of lineage-associated genes also change during the progression of lymphocytes from their most immature progenitors to the mature effector cell stage. These changes have allowed investigators to isolate and characterize populations of cells at distinct developmental stages and to determine their growth, survival, and differentiation requirements as well as their functional capabilities. These advances have contributed significantly to our understanding of the stages of lymphoid development; however, we are only beginning to appreciate the underlying mechanisms that regulate these changes in gene expression, developmental potential, and function.

Recently, studies of lymphoid lineage determination and differentiation have focused on the complex network of transcriptional regulatory proteins governing the developmental changes in gene expression observed in these cells. Studies of the regulation of transcription of the Ig and T-cell receptor (TCR) genes have been instrumental in the identification of transcription factors that function throughout lymphocyte development. Additional experiments from geneablated mice have been particularly informative in identifying the transcription factors that are important for the proper development of B and T lymphocytes (reviewed in Reya and Grosschedl, 1998). These studies have indicated that a combinatorial control mechanism exists for the regulation of many lymphoid-associated genes and for the continued progression through lymphoid development. In this review we focus

E PROTEINS: AN OVERVIEW

primarily on the role of one family of these transcriptional regulatory proteins, the E proteins, which bind to DNA elements termed E boxes found in the regulatory regions of many lymphocyte-specific genes. The E2A gene, which codes for two proteins E12 and E47, has been shown to play an essential role in B lymphopoiesis and is also required, in combination with other E proteins, for proper T lymphopoiesis (Bain et al., 1994; Bain et al., 1997a; Zhuang et al., 1994). Moreover, recent studies have suggested that E proteins may be instrumental in the establishment of the lineage choice made by multipotent progenitors during the development of the lymphoid lineages (Bain et al., 1997b; Heemskerk et al., 1997; Kee and Murre, 1998).

E PROTEINS: AN OVERVIEW The E2A gene products, E12 and E47, were first identified by their ability to bind to the E-box

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motif, CANNTG, found in the enhancers of many B- and T-lineage—associated genes, including the Ig heavy- and kappa light-chain genes (Henthorn et al., 1990; Murre et al., 1989a). These proteins, along with HEB and E2-2, are class I helix-loop-helix (HLH) proteins, also called E proteins, which are characterized by their wide tissue distribution and by their ability to form homodimers as well as heterodimers with other HLH proteins, particularly class II and class V HLH proteins (Fig. 16.2). Class II HLH proteins include tissue-restricted HLH proteins such as MyoD, myogenin, and NeuroD/Beta-2, which do not form homodimers but function as heterodimers with class I HLH proteins. Class III and Class IV HLH proteins contain the myc-related proteins, N-myc, c-myc, and L-myc, and myc-interacting proteins, such as max and mad, respectively. The class III and class IV HLH proteins are not considered further in this review. Class V HLH proteins include the four mammalian Id pro-

Figure 16.2. Classification and schematic representation of helix-loop-helix (HLH) proteins. The three classes of HLH proteins discussed in this review are represented. On the left side of the figure, examples of each class are given. On the right side of the figure, a schematic representation of the structure of each class is shown. The HLH domain is present in all three classes of proteins. The basic region, depicted in black adjacent to the HLH domain, which mediates binding to DNA, is present in class I and class II but not in class V proteins. The AD1 and LH transactivation domains are present only in class I HLH proteins.

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teins, Id1-4, which function as dominant negative regulators of bHLH proteins because they lack the basic region amino terminal to the HLH domain (Fig. 16.2) (Benezra et al., 1990; Sun et al., 1991). Class V HLH proteins are able to form heterodimers with both class I and II HLH proteins, but they inhibit the ability of the heterodimer to bind to DNA. The class V proteins have a wide tissue distribution, and individual members show both distinct and overlapping patterns of expression. However, within cells of the lymphoid system, Id2 and Id3 are the most frequently detected Class V HLH proteins (Cooper et al., 1997; Gretchen Bain, Barbara Kee, and Cornelis Murre, unpublished). HLH Structure and Function The HLH domain is composed of two amphipathic helices connected by a loop and is required for protein dimerization (Murre et al., 1989b). Both class I and class II HLH proteins contain a basic region immediately amino terminal to the HLH domain that mediates DNA binding (Fig. 16.2) (Murre et al., 1989a, 1994; Voronova and Baltimore, 1990). E12 and E47 arise by differential splicing of a single exon within the E2A gene that codes for the bHLH domain. These proteins share 80% sequence identity in their bHLH domains and, to date, no differences in their dimerization capacity or DNA sequence recognition preferences have been observed. However, E12 does appear to have a lower affinity for DNA due to the presence of an inhibitory region adjacent to the bHLH domain (Sun and Baltimore, 1991). This finding is consistent with the observation that the major E-box—binding activity in B-lineage cell lines is attributed to E47 homodimers even though E47 and E12 protein can be detected in these cells (Bain et al., 1993; Shen and Kadesch, 1995). The other class I HLH proteins, HEB and E2-2, are encoded by unique genes and share approximately 60% sequence identity with E12 and E47 within the bHLH domain (Henthorn et al., 1900; Hu et al., 1992). Transactivation by E2A Proteins The amino-terminal portion of the E2A proteins contains two well-characterized transactivation domains (Fig. 16.2). The loop-helix activation

domain (LH, or AD2), found between amino acids 279 and 342, is conserved between the class I HLH proteins E12, E47, HEB, E2-2, and the Drosophila homologue of E2A, daughterless (da). Deletion of this conserved region from an E47-, or E2-2-, Gal4 DNA-binding domain fusion protein results in a 70% decrease in transcriptional activity as detected by a 5X Gal4 CAT reporter construct in HeLa cells (Aronhheim et al., 1993; Quong et al., 1993). In addition, the LH domain alone can function as a potent transactivation domain when fused to the Gal4 DNA-binding domain. A second transactivation domain, activation domain 1 (AD1), has been identified within the amino-terminal 100 amino acids of the E2A proteins (Massari et al., 1996). This domain is also predicted to form a helix and is able to activate transcription from a Gal4 reporter construct when fused to the DNA-binding domain of Gal4. The AD1 domain is also conserved between the class I HLH proteins; it is 45% identical to the corresponding domain in HEB and 44% identical to that in E2-2. Interestingly, an alternatively spliced form of E47 that lacks the AD1 transactivation domain was isolated in a screen for E-box binding proteins (Henthorn et al., 1990). This finding raises the possibility that differential usage of the transactivation domains may occur during lymphocyte differentiation or activation. However, it remains to be determined whether the AD1 or LH domains play differential roles in the regulation of gene expression in lymphoid cells. The AD1 and LH activation domains are able to mediate transactivation in both mammalian and yeast cells, suggesting that they interact with cellular targets that are conserved through evolution (Massari et al., 1996; Quong et al., 1993). A number of recent papers have suggested an interaction between the E2A proteins and proteins with histone acetyltransferase (HAT) activity, an activity thought to play a central role in remodeling chromatin to facilitate transcription (reviewed in Kadonaga, 1998). It has been reported that the transcriptional coactivator p300/CBP, which contains both intrinsic HAT activity and associates with PCAF, which is also a HAT, can be found in gel shift complexes with appropriately spaced bHLH domains (Eckner et al., 1996). Moreover, microinjection of p300/CBP antibodies into myoblasts

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blocked the transcriptional activity of the myogenic bHLH proteins and myogenic differentiation. The significance of this observation is currently unclear given that the E2A bHLH domain alone is insufficient for activation of most E2A target genes (Kee and Murre, 1998). A second paper has reported that p300/CBP can augment transactivation by E2A and NeuroD/BETA2-Gal4 fusion proteins. Both of the transactivation domains of E2A were required for optimal transcriptional synergy with p300/CPB (Qui et al., 1998). However, biochemical and genetic studies indicate that the AD1 domain of E2A, and a related domain in a yeast HLH protein called Rtg3p, interact with members of an adapter complex containing histone acetyltransferase activity in yeast called the SAGA complex (Massari et al., 1999). Therefore, it has been postulated that the AD1 domain may function through a similar interaction with SAGA-related complexes in mammalian cells.

E PROTEINS IN DEVELOPMENT Heterodimers of E proteins and tissue-restricted, class II bHLH proteins play important roles in cell fate and differentiation decisions in a number of cell types. The Drosophila homologue of E2A, daughterless (da), is involved in the specification of the sensory organ precursor cell from epidermal cells during the development of the peripheral nervous system (Heitzler and Simpson, 1991). The decision of neuronal progenitors to develop into the sensory organ precursor (SOP) is positively regulated by the activity of da in conjunction with bHLH proteins encoded by the achaete-scute gene complex (Moscoso det Prado and Garcia-Bellido, 1984). In mammalian cells, heterodimers of E2A and class II HLH proteins also function in the development of a number of cell types. E2A proteins form active heterodimers with MyoD and Myf5, which are required to induce the expression of genes that are critical for the development of myocytes (Lassar and Weintraub, 1992). One of these myocyte-specific genes is myogenin, which is also a bHLH protein that can form heterodimers with E2A proteins (Weintraub et al., 1989). E2A/myogenin heterodimers have been shown to be involved in

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the regulation of differentiation of cells committed to the muscle lineage. Therefore, E2A protein participate in both commitment to the myogenic pathway and the subsequent differentiation of cells within that lineage. Heterodimers of E2A and the class II bHLH protein NeuroD/BETA2 are essential for the proper development of pancreatic cell lineages and may also be involved in neuronal development (Lee et al., 1995; Naya et al., 1997). Given that E2A proteins are components of heterodimers that are essential for the development of many cell types, it was surprising that the targeted deletion of the E2A gene, which resulted in the loss of both E12 and E47, did not lead to obvious alterations in the development of any of these lineages (Bain et al., 1994; Zhuang et al., 1994). It appears likely, therefore, that the alternative class I bHLH proteins, such as E2-2 and HEB, are able to compensate for the loss of E2A. In contrast to the normal development of most cell types in E2A-deficient mice, the absence of E2A proteins has a profound effect on the development of B lymphocytes. Interestingly, no class II bHLH proteins have been identified in immature B-lineage cells, and the predominant E-box binding complex in B-lineage cells is composed of E47 homodimers (Bain et al., 1993; Shen and Kadesch, 1995). Moreover, E2A proteins may form homodimers specifically in B-lineage cells due to lineage-specific hypophosphorylation of serine residues amino terminal to the bHLH domain (Sloan et al., 1996), or the formation of intramolecular disulfide bonds (Benezra, 1994). These B-lineage—restricted E2A homodimers play a unique and essential role in B lymphopoiesis. E Proteins in B-Cell Development B lymphocytes develop from multipotent progenitors in the embryonic liver and, post-nataly, in the bone marrow. Stages during B lymphopoiesis have been defined based on the sequential acquisition and loss of cell surface proteins and Ig gene rearrangements (reviewed in Rolink and Melchers, 1993; Kee and Paige, 1995). B-lineage cells can be identified by the expression of B220, the high-molecular-weight form of the protein-tyrosine phosphatase CD45, in conjunction with other stage-associated markers. The most immature pro-B lymphocytes

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express both B220 and CD43 and have their Ig heavy-chain locus in the germ-line configuration. However, this population is difficult to identify conclusively because a small population of non-B-lineage cells have been identified with this phenotype in the bone marrow (Rolink et al., 1996). Maturation of pro-B lymphocytes is associated with the acquisition of the cell surface proteins CD19, heat-stable antigen (HSA/ CD24), and BP-1/6C3 (aminopeptidase N) and the sequential rearrangement of the Ig heavychain locus. Once a functional Ig heavy-chain rearrangement is made, it is expressed in association with the surrogate light-chain proteins 5 and VpreB. Expression of the Ig/5/VpreB complex (pre-BCR) is required for progression of pro-B lymphocytes to the pre-B cell stage, which is characterized by the loss of CD43, reduced proliferation, a decrease in cell size, and the initiation of Ig light-chain gene rearrangement. The expression of a functional or Ig light chain leads to the expression of IgM on immature B cells followed by IgD on mature B cells, which subsequently exit the bone marrow. The development of mature B lymphocytes is dependent on the coordinated activities of a number of transcriptional regulatory proteins, many of which are reviewed in other chapters in this book (see Fig. 16.1) (reviewed in Singh, 1996; Reya and Grosschedl, 1998). The E2A gene products are required for proper B-cell development from a very early stage. In E2Adeficient mice, which lack both E12 and E47, a small number of B220> CD43> cells can be identified in the bone marrow, but expression of CD19 and rearrangement of the Ig heavychain locus is not detected (Bain et al., 1994; Zhuang et al., 1994). This observation suggests that B-cell development is arrested prior to the development of pro-B lymphocytes. In addition, recent studies have indicated that E2A proteins, in conjunction with other transcriptional regulatory proteins, also function in the regulation of expression of a number of genes involved in the transition of pro-B cells to the pre-B cell stage (Bain et al., 1997b; Kee and Murre, 1998; Sigvardsson et al., 1997). This transition is dependent on the functional rearrangement and cell surface expression of the pre-BCR in association with the signaling complex of Ig and Ig. The E2A gene products have been implicated in the regula-

tion of transcription of the Ig heavy-chain gene. The E2A proteins were originally cloned based on their ability to bind to, and activate transcription from, the E-box sequences found in the Ig heavy- and light-chain gene enhancers (Henthorn et al., 1990; Murre et al., 1989a). Expression of E47 in a pre-T-cell line led to initiation of transcription from a promoter upstream of the Ig heavy-chain constant regions, I, and an increase in rearrangement of the Ig D-J gene segments (Schlissel et al., & 1991). In addition, expression of E47 in a nonhematopoietic cell line led to the induction of I and TdT transcription (Choi et al., 1996). These observations suggest that E2A proteins play an essential role in the expression and rearrangement of the Ig heavy-chain gene. The E2A proteins have been implicated in the regulation of expression of the recombinase activating gene Rag-1, which is required, in combination with Rag-2, for the site-specific recombination of Ig and TCR gene segments. In the 70Z/3 macrophage line, transfection of E12, but not EBF, led to the expression of Rag-1 (Fig. 16.3) (Kee and Murre, 1998). Similarly, transfection of E47 into a pre-T-cell line led to an increase in expression of Rag-1 (Schlissel et al., 1991). Therefore, the E2A gene products are likely to play an important role in the ability to recombine the Ig and TCR genes. In addition, E2A proteins have been implicated in the expression of the surrogate light-chain proteins. E2A and EBF have been shown to synergistically activate the expression 5 and VpreB in the BaF/3 pro-B cell line (Sigvardsson et al., 1997). In the 70Z/3 macrophage cell line, expression of E12 induces the expression of 5 (Fig. 16.3) (Kee and Murre, 1998). However, in this cell line, EBF alone, in the absence of E2A proteins, can induce 5, suggesting that the E2A proteins are not absolutely required for 5 expression. EBF may be able to cooperate with other transcription factors that are expressed in the 70Z/3 macrophage line to activate the 5 promoter. Alternatively, high levels of expression of EBF alone may be sufficient to activate expression of 5. The data presented above indicate that the E2A proteins are able to regulate the expression of many genes, such as Rag-1, 5, and Ig heavy chain, whose protein products are required for progression from the pro-B- to pre-B-cell stage


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Figure 16.3. Potential targets of the E2A protein E12. Expression of E12 in the 70Z/3 macrophage cell line leads to the induction of EBF, Pax-5, 5, Rag-1, and IL7R. The arrows indicate that expression of a protein, either E12 or EBF, leads to expression of the gene indicated by the arrow. The dashed lines leading from E12 to 5 and Pax-5 indicate that this induction may be mediated through the induction of EBF.

of development. However, the absence of E2A leads to an arrest during B-cell lymphopoiesis prior to the pro-B-cell stage, indicating that novel targets of E2A must exist that are required for this early transition. Among these targets may be the transcription factors EBF and Pax5, both of which are required for B-cell development prior to the pro-B- to pre-B-cell transition (Figs. 16.1 and 16.3). This possibility is supported by the finding that the E2A protein E12 is able to induce the expression of EBF and Pax-5 in the 70Z/3 macrophage line (Kee and Murre, 1998). However, the arrest in B-cell lymphopoiesis observed in E2A-deficient mice occurs even earlier than that observed in Pax5—deficient mice and may be even earlier than that observed in EBF-deficient mice. In the absence of E2A, B lymphopoiesis is arrested prior to the development of a recently defined progenitor population (A2) expressing B220, CD43 without CD4, or CD19 (Bain and Murre, 1998; Li et al., 1997). This observation suggests that E2A proteins are required close to the time of B-lineage determination, although the A2 population appears to represent cells that are mostly committed to the B lineage (Allman et al., 1999). Recent experiments, using a cell-line model of lineage switching, suggest that E2A proteins are required for maintenance of the B-lineage com-

mitted state (Kee and Murre, 1998). The 70Z/3 pre-B cell line undergoes a spontaneous and irreversible loss of B-lineage characteristics and acquires characteristics of the macrophage lineage (Hara et al., 1990). This switch to the macrophage phenotype is associated with a loss of expression of both E2A and EBF. As mentioned above, ectopic expression of E12 in this macrophage line is sufficient to induce the expression of EBF and Pax-5 as well as IL7R, Rag-1, 5, and the ability to regulate the Igk light chain in response to mitogens (Kee and Murre, 1998). The ability of E47 to induce the B-lineage program in the 70Z/3 macrophage cell lines was not tested because the E47 protein could not be stably expressed in these cells (Barbara Kee, unpublished). Therefore, it remains to be determined whether the induction of B-lineage traits in this macrophage line is a function of both E2A proteins, or whether E12 is unique in its ability to activate this developmental program. E12 and E47 demonstrate distinct DNAbinding affinities, yet other functional differences between these proteins have not been identified. A transgenic mouse model was used to test whether E12 or E47 was sufficient to rescue B-cell development in E2A-deficient mice (Bain et al., 1997b). These experiments demonstrated that both E12 and E47 are sufficient to induce

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the formation of pro-B lymphocytes; however, only E47 was able to promote the further maturation of progenitors to the B-cell stage. Significantly, maximal numbers of mature B lymphocytes were observed only in E2A-deficient mice expressing both an E12 and an E47 transgene, suggesting that both proteins are essential for the proper development of B-lineage cells (Bain et al., 1997b). These findings also suggest that high-affinity DNA-binding activity of E proteins may not be as essential early in B-cell development as it is during the transition to the mature B-cell stage, given that E12 is sufficient to promote this early stage. The functional requirements for E47 at the pre-B- to mature B-cell transition remain to be determined. The E proteins HEB and E2-2 also play a role in the regulation of the early stages of B-cell development. In contrast to the targeted disruption of the E2A gene (see above), ablation of the HEB, or E2-2, gene does not significantly affect the development of B lymphocytes (Zhuang et al., 1996). However, mice that are transheterozygous for deletions of the E2-2, HEB, or E2A genes — that is, E2-2>\E2A>\, or HEB>\E2A>\ — produce fewer pro-B cells in the fetal liver than any of the single heterozygous mutants (Zhang et al., 1996). Therefore, while HEB and E2-2 are present in early B-cell progenitors, their presence is not sufficient to compensate for the absence of E2A during B-cell lymphopoiesis. Interestingly, it was demonstrated that when the E2A gene is replaced by an HEB cDNA, via a gene-targeting strategy, B-cell development proceeded normally (Zhuang et al., 1998). In contrast to the previous experiments, this result suggests that HEB is sufficient to replace the requirement for E2A during B-cell lymphopoiesis when it is placed under the control of the E2A regulatory sequences. Therefore, during Bcell development, either insufficient levels of HEB and E2-2 proteins are expressed or they are not expressed at the correct time to compensate for the absence of E2A. The requirement for E2A at the early stages of B-cell development is well established. However, E2A proteins are detected in B-lineage cells at all stages of development, although the levels are lowest in mature resting B lymphocytes (Melanie Quong, unpublished). The inability of E12 alone to promote the maturation of B-cell

progenitors into the mature B-cell pool suggests that E2A proteins, particularly E47, play additional roles at the later stages of B-cell differentiation (Bain et al., 1997b). E2A mRNA and protein are detected in the germinal center dark zones of the spleen consistent with a role for E2A protein in antigen-induced B-cell responses (Goldfarb et al., 1996; Roberts et al., 1993). Moreover, expression of Id1, an inhibitor of E2A DNA-binding activity, in the CH12.LX2 or 22D cell lines, inhibits the ability of these cells to undergo spontaneous, and IL4-induced, isotype switch recombination, respectively (Goldfarb et al., 1996). These findings suggest an essential role for E2A in isotype switching and possibly in other mature B-cell functions. Recently, a class II bHLH protein, ABF-1, has been identified that is expressed in mature activated human B lymphocytes and EBV transformed B-cell lines, and can form heterodimers with E2A proteins (Massari et al., 1998). Interestingly, ABF-1 is most closely related to the class II bHLH protein SCL/tal-1, a protein that can inhibit transactivation by E47 (Park and Sun, 1998). ABF-1 contains a transcriptional repression domain and can repress E47-induced activation of an E-box—containing reporter (Massari et al., 1998). Therefore, ABF-1 may function in the regulation of E2A activity in mature and activated B lymphocytes. At the present time, however, the function of ABF-1 in B-cell activation and differentiation remains unknown. E Proteins in T-Cell Development T cells develop in the specialized microenvironment of the thymus. Similar to studies of B-cell development, stages during T-cell development have been defined based on the sequential acquisition and loss of cell surface proteins and TCR gene rearrangements (reviewed in Shortman and Wu, 1996; Zuniga-Pflucker and Lenardo, 1996). Commitment to the T-cell developmental pathway is marked by the acquisition of the chain of the IL2 receptor, CD25, and the initiation of rearrangement at the TCR chain locus in cells that do not express the coreceptor proteins CD4 or CD8. Once a functional V -DJ rearrangement has been com@ @ pleted, the protein is expressed in association with pre-T, a protein that functions in an


analogous manner to the surrogate light chains during B-cell development. Association of the TCR chain with pre-T is thought to signal the cessation of recombination at the TCR chain locus, thereby mediating allelic exclusion and the activation of recombination at the TCR chain locus. During the process of TCR chain rearrangement, the CD8 coreceptor is upregulated on immature single positive (ISP) cells, after which the CD4 coreceptor is upregulated, leading to the development of a population of CD8>CD4> double positive (DP) thymocytes. Once a functional TCR is assembled, the DP thymocytes undergo positive selection (for a functional TCR) and negative selection (deleting thymocytes with too strong an affinity for self-antigens). During this stage, either CD4 or CD8 is downregulated, depending on the specificity of the TCR, leading to the development of a functional CD4>CD8\ or CD4\CD8> single positive (SP) T cell. T-cell development is regulated, in part, by the coordinate activities of a number of transcriptional regulatory proteins including the E proteins E2A and HEB, and other transcription factors such as Tcf-1, LEF-1, Gata-3, and Ets-1 (Fig. 16.1) (Bain et al., 1997b; Bories et al., 1995; Muthusamy et al., 1995; Okamura et al., 1998; Schilham et al., 1998; Ting et al., 1996; Yan et al., 1997). E2A-deficient mice display a 5—10— fold reduction in the total number of thymocytes compared to wild-type mice, and a similar reduction is observed in the number of splenic T cells (Bain et al., 1997). Thymuses from E2Adeficient mice show a significant decrease in the percentage of DP thymocytes and an absence of the DN CD44>CD25> thymocyte population. Moreover, the CD44>CD25\ population, which contains most of the thymic progenitors that have not yet undergone commitment to the T lineage, is increased in cell number. This representation of cellular phenotypes suggests that E2A proteins are required for the proper development of T-lineage-committed progenitors. However, unlike the requirement for E2A during B-cell development, the requirement for E2A proteins during T-lineage determination is not absolute. This leakiness in the E2A phenotype may be due to the expression of additional E proteins in T-cell precursors. This conclusion is supported by experiments using retroviral-mediated gene transfer of Id3 into thymic progenitors

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(Heemskerk et al., 1997). Expression of Id3 early in T-cell development inhibits the ability of these cells to give rise to both and T cells after transfer into fetal thymic organ cultures, indicating that the activity of E proteins is absolutely essential for the development of committed T-lineage cells. Both E47- and HEB-DNA-binding activity can be detected in thymus nuclear extracts (Bain et al., 1997a; Sawada and Littman, 1993). Therefore, the development of T lymphocytes in E2A-deficient mice may be the result of HEB expression in T-cell progenitors, which may form homodimers in the absence of E2A and activate transcription of E2A-dependent genes. Consistent with this interpretation, HEB-deficient mice also demonstrate an arrest in T-cell development at the DN stage (Zhuang et al., 1996). HEB-deficient mice also show a dramatic decrease in the number of DP thymocytes; however, unlike E2A-deficient mice, they do not show a decrease in total thymocyte numbers. Rather, the decrease in DP thymocytes correlates with a reciprocal increase in the percentage of DN thymocytes. In addition to the defects in early thymocyte development observed in the absence of E2A, E2A appears to play a role in the subsequent generation of SP thymocytes. This conclusion is based on the observation that the percentage of SP thymocytes is increased in the absence of E2A, with the percentage of CD8> T cells being increased more than CD4> (Bain et al., 1997a). This skewing toward CD8> T cells also observed in the spleens of E2A-deficient mice, although the total number of splenic T cells is reduced compared to wild-type littermate controls. A shift toward CD8> cells is also observed in HEB-deficient mice; however, the increase appears to be in the ISP CD8> population (Zhuang et al., 1996). At the present time, the mechanism leading to this increase in the percentage of SP thymocytes and the selective increase in CD8> T cells in E2A-deficient mice remains unresolved. A number of T-lineage—associated genes have E-box sequences in their regulatory elements including TCR and TCR and the CD4 gene (Duncan et al., 1996; Ho et al., 1989; Sawada et al., 1994; Takeda et al., 1990). However, few of the known T-lineage—associated genes have been shown to be dependent on the expression

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of E2A. A number of studies have shown that the E2A gene products are sufficient to activate Rag-1 expression in cell lines (Kee and Murre, 1998; Schlissel et al., 1991). Consistent with the idea that the Rag genes might be physiologic targets of E2A, analysis of RNA from E2Adeficient thymus indicates a reduction in both Rag1 and Rag2 expression (Gretchen Bain, unpublished). However, Rag-deficient mice show defects in thymocyte development after the DN CD44>CD25> stage, indicating that the decreased expression of recombinase-activating genes is not sufficient to account for the absence of CD44>CD25> thymocytes in E2A-deficient mice (Mombaerts et al., 1992; Shinkai et al., 1992). Therefore, novel targets of the E2A proteins must be required for the optimal development of the most immature T-cell progenitors. Recently, E2A proteins were shown to play a role in the development of T cells (Bain et al., 1999). T cells differ from T cells in a number of ways, including the timing of development, the use of distinct TCR genes, localization, and function (reviewed in Raulet, 1989). In particular, in the absence of E2A, T cells fail to develop in the secondary lymphoid organs and in the intraepithelial layers of the intestine. The T cells, which colonize different organs, often express unique combinations of V A and V genes and develop at distinct stages of development (Havran and Allison, 1998; Ito et al., 1998). In the thymus of adult E2A-deficient mice, rearrangements to V3, which are generally only observed during fetal development, are readily detected, suggesting that the normal regulation of this locus is disrupted. In contrast, rearrangements to a number of V and V genes that are used preferentially during adult thymopoiesis are significantly reduced in E2Adeficient thymocytes. This study suggests that the E2A proteins are required for the appropriate activation of gene rearrangements that normally occur during adult T-cell development. In addition, E2A proteins appear to be required for the inactivation of the recombination process at loci that rearrange preferentially during fetal development. Interestingly, mice that are heterozygous for the E2A deletion show alterations in the expression of V and V genes, suggesting that the regulation of these loci is sensitive to the dose of E2A (Bain et al., 1999). This dosage sensitivity may indicate that the

E2A proteins are rate limiting during the process of recombination of these TCR gene segments. E2A and T-Cell Lymphoma The E2A and Id proteins have been shown to be involved in the regulation of cell growth and survival. Id2 and Id3 are growth factor—regulated genes that can be induced by multiple stimuli including serum, protein kinase C, and phorbol esters (Christy et al., 1991; Deed et al., 1993; Tournay and Benezra, 1996). In fibroblasts the Id proteins are required for progression through the G1 phase of the cell cycle (Barone et al., 1994; Peverali et al., 1994). Conversely, overexpression of E12 or E47 inhibits cell cycle progression provided that the E2A proteins are expressed at least 4 hours before entry into G1. This E2A-mediated inhibition of the G1- to S-phase transition can be reversed by the addition of Id. E2A proteins have been shown to induce the expression of the cyclin-dependent kinase inhibitor p21, which may explain the ability of E2A to induce cell cycle arrest (Prabhu et al., 1997). Ectopic expression of Id3 in rat embryo fibroblasts (REF) promotes colony formation; however, Id3 does not synergize with ras or myc to transform these cells (Norton and Atherton, 1998). Interestingly, in addition to promoting proliferation, Id3 promotes apoptosis of REF, a process which can be inhibited by expression of Bcl-2, Bcl-xL or E47 (Norton and Atherton, 1998). Mutational analysis of Id3 indicates that the proliferation-inducing apoptotic activities of this protein are tightly linked. Interestingly, a number of recent studies have indicated that the cyclin-dependent kinase cdk2 can phosphorylate Id2 and Id3, abrogating the ability of these proteins to inhibit bHLH homoor heterodimer formation (Deed et al., 1997; Hara et al., 1997). Therefore, the interaction of Id with E2A may be regulated during the cell cycle. Id2, Id3, and the E proteins can be detected in developing lymphoid progenitors; however, the dynamics of the interaction between these proteins during cell cycle progression remains to be investigated. In contrast to the growth-promoting activity of Id proteins, E2A proteins appear to provide a tumor suppressor function during thymocyte development, since E2A-deficient mice develop


T-cell lymphomas (Bain et al., 1997a; Yan et al., 1997). Within the first few months of life, E2Adeficient mice begin to display an increase in thymocyte numbers with the emergence of monoclonal TCR gene rearrangements. By 3 to 6 months of age, most of these mice develop thymomas that express the chain of the IL2R (CD25), increased levels of c-myc, and heterogeneous levels of CD4 and CD8 (Bain et al., 1997a). Frequently these tumors are metastatic and, in addition to destroying the normal architecture of the thymus, invade the kidney, liver, lung, lymph nodes, and spleen. A number of E2A- and E47-deficient thymomas have been adapted to growth in vitro, providing a model system in which to investigate the role of E2A proteins in suppressing thymocyte transformation. Ectopic expression of E12 or E47 in E2A-deficient thymoma cell lines induced these cells to undergo rapid apoptosis (Engel and Murre, 1999). However, an E12 mutant that is unable to bind to DNA was unable to induce apoptosis in these lines (Engel and Murre, 1999). Surprisingly, E2A-mediated apoptosis did not appear to be associated with a decrease in cell cycle progression. However, it remains to be determined how the overexpression of E2A in these lymphoma cell lines relates to the normal function of E2A during thymic development. The most common genetic alteration in human T-cell acute lymphoblastic leukemia (TALL) involves the class II bHLH protein SCL/ tal-1. Tal-1 is normally expressed in, and required for, the development of immature multipotent hematopoietic progenitor cells (Porcher et al., 1996). However, transgenic expression of Tal-1 in the T-cell lineage, where it is not normally expressed, is sufficient to induce the formation of T-cell lymphomas (Condorelli et al., 1996; Kellieher et al., 1996). Tal-1 is unable to form homodimers but efficiently forms heterodimers with both E12 and E47. Tal-1/E47 heterodimers have been shown to bind to the E-box motif; however, these complexes are transcriptionally inert apparently due to a negative interaction between their transactivation domains (Park and Sun, 1998). Therefore, expression of tal-1 in T-lineage cells leads to the inhibition of E2A activity. The hypothesis that tal-1 leads to lymphoma development by inhibiting the activity of E2A was recently tested in the T-ALL cell line Jurkat

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(Park et al., 1999). The Jurkat cell line expresses tal-1 and E47 but demonstrates very low levels of activation of an E-box containing reporter construct. Expression of an altered form of tal-1 (E-T/2), which contains the tal-1 bHLH domain and the transactivation domains of E2A, in Jurkat cells results in dimeric complexes containing E47 and E-T/2, which activate transcription in a manner similar to that observed for E47 homodimers. Moreover, expression of E-T/ 2 in Jurkat cells reduced their clonogenic potential and resulted in aptoptosis. These findings support the hypothesis that tal-1 induces T-cell lymphomas by interfering with the tumor-suppressing activities of E2A. In this regard, it is interesting that another oncoprotein involved in T-ALL, lyl-1, can interfere with the normal functions of E2A. Lyl-1 can heterodimerize with E2A proteins and alter their DNA-binding specificity, thereby functionally removing E-box binding activity (Miyamoto et al., 1996). However, the mechanism by which E2A proteins suppress tumor formation during T-cell development remain to be determined. In addition, the possibility that E2A proteins play a similar function during B-lineage development has yet to be investigated.

CONCLUSION AND FUTURE DIRECTIONS The E proteins play essential roles in B- and T-lymphocyte development. However, a number of questions remain to be answered regarding the mechanism by which E proteins regulate these developmental processes. The genes regulated by E2A or HEB during the commitment of multipotent progenitors to the B and T lineage remain to be identified. As mentioned in the preceding sections, all of the known target genes of E proteins are required for the transition of pro-B or pro-T cells to the pre-B or pre-T cell stage. The identification of E-protein—regulated genes required for the development of committed B- and T-lineage cells will lead to a better understanding of how lineage restriction is achieved. Moreover, analysis of the mechanism by which these novel genes are regulated will provide insights into the interaction of E proteins with other key transcriptional regulators at the earliest stages of B- and T-cell development. In addition to its role in early B lymphopoiesis,

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E47 is also required for the development of mature B lymphocytes (Bain et al., 1997b). Similarly E12 or E47 functions in the regulation of the number of SP CD4 and CD8 cells in the thymus and spleen (Bain et al., 1997a). The mechanism by which E47 regulates these developmental stages and potential target genes has yet to be identified. Identification of E-protein target genes will provide insights into the mechanisms by which E proteins regulate many stages of lymphopoiesis. However, another important question remains to be addressed — that is, how is the expression or activity of the E proteins themselves regulated? One mechanism for regulating E-protein activity is through the Id proteins. The Id proteins, particularly Id2 and Id3, show dynamic patterns of expression during Band T-cell development (Cooper et al., 1997; Gretchen Bain, unpublished). These Id proteins can be regulated by signaling pathways initiated by a number of growth factors or cytokines, as well as by the cell cycle—associated kinase cdk2 (Christy et al., 1991; Deed et al., 1993, 1997). To date, little is known about the external pathways that could potentially regulate the activity of the E proteins. In this regard, it is interesting that the stages of B- and T-cell development affected by the absence of E2A are very similar to those affected by the absence of the chain of the IL7 receptor (IL7R) (Peschon et al., 1994). Fetal liver—derived lineage-unrestricted progenitors cultured in the absence of IL7 undergo B-lineage determination but show a pattern of expression of B-lineage—associated genes identical to that of E2A-deficient bone marrow cells (Kee and Paige, 1996). B-cell development in IL7R-deficient mice is arrested prior to the expression of CD19; however, some mature B cells do develop in these mice (Peschon et al., 1994). The thymus of IL7R-chain—deficient mice contains at least 100-fold fewer cells than a wildtype thymus, and essentially no CD44>CD25> cells can be detected. In addition, no T cells are observed in either heterozygous or homozygous IL7R-deficient mice (He and Malek, 1996; Maki et al., 1996). The striking similarities between E2A- and IL7R-deficient mice suggest that the activities of these two proteins may be related. One possible explanation for the similarities between these mice is that the activity of the E2A proteins is regulated by the signaling

pathway initiated by IL7R. A second possibility is that expression of IL7R could be regulated by the E proteins. IL7R expression is induced in the 70Z/3 macrophage line after transfection with E12; however, the E12 bHLH domain alone is able to activate IL7R expression in these cells, suggesting that this may not be a direct effect of E12 (Kee and Murre, 1998). Moreover, mRNA encoding IL7R is readily detected in the bone marrow of E2A-deficient mice, indicating that E2A is not required for IL7R expression (Bain et al., 1994). A third possibility is that E proteins and IL7R function in parallel but distinct pathways that are required for the same developmental processes. A number of other cytokines are required during B- and T-cell development that may modulate Id and E-protein activity. These factors include stem cell factor (SCF), or c-kit ligand and fl3/flk2, both of which synergize with IL7 to promote the earliest stages of B- and T-cell development (Billips et al., 1992; Kee et al., 1994; Mackarehtschian et al., 1995; Ray et al., 1996; Rodewald et al., 1997; Veiby et al., 1996). IL-1 and TNF may also play a role in early T lymphopoiesis, since the addition of neutralizing antibodies against these factors inhibits the development of CD25> cells in an in vitro culture system (Zuniga-Pflucker et al., 1995). Elucidation of the signaling pathways that regulate the activity of E protein and the identification of E-protein target genes will greatly increase our understanding of the mechanisms that regulate the development of both normal and malignant B and T lymphocytes.

ACKNOWLEDGMENTS We thank the members of the Murre lab for many helpful discussions. Barbara L. Kee was supported by a special fellowship from the Leukemia Society of America.

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CHAPTER 17


THE ROLE OF BCL-6 IN NORMAL LYMPHOID SYSTEM AND NON-HODGKIN’S LYMPHOMAS B. HILDA YE Department of Cell Biology, Comprehensive Cancer Center, Albert Einstein College of Medicine Bronx, New York

INTRODUCTION Non-Hodgkin’s lymphoma (NHL) is the most common form of lymphoid malignancy in the adult population. In the United States, more than 80% of these tumors are derived from mature B cells bearing surface immunoglobulin (Ig) molecules, while the rest of them originate from T cells. As mature lymphocytes can be found in vivo to exist in various differentiation and activation states, their malignant counterparts, the non-Hodgkin’s lymphomas, also come with a high degree of biological and clinical heterogeneity, suggesting multiple types/stages of target cells and multiple transformation pathways (Magrath, 1990). Understandably, traditional lymphoma classification systems that largely depend upon histological criteria, such as cell size, shape, and architecture of the tumor mass, are not able to provide consistent and uniform diagnosis in clinical practice. Concurrent existence of multiple classification sys-

tems also created confusion and difficulty in communication. To overcome these problems, the Revised European-American Lymphoma (REAL) classification was proposed to unify previous schemes and to incorporate genetic information along with immunophenotyping data (Harris et al., 1994). For immunologists, lymphoid malignancies post an interesting dilemma since they derive from immune cells that are responsible for maintaining the integrity of the body. As with all types of cancers, lymphomas are invariably clonal, meaning they represent the clonal outgrowth of a single immune cell that has accumulated multiple genetic lesions. Thus, if one considers various forms of lymphomas as transformed counterparts of normal B and T cells, these disease entities also provide valuable opportunities to elucidate the cellular mechanisms and molecular pathways governing normal lymphocyte differentiation and immune responses. In this regard, a number of common NHL


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THE ROLE OF BCL-6 IN NORMAL LYMPHOID SYSTEM AND NON-HODGKIN’S LYMPHOMAS

TABLE 17.1. Chromosomal Rearrangements and Target Genes in NHL

Rearrangement

Disease

Activated gene

Gene function

t(11;14)(q13;q32)

MCL

BCL-1/cyclin D1

Cell cycle regulator

t(14;18)(q32;q11)

FL

BCL-2

Apoptosis inhibitor

t(3;?)(q27;?)?

DLCL

BCL-6

Transcription factor

t(8;14)(q24;q32)@

BL

c-Myc


der(10)(q24)

CTCL

NF-B2/LYT-10


t(2;5)(p23;q35)

ALCL

NPM/ALK

T(9;?)(p13;?)?

LPL

PAX-5

Nucleolar phosphoprotein/tyrosine kinase Transcription factor

Mechanism of Alteration

References

Transcriptional deregulation Transcriptional deregulation Transcriptional deregulation Transcriptional deregulation Truncation/ deregulation Fusion protein

(Rosenberg et al., 1991) (Korsmeyer, 1992) (Ye, 2000)

Transcriptional deregulation

(Iida et al., 1996)

(Dalla-Favera, 1993) (Neri et al., 1991) (Morris et al., 1994)

MCL, mantle cell lymphoma; FL, follicular lymphoma; DLCL, diffuse large-cell lymphoma; MALT, mucosoaassociated lymphoma; BL, Burkitt’s lymphoma; CTCL, cutaneous T-cell lymphoma; ALCL, anaplastic large-cell lymphoma; LPL, lymphoplasmacytoid lymphoma. ?Various partner chromosomes can be involved in different cases. @Variant translocations are not listed. Source: Modified from Dalla-Favera et al., 1999.

subtypes, for example, the follicular center lymphoma (FL), diffuse large-cell lymphoma (DLCL), and Burkitt’s lymphoma, have been thought to derive from cells of the germinal center (GC) (Harris et al., 1994; Stein and Dallenbach, 1992), a dynamic structure in secondary lymphoid organs where T-cell—dependent antibody response is orchestrated and memory B cells are generated. In fact, immunologists have often relied on selected NHL cell lines as model systems to study various regulatory mechanisms governing differentiation and function of mature B cells in the GC. For molecular oncologists, non-Hodgkin’s lymphoma is an intriguing disease. Unlike many solid tumors, in particular those of the epithelial origin, where genome-wide, random genetic instability is very common (Johansson et al., 1996), B-cell NHLs seem to be able to maintain the integrity of the genome at all stages of the disease development. For example, microsatellite instability as the result of defective DNA mismatch repair is rarely observed in these tumors (Gamberi et al., 1997). In addition, not all

kinds of specific aberrations occur at the same frequency in NHL. Point mutations, gene amplifications and deletions have all been observed in these tumors, yet the hallmark genetic abnormality is balanced chromosomal translocations, which can be found in up to 90% of NHL patients. These translocations generally occur when Ig or T-cell receptor (TCR) genes undergo physiological rearrangements and often involve these genes themselves. Past studies of recurring genetic lesions in NHL and the corresponding oncogenes altered by them have provided valuable insights into the initiation and progression of these diseases. The specific genotype-phenotype association in many of the cases also provides a genetic basis for the wide histologic and clinical heterogeneity of these tumors. Table 17.1 summarizes chromosomal translocations and activated genes corresponding to them in various forms of NHL. In the high-grade Burkitt’s lymphomas, the c-Myc gene, which encodes a potent regulator of cellular proliferation, is invariably deregulated by immunoglobulin sequences translocated to its

INTRODUCTION

273

TABLE 17.2. Summary of Immunohistological Findings for AIDS-Related NHL Subtypes Marker Tumor Type Systemic AIDS-NHL SNCCL LNCCL IBLP AIDS-PEL AIDS-PCNSL LNCCL IBLP

Expression

BCL-6

Syndecan-1

LMP-1

BCL-2

; ; 9 9

9 9 ; ;

9 9 ; 9 or rare

? ? ? ?

; 9

9 ;/9

9 ;?

9 ;?

?A minority (2/7) of cases were negative for all three markers: BCL-6, LMP-1, and BCL-2. Source: Based on Carbone et al., 1998a; Larocca et al., 1998; Gaidano et al., 1998.

vicinity from one of the three Ig loci (DallaFavera, 1993). In the case of the low-grade mantle cell lymphomas, frequent t(11:14) translocation deregulates the expression of cyclin D1 involved in the control of cell cycle progression (Medeiros et al., 1990; Rosenberg et al., 1991). In the majority of FLs, the characteristic t(14;18) translocation leads to overexpression of BCL-2, an inhibitor of apoptosis (Korsmeyer, 1992). In line with the notion that tumorigenesis is a multistep process, more than one genetic alteration is often noted for individual NHL cases. A good example is p53 loss or mutation, which is acquired by a fraction of FL cases during their histologic transformation to become clinically more aggressive DLCLs (Lo Coco et al., 1994; Sander et al., 1993). It is probably not so surprising that many of these recurrent genetic events affect transcription factors that normally control the expression of multiple target genes directly involved in cell proliferation and differentiation. Presumably, by targeting these ‘‘master control genes,’’ a precancerous cell could benefit most from a minimum set of lesions in the genome. Although this observation can also apply to many other hematologic malignancies and solid tumors, NHLassociated genetic abnormalities have their own characteristics. First is the rather unique oncogene/tumor suppressor gene profile. Inactivation of p53, which is implicated in more than half of solid tumors, only occurs in low frequency in most NHL types with the exception of Burkitt’s lymphomas (Gaidano et al., 1991); Tu-

mor suppressor genes Rb and p16, which are frequently mutated or deleted in a variety of solid tumors and T-cell acute lymphoblastic leukemia, are rarely altered in NHL. Second is the prominent feature of transcriptional deregulation in NHL-associated translocations. This is in contrast with various lymphoid and myeloid leukemias, where chromsomal translocation yields either transcriptional deregulation or fusion protein production. Notably missing from these early studies is information regarding molecular pathogenesis of the DLCL. No frequent or specific genetic lesions were described for this disease, except a low incidence of rearrangements of c-MY C and BCL-2 (Weiss et al., 1987; Yunis et al., 1989). Since the high-grade Burkitt’s lymphomas usually respond well to current treatment protocol, and a significant fraction of follicular tumors eventually undergo histologic transformation to become DLCL, this lymphoma type accounts for nearly 80% of all NHL mortality even though only one-third of the initial NHL diagnoses fall into this category (Magrath, 1990). In other NHL types, the first clue to the molecular pathogenesis often starts from studying consistent translocations where one target gene is involved in translocations with a limited set of partner loci, as in the case of c-MY C and BCL-2. In DLCL, however, for unknown reasons, such consistent translocations are rare. Important molecular advances were not made until a group of cytogenetic abnormalities involving band 3q27 were recognized as recurrent

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events (Bastard et al., 1992; Offit et al., 1989). 3q27 translocation, which involves at least 34 different loci reported so far, is one of the two examples of the so-called promiscuous chromosomal translocations, the other being the ALL-1 gene on 11q23. The majority of the 3q27 translocations target the proto-oncogene BCL-6, leading to deregulated expression of a transcription factor normally expressed only during the GC stage of B-cell development. The remainder of this chapter reviews findings on BCL-6 translocations and mutations in various types of NHL, mechanisms of its deregulation in lymphomas, and the current understanding of its function in normal as well as tumor B cells. The clinical implications of BCL-6 gene abnormality and protein expression are also discussed.

BCL-6 TRANSLOCATIONS IN NHL Based upon cytogenetic observations of frequent 3q27 aberration in DLCL, the proto-oncogene BCL-6, also called LAZ3 and BCL-5, was identified at the vicinity of the 3q27 breakpoints by five groups in 1993 (Baron et al., 1993; Deweindt et al., 1993; Kerckaert et al., 1993; Miki et al., 1994a; Ye et al., 1993a, 1993b). Large panels of tumors representing various NHL types and other hematopoietic malignancies were subsequently screened for BCL-6 structural alterations. The results showed that rearrangement of BCL-6 in its 5´ flanking region is very common in DLCL (28—40%), less frequent in follicular lymphoma (6—15%) and marginal zone B-cell lymphoma (9%), and virtually absent in other lymphoid malignancies and solid tumors (Bastard et al., 1994; Lo Coco et al., 1994; Otsuki et al., 1995). BCL-6 rearrangement is also detectable in the acquired immunodeficiency syndrome (AIDS)-associated NHL, mainly in the AIDS-DLCL subtype (20%) independent of Epstein-Barr Virus (EBV) infection (Gaidano et al., 1994). In almost all cases, the BCL-6 rearranged tumors have a B cell phenotype. These data suggest that BCL-6 rearrangement is a frequent and specific molecular marker for DLCL. As illustrated in Figure 17.1A, the BCL-6 gene has 10 exons spanning 26 kb. Two transcription initiation sites were mapped to the noncoding exon 1, which is separated by a 10 kb

intron from the 38 bp noncoding exon 2. The protein-coding region of BCL-6 starts in exon 3 and ends in exon 10. The 3q27 breakpoints within the BCL-6 locus spread over a 10 kb region (MBR, major breakpoint region) spanning exon 1, with the majority of the breaks clustering in the 5 portion of the first intron. Since only the 5 regulatory sequences, which include the promoters in many cases, are removed from the gene and all the coding exons are left intact, one would predict that the functional consequence of BCL-6 translocations would be transcriptional deregulation as opposed to fusion protein production. Indeed, this was confirmed by molecular analyses of the BCL-6 transcripts and their protein product in various lymphomas with rearranged BCL-6 gene. In all cases analyzed, fusion transcripts were found that contained heterologous sequences fused to BCL-6 exons 2—10 while exon 1 sequence was missing, suggesting heterologous promoters were driving expression of BCL-6. For example, in the Ly8 cell line, the germ-line BCL-6 promoter is substituted by the I region promoter from Ig3 locus, which normally transcribes a germ-line transcript involved in Ig class switching. As the result, I3-BCL-6 fusion transcript is produced, which translates to a normal BCL-6 protein. In addition, no wildtype BCL-6 transcript is made in Ly8, indicating the germ-line BCL-6 allele is trancriptionally inactive (Ye et al., 1995). These data demonstrate that the cellular differentiation state of these lymphoma B cells is nonpermissive for BCL-6 expression and that translocation overrides this restriction by the mechanism of promoter substitution (Chen et al., 1998; Ye et al., 1995). Promoters that are juxtaposed to the BCL-6 locus by 3q27 translocations are not always B-cell specific. Both Ig heavy (Deweindt et al., 1993; Ye et al., 1993b) and light chain promoters (Miki et al., 1994b; Suzuki et al., 1994) can be used, as well as the promoter from a B-cell—specific coactivator BOB1/OCA-B (Galieque-Zouitina et al., 1996). In other instances, promoters from ubiquitously expressed genes are involved. They include histone H4 (Akasaka et al., 1997), small G protein TTF (Dallery et al., 1995), heat shock protein 89 (Xu et al., 2000), and L-plastin (GaliegueZouitina et al., 1999). It seems that in order to deregulate BCL-6, the coding region of the gene can be fused to many different kinds of promo-

BCL-6 MUTATION IN NORMAL AND NHL B CELLS

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Figure 17.1. A: Location of the major breakpoint cluster region and hypermutation region within the BCL-6 locus. Filled and empty boxes represent coding and noncoding exons, respectively. For viewing purposes, exons are drawn larger than introns. Two arrows above exon 1 indicate two transcription initiation sites. The 3 border for hypermutation region is not defined. Scale bar is for intron sequences only. B: Model illustrating promoter substitution in BCL-6 translocations. An active promoter of gene x from the heterologous locus is juxtaposed upstream of BCL-6 coding exons and replaces the wild-type BCL-6 promoter. As a result, a 5 fusion transcript is produced that retains the full coding capacity for a wild-type BCL-6 protein.

ters provided that these promoters are active in the tumor B cells. It is worth pointing out here that BCL-6 rearrangement and 3q27 alteration do not always agree with each other. The incidence of BCL-6 rearrangement in DLCL, as detected by Southern Blotting, greatly exceeds that of 3q27 aberration (8—12%) based on cytogenetic analysis (Bastard et al., 1992; Offit et al., 1989). It appears that a significant number of the BCL-6 alterations may have been overlooked by cytogenetic analysis possibly due to their submicroscopic nature and the fact that chromosomal aberrations involving telomeric region, such as 3q27, are more difficult to recognize with traditional cytogenetic methods. On the other hand, 30% of cases that carry 3q27 abnormalities do not show BCL-6 break within the MBR (Bastard et al., 1994; Michaud et al., 1997; Ye et al.,

1993a). Recent study suggests that there exists an alternative breakpoint region (ABR) 200— 270 kb telomeric and 5 to the BCL-6 gene (Butler et al., 1997b; Chen et al., 1998). In these cases, either BCL-6 could be affected remotely by structural alteration in ABR or there is a second B-cell lymphoma gene at 3q27. To address this issue, the vicinity of ABR should be searched for existence of new genes and the effect of ABR translocations on BCL-6 expression should be studied.

BCL-6 MUTATIONS IN NORMAL AND NHL B CELLS The coding region of BCL-6 is rarely mutated in lymphomas (Migliazza et al., 1995; Otsuki et al., 1995); in contrast, its 5 noncoding region dis-

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Figure 17.2. Schematic representation of BCL-6 protein structure. The BCL-6 protein is a 92—98 kD nuclear phosphoprotein. Its N-terminal POZ motif is responsible for protein-protein interactions including homodimerization and binding to nuclear corepressors SMRT and N-CoR. Sequence-specific DNA recognition is mediated through its C-terminal domain composed of six regularly spaced C H -type zinc fingers. Sequence similarities between the BCL-6 target site and the Stat6 sites have been proposed to be the bases for hyper-Th2 phenotype in the BCL-6 KO mice, while other BCL-6 target genes are currently unknown. Transrepression function of BCL-6 requires both the POZ domain and a portion of the intervening sequences.

plays remarkable structural instability. In addition to translocations and small intragenic deletions (Bernardin et al., 1997; Nakamura et al., 1999), this region is also targeted by highfrequency somatic mutations detectable in GCderived tumors as well as normal B cells (Migliazza et al., 1995; Shen et al., 1998). Initial evidence for such mutation came as a surprise finding when the first intron from a lymphoma cell line Ly1 was sequenced. Ly1 was originally though to have a rearranged BCL-6 gene demonstrated by an abnormal restriction pattern in the MBR region, yet genomic cloning demonstrated that both of its BCL-6 alleles were structurally normal. As it turned out, the first intron of Ly1 carried multiple sequence alterations in both alleles, some of which created new restrictions sites. Subsequently, more primary tumor samples and cell lines were screened for mutations by PCR-SSCP and direct sequencing (Migliazza et al., 1995). Within the 3.5 kb region including exon 1 and the 5 portion of the first intron (Figure 17.1A), multiple, somatically derived alterations were found that included point mutations, small deletions, and occasionally insertion. These somatic mutations, detected at the frequency of 1.4; 10\ to 1.6;10\ per base pair, were biallelic

and arose independently of translocation, since they were found in both the germ-line and translocated BCL-6 alleles as well in B-NHL samples without 3q27 translocation. These 5 BCL-6 mutations occur in 70% of DLCL and 45% of FL, but not in carcinomas or glioblastomas (Migliazza et al., 1995). More interestingly, BCL-6 5 mutations can also occur in normal germinal center B cells and memory B cells with similar characteristics, although the frequency is about 5-fold lower (Pasqualucci et al., 1998; Shen et al., 1998). These results indicate that BCL-6 5 mutations reflect a GC-associated physiological process rather than a lymphomaspecific phenomenon. The frequency and characteristics of BCL-6 5 mutations are reminiscent of somatic hypermutation of the Ig genes. Normally, mutations are introduced into the variable region (IgV) when B cells are going through GC to achieve antibody affinity maturation. In humans, such IgV hypermutations occur at the rate of 2 to 8;10\ per base pair (Klein et al., 1998). Based on their frequency, association with the GC, and other characteristics, BCL-6 mutations are likely to be generated by the IgV hypermutation mechanism (Migliazza et al., 1995; Pasqualucci et al., 1998; Shen et al., 1998). Relatively little is

PATTERN OF BCL-6 EXPRESSION; MULTIPLE CELL TYPES AND MULTI-LEVEL REGULATION

known about the exact components of the IgV hypermutation mechanism and how it is regulated in GC, except that DNA mismatch repair machineries are involved (Wiesendanger et al., 1998). Nevertheless, somatic hypermutation itself was generally assumed to be an Ig-specific phenomenon; BCL-6 is the only exception known to date. It is possible that some structural features of the BCL-6 5 region, which may be related to its primary sequence but more likely to its higher-order chromatin structure, lead to its recognition by the hypermutation machinery as a legitimate substrate. Whatever the deciding feature is that distinguishes BCL-6 from other actively transcribed genes in GC, BCL-6 should provide a valuable gene to explore the nature of the cis-regulatory element(s) involved in IgV hypermutation, since BCL-6 does not share sequence similarity with the IgV genes in their mutated regions. At the first glance, the functional significance of the BCL-6 mutations with respect to gene expression and tumorigenesis was not evident, since mutations target the noncoding region of the gene, and unlike the case of p53 and BRCA1, there do not appear to be any mutational ‘‘hot spots’’ in BCL-6. However, when mutated BCL6 alleles from B-NHL tumors and normal GC cells were tested in gene expression reporter assays, significant overexpression (3—18 fold) was observed from 4/12 DLCL-derived alleles, such that three out of six DLCL cases carried at least one overexpressed allele. In contrast, none of the GC-derived mutated sequences scored any transcriptional advantage in this assay (Pasqualucci et al., 1999). It is therefore possible that a variety of somatic mutations are created in BCL-6 when B cells travel through GC; later on, a subset of these may be selected and enriched in the GC-derived lymphoma B cells based upon their ability to deregulate BCL-6 expression.

PATTERN OF BCL-6 EXPRESSION; MULTIPLE CELL TYPES AND MULTI-LEVEL REGULATION BCL-6 mRNA can be detected at variable levels in many tissues and organs (Allman et al., 1996; Bajalica-Lagercrantz et al., 1997, 1998; Dew-

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eindt et al., 1993). However, its protein expression spectrum is far more restricted. Immunohistochemical studies of various lymphoid organs demonstrated that within the B-cell lineage, BCL-6 protein is only detected within the GC of the secondary lymphoid organs, but not in pre- and immature B cells or more differentiated progenies, such as memory or plasma cells (Allman et al., 1996; Cattoretti et al., 1995; Onizuka et al., 1995). In the T-cell lineage, it is expressed by a subset of CD4> T cells within the GC as well as in cortical thymocytes (Allman et al., 1996; Cattoretti et al., 1995; Onizuka et al., 1995). In the monocytic lineage, BCL-6 protein can be detected in activated macrophages (Ye et al., unpublished). These results suggest that BCL-6 expression is tightly regulated during lymphoid differentiation and that its downregulation in post-GC B cells may be necessary for further plasma/memory cell differentiation (Figure 17.3). In nonlymphoid tissues, BCL-6 protein could be detected in proliferating chondrocytes, apical supporting cells in the olfactory epithelium, mature myocytes, differentiated keratinocytes, and hair follicle matrix cells (Albagli-Curiel et al., 1998; Bajalica-Lagercrantz et al., 1998; Kanazawa et al., 1997). Therefore, BCL-6 protein appears to be restricted to cells at specific stages of differentiation in particular tissue types. In addition, discordant expression of BCL-6 mRNA and protein also suggests that BCL-6 expression is regulated by posttranscriptional as well as transcriptional mechanisms, a feature common to genes whose function is crucial to cell proliferation/survival. Posttranscriptional regulation of BCL-6 is especially intriguing. For example, it was shown that roughly the same amount of BCL-6 mRNA is expressed in GC and resting B cells; yet the former produce 3—34 fold more protein (Allman et al., 1996), suggesting that upregulation of BCL-6 protein in GC B cells is largely posttranscriptional. There are many examples of translational regulation through binding of proteins to cis-acting RNA motifs in the untranslated regions (UTR) of mRNAs (Klausner and Harford, 1989). Since the 1.3 kb 3 UTR of human and mouse BCL-6 are very well conserved, it is highly likely that this region of the BCL-6 message carries signal(s) important for translational regulation.

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Figure 17.3. BCL-6 expression in B cells of various differentiation stages and B-cell lymphomas. In B lineage, BCL-6 expression is restricted to B cells within GC. Cells prior to GC stage — that is pro-, pre-, and immature B cells — are BCL-6 negative. B cells that have emerged through GC — that is, plasma and memory B cells — all shut off BCL-6 expression. Many GC-derived lymphomas, including FL, DLCL, and BL, on the other hand, have retained BCL-6 protein either due to the translocated BCL-6 gene or simply reflecting their past GC history.

BCL-6 PROTEIN AS A TRANSCRIPTIONAL REPRESSOR BCL-6 encodes a 95 kDa phosphoprotein with six Kru¨ppel-type C H zinc fingers (ZF), typic ally found in sequence-specific transcription factors, and an evolutionarily conserved BTB/POZ (broad complex tramtrack bric-a-brac/poxvirus and zinc finger) domain at the N-terminus (Fig. 17.2). The BTB/POZ domain is a region of :120 amino acids found in a family of zincfinger transcription factors, a restricted number of actin-binding and poxvirus proteins (Albagli et al., 1996). This motif provides a protein-protein interaction interface for homodimeric, heterodimeric, as well as transcriptional activation and repression (Bardwell and Treisman, 1994; Chang et al., 1996; Dhordain et al., 1995; Kaplan and Calame, 1997; Seyfert et al., 1996). Using an in vitro PCR-based site selection procedure, a high-affinity DNA-binding site for BCL-6 has been identified that includes an 11 bp AT-rich sequence (TTCCTAGAAAG) for

binding specificity and some preferred peripheral sequences for binding affinity (Chang et al., 1996; Deweindt et al., 1995; Seyfert et al., 1996). It was also shown that BCL-6 has an autonomous transcription repression activity that requires the N-terminal POZ domain and a second repression domain localized in the middle portion of the molecule N-terminal to the zinc-finger motifs (Chang et al., 1996). The transcriptional repressor activity of BCL-6 is explained by the ability of its POZ domain to interact with corepressor molecules such as SMRT (silencing mediator of retinoid and thyroid receptor), N-CoR (nuclear corepressor of retinoid receptor) (Dhordain et al., 1997; Huynh and Bardwell, 1998), as well as with histone deacetylase 1 (HDAC1) (David et al., 1998). Therefore, in the current model, BCL-6 and other POZ-containing molecules, for example, PLZF, repress transcription by recruiting to the target promoter a HDAC through the corepressor/mSin3/HDAC complexes (David et al., 1998). This mechanism was previously dem-

REGULATORS AND EFFECTORS OF BCL-6 FUNCTION

onstrated for transcriptional repression by nuclear coreceptors and Myc-associated repressor (Mad). Currently, not much is known about the direct transcriptional target of BCL-6, except for the germ-line transcript involved in IgE class switching (see discussion in the following section). It is also not clear whether transcriptional repression as the result of direct DNA binding and recruitment of corepressor complexes is the only mechanism through which BCL-6 can regulate gene expression. For example, other POZ-containing proteins were implicated in chromatin remodeling and/or mediating longrange interactions between cis-regulatory elements (Katsani et al., 1999; Kim et al., 1999; Yoshida et al., 1999a).

REGULATORS AND EFFECTORS OF BCL-6 FUNCTION; UPSTREAM SIGNALS, TARGET GENES, AND INTERACTING PROTEINS As BCL-6 plays important roles in both normal and malignant B-cell development, mechanisms that regulate its expression or modulate its activity are important to its function. In nonlymphoid cell types, it was demonstrated that BCL6 is upregulated when undifferentiated myocytes and keratinocytes undergo terminal differentiation (Kumagai et al., 1999; Yoshida et al., 1996), although the specific signaling events/ molecules involved were not defined. For B cells, various stimuli involved in GC formation have been used to upregulate BCL-6 expression in naive B cells without success. Nevertheless, some of the signals capable of downregulating its expression have been defined. It is well established that activation of the CD40 pathway initiates signaling events essential for B-cell activation and proliferation (Lederman et al., 1996). Cross-linking of CD40 on Ramos cells, which are often used as GC model B cells, leads to rapid BCL-6 downregulation at the mRNA level (Allman et al., 1996; Cattoretti et al., 1997). Since EBV viral protein LMP1 resembles an activated CD40 molecule and uses the same downstream signaling pathway as CD40, it is not surprising that LMP1 can downregulate BCL-6 as well (Cattoretti et al., 1997), and that in group III Burkitt’s lymphoma cell lines where

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LMP1 is expressed, BCL-6 expression is extinguished. Another well-documented signaling pathway essential for differentiation of GC B cells is triggered through the surface antigen receptor (Mayumi et al., 1995). Anti-IgM treatment activates Ras-MAP kinase pathway in Ramos cells, resulting in BCL-6 phosphorylation and subsequent degradation of BCL-6 protein by the ubiquitin/proteasome-mediated processes (Niu et al., 1998). More studies are still needed to unravel the upstream signaling event(s) that upand downregulates BCL-6 in the GC. It is conceivable that BCL-6 may integrate in the nucleus various signals presented to B-cells in the GC environment and may be involved in the regulation of B-cell fate and differentiation. In normal GC B cells, BCL-6 may serve as a gatekeeper to prevent B cells from apoptosis and/or from differentiating into memory or plasma cells until a combination of proper signals is delivered. Another signaling molecule important for Bcell activation and growth is IL-4, which does not have a detectable influence on BCL-6 expression (Allman et al., 1996). Nevertheless, BCL-6 could affect events downstream of IL-4. This cross-talk was first suggested by the phenotype of BCL-6 knockout (KO) mice. Mice with defective BCL-6 function generated through homologous recombination in embryonic stem (ES) cells develop two major phenotypes. One is the lack of GC structures and impaired GC functions; the other is a severe hyper-Th2 inflammatory disease featuring over production of Th2 cytokines and abnormal Ig class switching to IgE and IgG1 (Dent et al., 1997; Ye et al., 1997). IL-4 is the key cytokine regulating these immune responses, and the DNA-binding site of BCL-6 shares homology with that of the IL-4responsive STAT (signal transducer and activator of transcription), Stat6. Therefore, it was proposed that BCL-6 could act as a negative regulator of IL-4 signaling by binding to a subset of Stat6-activated genes. Indeed, such a scenario was confirmed in a later study of the germ-line transcript I expression (Harris et al., 1999) . It was found that the BCL-6 recognizes a Stat6 binding site in I promoter and, by competitively binding to this site, inhibits IL-4— induced IgE class switching. Therefore, I qualifies as an authentic BCL-6 target gene.

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Remarkably, no other target genes have been reported for BCL-6 so far, although based on the KO phenotype, additional genes should exist that are directly involved in Th1/Th2 lineage differentiation. A recent study describes modulation of IL-4/ Stat6 signaling by interferon regulatory factor 4 (IRF-4, also known as Pip, LSIRF, or MUM1) and BCL-6 (Gupta et al., 1999). Assembly of three transcription factors, Stat6, IRF-4, and BCL-6, in the CD23b (low-affinity IgE receptor) regulatory region was studied. Both Stat6 and BCL-6 could recognize a single Stat6 site within this promoter, and both of them could interact directly with IRF-4, which binds to a nearby site, in vivo. Stat6 is activated by IL-4, BCL-6 is downregulated by CD40L, and IRF-4 can be upregulated by either IL-4 or CD40L alone and is superactivated when both signals are delivered. Therefore, it seems that IRF-4 serves as an anchor, which, depending on the nature of the signals delivered to the cell, recruits either Stat6 or BCL-6, leading to activation or suppression of the CD23b promoter. However, the importance of BCL-6 in regulating CD23b gene expression is not clear, since a study by Harris and colleagues using a larger segment of the CD23b regulatory region failed to detect any repression effect by BCL-6 (Harris et al., 1999). There are many examples where the activity of a transcription factor can be regulated by interacting protein partners. In addition to IRF4, two POZ domain—containing transcription factors have been described to be capable of interacting with BCL-6 in vivo. The zinc-finger transcription factor LRF (leukemia/lymphomarelated factor) was cloned based upon sequence homology with the POZ domain of PLZF (Davies et al., 1999). In vivo association between BCL-6 and LRF requires the POZ as well as the ZF domains from both proteins. It is not known whether LRF can recognize the DNA target sequence of BCL-6 or if it is a transcription repressor or activator. Another POZ-ZF protein, BAZF, was isolated based upon sequence homology with the ZF domain of BCL-6 (Okabe et al., 1998). The POZ and ZF domains as well as a short stretch of amino acid in the middle of the proteins are highly homologous. As a result, BAZF can readily heterodimerize with BCL-6 through the POZ domain and acts as a transcription repressor capable of binding

to the DNA target sequence of BCL-6. Since high-level BAZF expression is only found in heart and lung, and it is a transcription repressor as well, it remains to be determined whether BAZF has an important role in BCL-6—expressing lymphocytes, for example, the GC B cells. It seems that for both LRF and BAZF, it is important to establish their own biological roles and their influence on BCL-6 function within a physiologically relevant context.

THE ROLE OF BCL-6 IN NORMAL GERMINAL CENTER B CELLS AND B-CELL NHL The GC is a dynamic structure where T-dependent B-cell activation, Ig class switching, somatic hypermutation, and affinity maturation take place (Kelsoe, 1996; Liu et al., 1997). The decision for a particular B cell to either remain in the cell cycle, undergo apoptosis, or proceed to terminal differentiation to become plasma or memory B cells is the result of timing and integration of various signals. These include receptor-ligand interactions between B, T, and follicular dendritic cells as well as the local cytokine environment. As BCL-6 expression is specifically turned on when B cells enter the GC and then abruptly turned off when they exit the GC, upregulation of BCL-6 might be important for GC function and its downregulation might be necessary for post-GC differentiation. To study the function of BCL-6 in vivo, BCL-6 KO mice were created by homologous recombination. In these animals, B- and T-cell development was normal, yet these mice failed to develop GC and mounted a reduced antigenspecific antibody response with no evidence of Ig affinity maturation (Dent et al., 1997; Ye et al., 1997). This defect was attributed to the inability of the BCL-6 null B cells to proliferate and to participate in GC formation (Fukuda et al., 1997; Ye et al., 1997), suggesting that BCL-6 is required for GC progenitor B cells to either acquire or sustain a GC phenotype. On the other hand, since BCL-6 KO mice lack the GC, structure, and the proper cytokine/mitogen stimuli capable of inducing BCL-6 expression in resting B cells has not been identified, the exact function of BCL-6 during GC reactions is poorly understood.

THE ROLE OF BCL-6 IN T-CELL DIFFERENTIATION AND FUNCTION

An antiapoptotic role for BCL-6 was suggested by a recent study. As described above, surface IgM cross-linking eventually leads to apoptosis in the Ramos line. Shortly after antiIgM treatment, BCL-6 protein is degraded as the result of MAP kinase activation and phosphorylation. As BCL-6 degradation precedes the onset of apoptosis, and a phosphorylationdefective and thus ‘‘long-lived’’ BCL-6 mutant could delay and partially protect Ramos cells from anti-IgM—induced apoptosis, disappearance of BCL-6 might be an important intermediate event before final execution of the apoptosis program (Niu et al., 1999). While this result implies that the role of BCL-6 in normal GC B cells is, at least in part, to prevent apoptosis, one should be cautious to note that B cells experience different phases of activation and face different selection pressure within various microenvironments of GC (Liu et al., 1997). It is therefore conceivable that BCL-6 may play somewhat different roles in different subsets of GC B cells and that control of an apoptosis checkpoint may be just one of them. In B-cell NHL, ample genetic evidence points to the importance of BCL-6, yet its precise function remains poorly understood. Despite the fact that BCL-6 translocations involve many different partner chromosomal loci, in all cases analyzed, an active, heterologous promoter is fused upstream and in the same transcription orientation to the coding exons of BCL-6, resulting in fusion transcript encoding a wild-type BCL-6 protein, while the germ-line allele remains silent (Chen et al., 1998; Ye et al., 1995). These observations indicate that deregulated BCL-6 expression is specifically selected for during the clonal evolution and transformation process. As BCL-6 expression is so tightly restricted to the GC in normal B cells, it is not surprising that introduction of BCL-6 as a transgene into BCL-6 negative, activated B cells generally leads to cytotoxicity. Transgenic mouse studies based on traditional design also experienced technical hurdles presumably due to the same reason (Ye and Dalla-Favera, unpublished). Clearly, to directly demonstrate and elucidate the transforming activity of BCL-6, a GC-like experimental system has to be used, whether it is tissue culture—based or a transgenic animal model. On the other hand, as lymphomas with BCL-6 translocations produce

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the same wild-type protein, the oncogenic activity of BCL-6 should be related to its normal function in GC B cells, for example, hyperproliferation, resistance to certain apoptosis stimuli, or delayed differentiation.

THE ROLE OF BCL-6 IN T-CELL DIFFERENTIATION AND FUNCTION In the T lineage, BCL-6 protein is expressed by a subset of CD4> T cells within GC as well as in cortical thymocytes (Allman et al., 1996; Cattoretti et al., 1995; Onizuka et al., 1995). Mice deficient in BCL-6 function die at an early age of severe inflammatory diseases affecting multiple organ systems including the heart and lungs. This systemic disease is characterized by hyperproduction of Th2 cytokines IL-4, IL-5, and IL-13 in affected tissue sites accompanied by heavy infiltration of eosinophils and IgEbearing B cells, indicative of a Th2-biased inflammatory response (Dent et al., 1997; Fukuda et al., 1997; Ye et al., 1997). As IL-4 and its signal transducer Stat6 play an essential role in Th2 differentiation, and Stat6 and BCL-6 recognize similar DNA target sites, it was first hypothesized that BCL-6 could negatively regulate the Th2 pathway by binding to and suppressing a subset of Stat6 target genes. This attractive theory, unfortunately, was not supported by analysis of the IL-4\\BCL-6\\ and Stat6\\ BCL-6\\ mice (Dent et al., 1998), as these mice developed the same Th2-type inflammation of the heart and lungs and even the Th2 cytokine response typically seen in the BCL-6\\ mice, despite the fact that in vitro differentiation of Th2 cells from these animals was abolished as expected. This study suggests that BCL-6 controls Th2 responses in vivo through a pathway(s) independent of IL-4 and Stat6. A model system to study the development of Th cells during the course of a natural immune response is based on susceptibility to infection by Leishmania major. A prevailing Th1 response leads to healing while Th2 biased response results in progressive infection (Reiner and Locksley, 1995). On a resistant genetic background (C57BL/6;129 intercross), BCL-6\\ and IL4\\BCL-6\\ mice were highly susceptible to infection, as one would expect from their Th2

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bias. In contrast, the Stat6\\BCL-6\\ mice were resistant in this assay (Dent et al., 1999). Stat6 can act downstream of IL-4 as well as IL-13 (Takeda et al., 1996), and IL-13 has been suggested to play important roles in Th2 differentiation and inhibition of macrophage activation in L eishmania infection (Doherty et al., 1993; McKenzie et al., 1998). Therefore, Dent and co-workers suggested that IL-13 signaling through Stat6 may contribute to an exacerbated infection by Leishmania. One area that has not been thoroughly explored in these previous studies is the primary target cell type involved in the hyper-Th2 response in BCL-6 KO mice. As B-cell functions are severely compromised in these mice, most likely B cells are not involved. It is possible that BCL-6\\ T cells themselves have an intrinsic bias toward Th2. Alternatively, signals delivered to uncommitted CD4> T cells from other cell types are skewed toward Th2 in the absence of BCL-6. The transcriptional targets of BCL-6 that are directly involved in Th1/Th2 differentiation remain to be discovered.

ROLE OF BCL-6 IN NONLYMPHOID CELL TYPES Studies of human keratinocyte differentiation and mouse skeletal myogenesis revealed that BCL-6 protein can be induced when these cells acquire a more differentiated phenotype (Kumagai et al., 1999; Yoshida et al., 1996). It is not clear from these experiments whether upregulation of BCL-6 is absolutely required for the differentiation process, since there is no apparent developmental defects in either the skin or skeletal muscle in the BCL-6 KO mice. Still, it is possible that, analogous to the GCspecific phenotype of BCL-6 KO in the B lineage, the role of BCL-6 in these nonlymphoid tissues might be related to a specific cellular function of the differentiated cell types rather than their development. In line with this notion, BCL-6 was recently reported to play a role in protecting mature cardiac myocytes from eosinophilic inflammation commonly seen in BCL-6 KO mice (Yoshida et al., 1999b). Both differentiated myocytes and keratinocytes have terminally withdrawn from the cell cycle. However, BCL-6 protein can also be found in proliferating cells (GC B and T cells, chondrocytes,

and hair follicle matrix cells). Thus, the function of BCL-6 does not seem to be directly linked to proliferation or differentiation per se. BCL-6 might actively participate in a more flexible manner in the proliferation or differentiation process depending on the developmental/differentiation programs preexisting at the time when BCL-6 expression is activated.

BCL-6 PROTEIN EXPRESSION AS A HISTOGENETIC MARKER Most neoplastic cells of FLs, DLCLs, and BLs express the BCL-6 protein irrespective of the translocation status of the gene (Cattoretti et al., 1995; Onizuka et al., 1995; Otsuki et al., 1995). This could be an ‘‘activation and retention’’ phenomenon that simply reflects the past GC history of these tumors. Thus, protein detection is not informative of the BCL-6 genotype nor is it useful for differential diagnosis of these subtypes. Among low-grade B-cell lymphomas, however, immunostaining for BCL-6 proved useful for differentiating proliferation centers (BCL-2>/BCL-6\) from trapped germinal centers (BCL-2\/BCL-6>) in mantle cell lymphoma (MCL) (Falini et al., 1997). For MALT lymphomas, one group reported that while none of the 17 low-grade cases studied expressed the BCL-6 protein, all of the 8 high-grade cases examined showed p53 and/or BCL-6 overexpression (Ohshima et al., 1997; Yoshino and Akagi, 1998). This observation suggests that BCL-6 protein is not only a specific histogenetic marker for high-grade MALT but it also plays a functional role in the high-grade transformation process of MALT. It remains to be determined whether this interesting result will be confirmed by large-scale studies. BCL-6 protein also proved to be a useful marker in acquired immunodeficiency syndrome-related NHL (AIDS—NHL). Approximately 80% of these tumors arise systemically, while most of the remaining 20% arise in the central nervous system (PCNSL), with a small portion derived from the body cavity (primary effusion lymphoma or PEL). Only the third disease identity is known to be associated with infection of Kaposi’s sarcoma—associated herpesvirus (KSHV) (Knowles, 1997). The systemic type is classified into AIDS-related small

CLINICAL RELEVANCE OF BCL-6 TRANSLOCATIONS

noncleaved cell lymphoma (AIDS-SNCCL) and AIDS-related diffuse large-cell lymphoma (AIDS-DLCL). AIDS-DLCL, 20% of which carry BCL-6 translocations, may be further subdivided into large noncleaved cell (AIDSLNCCL) and large-cell immunoblastic (AIDSIBLP) subtypes. Based upon staining for BCL-6 as a marker for the GC B cell, and CD138/syndecan-1 as a marker for post-GC plasma cells, two distinct phenotypic categories were recognized (Table 17.2). The BCL-6>/syndecan\ group contained AIDS-SNCCL and AIDS-LNCCL while the opposite pattern was observed for AIDS-IBLP and AIDS-PEL (Carbone et al., 1998a), possibly reflecting different phenotypic origins of these lymphomas with respect to the GC. In PCNSL, BCL-6 staining was consistently detected in tumors with large noncleaved cells, whereas in the immunoblastic presentation, the staining was mutually exclusive with the expression of EBV protein LMP-1 and, in most cases, BCL-2 (Larocca et al., 1998). In addition to apparent pathological implications, these findings also provide a useful tool for differential diagnosis of PCNSL subgroups, which tends to be difficult and subtle based upon histology alone. In addition to B-cell lymphomas, BCL-6 protein was also detected in two related lymphoid diseases. Hodgkin’s disease (HD) is characterized histologically by a minority of true neoplastic cells in a background of benign inflammatory cells. After many years of debate, lineage origin of most of these neoplastic cells has recently been attributed to GC B cells (Gruss et al., 1997). Based upon characteristics of the neoplastic and reactive background cells, HD is divided into two major categories. Nodular lymphocyte predominance HD (NLPHD) generally follows an indolent course, whereas classic HD (CHD) is fatal without treatment (Gruss et al., 1997; Harris et al., 1994). It was reported that while tumor cells of NLPHD consistently display the BCL-6>/syndecan\ phenotype, CHD tumor cells either have the opposite staining pattern or are a mixture of the two in the same case (Carbone et al., 1998b). These results suggest that the GC phenotype is preserved by NLPHD tumor cells, in comparison to CHD, where the phenotype of the tumor cells can be modulated by the surrounding cellular background, especially CD40L> T cells, known to

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downregulate BCL-6 (Cattoretti et al., 1997). In peripheral T-cell neoplasms, which also comprise a number of subtypes (Harris et al., 1994), BCL-6 protein expression was found to be restricted to the CD30> anaplastic large-cell category (in 45% of the cases) irrespective of their antigenic phenotypes (T-cell or null-cell type) (Carbone et al., 1997). Since these lymphomas rarely display BCL-6 translocation, expression of the BCL-6 protein could simply reflect a BCL-6—positive history of the premalignant clones, which were presumed to be extrafollicular CD30> cells. Alternatively, BCL6 could be activated as part of the transformation process.

CLINICAL RELEVANCE OF BCL-6 TRANSLOCATIONS Molecular characterization of BCL-6 rearrangement and demonstration of its frequent involvement in aggressive B-cell lymphomas has generated considerable interest in exploring its diagnostic as well as prognostic value. Though it is predominantly detected in DLCL, the value of BCL-6 translocation as a disease-specific diagnostic marker is reduced by the fact that other subtypes do carry it at low frequencies. Still, persistence of tumor cells with BCL-6 translocation after treatment could be used as a marker to monitor minimum residual disease. Due to the increasing incidence of DLCL and the potential efficacy of high-dose chemotherapy regimens, identification of both favorable and unfavorable indices would be helpful in patient management. Sine the mid-1990s, at least five groups from the United States, Japan, France, and Italy have conducted retrospective analyses on the prognostic value of BCL-6 rearrangement (Bastard et al., 1994; Muramatsu et al., 1996, 1997; Offit et al., 1994, 1995; Pescarmona et al., 1997; Vitolo et al., 1998). The overall conclusions from these studies are controversial. Though the initial report strongly suggested BCL-6 rearrangement by itself to be a predictor of improved overall survival and freedom from disease progression, other groups have not confirmed this result. A number of factors could underlie these apparent differences. Treatment variation has always been an issue in prognostic studies of DLCL. Epidemiological variability of

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the disease and selection biases may influence the pathologic or cytogenetic composition of particular series. For example, only patients with known cytogenetic status were entered in one study (Bastard et al., 1994); and in the reports that failed to observe prognostic advantage of BCL-6, there were proportionally less extranodal presentations in the BCL-6 rearranged cases compared to the other studies. Detailed analysis of other known clinical and genetic markers associated with DLCL may also be necessary to reassess all of the panels used in these studies. As reported by two groups, when BCL-6 rearranged cases were further divided according to the presence or absence of additional genetic lesions, significant prognostic advantages were noted for the cohort without additional genetic abnormalities (Muramatsu et al., 1996; Offit et al., 1995). It seems that definition of the impact of individual prognostic markers will require prospective collection of large series of samples and combined analysis of various known clinical and genetic markers in the setting of uniform patient management.

CONCLUSION AND FUTURE DIRECTIONS Increasing recognition of 3q27 aberrations as a frequent genetic lesion in DLCL led to the cloning of BCL-6, a gene encoding a DNAbinding transcription factor highly expressed in GC B cells. Molecular analysis revealed a predominant association of BCL-6 translocation with the DLCL subtype of B-cell NHL, although it is also rearranged at a low frequency in FL and MCL. Translocations break the BCL-6 gene in its 5 MBR, resulting in deregulated transcription and production of a wild-type BCL-6 protein in tumor cells. In a minority of tumor cases where 3q27 breaks more than 200 kb telomeric to BCL-6, existence of another lymphoma gene cannot be ruled out. Somatic hypermutation in the MBR is a frequent event in lymphoma B cells as well as in normal GC B cells. It is postulated that these mutations arise through an IgV hypermutationrelated mechanism, and a subset of them capable of deregulating BCL-6 expression may be selectively enriched in B-cell NHL. The BCL-6 protein functions as a sequence-

specific transcription repressor by recruiting corepressor-associated HDAC complexes to the vicinity of promoters containing its target site. As the BCL-6 target site shares sequence similarity with that of Stat6, BCL-6 can negatively regulate a subset of IL-4—inducible genes such as I. BCL-6 can also interact with other transcription factors, for example, IRF-4, LRF, and BAZF, which potentially could influence its capability to regulate gene expression. Expression of BCL-6 itself is tightly regulated during lymphoid development and differentiation. In B cells, high-level BCL-6 expression is restricted to the GC. Signals operational during GC reactions have been found to regulate BCL-6 expression at the transcriptional as well as posttranslational levels. Through ablation of BCL-6 function in the mouse germ-line, BCL-6 KO animals were produced in which GC formation as well as GCdependent antibody responses were defective. Inflammatory reactions characteristic of Th1/ Th2 imbalance were also observed, possibly due to an overactive IL-4—independent pathway in the absence of BCL-6 repression, suggesting an important role of BCL-6 in non-B cells. Future studies should focus on the function of BCL-6 in the normal GC reaction as well as transformation of GC B cells. The molecular and cellular targets involved in BCL-6—mediated Th1/Th2 differentiation represent another issue that needs to be addressed. Identification of physiological, transcriptional targets of BCL-6 would be an important approach in both directions. Appropriate experimental approaches are also necessary including the controlled expression of BCL-6 in proper tissue culture systems and the development of transgenic animal models in which exogenous BCL-6 is expressed in a fashion mimicking translocated BCL-6 in human lymphoma patients. The significance of BCL-6 rearrangement as a favorable prognostic marker is an unsettled issue. Whereas the initial report pointed to a very strong association between BCL-6 rearrangement and favorable clinical outcome, subsequent studies did not substantiate this observation. This controversy calls for future studies with large prospective panels of lymphoma samples and inclusion of other relevant clinical and genetic markers. On a positive note, consistent results were obtained indicating that BCL-6

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CHAPTER 18


THE ROLE OF OCTAMER FACTORS AND THEIR COACTIVATORS IN THE LYMPHOID SYSTEM ERIC BERTOLINO Department of Molecular Genetics and Cell Biology, Howard Hughes Medical Institute, The University of Chicago

RALPH TIEDT AND PATRICK MATTHIAS Friedrich Miescher Institute, Basel, Switzerland

HARINDER SINGH Department of Molecular Genetics and Cell Biology, Howard Hughes Medical Institute, The University of Chicago

INTRODUCTION Promoters and enhancers found in higher eukaryotes are generally complex in structure, consisting of multiple-sequence motifs that serve as binding sites for numerous transcription factors (Tjian and Maniatis, 1994). Often, a binding site is recognized by different members of a family of regulatory proteins with identical or overlapping DNA-binding specificities. For example, octamer sites are recognized by several members of the POU domain family of transcription factors such as Oct-1, Oct-2, Oct-3/4, or Oct-6 (Ryan and Rosenfeld, 1997), GC boxes by Sp1, Sp2, or Sp3 (Kingsley and Winoto, 1992), and the GATA motif by GATA-1, GATA-2, GATA-3, GATA-4, or GATA-6 (Weiss and Orkin, 1995).

When two or more members of a regulatory protein family occur in the same cell type, the question arises as to which family member controls transcription of given target genes in vivo. Understanding how related transcription factors execute specific or redundant gene regulatory functions is an important step toward the dissection of genetic hierarchies controlling the development and function of organs such as the lymphoid system. Octamer transcription factors and their cognate-binding sites in control regions of lymphoid-specific genes constitute a paradigm for addressing the above question. A variety of molecular genetic and biochemical approaches have been employed in the study of this system and have led to new insights into the


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development and functioning of the lymphoid system.

OCT FACTORS AND THEIR COACTIVATORS The Octamer Motif The octamer motif ATGCAAAT was originally discovered in the promoters of immunoglobulin (Ig) genes (Falkner and Zachau, 1984; Parslow et al., 1984). It is also found in some Ig gene enhancers (reviewed in Staudt and Lenardo, 1991) as well as in the control regions of other B-cell—specific genes such as B29 and CD20 (Hermanson et al., 1989; Thevenin et al., 1993). The requirement for this motif in the B-cell— specific activity of Ig promoters has been demonstrated using transient transfection assays, in vitro transcription reactions, as well as transgenic mice (Bergman et al., 1984; Dreyfus et al., 1987; Jenuwein and Grosschedl, 1991; LeBowitz et al., 1988; Mason et al., 1985; Scheidereit et al., 1987; Wirth et al., 1987). Interestingly, multimerized octamer sites generate strong B-cell— specific enhancers (Gerster et al., 1987). In addition, functionally important octamer elements participate in the inducible activity of genes encoding T-cell cytokines such as the interleukin (IL)-2 gene (Durand et al., 1988; Ullman et al., 1991). Strikingly, octamer motifs are also present — and functionally essential — in the promoters of ubiquitously expressed genes such as the histone H2B gene (LaBella et al., 1988; Sive and Roeder, 1986) and the U2 and U6 snRNA genes (Lobo et al., 1990). Therefore, this motif provides a paradigm for how an identical sequence element can confer ubiquitous or cellspecific transcriptional activity. Octamer-Binding Proteins Lymphoid cells express the two related transcription factors Oct-1 and Oct-2. Both factors were discovered by assaying for DNA-binding proteins, which recognize the Ig octamer motif, in nuclear extracts from B-cell lines (Singh et al., 1986; Staudt et al., 1986). The Oct-1 protein is expressed in a wide variety of cell types, whereas Oct-2 protein expression is restricted to cells of

the lymphoid and central nervous systems (Gerster et al., 1987; He et al., 1989; Staudt et al., 1988; Sturm et al., 1988). The two factors have virtually indistinguishable DNA binding-specifities but are products of distinct genes (Clerc et al., 1988; Muller et al., 1988; Scheidereit et al., 1988; Staudt et al., 1988; Sturm et al., 1988). Oct-1 and Oct-2, along with Pit-1 and Unc-86, are founding members of the POU family of transcription factors (Herr et al., 1988). These proteins recognize DNA by means of a 150—160 amino acid POU domain (Fig. 18. 1) that consists of a conserved N-terminal POU-specific segment and a C-terminal homeodomain (reviewed in Ryan and Rosenfeld, 1997). A flexible linker between these two subdomains enables POU proteins to embrace the DNA by making contacts on both faces of the double helix (Jacobson et al., 1997; Klemm et al., 1994). The Oct-1 and Oct-2 POU domains are highly related (:90% amino acid identity), which accounts for their nearly identical DNA-binding properties. In B cells, alternative splicing of Oct-2 RNA results in the expression of two types of isoforms, Oct-2A and Oct-2B, which have distinct C-terminal regions (Schreiber et al., 1988). Oct-1 and Oct-2 proteins bear transactivation domains in their N- and C-terminal moieties (Fig. 18. 1). Both proteins have an N-terminal glutamine-rich activation domain with apparently similar properties. The C-terminal portions of Oct-1 and Oct-2A contain divergent activation domains that impart different transactivation properties to each protein. Thus, only Oct-1 is able to activate a U2 snRNA promoter, whereas activation from remote positions in B cells is a unique property of Oct-2A (Annweiler et al., 1992; Tanaka et al., 1992). Oct-2B is more similar to Oct-1 in its C-terminal region and consequently resembles the latter in its activation potential (Fig. 18. 1) (Tanaka et al., 1992). It should be noted that the POU DNA-binding domain also has transcription activation potential in certain contexts (Mittal et al., 1996; Muller-Immergluck et al., 1990; Shah et al., 1997). Residues present in a small acidic N-terminal segment of the Oct-1/2 POU domains (Sturm et al., 1988) have been implicated in activating transcription from Pol—II or Pol III—dependent promoters (Mittal et al., 1996; Muller-Immergluck et al., 1990). The Oct-1 POU domain

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Figure 18.1. Domain structure of Oct factors and the coactivator OBF-1. B cells express the Oct factors Oct-1 and Oct-2, the latter as two forms arising from alternative splicing. The DNA-binding POU domain in the middle consists of two halfs (blue boxes): the N-terminal POU-specific domain (POU ) and the C-terminal homeodomain (POU ), connected by a flexible linker. The N-termini of Oct-1 and Oct-2 contain glutamine-rich activation & domains (Q). Serine/threonine (in case of Oct-2 also proline)—rich activation domains are found (P S/T) C-terminal of the POU domain. The very C-terminus of Oct-1 resembles that of Oct-2B (yellow boxes), whereas Oct-2A has a unique C-terminus (small red box) that has been implicated in the specific ability of Oct-2A to activate transcription from remote positions. The B-cell—specific coactivator OBF-1 consists of an N-terminal part responsible for ternary complex formation and a C-terminal domain (green box) with transactivation potential.

stimulates transcription from the Pol III—dependent U6 promoter by recruiting the snRNA basal factor SNAPc (Ford et al., 1998). The identical DNA-binding properties but differing expression patterns of Oct-1 and Oct-2 led to the initial concept that Oct-1, being ubiquitously expressed, regulates the expression of octamer-dependent housekeeping genes (Fig. 18. 2A), whereas Oct-2 regulates transcription of those B-cell—specific genes that are dependent on octamer sites, most notably Ig genes (Muller et al., 1988; Schaffner, 1989; Scheidereit et al., 1987). This proposal, however, was challenged by in vitro transcription analyses that showed Oct-1 and Oct-2 were equally efficient at stimulating transcription in nuclear extracts from B cells (LeBowitz et al., 1988; Pierani et al., 1990). Subsequently, somatic cell gene targeting of the Oct-2 locus identified a pathway of B-cell—specific gene activation that is octamer site dependent but Oct-2 independent. Strikingly, in this Oct-2 mutant B-cell line, the activity of octamerdependent Ig promoters was not reduced and expression of endogenous Ig genes was also unimpaired (Feldhaus et al., 1993). Importantly, Ig genes were also shown to be transcribed at wild-type levels in developing B cells from mice in which the Oct-2 gene had been disrupted (Corcoran and Karvelas, 1994; Corcoran et al., 1993; see below). Thus, Oct-2 is not essential for

the developmental activation or maintenance of Ig gene transcription in B lymphocytes.

Another Player: The Coactivator OCA-B/OBF-1/Bob-1 In addition to the genetic evidence mentioned above, biochemical experiments indicated the presence, in B cells but not in HeLa cells, of a non-DNA—binding cofactor important for transcription activation through the octamer site (Luo et al., 1992). The cDNA for this cofactor was independently cloned by genetic screens (Gstaiger et al., 1995; Strubin et al., 1995) or following biochemical purification (Luo and Roeder, 1995) and has been called OBF-1, Bob1, or OCA-B. This novel B-cell—specific protein, which is proline-rich and lacks significant homology to other proteins, was found to associate specifically with Oct-1 and Oct-2 in an octamer site— dependent manner and to thereby enhance their transcription activation potential in transfection (Strubin et al., 1995) or in vitro transcription assays (Luo and Roeder, 1995) (Fig. 18. 2B). OBF-1 has a modular structure that includes an N-terminal Oct-interaction domain and a Cterminal transcription activation domain (Fig. 18. 1) (Gstaiger et al., 1996). Mutagenesis of the

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Figure 18.2. Mode of activation by Oct factors in different promoter/enhancer contexts. A: Ubiquitously expressed Oct-1 drives transcription from promoters of housekeeping genes such as histone 2B and snRNAs. B: Promoters of Ig and other B-cell—specific genes can be activated by a complex of either Oct-1 or Oct-2 together with the B-cell coactivator OBF-1, which increases the potential of Oct factors several-fold. C: Transactivation from enhancer contexts (remote positions) is a unique function of Oct-2A and it is mediated by its C-terminus. A B-cell—restricted activity (unidentified cofactor X) has been implicated in this process.

Oct-1 POU domain indicated that OBF-1 makes contacts with both POU subdomains, as well as with the DNA, thereby acting as a molecular clamp (Babb et al., 1997; Sauter and Matthias, 1998) (Fig. 18. 3). Recently, the cocrystal structure of an OCA-B peptide, the Oct-1 POU domain, and the cognate DNA site has confirmed the initial model (Chasman et al., 1999). During the process of binding, the other-

wise largely unstructured OBF-1 acquires a higher level of secondary structure, which might enhance its transactivation properties (Chang et al., 1999). The discovery and properties of OBF1 suggested an explanation for the Oct-2—independent expression of Ig genes in B cells. According to this view, the ubiquitous transcription factor Oct-1 can promote B-cell—specific transcription by interacting with the cell

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Figure 18.3. Model of the interaction surface between the POU domain/DNA complex and OBF-1. The model presented here is based on the study by Sauter and Matthias, 1998 and uses the coordinates of the Oct-1 POU domain/octamer motif cocrystal from Klemm et al., 1994. The modeling program Insight II was used to generate this picture. The two projections differ by a 90° rotation along the vertical axis. The octamer sequence DNA ATGCAAT is displayed in magenta. Backbone positions enhancing or eliminating association with OBF-1 when modified with a phosphothioate group are represented by green (p4, p5, p7, p8, and p9) or red balls (p6), respectively. Blue balls represent the backbone position p3 that was found not to influence interaction with OBF-1. The position of the methyl group on G ;3 that interferes with OBF-1 binding is indicated by a red ball to which an arrow points. The two central base pairs at positions ;5 and ;6 important for OBF-1 binding are represented with thicker lines. The POU domain is displayed in white. Residues whose substitution by alanine eliminated interaction with OBF-1 are indicated in red, and residues whose substitution by alanine did not interfere with OBF-1 association are depicted in blue. The POU linker that links both domains together is not visible in the crystal, but should be imagined to pass on the left of the DNA in projection (A) or in the back of projection (B).

type—restricted cofactor OBF-1 (Fig. 18. 2B). The Oct/OBF-1 system suggests a general mechanism by which the transcriptional activity of a ubiquitous regulator can be modulated through its interaction with a cell type—specific coactivator.

OCT FACTORS DURING PRIMARY B-CELL DEVELOPMENT The antigen-independent phase of B-cell development can be divided into three sequential stages: pro-B, pre-B, and immature B (Rolink and Melchers, 1991). In adult mice, these cells are generated in the bone marrow, where selfrenewing progenitors reside. Immature B cells can undergo positive as well as negative selection and then exit the bone marrow and home to

secondary lymphoid organs, primarily the spleen and lymph nodes, where they further differentiate to become mature B cells (Melchers et al., 1995). Developmentally regulated patterns of gene expression and DNA rearrangements are required for differentiation of pro-B cells into immature B cells. Therefore, the various stages of B-cell development are characterized by the rearrangement status of Ig genes as well as the expression of distinct combinations of intracellular or cell surface markers (Fig. 18. 4A).

Role of Oct Factors and OBF-1 During Early B-cell Development Gene targeting of the Oct-2 locus in mice has shown that this factor is not required for the antigen-independent phase of B-cell development

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Figure 18.4. Schemes for B-cell development and activation. A: Early B-cell development takes place in the bone marrow. A definitive role for Oct factors has not been demonstrated during this phase, but there is some evidence for a role of OBF-1 in the transition of immature B cells (IgM>, p130—140>) from the bone marrow to peripheral lymphoid organs. p130—140, which is recognized by a monoclonal antibody named 493, serves as a marker to distinguish immature (p130—140>) from mature (p130—140\) B cells (Rolink et al., 1998). B: B cells can be induced to proliferate and secrete antibody by bacterial LPS without the help of T cells. Cells from Oct2UU mice are impaired in this proliferative and differentiation response. C: The T-cell—dependent response occurs in germinal centers where B cells compete for antigen presented by follicular dendritic cells. The BCR is hypermutated, and the B cells with the highest affinity for antigen proliferate and advance to the plasma cell or memory B-cell state. Help from T cells is provided in the form of costimulatory signals such as CD40L and IL-4. Both OBF-1 and Oct-2 are involved in germinal center formation as indicated by deficiencies observed in the respective knockout mice.

OCT FACTORS DURING PRIMARY B-CELL DEVELOPMENT

but is involved in B-cell maturation (Corcoran and Karvelas, 1994; Corcoran et al., 1993). Oct2 executes its most critical function during B-cell activation and terminal differentiation into plasma cells (see below). Even though Oct-2 knockout mice die within a few hours of birth — for still unknown reasons — development of the B-cell compartment, which takes place in the fetal liver prior to birth, is largely unaffected (Corcoran et al., 1993). Newborn Oct-2UU mice contain normal numbers of pro- and pre-B cells but are somewhat deficient (50%) in immature B cells. However, Ig genes are properly rearranged and expressed. Reconstitution of the lymphoid compartment of SCID (lymphocyte-deficient) mice with fetal liver cells from Oct-2UU animals showed that Oct-2 is not required for normal Ig production by mature resting B cells. By contrast, defects were found in the later antigen-dependent phase of B-cell differentiation. In particular, Oct-2 mutant B cells were found to respond poorly to stimulation by bacterial lipopolysaccharide (LPS), apparently due to defective proliferation caused by a block in the G1 phase of the cell cycle (Corcoran and Karvelas, 1994) (Fig. 18. 4B). Unexpected results were also obtained from mice in which the OBF-1 gene had been disrupted. As was the case for the Oct-2 mutation, knockout of the OBF-1 gene did not result in a block to early B-cell development (Kim et al., 1996; Nielsen et al., 1996; Schubart et al., 1996a). During the antigen-independent phase, Ig gene rearrangement and expression were also not impaired. Thus, although OBF-1 protein is present at detectable levels in developing pro-B /pre-B cells, which express the marker B220 but are still negative for IgM expression, it does not appear to execute an essential function at these stages. While serum IgM levels were normal in OBF1 UU mice, the levels of secondary Ig isotypes were drastically lowered. Most strikingly, as detailed below, these mice have a dramatically impaired immune response. Recently, a novel function for OBF-1 has been delineated during B-cell maturation (Schubart et al., 2000): in OBF-1 deficient mice there is a reduction in the number of splenic B cells positive for the monoclonal antibody 493, which labels all stages of early B-cell development, including immature B

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cells. In a wild-type mouse, these 493 positive splenic B cells represent the population that recently entered the peripheral cell pool (Rolink et al., 1998). This finding suggests that OBF-1 might play an important role at the transition from the bone marrow to the spleen (Fig. 18. 4A). Moreover, when the OBF-1 mutation is combined with a mutation in the gene encoding Bruton’s tyrosine kinase (Xid), the defect is exacerbated and the double mutant animals virtually lack peripheral B cells (Schubart et al., 2000). On the basis that neither the loss of Oct-2 nor that of OBF-1 compromises Ig gene transcription during B-cell development, it was suggested that the two factors may be functionally redundant for activation of Ig genes (see Matthias, 1998). However, recent transplantation experiments with fetal liver cells from Oct-2/ OBF-1 double knockout mice have revealed that both factors are dispensable for Ig gene expression during the primary phase of B-cell development (Schubart, Massa, Rolink, and Matthias, manuscript in preparation). Thus, Oct-2 and OBF-1 are not likely to be functionally redundant during early B-cell development. Taken together, these findings argue for a predominant role of Oct-1 in primary B cells, perhaps in cooperation with an as yet unidentified cofactor. Consistent with this view, Oct-1 is expressed at significantly higher levels than Oct-2 in developing B cells (Klug and Singh, unpublished data; Miller et al. , 1991). The relative potency of Oct-1 and Oct-2 to regulate Ig gene transcription in B cells has been assessed using an equivalent pair of altered DNA-binding specificity mutant proteins (Shah et al., 1997). These experiments have revealed that Oct-1 is a more potent activator than Oct-2 when stimulating transcription from Ig promoters in conjunction with their cognate enhancers. Thus, the ubiquitous factor Oct-1 rather than the lymphoid-specific factors Oct-2 and OBF-1 is likely to regulate Ig gene transcription during early B-cell development.

Role of Oct Factors in Germ-Line V Gene Transcription and Rearrangement The rearrangement of Ig heavy- and light-chain genes is temporally regulated during B-cell de-

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velopment (Tonegawa, 1983). Ig variable (V) and constant (C) region gene segments are transcribed prior to undergoing DNA rearrangements, which lead to the assembly of complete Ig genes (reviewed in Sleckman et al., 1996). Transcripts emanating from germ-line — that is unrearranged — gene segments are not generally translated into protein and are therefore called sterile transcripts. The linkage between transcription and rearrangement of Ig gene segments has led to the formulation of the accessibility hypothesis (Alt et al. 1986), which suggests that elements controlling transcription are also regulating accessibility of Ig loci to the recombination machinery. The mechanism by which transcriptional regulatory sequences control recombination remains to be elucidated. An attractive possibility is that transcription factors upon binding to their cognate sites in promoters or enhancers recruit chromatin remodeling complexes and histone acetylases. These factors in turn alter chromatin structure, thereby making the DNA template available for rearrangement as well as transcription. As noted earlier, the octamer element is a conserved and essential transcriptional motif present in the promoters of Ig variable gene segments. Therefore, this element may also regulate V gene rearrangement. Given that V gene rearrangement can occur in the absence of Oct-2 or OBF-1, it is possible that Oct-1 is involved in regulating V gene transcription and rearrangement during B-cell development. In this regard, we note that the related POU domain transcription factor Pit-1 has been shown to interact with the histone acetylases CBP and p/CAF (Xu et al., 1998). The functions of Oct-1 in regulating V gene transcription or rearrangement remain to be genetically tested.

Role of the Octamer Element in Regulating B-Cell--Specific Gene Expression Octamer sites have also been implicated in the regulation of a number of other B-cell—specific gene promoters. For example, major histocompatibility complex (MHC) class II gene promoters contain functionally important octamer sites (Abdulkadir et al., 1995). MHC II genes are mainly expressed on antigen-presenting cells such as B cells, macrophages, and dendritic cells

and their expression is enhanced by interferon-. Studies with the prototypic human class II promoter from the HLA-DRA gene have established a role for Oct-2 and OBF-1 in its regulation. In the class II negative Jurkat T-cell line, which does not express Oct-2, expression of Oct-2A but not overexpression of Oct-1 stimulates the activity of the DRA promoter (Abdulkadir et al., 1995). The high-mobility-group protein HMG I(Y), which promotes Oct-2 binding to the octamer motif, synergistically stimulates transcription with Oct-2 from this promoter (Abdulkadir et al., 1998). Intriguingly, although OBF-1 also activates the DRA promoter, it can do so in the absence of the octamer site (Fontes et al., 1996). In this context, OBF-1 is apparently recruited to the DRA promoter by interacting with another B-cell—specific coactivator, CIITA, which is tethered to this promoter by transcription factors bound to the X box. B29, also known as Ig-, is a B-cell—specific member of the immunoglobulin gene superfamily and a part of the B-cell receptor signal transduction unit that is expressed throughout B-cell development beginning at the pro B-cell stage. A perfect octamer element is present at the 5 regulatory region of its promoter, thereby contributing to the tissue- specific expression of this gene (Hermanson et al., 1989). The CD20/B1 antigen gene promoter contains a variant octamer motif whose activity contributes to the stage-specific expression of this gene in B cells (Thevenin et al., 1993). This element supports the strong constitutive expression of CD20 in mature B cells and mediates phorbol ester—inducible expression of CD20 in a pre-B cell line. Oct-1 and Oct-2 can both specifically bind this divergent octamer site, although with lower affinity than to a consensus octamer. Octamer factors and Ets factors are involved in controlling the activity of the PU. 1 promoter (Chen et al., 1996; Kistler et al., 1995). PU. 1 is a hematopoietic-specific member of the Ets transcription factor family that is expressed in B and myeloid cells, but not in T cells (Klemsz et al., 1990). PU. 1 is required for the development of the lymphoid and myeloid lineages (McKercher et al., 1996; Scott et al., 1994; Singh et al., 2000). PU. 1 activates its own promoter through an Ets-binding site. In addition, the proximal pro-

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moter contains a divergent octamer site. Oct-1 and Oct-2 can bind specifically to this divergent octamer element in vitro and it is also specifically occupied in B cells in vivo. In macrophages the PU. 1-binding site predominantly contributes to promoter activity, whereas in B cells promoter activity is primarily dependent on the octamer motif (Chen et al., 1996). Moreover, OBF-1, which is only expressed in B cells and not in myeloid cells, can transactivate the PU. 1 promoter in HeLa and myeloid cells. It is likely that many other genes containing consensus or noncanonical octamer regulatory elements in their promoters may be target genes for Oct-1/ Oct-2 or OBF-1.

Oct-2--Dependent Genes As mentioned above, the B-cell—specific genes originally believed to be regulated by Oct-2, such as Ig genes or B29, were surprisingly not affected in their expression in Oct-2—deficient B cells (Corcoran and Karvelas, 1994; Corcoran et al., 1993; Feldhaus et al., 1993). An Abelson virus—transformed pre-B cell line obtained from Oct-2—deficient mice was used as a recipient for the expression of a conditionally activatable Oct-2 protein. This system enabled, by differential screening, the identification a set of genes critically dependent on Oct-2 for their expression. Using this system, genes encoding the membrane glycoprotein CD36, the monocyte/ neutrophil elastase inhibitor (mEI), as well as a cysteine-rich secreted protein (CRISP-3), were isolated as Oct-2 targets (Konig et al., 1995; Pfisterer et al., 1997). CD36 as well as mEI are expressed in all hematopoetic cell lines containing Oct-2, and CRISP-3 is expressed in pre-B cells, in bone marrow, and in the spleen. The function of CRISP-3 is unknown and it appears unlikely that any one of these genes can explain the phenotype of the Oct-2 knockout mice. This differential screening approach has also identified two novel genes, Nov1 and Nov 2, which are strictly dependent on Oct-2. While the function of these two genes has yet to be determined, their expression pattern in plasma cells for Nov1 or in B cells for Nov2 suggests that they could play a role during the antigen-dependent phase of B-cell differentiation.

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ROLES OF OCT FACTORS DURING ANTIGEN-DEPENDENT B-CELL DIFFERENTIATION Terminal Differentiation of B Cells B cells can be activated by T-cell—independent (TI) antigens, the best characterized of which is LPS, a compound that has the intrinsic ability to stimulate B cells to proliferate and secrete immunoglobulin without specific recognition by the B-cell receptor (Fig. 18. 4B). Other TI antigens of polymeric structure bind to the BCR and extensively cross-link it, thereby eliciting a similar response. By contrast, a T-cell—dependent response requires specific antigen recognition by the B-cell receptor (surface IgM) and by T-helper cells, to which antigen peptides must be presented in conjunction with MHC class II molecules. A fraction of the B cells differentiate to IgM-secreting plasma cells immediately, whereas others collaborate with activated T cells to establish germinal centers in the follicles (B-cell zones) of secondary lymphoid organs. In these specialized microenvironments, B cells undergo multiple rounds of proliferation, somatic hypermutation, and competition for antigen, a process called affinity maturation. There B cells also switch Ig isotype under the influence of cytokines released by T-helper cells, which, in addition, provide essential costimulatory signals such as CD40 ligand and IL-4. Finally, B cells with high-affinity Igs differentiate into plasma cells, which are specialized to secrete high amounts of Ig, or into memory B cells (Fig. 18. 4C).

Regulation of Ig Gene Transcription by Oct-1/2 and OBF-1 in Activated B Cells Oct-2 and OBF-1 expression is upregulated during B-cell activation and terminal differentiation. The increased levels of these factors may be important in sustaining the very high levels of Ig gene transcription characteristic of activated B and plasma cells. Oct-2 null mice, as well as SCID mice reconstituted with Oct-2 UU fetal liver progenitors, have significantly reduced serum Ig levels (Corcoran and Karvelas, 1994; Corcoran et al., 1993). This reduction has been attributed mostly to a B-cell proliferative defect

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upon antigen or LPS-mediated activation and not to a reduced Ig gene transcription per se. However, it remains possible that the severe reduction in serum Ig levels is partly due to lower levels of Ig gene transcription by Oct-2UU B cells in vivo. Somatic cell fusion experiments have also suggested a role for Oct-2 in regulating Ig gene promoter activity in B cells (Junker et al., 1990) and in maintaining the differentiated state of plasma cells (Radomska et al., 1994). In these latter experiments, extinction of Ig and other B-cell—specific genes, in a plasma cell ; T-cell hybrid, could be prevented by sustained expression of Oct-2, whose gene is also extinguished after cell fusion. Despite their essentially normal early B-cell development, OBF-1 UU mice show a dramatically impaired immune response to several antigens (Qin et al., 1998; Schubart et al., 1996a). While serum IgM levels are normal in these mice, a strong reduction in the secondary isotypes IgG as well as IgA was detected (Kim et al., 1996; Schubart et al., 1996a). However, the ability of OBF-1—deficient B cells to switch isotypes was found to be normal, suggesting that the defect might reflect an inefficient expression of switched genes. The DNA rearrangement that takes place during class switching brings the Ig 3 enhancer region closer to the VH promoter, and one might postulate that this leads to increased dependence of Ig expression on the coactivator OBF-1 (Kim et al., 1996). Recent results with transgenes support an involvement of OBF-1 in mediating activity of the Ig heavy-chain 3 enhancer: transgenic mice were generated harboring a reporter gene driven by either a VH Ig promoter or a -globin promoter, in conjunction with an Ig 3 enhancer (HS1-2). In an OBF-1UU background, cells from these mice showed reduced reporter expression specifically in case of the Ig promoter, suggesting that this promoter critically depends on OBF-1 when it functions with the Ig 3 enhancer (Andersson, Samuelsson, Matthias, and Pettersson, manuscript submitted).

Activation of Ig Enhancers The Ig enhancers (reviewed in Staudt and Lenardo, 1991) comprise the Ig heavy-chain gene intron enhancer (iE), a still exnding family of 3 Ig heavy-chain enhancers (HS1-4: 3E)

(Madisen and Groudine, 1994; Matthias and Baltimore, 1993; Pettersson et al., 1990), the k intronic enhancer (iE k), the 3 k enhancer (3E k), and the light-chain enhancers. Interestingly, several of these enhancers (iE, 3E, iEk) contain octamer-binding sites. Whereas the variable region gene promoter octamer sites are absolutely essential for Ig gene expression, the octamer site found in the Ig heavy-chain enhancer appears to make only a minor contribution to overall activity in transgenic mice (Jenuwein and Grosschedl, 1991). On the other hand, synthetic arrays of octamer elements generate potent B-cell—specific enhancers (Gerster et al., 1987; Seipel et al., 1992; Tanaka and Herr, 1994). In this context, Oct-2A, but not Oct-2B or Oct-1, stimulates transcription from these arrays, enabling promoter activation from large distances (Annweiler et al., 1992; Feldhaus et al., 1993; Muller-Immergluck et al., 1990; Pfisterer et al., 1994; Seipel et al., 1992). This ability of Oct-2A to transactivate from remote enhancer positions was mapped to the C-terminal activation domain (Annweiler et al., 1992; Friedl and Matthias, 1995) and relies on an as yet unidentified B-cell—specific cofactor distinct from OBF-1 (Pfisterer et al., 1995; Schubart et al., 1996b) (Fig. 18. 2C). As indicated above, Oct-2 levels are upregulated during terminal B-cell differentiation. Given the potential of Oct-2A to selectively activate transcription from large distances, it may promote high levels of Ig gene expression in plasma cells by contributing to the inducible activity of the 3 IgH enhancer region. Signaling pathways induced by LPS, IgM cross-linking, or CD40 ligation, which is critical for B-cell activation, induce the activity of the HS1-2 3IgH enhancer (Arulampalam et al., 1994; Grant et al., 1995, 1996; Linderson et al., 1997). This inducible activation of the enhancer correlates with increased occupancy of the octamer element as well as binding sites for Ets/AP-1 factors. Recently, the use of altered specificity Oct1/2 mutant proteins has been extended to examine their function from the 3 IgH enhancer region. These experiments have demonstrated that Oct-2A specifically stimulates transcription from the 3 IgH enhancer region in B cells (Tang and Sharp, 1999). Collectively, the various genetic and molecular studies lead to the view that Oct-1 and OBF-1 preferentially function at Ig gene promoters, whereas Oct-2 and a distinct

ROLE OF FACTORS BINDING TO THE OCTAMER SITE IN T CELLS

coactivator promote IgH gene transcription from the 3 enhancer region.

Involvement of NF-B, Oct-2, and OBF-1 in Germinal Center Formation Deficiencies in the formation of germinal centers (GCs) are observed in mice with targeted disruptions of p52/NF-B2 (Caamano et al., 1998; Franzoso et al., 1998), p50/p52 (Franzoso et al., 1997), OBF-1 (Kim et al., 1996; Nielsen et al., 1996; Schubart et al., 1996a), and Oct-2 (Schubart, Massa, Rolink, and Matthias, manuscript in preparation). In agreement with the importance of OBF-1 for GC formation, this protein was recently found to be highly expressed in these structures in immunized wild-type mice. Furthermore, IgM, CD40L, and IL-4 — signals that B cells receive in GCs — were able to induce OBF-1 expression in primary resting B cells (Qin et al., 1998). A selective role of the transcription factor Oct-2 was shown in the regulation of the chemokine receptor blr1 gene (Wolf et al., 1998). BLR1 is a major regulator of the microenvironmental homing of B cells in lymphoid organs. Both a NF-B motif and a noncanonical octamer motif confer cell type and differentiationspecific expression of the blr1 gene in human and murine B cells. Gene-targeted mice deficient in either Oct-2, OBF-1, or both NF, B subunits p50 and p52 show reduced or no expression of BLR1 (Wolf et al., 1998). A hallmark of the phenotype of blr1-deficient mice is the lack of primary lymphoid follicles and inappropriate formation of germinal centers in the spleen. It should be noted that blr1UU mice, despite their germinal center defects, are not impaired in their humoral response to a T-dependent antigen (Forster et al., 1996). Thus, while the germinal center defects in Oct-2 and OBF-1 mutant mice may be partly due to reduced BLR1 expression, the humoral response defects in these animals are likely due to impaired expression of other target genes, including Ig loci that have undergone class switching (see above). Interestingly, the Oct-2 gene itself seems to be downstream of NF-B (Bendall et al., 1997) and is under the control of several functionally important NF- elements (Tang, Klug, and Singh, manuscript in preparation). This suggests that NF-kB could act in a concerted way to activate Oct-2, and

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subsequently synergize with the latter and OBF1 in order to activate a set of genes, including blr1, which allow germinal center formation. Interleukin-6 and Retinoic Acid Signaling The synthesis and secretion of Ig is significantly increased through interleukin-6 (IL-6) signaling in human Epstein-Barr virus (EBV)-transformed B-cell lines or freshly isolated polyclonal B lymphocytes (Natkunam et al., 1994). These IL-6 induced cells are phenotypically similar to plasma cells in their ultrastructure and in their reduced expression of surface MHC class II. Coordinately with this differentiation, the amount and synthesis of Oct-2 are upregulated, suggesting that this factor might be important for the elevated Ig levels observed. Subsequently, if these cells are subject to prolonged IL-6 stimulation a down regulation of Ig and Oct-2 expression is observed. The proliferation and differentiation of many transformed or developing cells are influenced by retinoic acid (RA) (reviewed in Evans and Kaye, 1999). RA and its precursor molecule retinol (vitamin A) have been known as potent immunopotentiating agents since the early 1900s. RA can act directly on B cells and leads to an augmentation in Ig synthesis as well as an increase in IL-6 production in EBV-transformed B-cell lines (Ballow et al., 1996a). This cytokine secreted by EBV-transformed B cells can thereby act as an autocrine factor in increasing Ig and Oct-2 synthesis (Ballow et al., 1996a; Natkunam et al., 1994). Furthermore, stimulation of highly purified T cells from cord blood mononuclear cells with RA leads to enhanced IgM synthesis by cord blood B cells (Ballow et al., 1996b), suggesting that the stimulated T cells synthesize a cytokine acting on B cells to stimulate their Ig production.

ROLE OF FACTORS BINDING TO THE OCTAMER SITE IN T CELLS T-Cell Activation Leads to Induction of Oct-2 and OBF-1 Several genes that are induced after MHC-antigen encounter by the T-cell receptor (TCR), have functionally important octamers in their

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regulatory sequences. The promoters of the genes for the T-cell growth factors IL-2, IL-4 and IL-5, which are essential lymphokines required for the process of T-cell activation, proliferation, clonal expansion and differentiation, contain multiple binding sites for octamer factors (Kamps et al., 1990; Pfeuffer et al., 1994; Ullman et al., 1991). The T-cell signal transduction pathway initiated at the TCR leads to the activation of multiple transcription factors, most notably NFAT (Crabtree and Clipstone, 1994). TCR-dependent activation can be mimicked by the combination of phorbol myristate acetate (PMA), a protein kinase C activator, and the calcium ionophore ionomycin (Hutchinson and McCloskey, 1995). These agents induce OBF-1 in T-cell lines or in primary T lymphocytes (Sauter and Matthias, 1997; Zwilling et al., 1997) (Fig. 18. 5). Concomitantly, phosphorylation of OBF-1 on serine 184 in the C-terminal activation domain is observed, and this modification was reported to be important for function (Zwilling et al., 1997). It has been suggested that induction of OBF-1 may be important for the activation of octamer-dependent genes in T cells (Zwilling et al., 1997). Yet, the in vivo relevance of OBF-1 induction for T-cell function is unclear, since transfer of OBF-1UU T cells into nude mice showed that these T cells are fully competent to sustain a normal immune response (Schubart et al., 1996a). Likewise, OBF-1UU T cells are also normal when stimulated in vitro. The Oct-2 gene is expressed in CD4> and CD8> T cells and is upregulated upon antigen stimulation (Kang et al., 1992). The kinetics of Oct-2 induction suggest that it may regulate late gene expression in activated T lymphocytes. However, Oct-2 UU T cells also appear to function normally during an immune response (Humbert and Corcoran, 1997). Thus, although both Oct-2 and OBF-1 are induced during T-cell activation, genetic analysis has yet to reveal any major function for these regulators in T cells.

Octamer-Dependent IL-2 and IL-4 Gene Regulation As mentioned above, the octamer-binding sites of the IL-2 and IL-4 promoters constitute important and conserved upstream promoter el-

ements to which Oct-1 and Oct-2 can bind (Pfeuffer et al., 1994). Multimerized copies of the IL-2 and IL-4 promoter elements encompassing the octamer sites act as inducible enhancers in T cells; their inducibility is inhibited by the immunosuppressant cyclosporin A (CsA). After antigenic stimulation of T lymphocytes, the IL-2 gene is transcriptionally activated. This is mediated by NF-AT sites and by a composite octamer and AP-1 site that binds Oct-1 and/or Oct-2 together with JunD and c-Jun (Ullman et al., 1991, 1993). Before their identification as JunD and c-Jun, the protein(s) binding to this composite AP-1 siter had been refered to as OAP40 (Ullman et al., 1991). In this arrangement, JunD and c-Jun reduce the rate of dissociation of Oct-1 from its cognate-binding site. Mutation of either site impairs the induction of the IL-2 promoter (Pfeuffer et al., 1994) and by increasing the distance between the octamer and the AP-1 site, disruption of the promoter structure results in a severe reduction of inducible promoter activity, and also in the loss of suppression by CsA or stimulation by the Ca>dependent phosphatase calcineurin (CN). This suggests that either a factor downstream of CN is recruited by the Oct/Jun complex or that Oct/Jun is directly dephosphorylated by CN in a way dependent on the close association of the two transcription factors (see Figure 18. 5). Within the IL-4 promoter, octamer and noncanonical AP-1—binding motifs are separated by a binding site for the nuclear factor of activated T cells (NF-AT). In this promoter, binding of NF-AT to the IL-4 promoter site inhibits the simultaneous binding of Oct factors and it seems that in this case Oct-1 plays a lesser role for induction. IL-5 Gene Regulation The mouse EL4 T-cell line can, under certain conditions, produce IL-5 transcripts, and costimulation of EL4 cells by phorbol myristate acetate (PMA), the Ca> ionophore A23187, and the cAMP analog Bt2cAMP can lead to activation of a transfected human IL-5 gene promoter (Gruart-Gouilleux et al., 1995). The IL-5 promoter contains a distal positive regulatory element with a perfect octamer motif to which Oct-1, Oct-2A, and Oct-2B can bind in vitro. In one study, this octamer motif sup-

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Figure 18.5. Schematic representation of signaling pathways implicated in IL-2 gene activation. Antigen-MHC recognition by the T-cell receptor (TCR) triggers several signaling cascades including the Ras pathway and a PLC-dependent pathway that leads to an increase of intracellular Ca> concentration as well as PKC activation. Ras upregulates levels of the AP-1 (Fos/Jun) transcription factors, whereas Ca> activates the phosphatase Calcineurin, which in turn mediates NF-AT translocation to the nucleus. The OBF-1 gene is also induced during T-cell activation. An Oct-1/OBF-1/Jun complex is suggested to inducibly assemble at the IL-2 promoter in a calcineurin-dependent manner. OBF-1 is induced in T cells by PMA/ionomycin, which mimick PKC and Calcineurin activation. The role of OBF-1 in T-cell activation is unknown. As shown in the model, it could enhance the transcriptional potency of Oct-1 in the Oct/Jun complex. Cyclosporin A (CsA) and FK506 are specific inhibitors of Calcineurin that totally block IL-2 gene induction.

ported 90% of the activity in stimulated EL4 cells (Gruart-Gouilleux et al., 1995). In another study, a second octamer element in the promoter-proximal region of the IL-5 promoter was also found to be important for activity (Thomas et al., 1999). This second element bound Oct-1 in a constitutive manner, whereas binding of Oct-2 or AP-1 was induced in response to cell activation by PMA/A23187.

Inhibition of Octamer-Dependent Transcription in T Cells by TGF- and Retinoic Acid Transforming growth-factor (TGF-) has been implicated in the growth inhibition of several different cell types of the immune system, including T lymphocytes. For example, IL-2 gene expression is blocked following TGF-

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treatment Brabletz et al., 1993). At least part of this inhibition by TGF- appears to be mediated by the octamer sites, while the AP-1 site is not impaired in EL4 T-lymphoma cells. This inhibition by TGF- occurs without impairment of the DNA-binding capacity of Oct factors and therefore may take place at the level of posttranslational modification(s) or at the level of a cofactor, for example, OBF-1 (which is induced in T cells and highly expressed in EL4 cells). The induction of the IL-2 gene is also inhibited by RA (Felli et al., 1991), and here, too, RA-responsive elements of the IL-2 promoter were mapped to sequences containing an octamer motif. As in the case of TGF-, RA does not decrease the DNA-binding capability of Oct-1. Direct interaction between Oct-1 and members of the steroid receptor superfamily, including RA receptors, have been previously reported in several systems. Interaction between Oct and the retinoid X receptor (RXR) negatively regulates nuclear hormone signaling by reducing the binding of the thyroid hormone (TR) receptor and RXR to TR-responsive elements (Kakizawa et al., 1999). Conversely, glucocorticoid receptors (GR) and androgen receptor antagonize octamer-dependent Oct-1 or Oct-2 function on a wide array of promoters (Chandran et al., 1999; Kutoh et al., 1992; Song et al., 1998; Subramaniam et al., 1998; Wieland et al., 1991). Surprisingly, this repression does not involve steroid receptor DNA binding. The reported interaction between steroid receptors and the homeo-specific subdomain of the POU domain might explain the repressor activities observed on promoters lacking an optimal steroid receptor binding site (Wang et al., 1999). Alternatively, competition between the POU and the steroid receptor superfamilies for limiting chromatin remodeling cofactors could be invoked (Xu et al., 1998). In other cases, the presence of both GR and octamer sites can result in strong transcriptional synergism (Prefontaine et al., 1998; Wieland et al., 1991). Thus, the convergence of ligand-dependent and ligand-independent regulatory pathways may be part of a widely used biological strategy allowing functionally synergistic or antagonistic actions to occur at various gene promoters, with consequences on cell proliferation and differentiation (Bertolino et al., 1995; Chen et al., 1995).

Transformation of T Cells by the Oct-1/2 POU Domain When they are expressed in T cells of transgenic mice, the POU domains of Oct-1 or Oct-2 surprisingly lead to malignant lymphomas (Qin et al., 1994). In this case, the basis for oncogenic transformation by these POU domains has been postulated to reside in the generation of selective and sequence-specific dominant negative mutants. However, the molecular targets of POU domain action have not been identified, and the link between putative repression of tumor suppressor genes and malignant transformation is a matter of conjecture. It should be noted that expression of an Oct-2 RNA encoding only the POU domain, mini Oct-2, has been detected in the olfactory bulb of the mouse adult brain. On the basis of transfection experiments, it has been argued that this putative protein might serve as an octamer site—dependent repressor (Stoykova et al., 1992), but also in this case it is a hypothesis awaiting molecular confirmation. As described earlier, the POU domain has been shown to function as an activator in several transcriptional contexts rather than as a repressor. Thus, the transforming potential of the POU domain may reside in its ability to activate rather than to repress transcription.

CONCLUSION Functional redundancies arising from the principles of combinatorial gene regulation often make it difficult to assign specific functions in the regulation of a gene to a particular transcription factor. DNA motifs that can be bound by two or more related factors in a cell add another level of complexity. Gene knockouts combined with other molecular genetic approaches, including ectopic expression and use of altered specificity mutants, enable a comprehensive analysis of the biological and molecular functions of related transcription factors. Since the discovery of the octamer site and its functional importance in B cells, considerable effort has been devoted to characterize transcription factors that bind this element and to understand their mode of action. The ubiquitous Oct-1 and the largely B-cell—restricted Oct2 were identified as direct DNA-binding factors

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ACKNOWLEDGMENT This work represents an equal contribution from our two laboratories.

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Tanaka, M., Lai, J. S., and Herr, W. (1992). Promoterselective activation domains in Oct-1 and Oct-2 direct differential activation of an snRNA and mRNA promoter. Cell 68, 755—767. Tang, H., and Sharp, P. A. (1999). Transcriptional regulation of the murine 3 IgH enhancer by OCT2. Immunity 11, 517—526. Thevenin, C., Lucas, B. P., Kozlow, E. J., and Kehrl, J. H. (1993). Cell type— and stage—specific expression of the CD20/B1 antigen correlates with the activity of a diverged octamer DNA motif present in its promoter. J. Biol. Chem. 268, 5949—5956. Thomas, M. A., Mordvinov, V. A., and Sanderson, C. J. (1999). The activity of the human interleukin-5 conserved lymphokine element 0 is regulated by octamer factors in human cells. Eur. J. Biochem. 265, 300—307. Tjian, R., and Maniatis, T. (1994). Transcriptional activation: a complex puzzle with few easy pieces. Cell 77, 5—8. Tonegawa, S. (1983). Somatic generation of antibody diversity. Nature 302, 575—581. Ullman, K. S., Flanagan, W. M., Edwards, C. A., and Crabtree, G. R. (1991). Activation of early gene expression in T lymphocytes by Oct-1 and an inducible protein, OAP40. Science 254, 558—562. Ullman, K. S., Northrop, J. P., Admon, A., and Crabtree, G. R. (1993). Jun family members are controlled by a calcium-regulated, cyclosporin A— sensitive signaling pathway in activated T lymphocytes. Genes Dev. 7, 188—196. Wang, J. M., Prefontaine, G. G., Lemieux, M. E., Pope, L., Akimenko, M. A., and Hach, R. J. (1999). Developmental effects of ectopic expression of the glucocorticoid receptor DNA binding domain are alleviated by an amino acid substitution that interferes with homeodomain binding. Mol. Cell. Biol. 19, 7106—7122. Weiss, M. J., and Orkin, S. H. (1995). GATA transcription factors: key regulators of hematopoiesis. Exp. Hematol. 23, 99—107. Wieland, S., Dobbeling, U., and Rusconi, S. (1991). Interference and synergism of glucocorticoid receptor and octamer factors. EMBO J. 10, 2513— 2521. Wirth, T., Staudt, L., and Baltimore, D. (1987). An octamer oligonucleotide upstream of a TATA motif is sufficient for lymphoid-specific promoter activity. Nature 329, 174—178. Wolf, I., Pevzner, V., Kaiser, E., Bernhardt, G., Claudio, E., Siebenlist, U., Forster, R., and Lipp, M. (1998). Downstream activation of a TATA-less promoter by Oct-2, Bob1, and NF-kappaB directs

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CHAPTER 19


THE ROLE OF EARLY B-CELL FACTOR IN B-LYMPHOCYTE DEVELOPMENT MIKAEL SIGVARDSSON The Laboratory for Cellular Differentiation Studies, The Department for Stem Cell Biology, Lund University, Lund, Sweden

INTRODUCTION The development of the B-lymphoid lineage is a complicated process that can be divided into several well-defined stages based on expression of developmentally regulated genes and the recombination status of the immunoglobulin (Ig) locus (Fig. 19.1) (Ghia et al., 1998; Hardy et al., 1991; Li et al., 1996). The earliest characterized precursor cells express the surface molecules AA4.1, B220, and CD43. They produce mRNA coding for the component of the B-cell receptor complex (B29) (Hombach et al., 1990a, 1990b) and sterile germ-line transcripts from the immunoglobulin heavy-chain (IgH) locus. This stage precedes the Igr ecombination events and therefore at this stage both the IgH and immunoglobulin light-chain (IgL) genes remain in their germ-line configuration. Subsequent differentiation results in cells that express the surface molecule heat-stable antigen (HSA). These cells also express the component of the B-cell receptor complex (mb-1), the surrogate light-chain genes, 5 and VpreB, as well as the recombination activating genes Rag1 and

Rag2. The expression of Rag genes allows for the initiation of the IgH recombination events and joining of D segments to J segments. DJ joining is followed by VDJ joining and exposure of a pre-B-cell receptor composed of the IgH chain and the surrogate light chains on the cell surface. This event is proposed to signal to the cell that a functional IgH rearrangement has occurred, which allows for proliferation and initiation of IgL recombination events. Once a functional light-chain rearrangement has been generated, the cell becomes an IgM-positive immature Bcell that may become an IgM, IgD surfacepositive mature B-cell, unless negatively selected due to autoimmune specificity. Terminal differentiation of the mature B-cell into the Ig-secreting plasma cell requires activation through the B-cell receptor in combination with stimulatory actions from helper T cells. Progression of the B-lymphoid differentiation pathway is critically dependent on correct regulation of gene expression by a number of transcription regulatory factors suggested as essential for B-cell development (Georgopoulos, 1997; Reya and Grosschedl, 1998; Singh, 1996). One of these is early


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Figure 19.1. A scheme indicating the expression of EBF and suggested EBF target genes during B-cell development. Markers used to define developmental stages are indicated on top of the figure above the corresponding cells. Development blocks induced by homologous disruption of genes discussed in the text are indicated by crosses. The potential target genes of EBF are listed on the left margin underneath EBF. Ig, immunoglobulin; IgH, immunoglobulin heavy chain; IgL, immunoglobulin light chain; HSA, heat-stable antigen; D; diversity segments; J, joining segments; BCR, B-cell receptor; EBF, early B-cell factor; Il-7r, interleukin-7 receptor ; BSAP, B-cell—specific activator protein; Blk, B-lymphoid kinase.

B-cell factor (EBF) (Hagman et al., 1993; Wang and Reed, 1993), since mice devoid of this transcription factor display a complete block of B-lymphocyte development (Lin and Grosschedl, 1995). EBF was first defined as a protein that interacts with a functionally important site in the mb-1 promoter, AGACTCAAGGGAAT (Fig. 19.3) (Feldhaus et al., 1992; Hagman et al., 1991; Travis et al., 1991). The composition of this binding site resembled that of the transcription factor Lyf/Ikaros (GGGAA) (Hahm et al., 1994; Chapter 12) that interacts with the terminal deoxynucleotide transferase (TdT) promoter (Lo et al., 1991), resulting in the protein also being

denoted B-Lyf (Feldhaus et al., 1992). EBF was purified by affinity chromatography from a 38B9 pre-B-cell extract based on its interaction with the mb-1 promoter (Travis et al., 1993). Subsequent amino acid sequencing and cloning revealed that EBF was a 65 kD polypeptide with low homology to other, at that time, known transcription factors (Hagman et al., 1993) with the exception of rat Olf-1 (Wang and Reed, 1993). This factor was cloned independently by yeast one hybrid screening for factors interacting with the cyclic nucleotide—activated ion channel (OcNC) promoter (Wang and Reed, 1993). Biochemical characterization of EBF revealed that the protein interacts with DNA as a

INTRODUCTION

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Figure 19.2. Schematic drawing showing functional domains of the EBF protein (Hagman et al., 1995). The DNA-binding domain is indicated in black and the amino acids involved in the formation of the Zn coordination motif are also indicated. The dimerization domains are indicated in light gray and the amino acids homologous to helix 2 of b-hlh proteins are confined to shaded boxes. The dark gray area represents the carboxy-terminal transactivation domain of EBF.

homodimer via an amino-terminal Zn coordination motif (Hagman et al., 1993, 1995). This region also contains a context-dependent transactivation domain, which together with a second carboxy-terminal region constitutes the transactivation ability of EBF (Hagman et al., 1995). Homodimerization is mediated by 2 putative helixes with homology to helix 2 of the basic helix-loop-helix (b-hlh) transcription factor family (Fig. 19.2) (Hagman et al., 1993, 1995). Thus EBF/Olf-1 has been suggested to constitute a subgroup of the helix-loop-helix family of transcription factors. EBF is expressed in B-cell progenitors and in mature B cells, but not in terminally differentiated plasma cells (Hagman et al., 1993). The factor is also expressed in neural (Wang and Reed, 1993), stromal, and adipose tissue (Hagman et al., 1993), but the exact role for EBF in these tissues is still rather unclear. Later studies of EBF in nerve cells have revealed the existence of two EBF-related proteins expressed mainly in olfactory neurons, EBF2 and EBF3 (Wang et al., 1997). It has been suggested that these factors are redundant to EBF, which may explain the lack of an apparent neurological phenotype in mice deficient of EBF. This work has also identified an EBF interaction partner, Roaz, expressed in CNS but also in spleen. Roaz is a Zn-finger protein containing 29 potential Zn-finger domains of the TFIIIA type (Tsai and

Reed, 1997, 1998) and appears to impair the ability of EBF to activate olfactory neuronspecific promoters (Tsai and Reed, 1997, 1998). However, no function of Roaz in B-cell development has yet been reported. EBF homologues have been cloned in a number of species. Mouse (Hagman et al., 1993), rat (Wang and Reed, 1993), and human EBF (Gisler et al. 2000) have been cloned, showing a high degree of amino acid conservation. EBF-related proteins have also been isolated from Drosophila (Crozatier et al., 1996), Xenopus (Dubois et al., 1998), zebra fish (Bally-Cuif et al., 1998), and Caenorhabditis elegans (Prasad et al., 1998). The Drosophila homologue, collier, appears to play a role in head development and hedgehog signaling in the fly embryo (Crozatier et al., 1996; Vervoort et al., 1999), while the C. elegans protein Unc-3 and the Xenopus Xcoe2 appear to be involved in the formation of the nervous system in the worm (Prasad et al., 1998) and frog (Dubois et al., 1998), respectively. Thus, EBF belongs to an evolutionary conserved family of proteins involved in the development of specific structures or cell lineages. The focus of this chapter is the role of EBF in B-cell development, where the work from several laboratories has allowed for the identification of several genetic targets and interaction partners, making EBF one of the best-characterized transcription factors in early B-cell development.

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B-CELL DEVELOPMENT IN THE ABSENCE OF EBF A powerful tool to investigate the functional roles of transcription factors in B-cell development has been targeted disruptions of genes encoding these proteins. Disruptions of the genes encoding c-myb (Mucenski et al., 1991); Chapter 29, SCL/tal1 (Porcher et al., 1996; Chapter 4), GATA-2 (Tsai et al., 1994; Chapter 1), AML1 (Okuda et al., 1996; Chapter 6), and PU.1 (Scott et al., 1994, 1997; Chapter 13) affects heamatopoesis broadly, while mice carrying disruptions of genes encoding IKAROS (Georgopoulos et al., 1994; Wang et al., 1996; Chapter 12), E47/12 (Bain et al., 1994, 1997; Zhuang et al., 1994; Chapter 16), Sox-4 (Schilham et al., 1996), BSAP (Urbańek et al., 1994; Chapter 14) or EBF (Lin and Grosschedl, 1995) display phenotypes that are more restricted to the lymphoid compartment of the hematopoetic system. In mice deficient for EBF, there is a complete block of B-cell differentiation at the pro-B cell stage (Lin and Grosschedl, 1995). Bone marrow from these mice contains cells with surface expression of B220 and high levels of CD43. However, no cells with surface expression of BP-1 or IgM were detected. The developmental block was also confirmed by RT-PCR analysis, since no expression of the pre-B cell markers 5 or VpreB could be detected. Furthermore, no Rag expression or Ig gene rearrangements were present in these pro-B cells. This suggests that B-cell development in the absence of EBF is blocked at the pro-B cell stage in fraction A (B220>, CD43 high, HSA low cells) according to Hardy et al. (Hardy et al., 1991). A similar or earlier differentiation block is observed in mice carrying a targeted disruption of the E2A gene (Bain et al., 1994; Zhuang et al., 1994; Chapter 16). This gene encodes the E47 and E12 b-hlh transcription factors suggested to control a large number of genes relevant for lymphocyte development (Bain and Murre, 1998; Murre et al., 1994). The early differentiation block in these mice suggests that E2A proteins are located upstream of EBF in the transcription factor hierarchy of B-cell development. This is also supported by the finding that EBF expression can be induced by ectopic expression of E12 in dedifferentiated 70Z/3 pre-B cells (Kee and Murre, 1998). However, the pres-

ence of low amounts of EBF mRNA in total bone marrow from E2A-deficient mice (Bain et al., 1997) and reduced expression of E2A gene products in B220> bone marrow cells from mice lacking EBF (Lin and Grosschedl, 1995) raise the possibility that E2A proteins and EBF are independently required for B-cell development. The EBF expression detected in E2A-deficient mice could, however, be a result of stroma cell contamination, making it rather difficult to be absolutely definitive on this issue. Independent and collaborative functions of EBF and E47 in late pro-B cells are suggested from the finding that mice trans-heterozygote for mutations in the EBF and E2A genes display a more dramatic pro-B cell, differentiation block than any of the single-heterozygote mutant mice (O’Riordan and Grosschedl, 1999). Interestingly, the observed differentiation block occurs at a later differentiation stage, fraction B (B220>, CD43>, HSA>, and BP-1\), according to Hardy (Hardy et al., 1991), than in the homozygote single mutants that display early pro-B, fraction A, developmental blocks (Bain et al., 1994; Lin and Grosschedl, 1995; Zhuang et al., 1994). This suggests that EBF and E2A proteins may act in a cooperative manner to promote B-cell development, possibly by sharing genetic targets such as the surrogate lightchain genes (O’Riordan and Grosschedl, 1999; Sigvardsson et al., 1997). An early B-cell differentiation block is also observed in mice with a disruption of the interleukin-7 (Il-7) receptor- gene (Fig. 19.1) (Peschon et al., 1994). However, expression of the Il-7 receptor in B220> bone marrow cells (Lin and Grosschedl, 1995) and the apparently normal T-cell development in EBF-deficient mice (Lin and Grosschedl, 1995), as opposed to Il-7 receptor—deficient mice (Maraskovsky et al., 1996; Peschon et al., 1994), argues against disrupted Il-7 signaling as an explanation for the phenotype of EBF null mice. A slightly later differentiation block is observed in mice deficient in the high-mobilty group protein Sox-4 (Fig. 19.1) (Schilham et al., 1996; van de Wetering et al., 1993). This block appears to be a result of impaired expansion of the B-cell progenitors (Schilham et al., 1996), similar to what is observed in the absence of the Il-7 receptor (Peschon et al., 1994). This is in contrast to the phenotype observed in EBF-deficient mice, since

GENETIC TARGETS FOR EBF

their bone marrow contains a large number of progenitor cells, suggesting that the phenotype is a result of a differentiation block rather than a lack of pro-B cell expansion (Lin and Grosschedl, 1995). The relationship between Sox-4 and EBF has not been extensively studied, but their different effects on B-cell development imply that they act independently to promote B-cell differentiation. Interestingly, the pro-B cells in EBF-deficient mice do not express the paired domain transcription factor B-cell specific activator protein (BSAP) (Chapter 14; Lin and Grosschedl, 1995), indicating that this factor is positioned downstream of EBF in the developmental hierarchy of B cells. This is also supported by the findings that B-cell development in BSAP-deficient mice is arrested at a later differentiation stage than in EBF-deficient mice (Fig. 19.1) (Nutt et al., 1997; Urbańek et al., 1994). Furthermore, pro-B cells from mice devoid of BSAP express high levels of EBF (Nutt et al., 1997). Ectopic expression of EBF has also been suggested to induce endogenous BSAP expression in dedifferentiated 70Z/3 cells (Kee and Murre, 1998) and the pax-5 (BSAP) promoter in fibroblasts (O’Riordan and Grosschedl, 1999), indicating that BSAP may be a direct target for EBF. Even though homologous disruption of the EBF gene has provided evidence that the progression of B-cell differentiation is critically dependent on EBF, the underlying molecular mechanism will remain unclear until the genetic targets that are essential for pro-B cell development are identified.

GENETIC TARGETS FOR EBF The first defined transcriptional control element demonstrated to interact with EBF was within the promoter of the mb-1 gene (Fig. 19.3) (Hagman et al., 1991, 1993; Travis et al., 1991), encoding the Ig component of the B-cell receptor (BCR) (Hombach et al., 1990a, 1990b). Mutations in the EBF-binding site reduced the functional activity of the promoter to about 30% of the full function in transient transfection experiments (Hagman et al., 1991). This is comparable to what was observed when either the BSAP- or the Ets-binding sites in the promoter were disrupted (Fitzsimmons et al., 1996). Inter-

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estingly, the importance of the EBF-binding site is lower in mature B cells (Travis et al., 1991), implying that the requirements for transcriptional activation of the mb-1 promoter may be altered during B-cell development. EBF was also shown to interact with the promoter of the B29 gene (Fig. 19.3) (Akerblad et al., 1999, encoding the component of the BCR (Campbell et al., 1991; Hombach et al., 1990). The B29 gene is expressed from the earliest stages of B-cell development to the plasma cell stage (Hermanson et al., 1988; Li et al., 1996). The B29 promoter (Omori and Wall, 1993; Thompson et al., 1996) contains three independent binding sites for EBF, two of which stimulated cooperative interaction of two EBF homodimers to the promoter (Akerblad et al., 1999). Furthermore, ectopic expression of EBF in epitheloid HeLa cells both induced transcription of a reporter gene under the control of the B29 promoter and the endogenous B29 gene in the same cells (Akerblad et al., 1999). The EBFbinding sites were also important for the full function of the promoter in pre-B cell lines, since the activity of the B29 promoter was impaired by mutations of the EBF-binding sites (Akerblad et al., 1999). Such a functional impairment was not observed when the same mutated promoters were introduced into cell lines representing later B-cell differentiation stages (Akerblad et al., 1999). Thus, it appears as if EBF is a key regulator of the B29 gene in early stages of B-cell development, while other transacting factors control expression at later developmental stages (Omori and Wall, 1993). A role for EBF in the regulation of the B29 gene is also supported by the finding that mRNA coding for Ig could be detected in bone marrow from mice deficient in E2A proteins (Bain et al., 1994; Zhuang et al., 1994), but not in mice lacking EBF (Lin and Grosschedl, 1995). This is intriguing, since the developmental block of B cells in E2A-deficient mice appears to be earlier than in mice lacking EBF (Bain et al., 1994; Lin and Grosschedl, 1995; Zhuang et al., 1994). Thus, EBF-deficient mice appear to have a specific reduction of B29 expression in their B-cell progenitors, supporting the conclusion that EBF is involved in B29 transcription during normal B-cell development. EBF has also been shown to interact with the promoter region of the gene encoding the B-

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Figure 19.3. Partial sequences of EBF target gene promoters highlighting the position and composition of characterized EBF-binding sites. The top sequence shows a consensus EBF-binding site (Travis et al., 1993), and homology to this site among the target promoters are indicated by *. The number at the 5 end of each promoter sequence indicates the distance from the major transcription start site. The target gene promoters that are shown are mb-1 (Hagman et al., 1991; Travis et al., 1991), B29 (Akerblad et al., 1999; Omori and Wall, 1993), B-lymphoid kinase (Blk) (Akerblad and Sigvardsson, 1999; Dymecki et al., 1992), 5 (Martensson and Martensson, 1997; Sigvardsson et al., 1997, Sigvardsson, unpublished observation), and VpreB (Kudo and Melchers, 1987; Persson et al., 1998).

lymphoid kinase (Blk) (Fig. 19.3) (Åkerblad and Sigvardsson, 1999; Dymecki et al., 1990, 1992). Blk is a member of scr family of kinases expressed from the pro-B until the mature B-cell stage, but not in the terminally differentiated plasma cells (Fig. 19.1) (Dymecki et al., 1990). The Blk kinase interacts with the cytoplasmatic domains of Ig and Ig to participate in BCR signaling (Lin and Justement, 1992). EBF binds to and activates transcription from a site in the

Blk promoter protected from in vivo methylation in pre-B cells (Åkerblad and Sigvardsson, 1999), implying that Blk is a direct target for EBF activation. Analogous to what was observed for the mb-1 and B29 promoters, transient transfections with reporter genes controlled by either wild type or EBF-site mutated Blk promoters suggested that functional EBF-binding was important for full promoter activity in pre-B but not B-cell lines. The Blk promoter was

GENETIC TARGETS FOR EBF

also shown to interact with BSAP (Zwollo and Desiderio, 1994; Zwollo et al., 1998) and ectopic expression of BSAP and EBF in epitheloid cells resulted in cooperative activation of the Blk promoter (Åkerblad and Sigvardsson, 1999). This is of special interest, since EBF and BSAP have been suggested to share several additional genetic targets such as mb-1 (Fitzsimmons et al., 1996; Hagman et al., 1991), VpreB, and 5 (Tian et al., 1997). The VpreB and 5 genes encode the pre-B— cell specific surrogate light-chain components (Kudo and Melchers, 1987; Sakaguchi and Melchers, 1986) that interact with the newly rearranged Ig heavy chain on the surface of the pre-B cell (Karasuyama et al., 1990). The formation of this pre-BCR has been shown to be important for the progression from the early to the late pre-B cell stage and for initiation of Ig light-chain rearrangements (Fig. 19.1) (Karasuyama et al., 1994; Kitamura et al., 1992). Transcription from the 5 gene was induced upon stable ectopic expression of EBF in the bone marrow—derived cell line Ba/F3 Sigvardsson et al., 1997) and in dedifferentiated 70Z/3 pre-B cells (Kee and Murre, 1998). This, together with the finding that the 5 promoter contains at least three functionally important EBF-binding sites within 300 base pairs 5 of the major transcriptional start site (Fig. 19.2) (Martensson and Martensson, 1997; Sigvardsson et al., 1997), suggest that this gene is a direct target for EBF. Furthermore, ectopic expression of EBF induced transcription of the 5 promoter in both hematopoetic Ba/F3 cells and in epitheloid HeLa cells (Sigvardsson et al., 1997). Expression of endogenous VpreB encoding transcripts was also induced in Ba/F3 cells stably transfected with EBF (Sigvardsson et al., 1997). VpreB is encoded by either of two highly homologous (Kudo and Melchers, 1987) and apparently redundant genes (Dul et al., 1996) in the mouse. The VpreB1 gene is located 4.6 Kb pairs 5 of the 5 gene (Kudo and Melchers, 1987), while VpreB2 is located at a distal position on the same chromosome (16) (Kudo and Melchers, 1987; Kudo et al., 1987). The homology between the two VpreB genes is also conserved 5 of the coding regions (Kudo and Melchers, 1987), suggesting that they are regulated in a similar fashion (Okabe et al., 1992).

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The VpreB1 promoter was shown to contain EBF-binding sites 130 base pairs 5 of the major transcriptional start site (Fig. 19.3) (Persson et al., 1998), suggesting that VpreB1 is a direct target for EBF activation. The expression of the surrogate light-chain genes in the EBF transfected Ba/F3 cells was upregulated dramatically by coexpression of a homodimer of the E2A-encoded b-hlh protein E47 (Sigvardsson et al., 1997). E47 forms a B-cell—specific homodimer (Bain et al., 1993) due to tissue-specific dephosphorylation of the protein (Sloan et al., 1996). The synergistic activity of E47 and EBF was also shown to occur directly on the 5 promoter (Sigvardsson et al., 1997). A different function for EBF was suggested by the finding that ectopic expression of EBF in plasmacytoma cells represses the function of the IgH intron- (Akerblad et al., 1996) and 3 enhancers as well the Ig intron and 3 enhancers (Akerblad and Sigvardsson, unpublished observations). This finding indicates that EBF may play a dual role, involved in positive regulation of early and negative regulation of latestage B-lineage genes. Such a repressor/activator function is also proposed for BSAP. BSAP acts as an activator of Blk (Zwollo and Desiderio, 1994; Zwollo et al., 1998), mb-1 (Fitzsimmons et al., 1996), Lef-1 (Nutt et al., 1998), and CD19 (Kozmik et al., 1992), while it represses the function of plasma cell—restricted control elements such as the Ig3 enhancer (Neurath et al., 1994) and the J-chain promoter (Rinkenberger et al., 1996). An altered function of EBF during B-cell development is also indicated by the findings that the factor is not as critical for target gene promoter activity in mature B as compared to pre-B cells (Akerblad and Sigvardsson, 1999; Akerblad et al., 1999; Hagman et al., 1991). There are, however, no reports to date examining the exact role of EBF as a repressor or modulator of late-stage genes. EBF is also implicated in the regulation of expression of the transcription factor BSAP due to its ability to activate the gene in dedifferentiated 70Z/3 pre-B cells (Kee and Murre, 1998), further expanding the number of genetic targets for EBF. Despite the identification of many genetic targets for EBF, there is still not any specific target gene that explains the early differentiation block observed in EBF-deficient mice (Fig. 19.1) (Liberg and Sigvardsson, 1999). The

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effect may be a result of a combined loss of several genes defining the pre-B cell stage, some of which are still to be explored.

CONCLUSION The collected findings suggest that EBF has a key role in early B-cell development. However, the presence of B-cell progenitors with expression of B220, CD43-sterile Ig transcripts, and Il-7 receptor in EBF-deficient mice (Lin and Grosschedl, 1995) indicates that EBF is not a fatedetermining factor in B-cell development. This is also supported by the rather broad expression pattern of EBF that extends into stroma cells, adipocytes (Hagman et al., 1993), and the CNS (Wang and Reed, 1993). Such an expression pattern may be incompatible with a classical fate-determining factor such as MyoD (Weintraub et al., 1991). The function of EBF in cell fate determination could be achieved by interactions with other transcription factors, which together create specific combinations that induce lineage commitment. One such possible combination in B-cell development could be high levels of b-HLH proteins such as E47 in combination with EBF. Alternatively, the data accumulated to date may be interpreted to indicate that EBF is essential for the progression of, rather than the initiation of, B-cell development. The current knowledge of the function of EBF in B-cell development has been extracted mainly from experiments in mice. However, the large number of similarities in B-cell development between humans and mice (Ghia et al., 1998), the high homology between mouse and human EBF (Gisler et al. 2000), as well as the fact that the promoters of several EBF target genes are conserved (Dymecki et al., 1992; Leduc and Cogne, 1996; Lin et al., 1995; Omori and Wall, 1993; Thompson et al., 1996; Travis et al., 1991), suggest an important role for EBF in human B-cell development. In the mouse, EBF has been suggested to be a key factor in the determination of the B as opposed to the myeloid lineage, because ectopic expression of EBF in 70Z/3 pre-B cells converted into macrophages resulted in a partial restoration of the pre-B-cell phenotype (Kee and Murre, 1998). This may be of interest in human malignancies where the conversion of B

lymphomas into mixed phenotype or myeloidlike cells is associated with lower remission rates and poor prognosis (Hurwitz and Mirro, 1990). Furthermore, the human chromosomal location of EBF at chromosome 5 band q34 (Milatovich et al., 1994), a region affected in numerous myeloid leukemias (Mitelman et al., 1997), motivates future examination of a potential role for EBF in human disease.

ACKNOWLEDGMENTS I wish to thank Peter Åkerblad and David Liberg for critical reading of the manuscript. This work was funded by the Swedish Medical Research Council, the Swedish Cancer Foundation, the Royal Physiographic Society, and the Magnus Berwalls, Kocks, OJsterlunds, the American Cancer Society and Crafoord foundations.

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PART IV


TRANSCRIPTION FACTORS INVOLVED IN LEUKEMIA DUE TO CHROMOSAL TRANSLOCATION

CHAPTER 20


THE ROLE OF RAR AND ITS FUSION PARTNERS IN ACUTE PROMYELOCYTIC LEUKEMIA ARI MELNICK AND JONATHAN D. LICHT Derald H. Ruttenberg Cancer Center and Department of Medicine, Mount Sinai School of Medicine

INTRODUCTION Since the 1990s, the elucidation of the molecular basis of acute promyelocytic leukemia (APL) emerged as a paradigm for translational research. APL is uniquely responsive to all-trans retinoic acid (ATRA), and clinical trials indicated that ATRA induced complete remissions by differentiation and eventual elimination of the malignant clone (reviewed in Degos, 1994; Fenaux et al., 1997; Fenaux and Degos, 1997; Grignani et al., 1994; Huang et al., 1987, 1988; Tallman, 1996; Warrell et al., 1993). In 1991, it was discovered that translocation (15:17) of APL (Rowley et al., 1977) fused the retinoic acid receptor alpha (RAR) gene to the promyelocytic leukemia (PML) gene, yielding the PMLRAR fusion protein (Alcalay et al., 1991; Chang et al., 1992a; de The et al., 1990a; Goddard et al., 1991; Kakizuka et al., 1991; Pandolfi et al., 1991). This suggested that disruption of RAR function was the major cause of APL. APL is now associated with five different gene rearrangements, fusing RAR to the PML, pro-

myelocytic leukemia zinc finger (PLZF), nucleophosmin (NPM), nuclear matrix—associated (NuMA) and STAT5b genes (Fig. 20.1), leading to the formation of reciprocal fusion proteins (N-RAR and RAR-N). Disprution of both RAR and the partner gene function may both play roles in the pathogenesis of the disease.

THE RETINOIC ACID RECEPTOR Retinoids may be key for myeloid differentiation. Vitamin A—deficient mice and humans have defects in hematopoiesis (Hodges et al., 1977; Wolbach and Howe, 1925), and retinoids preferentially stimulate myelopoiesis (Douer and Koeffler, 1982; Gratas et al., 1993). In the early 1980s, ATRA was found to induce differentiation of the HL60 myeloid cell lines (Breitman et al., 1980) and primary cells from patients with APL (Breitman et al., 1981). The RARs and other members of the nuclear receptor superfamily (Evans, 1988; Glass et al., 1991;


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328

THE ROLE OF RAR AND ITS FUSION PARTNERS IN APL

Figure 20.1. The five chromosomal translocations associated with APL result in fusion proteins in which the B—F domains of RAR, including the DNA-binding and ligand-binding domains of protein, are linked C-terminal to four different nuclear proteins containing self-association domains.

Mangelsdorf and Evans, 1995) were cloned in the late 1980s. Of these, RAR is identified with myeloid development (de The et al., 1989; Gallagher et al., 1989; Largman et al., 1989).

THE TRANSCRIPTIONAL FUNCTION OF RAR RAR and other nuclear receptors contain six evolutionarily conserved domains (A—F). The highly conserved C domain contains two C C zinc-finger motifs that bind to retinoic acid response elements (RARE), which consist of a direct repeat (A/G)G(G/T)TCA separated by two or five nucleotides (reviewed in Chambon, 1996). RAR binds the promoters of many genes, including those of RARs themselves (de The et al., 1990b; Lehmann et al., 1992; Leroy et al., 1991a, 1991b; Sucov et al., 1990). RAR binds to DNA as a heterodimer along with the

related retinoid X receptor protein (RXR) (Gudas, 1994; Naar et al., 1991; Perlmann et al., 1996; Umesono et al., 1991; Zechel et al., 1994). Transcription by RAR is stimulated by ATRA, while its partner RXR responds to ATRA or 9-cis retinoic acid (Mangelsdorf et al., 1992). RAR and other nuclear receptors contain two transcriptional activation domains, AF-1 and AF2, which cooperate to activate transcription (Nagpal et al., 1992). AF-1, contained within the N-terminal A/B domain, is ligand independent (Nagpal et al., 1992, 1993). Through alternative promoter usage, the RAR protein can have two different A domains (A1 or A2). The C-terminal E domain of RAR contains the AF2 ligand-binding and transcriptional activation domain and a dimerization interface for RXR (Nagpal et al., 1993; Perlmann et al., 1996; Zechel et al., 1994). RARs modulate transcription through interaction with cofactors. The AF-2 domain of the protein associates with corepressor molecules in

RAR TARGET GENES

the absence of ligand. These corepressors, NCoR and SMRT (Chen and Evans, 1995; Horlein et al., 1995), are part of a multiprotein repressor complex also containing the Sin3A corepressor and histone deacetylases (Pazin and Kadonaga, 1997, and references therein). This suggests that RARs may silence certain promoters by alterations in chromatin configuration. In the presence of ligand, the conformation of the AF2 domain changes, making new residues available to bind to coactivator proteins (Bourguet et al., 1995a 1995b; Chambon, 1996; Renaud et al., 1995). Such coactivators include TIF1 (LeDouarin et al., 1995; vom Baur et al., 1996) related to the PML protein associated with t(15;17)-associated APL (see below), Trip1/ sug1 (Lee, J. W. et al., 1995), Tif2 (Voegel et al., 1996), ACTR (Chen, H. et al., 1997), Src-1 (Onate et al., 1995), TAF (Mengus et al., '' 1997), CBP (Chakravarti et al., 1996; Kamei et al., 1996; Yao et al., 1996), members of the DRIP complex (Naar et al., 1999; Rachez et al., 1999), and most recently PML itself (Zhong et al., 1999). These factors potentially function as a bridge between ligand-bound RAR and basal factors. They may also function to unwind DNA and to remodel nucleosomes through swi/snf, ATP-dependent mechanisms, and acetylation of chromatin (Bhattacharyya et al., 1997; Minucci and Pelicci, 1999; Wade and Wolffe, 1997). The use of synthetic ligands specific for RAR and RXR indicate that RAR/RXR complexes mediate differentiation by ATRA (Benedetti et al., 1996; Chen, J. Y. et al., 1996; Dawson et al., 1994; Kizaki et al., 1994) whereas RXR/RXR cannot. RARs also repress transcription by other activators such as AP1 (Horvai et al., 1997; Janknecht and Hunter, 1996; Kamei et al., 1996; Pfahl, 1993). However, artificial ligands that inhibit AP1 activity but fail to stimulate RARE-mediated transcription fail to induce myeloid differentiation (Chen, J. Y. et al., 1996; Kizaki et al., 1996).

RAR AND MYELOID DIFFERENTIATION The importance of RAR in myeloid differentiation was empasized by the development of an ATRA-resistant HL60 cell line that harbored a dominant negative mutant RAR with a truncation within the C-terminal AF-2 domain (Col-

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lins et al., 1990; Damm et al., 1993; Robertson et al., 1992a). Such a mutant is believed to sequester the RXR protein into active complexes. Differentiation of these cells under the influence of ATRA was restored by infection with a retrovirus expressing wild-type RAR, RAR, or RAR (Collins et al., 1990; Robertson et al., 1992b). RAR may help program normal hematopoietic development. Erythroid induction of multipotent FDCP mixA4 cells by erythropoietin was correlated with repression of RAR expression while myeloid differentiation induced by G-CSF was correlated with upregulation of RAR (Zelent et al., 1997). Introduction of an RAR mutant, with a deletion in the ligandbinding domain, into a multipotential hematopoietic cell line altered differentiation from the granulocyte/monocyte to the mast cell lineage (Tsai et al., 1992). GM-CSF—mediated myeloid differentiation of these cells was blocked at the promyelocyte stage (Tsai and Collins, 1993). Though truncation of the RAR within the ligand-binding domain has a profound effect on myeloid differentiation, this type of mutation was not identified in a series of 118 specimens of human cancer, including a number of fresh APL specimens (Morosetti et al., 1996). The notion that the dominant negative RAR functions by sequestration of RXR was supported by the finding that overexpression of wild-type RAR (Du et al., 1999; Onodera et al., 1995) led to the accumulation of promyelocytic colonies derived from murine marrow, which differentiated in response to ATRA. Overexpression of wild-type RAR, C-terminal truncated forms of RAR, and fusion proteins consisting of partners fused to the N-terminus of RAR, as in APL, blocks myeloid differentiation at the promyelocyte stage when cells are grown at physiological levels (10\ M) of ATRA. Only pharmacological levels of ATRA (10\— 10\ M) can overcome this. A key issue is the identity of the gene(s) required for myeloid differentiation whose expression is blocked in APL.

RAR TARGET GENES ATRA treatment of myeloid and other cells induces multiple classes of genes (Table 20.1),

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TABLE 20.1. Genes Potentially Regulated by RAR in Myeloid Differentiation Class of Genes Regulators of the cell cycle Cell surface adhesion molecules

Intrinsic host defense systems and extrinsic cytokines Neutrophil granule proteins

Colony-stimulating factors Colony-stimulating factor receptors Regulators of apoptosis and terminal cell division Structural proteins, enzymes, chromatin components Signal transduction molecules Protein degradation Clotting factors

Transcription factors

Examples/References Cyclins, cyclin-dependent kinases, CDK inhibitors (Brooks et al., 1996; Burger et al., 1994; Liu et al., 1996) CD11b, CD18 (Benedetti et al., 1996; Di Noto et al., 1994; Hickstein et al., 1992; Pahl et al., 1992; Rosmarin et al., 1992) Monocyte chemo-attractant factor, interleukins (Burn et al., 1994; Grande et al., 1995) Defensin, secondary granule proteins, leukocyte alkaline phosphatase, lactoferrin (Garattini and Gianni, 1996; Gianni et al., 1994; Herwig et al., 1996; Khanna-Gupta et al., 1994; Lee et al., 1995) IL-1b, IL-8, G-CSF (Dubois et al., 1994a, 1994b; Matikainen et al., 1994) M-CSFR, G-CSFR (Hsu et al., 1993; Tkatch et al., 1995) Transglutaminase II (Benedetti et al., 1996; Nagy et al., 1996), bcl2 (Dipietrantonio et al., 1996) (Davies et al., 1985; Glass et al., 1991; Gudas, 1994; Mader et al., 1994; Scott et al., 1996), CRABPII (Astrom et al., 1994; Delva et al., 1993). Calmodulin kinase (Lawson et al., 1999), GPI-linked protein (Mao et al., 1996) LMP7 and UBE1L-ubiquitnation pathway (Tamayo et al., 1999) Thrombomodulin, tissue factor, urokinase, tissue plasminogen activator and its inhibitors (Falanga et al., 1995; Koyama et al., 1994; Tapiovaara et al., 1994) RARs (Chomienne et al., 1991; Gudas, 1994; Leroy et al., 1991b), STATs (Gianni et al., 1997), Hox genes (Krumlauf, 1994; Marshall et al., 1996), C/EBP-, STAT1 (Gianni et al., 1997), Jem1 (Duprez et al., 1997)

Adapted from (Melnick and Licht, 1999).

expressed immediately after ATRA treatment or after a delay. This is accompanied by inhibition of cell growth, induction of terminal differentiation, and production of a mature neutrophil. The retinoic acid syndrome encountered during treatment of APL with ATRA, characterized by a rise in leukocyte count, fever, and pulmonary infiltrates, may be due to the increased adhesive characteristics of the differentiating granulocytes and secretion of cytokines (Frankel et al., 1992). ATRA also represses expression of procoagulants found in the promyleocyte, explaining why ATRA treatment of APL relives the caogulopathy associated with the disease (reviewed in Barbui et al., 1998). The initial waves of leukocytes found in APL patients are derived from the malignant clone (Vyas et al., 1996) and

function normally in vitro to kill pathogens (Glasser et al., 1994), despite abnormalities of secondary granules (Khanna-Gupta et al., 1994; Miyauchi et al., 1997). Many of the initial target genes of RAR are themselves transcription factors (Gudas, 1994). RAREs are present in the promoters of the RARs, and ATRA treatment of APL cells induces the mRNA for RAR (Chomienne et al., 1991; Leroy et al., 1991b). One way that ATRA may induce myeloid differentiation may be to upregulate the RAR/RXR complexes and induce the degradation of PML-RAR (Raelson et al., 1996; Yoshida et al., 1996), shifting the balance toward the wild-type receptor. Many hox genes (see Chapter 9 in this book) expressed in myeloid cell lines in a regulated manner (Magli et

PML

al., 1991) contain RAREs and may be directly regulated by RARs (Frasch et al., 1995; Langston and Gudas, 1992; Langston et al., 1997; Marshall et al., 1994; Ogura and Evans, 1995). ATRA rapidly induces transcription of the IFN regulatory factor-1 (IRF-1), which activates expression of IFN and other genes (Matikainen et al., 1996). ATRA induction of the IRF-1 promoter is mediated by a GAS (-IFN activation sequence) (Pelicano et al., 1997) that binds STAT proteins (Darnell, 1997; see Chapter 15). ATRA rapidly induces the STAT1 expression, tyrosine phosphorylation of STAT1, leading to increased binding of an IFN-responsive element (IRE) (Gianni et al., 1997). Recently, C/EBP-, a basic-zipper transcription factor that recognizes CCAAT sequences, was found to be rapidly induced by ATRA. C/EBP- is the only C/EBP factor expressed in the APL cell line NB4, suggesting a role in promyelocyte differentiation (Antonson et al., 1996; Chih et al., 1997; Morosetti et al., 1997; Yamanaka et al., 1997). HL60 cells that express the PML-RAR oncoprotein show reduced C/ EBP- expression in the absence of ATRA, and expression is restored with ATRA treatment (Park et al., 1999). Hence C/EBP- may be a model target gene of the PML-RAR fusion protein. ATRA alters the cell cycle as it causes differentiation (Drach et al., 1993; Zhu et al., 1995), inducing G1 arrest and the accumulation of hypophosphorylated Rb protein (Brooks et al., 1996). ATRA induces the expression of the p215$! cyclin-dependent kinase inhibitor in myeloid cells (Bocchia et al., 1997; Liu et al., 1996). RAR, in combination with RXR, binds and activates the p21 promoter in a liganddependent manner. Therefore, p21 meets criteria for a bona fide RAR target gene whose expression could decrease leukemic cell proliferation. Other recently identified putative RAR targets are noted in Table 20.1.

nine exons encoding mRNAs of 4.6, 3.0, and 2.1 kb. Alternative splicing of C-terminal exons yields up to 20 different isoforms of the protein (Chang et al., 1992a, 1992b; de The et al., 1991; Fagioli et al., 1992; Goddard et al., 1991; Grignani et al., 1994; Kakizuka et al., 1991; Stadler et al., 1995). The longest cDNA open reading frame yields a 560 Aa polypeptide with a predicted MW of 70 kDa (de The et al., 1990a; Fagioli et al., 1992). The PML protein has several domains:

· A cystein-rich region (Aa 57-222) with

·

· · ·

PML PML Gene and Protein Structure The t(15;17) rearrangement involving the PML gene on chromosome 15q22 is found in about 98% of cases of APL (Grignani et al., 1994). The PML gene is spread over 35 kb and contains

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three structures. The first is the RING finger with the configuration C HC (Aa 57—91) (reviewed in Freemont, 1993; Reddy and Etkin, 1992; Saurin et al., 1996), a protein interaction motif, spherically organized around two Zn>> ions that coordinately bind cysteine and histidine (Borden and Freemont, 1996; Borden et al., 1995). The following two are called B-box zinc fingers (Aa 140—161 and Aa 189—222) (de The et al., 1991; Fagioli et al., 1992; Goddard et al., 1991; Kakizuka et al., 1991). The RING finger/B-box region is involved in localization of PML into nuclear bodies (NB) (Borden et al., 1996; Grignani et al., 1994, 1996). A helical coiled-coil region (Aa 229—360) consisting of eight heptad repeats. This region is responsible for multimerization of PML, heterodimerization with PMLRAR and plays a role in NB localization (Grignani et al., 1996; Kastner et al., 1992; Le et al., 1996; Perez et al., 1993). An N-terminal proline-rich sequence (Aa 1—46) that binds viral proteins (Borden et al., 1998, 1998b). A basic sequence containing a nuclear localization signal (Aa 476—490) (Kastner et al., 1992; Le et al., 1996). An acidic C-terminal Ser/Pro-rich domain of unknown function (Chang et al., 1992b; de The et al., 1991; Fagioli et al., 1992; Goddard et al., 1991; Kakizuka et al., 1991; Le et al., 1996).

PML, expressed after transfection, is found as a set of 70—100 kDa protein bands (Chang et al., 1995; Daniel et al., 1993; Flenghi et al., 1995).

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Endogenous PML is detected as a 90 kDa species along with a variety of other protein species (150—50 kDa), due to alternative splicing and covalent modifications (Chang et al., 1995; Chelbi-Alix et al., 1995; Grotzinger et al., 1996b; Kamitani et al., 1998a, 1998b; Mu¨ller et al., 1998a; Sternsdorf et al., 1997). PML is phosphorylated on serine, and to a lesser extent on tyrosine residues (Chang et al., 1993; Grignani et al., 1993, 1994; Kastner et al., 1992). Some of the sites may be cell cycle dependent (Mu et al., 1996), and PML was found to be an in vitro substrate for phosphorylation by cyclin A/cdk2 (Chang et al., 1995; Mu et al., 1996). PML Expression PML mRNA is widely expressed, and the pattern of PML protein expression in tissues is complex and controversial (Grignani et al., 1994; Terris et al., 1995). In addition, PML mRNA and protein expression are often not concordant. PML is expressed in inflammatory diseases such as psoriasis and hepatitis, inflammatory cells surrounding epithelial cancers and Hodgkin’s disease, lesions of graft versus host disease, and activated fibroblasts (Aractingi et al., 1997; Gambacorta et al., 1996; Koken et al., 1995; Terris et al., 1995). This suggests induction of PML by soluble factors such as IFNs (see below). Some groups found a correlation between the level of PML expression and degree of dysplasia in precancerous breast and cervical lesions. When the breast tumors became invasive, PML expression decreased (Gambacorta et al., 1996; Koken et al., 1995; Terris et al., 1995). PML delocalization can be associated with neoplasia. Hepatic carcinoma was associated with mislocalization of PML in the cytoplasm rather than the nucleus (Terris et al., 1995). PML is induced by hormones such as estrogen and cytokines such as IFN (Chelbi-Alix et al., 1995; Koken et al., 1995; Lavau and Dejean, 1994; Nason-Burchenal et al., 1996; Terris et al., 1995). PML is expressed in myeloid precursors (Daniel et al., 1993; Nason-Burchenal et al., 1996) and in circulating monocytes and granulocytes with IFN inducing its expression. This suggests an early role for the protein in myeloid differentiation and later in host defense (Daniel et al., 1993; Flenghi et al., 1995; Gambacorta et al., 1996; Terris et al., 1995; Wang et al., 1996).

Nuclear Bodies and PML Expression One of the most striking features of PML is its speckled localization to discrete nuclear domains termed PODs (PML oncogenic domains), ND10 (nuclear domain—10), or nuclear bodies (NB) (Daniel et al., 1993; Dyck et al., 1994; Kastner et al., 1992; Maul et al., 1995). Recent knockout data indicate that proper formation of the NB requires the presence of PML (Wang et al., 1998; Ishovetal, 1999; Zhong et al., 2000). These 0.3—0.5 m structures, usually 10— 20 per cell, were originally described in 1960 (de Th et al., 1960) and are detected by human autoimmune antisera (Andre et al., 1996; Brasch and Ochs, 1992; Dyck et al., 1994; Koken et al., 1994; Szostecki et al., 1997; Weis et al., 1994; Zuchner et al., 1997). Nuclear bodies vary in both size and number in different cell types. Their presence is roughly proportional to the rate of protein synthesis and inversely proportional to differentiation (Lam et al., 1995). Strikingly, PML is delocalized from the NBs to a microspeckled nuclear pattern in t(15;17) APL cells and relocalizes to the NB after ATRA treatment (Daniel et al., 1993; Dyck et al., 1994; Koken et al., 1994). In the NB, PML and other proteins surround an electron dense core that may contain ribonucleic acid (Doucas and Evans, 1996; Koken et al., 1994; Weis et al., 1994). NBs are associated with the nuclear matrix, which plays a role in trafficking of molecules and organization of chromatin within the nucleus. In initial studies, the NBs did not overlap with spliceosomes, centromeres, sites of RNA transcription (Dyck et al., 1994; Grande et al., 1996; Weis et al., 1994), or the expression pattern of sequence-specific or general transcription factors (Grande et al., 1996). This appeared to exclude a transcriptional role for the NB. However, more recent studies found nascent mRNA in the center of the NB structure (LaMorte et al., 1998) and that the transcriptional coactivator CBP was found colocalized in the NB with PML, which suggests that the NB may play a role in stimulating transcription. Whether the NB might be a site of transcriptional initiation, elongation, or processing of the new mRNA transcript is unknown. PML-containing nuclear bodies do not colocalize with sites of nascent DNA (Dyck et al., 1994; Weis et al., 1994) except during mid-S

PML

phase when they are found adjacent to replication sites (Grande et al., 1996). PML can also be found in the nucleoplasm in a diffuse staining pattern (Maul et al., 1995) as well as in a cytoplasmic granular pattern (Flenghi et al., 1995; Nason-Burchenal et al., 1996). Although the exact site and mechanism of PML is still unclear, nuclear expression of PML is essential for its biological function, since PML mutants lacking the NLS cannot suppress oncogenic transformation (Le et al., 1996). However, certain spliced isoforms of PML, devoid of the C-terminal NLS, are found exclusively in the cytoplasm (Flenghi et al., 1995), and others that do contain the NLS can be found in both the nucleus and cytoplasm. Hence a cytoplasmic role for the protein is not ruled out. The partition of PML between the nucleoplasm and the NBs may be controlled by processes such as differential phosphorylation and conjugation of PML to a ubiquitin-like molecule, SUMO-1 (Mu¨ller et al., 1998a). The presence of the RING finger, B-box motifs, and coiled-coil motifs is necessary for PML to properly localize in the NBs (Le et al., 1996). Mutants lacking these structures sequestered normal PML from the NBs in a dominant negative manner (Flenghi et al., 1995; Le et al., 1996). In addition, forms of PML with mutations in critical cysteine residues required for normal protein folding failed to localize in the NBs (Borden et al., 1995, 1996; Kastner et al., 1992). PML expression and NB structure are dynamic. Augmenting the cellular levels of PML by transfection or IFN treatment increased the size and number of nuclear bodies, possibly due to deposition of PML and recruitment of other proteins (Chelbi-Alix et al., 1995; Koken et al., 1994; Lavau et al., 1995; Maul et al., 1995; Nason-Burchenal et al., 1996). In contrast, PML-RAR caused disappearance of nuclear bodies from the nucleus (Koken et al., 1994). PML expression changes across the cell cycle. Cells in G exhibit few nuclear bodies and show weak staining for PML, while the number of NBs and intensity of expression rise in cycling cells. As cells progress through S to G2 phase, PML disperses to multiple smaller dots and gradually fades, with two or three of these residual structures left in mitosis (Chang et al., 1995; Koken et al., 1995; Terris et al., 1995). Prolonged amino acid starvation (Kamei, 1996)

333

or induction of cell senescence (Jiang and Ringertz, 1997) induces the coalescence of the NBs into two or three large structures with the normal pattern restored by the readdition of nutrients (Kamei, 1996). The pattern of PML expression also responds to cell stresses such as heat shock (Maul et al., 1995), -irradiation (Doucas and Evans, 1996), and viral infection. How these changes relate to the function of the nuclear body is unknown. PML and Transcription Transcriptional properties of PML have been controversial. PML inhibited transcription of the MDR- and EGF-receptor (EGF-R) promoters (Mu et al., 1994) and enhanced transcription of the CD18 and TAP-1 promoters (Mu et al., 1994; Zheng et al., 1998). Furthermore, PML increased the transcriptional activity of the progesterone receptor, the mineralocorticoid, glucocorticoid (GR), and androgen receptors, but not the RARs (Guiochon-Mantel et al., 1995), although a more recent report did indicate that PML could coactivate with RAR (Zhong et al., 1999). The physiological relevance of this interaction must be tempered by a report that PML does not colocalize with the GR or with RNA polymerase II (Grande et al., 1996). A recent report also indicated that PML can stimulate transcription mediated by fos and jun (Vallian et al., 1998). In contrast, GAL4-PML fusions repressed transcription (Kuehnle et al., 1997; Vallian et al., 1997), though an earlier publication did not find this effect (Kastner et al., 1992). The coiled-coil, not the RING-finger, motif was required for repression, and the nucleoplasmic fraction of PML may be responsible for this effect. It is tempting to speculate that PML, like PLZF (see below), interacts with histone deacetylase corepressors. It was recently found that PML repression of the EGF-R promoter was mediated through Sp1 sites and that PML inhibited DNA binding by Sp1 (Vallian et al., 1998a). These data suggest that under certain circumstances, the PML protein could modulate gene expression by direct interaction with specific transcription factors. This function might be abrogated by PML-RAR, which when coexpressed with Gal4-PML, leads to simulation of transcription rather than repression (Vallian et al., 1997).

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PML Partner Proteins Nearly 15 natural components of the nuclear body have been identified. The diverse nature of these proteins and the fact that they are often found in other subnuclear patterns suggests that the NBs may not be a site of active cellular metabolism but rather a storage site for nuclear proteins. However, several of these proteins are induced by IFN, including PML, SP100, and PLZF (Bloch et al., 1996; Gongora et al., 1997; Grotzinger et al., 1996a, 1996b; Koken et al., 1997), pointing to the notion that the NBs play a role in cellular proliferation and/or the antiviral response. Only a few proteins are known to actually bind directly to PML. The first of these identified is SUMO-1, originally called PIC1 (Boddy et al., 1996), also called sentrin and UBL-1 (Mahajan et al., 1997; Shen et al., 1996). SUMO-1 is a ubiquitously expressed 11.5 kDa peptide that contains a ubiquitin homology (UbH) domain (Boddy et al., 1996) and covalently attaches to proteins, suggesting that SUMO-1 may play a role in protein turnover (Gong et al., 1997). One group found that overexpression of SUMO-1 protected cells from fas/apo and TNF-induced apoptosis (Okura et al., 1996). Given that PML is a growth suppressor, it is possible that PML sequesters SUMO-1, promoting apoptosis and balancing the function of SUMO-1. However, it is more likely that SUMO-1 modifies the function of PML and other components of the nucleus. SUMO-1 binds to PML in the RING-finger motif, the B-box domain, and the more Cterminal nuclear localization signal of PML (Kamitani et al., 1998a, 1998b; Muller et al., 1998a; Sternsdorf et al., 1997), and it was suggested that SUMO-1 modification targets PML to the NB. The PLZF protein may be a key interaction partner of PML. The PLZF protein colocalizes with PML in myeloid cells (Koken et al., 1997). Colocalization was incomplete, and one group estimated only about 30% overlap of PML and PLZF (Ruthardt et al., 1998). Nevertheless, interaction between PML and PLZF was demonstrated by coprecipitation and was found to be mediated by the coiled-coil domain of PML and the first two zinc fingers of PLZF (A. Zelent, personal communication). PLZF could be de-

localized to a microspeckled pattern in NB4 cells and reverted to the NBs upon treatment with retinoic acid. These findings suggest that PML may be involved the transcriptional activity of PLZF or that PML could sequester PLZF within the NBs (Koken et al., 1997). Thus, PML and PLZF may impact on a common pathway in APL. PML can also be coprecipitated with a small amount of the underphosphorylated form of Rb but not the p107 or p130 (Alcalay et al., 1998). The functional consequences of this interaction in growth control are uncertain; however, it is possible that PML inhibits cell growth by limiting RB phosphorylation. Finally, novel functions for PML are suggested by its association with the ribosomal P proteins (Borden et al., 1998b) and EF-1 (Boddy et al., 1996), which are involved in the processes of translation and RNA transport, respectively (Uchiumi and Ogata, 1986; Uchiumi et al., 1987; Yacoub et al., 1996). The nucleophosmin (NPM) protein, fused to RAR in t(5;17) APL (Redner et al., 1997), is involved in ribosome biogenesis and shuttling ribonucleoproteins between the nucleus and cytoplasm (Borer et al., 1989; Dumbar et al., 1989; Yung et al., 1985a). This places both PML and NPM in a single fuctional axis. PML, Interferon, and Viral Infection PML is IFN responsive and may be a primary target of IFN action (Chelbi-Alix et al., 1995; Lavau and Dejean, 1994; Nason-Burchenal et al., 1996; Stadler et al., 1995). STATs induce expression of multiple components of the NB, including Sp100 (Grotzinger et al., 1996a, 1996b). PML may mediate the antiproliferative and antiviral effects of IFN (Stadler et al., 1995). One of the earliest effects of viral infections is the targeting of viral products to NBs, often resulting in the reorganization of PML expression. This effect was observed for adenovirus (Doucas et al., 1996; Puvion-Dutilleul et al., 1995), herpes simplex virus (Everett et al., 1995), and Epstein-Barr virus (Jiang et al., 1996; Szekely et al., 1996). Though the data are conflicting, PML may be required for the antiviral effect of interferon on some viruses (Chelbi-Alix et al., 1998). Viruses may target and disrupt the nuclear body in order to abrogate an apoptotic program. The delocalization of NB proteins

PML

caused by PML-RAR expression could mimic viral infection, resulting in uncontrolled cellular proliferation (Doucas et al., 1996). PML, Growth Suppression, and Apoptosis NB4 cells selected for expression of exogenous PML harbored mutant forms of PML, suggesting that the wild-type protein was toxic (Ahn et al., 1995; Mu et al., 1994) and PML expression suppressed the ability of the cells to form colonies in soft agar (Mu et al., 1994). PML-overexpressing NB4 cells also yielded smaller tumors in nude mice. Another group demonstrated that even low levels of PML were toxic to NB4 cells, suggesting that the reduced dosage of PML in APL cells could contribute to uncontrolled growth (Ahn et al., 1995). PML also inhibited transformation by Ha-ras and other oncogenes (Koken et al., 1995; Liu et al., 1995; Mu et al., 1994). In HeLa and breast cancer cells, PML arrested cells in G1 (Mu et al., 1997) and decreased cyclins and cdks (Le et al., 1998). A structure/function analysis of the PML protein indicated that deletion or mutation of the RING-finger motif of PML abrogates both NB formation and growth suppression (Borden et al., 1995, 1997; Kastner et al., 1992; Le et al., 1996). Deletion of the coiled-coil motif yielded a diffuse nuclear pattern of expression and no growth suppression, while deletion of the NLS of PML or expression of a splice variant of PML led to a granular cytoplasmic pattern of expression and abolished growth suppression (Fagioli et al., 1998). Correct localization of PML appears to play an important role in apoptosis and cell growth. PML may promote cell death by novel mechanisms. PML interaction with the P0 ribosomal protein (Borden et al., 1998b) could target the 28S rRNA for cleavage, an important step during apoptosis. PML expression yields cell death, which is not associated with the usual chromatin condensation or activation of caspase 3 (Quignon et al., 1998). Paradoxically, a caspase inhibitor accentuated PML-mediated apoptosis and increased PML expression. Experiments in PML null animals yielded different results with such cells showing decreased apoptosis of T cells after -irradiation and fas ligand treatment (Wang et al., 1998b). Furthermore, PML null animals were resistant to ce-

335

ramide-, TNF- and IFN-mediated apoptosis. Therefore, high expression of PML may induce apoptosis in the absence of caspase activation, whereas low levels may be required for normal caspase activation (Hess and Korsemeyer, 1998). Recent data suggest that PML may regulate fas-mediated apoptosis through interaction with Daxx, a putative transcriptional regulator (Torii et al., 1999). Experiments in PML null animals supports a tumor suppressor function for the protein (Wang et al., 1996, 1998). Such mice were very susceptible to fungal or bacterial infections (Wang et al., 1998c), suggesting a functional defect in the inflammatory cells. Circulating and bone marrow mature myeloid cell counts were modestly decreased, suggesting that PML may be involved in efficient terminal differentiation of these cells. Skin of PML\\ animals treated with DMBA formed increased numbers of papillomas while animals treated with systematically DMBA developed lymphomas at an increased rate. PML null murine embryo fibroblasts proliferated more rapidly than wild-type fibroblasts and more readily formed colonies in soft agar. In addition, PML null cells seemed immune to the growth-suppressive effects of ATRA and IFN (Wang et al., 1998c). An important target gene of RAR, p21WAF1/CIP1 could not be upregulated in the PML\\ fibroblasts. Thus, PML might be required for certain pathways of retinoid signaling. PML may also influence tumor immunity. PML stimulates expression of MHC class I antigens (Zheng et al., 1998). Hence disruption of PML expression might lead to decreased presentation of antigens and defective immune surveillance for tumors. Lastly, PML is induced by ionizing radiation (Chan et al., 1997) or a DNAdamaging agent, suggesting that PML might be considered a GADD (growth arrest and DNA damage) gene (Fornace et al., 1992). Very recent studies implicated PML in cellular senescence and the p53 pathway (Febeyre et al., 2000, Pearson et al., 2000). The ras oncogene induced expression of PML during cell senescence. In addition, PML deficient cells did not undergo growth arrest and senescence in the presence of oncogenes. This finding was correlated with a lack of p53 acetylation and function and suggested that the PML-NB could be a site of protein modification, possibly by CBP.

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In view of the emerging evidence, it is reasonable to describe PML as a tumor-suppressor protein, involved in the growth suppression, differentiation, and immune response pathways of certain cytokines such as IFN. The mechanism by which PML encourages growth arrest is unclear. PML induces apoptosis in the absence of new protein synthesis (Quignon et al., 1998) and may act in both a caspase-dependent and -independent manner (Hess and Korsemeyer, 1998), suggesting that its role in transcription may be secondary to its role in growth control. Apoptosis by PML is intimately related to its localization in the NB organelle. Delocalization of PML by PML-RAR may be a critical step in the pathogenesis of APL. Supporting this notion, PML-RAR inhibited fas-mediated suppression of myeloid growth. When expressed in the PML>\ background, PML-RAR further inhibited apoptosis, suggesting that PML-RAR works in part by subverting normal PML function (Wang et al., 1998c). A balanced model indicates that PML-RAR is leukemogenic, with wild-type PML acting to oppose the effect. In accordance with this, mice harboring a PMLRAR transgene developed APL rapidly in the PML null background, and APL developed with increased latency in PML\> and PML>> mice (Wang et al., 1998a).

PML-RAR Structure Translocation (15;17) fuses the RAR and PML genes and generates a PML-RAR fusion transcript (Baranger et al., 1993; Borrow et al., 1994; Goddard et al., 1991; Hiorns et al., 1994; Lo Coco et al., 1992; McKinney et al., 1994). Comparison of the PML-RAR cDNA structures obtained by multiple groups showed variation in the amount of PML sequences included in the fusion protein (Chang et al., 1992a; de The et al., 1991; Kakizuka et al., 1991; Kastner et al., 1992; Pandolfi et al., 1991). The RAR portion was invariant, containing the DNA-binding and ligand-binding motifs (B—F domains) (Pandolfi et al., 1992). The PML sequence variation seen among patients was generated by heterogeneous breakpoint cluster regions (bcr1, bcr2, bcr3) as well as by alternative splicing (Chang et al., 1992a, 1993b; Dong et al., 1993; Geng et al., 1993;

Pandolfi et al., 1992; Tong et al., 1992). The most 5 breakpoint, bcr3, yields short PML-RAR fusion proteins (PML(S)-RAR). Bcr1 is more 3 and yields 554 amino acids of PML to RAR (PML(L)-RAR). Breakage in bcr2 involves sites in and around exon 6 of PML and leads to an intermediate length of PML sequence (PML(V)RAR). In general, 70% of patients exhibit PML(L)-RAR, 20% PML(S)-RAR, and 10% PML(V)-RAR (Fenaux and Chomienne, 1996; Slack et al., 1997), with PML(S)-RAR and PML(L)-RAR representing extremes of contiguous PML sequence fused to RAR. Internal splicing of portions of PML exon 3 led in one patient to a small PML-RAR fusion protein that contained the RING fingers, B boxes, and the first two portions of the -helical coiled-coil domain, representing the minimal PML moiety required for oncogenicity (Pandolfi et al., 1992). Detection of the PML-RAR fusion transcript by RT/PCR is a sensitive (Chen et al., 1992) and specific test for the diagnosis of APL and can be used to measure minimal residual disease after chemotherapy, differentiation therapy, and bone marrow transplantation (reviewed in Lo Coco et al., 1999). Reappearance of PML-RAR transcripts in the marrow often precedes a frank leukemic relapse. The mechanism by which the t(15;17) translocation occurs is not clear; however, a recent study found that the PML and RAR genes are physically adjacent to each other in chromatin (Neves et al., 1999), this perhaps explaining the tendency of the two genes to rearrange. The PML(L)-RAR fusion transcript yields proteins of 110 and 120 kDa and PML(S)RAR species of 103 and 90 kDa (Chang et al., 1995; Jansen et al., 1995; Kastner et al., 1992), possibly resulting from alternative-start codons (Chang et al., 1992a, 1992b; de The et al., 1991; Kakizuka et al., 1991; Kastner et al., 1992; Pandolfi et al., 1991) and posttranslational modifications (Jansen et al., 1995; Kastner et al., 1992). In APL cells, PML-RAR is a major form of the retinoid receptor (Jansen et al., 1995; Pandolfi et al., 1992). Protein-Protein Interactions PML-RAR oncoprotein, an aberrant retinoid receptor with altered DNA-binding activity (Chang et al., 1992b; de The et al., 1991; Kakizuka et al., 1991; Kastner et al., 1992;

PML-RAR

Pandolfi et al., 1991; Perez et al., 1993), can bind RAREs as a homodimer (Jansen et al., 1995; Perez et al., 1993) whereas wild-type RAR cannot (Leid et al., 1992). Homodimerization requires the coiled-coil domain of PML, which also mediates PML-RAR/PML association. PML-RAR homodimers are distinct DNAbinding species in NB4 cells (Jansen et al., 1995) and display weaker affinity for certain artificial and natural RARE sites than RAR/RXR heterodimers (Jansen et al., 1995). When combined with RXR, PML-RAR preferentially forms multimeric complexes on the RARE (Jansen et al., 1995; Licht et al., 1996; Perez et al., 1993). Hence the existence of the PML-RAR homodimer complex in NB4 extracts probably reflects the high level of expression of PMLRAR relative to wild-type RAR and RXR in APL cells. When bound to RAREs along with RXR, PML-RAR displays the same bindingsite preference as wild-type RAR. Multimeric complexes from transfected cells may reflect the ability of PML-RAR/RXR heteromers on one DNA-binding site to associate through the PML coiled-coil domain with heteromers on other sites. In the cell, this could reflect the ability of PML-RAR to sequester RXR. In addition, PML-RAR can be purified from cells as a 600—1300 kDa complex, supporting the idea that PML-RAR multimerizes and associates with other proteins (Benedetti et al., 1997; Nervi et al., 1992). Reinforcing these studies, confocal microscopy showed that PML-RAR draws RXR away from its usual subnuclear localization (Kastner et al., 1992). Hence, PMLRAR may affect ATRA-mediated signaling by: (1) binding of PML-RAR homodimers to novel genes, (2) binding of PML-RAR with RXR to RAR target genes in competition with RAR, and (3) sequestration of RXR via high levels of PML-RAR in APL cells. Transcriptional Activity of the PML-RAR Fusion Protein Many groups observed that in the absence of ATRA, PML-RAR represses transcription from RAREs to a greater extent than RAR (Kakizuka et al., 1991; Kastner et al., 1992; Rousselot et al., 1994). There were conflicting reports regarding transcriptional activation by PML-RAR. In some reports, both PML-RAR (S) and (L) stimulated ATRA-mediated transac-

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tivation more strongly than RAR (Kakizuka et al., 1991; Pandolfi et al., 1991) whereas others found that PML-RAR activated weakly or not at all (Kastner et al., 1992). When coexpressed with RAR, PML-RAR behaved in a dominant negative fashion, reducing activation by RAR alone (de The et al., 1991; Rousselot et al., 1994), which could be due in part to sequestration of the essential RAR cofactor RXR as well as PML, which may also potentiate RAR function. RAR and PML-RAR possess similar dissociation constants for ATRA binding (:10\ M) (Kastner et al., 1992; Nervi et al., 1992). Hence the altered transcriptional activity of the PML-RAR fusion protein is not related to impaired ability to bind ATRA but may be do to an inability of ATRA to alter the PMLRAR conformation and change it into an activator. The impaired ability of the PML-RAR protein to activate certain promoters is not likely due to deletion of the A domain of RAR, but is probably due to the ability of PML-RAR to bind the corepressors SMRT and N-CoR more tightly than wild-type RAR requiring pharmacologic doses of ATRA (10\ M) for disassociation. This is in marked contrast to the physiologic heterodimer of RAR-RXR, which releases corepressors at 10\ M ATRA (Grignani et al., 1998; Guidez et al., 1998; Hong et al., 1997; Lin et al., 1998). The chimeric protein had increased affinity for SMRT and N-CoR despite the fact that neither corepressor bound to PML in vitro nor colocalized in vivo. This may be explained by the finding that PML-RAR homodimers bind two co-repressor molecules, while RAR/RXR heterodimers only bind one (Lin and Evans, 2000). The corepressors N-CoR and SMRT are part of a multiprotein complex that includes histone deacetylases (HDACs). Deacetylation of histones alters the conformation of chromatin and its accessibility to the transcriptional machinery, resulting in transcriptional silencing (Pazin and Kadonaga, 1997). PML-RAR associates with HDAC1 (histone deacetylase 1) (Grignani et al., 1998; Lin et al., 1998), and this may be the basis of the block of critical myeloid gene expression at physiological doses of ATRA. Sodium butyrate (Candido et al., 1978) or trichostatin A (TSA) (Yoshida et al., 1995) relieves the ability of PML-RAR to inhibit transcription and stimulate activation mediated by PML-RAR

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(Grignani et al., 1998; Guidez et al., 1998; He et al., 1998a; Lin et al., 1998). This could explain the synergistic effect of sodium butyrate and ATRA to accelerate differentiation of APL (Chen et al., 1994; Guidez et al., 1998; He et al., 1998a). PML-RAR also affects other important transcriptional pathways. RAR inhibits transcriptional activation by AP1 protein, possibly by a competition for p300 and/or CBP (Chakravarti et al., 1996; Kamei et al., 1996). Paradoxically, PML-RAR stimulates AP1 activity in the presence of ATRA (Doucas et al., 1993), though the mechanism for this is unclear. PML-RAR inhibits transcription by the vitamin D receptor (VDR) as well as the peroxisome proliferator (PPAR) by sequestration of RXR (Jansen et al., 1995; Perez et al., 1993). Lastly, while RAR and STAT1 synergistically stimulate transcription thorugh STAT-binding sites, PML-RAR cannot (Gianni et al., 1997). Distillation of these studies indicate that PML-RAR has substantially different transcriptional properties from wild-type RAR. The most important is its ability to act as a dominant negative form of RAR, inhibiting RAR target gene expression in the absense of ATRA and having an altered ability to activate RAR target genes in the presence of ATRA. The molecular basis of this phenomenon appears to be the ability of the protein to bind to corepressor complexes with more avidity than the wild-type receptor. PML-RAR and Retinoid Resistance ATRA-resistant APL cell lines derived by X-ray mutagenesis (Dermime et al., 1993) were found to lose expression of the PML-RAR protein (Dermime et al., 1993) due to a protease activity (Fanelli et al., 1997). This highlights the importance of the fusion protein both in generating APL phenotype and in mediating the ATRA sensitivity, and implies that there are secondary changes in APL cells that maintain the transformed phenotype. Resistant APL cells were also generated by prolonged culture of NB4 cells (Lanotte et al., 1991) in ATRA (Duprez et al., 1992; Rosenauer et al., 1996). Such cell lines harbor missense mutation in the ligand-binding domain PML-RAR (Shao et al., 1997) and were defective for transcriptional activity, suggesting that therapeutic response to ATRA in

APL is dependent on transcriptional activation by the fusion protein. However, some RAR targets can be induced in these cells, which indicates that the remaining endogenous RARs within the cell can activate a subset of target genes, but the genes most critical for cell differentiation continue to be inhibited by PMLRAR. These types of mutations are clinically relevant, as recent studies revealed mutations in the ligand-binding domain or adjacent AF-2 region in nearly 15% of patients, particularly after prolonged ATRA treatment (Ding et al., 1998; Imaizumi et al., 1998), indicating that ATRA treatment selects for clones with defects in PML-RAR (Ding et al., 1998). PML-RAR and the Nuclear Body Whereas PML is localized in 6—30 large nuclear bodies/cell (Dyck et al., 1994), in APL it is delocalized to 100 small (0.1 m) microspeckles (Weis et al., 1994) due to the ability of PML to heterodimerize with PML-RAR through the coiled-coil motif (Dyck et al., 1994; Kastner et al., 1992). PML-RAR draws other nuclear proteins including SP100 (Koken et al., 1994), PLZF (Koken et al., 1997), RXR (Weis et al., 1994), and Rb (Alcalay et al., 1998) into the microspeckled structure as well. These microspeckles colocalize with nascent RNA, signifying the transcriptional function of PML-RAR (Dyck et al., 1994; Grande et al., 1996). In at least one set of studies, a large proportion of PML-RAR fusion was localized in the cytoplasm rather than the nucleus (Daniel et al., 1993), consistent with the notion that PMLRAR could draw critical factors away from loci controlled by RAR. ATRA treatment of APL cells relocalizes the PML protein into the wild-type nuclear body configuration (Daniel et al., 1993; Duprez et al., 1996; Dyck et al., 1994; Koken et al., 1994; Weis et al., 1994). This is largely due to degradation of PML-RAR (Duprez et al., 1996a; Dyck et al., 1994; Raelson et al., 1996; Yoshida et al., 1996; Zhu et al., 1997) through the action of the proteosome (Fanelli et al., 1997), likely by the induction of a caspase 3—like activity after ATRA treatment (Nervi et al., 1998). The specific cleavage of PML-RAR occurs C-terminal to the the RING, B-boxes, and coiled-coil motifs of PML, yielding a product recognized by RAR

PML-RAR

antibodies that contains residual PML sequences (Raelson et al., 1996; Yoshida et al., 1996). The resulting protein could be predicted to be unable to bind wild-type PML, which would then be released and free to form its usual macromolecular complex in the NB. The remaining truncated PML-RAR protein might function in a similar fashion as wild-type RAR, activating its target genes and no longer sequestering other proteins critical for cell differentiation through the N-terminal PML moiety. However, the PML(S)-RAR isoform does not contain the sequences required for caspase cleavage and does not undergo degradation after ATRA, yet these patients respond to ATRA therapy (Nervi et al., 1998; Slack and Yu, 1998). In addition, in a model system ATRA could induce differentiation in the presence of caspase inhibitors, suggesting that the degradation of PML-RAR may not be essential (Nervi et al., 1998). Arsenic trioxide (As O ), a component of traditional Chinese medicine, induces complete clinical remission of APL in ATRA-resistant patients (Shen et al., 1997) associated with rapid formation of wild-type NBs within 6 hours followed by loss of PML expression (Andre et al., 1996; Chen, G. Q. et al., 1996; Mu¨ller et al., 1998b; Zhu et al., 1997). During this process, both PML and PML-RAR are targeted to the nuclear body and then rapidly degraded (Gianni et al., 1998; Shao et al., 1998). As O increased transfer of PML from the nucleoplasm to the nuclear matrix (Mu¨ller et al., 1998b; Zhu et al., 1997) and raised PML levels within the NB, accelerating apoptosis (Quignon et al., 1998). This was associated with linkage of the SUMO1 molecule to PML-RAR (Kamitani et al., 1998a, 1998b; Sternsdorf et al., 1999). As O induces degradation of PML-RAR even in APL cell lines resistant to ATRA (Chen et al., 1997; Gianni et al., 1998; Shao et al., 1998; Wang et al., 1998d). As O treatment correlates with only partial differentiation of the promyelocytes and predominantly the induction of apoptosis (Chen, G. Q. et al., 1997; Gianni et al., 1998; Shao et al., 1998; Zhu et al., 1997). Simultaneous treatment with ATRA and As O enhanced differentiation and apoptosis of NB4 cells (Gianni et al., 1998), and enhanced survival of animals harboring APL (Lallemand-Breitenbach et al., 1999). However, this was not the

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case in fresh human APL cells (Shao et al., 1998), making it uncertain whether ATRA and arsenic might best be used concomitantly or as sequential agents in APL. Antimony, a metal in the same column of the periodic table as arsenic, can induce the degradation of PML and can induce apoptosis (Mu¨ller et al., 1998b), suggesting a common mechanism of action by these heavy metals. Hence the treatment of t(15;17)-associated APL is always related to degradation of PMLRAR and restoration of the NB. However, the complete lack of PML-RAR after As O treat ment leads primarily to apoptosis, while the residual PML-RAR fragment present after ATRA treatment, in combination with residual RAR, induces the genes critical for cell differentiation. Disruption of the nuclear body in t(15;17) APL may not be absolutely required for the pathogenesis of APL, since PML is in the wild-type configuration in variant forms of APL. Thus, it may be the degradation of PMLRAR that may be most critical for the induction of differentiation in APL. Cellular Models of PML-RAR Action Cellular models of PML-RAR function have been hampered by the toxicity of the fusion protein (Ferrucci et al., 1997; Grignani et al., 1993b; Lavau et al., 1996). A successful model was constructed in the monocytoid U937 cell (Grignani et al., 1993b). Cells expressing PMLRAR fail to differentiate in response to ATRA or vitamin D ; TGF (Grignani et al., 1993b; Testa et al., 1994). Under physiological concentrations of ATRA (10\ M), PML-RAR expression was associated with an increase in cell growth rate. However, when treated with 10\ M ATRA, PML-RAR expression was associated with decreased cell growth due to increased apoptosis and increased differentiation. PML-RAR also blocked apoptosis in response to TNF (Testa et al., 1998), and in another cell model PML-RAR protected cells from G-CSF withdrawal (Rogaia et al., 1995; Slack and Yu, 1998). This suggests that PML-RAR functions mainly to promote cell survival. Furthermore, ablation of PML-RAR expression in NB4 APL cells induces apoptosis (Nason-Burchenal et al., 1998a, 1998b). The fraction of cycling cells in APL is relatively low; thus, the persistence of

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cells due to the antiapoptotic effects of PMLRAR could be critical. After ATRA or arsenic treatment, PML-RAR degradation (Raelson et al., 1996; Zhu et al., 1997) would be expected to lead to increased apoptosis (Gianni et al., 1998; Kitamura et al., 1997; Shao et al., 1998; Shen et al., 1997; Zhu et al., 1997). After ATRA treatment and prior to its degradation, PML-RAR likely activates genes such as p21 (Liu et al., 1996), blocking cell division. Arsenic also activates p21 by an unknown mechanism (Gianni et al., 1998). The U937 model was used to determine which structural features of the PML-RAR fusion protein were critical for effects on cell growth and differentiation (Grignani et al., 1996). Deletion analysis showed that the first coiled-coil motif of PML-RAR was required for its ability to block differentiation, but deletion of this segment did not affect PML/PML-RAR interaction, NB disruption, or RARE-dependent trans-activation. This suggests that disruption of PML within the nuclear body is not critical for the action of PML-RAR. Another model was created by transient expression of PML-RAR in HL60 cells. This inhibited granulocytic differentiation induced by ATRA and vitamin D but not granulocytic differentiation induced by DMSO or monocytic differentiation induced by phorbol ester. These results support a relatively specific mechanism of action for PML-RAR upon nuclear receptor pathways. However, NB4 cells cannot differentiate in response to vitamin D (Chen et al., 1994; Testa et al., 1994), but when ATRA and D are added in concert or sequentially (ATRA first), marked differentiation and inhibition of proliferation occurs. In addition, NB4 cells are resistant to polar compounds such as sodium butyrate or HMBA unless pretreated with ATRA for a period as brief as 30 minutes (Chen et al., 1994). Thus, PML-RAR may affect nonnuclear receptor differentiation pathways as well. PMLRAR was shown to enhance the proliferation of murine bone marrow progenitor cells after retroviral transfer, allowing these cells to be serially plated ex vivo. The cells remained growth factor dependent, however, suggesting that PML-RAR on its own cannot completely transform the cells (Du et al., 1999). From these data it can be concluded that PML-RAR functions primarily by inhibiting apoptosis of cells. The exact mechanism by which this occurs is

unclear and may be related to the inhibition of the function of RAR and other nuclear receptors.

Mouse Models of PML-RAR Function The first transgenic mouse created that expressed the PML-RAR fusion utilized the CD11b promoter, and these mice did not develop APL or a preleukemic syndrome but did show a defect in myeloid response to cytokines and profound neutropenia after sublethal irradiation, implying that PML-RAR impaired myeloid development (Early et al., 1996). Another transgenic murine model expressed the PML-RAR transgene from the metallothionine promoter (David et al., 1997). The investigators had difficulty obtaining transgenic founders, likely reflecting the poor tolerance of PMLRAR by nonhematopoietic cells. One animal line expressed PML-RAR only in the liver, and these animals developed liver pathology including hepatocellular carcinoma. These experiments confirmed the oncogenic nature of PMLRAR. Two groups expressed PML-RAR from the cathepsin G promoter (Grisolano et al., 1997; He et al., 1997), and the resulting mice developed a preleukemic syndrome (Grisolano et al., 1997) followed by the development of acute leukemia in 10—30% of animals with a median latency of 300 days. However, these animals did not accumulate a large number of promyelocytes like human APL (He et al., 1997). ATRA treatment led to an initial increase in peripheral white count, reminiscent of the ‘‘retinoic acid syndrome’’ (Frankel et al., 1992) followed by a drop in leukocyte count and the appearance of differentiated neutrophils. It is not clear whether this was due to differentiation of the malignant cells or apoptosis of these cells (Grisolano et al., 1997), and these animals offer an imperfect model for differentiation therapy. As in humans (Fenaux et al., 1997; Fenaux and Degos, 1997), all mice relapsed even with the continuation of ATRA, suggesting that other oncogenic lesions must have accumulated in these mice during the long latent period before the development of overt disease (He et al., 1998a). An excellent model of APL was generated by use of the MRP8 promoter (Lagasse and Weissman, 1992), which is expressed at the

PLZF

promyelocyte to metamyelocyte stage and continues to be active in mature neutrophils. After a preleukemic phase of about 6 months, about one-third of transgenic mice developed APL with all of the characteristics of the human disease, including bleeding, anemia, thrombocytopenia, and a low leukocyte count with a median latency of 6 months (Brown et al., 1997). When these cells were placed into culture and treated with ATRA, differentiated neutrophils were observed. Suggested Model of PML-RAR Action in APL Myeloid differentiation usually occurs at physiological levels of ATRA (10\ M), which activates key RAR target genes. PML-RAR (Guidez et al., 1998; He et al., 1998a) is a poor activator of RAR target genes and would overwhelm the tonic effect of RAR to induce myeloid differentiation. With pharmacological doses of ATRA, the PML-RAR fusion releases the corepressors and stimulates transcription of target genes that allow myeloid development to proceed (Figs. 20.2 and 20.3). Furthermore, the PML-RAR protein is degraded (Raelson et al., 1996; Yoshida et al., 1996) and wild-type RAR is upregulated (Chomienne et al., 1991), shifting the balance of power of RARs in the cell from PML-RAR to RAR. A PML-RAR/RXR heterodimer is probably the mediator of differentiation, since this process is synergistically stimulated by a combination of RAR- and RXR-specific ligands (Chen, J. Y. et al., 1996; Kizaki et al., 1996). In addition, brief treatment of NB4 cells or fresh APL cells with ATRA allows potent differentiation of APL cells to proceed in the presence of other agents such as hexamethylene bis-acetamide (HMBA), cyclic AMP, and vitamin D (Chen, A. et al., 1994; Ruchaud et al., 1994), suggesting that rapid transcriptional events prior to PML-RAR degradation mediate differentiation. These events might also include activation of other nuclear receptors, STATs, and AP1. In contrast, arsenic degrades PML-RAR fusion without RAR-mediated signaling. Modest differentiation might occur in this case by low-level signaling through the endogenous RAR. A component in the pathogenesis of APL may include the delocalization of one or more key proteins from the NB.

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Current evidence points away from this being PML itself. RAR-PML The reciprocal RAR-PML fusion generated in t(15;17) (Alcalay et al., 1992; Chang et al., 1992b) is present in 70—80% of APL cases (Alcalay et al., 1992; Grimwade et al., 1996) but in general does not appear to be required for the development of APL. Two transcripts can be generated from the alternative RAR promoters, of which RAR1PML was the most common (Li et al., 1997). RAR-PML contains the A1 or A2 domain of the RAR protein fused to a variable portion of the PML protein, due to alternative splicing, including the serine-rich C-terminal domain. It is hard to predict the effects of RAR-PML, since the role of the C-terminus of PML is unknown. There were cases of APL associated with nonreciprocal fusions of the PML and RAR genes that did not generate a RAR-PML fusion gene (Borrow et al., 1994; Hiorns et al., 1994; McKinney et al., 1994). Furthermore, there is no difference in ATRA sensitivity or clinical outcomes of patients who do or do not harbor the RARPML transcript (Grimwade et al., 1996; Li et al., 1997). Transgenic mice harboring the RARPML fusion did not develop leukemia, but when crossed with PML-RAR mice, leukemia developed with increased frequency (Pollock et al., 1999). Hence, the RAR-PML may modestly contribute to the disease process. PLZF The PLZF Gene The promyelocytic leukemia zinc-finger (PLZF) gene was initially identified by its rearrangement in an APL with t(11;17)(q23;q21) (Chen, Z. et al., 1993; Chen, S. J. et al., 1993; Chen et al., 1991). Since then, a total of 16 cases of this form of APL, fusing the PLZF and RAR genes, were described (Culligan et al., 1998; Grimwade et al., 1997, 1998; Guidez et al., 1994; Licht et al., 1995). In general, these six patients were resistant to ATRA and chemotherapy, and leukemic cells from these patients could not be induced to differentiate with ATRA in vitro (Guidez et al., 1994; Licht et al., 1995). Translocation (11;17)(q23;q21)-APL is unique in its resistance to ATRA; one patient was successfully treated

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Figure 20.2. Suggested model of PML-RAR action in APL. A: At 10\ M ATRA, PML-RAR prevents activation of key target genes required for myeloid differentiation by sequestration of RXR and other RAR cofactors, inhibiting normal RAR function. In addition, PML-RAR may bind to RAR targets as a homodimer or as a heterodimer with RXR and inhibit transcription of these genes by recruitment of corepressor/histone deacetylase complexes. PML-RAR also may affect transcription mediated by AP1 and IFN-responsive factors, and can sequester PLZF and potentially affect its function. PML-RAR prevents apoptosis through unknown

PLZF

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Figure 20.3. t(15;17) APL is sensitive to ATRA. At 10\ M ATRA, PML-RAR has aberrantly high affinity for corepressors relative to wild-type RAR and may bind and repress the transcription of key RAR target genes. Upon the addition of pharmacological doses of ATRA, the corepressors are released and coactivators may bind to PML-RAR in a manner similar to wild-type RAR.

with a combination of ATRA and G-CSF (Jansen et al., 1999) and another patient achieved a remission with concurrent ATRA and chemotherapy (Culligan et al., 1998). A very recent report from a European Working group showed that ten patients with t(11;17)-APL treated with chemotherapy, six of whom also received ATRA, achieved a complete remission (Grimwade et al., 2000), possibly mitigating the initial reports of the poor prognosis of this disease. The PLZF gene encodes for a zinc-finger transcription factor (Chen, Z. et al., 1993; Cook et al., 1995; Licht et al., 1996; Reid et al., 1995; van Schothorst et al., 1999; Zhang et al., 1999) of 673 amino acids with nine Kru¨ppel-like C H zinc-finger domains. The N-terminal 118 amino acids encode a POZ (pox virus and zinc finger)

or BTB (broad complex, tramtrack, bric a brac) domain. The POZ/BTB domain mediates protein self-association and heterotypic associations (Bardwell and Treisman, 1994), and acts as a transcriptional repression domain (Albagli et al., 1996; Chang et al., 1996; Numoto et al., 1993; Oyake et al., 1996; Seyfert et al., 1996). The POZ/BTB is involved in chromatin remodeling (Albagli et al., 1995; Raff et al., 1995) and transcriptional repression through interaction with histone deacteylase. The PLZF POZ/BTB domain forms a tight, highly intertwined dimer (Ahmad et al., 1998; Li, Xi et al., 1997, 1999). The top portion of the dimer structure forms a groove exposed to solvent that is lined with conserved charged amino acids, potentially representing a peptide-binding site.

mechanisms and delocalizes PML and other proteins from the nuclear body, though the importance of this is uncertain as the NB is normal in the other forms of APL. B: In the presence of pharmacological doses of ATRA, the PML-RAR fusion is degraded and releases PML and other cofactors. The NB structure is restored. A residual fragment of the PML-RAR fusion and/or the wild-type RAR, which is upregulated in response to ATRA, can then stimulate transcription of myeloid target genes. The blockade of other signaling pathways is released and the antiapoptotic effect of PML-RAR is lost. As a result terminal cell differentiation can proceed. (Adapted from Melnick and Licht, 1999.)

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PLZF Nuclear Localization PLZF is localized to the nucleus and (Licht et al., 1996; Reid et al., 1995) phosphorylated on serine and threonine residues (Ball et al., 1999). Immunofluorescent microscopy showed PLZF in a pattern of :50 small nuclear speckles (Licht et al., 1996) dependent on the presence of the POZ/BTB (Duong et al., 1996). PML and PLZF could partially colocalize in nuclear body structures (Koken et al., 1997). The PLZFRAR fusion did not colocalize with PML or delocalize PML from nuclear body structures (Koken et al., 1997), indicating that disruption of the PML NB is not required for the development of APL. In contrast, in t(15;17) APL, PLZF is delocalized into a microspeckled pattern identical to PML-RAR (He et al., 1998a; Koken et al., 1997). Delocalization of PLZF was also found in t(5;17)-APL associated with the NPM-RAR protein (Hummel et al., 1999).

PLZF Expression PLZF mRNA is expressed in undifferentiated myeloid cell lines and at lower levels in more differentiated erythroleukemia, promyelocytic and monocytic cell lines, and peripheral blood mononuclear cells (Chen, Z. et al., 1993; Reid et al., 1995). PLZF is downregulated during differentiation of NB4 and HL60 cells (Chen, Z. et al., 1993; A. Chen, S. Waxman, and J. Licht, unpublished) but is upregulated in the MDS cell line after treatment with calcium ionophore, perhaps recapitulating some aspect of monocyte development (Licht et al., 1996). In CD34> human progenitor cells could be immunostained with PLZF antisera in a distinct nuclear speckled pattern (Reid et al., 1995). When such cells were placed into culture and allowed to differentiate, PLZF levels transiently increased, then declined (C. Labbaye, personal communication). PLZF expression may be important for the maintenance or survival of hematopoietic stem cells and/or early progenitors downregulated with differentiation and reexpressed in monocytes. During mouse embryogenesis, PLZF is expressed in a segmental pattern (Avantaggiato et al., 1995) in the nervous system, a pattern similar to the hoxb2 gene (Cook et al., 1995). A PLZF site was found in the hoxb2 5 flanking region, and PLZF could repress the hoxb2 pro-

moter in cotransfection assays (Ivins and Zelent, 1998). PLZF expression is also notable in the limb buds, where hox genes help guide development (Avantaggiato et al., 1995; Cook et al., 1995) and PLZF knockout animals (Barna et al., 2000) show limb defects, possibly due to Hox gene deregulation. Homozygous PLZF null mice have not exhibited an obvious hematopoietic phenotype nor have they developed leukemia or other tumors. This does not rule out a role for PLZF in hematopoiesis. FAZF/TZFP, a gene highly similar to PLZF, might partially compensate for the lack of PLZF during development (Hoatlin et al., 1999; Lin et al., 1999). Transcriptional Function of PLZF From site selection and other experiments (Ball et al., 1999; Li, J. Y. et al., 1997; Sitterlin et al., 1997), a relatively loose consensus sequence for PLZF binding of GT AGT can be derived. !! Such sites can be recognized by the C-terminal seven zinc fingers retained in the RAR-PLZF fusion protein (Ivins and Zelent, 1998; Li, J. Y. et al., 1997; Sitterlin et al., 1997). The FAZF protein, which only has 3 ZF motifs, very similar to PLZF, can also bind to the PLZF site. This suggests that the N-terminal zinc fingers of PLZF might not bind to DNA but may participate in protein-protein interactions. Reporter genes containing PLZF sites (David et al., 1998; Guidez et al., 1998; Li, J. Y. et al., 1997) are repressed by coexpressed PLZF protein. In contrast, the RAR-PLZF fusion protein activates such reporters while PLZF-RAR has no effect. Hence the RAR-PLZF protein could act in a dominant negative manner, binding and altering transcription of PLZF target genes. The PLZF protein was found to contain two separable transcriptional repression domains, one of which overlaps the POZ/BTB domain (Li, J. Y. et al., 1997). We and others recently created missense mutations in the POZ/BTB domain that abrogated repression and that will aid in the molecular characterization of the POZ/BTB domain (Li et al., 1999; Melnick et al., 2000). PLZF interacts with the corepressors N-CoR, SMRT, Sin3A, and HDAC1 (histone deacetylase 1) (David et al., 1998; Guidez et al., 1998; He et al., 1998a; Hong et al., 1997; Lin et al., 1998; Wong and Privalsky, 1998a), in large part through the POZ/BTB domain of PLZF

PLZF-RAR

(David et al., 1998; Grignani et al., 1998; Wong and Privalsky, 1998a). We recently found that the ETO protein that is fused to AML1 in t(8;21)-associated AML is another PLZF corepressor that primarily binds to PLZF, through the second repression domain (Melnick et al., 2000). Repression by PLZF was augmented by corepressors and partially blocked by the HDAC inhibitor trichostatin A (Wong and Privalsky, 1998a, 1998b; Yoshida et al., 1995). Additional mechanisms could also be at play. Our group found that PLZF formed a DNA protein complex with a molecular weight of nearly 600 kDa that contained cdc2 (Ball et al., 1999), which was implicated in transcriptional repression by phosphorylation of basal transcription factors (Long et al., 1998). Growth Suppression by PLZF PLZF, like PML, can repress cell growth. 32DCL3 cells, overexpressing the PLZF protein, were highly growth inhibited, accumulated in G1, traversed S phase slowly, and had an increased rate of apoptosis (Shaknovich et al., 1998). PLZF expression inhibited myeloid differentiation induced by G-CSF or GM-CSF and led to the upregulation of the early hematopoietic marker Sca1 in the 32Dcl3 cells. Acute infection of myeloid cells with a PLZF-containing retrovirus was associated with growth arrest of cells in the S phase of the cell cycle, implicating cyclin A as a potential target. Fibroblasts expressing PLZF showed blunted induction of cyclin A when stimulated into the cell cycle. PLZF can bind and repress the cyclin A2 promoter (Yeyati et al., 1999) and growth suppression by PLZF was overcome by enforced expresion of cyclin A2. This suggests that PLZF inhibits growth by altering the expression of cell cycle regulators. PLZF also protected 32D cells (Shaknovich et al., 1998) from apoptosis due to factor withdrawal. This has led us to hypothesize that PLZF plays a role in quiescence and resistance to apoptosis exhibited by hematopoietic stem cells. Downregulation of PLZF during myeloid differentiation may be accompanied by cycles of committed cell division. It is possible that PLZF is a tumor suppressor disrupted in t(11;17)(q23;q21)-APL. The resulting PLZF-RAR fusion proteins may act as dominant negative inhibitors of PLZF.

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PLZF-RAR The t(11;17)(q23;q21) fusion yields two reciprocal transcripts (Grimwade et al., 1997; Licht et al., 1995). The resulting PLZF-RAR fusion contains the entire N-terminal transcriptional effector region of PLZF and the first two zinc fingers, and, in one case due to an alternative 3 breakpoint in the PLZF gene, three zinc fingers. In four of seven cases tested, a reciprocal RARPLZF transcript was detected, linking the A activation domain of RAR (Nagpal et al., 1992) to the last seven PLZF zinc fingers. PML-RAR and PLZF-RAR have some similarities as aberrant receptors. Both fusion proteins can bind as homodimers to retinoic acid response elements (RAREs) (Duong et al., 1996; Licht et al., 1996; Perez et al., 1993). In PML-RAR this binding is mediated by the coiled-coil motif (Perez et al., 1993), and in PLZF by the POZ/ BTB domain (Duong et al., 1996). In combination with RXR, PLZF-RAR forms multiple DNA protein complexes, but the PLZF-RAR/ RXR heterodimer binds to RAREs with higher affinity than PLZF-RAR homodimers (Licht et al., 1996). The PLZF-RAR/RXR heterodimer bound to the RARE less efficiently than wildtype RAR (Licht et al., 1996), perhaps due to the ability of the POZ/BTB domain to multimerize and preclude efficient DNA binding (Bardwell and Treisman, 1994). Hence PLZFRAR might sequester limiting amounts of RXR, an essential cofactor for RAR function. Like PML-RAR PLZF-RAR (Chen, Z. et al., 1994; Duong et al., 1996; Licht et al., 1996) is a dominant negative inhibitor of wild-type RAR. PLZF-RAR is a relatively weak transactivator (Chen, Z. et al., 1994; Licht et al., 1996). This is predominantly due to the high affinity of PLZF-RAR for the SMRT, NCoR, and Sin3A corepressors and HDAC1 (David et al., 1998; Grignani et al., 1998; Guidez et al., 1998; He et al., 1998a; Hong et al., 1997; Lin et al., 1998). However, while PML-RAR releases these factors in the presence of 10\ M ATRA, PLZF-RAR still retains them (Collins, 1998; Grignani et al., 1998; Guidez et al., 1998a; He et al., 1998a; Lin et al., 1998). This is due to the ability of the PLZF POZ/BTB domain to bind the corepressors in an ATRA-insensitive manner (Grignani et al., 1998; Lin et al., 1998). HDAC inhibitors, such as trichostatin A and sodium

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Figure 20.4. Potential mechanism of relative resistance of t(11;17)(q23;q21)-associated APL to ATRA. Axis 1: PLZF itself being a transcriptional repressor binds to corepressor molecules through two repression domains contained in the PLZF-RAR fusion protein. Even in the presence of 10\ M ATRA, this fusion protein will continue to retain corepressors and inhibit RAR targets. Axis 2: PLZF is an inhibitor of cell growth, potential through its ability to inhibit cyclin A and other growth regulators. Cells harboring t(11;17)(q23;q21) are hemizygous for PLZF, which may lead to disinhibition of PLZF targets and increased cell growth. Axis 3: RAR-PLZF can activate genes usually repressed by PLZF and actively stimulate cell growth.

butyrate, allowed PLZF-RAR to respond to ATRA by inactivating the corepressor complex bound to the POZ/BTB domain (Grignani et al., 1998; Lin et al., 1998) (Fig. 20.4). PLZF-RAR inhibited transactivation by the wild-type receptor (Chen, Z. et al., 1994; Duong et al., 1996; Licht et al., 1996), with this effect partially relieved by overexpression of RXR, consistent with the notion that the aberrant receptors block myeloid differentiation at least partly by limiting the ability of RAR to bind with RXR to its targets (Licht et al., 1996). Deletion mapping of PLZF-RAR protein also revealed that dominant negative activity was dependent on the presence of the POZ/BTB domain (Duong et al., 1996). This region is also required for self-association of PLZF-RAR and for formation of multimers that could sequester RXR. Curiously, inhibition of wild-type RAR function was partially dependent on the presence of the first two PLZF zinc fingers, which are present in the fusion protein and are the binding site for PML. When the POZ/BTB domain and first two PLZF zinc fingers were

deleted from the fusion protein, PLZF-RAR became an efficient activator of ATRA-mediated transcription. The dominant negative effect of PLZF-RAR also suggests that it may work by sequestering RAR transcriptional coactivators such as TIF1 or CBP in an inactive conformation, drawing them from RAR target genes. This provides another explanation for why RXR only partially rescues the dominant negative, effect of PLZFRAR. However, the hypothesis that PLZFRAR binds to RAR corepressors (Chen and Evans, 1995; Chen, J. D. et al., 1996; Horlein et al., 1995), inappropriately repressing RAR target genes in the absence of ligand, is more likely. In keeping with this belief, PLZF-RAR like PML-RAR, inhibited the activity of a RAREcontaining promoter in the absence of exogenous ATRA (He et al., 1998a). Finally, PLZF-RAR could in theory also affect the function of wild-type PLZF. In fact, PLZFRAR and PLZF can preferentially heterodimerize to the formation of PLZF homomeric complexes (Duong et al., 1996). Hence, high-

NUCLEOPHOSMIN

level expression of PLZF-RAR in t(11;17)(q23;q21) blasts might sequester PLZF from binding to its natural target genes and/or binding to limiting quantities of PLZF transcriptional cofactors (Ruthardt et al., 1997).

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tion, treatment of these cells with ATRA led to the degradation of PLZF-RAR, lifting the block to induction of RAR targets, yet did not induce differentiation. (Koken et al., 1999). This implicates the reciprocal RAR-PLZF protein in the aggressive nature of this form of APL.

Models of t(11;17)(q23;q21) APL PLZF-RAR like PML-RAR, is toxic to many cell types (R. Shaknovich and J. Licht, unpublished); however, one group found that the fusion protein blocked the ability of leukemic cells to differentiate (Ruthardt et al., 1997). While reintroduction of wild-type RAR or PML-RAR into a mutant HL-60 devoid of endogenous RAR restored the ability of the cells to differentiate in response to ATRA, PLZF-RAR only mediated partial differentiation. This inability to mediate ATRA signaling may help explain the resistant clinical phenotype of t(11;17)(q23;q21)-associated APL. More physiologically relevant data indicate that PLZF-RAR on its own may not fully account for the ATRA resistance of t(11;17)(q23;q21) APL. Marrow progenitor cells infected with a PLZF-RAR retrovirus are able to be serially passaged ex vivo, but upon treatment of these cells with ATRA, proliferation ceased and differentiation ensued (Du et al., 1999), suggesting that the retinoic acid resistance due to expression of PLZF-RAR is not absolute. Transgenic animals harboring PLZF-RAR developed a CML-like syndrome rather than APL (Cheng et al., 1999; He et al., 1998a) after a preleukemic phase, suggesting that secondary mutations are required for transformation. Unlike PML-RAR transgenic mice, the PLZFRAR mice did not achieve durable complete remission after ATRA treatment, although cells did show some evidence of differentiation and leukemic cells from the mice treated with ATRA readily differentiated ex vivo (He et al., 1998a). The relative insensitivity of the disease correlates with the impaired ability of PLZF-RAR to transactivate due to binding of corepressors. This idea was solidified by the finding that the histone deacetylase inhibitor and ATRA synergistically induced differentiation of these leukemic cells. The animal model does not explain the poor response of some t(11;17)(q23;q21) patients to chemotherapy and the resistance of fresh APL cells to high doses of ATRA in vitro (Guidez et al., 1994; Licht et al., 1995) In addi-

RAR-PLZF The reciprocal transcript encoding RAR-PLZF is consistently expressed in all t(11;17)(q23;q21) patients tested (Grimwade et al., 1997; Licht et al., 1995). These seven zinc fingers of RARaPLZF can bind to the same site as full-length PLZF (Ball et al., 1999; Li, J. Y. et al., 1997). While PLZF represses gene transcription, RAR-PLZF activates transcription through this site (Li, J. Y. et al., 1997). Whereas PLZF is a growth suppressor, RAR-PLZF activates transcription, activates expression of cyclin A2 (Yeyati et al., 1999), and enhances cell growth (P. Yeyati, R. Shaknovich, and J Licht, unpublished). Hence t(11;17)(q23;q21) may be a therapy-resistant disease due to the presence of two oncogenes. PLZF-RAR blocks the action of ATRA while RAR-PLZF may activate cell cycle regulators, blocking the antiproliferative effects of ATRA. In accordance with this idea, mice harboring the RAR-PLZF protein develop a myeloproliferative syndrome (He et al., 1998b). Hence this form of APL is likely resistant to therapy by subversion of the RAR and PLZF pathways (Fig. 20.4).

NUCLEOPHOSMIN In t(5;17)-associated APL, RAR is translocated to a region on chromosome 5q35 encoding the ubiquitously expressed and evolutionarily conserved nucleophosmin gene (NPM) (Brunel et al., 1995; Chan, P. et al., 1989; Redner et al., 1996b). NPM was initially isolated as a nucleolar phosphoprotein in hepatoma cells (Hernandez-Verdun et al., 1982; Olson et al., 1974; Verdun, 1983). The human NPM gene spans 25 kb, consists of 12 exons (Chan, P. K. et al., 1997), and alternative splicing yields two major isoforms differing in their C-terminal region (Chan, P. K. et al., 1997; Chan et al., 1989; Chang and Olson, 1989; Lee and Welch, 1997). Major structural features of NPM include two Asp/Glu-rich acidic domains, which may serve

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as binding sites for basic regions of other proteins; a bipartite nuclear localization signal (NLS); a metal-binding motif; an ATP-binding site; phosphorylation sites for cdc2 kinase and casein kinase II, and a binding site for proteins that contain nucleolar localization signals (Adachi et al., 1993; Chan, P., 1989; Chan, P. et al., 1986a, 1986b; Chan, W. et al., 1989; Chang et al., 1988; Chang et al., 1998; Li et al., 1996; Marasco et al., 1994; Miyazaki et al., 1995; Szebeni et al., 1995; Valdez et al., 1994). Notably, both isoforms reversibly multimerize to a hexameric state (Chan and Chan, 1995) via the N-terminal domain (Liu and Chan, 1991). NPM is localized most prominently to areas of the nucleolus associated with ribonucleoprotein (RNP) processing (Biggiogera et al., 1989; Dumbar et al., 1989; Smetana et al., 1984; Spector et al., 1984; Yung et al., 1985a, 1985b). NPM functions as part of a transport system used by ribosomal precursors and other proteins to shuttle between the cytoplasm and nucleolus (Borer et al., 1989; Chang et al., 1998; Szebeni et al., 1997). NPM levels are increased in proliferating cells (Derenzini et al., 1995a, 1995b) and leukemic blasts (Kondo et al., 1997), perhaps due to the consequence of increased requirements for ribosomal precursors. However, overexpression of NPM transformed NIH 3T3 cells, suggesting that NPM could actively control cell growth (Kondo et al., 1997). One explanation could be that NPM binds to the tumor suppressor IRF-1 and inhibits its ability to activate genes that mediate the antiproliferative effect of IFN (Kondo et al., 1997). Thus, NPM could behave as an anti-tumor suppressor. NPM binds to transcription factor YY1, involved in cell growth, changing it from a transcriptional repressor to an activator (Inouye and Seto, 1994; Shi et al., 1997). ATRA-induced differentiation of HL60 cells, but not growth arrest by serum withdrawal, resulted in downregulation of NPM (Hsu and Yung, 1998). When cellular NPM levels were decreased by an antisense oligonucleotide, there was potentiation of the ATRAinduced differentiation (Hsu and Yung, 1998). Growth-suppressive IRF-1 is upregulated by ATRA during myeloid differentiation, in opposition to the effect of NPM (Matikainen et al., 1996). These results support a possible role for NPM in the control of cellular growth and

differentiation and hint at involvement in retinoid and IFN pathway regulatory crosstalk. NPM expression peaks at S or G2 phase and is minimal in cells at G (De Angelis et al., 1997; Feuerstein et al., 1988a, 1988b; Sirri et al., 1997). This might be related to the fact that NPM specifically stimulates the activity of DNA polymerase (Takemura et al., 1994) or may simply be a reflection of the metabolic demand of the cell. During M-phase progression, NPM associates with perichromosomal regions and prenucleolar bodies, thus functionally linking the processes of nucleolar disassembly to mitotic chromosome condensation (Peter et al., 1990). NPM is preferentially dephosphorylated and degraded during apoptosis and cell damage (Tawfic et al., 1993, 1995). NPM reversibly delocalizes from the nucleolus to the nucleoplasm (Yung et al., 1985a, 1985b) when cells are exposed to conditions that discourage DNA or RNA synthesis or encourage terminal cell division (Chan et al., 1987; Chan et al., 1996; Yung et al., 1990; Yung et al., 1986, 1991). Thus, NPM, like PML, undergoes changes with cell stress, indicating that both proteins may measure or control cell homeostatic processes. NPM is fused to genes other then RAR in hematologic malignancies as in the t(2;5)(p23;q35) translocation found in Ki-1> anaplastic large-cell lymphoma (ALCL) (Ladanyi et al., 1994; Morris et al., 1994; Shiota et al., 1995). In this situation, NPM is linked to Alk, a gene encoding a membrane spanning tyrosine kinase (Morris et al., 1994) normally not expressed in lymphoid tissue (Iwahara et al., 1997). As a result, the ubquitously expressed NPM gene drives the expression of an aberrant fusion tryosine kinase, (Bischof et al., 1997; Fujimoto et al., 1996; Kuefer et al., 1997; Mason et al., 1998; Shiota et al., 1995; Wellmann et al., 1997; Yee et al., 1996). In the t(3;5)(q25.1;q34) translocation, found in myelodysplasia and M6-AML, a larger 175 amino acid portion of the NPM gene is linked to the MLF1 gene encoding an abundant cytoplasmic protein of unknown function (Chan, P. X. et al., 1997; Yoneda-Kato et al., 1996). NPM-RAR The t(5;17)(q35;q21) translocation was first described in a 2-year-old girl with APL (Corey et

NPM-RAR

al., 1994) who achieved cytogenetic remission after treatment with ATRA and chemotherapy. Blasts from this index patient, when thawed and treated with ATRA, differentiated into mature granulocytes (Redner et al., 1997a). Subsequently, at least two other cases were described (Brunel et al., 1995) one of whom has had a prolonged survival after ATRA and bone marrow transplantation (Hummel et al., 1999). This gene rearrangement joins the NPM gene 5 to exon 3 of RAR, in a manner similar to the other forms of APL (Brunel et al., 1995; Redner et al., 1997a), yielding two isoforms of the chimeric oncoprotein. Both forms contain the oligomerization domain of NPM as well as its active promoter. Like PML-RAR and PLZF-RAR the NPM-RAR fusion acts as a ligand-dependent transcriptional activator when coexpressed with reporter genes containing RAREs (Redner et al., 1996b), yet can act as a dominant negative inhibitor of wild-type RAR through abberant interaction with corepressor molecules (Redner et al., 2000). Enforced expression of NPM-RAR in U937 cells blocked monocytic differentiation in response to vitamin D and TGF (Redner et al., 1996a). It was further found that PMLRAR, PLZF-RAR, and NPM-RAR could enhance the proliferation of primitive marrow progenitor cells. Treatment of these cells with ATRA induced differentiation and inhibited cell growth (Du et al., 1999). A transgenic model of t(5;17)-APL was recently created utilizing the cathepsin G promoter. These mice developed an APL-like syndrome after a latent period, and blasts derived from these animals were ATRA sensitive (Cheng et al., 1999). NPM-RAR is expressed in a microspeckled pattern throughout the nucleus similar to PMLRAR and PLZF-RAR (Hummel et al., 1999), and APL blasts from a t(5;17) patient exhibited a normal NB configuration (Hummel et al., 1999), supplying additional evidence that disruption of the nuclear body is not required for the pathogenesis of APL. However, PLZF was delocalized in t(5;17) APL cells in a pattern distinct from its wild-type distribution in progenitors (Hummel et al., 1999), lending support for a broad role for PLZF protein in APL. A reciprocal RAR-NPM mRNA was identified in the index t(5;17) patient, leading to fusion of the A domain of RAR to the acidic domains, NLS, and the rest of the C-terminus of NPM (Redner

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et al., 1996b), though the importance of this putative protein is unknown. Nuclear Matrix‒Mitotic Apparatus Protein (NuMA) NuMA is an abundant, conserved, and ubiquitously expressed protein involved in the completion of mitosis and reformation of nuclei in the postmitotic daughter cells and a component of the interphase nucleus (Compton and Cleveland, 1994; Compton et al., 1992; He et al., 1995; Kallajoki et al., 1991, 1992; Saredi et al., 1996). The NuMA gene, located on chromosome 11q13 (Sparks et al., 1993), encodes a protein of 2115 amino acids with a molecular weight of approximately 230 kDa, and by alternative splicing yields 1776aa and 1763aa proteins (Tang et al., 1993, 1994). The NuMA protein is divided into two globular domains at either end of the protein, with a central coiled region of 1485 Aa (Compton et al., 1992; Maekawa and Kuriyama, 1993; Parry, 1994; Yang et al., 1992). The coiled motifs likely mediate protein homoand heteroassociation (Harborth et al., 1995; Yang et al., 1992). The C-terminal region contains basic sequences, motifs for phosphorylation by cdc2, and other kinases (Hsu and Yeh, 1996; Yang et al., 1992), and sequences that confer localization to the nucleus (NLS) and mitotic spindle (Compton and Cleveland, 1993; Gueth-Hallonet et al., 1996; Maekawa and Kuriyama, 1993; Tang et al., 1994; Yang et al., 1992; Yang and Snyder, 1992). NuMA is regulated across the cell cycle by post-translational modifications. For example, NuMA is phosphorylated by the cdc2/cyclin B regulatory kinase at the initiation of mitosis (Hsu and Yeh, 1996; Saredi et al., 1997) and associates with the spindle microtubules. NuMA may be required for the formation of the nuclear spindle and the organization of daughter nuclei as cell division ends (Kallajoki et al., 1991, 1993; Lyderson and Pettijohn, 1980; Price and Pettijohn, 1986; Tousson et al., 1991; Yang and Snyder, 1992). At the end of mitosis, NuMA undergoes proteolytic cleavage and dephosphorylation (Hsu and Yeh, 1996; Saredi et al., 1997). As cells progress toward G1, the remaining NuMA reverts to an insoluble form, yielding a fibrous network that may play a structural role during interphase. In interphase cells,

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NuMA is localized in diffuse and speckled nuclear patterns (Kallajoki et al., 1991; Kempf et al., 1994; Lyderson and Pettijohn, 1980; Maekawa et al., 1991), and like the other APL fusion partner proteins, it is associated with the nuclear matrix (Zeng et al., 1994). NuMA specifically attaches to DNA matrix attachment regions (MAR), which are important for chromatin compaction and isolation of transcriptionally active loops of DNA (Luderus et al., 1994; Tsutsui et al., 1993) and hence could participate in the regulation of transcription. NuMA is an early target for proteolysis by caspase-3 and caspase-6 (Zweyer et al., 1997), yielding a proteolytic product (Casiano et al., 1996; Hsu and Yeh, 1996; Sodja et al., 1997) that could act as a dominant negative, disrupting normal nuclear structure. Release of NuMA from the DNA matrix attachment regions may facilitate the DNA fragmentation characteristic of apoptosis (Gueth-Hallonet et al., 1997). In summary, NuMA is a structural component of the cell that responds to cell cycle signals on cue rather than a controlling factor in cell proliferation. It is unclear whether inhibition of NuMA function might contribute to oncogenesis.

NuMA-RAR The NuMA-RAR fusion protein was described in a 6-month-old boy with APL harboring a translocation t(11;17)(q13;q21) (Wells et al., 1996). The patient had a complete remission after ATRA therapy and was disease-free at 24; months after a bone marrow transplant. (Wells et al., 1996). The t(11;17)(q13;q21) results in a 2286 Aa protein predicted to consist of 1883 amino acids of NuMA including the N-terminal globular and coiled-coil domains of NuMA fused to RAR domains B—F, as in all the other RAR fusion proteins (Wells et al., 1997). NuMA-RAR localized to sheetlike nuclear aggregates in the patient’s leukemic cells, but NB structure and PML staining were unperturbed. Introduction of C-terminal mutants of NuMA into cells completely disrupt mitosis (Compton and Cleveland, 1993; Maekawa and Kuriyama, 1993), and it is surprising that the existence of NuMA-RAR is even compatible with cell division. The most likely mechanism of action of the NuMA-RAR is interference with nuclear recep-

tor function. Although no functional data for the NuMA-RAR fusion are yet available, it is probable that, like the other RAR fusion proteins, NuMA-RAR is a dominant negative retinoid receptor. A fundamental common denominator among APL fusion proteins is their ability to form higher-order complexes. Like PML, PLZF, and NPM, NuMA can multimerize (Harborth et al., 1995; Yang et al., 1992). NuMA-RAR might thus sequester RAR partner proteins or have aberrant affinity for nuclear receptor coactivators or corepressors. Like PML, NuMA is a component of the nuclear matrix and NuMA-RAR might further inhibit RAR function by confinement of RAR cofactors in a nuclear compartment apart from wildtype RAR. It is unknown whether a reciprocal RAR-NuMA transcript is expressed in this disease. In fact, there might be selection against this protein as it could represent a detriment to mitosis.

STAT5b-RAR The newest protein identified as a fusion partner with RAR is STAT5b , isolated from a patient with M1 AML but with a proportion of blasts exhibiting microgranular APL morphology (Arnould et al., 1999; Jonveaux et al., 1996). Leukemic blasts from the patient failed to respond to ATRA in vitro. As reviewed in Chapter 15, the seven members of the mammalian STAT proteins are transcription factors that are resident in the cytoplasm until they are phosphorylated on specific tyrosine residues after the triggering of a cell by various cytokines. Cytokine binding to specific receptors allows for dimerization, and for receptors with intrinsic kinase activity, cross-phosphorylation and activation of the tyrosine kinase activity of the receptor. Receptors without intrinsic kinase activity are noncovalently attached to JAK kinases, which when brought into opposition, can in a similar way cross-phosphorylate and activate each other. STAT5b, like other STAT proteins, binds to the JAK kinase through an SH2 domain and then is itself tyrosine phosphorylated at a C-terminal residue. STAT5b can then homodimerize, migrate to the nucelus, and bind to a GAS element (Darnell, 1997). STAT5b is widely expressed in a number of

CONCLUSION

tissues, including hematopoietic progenitors, and can be activated by a number of cytokines including prolactin, GM-CSF, EPO, and IL-3 (Chretien et al., 1996; Dong et al., 1998; Mui et al., 1996). STAT5b is very similar to STAT5a but differs in the C-terminal activation domain (Lin et al., 1996). STAT5 was originally identified as a factor important in mammary gland response to prolactin, and knockout studies showed that STAT5a is required for breast development and lactation (Teglund et al., 1998). STAT5b null mice have a defective response to growth hormone, no overt quantitative hematopoietic defects, but a defect in NK-cell killing (Imada et al., 1998). Compound null mice have a severe defect in response to CSFs and a deficiency in T cells (Teglund et al., 1998). STAT5b target genes relevant to hematopoiesis include c-myc and IL2R (Matikainen et al., 1999), and STAT5 activation was recently identified as a crucial function of the bcr-abl oncoprotein (Nieborowska-Skorska et al., 1999). Hence, like PLZF, STAT5b represents a transcription factor fused to the RAR, which itself may play a role in normal and malignant blood development. The STAT5b gene is localized on chromosome 17q21.1-21.1, like RAR, and the two genes are estimated to be about 3 megabases apart. The patient with the STAT5b-RAR fusion had a deletion of chromosome 17, leading to the creation of a fusion intron linking the STAT5b and RAR loci, and no reciprocal RAR-STAT5b transcript was detected. The breakpoint in the RAR locus was within the same highly localized region found in most cases of APL associated with t(15;17). The STAT5b-RARa fusion protein contains the majority of the STAT5b protein. The Nterminus consists of a coiled-coil region that mediates dimerization and can interact with a protein called Nmi1, which enhances STAT interaction with the p300/CBP coactivators (Zhu et al., 1999). This domain might allow the STAT5b-RAR to sequester coactivators from the remaining wild-type receptor in the cell. Located centrally in the protein is a DNAbinding domain and more C-terminal a SH3 domain followed by a truncated SH2 domain. The STAT proteins dimerize through their SH2 domains after they become phosphorylated. The C-terminal Y699 required for STAT5b activa-

351

tion is missing in the fusion protein as is a more C-terminal serine residue whose phosphorylation activates STAT5b transcription. Hence it is unclear whether this molecule would act as a homodimer like the other APL fusion proteins. The N-terminal portion of the STAT5b SH2 domain remains in the fusion protein and could compete with wild-type STAT5b for docking with cytokine receptors. However, in the index patient, the STAT5b protein was localized in the nucleus, whereas in control marrow cells STAT5b was localized to the cytoplasm (Arnould et al., 1999). Thus, it is not yet clear whether this truncated STAT5b fusion protein can homoor heterodimerize with other STAT proteins and constitutively activate STAT target genes. It is also not known whether the STAT5bRAR fusion is a dominant negative form of RAR and whether it has aberrant affinity for coactivators or corepressors which might explain the therapy resistance of the index patient.

CONCLUSION There are three axes to be investigated in understanding the pathogenesis of APL. First, in all cases of APL, RAR is fused to partner proteins, which can multimerize. Tables 20.2 and 20.3 summarize the properties of the partner proteins and the fusion proteins. Multimerization may mediate the abnormally high affinity of the fusion proteins for corepressors. PLZF itself binds to corepressors, prohibiting their full release at the pharmacological dose of ATRA, leading to a resistant form of APL. These fusion proteins block the activation of critical genes required for myeloid differentiation. The fusion proteins may also affect other transcriptional pathways via protein-protein interactions. Second, a loss of a dose of the N-partner gene could disable a growth-suppressive pathway in the cell. Furthermore, the N-RAR fusion can sequester the normal N product. Third, the reciprocal RAR-N fusion protein could act to sequester RAR cofactors and affect RAR function, and could act as a second dominant negative protein to inhibit the function of the N protein.

352

N-terminal pro-rich RING and B-box coiled-coil Variable acidic C-terminal region Through the coiled domain

Through the coiled-coil N-terminal pro-rich domain, and possibly RING and B-box domains Ser and Tyr residues Substrate of cyclin A/cdk2 has casein kinase II sites Mainly in nuclear bodies Also in cytoplasm and other nuclear regions Associates with nuclear matrix Present in inflammatory tissues, myeloid precursor cells Induced by IFNs

Increases in G to G transition and decreases as cells progress to S phase Transcriptional repression and activation Involved in retinoid

Secondary structure motifs

Heterologous interaction

Cell cycle

Transcription and RNA metabolism

Nuclear matrix association Expression pattern

Nuclear localization

Phosphorylation

Homodimer

Up to 20 splicing products identified

Isoforms

PML

PLZF

NPM

Possible through the nuclear bodies CD34> progenitors, macrophages, mouse embryo, CNS, liver, heart, kidney, limb, and tail buds Blocks cells in G /S correlating with downregulation of cyclin A Transcriptional repression

Nuclear speckles, partial overlap with PML

Through the BTB/POZ domain, a second repression domain, and the first two zinc fingers Ser and Thr residues Possible cdc2 phosphorylation

Through the BTB/POZ domain

Modulates transcriptional effects of YY1 and IRF-1

Peaks at onset of S phase, declines as cells enter G

One isoform associates with the matrix Ubiquitous

Principally nucleolar, also nucleoplasm Shuttles to cytoplasm

Casein kinase II, nuclear kinase II, PKC, and cdc2 kinase

Forms hexamers through N- and C-terminal portion Through the acidic domains and C-terminal region

Two major isoforms A third associates with nuclear matrix N-terminal Two acidic domains, BTB/POZ domain one metal-binding C-terminal Kru¨ppel-like motif, two NLS, zinc fingers ATP-binding site

One

TABLE 20.2. Comparison of RAR Fusion Partner Proteins

Transcriptional activation

Phosphorylated by cytokines that drive the cell into S phase

Widely expressed, breast, liver, hematopoetic progenitors, T cells, NK cells

Ubiquitous, except in some terminally differentiated cells Phosphorylated in G /M Essential for M phase Colocalizes and coprecipitates with snRNPs and splicing complexes

?

Basal states in cytoplasm, to nucleus after phosphorylation

By receptor tyrosine kinases and Jak kinases

Through N-terminal coiled, coiled motif

Through C-terminal SH2 domains

Coiled-coil, DNA-binding domain

Two isoforms due to proteolytic cleavage

STAT5b

During interphase

Cdc2 kinases, cyclic AMP kinase PKC, Ca/calmodulin kinase Interphase — diffuse and speckled Mitosis — binds spindle poles

Through C-terminal domain

Through coiled-coil domain

Central coiled-coil flanked by two globular domains

At least three

NuMA

353

Function

Knockout data

Miscellaneous

Apoptosis

Action on cell growth

Protein partners

Knockout mice susceptible to infections, susceptible to transforming agents, lack IFN-induced growth suppression, defective induction of p21 by ATRA Involved in growth suppression, differentiation, and immune response pathways Possible role in translation Transcriptional modulator

receptor signaling Possible role in translation SUMO-1, PLZF Rb, L7 leucine zipper, EF-1, ribosomal P proteins Daxx, and others Growth suppression in NB4, HeLa, and CHO cells blocks transformation in rat embryo fibroblasts and 3T3 cells Removal delays apoptosis Association with SUMO-1 and targeting by As O imply role in apoptosis Targeted during certain viral infections

Growth suppressor, transcriptional repressor, control of developmental programs and differentiation, possibly through hox genes

Musculoskeletal and limb defects, impaired spematogenesis, T-cell lymphopenia

DNA-binding site GT AGT !!

Promotes cell survival with factor withdrawal

Growth suppression differentiation block

PML, LRF, SMRT, N-Cor, HDAC, sin3a, sin3b, Rb, cdc2, ETO

Ribonucleoprotein maturation and transport, shuttle proteins between cytoplasm and nucleolus Transcriptional modulator Role in DNA recombination

None published to date

Nuclei acid binding

Becomes hypophosphorylated and degraded during apoptosis

Expression highest in tumor cells, overexpression causes 3T3 cells to transform

Rev, Tat, Tex, nucleolin, p120, YY1, IRF-1

Involved in ribosome biogenesis

Structural role in interphase and in particular mitotic cells Major target of apoptosis program

Attaches to DNA matrix attachment regions (MAR) None published to date

Specifically targeted for proteolysis by caspases

Required for proper completion of mitosis

Tubulin and mitotic spindle

Transcriptional activator implicated in hematopoletic and immune cell growth proliferation and function

STAT5b null mice have deficiency in NK cell function STAT5a/b null mice have defective cytokine responses

Binds -interferon response element

STAT5 activation prevents apoptosis

Stimulates cell growth required for immune function

STAT5b, Nmi, p300, CBP, protein phosphatase

354

PML, RXR, SMRT, N-Cor, HDAC1

Dominant negative for retinoids Avid binding to corepressors, relieved only by high-dose ATRA Sensitive to ATRA

Relocalizes PML to NBs, degrades PML-RAR, upregulates RAR, differentiation

Heterologous interactions

Transcriptional effects

Effects of ATRA

ATRA sensitivity

Homodimerization

Localizes to :100 microspeckles, which are distinct from NBs May also localize in the cytoplasm Through coiled-coil domain

Three breakpoint clusters result in three major forms All variants contain RING, B-box, coiled-coil

Nuclear localization

N-protein structures

Breakpoint variants

PML-RAR

PLZF, RXR, SMRT, N-Cor, HDAC1, sin3A Dominant negative for retinoids Avid binding to corepressors, not relieved by high-dose ATRA Generally insensitive to ATRA as a single agent ATRA degrades PLZF-RAR but does not induce differentiation ATRA ; HDAC inhibitor suppress growth and promote differentiation

Through the POZ/BTB domain

Localized to microspeckles, not to nuclear bodies or cytoplasm

POZ/BTB, first two zinc fingers

Most cases include first two zinc fingers

PLZF-RAR

TABLE 20.3. Comparison of N-Protein/RAR Fusion Products

Induces differentiation and inhibits growth

Sensitive to ATRA

ATRA-dependent transactivation

Presumably through oligomerization domain SMRT, RAC3, RXR

Microspeckled pattern

Oligomerization domain, metal-binding domain, first acidic domain

Two fusion cDNAs Alternative splicing

NPM-RAR

Not yet reported

Sensitive to ATRA

Unknown Possible interaction with RXR Unknown

Presumably through NuMA coiled domain

Sheetlike aggregates

N-terminal globular domain and central cooled region

Only one breakpoint

NuMA-RAR

Not reported

Insensitive

Unknown

JAKs, Nmi, p300/CBP

Possibly through SH2 domain

Coiled-coil domain, DNA-binding domain, SH3 domain, part of SH2 domain Nuclear microspeckled

One breakpoint

STAT5b

355

Multimerization, sequestration of RXR and other factors Increased affinity for corepressors Transcriptional effects on target genes Interference with PLZF actions Present in 70—80% of cases Unclear role in leukemogenesis

Model

Reciprocal translocation

Miscellaneous

All develop disease after a protracted prodromal period Several models Closest to human APL is MRP8 promoter construct Animals respond to ATRA Resistance to ATRA caused by mutations in ligand-binding domain

Induces CR in APL, rapid degradation of PML-RAR and apoptosis Inhibits differentiation, protects from apoptosis, enhances proliferation, does not transform cells

Transgenic models

Transfected cell models

Effects of arsenic

Multimerization, sequestration of RXR and other factors Increased affinity for corepressors Transcriptional effects on target genes Reciprocal fusion may play a role Present in almost all cases tested Activates PLZF target genes and induces cell proliferation

May bind to different RAREs compared to PML-RAR

Blocks differentiation Fails to increase sensitivity to ATRA Murine marrow progenitors could be serially passaged Mice develop CML-like syndrome earlier than PML-RAR animals Poor response to ATRA

Fails to degrade PLZF-RAR or induce apoptosis

Identified in the index case Actions still unknown

Does not localize to NB Does not delocalize PML but does delocalize PLZF Multimerization, sequestration of RXR and other factors Transcriptional effects on target genes Interference with PLZF actions

APL sensitive to ATRA

Blocked differentiation and enhanced proliferation

Not yet reported

Not yet reported

Multimerization, sequestration of RXR and other factors Interference with apoptosis program

Does not localize to NB Does not delocalize PML

Not yet reported

Not yet reported

Not yet reported

Not present

Sequestration of coactivators, RXR

Could also bind to STAT target genes

Not yet reported

Not yet reported

Not reported

356


Many open questions remain in the field, including:

specifically binds to nucleolar shuttle protein B23. J. Biol. Chem. 268, 13,930—13,934.

· What is the identity of RAR target genes

Ahmad, K. F., Engel, C. K., and Prive, G. G. (1998). Crystal structure of the BTB domain from PLZF. Proc. Natl. Acad. Sci. USA 95, 12,123—12,128.

required for myeloid differentiation?

· What is the mechanism of action of the · · · · ·

PML protein in transcription, apoptosis, and growth control? What is the role of PLZF in hematopoiesis and what are its target genes? How do RAR fusion proteins transform cells? How does arsenic cause the apoptosis of APL cells? Do RAR-N reciprocal fusion proteins play a role in APL? Can manipulation of caspases or IFN pathways offer new therapies for leukemia?

The study of the molecular pathogenesis of APL is at the forefront of the application of molecular biology to clinical medicine, as this disease is the paradigm for successful differentiation therapy. The study of the resistant and sensitive forms of APL yielded an appreciation of the importance of transcriptional repression by histone deacetylation in the development of the disease. This led to the recent use of the deacetylase inhibitor sodium butyrate as a form of targeted transcription therapy in a patient with resistant APL (Warrell et al., 1998). With the development of animal and cell models of sensitive and resistant forms of APL, continued insights leading to the development of more effective therapies for this fascinating disease can be expected. ACKNOWLEDGMENTS This work was supported by NIH R01 CA 59936 (JDL) and American Cancer Society Award DHP 160 (JDL). JDL is a scholar of the Leukemia and Lymphoma Society. AM is supported by NIH K08 CA73762. REFERENCES Adachi, Y., Copeland, T., Hatanake, M., and Oroszlan, S. (1993). Nucleolar targeting signal of rex orotein of human T-cell leukemia virus type I

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CHAPTER 21


THE LEUKEMOGENIC FUNCTION OF THE inv(16) FUSION GENE CBFB-MYH11 P. PAUL LIU, LUCIO H. CASTILLA, AND NEERAJ ADYA Oncogenesis and Development Section, Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health

INTRODUCTION Chromosome 16 inversion, inv(16)(p13;q22), and the related t(16;16) rearrangement are among the most frequent chromosome translocations in acute myeloid leukemias (AML) (Look, 1997). Inv(16) is found in almost 100% of patients with the M4Eo subtype of AML and has also been identified in other subtypes of AML. In many cases, inv(16) is the only chromosome change detected in leukemic cells, suggesting that it plays a very important role during leukemogenesis. In 1993, we identified the breakpoints of inv(16) and the related t(16;16) in AML M4Eo patients by positional cloning (Liu et al., 1993a, 1993b) (Fig. 21.1A). The q-arm breakpoints are within the introns of CBFB and the p-arm breakpoints are within the introns of MYH11. CBFB codes for the -subunit (CBF) of the heterodimeric DNA-binding protein CBF (corebinding factor, also known as PEBP2). MYH11 encodes the smooth muscle myosin heavy chain (SMMHC). Both genes are transcribed in the

centromere to telomere direction, so two reciprocal fusion genes, CBFB-MYH11 and MYH11CBFB, can potentially be generated and transcribed. However, CBFB-MYH11 has been considered as the relevant fusion, since (1) the CBFB-MYH11 fusion transcript can always be detected in inv(16) patients; (2) up to 30% of patients have deletions of MYH11 proximal to the p-arm breakpoint, in addition to the inversion, which prevents the generation of a MYH11-CBFB fusion transcript (Kuss et al., 1994; Marlton et al., 1995); and (3) the MYH11 promoter is most likely inactive in hematopoietic cells (Miano et al., 1994). In fact, we and others failed to detect the expression of the MYH11-CBFB fusion (Asou et al., 1998; Liu et al., 1993b). Multiple fusion combinations have been identified due to heterogeneity of inversion breakpoints (Liu et al., 1995). With the exception of a few cases, the CBFB breakpoints are located in intron 5. For MYH11, more than eight different breakpoints have been described, but about 80% of them are within intron A (Liu et al., 1995). Importantly, the reading frame of


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Figure 21.1. Molecular level defects generated by chromosome 16 inversion. A: Inv(16) breaks the CBFB gene on 16q22 and the MYH11 gene on 16p13 and results in a fusion between the two genes. The CBFB-MYH11 fusion gene is functional and can be transcribed into CBFB-MYH11 fusion mRNA. B: The breakpoints in the CBFB gene removes the coding sequence for the last 22aa of CBF, leaving its CBF-binding domain intact. The breakpoints in the MYH11 gene are in the region coding for the coiled-coil repeats of the myosin tail. The resulting chimeric protein can therefore bind to CBF proteins and form homodimers and multimers.

MYH11 is preserved in all the fusion combinations; therefore, a chimeric protein composed of N-terminal CBF and C-terminal SMMHC sequences can be produced in each type of fusion. All the in vitro and animal studies reported so far have used the type A CBFB-MYH11 fusion gene, the most common fusion generated by combining CBFB exons 1—5 with MYH11 exons after intron A (encoding 165aa of CBF and 446aa of SMMHC). This chapter reviews recent studies on CBFB-MYH11 that have led to a better understanding of how it contributes to the multistep progression of leukemia.

CHARACTERISTICS OF PROTEINS CBF AND SMMHC CBF is a relatively small protein with its longest isoform at 187aa. The protein sequence

reveals very little functional information, since it does not contain recognizable functional domains found in other proteins nor is it homologous to any protein in the GenBank other than its orthologs in other species. The only known function for this protein is its association with the -subunits of CBF, which binds to a core sequence TGTGGT, initially found in the enhancers of MMLV and polyomavirus, and later in the regulatory regions of many cellular genes (see Chapter 6 in this book). CBF does not bind to DNA directly, but enhances the binding affinity of the CBF proteins. This enhancement seems necessary for the full function of at least one of the CBF proteins, as the phenotypes of Cbfb and Cbfa2 knockout mice are almost identical to each other (Niki et al., 1997; Okuda et al., 1996; Sasaki et al., 1996; Wang et al., 1996a, 1996b). The fact that one of the three CBF proteins, CBF2, also known as

BIOCHEMICAL AND CELLULAR STUDIES

AML1 and PEBP2B, is also a frequent target of chromosome translocations in human leukemias (see other chapters in this book) underscores the importance of the CBF heterodimer in leukemogenesis. With a few exceptions, the inv(16) breakpoints in the CBFB gene are localized in intron 5, removing only the last 22 amino acids of CBF from the CBF-SMMHC fusion. According to in vitro protein-protein interaction and ES cell differentiation studies, CBF1-165, the portion retained in the CBF-SMMHC chimeric proteins, is functionally equivalent to the full-length protein (Huang et al., 1998). Therefore it seems likely that an additional functional unit rather than the truncation of 22aa of CBF underlies the pathogenic activity of the fusion protein. SMMHC is normally found in the muscle fibers in smooth muscle cells. As a component of a highly organized cellular motor complex, the protein is large and multimodular. The Nterminal portion of the protein folds into a globular head and contains domains for actin interaction as well as ATPase acitivity. The C-terminal portion of the protein forms a long -helical rod that is subdivided into the S-2 region and the most C-terminal light meromyosin (LMM) region, which is composed of tandem repeats of the coiled-coil domain. The coiled-coil domain contains a heptad sequence repeat, (a-b-c-d-e-f-g) , with the a and d residues L being hydrophobic (McLachlan and Karn, 1983; McLachlan et al., 1975). In addition, groups of 4 heptad repeats form higher-order repeats of 28 residues. This coiled-coil domain is capable of spontaneous dimerization through the hydrophobic residues. The rodlike tail is also responsible for filament assembly, an organizational process involving multiple myosin molecules. All the inv(16) breakpoints discovered so far disrupt the MYH11 gene in the LMM region. Therefore, the difference among various fusion combinations is mainly the copy number of the coiled-coil repeat retained in the fusion, with the type A fusion retaining the fewest. In summary, the chimeric CBF-SMMHC protein retains the full function of CBF and acquires the dimerization/multimerization ability from the SMMHC tail (Fig. 21.1B). The fact that both CBFB and one of the CBFA genes are

381

involved in human leukemia in similar fashions suggests that CBF function is the likely target of the CBF-SMMHC fusion protein. The ability of the CBF-SMMHC protein to disrupt normal CBF function may come from the SMMHC tail. Several years ago, we proposed three possible models for the mechanism of CBFSMMHC pathogenesis: dominant superactivation, dominant negative sequestration, and dominant interference (Liu et al., 1995). All of the models are dominant, since inv(16) disrupts only one set of CBFB and MYH11 genes, with a normal copy of the CBF gene present and expressed in the leukemic cells.

BIOCHEMICAL AND CELLULAR STUDIES Analysis of DNA-protein and protein-protein interactions using proteins generated in vitro demonstrated that, as predicted, CBFSMMHC can bind to CBF proteins with equal efficiency as the wild-type CBF, and can dimerize spontaneously (Adya et al., 1998; Liu et al., 1994). In addition, CBF-SMMHC does not change the pattern of DNA contact region by CBF in the DNA footprinting assays using DNase or hydroxy radical (Adya and Liu, unpublished results). However, in protein-DNA interaction studies with cell extracts from transfected myeloid and lymphoid cells, CBFSMMHC seems to decrease the binding of CBF to DNA (Cao et al., 1997). This difference between in vitro and in vivo behaviors of the protein is consistent with the finding that CBF-SMMHC can sequester CBF2 in the cytoplasm, as described below. As summarized in Table 21.1, several groups have studied the subcellular localization of CBF2, CBF, and CBF-SMMHC, when expressed individually or in combination. In transient transfection experiments, we and others have observed that normal CBF2 is nuclear (Adya et al., 1998; Lu et al., 1995). This has been confirmed not only in cell culture systems but also in murine embryonic fibroblasts (MEF) with a knocked-in Cbfa2-lacZ gene, where the protein is expressed at the physiological level, thereby eliminating the potential effect of overexpression in the cell culture systems (Adya et al., 1998).

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THE LEUKEMOGENIC FUNCTION OF THE inv(16) FUSION GENE CBFB-MYH11

TABLE 21.1. Summary of Subcellular Localization of CBF, CBF2, and CBF-SMMHC from the Literature Subcellular Localization Expressed Proteins CBF2

Cell Type NIH3T3 MEFB

Adya/Liu? Nuclear

NIH3T3 REF52 CBF

NIH3T3

Lu/Kanno@ NDC Nuclear

Cytoplasmic;nuclear (diffuse)

Cytoplasmic (diffuse)

REF52 Muscle cells

Cytoplasmic;nuclear (some in stress fiber) into nucleus

CBF2;CBF

NIH3T3

CBF-SMMHC

AML-M4Eo Mainly cytoplasmic TransgenicD speckles in nucleus NIH3T3

into nucleus with truncated

Cytoplasmic

Myoblast NIH3T3 MEF?

ND

Mainly cytoplasmic stress fibers

NIH3T3 REF52 Jurkat

CBF2; CBF-SMMHC

Chiba/TanakaA

Mainly cytoplasmic Disrupts stress fibers Sequestration of CBF2 to cytosol

REF52 Jurkat

ND Sequestration of CBF2 to cytosol

?Adya et al., 1998; Liu et al., 1996. @Lu et al., 1995; Kanno et al., 1998. AChiba et al., 1997; Tanaka et al., 1997, 1998. BMouse embryonic fibroblasts with a knocked-in Cbfb-MYH11 and a knocked-in cbfa2-lacZ. CND: not done. DKogan et al., 1998.

In NIH3T3 cells, CBF is mainly in the cytoplasm and distributed in a diffuse pattern (Adya et al., 1998; Lu et al., 1995). In addition, a fraction of CBF in the cytoplasm was found to be associated with actin stress fibers in a rat embryonic fibroblast cell line REF52 and in muscle cells (Chiba et al., 1997; Tanaka et al., 1997). This association between CBF and the stress fibers appears to be inefficient, since most CBF is diffusely distributed in the cytoplasm (Tanaka et al., 1997). We have investigated and failed to observe this cytoskeletal association (Adya et al., 1998). Using immunofluorescent staining and EGFP-fusion proteins, we have demonstrated that coexpression of CBF2 and CBF in NIH 3T3 cells resulted in the transportation of CBF into nucleus, presumably by CBF2 (Adya et al.,

1998). This transportation is consistent with the model that CBF2 requires the presence of CBF to be fully functional as a transcription factor. In our experiments, full-length CBF2 was able to transport CBF, in a dose-dependent fashion. However, another group reported that only a truncated CBF2 protein without sequences C-terminal to the runt domain can transport CBF in NIH 3T3 cells (see Table 21.1 and the reference therein). This discrepancy may result from different experimental settings. It is likely that full-length CBF2 is capable of transporting CBF, albeit at low efficiency, while C-terminal truncation of CBF2 enhances this transportation. CBF-SMMHC is also mostly cytoplasmic in both fibroblasts in culture (Adya et al., 1998; Lu et al., 1995) and in human inv(16)> leukemic

FUNCTIONAL STUDIES

cells (Liu et al., 1996). In fibroblasts with rich cytoskeletal structure, the protein is distributed along stress-fiber filaments and aggregates, which colocalize with actin (Adya et al., 1998). Since CBF-SMMHC does not contain the actin-interacting head region of myosin heavy chain, this colocalization presumably results from participation in filament assembly with cellular nonmuscle myosin heavy chain through their similar tail domains. In addition, some CBF-SMMHC molecules can be detected in the nucleus, forming speckled or rodlike structures (Wijmenga et al., 1996), which are most likely multimers of the protein generated through myosin tail association. Most importantly, CBF-SMMHC can sequester CBF2 in the cytoplasm, or can form aggregates with CBF2 in the nucleus, upon coexpression (Adya et al., 1998; Kanno et al., 1998). This sequestration has been demonstrated in cultured NIH3T3, REF52, and Jurkat cells, after transfection of cDNA constructs (Table 21.1). It has also been observed in primary fibroblasts from embryos resulting from matings between the Cbfa2-lacZ mice and chimeras with a knocked-in Cbfb-MYH11 gene (see below) (Adya et al., 1998). Again, this in vivo demonstration provides more compelling evidence for CBF2 sequestration by CBFSMMHC. Since this sequestration will trap CBF2 in the cytoplasm, or maybe in an abnormal nuclear compartment, it may explain the reduced DNA binding by CBF2 in cells expressing CBF-SMMHC, as mentioned above, and would support the dominant negative model for CBF-SMMHC leukemogenesis. It is still not clear how the sequestration could be dominant, since CBF-SMMHC was not shown to have increased affinity for CBF2. One possibility is that CBF-SMMHC is more abundant in the cell than CBF, because CBFSMMHC is more stable due to the myosin tail. Another possibility is that partial CBF2 sequestration is enough to abrogate CBF function, since CBF2 concentration may be limiting in hematopoietic progenitor cells (Wang et al., 1996a). The importance of the functional domains of the CBF-SMMHC protein has also been investigated by analysis of deletion constructs (Adya et al., 1998). It was demonstrated that deleting the N-terminal aa2—11 of the protein, a region required for CBF-interaction, or the C-ter-

383

minal dimerization domain, abrogates sequestration of CBF2 by CBF-SMMHC (Adya et al., 1998). Interestingly, a CBF-SMMHC construct without the C-terminal 95aa, which can form dimers but with much reduced ability to form filaments, still sequesters CBF2 efficiently (Adya et al., 1998). This result suggests that mutimerization or filament assembly may not be required for CBF2 sequestration. Some CBF2 and CBF-SMMHC can be observed in the nucleus as well. When they are present at high concentration, a rodlike structure forms, presumably resulting from multimerization through the myosin tail. At physiological levels, these proteins may still be able to bind DNA. It was recently observed that CBFSMMHC could in combination with CBF2 drastically suppress the p21 promoter (driving the expression of the cell cycle inhibitor p21) in NIH3T3 cells, which suggests that CBFSMMHC may also interfere with transcription at the DNA level (Lutterbach et al., 1999).

FUNCTIONAL STUDIES Cell lineage studies of clinical leukemia samples suggest that CBF-SMMHC interferes with lineage determination and differentiation. A large proportion of inv(16)> leukemic cells are CD34> and c-kit>, indicating the involvement of stem cells or early progenitors (Osato et al., 1997). More than half of inv(16) leukemia cases have bone marrow eosinophilia, characterized by abnormal eosinophilic blasts with both eosinophilic and basophilic granules. Such cells are believed to be part of the malignant cell population since they contain inversion 16 (Haferlach et al., 1996). In addition, lymphocytic cell markers, such as CD2 and TdT, are frequently detected (Adriaansen et al., 1993) in these leukemic cells. Therefore, CBF-SMMHC may direct aberrant differentiation along the eosinophilic lineage, with expressions of genes from other lineages. Functionally, CBF-SMMHC was found to mostly inhibit CBF-mediated transactivation in transient transfection assays. This inhibition has been demonstrated for Mo-MLV enhancer (Liu et al., 1994), the T-cell—specific T-cell receptor enhancer (Tanaka et al., 1998), an artificial reporter construct with four copies of the CBF

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site in the myeloid-specific myeloperoxidase gene (Cao et al., 1998), and the myeloid-specific M-CSF-R (Kanno et al., 1998) and neutrophil protein-3 enhancers (Westendorf et al., 1998). Again, the inhibition of these CBF transactivation functions is dependent on the presence of both the CBF-interacting domain and the SMMHC tail. Another study showed that GATA-1 is expressed at lower levels in leukemia cells with inv(16) than those without inv(16), but it is not known whether GATA-1 gene is a direct target of CBF (Patmasiriwat et al., 1996). These findings are consistent with the model of dominant suppression of CBF function by CBF-SMMHC. It is unlikely, however, that any of these genes plays an important role in myeloid leukemia transformation, since they are normally expressed in late stages of myeloid differentiation or in other lineages. When expressed in cultured myeloid and lymphoid cells from an inducible viral vector, CBF-SMMHC was found to increase cell generation time, decrease tritiated thymidine incorporation into DNA, and inhibit G1 to S cell cycle transition at the restriction point (Cao et al., 1997). Consistent with this observation is the finding that Rb phosphorylation is reduced by CBF-SMMHC expression and that p21 protein level is increased in 32D c13 cells expressing CBF-SMMHC (Cao et al., 1997). It is interesting to note that p21 protein is increased in 32D c13 cells while the promoter of the p21 gene was found to be suppressed by CBF-SMMHC. It is possible that the effect of CBF-SMMHC on p21 promoter is cell type dependent. This inhibition of cell cycle progression also requires the N-terminal CBF-interaction and C-terminal dimerization domains (Cao et al., 1998). These data seem to be paradoxical regarding the leukemogenic role of CBF-SMMHC. One possible explanation is that this inhibition of cell cycle is related to the blockage of differentiation by CBF-SMMHC, as observed in the mouse model (see below). However, granulocytic differentiation of the cultured myeloid cell line 32D in G-CSF was not affected by the presence of CBF-SMMHC. It was proposed that additional genetic mutations that stimulate G1 could bypass the growth inhibitory effect of CBF-SMMHC, potentiating its ability to block differentiation or to block apoptosis (Cao et al., 1997). Indeed, exogenous cdk4 can par-

tially overcome growth inhibition by CBFSMMHC (Lou et al., 1998). Alternatively, the data could be reflective of CBF-SMMHC function under a specific set of experimental conditions, which may or may not correlate with its leukemogenic potential. Similar paradoxical effects on cell proliferation have been documented for other oncogenes such as c-myc and E2APBX1 (Dedera et al., 1993; Evan et al., 1992). More recently, it was reported that CBFSMMHC reduces p53 induction by DNA damage in cultured Ba/F3 cells, a murine pro-B cell line (Britos-Bray et al., 1998). Moreover, apoptosis of Ba/F3 cells induced by DNA damage is slowed by CBF-SMMHC, even though apoptosis induced by IL-3 withdrawal is not affected. Again, the effect is shown to be a CBFspecific function, since cells expressing CBFSMMHC with a deletion of the N-terminal CBF-interacting domain do not attenuate p53 induction. On the other hand, the induction of p21 expression by DNA damage is not affected by the presence of CBF-SMMHC, nor does it affect the expression of other apoptotic pathway genes, such as Bcl-2, Bcl-Xl, Bcl-Xs, and Bax. These data thus suggest that CBF-SMMHC may also contribute to leukemogenesis through attenuation of p53 induction from DNA damage, which may lead to the accumulation of additional mutations. Despite the effects of CBF-SMMHC on apoptosis and cell cycle regulation, the protein is not transforming in standard cell culture assays: NIH3T3 cells expressing the protein do not form foci and cannot produce colonies when grown on soft agar (Liu et al., unpublished data). Functional studies of CBF-SMMHC, as reviewed here, suggest that it contributes to leukemogenesis by affecting normal functions of CBF.

TRANSGENIC MOUSE MODELS Mouse models have been very useful for the study of genetic and cellular processes associated with leukemogenesis and for designing and testing new interventions of leukemia. Mice develop lymphoma and lymphocytic leukemia spontaneously. Hematological malignancies can also be induced by viruses, chemical mutagens, or radiation. More importantly, specific on-

KNOCK-IN MOUSE MODEL

cogenes can be introduced into the mouse genome by transgenic technologies and their ability to induce leukemia can then be studied. We have attempted to generate mouse models for CBFB-MYH11—associated leukemia through both retroviral infection and transgenesis. Bone marrow transplantation with hematopoietic cells infected with a retroviral construct containing the CBFB-MYH11 gene resulted in poor engraftment, even with high titers of the virus. A transient leukocytosis was observed, but no leukemia was induced (Liu et al., unpublished results). Transgenic mice were also produced, after pronuclear injection of a plasmid CBFB-MYH11 construct controlled by the CMV promoter. The expression of the gene was low but detectable in the bone marrow, and quite high in other tissues, such as heart, muscle, and kidney. Again, no leukemia was induced in those transgenic mice (Liu et al., unpublished results). Kogan and colleagues reported the generation of transgenic mice with the CBFBMYH11 gene driven by the promoter of a myeloid-specific gene, MRP8 (Kogan et al., 1998). The S100-like calcium-binding protein Mrp8 is expressed in immature myeloid cells as well as circulating neutrophils and monocytes (Lagasse and Weissman, 1992), but not hematopoietic stem cells (Kogan et al., 1998). The transgenic mice expressed the fusion protein at high levels in the bone marrow, but not in the spleen, thymus, and liver. By immunofluorescent staining the fusion protein was detected in neutrophils (Kogan et al., 1998). The staining was mostly in the nucleus, with speckled and rodlike staining patterns, but some staining was seen in the cytoplasm as well. However, the transgenic mice were healthy and no malignancies above background rate were observed in over 100 transgenic mice from two independent transgenic lines for more than 15 months. Detailed analysis of myeloid differentiation in the bone marrow and in culture revealed a shift toward more immature neutrophilic cells from mature ones, but the mice were able to maintain normal numbers of neutrophils in the peripheral blood. Close examination of the circulating neutrophils revealed subtle changes of the nuclear morphology, the significance of which is unclear. The hMRP8-CBF-MYH11 mice were also crossed with mice transgenic for activated

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NRAS (NRASG12D) under the control of the hMRP8 promoter. Again, there was a relative increase of immature neutrophils in the bone marrow, and the mature neutrophils showed some dysplastic nuclear features, but no leukemia developed in these double transgenic mice over the course of 15 months. The failure to produce a leukemic phenotype in these mice may be caused by inadequate expression of the CBFB-MYH11 gene. Alternatively, the hMRP8 promoter may have expressed the gene in the wrong target cell population or to cells at the wrong stage of differentiation. Although the same group using the same promoter successfully induced leukemia in mice transgenic for the PML-RARA gene, the frequency of leukemia development in those mice was very low (Brown et al., 1997). Taken together, these studies also suggest that the fusion gene CBFB-MYH11 by itself may not be enough to induce murine leukemia and may require cooperating events.

KNOCK-IN MOUSE MODEL Since no leukemia has yet been generated with transgenic or retroviral approaches, and the appropriate expression of the gene seems to be critical, we designed a strategy to express CBFBMYH11 in mice mimicking the natural setting (Castilla et al., 1996). In the approach, a mouse Cbfb exon 5—human MYH11 fusion fragment was engineered to replace the endogenous Cbfb exon 5 in embryonic stem (ES) cells by homologous recombination. The approach has been dubbed ‘‘knock-in,’’ since we were inserting a replacement fragment of a gene into the locus rather than deleting the entire gene. Through knock-in technology, one copy of the mouse Cbfb gene is replaced by a Cbfb-MYH11 fusion and its expression is controlled by the endogenous regulatory sequences for Cbfb. This way, we produced murine ES cells with a CBFB gene composition (Cbfb>/Cbfb-MYH11) closely resembling that in the human leukemic cells with an inv(16). Such Cbfb>/Cbfb-MYH11 ES cells were used to produce chimeric mice (Castilla et al., 1996). Despite good contribution of the ES cells to other tissues of the chimeras, the contribution to the hematopoietic tissues was poor: by G-6-PD

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TABLE 21.2. Phenotype Comparison Between Cbf Gene Knockouts and Cbf Leukemia Gene Knock-ins Cbfa2\\ ? Embryonic lethality CNS hemorrhage Definitive hematopoiesis Primitive hematopoiesis

E11.5—12.5 Yes Absent Normal

Cbfb\\ @ E12.5—13.5 Yes Absent Normal

Cbfa2;/Aml1-ETO A E12.5 Yes Absent Normal

Cbfb;/Cbfb-MYH111 B E12.5 Yes Absent Maturation block

?Okuda et al., 1996; Wang et al., 1996a. @Wang et al., 1996b; Sasaki et al., 1996. AYergeau et al., 1997. BCastilla et al., 1996.

polymorphism analysis there were no ES-derived cells in the bone marrow, spleen, thymus, and peripheral blood. In addition, a PCR reaction for the neo gene (present in the knock-in construct) was negative using DNA from nucleated blood cells. Therefore, the Cbfb>/Cbfb-MYH11 ES cells seemed to have a selective inability to populate the hematopoietic tissues. However, it is not clear whether this deficiency results from a homing defect of hematopoietic stem cells or a differentiation defect after engraftment, since the G-6-PD assay is not sensitive enough to detect the presence of small numbers of ES-derived cells. When the Cbfb>/Cbfb-MYH11 chimeras were bred to wild-type females, no F1 mice heterozygous for the fusion gene were born (Castilla et al., 1996). The F1 embryos were analyzed and embryos with the knocked-in fusion gene were found to have died around midgestation (E12.5). The overall development of the embryos was normal up to E12.5, suggesting that CBFSMMHC does not interfere with normal embryogenesis. However, the embryos suffered from massive hemorrhage, mostly in the periventricular regions of the brain and the spinal cord, the cranial nerve ganglia, and the dorsal root ganglia. Such hemorrhage may have resulted from vascular defects in the nervous systems, with necrosis of the endothelial cells (Wang et al., 1996a). More interestingly, definitive hematopoiesis (taking place in the fetal liver) is absent in the Cbfb-MYH11 heterozygous embryos. Consistent with this in vivo observation, there were 30—100 fold fewer hematopoietic colonies generated in vitro with cells from the fetal liver and the yolk sac of the Cbfb>/Cbfb-MYH11 embryos than from those of the

wild-type embryos. This set of phenotypic abnormalities is remarkably similar to that of embryos with homozygous deletions of the Cbfb gene or the Aml1/Cbfa2 gene (Okuda et al., 1996; Sasaki et al., 1996; Wang et al., 1996a, 1996b), thus providing very strong genetic evidence for the hypothesis that Cbfb-MYH11 is a dominant negative regulator of CBF function (Table 21.2). In addition, the maturation of yolk sac—derived primitive erythrocytes was delayed, which is unique to the Cbfb>/Cbfb-MYH11 embryos. The mechanism for this maturation interference is still unresolved. In order to study the effect of CBF-SMMHC on adult hematopoiesis and its contribution to leukemia formation, two approaches can be taken to overcome the lethality of the Cbfb>/Cbfb-MYH11 embryos. First, a conditional knock-in can be generated. The bacterial Crelox system can be used to activate the CbfbMYH11 allele in specific tissues and at specific times during development. This way, we can target the expression of the fusion gene to hematopoietic cells in the adult bone marrow. Second, the Cbfb>/Cbfb-MYH11 chimeras can be studied. As mentioned above, we could not detect the presence of the fusion gene in the hematopoietic tissues in the chimeras using the G-6-PD assay. The result indicates poor contribution to hematopoietic tissues, but cannot rule out the presence of Cbfb>/Cbfb-MYH11 cells in the bone marrow. The population of such cells may be very small, since they may have a differentiation defect and could remain undetectable due to the low sensitivity of the protein assay. Indeed, we found the presence of the ES-cell-derived mature erythrocytes in the blood of the chimeras with a

CONCLUSIONS AND FUTURE DIRECTIONS

globin polymorphism assay, which can distinguish those of strain 129 origin (the ES cells) from those of strain C57BL/6 origin (the blastocysts). The percentage of ES-derived erythrocytes in the blood of these chimeras ranged from zero to over 80% (Castilla et al., 1999). In contrast, PCR reactions continued to be negative for the neo gene with DNA from the blood nucleated cells of the same chimeras. The results are consistent with our hypothesis that a small population of the ES-derived stem cells is present in the bone marrow of the Cbfb>/Cbfb-MYH11 chimeras. In addition, these stem cells are blocked selectively for myeloid and lymphoid differentiation. A recent study demonstrates that Cbfa2 is expressed only in the myeloid and lymphoid lineages of adult hematopoiesis, but not in the erythroid lineage (Corsetti and Calabi, 1997). This would explain why we observed a selective blockage of myeloid and lymphoid differentiation but not erythroid differentiation in our Cbfb>/Cbfb-MYH11 chimeras. Moreover, 45% of the Cbfb>/Cbfb-MYH11 chimeras developed leukemia or lymphoma between 12 and 26 months of age (Castilla et al., unpublished results). The malignant cells contain the Cbfb-MYH11 gene, as detected by PCR and Western blot analysis. Therefore, Cbfb-MYH11 can induce hematologic malignancies in the chimeric mice, but the long latency suggests that additional steps are required for full transformation. To test this two-hit hypothesis, we injected the Cbfb>/Cbfb-MYH11 chimeras intraperitoneally with a single sublethal dose (100mg/kg) of

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N-ethyl-N-nitrosourea (ENU), a potent DNA mutagen. Two to six months later, 84% of the ENU-treated Cbfb>/Cbfb-MYH11 chimeras developed acute myelomonocytic leukemia, whereas none of the ENU-treated control animals did (Castilla et al., 1999). The leukemic cells contained the Cbfb-MYH11 fusion gene and therefore were derived from the hidden Cbfb>/Cbfb-MYH11 stem cells in the bone marrow. Interestingly, the atypical eosinophils found in human AML M4Eo patients (Bitter et al., 1984) can also be identified in some of the mice (Fig. 21.2). In addition, the leukemic cells frequently infiltrated the meningeal membrane in the chimeric mice, again a typical finding in human AML M4Eo cases (Glass et al., 1987; Holmes et al., 1985). These findings confirmed the two-hit hypothesis and established an efficient and fairly accurate model for the leukemogenic contribution by the Cbfb-MYH11 fusion gene.

CONCLUSIONS AND FUTURE DIRECTIONS In summary, much has been learned about the inversion 16 fusion gene CBFB-MYH11 and its encoded protein CBF-SMMHC since the cloning of the inversion 16 breakpoints in 1993. The biochemical and molecular biological studies have indicated that CBF-SMMHC can suppress CBF function, with sequestration of CBF proteins as one mechanism. Our mouse model generated through homologous recombination confirmed in vivo that CBF-SMMHC represses CBF function dominantly and interferes with embryonic hematopoiesis. Moreover,

Figure 21.2. Leukemic cells in the Cbfb-MYH11 chimeric mice resemble the abnormal eosinophils in inv(16)> AML M4Eo. The cell on the left is from a chimeric mouse and the cell on the right is from a patient with inv(16)> AML M4Eo. Typical M4Eo eosinophils have nuclear features of monocytes while their cytoplasm contains both eosinophilic and basophilic granules. The right-hand panel is derived from slide C2002 from the American Society of Hematology Slide Bank. Used with permission.

Figure 21.3. Hematopoietic blockage and leukemogenesis by CBFB-MYH11. A: Normal expression pattern of CBF in adult hematopoiesis. B: CBFB-MYH11 blocks maturation of myeloid and lymphoid lineages. The exact point of blockage is unclear. C: Full leukemic transformation takes place after additional genetic alterations.

the mouse model demonstrated that CBFSMMHC blocks myeloid and lymphoid differentiation during adult hematopoiesis, which serves as an initiation step toward leukemia formation (Fig. 21.3). The picture therefore emerges that CBFBMYH11 is an oncogene for a specific type of leukemia rather than a generalized oncogene that can transform cells of multiple tissues. This specificity most likely resides in the ability of CBF-SMMHC to suppress CBF2 function. In a broad sense, our results suggest that CBF2 is a tumor suppressor for acute myeloid leukemia. In fact, biallelic and heterozygous point mutations in the AML1/CBFA2 gene have been detected in several myeloid leukemia cases (Osato et al., 1999). The mutations are either truncations or missense mutations resulting in functional defect. We are conducting ENU mutagenesis studies using Cbfa2\\chimeras (Cbfa2\\ is also embryonic lethal). If we can induce acute myeloid leukemia in such Cbfa2\\ mice by ENU treatment, a link between loss of CBF function and leukemia development will be substantially strengthened. It is also clear from our mouse model that interruption of CBF function is just one of two

or more steps toward full leukemogenesis (Fig. 21.3). It will be very important to identify what genes can cooperate with CBFB-MYH11 for leukemic transformation. Such cooperating genes are likely involved in the control of cell cycle progression and apoptosis, since CBFBMYH11 mainly blocks differentiation. No consistent mutations or genetic alterations have been identified in human inv(16)> leukemia cases. The murine ENU chimera model is a less than ideal choice for such studies, since ENU generates only point mutations, which are very difficult to detect. Therefore, we are trying to induce leukemia in our chimeras by retroviral insertional mutagenesis. The retrovirus we are using does not induce leukemia in wild-type mice. Preliminary data indicate that the retrovirus can induce acute myeloid leukemia in our Cbfb>/Cbfb-MYH11 chimeras with high efficiency (Castilla and Liu, unpublished results). Therefore, we will be able to identify the cooperating genes rapidly in this system through cloning viral insertion sites. Since CBF functions by regulating transcription, it is obviously of great importance to identify the target genes of CBF that are involved in the pathogenesis of leukemia. As al-

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ready mentioned, only a few potential target genes have been identified, with their expressions inhibited by CBF-SMMHC. A systematic screening for target genes using technologies such as the cDNA microarray is needed to identify true target genes, which will enhance our understanding of the mechanism through which CBF-SMMHC contributes to leukemogenesis, and provide new directions for clinical management. CBF-SMMHC may have functions other than suppressing CBF. As mentioned above, its effect on cell cycle control and apoptosis needs to be explored further. In addition, CBFSMMHC may interact with other proteins through its myosin tail that contributes to its leukemogenic potential. CBF-SMMHC may also interfere with DNA binding or complex assembly by other transcription factors. Finally, it is our ultimate goal to design better treatment and to cure leukemia. Several scenarios can already be considered based on our current knowledge. Gene transfer technology can be employed to deliver antisense constructs to the CBFB-MYH11 fusion gene. It can also be used to overexpress the AML1 gene, in the hope of overcoming the dominant suppression by CBFB-MYH11. Since differentiation blockage is an important step during leukemogenesis, cytokines and other reagents (such as histone deacetylase inhibitors) may be used to induce differentiation. Our mouse leukemia model can be used to test these different strategies. In the near future, additional strategies can be generated after the CBFB-MYH11 target genes are identified.

ACKNOWLEDGMENTS We would like to thank Alan Friedman and Jennifer Puck for critical reading of the manuscript, and Scott Hiebert for sharing unpublished results. The AML M4Eo cell in Figure 21.2 was from slide C2002 of the American Society of Hematology Slide Bank, used with permission.

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Patmasiriwat, P., Fraizer, G. C., Claxton, D., Kantarjian, H., and Saunders, G. F. (1996). Expression pattern of WT1 and GATA-1 in AML with chromosome 16q22 abnormalities. Leukemia 10, 1127— 1133. Sasaki, K., Yagi, H., Bronson, R. T., Tominaga, K., Matsunashi, T., Deguchi, K., Tani, Y., Kishimoto, T., and Komori, T. (1996). Absence of fetal liver hematopoiesis in mice deficient in transcriptional coactivator core binding factor beta. Proc. Natl. Acad. Sci. USA 93, 12,359—12,363. Tanaka, Y., Watanabe, T., Chiba, N., Niki, M., Kuroiwa, Y., Nishihira, T., Satomi, S., Ito, Y., and Satake, M. (1997). The protooncogene product, PEBP2beta/CBFbeta, is mainly located in the cytoplasm and has an affinity with cytoskeletal structures. Oncogene 15, 677—683. Tanaka, Y., Fujii, M., Hayashi, K., Chiba, N., Akaishi, T., Shineha, R., Nishihira, T., Satomi, S., Ito, Y., Watanabe, T., and Satake, M. (1998). The chimeric protein, PEBP2 beta/CBF beta-SMMHC, disorganizes cytoplasmic stress fibers and inhibits transcriptional activation. Oncogene 17, 699—708. Wang, Q., Stacy, T., Binder, M., Marin-Padilla, M., Sharpe, A. H., and Speck, N. A. (1996a). Disruption of the Cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis. Proc. Natl. Acad. Sci. USA 93, 3444—3449. Wang, Q., Stacy, T., Miller, J. D., Lewis, A. F., Gu, T. L., Huang, X., Bushweller, J. H., Bories, J. C., Alt, F. W., Ryan, G., Liu, P. P., Wynshaw-Boris, A., Binder, M., Marin-Padilla, M., Sharpe, A. H., and Speck, N. A. (1996b). The CBF beta subunit is essential for CBF alpha2 (AML1) function in vivo. Cell 87, 697—708.

Okuda, T., van Deursen, J., Hiebert, S. W., Grosveld, G., and Downing, J. R. (1996). AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis. Cell 84, 321—330.

Westendorf, J. J., Yamamoto, C. M., Lenny, N., Downing, J. R., Selsted, M. E., and Hiebert, S. W. (1998). The t(8;21) fusion product, AML-1—ETO, associates with C/EBP-alpha, inhibits C/EBP-alpha-dependent transcription, and blocks granulocytic differentiation. Mol. Cell. Biol. 18, 322—333.

Osato, M., Asou, N., Okubo, T., Nishimura, S., Yamasaki, H., Era, T., Suzushima, H., Kawano, F., Matsuoka, R., Oka, H., Bae, S. C., Ito, Y., and Takatsuki, K. (1997). Myelomonoblastic leukaemia cells carrying the PEBP2 beta/MYH11 fusion gene are CD34, c-KIT> immature cells. Br. J. Haematol. 97, 656—658.

Wijmenga, C., Gregory, P. E., Hajra, A., Schrock, E., Ried, T., Eils, R., Liu, P. P., and Collins, F. S. (1996). Core binding factor beta—smooth muscle myosin heavy chain chimeric protein involved in acute myeloid leukemia forms unusual nuclear rod-like structures in transformed NIH 3T3 cells. Proc. Natl. Acad. Sci. USA 93, 1630—1635.

Osato, M., Asou, N., Abdalla, E., Hoshino, K., Yamasaki, H., Okubo, T., Suzushima, H., Takatsuki, K., Kanno, T., Shigesada, K., and Ito, Y. (1999). Biallelic and heterozygous point mutations in the runt domain of the AML1/PEBP2 alphaB gene associated with myeloblastic leukemias. Blood 93, 1817—1824.

Yergeau, D. A., Hetherington, C. J., Wang, Q., Zhang, P., Sharpe, A. H., Binder, M., Marin-Padilla, M., Tenen, D. G., Speck, N. A., and Zhang, D. E. (1997) Embryonic lethality and impairment of haematopoiesis in mice heterozygous for an AML1ETO fusion gene. Nat. Genet. 15, 303—306.

CHAPTER 22


EVI1 REARRANGEMENTS IN MALIGNANT HEMATOPOIESIS GIUSEPPINA NUCIFORA Oncology Institute and Department of Medicine Loyola University Medical Center

INTRODUCTION Hematopoiesis is the result of a complex balance between stem cell replication and lineage-specific differentiation that is regulated by a large number of cytokines promoting the differentiation, survival, or proliferation of hematopoietic cells. The lifelong maintenance and regenerative capacity of the hematopoietic system depend on the correct expression of key regulatory proteins that govern self-renewal, lineage commitment, and differentiation of hematopoietic stem cells and progenitors. The genes encoding these proteins are frequently targeted by chromosomal abnormalities that alter either the biochemical structure or their level of expression, often leading to leukemia (Baron et al., 1993; Chen et al., 1993; Nucifora and Rowley, 1995; Peters et al., 1997; Ye et al., 1993). These abnormalities present only in the leukemic clones, are usually caused by environmental factors and consist of structural aberrations such as chromosomal translocations, inversion, deletions or duplications, and numerical aberrations, with gain or

loss of entire chromosomes. Because of the frequency with which specific abnormalities occur in human leukemia, and because they are associated with very specific types of leukemia, it has long been suggested that key regulatory genes active in hematopoiesis are disrupted at the site of the chromosomal rearrangement. Indeed, after molecular cloning and analysis, it was determined that chromosomal abnormalities disrupt or mutate several genes that are necessary for the ontogeny of the hematopoietic system during early embryonic development (AML1, acute myeloid leukemia 1), at a later stage, when genes are necessary to maintain and to differentiate the hematopoietic stem cell into lineage-committed progenitors (T EL , translocation ets leukemia), or into mature blood cell types (E2A, kappa E2 activator). Chromosome band 3q26 contains the locus of two genes, MDS1/EVI1 (myelodysplastic syndrome 1, ecoptropic viral integration 1) and EVI1, encoding nuclear factors with two sets of DNA-binding zinc fingers. MDS1/EVI1 is identical to EVI1 aside from the N-terminus exten-


393

394

EVI1 REARRANGEMENTS IN MALIGNANT HEMATOPOIESIS

sion, which is missing in EVI1, but the two proteins have opposite functions as transcription factors and as regulators of hematopoietic cell differentiation and growth. The two genes are often activated inappropriately in leukemic cells by different mechanisms involving chromosomal rearrangements at 3q26: whereas EVI1 is inappropriately overexpressed in frequent rearrangements simultaneously involving bands 3q21 and 3q26, MDS1/EVI1 is activated by gene fusion, leading to protein fusion. In both cases, the inappropriate activation of the two genes has been associated with the development or the progression of acute myeloid leukemia or myelodysplastic syndrome and chronic myelogenous leukemia. In this chapter, the current information on the two genes and their involvement in hematopoiesis and myeloid leukemia is reviewed.

IDENTIFICATION OF EVI1 Several proteins that are active in normal hematopoiesis or that are inappropriately activated in leukemia by chromosomal rearrangements are transcription activators or repressors that belong to the group of the Kru¨ppel family of proteins, characterized by one or more regions of tandemly repeated DNA-binding zinc-finger domains of the Cys -His type. One Kru¨ppel like gene associated with genesis or progression of human myeloid leukemia is Evi1, originally identified during studies of mouse insertional activation by replication-competent, nonacute transforming retroviruses leading to murine myeloid transformation. Molecular analysis of retroviral integration identified in myeloid tumors of AKXD mice (Mucenski et al., 1988) showed that the retroviral insertion sites were clustered in two well-defined regions upstream of a novel gene, and that they activated the expression of this gene, which was called Evi1, by the retroviral long-terminal repeat (LTR) (Morishita et al., 1988). The preferential integration site closest to the coding region of the gene was either within a 2 kb stretch of DNA, which encompasses exon 1 of Evi1, or between the noncoding exons 1 and 2 of the gene, and it was suggested that Evi1 transcription could be initiated from the retroviral promoter inserted into this region (Morishita et al., 1988). Less

frequently, Evi1 expression was also activated from a second insertion site in the Cb-1/Fim3 locus, located 90 kb upstream of exon 1. This integration site was independently identified by two separate groups (Bartholomew et al., 1989; Bordereaux et al., 1987). In the mouse, this locus is genetically linked to Evi1, as no recombination was observed in interspecific backcrossed mice. Because retroviral insertions at this site act from such a distance to activate Evi1 expression, the activation probably occurs through the viral enhancer.

EVI1 STRUCTURE EVI1 is a relatively large protein consisting of 1003 amino acids. The human and murine proteins are remarkably conserved, with 91% and 94% overall identity in the nucleotide and amino acid sequence respectively (Morishita et al., 1990a). Although the predicted translation start site of the human and murine cDNAs is in the third exon, which contains the first in-frame methionine codon, the DNA sequence of the second exon is in frame and highly conserved between the two species (90%). EVI1 is evolutionarily conserved, and is about 60 to 80% identical to limited regions of the Caenorhabditis elegans differentiation protein Egl-43 involved in migration of hermaphrodite sensory neurons during embryonic development (Garriga et al., 1993). This surprisingly high conservation between the two proteins suggested that they could be involved in similar functions maintained through evolution (Garriga et al., 1993). EVI1 is a DNA-binding protein (Matsugi et al., 1990; Perkins et al., 1991a) with two domains containing sets of seven and three repeats of the zinc-finger motif (Morishita et al., 1988). The domain consisting of seven zinc-finger repeats is located in the amino terminus, whereas the second domain of three zinc-finger motifs is found toward the carboxyl terminus (Fig. 22.1). Most of the zinc fingers are of the Cys His Kru¨ppel type (Table 22.1); however, the first and the seventh motifs of the proximal domain contain the unusual motif Cys HisCys. It has been hypothesized that in some cases this motif mediates protein-protein interaction rather than DNA binding, such as with the enhancer-binding protein PRDII (Fan and

EVI1 STRUCTURE

395

Figure 22.1. Diagram of the predicted structure of the EVI1 and MDS1/EVI1 genes. The thick line indicates the coding region of the two cDNA. EVI1 is shown at the top. The thin line represents the first two exons (1 and 2) and part of the third exon (3) that are not translated in the EVI1 protein. The short vertical lines show the boundaries of exon 2. The two vertically striped boxes indicate the zinc-finger domains (Zn). The black segment (RD) shows the approximate position of the proline-rich repression domain. The light gray box encodes the acidic domain (AD) at the 3 end of the cDNA. MDS1/EVI1 is shown at the bottom: it contains the same domains described for EVI1, including the two zinc-finger domains, the proline-rich repression domain, and the acidic domain. In addition, MDS1/EVI1 contains a 5 extension (diagonal stripes). The approximate position of the PR domain (that includes exon 2 and part of exon 3) is shown as a line below the 5 end of MDS1/EVI1.

Maniatis, 1990), the histone acetyltransferase— related factor MOZ (Borrow et al., 1996), and the GATA1-binding factor FOG (Tsang et al., 1997). A comparison between the Cys HisCis type of zinc fingers for EVI1 and other proteins is shown in Table 22.2. The Kru¨ppel type of zinc finger is frequently seen in transcription factors involved in other leukemia-associated chromosomal translocations, such as BCL6, identified in several rearrangements at 3q27 (Baron et al., 1993; Chang et al., 1996; Ye et al., 1993), and

PLZF, fused to RAR in the t(11;17) (Chen et al., 1993). The biological DNA target sequence of EVI1 is not known. However, several investigators have reported precise DNA consensus sequence to which the protein efficiently binds in vitro either through the proximal or the distal domains of zinc fingers. The first zinc-finger domain of EVI1 recognizes a consensus of 15 nucleotides consisting of GA(C/T)AAGA(T/C)AAGATAA (Delwel et al., 1993). The first three

TABLE 22.1. Alignment of the Amino Acid Sequence of EVI1 Zinc Fingers? Zinc-Finger Number Proximal domain 1 2 3 4 5 6 7 Distal domain 8 9 10

Sequence

YRCEDCDQLFESKAELADHQKFPCSTP QECKECDQVFPDLQSLEKHMLSHTEER YKCDQCPKAFNWKSNLIRHQMSHDSGK YECENCAKVFTDPSNLQRHIRSQHVGA ACPECGKTFATSSGLKQQHKHIHSSV FICEVCHKSYTQFSNLCRHKRMHADCR IKCKDCGQMFSTTSSLNKHRRFCEGKN YTCRYCGKIFPRSANLTRHLRTHTGEQ YRCKYCDRSFSISSNLQRHVRNIHNKE FKCHLCYRCFGQQTNLDRHLKKHENG

?A normal alternatively spliced form of EVI1 (and presumably of MDS1/EVI1) lacks the zinc fingers 6 and 7.

396


TABLE 22.2. Alignment of the First and Seventh Zinc Fingers of EVI1 with Predicted Protein-Binding Zinc Fingers of Other Nuclear Factors? Protein EVI1 F1 EVI1 F7 PRDII MOZ FOG

Zinc Fingers YRCEDCDQLFESKAELADHQKFPCSTPHSAFS IKCKDCGQMFSTTSSLNKHRRFCEGKNHFAA FECETCRNRYRKLENFENHKKFYCSELHGPK YLCEFCLKYMKSRTILQQHMKKCGWFHPP TLCEACNIRFSRHETYTVHKRYYCASRH

?F1: First zinc finger of EVI1. F7: seventh zinc finger of EVI1. Conserved Cys and His residues that form the zinc-finger domain are shown in bold and are underlined. Other His residues that are conserved in other proteins are shown in bold but are not underlined.

fingers of the proximal domain do not bind the above sequence, but contribute to binding by conferring a relative specificity for GACAA versus GATAA in the first position (Delwel et al., 1993). The second set of three zinc fingers binds to the consensus sequence GAAGATGAG (Funabiki et al., 1994). EVI1 migrates with an apparent mass of 145 kDa, but it is also expressed as an alternatively spliced 88 kDa protein missing zinc fingers 6 and 7 and a large part of the central region. This shorter form of EVI1 is consistently detected by Western and Northern blot analyses of human (Morishita et al., 1990a) and murine (Bordereaux et al., 1990) tissues. The physiological role of this shorter EVI1 is not clear, but its expression is induced approximately 16-fold by elevated levels of intracellular cAMP and is downregulated by TPA in the human renal cell carcinoma cell line A704 (Bartholomew and Clark, 1993). EVI1 also contains a highly acidic domain at the carboxyl terminus (Fig. 22.1). Although acidic domains are often identified in transcription factors and usually are part of transcription activation domains, there are no data that indicate conclusively that the acidic domain of EVI1 is necessary for transcription regulation.

EVI1 EXPRESSION AND FUNCTION Several investigators have analyzed the pattern of expression of Evi1 during murine embryonic development and in adult human and murine

tissues. Perkins and co-workers (1991b) first showed that, in the mouse embryo, Evi1 is detected at high levels in the urinary system, in the Mullerian ducts, in the lung, and in the heart. In the mouse, the expression of Evi1 was also detected in the developing limbs, primary fetal cells, differentiating red blood cells, and developing oocytes in the ovary (Morishita et al., 1990a, b; Perkins et al., 1991b). Because of the spatial and temporal restricted pattern of expression of Evi1, it was suggested that this gene plays an important role in mouse development (Perkins et al., 1991b) and could be involved in organogenesis, cell migration, cell growth, response to physiological state, and differentiation. Northern blot analysis of adult human tissues has shown that EVI1 is abundantly expressed in a large number of tissues and organs, including kidney, lung, pancreas, and ovaries, and to a lesser extent in several other tissues, including skeletal muscle, heart, brain, and placenta (Fears et al., 1996). As would be expected, given the pleiotropic pattern of expression of this gene during embryonic development, the distruption of the fulllength Evi1 by targeted mutagenesis of embryonic stem cells results in a wide range of defects and in the death in utero of mutant embryos at approximately E10.5 (Hoyt et al., 1997). Homozygous murine embryos with a disrupted Evi1 gene were readily identified by hemorrhaging and malformation of the paraxial mesoderm. Histological analysis revealed widespread hypocellularity, defective myotome formation, defects in the neural ectoderm, and failure of the

INVOLVEMENT OF EVI1 IN MYELOID LEUKEMIAS

397

Figure 22.2. Map of the locus at 3q26 targeted by rearrangements leading to myeloid leukemia. The restriction enzymes used for the mapping are BssHII (B) and Sf I (S). The numbers between the restriction sites show the approximate size of the fragments in kb. The horizontal line indicates the genomic region at 3q26 containing several genes involved in chromosomal rearrangements. The boxes represent the relevant exons of the genes. Predicted initiation of translation (ATG) and stop codons (TAA) are indicated at the top of the boxes. Gray boxes represent the first (ATG), the central, and the two alternative last (TAA) coding exons of MDS1. Empty boxes represent the first three exons of EVI1 and the last translated exon of EVI1. Several intervening exons of EVI1 are not shown. Additional coding or noncoding exons of the two genes are not indicated. The arrows show the direction of the genes’ transcription (two horizontal arrows represent the two alternative splice variants of MDS1). The arrow below the diagram indicates the splicing (dotted line) necessary to express the MDS1/EVI1 transcript. The breakpoint (bp) regions in myeloid leukemia are indicated below the diagram by large arrows.

peripheral nervous system to develop. The cellular proliferation defects noted in mutant embryos would support the involvement of Evi1 in dysregulated cell growth observed in retrovirusinduced leukemias.

INVOLVEMENT OF EVI1 IN MYELOID LEUKEMIAS In the mouse, the activation of Evi1 occurs by retroviral insertion in the Evi1 locus. In humans there are no confirmed reports of retrovirus integration near this gene, and the inappropriate activation of EVI1 appears to occur mostly by chromosomal rearrangements involving band 3q26, where the gene is located, leading to development or progress of myeloid leukemias (Fichelson et al., 1992; Jenkins et al., 1989). The most frequent rearrangements of the EVI1 locus are those that involve simultaneously two bands of chromosome 3, bands 3q21 and 3q26, and result in the t(3;3)(q21q26) and in the

inv(3)(q21q26). By using pulsed field gel electrophoresis (PFGE) and fluorescence in situ hybridization (FISH) analyses, the breakpoint of the 3;3 translocations have been mapped to a very large genomic region 13—300 kb upstream of EVI1, whereas the breakpoints of the inv(3) are clustered in a smaller region at the 3 of EVI1 (Morishita et al., 1992a; Suzukawa et al., 1994) (Fig. 22.2). This defined orientation of the two breakpoints on chromosome 3 suggested that the activation could occur through inappropriate juxtaposition of EVI1 to the regulatory sequence of a gene located at 3q21. After cloning and molecular analysis of breakpoint junctions of the t(3;3) and inv(3), it was determined that both these rearrangements juxtapose the strong enhancer of the Ribophorin I gene to the coding region of EVI1 (Suzukawa et al., 1994). In general, defects of 3q in bands q21 and q26 have been reported in patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Patients who inappropriately

398


Figure 22.3. Diagram of normal and fusion cDNA. The two arrows indicate the region of chromosomal breakpoints involving EVI1 (see also Fig. 22.1). The vertical dotted line shows the cDNA breaks and the fusion junction. BD and TD: DNA-binding domain and transactivation domain of AML1. HLH and ETS: oligomerization domain (HLH) and DNA-binding domain (ETS) of TEL. The chromosomal location of the normal and fusion genes and the chromosomal rearrangements involved are also indicated.

express EVI1 often have monosomy of chromosome 7 or deletions of the long arm of chromosome 7, and, less frequently, deletions in chromosome 5. The most characteristic clinical features of these patients are elevated platelet counts, marked hyperplasia with dysplasia of megakaryocytes, and poor prognosis. Although disturbance of thrombopoiesis is not systematically observed in all patients with 3q21q26 rearrangements, the frequency of patients with these characteristics brings further evidence to the existence of a cytogenetic syndrome involving bands q21 and q26 simultaneously and referred to as q21q26 syndrome (Bouscary et al., 1995; Dreyfus et al., 1995; Jenkins et al., 1989; Jotterand et al., 1992; Pintado et al., 1985). Because monosomy 7 has been detected in AML patients who do not express EVI1, alteration in chromosome 7 may contribute to the evolution of AMLs that already involve EVI1 (Kreider et al., et al., 1993). There are no unique morphologic characteristics in cells that express EVI1, and the leukemic cells have been described as FAB M0, M1, or M2 (Kreider et al., 1993). Some of the cells examined were CD34>, suggesting that they were immature myeloid cells. This agrees with the immature phenotype characteristic of murine myeloid cells expressing Evi1.

Activation of EVI1 also occurs as a result of translocations involving a chromosome other than 3. The chromosomal breakpoint of two such cases have been cloned, that of the t(3;21)(q26;q22) and that of the t(3;12)(q26;p13) (Nucifora et al., 1993a,b; Peters et al., 1997) (Figs. 22.2 and 22.3). These translocations have been reported in therapy-related MDS and AML, or in chronic myelogenous leukemia (CML) during the blast crisis (BC) (Bitter et al., 1985; Coyle et al., 1988; Raynaud et al., 1996; Rubin et al., 1987; Rubin et al., 1990; Schneider et al., 1991); however, a t(3;21) was detected in a patient with de novo childhood AML (Johansson et al., 1996). The 3;21 translocation is unique in that it results consistently in the simultaneous expression of multiple fusion genes between the DNA-binding domain of the transcription factor AML1, also known as CBFA2 (Miyoshi et al., 1991; Ogawa et al., 1993; Wang and Speck, 1992), and three genes located at 3q26. The three genes are EVI1, EAP (EBV-associated protein) encoding the ribosomal protein L22, and MDS1 (myelodysplastic syndrome 1), which spans several hundred kb but encodes a small protein of about 14 kDa (Nucifora et al., 1993b, 1994; Sacchi et al., 1994). The fusion transcripts that result from this translocation

EVI1 AND MDS1/EVI1

are AML1/EAP, AML1/MDS1, and AML1/ MDS1/EVI1 containing MDS1 and EVI1 spliced together downstream of AML1 (Fig. 22.3) (Nucifora et al., 1994). After molecular cloning and sequence analysis, it was determined that, in AML1/MDS1/EVI1, AML1 was fused in frame to almost the entire MDS1, which in turn was spliced in frame to the second, conserved exon of EVI1, as shown in Fig. 22.3 (Mitani et al., 1994; Nucifora et al., 1994). AML1 is a transcription activator necessary to fetal liver hematopoiesis that also regulates several genes activated during adult hematopoietic differentiation. The strong, positive activity of AML1 in transcription requires the interaction between the coactivator p300 and the C-terminus of AML1 (Kitabayashi et al., 1998), which is lost following the t(3;21) and gene fusion (Nucifora et al., 1994). In the fushion protein, the DNA-binding domain of AML1 is maintained (Fig. 22.3), and therefore AML1/MDS1/ EVI1 is a bifunctional transcription factor that can recognize promoters containing binding sites for AML1 and EVI1 (Sood et al., 1999; Zent et al., 1996). The second chromosomal translocation that has been cloned is the t(3;12), which results in gene fusion between TEL and EVI1 (Peters et al., 1997). TEL, located on chromosome band 12p13, encodes a strong transcription repressor (Chakrabarti et al., 1999) that interacts with the corepressors mSin3 and SMRT (Chakrabarti and Nucifora, 1999). TEL is involved in many recurring translocations observed in leukemia and in some sarcomas, and is often deleted in leukemia patients (Buijis et al., 1995; Golub et al., 1994; Romana et al., 1995). In contrast to the majority of other translocations involving TEL, in the t(3;12) the contribution of TEL to the fusion gene is limited to exons encoding the first 50 amino acids. This region has no clear function, and it was suggested that TEL could provide just the promoter activity (Peters et al., 1997). As in the t(3;21), cells with a t(3;12) chromosomal fusion express transcripts that join TEL and EVI1 either directly or through MDS1. However, DNA-sequence analysis of the fusion transcripts junction indicates that the reading frame is maintained only in the transcripts that also include MDS1 (Peters et al., 1997). Finally, it was reported that in cells with a t(3;12) or t(3;21) in addition to the fusion

399

genes, EVI1 can also be activated from an unidentified promoter, resulting in the inappropriate expression of the protein (Nucifora et al., 1994; Peters et al., 1997). Activation of EVI1 was also reported in CML-BC patients with apparently normal chromosome 3 (Carapeti et al., 1996; Ogawa et al., 1996; Russell et al., 1993; Russel et al., 1994), and results from several groups would indicate that a correlation exists between the activation of EVI1 (either as a fusion protein or by inappropriate expression) and the progression of CML from the chronic to the acute phase. In addition, expression of EVI1 has been reported in ovarian and other cancers and in promyelocytic leukemia (Brooks et al., 1996; Xi et al., 1997). In addition to the t(3;12) and t(3;21), EVI1 is involved at the breakpoint of several other chromosomal translocations that have not been cloned yet, such as the t(2;3)(p13;q26), t(2;3)(q23;26), t(3;7)(q26;q22), t(3;13)(q27;q13—14), and t(3;17)(q26;q22) (Asou et al., 1996; Desangles et al., 1995; Fleishman et al., 1996; Kwong et al., 1992; Levaltier et al., 1996; Mugneret et al., 1992; Yu et al., 1996). Although these rearrangements result in the inappropriate expression of EVI1, as determined by RT-PCR of the 3 region of the gene, it is not yet known whether EVI1 or a fusion gene that includes EVI1 is inappropriately expressed. Table 22.3 summarizes the location of breakpoints in the rearrangements involving EVI1 that have been reported.

EVI1 AND MDS1/EVI1 By using PFGE and FISH analyses, MDS1 was mapped on chromosome band 3q26 about 300— 400 kb upstream of the first exon of EVI1 (Nucifora et al., 1994) (Fig. 22.2). Analysis of multiple tissue Northern blots with an MDS1specific probe identified two large transcripts of 5.8 and 6.2 kb, identical in size to those observed with an EVI1 probe, and, in addition, three small transcripts of 1, 1.5, and 2.0 kb not detected with an EVI1 probe (Fears et al., 1996). These results suggested that MDS1/EVI1 presumably resulted from the splicing of a small new gene, MDS1, to the second exon of EVI1 (Fears et al., 1996). The pattern of expression of

400


TABLE 22.3. Chromosomal Rearrangements That Result in the Inappropriate Expression of EVI1 Chromosomal Rearrangement t(2;3)(p15;q26) inv(3)(q21q26) t(3;3)(q21;q26) t(3;7)(q27;q22) t(3;12)(q26;p12) t(3;13)(q26;q13-14) t(3;17)(q26;q22) t(3;21)(q26;q22)

Disease

EVI1 Expression?

EVI1 Fusion

Reference

; ; ; ; ; ; ; ;

n.d. Ribophorin/EVI1 Ribophorin/EVI1 n.d. TEL/MDS1/EVI1 n.d. n.d. AMLI/MDS1/EVI1

Kwong et al., 1992 Susukawa et al., 1994 Suzukawa et al., 1994 Asou et al., 1996 Peters et al., 1997 Yu et al., 1996 Mugneret et al., 1992 Nucifora et al., 1994

CML-BC, t-MDS MDS/AML, CML-BC MDS/AML, CML-BC AML MDS/AML AML MDS CML-BC, AML, t-MDS/AML

?EVI1 expression measured by Northern blot hybridization or by RT-PCR analysis. Because of the probes or primers used, these analyses cannot distinguish between EVI1 and MDS1/EVI1. n.d., not determined.

MDS1/EVI1 in adult tissues is identical to that of EVI1, and several MDS1/EVI1 cDNA clones were easily isolated and identified from normal human pancreas and kidney libraries, confirming the existence of this gene in normal tissue. Analysis of the sequence showed that the predicted translation of MDS1/EVI1 added 188 amino acids upstream of the start site of EVI1 in the third exon (Fears et al., 1996), of which 63 codons were derived from the second exon and from the nontranslated part of the third exon of EVI1, and the remaining 125 codons were from the MDS1 gene. This new amino acid extension of EVI1 has about 40% homology (Fears et al., 1996) to the N-terminus of seemingly unrelated proteins such as the retinoblastoma-binding protein RIZ (Buyse et al., 1995), the B-cell factor B-LIMP-1 necessary for plasma cell differentiation (Turner et al., 1994), and the C. elegans differentiation factor Egl-43 (Garriga et al., 1993). Recently, Huang et al. (1998) determined that the homology extends to include the SET domain functioning in chromatin-mediated gene expression (Yu et al., 1995), the ALL/HRX/ MLL protein (Gu et al., 1992), and the Drosophila proteins ASH1 (Nislow et al., 1997) and Su(Var)3—9 (Tschiersch et al., 1994) (Fig. 22.4). The homology is concentrated mainly within three blocks of about 15 amino acids each, which are evenly spaced (Fig. 22.4). The domain was originally named the PR domain (Fears et al., 1996), and its conservation among the various proteins would suggest it has a common

function in different types of cells. The identification of the cDNAs of MDS1, EVI1, and MDS1/ EVI1 and the overall homology of MDS1/EVI1 to Egl-43 raise the question of the nature and relationship of these genes. Recent RT-PCR analysis of murine embryonic stem (ES) cells suggests that the two genes are differentially regulated and transcribed during in vitro development of embryoid bodies (EBs) (Sitailo et al., 1999; Wimmer et al., 1998), suggesting that transcription of Evi1 could start from an internal promoter. Results from our group indicate that whereas the expression of Mds1/Evi1 is low in undifferentiated ES cells and reaches a maximum after 10—11 days of differentiation, Evi1 is not detected in undifferentiated ES cells, but appears 4—5 days later and reaches the highest level of expression at days 10—12 of differentiation (Sitailo et al., 1999). Although EVI1 or MDS1/EVI1 have not been detected in human peripheral blood leukocytes, the analysis of single, differentiated, hematopoietic ES colonies confirmed that both genes are transiently expressed during in vitro murine hematopoietic differentiation (Sitailo et al., 1999).

TRANSCRIPTION ACTIVITY OF EVI1 AND MDS1/EVI1 Several groups have reported on the function of EVI1 as a transcription regulator. The assays were performed in vitro, and although some-

EVI1 AND MDS1/EVI1

401

Figure 22.4. The PR domain has partial homology to the SET domain of other proteins. Only identical residues (gray background) are shaded. Dashes indicate sequence gaps. Conserved blocks A, B, and C are underlined. The boxed Met residue is the predicted translation start site of EVI1. All the region upstream of this residue is lost following rearrangements that lead to overexpression of EVI1.

times results from several groups seem to be in contradiction, they clearly indicate that gene regulation by EVI1 is quite complex. It was reported that EVI1 can activate by 3-fold a promoter containing the target sites of both groups of zinc fingers; however, the transcrip-

tion activation depends on the binding of the carboxyl-terminal domain of zinc fingers to the second DNA consensus site, and not on binding of the amino domain of zinc fingers to the first DNA consensus site (Morishita et al., 1995). In contrast, studies using the CAT reporter gene,

402


under the control of either the first or the second EVI1 target site inserted upstream of the herpes simplex virus (HSV) thymidine kinase (tk) promoter, indicated that EVI1 dramatically represents the activity of the reporter gene (Kreider et al., 1993; Lopingco and Perkins, 1992). The studies showing that EVI1 is a transcription repressor were confirmed by Bartholomew and co-workers (1997) using a similar artificial promoter in which the zinc-finger consensus site was cloned either upstream of the HSV tk promoter or the adenovirus minimal promoter. These investigators also reported on an extensive analysis of N-terminal and C-terminal deletion mutants that resulted in the identification of a specific region between the two zinc-finger domains (Fig. 22.1) that is associated with the repression activity (Bartholomew et al., 1997). Results from our group indicate that this region associates with the corepressors mSin3 and SMRT (Sood and Nucifora, unpublished results); others found that this region associates with the corepressor CtBP (Turner and Crossley, 1998). In separate studies, the transcription activity of EVI1 was compared to that of MDS1/EVI1 using a genomic promoter containing several repeats of the AGATA sequence (Soderholm et al., 1997). These studies confirmed that EVI1 is a strong repressor, as previously shown by Kreider and colleagues (1993), but in addition they showed that MDS1/EVI1 is a very strong activator of that promoter. It is of interest to note that the EVI1 repression domain identified by Bartholomew and co-workers (1997), and that associates with CtBP (Turner and Crossley, 1998) or mSin3 and SMRT (Sood and Nucifora, unpublished results), is present also in MDSI/ EVI1, but apparently is not dominant. The extent of promoter activation by MDS1/EVI1 is comparable to that observed with the erythroid factor GATA-1 (Soderholm et al., 1997), and, when expressed together, EVI1 can inhibit promoter activation by MDS1/EVI1 and GATA-1 (Soderholm et al., 1997). The transcription activity of MDS1/EVI1 was localized to the PR domain, which can also function independently as a transcription activation domain when fused to a heterologous DNA-binding domain such as that of Gal4. Analysis of deletion mutants showed that in order to maintain the transcription activity of the promoter, the PR domain

must be intact and must include the region indicated as block C (Fig. 22.4), containing the second and part of the third exon of EVI1, not translated in the E4I1 protein (Soderholm et al., 1997). Although MDS1/EVI1 is a strong activator, the fusion protein AML1/MDS1/EVI1 is strong repressor that can bind and repress promoters depending on either AML1 or MDS1/EVI1 for activation (Sood et al., 1999; Zent et al., 1996). Although the available evidence strongly indicates that EVI1 and MDS1/ EVI1 are transcription factors, all the data presented have been obtained with artificial promoters, and the nature of the biological genes regulated by the two proteins is not known. Recently, Kim and co-workers used a two-step selection strategy to construct a sublibrary of genomic fragments containing a significant fraction of the Evi1 binding sites present in the murine genome, and they also isolated several cDNA clones that are predicted to be regulated by Evi1 (Kim et al., 1998).

ROLE OF EVI1 AND MDS1/EVI1 IN IN VITRO HEMATOPOIETIC DIFFERENTIATION AND IN CONTROL OF CELL GROWTH The forced expression of EVI1 in murine bone marrow cells inhibits terminal differentiation in granulocytes, erythroid cells, and bone marrow progenitor cells (Kreider et al., 1993; Morishita et al., 1992b). Because EVI1 can repress GATA-1—dependent transactivation in transient transfection CAT assays (Kreider et al., 1993), it was suggested that the block in erythroid differentiation may be caused by transcription repression of a subset of GATA-1 target genes (Kreider et al., 1993). Additional studies were performed with the murine hematopoietic cells 32Dcl3, which are interleukin (IL-3) dependent for growth but differentiate into granulocytes in response to granulocytic colony-stimulating factor (G-CSF) (Greenberger et al., 1983), and it was shown that the forced expression of EVI1 in these cells leads to a block of G-CSF—dependent differentiation, and to the inability of the cells to express myeloperoxidase and to differentiate to granulocytes, resulting in cell death (Morishita et al., , 1992b; Kreider et al., 1993; Sood et al., 1999). In contrast to EVI1, the forced expression

CONCLUSION

403

TABLE 22.4. Effects of MDS1/EVI1, and AML1/MDS1/EVI1 on the Response of 32Dcl3 Cells to TGF-1 Gene

TGF-1

G-CSF

Reference

None

Growth suppression

Granulocytic differentiation

MDS1/EVI1

Enhanced growth suppression Inhibition of growth suppression Inhibition of growth suppression

Granulocytic differentiation

Morishita et al., 1992b, Sood et al., 1999 Sood et al., 1999

No response. Cell death

Sood et al., 1999

No response. Cell death

Sood et al., 1999

EVI1 AML1/MDS1/EVI1

of MDS1/EVI1 has no effect on the granulocytic differentiation of the 32Dc13 cells, which, in the presence of G-CSF, develop myeloperoxidaserich granules and complete their differentiation as unaltered control cells (Sood et al., 1999). Furthermore, expression of MDS1/EVI1 in EVI1-32Dc13 cells can abrogate the differentiation block in response to G-CSF (Sitailo, unpublished data). More recently, the forced expression of EVI1 has also been studied in undifferentiated and differentiated murine ES cells. The results indicated that EVI1, but not MDS1/EVI1, accelerates the rate of growth of the ES cells and strongly favors hematopoietic differentiation along the megakaryocytic lineage (Sitailo et al., 1999). EVI1 and MDS1/EVI1 also have opposite effects in the response of the 32Dc13 cells to factors that control cell replication such as TGF-1, which normally reduces the growth of the 32Dc13 cells by about 50% (Ohta et al., 1987). As it was shown independently by two separate groups, the forced expression of EVI1 abrogates the growth inhibition that is induced by TGF-1 (Kurokawa et al., 1998; Sood et al., 1999); however, in contrast to EVI1, MDS1/ EVI1 seems to increase the sensitivity of the 32Dcl3 cells to TGF-1—induced growth inhibition in liquid culture and in semisolid methylcellulose-based medium (Sood et al., 1999). The mechanisms by which EVI1 blocks the inhibitory signaling of TGF-1 are not clear. It was proposed that this effect could result from the recruitment of SMAD3 by EVI1 through direct interaction, thus limiting the ability of small protein to regulate promoters of genes necessary to complete the TGF-1 signaling (Kurokawa

et al., 1998). However, it was also shown that MDS1/EVI1 interacts with SMAD3 as efficiently as EVI1 (Sood et al., 1999); therefore, it is likely that other mechanisms are also involved in the altered response induced by the two proteins to TGF-1 signaling. Because the two proteins have opposite transactivation properties, it is possible that they regulate divergentspecific downstream target genes involved in replication and/or differentiation, resulting in the effects just described for the ES cells and the 32Dcl3 cells (summarized in Table 22.4). This possibility is supported by the results obtained with the repressor AML1/MDS1/EVI1, which, when expressed in 32Dcl3 cells, has the same effect as EVI1 in interfering with the response of the cells to G-CSF and TGF-1 signaling (Sood et al., 1999).

CONCLUSION Since the identification of EVI1 as a gene associated with myeloid leukemia when inappropriately expressed, its involvement in myeloid disorders in humans and in mice has been well documented. However, despite the effort of several investigators, not much progress has been made in the understanding of the role of this gene in normal or malignant hematopoiesis. In addition, the work by Hoyt and co-workers (1997) has clearly indicated that this gene has a pleiotropic role during development, being involved in the development of several organs, and not exclusively in hematopoiesis. The identification of MDS1/EVI1 has considerably complicated the study of this gene, in that it now

404


appears that the locus can express two proteins with opposite properties, one of which is overexpressed in myeloid leukemia. Results from our group suggest that EVI1 and MDS1/EVI1 are expressed in early CD34> human cells. Given the opposite properties of the two proteins, it would be tempting to speculate that MDS1/ EVI1 and EVI1 are involved in the control of cell replication and differentiation in opposite ways, and that perhaps the expression of both proteins is needed for the fine-tuning of these two processes. The homology between the PR domain of MDS1/EVI1 and the SET domain increases the complexity of the two proteins and the understanding of their individual role. SET domain proteins have chromatin structure—related functions, and the presence of this domain would indicate another possible role for MDS1/ EVI1, clearly not shared by EVI1.

ACKNOWLEDGMENTS I wish to apologize to those investigators I failed to cite because of space limitations. I thank all the enthusiastic students and postdoctoral fellows who worked and still work in my laboratory on the EVI1 gene. Supported by National Institutes of Health grants CA 67189 and CA 72675 (G.N.). G.N. is a scholar of the Leukemia and Lymphoma Society.

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Perkins, A. S., Fishel, R., Jenkins, N. A., and Copeland, N. G. (1991a). Evi1, a murine zinc finger proto-oncogene, encodes a sequence specific DNAbinding protein. Mol Cell. Biol 11, 2665—2674.

Sacchi, N., Nisson, P. E., Watkins, P. C., Faustinella, F., Wijsman, J., and Hagemeijer, A. (1994). AML1 fusion transcripts in t(3;21) positive leukemia: evidence of molecular heterogeneity and usage of splicing sites frequently involved in the generation of normal AML1 transcripts. Genes Chrom. Cancer 11, 226—236.

Perkins, A. S., Mercer, J. A., Jenkins, N. A., and Copeland, N. G. (1991b). Patterns of EVI1 expression in embryonic and adult tissues suggest

Schneider, N., Bowman, W., and Frenkel, E. (1991). Translocation (3;21)(q26;q22) in secondary leukemia. Ann. Genet. 34, 256—263.

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Sitailo, S., Sood, R., Barton, K., and Nucifora, G. (1999). Forced expression of the leukemia-associated gene EVI in ES cells: a model for myeloid leukemia with 3q26 rearrangements. Leukemia 13, 1639—1645. Soderholm, J., Kobayashi, H., Mathieu, C., Rowley, J. D., and Nucifora, G. (1997). The leukemia-associated gene MDSI/EVI1 is a new type of GATAbinding transactivator. Leukemia 11, 352—358. Sood, R., TalwarTrikha, A., Chakrabarti, S. R., and Nucifora, G. (1999). MDS1/EVI1 enhances TGF1 signaling and strengthens its growth-inhibitory effect, but the leukemia-associated fusion protein AML1/MD21/EVI1, product of the t(3;21), abrogates growth-inhibition in response to TGF-1. Leukemia 13, 348—357. Suzukawa, K., Parganas, E., Gajjar, A., Abe, T., Takahashi, S., Tani, K., Asano, S., Asou, H., Kamada, N., Yokota, J., Morishita, K., and Ihle, J. N. (1994). Identification of a breakpoint cluster region 3 of the Ribophorin I gene at 3q21 associated with the transcriptional activation of the EVI1 gene in acute myelogenous leukemias with inv(3)(q21q26). Blood 84, 2681—2688. Tsang, A. P., Visvader, J. E., Turner, C. A., Fujiwara, Y., Yu, C., Weiss, M. J., Crossley, M., and Orkin, S. H. (1997). FOG, a multitype zinc finger protein, acts as a cofactor for transcription factor GATA-1 in erythroid and megakaryocytic differentiation. Cell 90, 109—119. Tschiersch, B., Hoffmann, A., Krauss, V., Dorn, R., Korge, G., and Reuter, G. (1994). The protein encoded by the Drosophila position-effect variegation suppressor gene Su(var)3-9 combines domains of antagonistic regulators of homeotic gene complexes. EMBO J. 13, 3822—3831. Turner, C. A., Mack, D. H., and Davis, M. M. (1994). Blimp 1, a novel zinc finger containing protein that can drive the maturation of the B lymphocytes into immunoglobulin secreting cells. Cell 77, 297—306.

Turner, J., and Crossley, M. (1998). Cloning and characterization of mCtBP2, a co-repressor that associates with basic Kru¨ppel-like factor and other mammalian transcriptional regulators. EMBO J. 17, 5129—5140. Wang, S., and Speck, N. A. (1992). Purification of core-binding factor, a protein that binds the conserved core site in murine leukemia virus enhancers. Mol. Cell. Biol. 12, 89—102. Wimmer, K., Vinatzer, U., Zwirn, P., Fonatsch, C., and Wieser, R. (1998). Comparative expression analysis of the antagonistic transcription factors EVI1 and MDS1-EVI1 in murine tissues and during in vitro hematopoietic differentiation. Biochem. Biophys. Res. Commun. 252, 691—696. Ye, B. H., Rao, P. H., LoCoco, F., and Dalla Favera, R. (1993). Cloning of BCL6, the locus involved in chromosomal translocations affecting band 3q27 in B-cell lymphoma. Cancer Res. 53, 2732—2735. Yu, B. D., Hess, J. L., Horning, S. E., Brown, G. A., Korsmeyer, S. J. (1995). Altered Hox expression and segmental identity in MLL-mutant mice. Nature 378, 505—508. Yu, Fu, Y., Sadamura, S., Ishikura, H., Abe, Y., Katsumo, M., Nishimura, J., and Nawata, H. (1996). Expression of EVI1 and the retinoblastoma genes in acute myelogenous leukemia with t(3;13)(q26;q13—14). Am. J. Hematol. 53, 30—34. Xi, Z. F., Russell, M., Woodward, S., Thompson, F., Wagner, L., and Taetle, R. (1997). Expression of the Zn finger gene EVI1 in acute promyelocytic leukemia. Leukemia 11, 212—220. Zent, C., Kim, N., Hiebert, S., Zhang, D.-E., Tenen, D., Rowley, J. D., and Nucifora, G. (1996). Rearrangement of the AML1/CBFA2 gene in myeloid leukemia with the 3;21 translocation: expression of co-existing multiple chimeric genes with similar functions as transcriptional repressors, but with opposite tumorigenic properties. Curr. Topics Microbiol. Immunol. 211, 243—252.

CHAPTER 23


t(8;21) AML AND THE AML1/ETO FUSION GENE: FROM CLINICAL SYNDROME TO PARADIGM FOR THE MOLECULAR BASIS OF ACUTE LEUKEMIA RICHARD C. FRANK AND STEPHEN D. NIMER Laboratory of Molecular Aspects of Hematopoiesis, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA

INTRODUCTION The generation of fusion transcription factors by nonrandom chromosomal abnormalities is the most prevalent molecular abnormality in human acute leukemia (Look, 1997). These chimeric proteins appear to act by interfering with the function of their wild-type constituent proteins and through the acquisition of novel gain-offunctions. The heterodimeric transcription factor AML1:CBF is targeted with a highfrequency by cytogenetic abnormalities in acute myeloid and lymphoid leukemias, indicating that dysregulated AML1 function may underlie the pathogenesis of a variety of acute leukemias. This review details our current understanding of the function of the 8;21 translocation protein, AML1/ETO, the first characterized and most frequently encountered AML1fusion protein.

BIOLOGIC AND GENETIC FEATURES OF t(8;21) AML The application of Giemsa-banding techniques to the analysis of chromosomal abnormalities in acute myelogenous leukemia (AML) identified a high percentage of patients with a recurring translocation involving chromosomes 8 and 21 (Rowley, 1973; Trujillo et al., 1979). It was also recognized that t(8;21) leukemias constitute a distinct subtype of AML, exhibiting the following common features: (1) a characteristic bone marrow morphology, containing myeloperoxidase-positive blasts with prominent Auer rods, and some degree of differentiation, which may be dysplastic; (2) an associated bone marrow eosinophilia; (3) a tendency to form granulocytic sarcomas; and (4) a high remission rate with chemotherapy and a better-than-average prognosis in adults but not in children (Mrozek et al., 1997; Swirsky et al., 1984). The t(8;21)(q22;q22) is the most common structural chromosomal abnormality in AML, constituting 40% of FAB-M2 cases with a


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t(8;21) AML AND THE AML1/ETO FUSION GENE

Figure 23.1. Structure-function relationships in the AML1/ETO fusion protein. The RHD mediates interaction with DNA and with the CBF protein. The EID region (ets interaction domain) is in the C-terminal part of the Runt homology domain (RHD) and mediates interactions with various ets proteins. The ETO part of AML1/ETO includes nervy homology regions (NHR) 1—4 (striped boxes). NHR1 has homology with the TAF110 protein (TAF, TATA—binding protein (TBP)—associated factor); NHR2 mediates homo- and heterodimerization among AML1/ETO and ETO proteins; NHR4 contains the zinc-finger domain that mediates specific interactions with the nuclear corepressor (N-CoR) protein.

karyotypic abnormality and 10—15% of all cases of AML (Mitelman and Heim, 1992). Rarely, an AML1/ETO fusion transcript can be detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in clinical samples that have the classical morphologic features of t(8;21) AML but lack the cytogenetic abnormality (Nucifora et al., 1994b). It is important that AML cases with the 8;21 abnormality be correctly identified because such patients have a high potential for cure when treated with high doses of cytosine arabinsoside (Ara-C) as consolidation therapy (Mrozek et al., 1997). The loss of a sex chromosome is a common additional cytogenetic abnormality in t(8;21) leukemias, but it does not clearly impact on prognosis (Schoch et al., 1996). Poor prognostic features associated with t(8;21) leukemias include CD56 expression (Baer et al., 1997), extramedullary disease (Byrd et al., 1997), and a 9q- abnormality (Schoch et al., 1996). The 8;21 translocation rearranges the AML1 gene (AKA RUNX1/CBFA2/PEBP2B) from its normal location on chromosome 21 to chromosome 8, where it is fused to nearly all of the ETO gene (eight twenty-one), also called MTG8 (myeloid translocation gene on chromosome 8) (Miyoshi et al., 1993). The AML1 breakpoints cluster within a single intron of the AML1 gene, between exons 5 and 6, and the AML1/ETO fusion gene is transcribed from the AML1 promoter in a telomere to centromere direction on the der8 chromosome (Shimizu et al., 1992). Identical fusion transcripts have been found in all patients analyzed thus far, which can be easily detected by RT-PCR (Downing et al.,

1993). As shown in Figure 23.1, the chimeric AML1/ETO protein is 752aa (:95 kD) and contains the first 177aa of AML1, including the Runt homology domain (RHD), joined to nearly all of ETO (575aa) (Miyoshi et al., 1993). Structural alterations of the AML1 gene are seen in several other chromosomal abnormalities associated with hematologic malignancies (see other chapters in this book). A second AML1/ETO-related fusion gene has been found in two cases of AML with a t(16;21), which joins AML1 to an ETO homolog, MTG16, resulting in the formation of an AML1/MTG16 fusion gene (Gamou et al., 1998). Translocations of the AML1 gene also occur in the t(3;21), associated with blastic crisis of CML or therapy-related AML and MDS, variably resulting in AML1/ EVI-1, AML1/MDS1, or AML1/EAP fusion genes (Mitani et al., 1994; Nucifora et al., 1994a). The t(12;21), which is the most frequent chromosomal abnormality in pediatric ALL and is often cytogenetically cryptic but detectable by RT-PCR, results in a TEL/AML1 fusion gene (Golub et al., 1995; Romana et al., 1995). Several balanced translocations involving chromosome 21q22 (and the AML1 gene) and chromosomes 17q11.2, 5q13, 1p36, 12q24, 15q22, and 14q22 have been found in patients with de novo and therapy-related (with topoisomerase II inhibitors) AML and MDS (Roulston et al., 1998). The AML1:CBF complex is also targeted by the commonly occurring inv(16) abnormality, associated with the M4Eo subtype of AML, which generates a CBF:MYH11 fusion gene (Liu et al., 1993). CBF:MYH11 has been

AML1 IS A MEMBER OF THE RUNT FAMILY OF TRANSCRIPTION FACTORS

shown to inhibit AML1 function, possibly by interfering with the nuclear localization of AML1 (Adya et al., 1998). Furthermore, point mutations clustered within the Runt domain of the AML1 gene, which would disrupt the function of the protein, have recently been detected in 5% of AML patient samples (although the biological significance of heterozygous loss of AML function is unclear) (Osato et al., 1999). A recent report (Song et al., 1999) describes a family with a familial predisposition to develop leukemia (FPD) in which affected but not unaffected family members have loss of one copy of the AML1 gene. Therefore, AML1 function may be impaired through multiple mechanisms, thereby contributing to the leukemic phenotype.

AML1 IS A MEMBER OF THE RUNT FAMILY OF TRANSCRIPTION FACTORS Identification of AML1 and Related Genes The AML1 gene was isolated by Miyoshi and colleagues from a human bone marrow cDNA library derived from t(8;21)-positive leukemic blasts (Miyoshi et al., 1991). The AML1 cDNA encodes a protein of 250 amino acids (referred to as AML1A) that contains an evolutionary conserved 128aa region with homology to the

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Drosophila transcription factor Runt (the Runt homology domain, RHD). Runt is involved in regulating segmentation, sex determination, and neurogenesis (Duffy and Gergen, 1994). It was soon recognized that AML1 encodes the subunit of the previously characterized murine heterodimeric transcription factors PEBP2 (polyoma enhancer-binding protein) and CBF (core-binding factor) (Speck and Stacy, 1995), which are discussed in Chapter 6. The subunit of the PEBP2 or CBF complex, called PEBP2/CBF, increases the affinity of the subunit for binding DNA (Ogawa et al., 1993; Speck and Stacy, 1995; Wang et al., 1993). Importantly, it was determined by Meyers and co-workers that AML1 proteins bind to the same core motif, TGT/cGGT, as do the PEBP2/ CBF proteins (Meyers et al., 1993); this recognition site is contained in the regulatory regions of many genes active in hematopoietic cells (Meyers and Hiebert, 1995). The AML1 gene consists of nine exons spanning 150 kb (Miyoshi et al., 1995) and encodes three major isoforms: AML1A, AML1B: AML1c (480aa), and AML1b (453aa), which arise from alternative splicing and the differential use of two promoters and three polyadenylation sites (Fig. 23.2) (Meyers et al., 1995; Miyoshi et al., 1995). Two additional AML1related genes have been cloned: AML2, which is located on chromosome 1p36, and AML3,

Figure 23.2. Schematic diagram of AML1 isoforms. AML1A, AML1B, AML1c, and AML1b represent the major isoforms resulting from alternative splicing of the AML1 gene. RHD, Runt homology domain; TAD, transactivation domain. The VWRPY motif mediates protein interactions with the groucho/TLE family of transcriptional repressors. The arrow indicates the point of fusion with ETO in the t(8;21).

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which is located on chromosome 6p21. These proteins share greater than 90% homology with AML1 within the RHD and 60% overall homology (Levanon et al., 1994). It appears that AML1 (mainly AML1B) and AML2 are the predominant AML proteins found in myeloid and lymphoid cell lines and in murine thymus and spleen (Meyers et al., 1996). Translocations involving AML2 or AML3 have not yet been reported, so their role, if any, in leukemogenesis is unclear. However, several lines of evidence have proven a critical role for AML3 (also known as CBFA1 and Osf2) in osteogenesis: AML3 is expressed exclusively in murine osteoblastic cells and adult bone, and AML3 can transcriptionally activate the osteocalcin gene promoter (Ducy et al., 1997). Furthermore, AML3 knockout mice exhibit a complete lack of bone formation (Rodan and Harada, 1997) and heterozygous mutations in the AML3 gene in humans cause cleidocranial dysplasia (CCD), an autosomal-recessive disorder associated with severe deformities in skeletal patterning and growth (Lee et al., 1997; Rodan and Harada, 1997). These knockout studies demonstrate the lack of functional overlap of AML1, AML3, and AML2. AML1 Is a Complex Transcriptional Regulator Required for Blood System Development An intact AML1:CBF complex is essential for normal hematopoietic development. Knockout mice that lack either AML1 or CBF develop yolk sac hematopoiesis but fail to generate definitive fetal liver hematopoiesis and die between days E11.5 and E13.5 with a distinct pattern of central nervous system hemorrhage (Okuda et al., 1996; Wang et al., 1996). Replacement of only the AML1B isoform may be sufficient to restore AML1 function, as demonstrated recently by Okuda and colleagues, who performed a targeted knock-in of AML1B into AML1-deficient ES cells and restored their ability to differentiate into normal hematopoietic lineages (Okuda et al., 1998b). Similar to their Drosophila Runt protein counterparts, AML1 proteins can activate or repress target genes, depending upon the promoter and cell contexts used for transcriptional studies. To some extent, the functional domains

present in the various AML1 isoforms determine their transcriptional abilities. For example, all three AML1 isoforms contain the RHD, but only AML1B and AML1b contain a C-terminal proline/serine/threonine-rich region, which has been demonstrated to function as a transcriptional activation domain (Fig. 23.2) (Bae et al., 1994; Meyers et al., 1995). Transient transfection studies have shown that AML1B or AML1b activate transcription from multiple hematopoietic-specific promoters, including the T-cell receptor chain genes (, , , ), CD3, myeloperoxidase, granzyme B, neutrophil elastase, granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-3, and macrophagecolony stimulating factor receptor genes (reviewed in Meyers and Hiebert, 1995). AML1B/b can function synergistically in a multifactor activation complex: in the TCR enhanceosome, AML1 can interact with CREB/ATF, LEF-1, Ets-1, and the coactivator ALY (Bruhn et al., 1997; Giese et al., 1995). AML1B can also interact with PU.1 and C/EBP to regulate the M-CSFR promoter (Petrovick et al., 1998), and with MEF to regulate the IL-3 promoter (Mao et al., 1999). In addition, AML1b can interact directly with the CBP/p300 coactivators, via a C-terminal region distal to the RHD (Kitabayashi et al., 1998b). Thus, AML1 is considered to be an enhancer-organizer protein, which is critical to the proper assembly of multiprotein transcriptional activating complexes. In contrast to the longer AML1 isoforms, AML1A lacks a p300 interaction domain and has been transcriptionally silent in many transfection studies. We have found that AML1A can transactivate the IL-3 promoter in T cells, although less well than AML1B (Uchida et al., 1997). In contrast, Tanaka and co-workers showed that AML1A could function as a negative regulator of AML1b transactivation in 32D cells and could also block granulocytic differentiation (Tanaka et al., 1995). These findings emphasize the context-dependent activities of AML1 proteins. In addition to AML1A, a novel AML1 isoform, AML1N, which contains an N-terminal truncation that deletes part of the RHD, has been found to interfere with AML1B/b transactivation (Zhang et al., 1997b). These studies raise the possibility that the different spliced forms of AML1 may function antagonistic-

ETO: FOUNDING MEMBER OF A GROWING PROTEIN FAMILY

ally to modulate the regulation of AML1 target genes in certain situations. AML1A and AML1N transcripts, however, represent a small percentage of the total AML1 transcripts present in most cells analyzed thus far; therefore, further studies are required before conclusions regarding the biological relevance of their antagonistic properties can be made. To identify unknown target genes of AML1, Harada and colleagues performed representational difference analysis (RDA) on mRNA from wild-type and AML1-deficient ES cells, and identified a novel gene, HERF1 (hematopoietic RING finger 1), whose expression required functional AML1 (Harada et al., 1999). HERF1 contains a tripartite RING-finger B-box coiledcoil domain, present in the PML and TIF1 proteins, and was shown to be required for the normal differentiation of MEL cells (Harada et al., 1999). Questions regarding the mode of regulation of HERF1 by AML1 (direct or indirect) and the importance of HERF1 to AML1 biologic activity remain to be answered. The discovery of additional AML1 target genes is a major focus of investigators attempting to elucidate how AML1 exerts its effects on hematopoietic development and function. Despite the abundant examples of transcriptional activation by AML1 proteins, when fulllength AML1 or PEBP2B is fused to a Gal4 DNA-binding domain, they repress transcription from a Gal4 response element, suggesting that, like Runt proteins, AML1 proteins possess intrinsic repressor functions (Aronson et al., 1997). Both Runt and AML1 contain a C-terminal pentapeptide motif VWRPY, which mediates direct protein-protein interactions with the Groucho family of transcriptional repressors (Fig. 23.2) (Aronson et al., 1997; Fisher and Caudy, 1998). AML1b has been shown to interact with TLE1, a human homolog of Drosophila Groucho, and overexpression of TLE repressed AML1-dependent activation of hematopoieticspecific promoters (Imai et al., 1998; Levanon et al., 1998). Therefore, the ability of AML1 proteins to function as either transcriptional activators or repressors may depend on the levels of TLE/Groucho proteins in the cell; confirmation of this hypothesis awaits further investigation. Transcription factor (TF) function is commonly regulated by phosphorylation events gen-

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erated by signal cascades that are initiated by the binding of cytokines or other extracellular stimuli to their cell surface receptors. Acetylation and ubiquitination are other methods of regulating TF activity. The potential regulation of AML1 function through phosphorylation or acetylation has not been extensively explored, although there is in vitro evidence that ERK (extracellular signal-related kinase) — mediated phosphorylation of residues ser249 and ser266 may play a role in potentiating transactivation by AML1 (Tanaka et al., 1996). It is also possible that the interaction of AML1 with TLE/ Groucho may be subject to cell signaling, adding an additional layer of complexity to transcriptional regulation by AML1 proteins (Fisher and Caudy, 1998). Thus, the context-dependent function of AML1 proteins, and their ability to either activate or repress a given target gene, likely relates to the presence and levels of cooperating transcription factors, coactivators and corepressor molecules, and the activation states of these molecules in the biological system being studied.

ETO: FOUNDING MEMBER OF A GROWING PROTEIN FAMILY AND PART OF A MULTIPROTEIN TRANSCRIPTIONAL REPRESSION COMPLEX ETO Members and Function The fusion partner of AML1 in the t(8;21), ETO (eight twenty-one), was initially identified by Erickson and co-workers using a chromosome 8 genomic probe to screen a cDNA library derived from t(8;21) AML blasts (Erickson et al., 1992). Partial identification of the ETO amino acids that were fused in-frame in the fulllength AML1/ETO protein were reported (Erickson et al., 1992), and Miyoshi and colleagues performed 5 RACE to identify the complete AML1/ETO cDNA sequence and clone the wild-type ETO cDNA, which they termed MTG8 (myeloid translocation gene on chromosome 8) (Miyoshi et al., 1993). Although not used by investigators thus far, the Human and Mouse Genome Workshop approved symbol for the ETO/MTG8 gene is CBFA2T1. The human ETO gene consists of 13 exons distributed over 87 kb of genomic DNA (Wolford

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Figure 23.3. Model for transcriptional repression by AML1/ETO. Gray circles represent the different components of AML1/ETO, , AML1 portion; 1—3, NHR 1—3; ZF, zinc-finger domain (NHR4). , CBF protein, which heterodimerizes with AML1/ETO via the RHD. The ZF recruits a copressor complex containing N-CoR/SMRT/ mSin3A/HDAC(HD) proteins, which results in local histone deacetylation and chromatin silencing. The NHR2 region can heterodimerize with ETO and may be able to recruit another corepressor complex.

and Prochazka, 1998). Two N-terminal alternatively spliced forms of the MTG8 cDNA were initially recognized, MTG8a and b, which encode proteins of 577 and 604aa, respectively (Miyoshi et al., 1993). Northern blot analysis indicates that ETO is not expressed in mouse spleen or thymus, but is highly expressed in the brain and in lesser amounts in the testis, ovary, lung, and heart (Miyoshi et al., 1993). ETO proteins are present in the HEL (erythroleukemia), Raji (Burkitt’s lymphoma) (Miyoshi et al., 1993), K562 (CML), and Kasumi-1 [t(8;21) containing AML] (Erickson et al., 1996) human cell lines and also in human megakaryocytes and CD34> human peripheral blood stem cells (Erickson et al., 1996). ETO has not been shown to be able to bind DNA but it may function as a transcriptional regulator, based on its homology to the Drosophila transcription factor nervy, identified as a target gene of the homeotic gene ultrabithorax (Feinstein et al., 1995). There is an overall 30% similarity between ETO and nervy at the amino acid level, but a 45—71% identity at four distinct regions, termed nervy homology regions (NHR) 1—4 (Fig. 23.3) (Kitabayashi et al., 1998a).

NHR1 (120—216aa) includes a region of homology with the Drosophila TAF110 protein (and human TAF105 and TAF130); the function of this motif remains unknown. dTAF110 can physically interact with Sp1 to activate transcription, but the region of homology with ETO is not sufficient for this interaction (Hoey et al., 1993). NHR2 (352—378) has been modeled as an amphipathic -helix (Lenny et al., 1995) and was recently shown to mediate homodimerization among ETO proteins (Davis et al., 1999; Kitabayashi et al., 1998a). The function of NHR3 (444—492) is unknown, but NHR4 (515—552) contains a zinc-finger domain that is the most highly conserved region between ETO and nervy (27 out of 38 residues are identical). This region contains one GATA-like CCCC-type zinc finger and one CCHC-type zinc finger, which is found in the nucleocapsid protein of all known retroviruses (Henderson et al., 1981). Homologous regions have been found in the Drosophila DEAF-1 TF (Gross and McGinnis, 1996); the rat, mouse and Caenorhabditis elegans RP-8 apoptosis-associated protein (Owens et al., 1991); and the mouse T-cell—related BOP protein (Hwang and Gottlieb, 1997). The zinc-

ETO: FOUNDING MEMBER OF A GROWING PROTEIN FAMILY

TABLE 23.1 The ETO Gene Family Common Name

HMGW? Symbol

Chromosome Location

ETO, MTG8 EHT, MTGR1 MTG16, MTGR2

CBFA2T1 CBFA2T2 CBFA2T3

8q22 20q11 16q24

?Human and Mouse Genome Workshop.

finger domain has also been termed the MYND motif (for myeloid, Nervy, DEAF-1) (Gross and McGinnis, 1996), and both fingers within this region are necessary for ETO to physically interact with the nuclear corepressor protein NCoR, as discussed below. Presently, the human ETO gene family contains two additional members, MTGR1 (MTG8-related protein), which is located on chromosome 20q11 (Kitabayashi et al., 1998a) [also called EHT (Fracchiolla et al., 1998)], and MTGR2 (also referred to as MTG16), which is located on chromosome 16q24 (Table 23.1) (Calabi and Cilli, 1998; Gamou et al., 1998). The MTGR1 cDNA was discovered in a search of the GenBank EST database for ETO homologs and encodes a 604aa protein with 68% identity to MTG8; Northern blotting has shown it to be ubiquitously expressed in fetal and adult human and mouse tissues, with the highest levels being found in the brain (Kitabayashi et al., 1998a). The expression pattern of MTGR2 mRNA has been less well characterized, but it is detectable in human thymus (Calabi and Cilli, 1998). A novel murine ETO family member has been isolated, ETO-2, which may represent the mouse homolog of a fourth ETO gene (Davis et al., 1999). Aside from the ability of ETO to physically interact with the corepressor complex, little is known about the normal function of ETO family members in hematopoietic or nonhematopoietic cells; a knockout mouse model has not yet been reported. A potential role of ETO proteins in cellular transformation has been reported, in which expression of ETO in NIH3T3 cells resulted in the formation of a low number of soft agar colonies (Wang et al., 1997). A second group failed to detect a transforming effect of ETO alone in NIH3T3 cells, although ETO could cooperate

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with v-Ha-ras for transformation (Sueoka et al., 1998). We established a transformation assay in which AML1/ETO scored positive for in vitro anchorage-independent growth and in vivo nude mouse tumor formation; ETO scored negatively in this assay (Frank et al., 1999). Besides the involvement of ETO in the t(8;21), the MTGR2 gene has been found joined to AML1 in a t(16;21) translocation in two cases of AML (Gamou et al., 1998). Clearly, fusion of ETO or MTGR2 to AML1 may alter the expression of AML1 target genes and contribute to neoplastic transformation. Whether wild-type ETO proteins play a role in transformation or can affect the expression of specific target genes has not been established. Transcriptional Effects of ETO ETO proteins have not been shown to be able to directly bind DNA, but evidence for their involvement in transcriptional regulation stems from their predominantly nuclear localization (Davis et al., 1999; Erickson et al., 1996) and especially from the discovery that ETO can directly bind to the corepressor molecules NCoR (nuclear receptor corepressor) and SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) in yeast two-hybrid screens (Gelmetti et al., 1998; Lutterbach et al., 1998b; Wang et al., 1998). The mSin3A and histone deacetylase (HDAC) 1 and 2 proteins are also found in this complex, linking ETO to transcriptional repression resulting from histone deacetylation. Wang and co-workers demonstrated that a Gal4-DNA—binding domain (DBD)ETO fusion protein repressed transcription from a plasmid containing four Gal4 sites upstream of the thymidine kinase promoter, establishing the repressive function of ETO when it is recruited to DNA (Wang et al., 1998). By extension, this activity of ETO is presumed to be the mechanism of transcriptional repression by AML1/ETO (see below). ETO proteins can heterodimerize with AML1/ ETO via the NHR2 domain (Davis et al., 1999), and AML1/ETO and ETO or MTGR1 can be coimmunoprecipitated from t(8;21) containing leukemia cells (Kitabayashi et al., 1998a). A clear idea of the effects of ETO homo- versus heterodimerization on AML1/ETO function has not been generated. The MTGR1 protein was

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found to enhance transcriptional repression of the T-cell receptor chain (TCR)-enhancer by AML1/ETO (Kitabayashi et al., 1998a). Expression of ETO had no effect on repression of the MDR-1 promoter by AML1/ETO, and when expressed at high levels, ETO caused loss of AML1/ETO-mediated repression (Lutterbach et al., 1998a). Further studies are needed to clarify how these protein-protein interactions regulate AML1/ETO function.

THE ROLE OF AML1/ETO IN LEUKEMOGENESIS AML1/ETO Is a Dominant Transcriptional Repressor Initial insights into the function of the AML1/ ETO fusion protein were gained from transient transfection studies using reporter genes regulated by promoters that contain AML1-binding sites. These studies generally demonstrated that the longer isoform of AML1, AML1B (Fig. 23.3 and preceding text), could activate transcription, whereas AML1/ETO both repressed basal transcription and dominantly repressed AML1B-dependent transactivation, even when transfected at lower levels than AML1B (Frank et al., 1995; Meyers et al., 1995). AML1 (1-177), which contains the part of AML1 contained in AML1/ ETO, does not repress AML1B transactivation, indicating that ETO sequences are also important for transrepression by the fusion protein (Uchida et al., 1997). Additional mutagenesis studies showed that the RHD, which is responsible for binding DNA and interacting with the CBF protein, and C-terminal ETO amino acids are essential for transrepression (Fig. 23.1) (Lenny et al., 1995). These same functional regions overlap with those necessary for transformation of NIH3T3 cells (Frank et al., 1999) and for blocking differentiation of a murine myeloid progenitor cell line (Kitabayashi et al., 1998a), suggesting a close relationship between transformation and transcriptional repression of AML1 target genes by AML1/ETO (Fig. 23.1). It was found that substoichiometric levels of AML1/ETO, compared to AML1B, were sufficient to cause profound transcriptional repression and that mutations in the DNA-binding RHD eliminated this function. These findings

led to the hypothesis that AML1/ETO-mediated repression is an active process, in which the fusion protein does not simply prevent AML1 from binding to its gene targets. This hypothesis appears to be correct, as AML1/ETO has been shown to recruit corepressor molecules to specific regulatory regions. Both ETO and AML1/ETO bind directly to the nuclear corepressor molecules N-CoR and SMRT in a multiprotein transcriptional repression complex that includes the mSin3A and histone deacetylase (HDAC) proteins (Lutterbach et al., 1998b; Wang et al., 1998). The recruitment of this complex results in local histone hypoacetylation, chromatin condensation, and transcriptional repression (Fig. 23.3) (Pazin and Kadonaga, 1997). This corepressor complex has been demonstrated to underlie the mechanism of transcriptional repression by the mad/max complex and by unliganded nuclear hormone receptors (Pazin and Kadonaga, 1997; Schreiber-Agus and DePinho, 1998). An N-CoR/SMRT interaction domain within AML1/ETO was localized to a C-terminal region, distal to residue 469, and specifically involves the zinc-finger domain (also termed NHR4, see earlier in this chapter) (Fig. 23.1) (Gelmetti et al., 1998; Lutterbach et al., 1998b). This region was previously found to be dispensable for the repressor activity of AML1/ETO on the TCR enhancer (Lenny et al., 1995), implying that the corepressor complex may not be the sole mediator of repression by AML1/ETO. Yet the NHR4 region is required for repression of the MDR-1 promoter, and HDAC inhibitors, such as trichostatin A, partially relieve AML1/ ETO-mediated repression when included in transfection experiments, confirming the importance of the corepressor complex to this process (Lutterbach et al., 1998a, 1998b). These data are resolvable and compatible with a mechanism of active repression, based on the finding that a zinc-finger region mutant of AML1/ETO can still bind N-CoR and mSin3A in vivo (Lutterbach et al., 1998b), indicating that other regions of the fusion protein can also interact with corepressor molecules. Furthermore, AML1/ETO and ETO can heterodimerize via their ETO NHR2 regions (Davis et al., 1999), so a mutant AML1/ETO protein that lacks a zinc-finger motif (and also an AML1/ETO mutant protein containing only 1-469aa) could potentially recruit the HDAC complex through interactions

THE ROLE OF AML1/ETO IN LEUKEMOGENESIS

with wild-type ETO proteins (diagramed in Fig. 23.3). AML1/ETO can repress the activation of gene expression by transcription factors other than AML1. AML1/ETO can physically associate with C/EBP, via the RHD, and block its activation of the rat NP-3 (neutrophil protein-3, a defensin found in the primary granules of neutrophils) promoter, which contains adjacent AML1-binding sites (Westendorf et al., 1998). C/EBP is an important regulator of myeloid differentiation, and blocking its transcriptional activities may be one of the mechanisms by which AML1/ETO prevents myeloid differentiation (Zhang et al., 1997a). AML1/ETO has also been shown to be able to inhibit the transcriptional activities of different ETS proteins, which play diverse roles in myeloid and lymphoid cell function (Tenen et al., 1997). We have shown that AML1/ETO blocks transcriptional activation by MEF (myeloid elf-like factor), an ETS member with potent transactivator function and significant homology to the E74 subfamily of ETS proteins (Mao et al., 1999; Miyazaki et al., 1996). Repression of MEF targets by AML1/ETO can occur even in the absence of DNA binding by AML1/ETO, but it requires an ets interaction domain (EID) in the C-terminus of the RHD, which is required for the AML1/ETO:MEF physical interaction (Fig. 23.1) (Mao et al., 1999). AML1/ETO can also block ets-1 activation of the MDR1 promoter, but whether this repression requires an intact DNA-binding domain has not been determined (Lutterbach et al., 1998a). Thus, through various mechanisms involving direct DNA binding and protein-protein interactions with other transcription factors, AML1/ETO could potentially affect the expression of a broad range of genes important to normal cell growth and differentiation.

Cellular Functions of AML1/ETO The precise role and mechanism of action of AML1/ETO in the pathogenesis of AML remains largely unknown, in part because mouse genetic models have resulted in embryonic lethality (discussed below). The invariant transcripts found in clinical samples and the recent discovery of a second AML1/ETO-related fu-

417

sion gene (see earlier in this chapter) suggest a prominent role for AML1/ETO in leukemogenesis. However, additional mutations may be required for AML1/ETO to cause leukemia, as is suggested by the presence of the t(8;21) in the clinical setting of the myelodysplastic syndrome (considered a clonal but ‘‘preleukemic’’ disorder) (Taj et al., 1995). Also, persistent AML1/ETO expression can be detected by RT-PCR in many patients in clinical complete remissions, some for more than 10 years (Nucifora et al., 1993). Alternatively, as suggested by quantitative RTPCR data, this could indicate that a threshold level of AML1/ETO expression is required for its full biological effect (Marcucci et al., 1998). Nonetheless, ample experimental evidence exists to implicate the fusion protein in the processes of blocking hematopoietic cell differentiation and promoting cellular transformation. AML1/ETO knock-in mice (AML1/ETO/;) were created by Yergeau and colleagues (1997) and by Okuda and colleagues (1998a), by inserting the AML1/ETO cDNA into the AML1 locus, mimicking the balanced expression of AML1 and AML1/ETO seen in 8;21 leukemia cells. In both cases, the resulting phenotype was embryonic lethality at E12.5—13.5 due to a lack of definitive hematopoiesis in the fetal liver and multiple areas of central nervous system hemorrhage. This phenotype is nearly identical to that which occurs in the AML1 knockout mice (AML1\\), consistent with the effect of AML1/ETO as a dominant repressor of AML1 function (Okuda et al., 1996; Wang et al., 1996). Differences in the growth properties of yolk sac—derived cells from AML knockout versus AML1/ETO knock-in animals were observed in in vitro hematopoietic colony assays: Whereas AML1\\ cells did not give rise to any hematopoietic colonies, AML1/ETO> cells gave rise to normal numbers of solely macrophage colonies in one study (Yergeau et al., 1997) and rare myeloid colonies in a second study (Okuda et al., 1998a). Moreover, hematopoietic cells derived from the fetal livers of E11.5—12.5 AML1/ ETO/> mice, although few in number, gave rise to large multilineage colonies that exhibited significant dysplasia. When the normal and AML1/ETO/> fetal liver cells were serially replated, the normal cells lost colony-forming abiity after the seventh passage, whereas the AML1/ETO/> cells formed colonies through

418


passage 20 and continuously proliferated in liquid culture in the presence of growth factors (Okuda et al., 1998a). The increased self-renewal capacity of hematopoietic progenitors expressing AML1/ETO was confirmed in experiments using retrovirally transduced mouse bone marrow cells. However, the AML1/ETO-expressing cell lines were not able to generate leukemia when transplanted into mice (Okuda et al., 1998a). These studies show that AML1/ETO can increase the growth potential of hematopoietic stem cells and generate dysplastic hematopoiesis in vitro. The lack of in vivo leukemia development may reflect a ‘‘true’’ biological requirement of AML1/ ETO for a cooperating mutation to induce leukemia. Alternatively, the progenitor cells targeted experimentally may not represent the same progenitor cell that acquires the t(8;21) in humans. Differences in the hematopoietic stem cells between human and murine species or of the bone marrow microenvironment could also account for the lack of full transformation. Long-term expression of AML1/ETO in human CD34> cells has not yet been reported, but such experiments might yield different results than those obtained by expressing AML1/ETO in murine hematopoietic cells. The use of different in vitro systems has uncovered diverse functions for AML1/ETO. We have demonstrated the oncogenic potential of AML1/ETO in an NIH3T3 transformation assay, in which expression of the fusion protein but not the wild-type AML1 or ETO proteins conferred anchorage-independent growth properties and in vivo tumorigenicity to the cells (Frank et al., 1999). Interestingly, and paralleling the findings in the mouse models described above, NIH3T3 cells expressing AML1/ETO exhibited features of both cell death and transformation (Frank et al., 1999). Other investigators have found that AML1b (Kurokawa et al., 1996) or ETO (Wang et al., 1997) can transform NIH3T3 cells. Transformation assay conditions may vary from one lab to another, but when we tested the activities of AML1/ETO, AML1A, AML1B, and ETO in a uniform assay, we observed that only AML1/ETO had oncogenic potential (Frank et al., 1999). These findings do not eliminate the possibility that, under certain conditions, the wild-type proteins could exhibit oncogenic characteristics, but they do suggest

that AML1/ETO possesses greater oncogenic capabilities than its wild-type constituents. Differences in the level of AML1 or ETO protein expression do not appear to account for the disparate results of these experiments. Expression of AML1/ETO in the murine myeloid progenitor cell lines 32Dcl3 (Ahn et al., 1998) and L-G (Kitabayashi et al., 1998a) blocked the normal differentiation response to G-GSF, although growth factor—independent growth was not obtained; antisense oligonucleotides directed against the AML1/ETO mRNA junction increased the differentiation response of a t(8;21) cell line to PMA (Sakakura et al., 1994), further suggesting a direct ability to block differentiation. There is also evidence that AML1/ETO may function as an anti-apoptotic factor: AML1/ETO can transcriptionally activate the BCL-2 gene promoter (Klampfer et al., 1996) and ribozymes targeted against the chimeric mRNA induce apoptosis when expressed in an t(8;21) AML cell line (Matsushita et al., 1999).

GAIN-OF-FUNCTION PROPERTIES OF AML1/ETO In addition to the numerous inhibitory effects of AML1/ETO on AML1 function, novel gain-offunction properties that are independent of AML1 activity have been described. AML1/ ETO can activate the BCL-2 promoter (Klampfer et al., 1996) and can upregulate bcl-2 gene expression under steady-state conditions and upon growth factor withdrawal (our unpublished data). AML1/ETO can also synergize with AML1B to activate the M-CSFR promoter, possibly due to titration of repressor molecules, since high levels of AML1/ETO no longer activate the promoter (Rhoades et al., 1996). We have demonstrated that AML1/ETO can activate both AP-1—responsive reporter gene constructs (such as the collagenase type-1 promoter linked to the luciferase gene) and endogenous target genes (such as c-Jun) in NIH3T3 cells (Frank et al., 1999) as well as in hematopoietic cell lines (unpublished data). This effect is independent of the presence of AML1-binding sites in the promoters of the target genes, but likely results from the indirect activation of c-Jun as a result of expression of AML1/ETO.

REFERENCES

Studies performed in NIH3T3 cells directly correlated the ability of wild-type or deletion mutant AML1/ETO fusion proteins to transform NIH3T3 cells, with the ability to increase levels of the N-terminally phosphorylated form of cJun and to activate c-Jun—dependent transcription (Frank et al., 1999). The kinase activity of the c-Jun amino terminal kinase (JNK) is also increased in NIH3T3 cells expressing AML1/ ETO (unpublished data), which can account for the elevated levels of phosphorylated c-Jun observed in these cells. The mechanism underlying the activation of JNK as a result of expression of AML1/ETO is under investigation, but it might involve the transcriptional repression of a phosphatase involved in JNK regulation, or the stabilization of JNK through inhibition of the proteasome pathway (see below). A link between AML1/ETO and the proteasome pathway has been made by Liu and coworkers: AML1/ETO knock-in murine embryonic stem (ES) cells were found to exhibit upregulation of a novel ubiquitin-specific protease, UBP43, which was not found in normal ES cells (Liu et al., 1999). The mechanism by which AML1/ETO upregulates UBP43 is under investigation. The targets of UBP43 are unknown, but its overexpression blocks cytokineinduced terminal differentiation of the M1 leukemia cell line, suggesting a potential role in mediating the effects of AML1/ETO (Liu et al., 1999). Inhibition of the proteasome pathway can increase JNK activity (Merlin et al., 1998); therefore, upregulation of UBP43 (or inhibition of other ubiquitin-regulating enzymes) could account for the upregulation of JNK activity found in AML1/ETO-expressing cells.

CONCLUSIONS AND FUTURE DIRECTIONS Tremendous progress has followed the identification of the AML1 gene as being involved in the 8;21 translocation. Dysregulation of the AML1:CBF complex has been found in other types of acute myeloid leukemia and in childhood acute lymphoid leukemia (see other chapters in this book). Investigation into the function and mechanism of action of AML1/ETO has revealed that it acts as a dominant transcriptional inhibitor of AML1-dependent gene activation and that this repression, at least in part,

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involves recruitment of a corepressor complex that contains histone deacetylase activity. In addition, the cellular effects of AML1/ETO in vitro demonstrate that the fusion protein possesses unique activities, independent of AML1 or ETO function. Several key questions about t(8;21) leukemias include:

· What regulates the expression of the AML1/ETO, AML1, and ETO genes?

· Can AML1/ETO alone cause leukemia or are other mutations required? If so, what are the nature of these mutations?

· What are the critical target genes of AML1 and AML1/ETO that execute their functions?

· Is the activity of AML1/ETO subject to regulation by an intracellular signaling pathway, and if so, can pharmacologic manipulation of this pathway disrupt this activity?

· Can blocking AML1/ETO function and/or expression be of clinical use in the treatment of 8;21 leukemias? Clearly, although much has been discovered regarding the many functions of AML1 and AML1/ETO, much remains to be learned before the knowledge gained from basic science discoveries can be translated into effective targeted therapies for the treatment of t(8;21) leukemia and other acute leukemias characterized by alterations of the AML1:CBF complex.

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CHAPTER 24


TEL/ETV6 GENE REARRANGEMENTS IN HUMAN LEUKEMIAS EMA ANASTASIADOU, MICHAEL H. TOMASSON, DAVID W. STERNBERG, TODD R. GOLUB, AND D. GARY GILLILAND Howard Hughes Medical Institute, Brigham and Women’s Hospital, Harvard Medical School and Harvard Institute of Medicine

THE TEL GENE PRODUCT The T EL gene (translocation, ets, leukemia) is a member of the ETS family of transcription factors and is also known by its Genbank designation ETV6 (ets translocation variant 6). It was cloned as a fusion partner of the platelet-derived growth factor receptor gene (PDGFR) in a patient with chronic myelomonocytic leukemia (CMML) and t(5;12)q33;p13) (Golub et al., 1994). The TEL protein contains two functional domains, the pointed (PNT domain) and the DNA-binding or ETS domain. The latter binds to consensus DNA sequences characteristic of the ETS family of transcription factors. The PNT domain is highly conserved in a subset of ETS family members, but prior to the cloning and characterization of TEL, the function of the PNT domain was poorly understood. Although deletion of the PNT domain in other ETS family members impaired transactivation, the PNT domain was specifically not thought to represent a dimerization or oligomerization interface.

Insights gained from the analysis of the structure function relationships of the TEL/ PDGFR fusion have demonstrated that in TEL the PNT domain serves as a dimerization or oligomerization motif. TEL/PDGFR expression is driven by the TEL promoter, and incorporates the amino terminal PNT domain of TEL fused in frame to the transmembrane and tyrosine kinase domain of PDGFR. In the context of the TEL/PDGFR fusion, the PNT domain oligomerizes and constitutively activates the tyrosine kinase domain of the PDGFR (Carroll et al., 1996; Jousset et al., 1997). The PNT domain is required for transformation of cultured hematopoietic cell lines, as well as for primary hematopoietic progenitor cells in murine bone marrow transplant assays of transformation (Carroll et al., 1996; Tomasson et al., 2000). Although the TEL PNT domain is an oligomerization motif in the context of the native TEL protein as well as the TEL/PDGFR fusion, the PNT domain of other ETS family


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members, though highly conserved, does not appear to serve this function. In domain-swapping experiments, for example, the TEL PNT domain can mediate oligomerization of other ETS family members, but the converse is not true (Jousset et al., 1997). The structural basis for this apparent disparity in function of this conserved motif is not clear. The NMR structure of the ETS1 PNT domain, a monomer in solution, was recently solved, but it did not provide insights into the oligomerization function of the TEL PNT domain (Slupsky et al., 1998). It is possible that posttranslational modification of the domain is required for oligomerization in some ETS family members. In addition, the PNT domain shares homology with a SAM (sterile alpha motif) domain that can serve as a homo- and hetero-oligomerization module. Further structural analysis of the TEL PNT domain will be necessary to definitively address this question and to determine the stoichiometry of the PNT domain complexes.

THE ROLE OF TEL IN DEVELOPMENT AND NORMAL HEMATOPOIESIS Targeted disruption of the Tel locus by homologous recombination has provided useful insights into the function of Tel in development and during hematopoiesis. Tel heterozygotes are healthy, fertile, and without any discernible phenotype. Tel-null mice die between E10.5 and E11.5 with defective yolk sac angiogenesis and intraembryonic apoptosis of mesenchymal cells and neural cells (Wang et al., 1997). Two-thirds of the Tel-deficient yolk sacs at E9.5 lack vitelline vessels yet possess capillaries, indicative of normal vasculogenesis. In the remaining onethird of Tel-deficient yolk sacs, vitelline vessels form but regress by E10.5, suggesting that Tel is required for maintenance of the developing vascular network. Yolk sac hematopoiesis is unaffected in the Tel\\embryos. However, analysis of chimeric animals generated using Tel\\ES cells has demonstrated an unusual hematopoietic phenotype (Wang et al., 1998). Tel is not required for the intrinsic proliferation and/ or differentiation of adult-type hematopoietic lineages in the yolk sac and fetal liver. However, analysis of chimeric animals constructed using Tel\\ES cells demonstrates that Tel function is

essential for the establishment of hematopoiesis of all lineages in the bone marrow. These data identify a critical role for Tel in the normal transition of hematopoietic activity from fetal liver to bone marrow. Tel is thus the first transcription factor required specifically for hematopoiesis within the bone marrow (Wang et al., 1998).

THE ROLE OF TEL IN HEMATOLOGIC MALIGNANCIES AND OTHER HUMAN CANCERS The T EL gene is now known to be involved in more than 40 different chromosomal translocations associated with hematologic malignancies, primarily leukemias (Odero et al., 1999) (Table 24.1). In addition, the TEL/TRKC fusion has been reported and associated with nonhematologic malignancies including congenital fibrosarcoma (Knezevich et al., 1998b) and mesoblastic nephroma (Knezevich et al., 1998a; Rubin et al., 1998). In addition to promiscuity of its involvement in translocations, TEL is notable for the striking diversity of its fusion partners that include receptor and nonreceptor tyrosine kinases, transcription factors, and homeobox genes (reviewed in Golub and Gilliland, 1997; Golub et al.,1996b). TEL also contributes different functional domains to these various fusion proteins. For example, the TEL PNT oligomerization motif is expressed in fusion proteins with diverse partners such as PDGFR receptor noted above (Golub et al., 1994); other receptor and nonreceptor tyrosine kinases such as ABL (Golub et al., 1996a; Papadopoulos et al., 1995), TRKC (Eguchi, 1999; Knezevich et al., 1998a, 1998b; Rubin et al., 1998), and JAK2 (Lacronique et al., 1997; Peeters et al., 1997); and the ABL-related tyrosine kinase ARG (Cazzaniga et al., 1999; Iijima et al., 2000). TEL fusions with transcription factors including the TEL/AML1 fusion (Golub et al., 1995; Romana et al., 1995), TEL/EVI1 (Peeters et al., 1997b) and TEL/CDX2 (Chase et al., 1999). Furthermore, the TEL DNA-binding domain is expressed as a fusion partner of proteins such as the product of the MN1 gene in the t(12;22) (Buijs et al., 1995), and the BTL/TEL fusion (Cools et al., 1999). It appears that the primary

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TABLE 24.1. Cloned Chromosomal Translocations Involving the TEL Gene Translocation

TEL Fusion

Phenotype?

Reference

t(5;12)(q33;p13) t(9;12)(q43;p13)

Tyrosine Kinases TEL/PDGFR TEL/ABL

CMML AML, ALL

t(9;12)(p24;p13)

TEL/JAK2

t(12;15)(p13;q25)

TEL/TRKC

t(1;12)(q25;p13)

TEL/ARG

Golub et al., 1994 Golub et al., 1995 Papadopoulos et al., 1995 Lacronique et al., 1997 Peeters et al., 1997a Knezevich et al., 1998a Knezevich et al., 1998b Rubgin et al. 1998, Eguchi et al., 1999 Cazzaniga et al., 1999, Iijima, 2000

t(12;21)(p13;q22) t(3;12)(q26;p13) t(12;13)(p13;q12)

Transcription Factors TEL/AML1 pediatric B-cell ALL TEL/EVI1 MPD TEL/CDX2 AML

Golub et al., 1995, Romana et al., 1995a,b Peeters et al., 1997b Chase et al., 1999

t(12;22)(p13;q11) t(4;12)(q11-1;p13)

Unknown function MN1/TEL CHIC2/TEL

AML, MDS AML

Buijs et al., 1995 Cools et al., 1999

t(5;12)(q31;p13)

CoA synthetases TEL/ACS2

MDS(RAEB)

Yagasaki, 1999

t(1;12)(q21;p13)

Hypoxia response TEL/ARNT

AML-M2

Salomon-Nguyen et al., 1999

atypical CML, T-ALL B-ALL AML, congenital fibrosarcoma, mesoblastic nephroma AML

?Acute myelogenous leukemia, AML; myelodysplastic syndrome, MDS; acute lymphoblastic leukemia, ALL; refractory anemia with excess blasts, RAEB; chronic myelomonocytic leukemia, CMML; chronic myelogenous leukemia, CML; myeloproliferative disorder, MPD.

contribution of the TEL gene in some translocations is an active promoter, as in the TEL/ EVI1 fusions (Peeters et al., 1997b), in which only the first exon of TEL is expressed in the fusion, or the TEL/CDX2 fusion (Chase et al., 1999), in which the first three exons of TEL are expressed, but there is disruption of the TEL PNT domain that would likely abrogate oligomerization function. Several unusual TEL fusion partners have recently been identified, including the long-chain fatty acyl CoA synthetase 2 (ACS2 gene) and the aryl hydrocarbon receptor nuclear transporter (ARNT), which is involved in the cellular response to hypoxia (Salomon-Nguyen et al., 1999). The mechanisms by which the TEL/ACS2 or TEL/ARNT fusions contribute to pathogenesis of leukemia are unknown. Finally, as discussed below, loss of TEL function may contribute to pathogenesis of leukemias in some circumstances. Specifically, the residual TEL allele is deleted in ALL pa-

tients that harbor the TEL/AML1 gene rearrangement (Cave et al., 1997; Filatov et al., 1996; Golub et al., 1995; Kim et al., 1996; Nishimura et al., 1999; Raynaud et al., 1996b; Romana et al., 1995a, 1995b; Wlodarska et al., 1996). The diversity of involvement of TEL functional domains contrasts with proteins such as MLL, which is also involved in a broad spectrum of chromosomal translocations (Rowley, 1999). Chromosomal translocations involving MLL on chromosome 11q23 occur invariably, resulting in expression of fusion transcripts in which the amino terminus of MLL is fused to a partner gene.

T EL/TYROSINE KINASE FUSIONS IN HUMAN LEUKEMIA There are four different chromosomal translocations that result in fusion of 5 sequences from

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the TEL gene to tyrosine kinase partner genes including PDGFR, ABL, JAK2 TRKC, and ARG (Cazzaniga et al., 1999; Eguchi et al., 1999; Golub et al., 1994, Golub et al., 1996a; Iijima et al., 2000; Knezevich, 1998a, 1998b; Lacronique et al., 1997; Papadopoulos et al., 1995; Peeters et al., 1997a; Rubin et al., 1998). In each case, the TEL PNT domain is thought to oligomerize and constitutively activate the respective tyrosine kinase domain. Detailed analysis of the signal transduction has provided insights into the molecular mechanisms of transformation mediated by these fusion proteins.

TEL/PDGFR TEL was cloned as the fusion partner of PDGFR in patients in CMML and t(5;12)(q33;p13) (Golub et al., 1994). The TEL PNT domain serves to oligomerize and constitutively activate the PDGFR tyrosine kinase domain. PNT deletion mutants or point mutations that block PDGFR kinase activity abrogate transformation by TEL/PDGFR in both cultured hematopoeitic cell lines and in murine bone marrow transplant assays of transformation (Carroll et al., 1996). Taken together, these data indicate that PDGFR tyrosine kinase activity is required for transformation, and provide a rationale for the identification of signal transduction pathways that are activated by the TEL/PDGFR fusion. Tyrosine to phenylalanine substitution mutations in the PDGFR moiety have provided

insights into the signal transduction pathways that are activated by TEL/PDGFR (Sternberg et al., 1999). Mutant F2 lacks the juxtamembrane Y579 and Y581, F5 lacks the kinase inset Y740, Y751, Y771, and C-terminal Y1009 and Y1021, F7 lacks all 7 of these tyrosine residues, and F8 lacks an additional kinase insert Y716 (Figure 24.1). TEL/PDGFR F2, F5, F7, and F8 mutants are each active as a protein tyrosine kinase, and transform Ba/F3 cells for IL3-independent growth. TEL/PDGFR and F2 associate with the p85 subunit of PI 3-kinase, and phosphorylate PLC and PI 3-kinase, but like the native PDGFR these interactions are lost by mutation of the kinase insert and D-terminal tyrosine residues in F5, F7, and F8. Thus, PLC and PI 3-kinase activation are dispensable for transformation of Ba/F3 cells. In addition, although both TEL/PDGFR and F5 are potent activators of Stat5, F2, F7, and F8 only weakly activate Stat5 in culture hematopoietic cell lines (Sternberg et al., 1999). A murine bone marrow transplant (BMT) assay system has been employed to characterize the transforming properties of TEL/PDGFR and related mutants in primary bone marrow cells (Tomasson et al., 2000). TEL/PDGFR causes a rapidly fatal myeloproliferative disease that has phenotypic similarities to human CMML. TEL/PDGFR transplanted mice develop leukocytosis with Gr-1> granulocytes, splenomegaly, extramedullary hematopoiesis in spleen and liver, and bone marrow fibrosis, but do not develop lymphoproliferative disease. Control BMT experiments further demonstrate

Figure 24.1. TEL/PDGFR fusion and F2, F5, F7, and F8 tyrosine to phenylalanine substitution mutations.

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Figure 24.2. Kaplan-Meier survival curves for animals transplanted with TEL/PDGFR and related mutants. Solid lines indicate myeloproliferative disease caused by the TEL/PDGFR and F5 mutant, whereas the dotted lines indicate the F2, F7, and F8 mutants caused only a lymphoproliferative phenotype.

that kinase inactive mutants of TEL/PDGFR do not cause hematologic disease. Although all F mutants confer factor-independent growth to Ba/F3 cells, different F mutants generate markedly different phenotypes in the murine BMT assay of transformation. TEL/PDGFR and F5 cause a short latency myeloproliferative phenotype, whereas the F2, F7, and F8 mutants cause a long latency T-cell lymphoblastic lymphoma (Fig. 24.2). It thus appears that the juxtamembrane residues 579/581 are critical for development of the myeloproliferative phenotype. In addition, the ability to cause the myeloproliferative phenotype corresponds to the ability of TEL/PDGFR and F5 to activate Stat5, whereas F2, F7, and F8 cause a lymphoproliferative disease and do not activate Stat5 (Tomasson et al., 2000). There is an absolute requirement for TEL/ PDGFR tyrosine kinase activity for transformation in cultured hematopoeitic cells and in the murine BMT assay. These data indicate that specific inhibition of the TEL/PDGFR kinase

should inhibit transformation in these systems. CGP57148, now known as STI571, is a 2phenylaminopyrimidine that inhibits the ABL and PDGFR tyrosine kinases with an IC50 of approximately 0.3 uM, but has no effect on tyrosine kinase activity of a spectrum of other receptor and nonreceptor tyrosine kinases or serine/threonine kinases. CGP57148 specifically inhibits Ba/F3 cells transformed by TEL/ PDGFR, TEL/ABL, or BCR/ABL (Carroll et al., 1997). Inhibition is not due to nonspecific toxicity, since addition of IL-3 restores growth of TEL/PDGFR cells treated with CGP57148. As a further test of CGP57148 activity in TEL/PDGFR disease in primary hematopoietic cells, transgenic lines have been prepared in which TEL/PDGFR expression was directed by the immunoglobulin enhancer/promoter Eu (Tomasson et al., 1999). These mice develop clonal lymphoblastic lymphoma. These data indicate that TEL/PDGFR is capable of causing lymphoid disease, but the clonal nature

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of the lymphoma suggests that acquisition of mutations in addition to the germ-line TEL/ PDGFR mutation is necessary for this phenotype. As such, this transgenic model can also serve as a model for disease progression mediated by TEL/PDGFR. The disease is readily transplantable into secondary recipient animals. CGP57148 administration to these mice causes a statistically significant prolongation in survival in these animals. Perhaps of greater incidence is that CGP57148 also prolongs survival in the secondary transplant recipients, indicating that CGP576148 is active even in cells that have acquired mutations in addition to TEL/ PDGFR (Tomasson et al., 1999). These observations have implications for therapeutic applications of CGP57148/STI571 not only for stable-phase CML or CMML, but also for therapy for CML in blast crisis, or CMML that has progressed to acute leukemia.

TEL/JAK2 Signal transduction data from analysis of the TEL/PDGFR fusion suggests that activation of Stat5 might be an important determinant of transformation mediated by tyrosine kinase fusions, a hypothesis that is supported by the observation that both BCR/ABL and TEL/ABL are potent activators of Stat5. Perhaps the most compelling evidence that supports a role for Stat5 in transformation by tyrosine kinases has been generated from the analysis of the TEL/ JAK2 fusion protein. TEL/JAK2 is expressed as a consequence of a rare t(9;12)(p24;p13) translocation observed in patients with atypical CML, T-cell ALL, and preB-cell ALL JAK2 (Lacronique et al., 1997; Peeters et al., 1997a). Like the other TEL-tyrosine kinase fusions, the TEL PNT domain is fused to the JAK2 tyrosine kinase domain and serves to constitutively activate JAK2. TEL/JAK2 confers factor-independent growth to Ba/F3 cells, and causes a myeloand lymphoproliferative disease in the murine BMT assay (Schwaller et al., 1998). JAK2 normally activates members of the STAT (signal transducers and activators of transcription) family of latent cytoplasmic transcription factors upon ligand stimulation of cytokine receptors. JAK2 is activated upon association with the cytoplasmic domain of the dimerized

receptor scaffold, and phosphorylates tyrosine residues that serve as docking sites for STAT proteins. JAK2 then phosphorylates STATs, inducing dimerization and shuttling to the nucleus where Stat-inducible transcription is activated. TEL/JAK2 is localized to the cytoplasm of cells (Schwaller et al., 1998), and lacks both an association with a receptor scaffold as well as approximately two-thirds of the coding sequence of native JAK2. It is therefore somewhat surprising that TEL/JAK2 efficiently tyrosine phosphorylates Stat5, but not other Stat family members, in transformed Ba/F3 cells. To test the requirement for Stat5 in TEL/JAK2-mediated transformation, murine BMT experiments have been performed in which TEL/JAK2 is introduced into Stat5a/b\\bone marrow cells by retroviral transduction. These mice do not develop hematopoietic malignancy, indicating that STAT5a/b are required for TEL/JAK2-mediated transformation. Complementation experiments in which both TEL/JAK2 and wild-type STAT5a are expressed as a consequence of retroviral transduction recapitulates the myeloproliferative phenotype, indicating that the inability of TEL/ JAK to cause disease in the STAT5a/b\\background can be fully complemented by reintroduction of STAT5a (Schwaller et al., 1999). To determine whether activation of STAT5a is sufficient to cause a myeloproliferative phenotype, BMT experiments have been performed in which a constitutively active mutant of Stat5a is expressed in bone marrow cells by retroviral transduction. These animals develop a myeloproliferative phenotype that is quite similar to that induced by TEL/JAK2 (Schwaller et al., 1999). Taken together, these data indicate that activation of Stat5a/b is both necessary and sufficient for efficient induction of myeloproliferative disease in the BMT model. In addition, these data suggest that Stat5a/b may be a reasonable therapeutic target for hematologic malignancies associated with constitutively activated tyrosine kinases.

TEL/ABL TEL/ABL is expressed as a consequence of another rare translocation t(9;12)(q34;p13) associated both with atypical CML and with childhood B-cell ALL (Golub et al., 1996a;

THE TEL/AML1 FUSION IN PEDIATRIC ALL

431

Figure 24.3. Structure of the TEL/AML1 and AML1/ETO fusions. RHD indicates the DNA-binding Runt homology domain. PNT indicates the pointed domain of TEL, which is a self-association motif.

Papadopoulos et al., 1995). The fusion occurs at ABL exon 2, the same junction of ABL involved in the BCR/ABL fusion associated with CML and t(9;22)(q34;q22). In all respects, TEL/ABL appears to mimic the biologic properties of the BCR/ABL fusion protein. Both are constitutively active tyrosine kinases that require the BCR coiled-coil and TEL PNT domain, respectively, for kinase activation and transformation, and are localized to actin filaments of transformed hematopoietic cells. Both confer factor-independent growth to Ba/F3 cells, and the signaling pathways and intermediates that are activated by TEL/ABL and BCR/ABL are nearly identical, as in the case of other TEL/tyrosine kinase fusions include STAT5 (Okuda K. et al., 1996).

TEL/TRKC A striking counterpoint to the role of STAT5 in transformation mediated by TEL—tyrosine kinase fusions is the TEL/TRKC fusion that has been associated both with AML (Eguchi, 1999) and the nonhematologic congenital fibrosarcomas and mesoblastic nephromas (Knezevich, 1998a, 1998b; Rubin et al., 1998). TRKC is a neurotrophic growth factor receptor that is expressed primarily in neural tissue. The consequence of t(12;15)(p13;q25) is expression of the TEL/TRKC fusion, which like the other tyrosine kinase fusions, generates a constitutively active tyrosine kinase that transforms Ba/F3 cells and causes a myeloproliferative syndrome in the murine BMT assay. However, in contrast to all other tyrosine kinase fusions associated with hematologic malignancy that have been examined to date, Stat5 is not activated by TEL/TRKC, nor are other Stat family members (Liu et al., 2000). Although these findings need to be corroborated by murine BMT into Stat5a/

b-deficient bone marrow cells, they indicate that myeloproliferative disease mediated by some tyrosine kinase fusions can bypass Stat5 activation. Further analysis of the structure function relationships of these transforming oncoproteins will be required to fully elucidate which pathways are critical for transformation, and thus represent bona fide targets for drug development. In addition, genomic approaches to identification of target genes of these fusion proteins should provide further insight into mechanisms of transformation and novel therapeutic approaches. At a minimum, it is critical that transformation be assessed and evaluated in the context of primary hematopoietic progenitors, given the disparity in transforming properties that have been observed when comparing cultured hematopoeitic cells with the murine BMT assay. In addition, the availability of a spectrum of knockout mice deficient in putative downstream effectors of transformation should provide valuable information on critical pathways for transformation.

THE TEL/AML1 FUSION IN PEDIATRIC ALL After cloning of the TEL gene as the fusion partner of PDGFR in chronic myelomonocytic leukemia, we investigated the potential involvement of TEL in lymphoid malignancies with rearrangements at chromosome band 12p13. We identified two children with acute lymphoblastic leukemia (ALL) in which the TEL gene was fused to a gene on chromosome 21, although a t(12;21) translocation was not apparent on cytogenetic analysis (Golub et al., 1995). An anchored PCR strategy revealed that TEL was fused to the AML1 gene on chromosome band 21q22. Identical results were simulta-

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Figure 24.4. Wild-type AML1 or TEL/AML1 were transfected into C33A cells together with the T-cell receptor enhancer element containing AML1-binding sites, upstream of a CAT reporter. In this representative example, AML1 activates the reporter 6-fold, but fusion of TEL to AML1 results in transcriptional repression.

neously reported by Bernard and colleagues (Romana et al., 1995a). The TEL/AML1 fusion is unique for a number of reasons. First, in contrast with the AML1/ETO and AML1/EVI1 fusions associated with myeloid malignancies, TEL/AML1 is exclusively associated with acute lymphoblastic leukemia (Loh et al., 1998). Second, as illustrated in Figure 24.3, the TEL/ AML1 chimera differs from the myeloid leukemia—associated AML1/ETO in that the entire AML1 transactivation domain is preserved in the TEL/AML1 fusion, whereas it is lost in AML1/ETO. Of the four initially reported cases of TEL/AML1 fusion, all had deletion of the other (nontranslocated) TEL allele (Golub et al., 1995; Romana et al., 1995a), a finding that has subsequently been confirmed in the majority of cases of pediatric ALL with the TEL/AML1 gene rearrangement (Cave et al., 1997; Filatov et al., 1996; Kim et al., 1996; Nishimura, 1999; Raynaud et al., 1996b; Romana et al., 1995b; Wlodarska et al., 1996). Several groups have addressed the mechanism of the TEL/AML1 gene rearrangement by analysis of the genomic fusion sequences. Although there is variability in the genomic breakpoints, the fusion sequences exhibit characteristic signs of nonhomologous end joining, compatible with nonhomologous recombination involving imprecise constitutive repair processes following double-stranded DNA breaks (Wiemel and Greaves, 1999). It has also been demonstrated that the TEL/AML1 translocation breakpoints cluster near a purine/py-

rimidine repeat region in the TEL gene (Thandla, 1999).

TEL-AML1 INHIBITS THE FUNCTION OF THE CORE-BINDING FACTOR COMPLEX ESSENTIAL FOR HEMATOPOIESIS The AML1 gene (also known as CBFA2 and RUNX1) on chromosome 21 encodes the subunit of the dimeric transcription factor CBF (core-binding factor; see Chapter 6 in this book). AML1 is responsible for binding DNA, for interacting with the subunit of CBF, and for inducing transcriptional activation of hematopoietic target genes including IL-3, GM-CSF, CSF1-R, TCR enhancer, and the immunoglobulin heavy-chain enhancer/promoter. Mice deficient in either AML1 or CBF- lack definitive hematopoiesis, further emphasizing the importance of the CBF pathway. CBF is a frequent target of chromosomal translocations in human leukemia (Friedman, 1999; Lo Coco and Diveria, 1997; Rowley, 1999; Speck et al., 1999). AML1 is rearranged in the t(3;21) and t(8;21) translocations associated with myeloid leukemias. In addition, an inversion of chromosome 16 associated with M4 AML results in rearrangement of CBF- . Taken together, rearrangements of components of the CBF complex account for nearly one-third of all human acute leukemias. Several lines of evidence indicate that the AML1 fusion proteins are dominant negative forms of the AML1 protein. Expression of AML1/ETO in

TEL/AML1 FUSION PROTEIN IS NOT SUFFICIENT FOR TRANSFORMATION

433

Figure 24.5. TEL/AML1 exerts its dominant negative effect on transcription through recruitment of the nuclear corepressor/histone deacetylase complex.

transfection experiments inhibits transactivation by native AML1 (Hiebert et al., 1996). The most elegant demonstration of dominant negative activity has come from analysis of the role of CBF in development. Mice deficient for either AML1 or for CBF have similar phenotypes characterized by early embryonic lethality associated with CNS hemorrhage, and the absence of definitive hematopoeisis (Okuda, T. et al., 1996; Wang et al., 1996a, 1996b). However, heterozygous animals (AML1>\ or CBF>\) have no discernible phenotype. When knock-in mice are constructed that express the AML1/ETO fusion from the endogenous AML1 locus in the context of a normal AML1 allele, the phenotype is identical to that of the AML1\\or CBF\\knock-out mice (Okuda et al., 1998; Yergeau et al., 1997). That is, AML1/ETO is a dominant negative inhibitor of native AML1 during development. Similar data have been obtained from the CBF/SMMHC knock-in (Castilla et al., 1996). Recent data indicate that the ETO moiety of the AML1/ETO fusion protein recruits the nuclear corepressor/ histone deacetylase complex to promoters, providing a mechanistic explanation for dominant negative effects on CBF promoters (Gelmetti et al., 1998; Lutterbach et al., 1998). Like AML1/ETO, TEL/AML1 also acts as a dominant negative inhibitor of the AML1 (Hiebert et al., 1996). As shown in Figure 24.4, TEL/AML1 alone represses basal transcription from a TCR promoter and abrogates transactivation mediated by AML1. TEL/AML1 also represses transcription from the IL-3 (Uchida et al., 1999), the M-CSF receptor (Fears et al., 1997), the CR1, (Song et al., 1999), and the AML2 promoters (Meyers S, 1996). Recent data indicate that TEL/AML1 exerts its dominant negative effect through recruitment of the nuclear corepressor complex, in a manner analogous to that of AML1/ETO. Further analysis will

be necessary to determine whether a TEL/ AML1 knock-in to the TEL locus also generates an embryonic lethal phenotype that recapitulates the AML1 null phenotype (Fenrick et al., 1999). Taken together these data indicate that the CBF-related fusion proteins have similar structure function relationships. The consequence of chromosomal translocations involving corebinding factor, including the TEL/AML1, AML1/ETO, CBFMYH11, and AML1/EVI1, is expression of dominant negative inhibitors of core-binding factor-mediated transactivation.

EXPRESSION OF THE TEL/AML1 FUSION PROTEIN IS NOT SUFFICIENT FOR TRANSFORMATION It is certain that the CBF fusions are necessary for the acute leukemia phenotype in humans. However, it is equally clear that the CBF fusions are not sufficient to transform hematopoietic progenitor cells. Perhaps the most compelling data for multiple-step pathogenesis of TEL/ AML1 leukemia have come from the analysis of identical twins who develop TEL/AML1 leukemia during childhood. Greaves and colleagues have cloned the genomic breakpoints from sets of such twins, and have demonstrated using PCR analysis of Guthrie cards that the TEL/AML1 fusion was present in utero. The fact that these twin pairs develop ALL years after birth, and at different times after birth, provides strong support for the hypothesis that additional acquired mutations are required, along with the TEL/AML1 gene rearrangement, to give rise to the ALL phenotype (Ford et al., 1998; Wiemels et al., 1999a, 1999b). There is also experimental evidence that TEL/ AML1 expression alone is not adequate to

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Figure 24.6. The t(12;21) that results in fusion of the TEL and AML1 genes is associated with del 12p on the residual chromosome 12. TEL and the cyclin-dependent kinase inhibitor CDNK2, also known as p27/Kip1, are involved in the deletion in most cases of pediatric ALL. In addition, about 10—12% of TEL/AML1-positive cases are p16 deficient.

transform hematopoietic cells. For example, in contrast with TEL-tyrosine kinase fusions associated with human leukemias, the TEL/AML1 fusion protein does not confer factor-independent growth to the murine hematopoietic cell lines Ba/F3, 32D, or the IL-7 dependent preB cell line IXN/2B, nor does TEL/AML1 transform fibroblasts. Furthermore, TEL/AML1 does not cause hematologic malignancy in a murine bone marrow transplant model of leukemia, nor when expressed in transgenic mice under the control of the immunoglobulin enhancer promoter (DGG, unpublished data).

IF TEL/AML1 ALONE IS NOT SUFFICIENT FOR TRANSFORMATION, WHAT IS MISSING? There are several possible explanations why TEL/AML1 expression alone is insufficient for transformation. First, the TEL/AML1 gene rearrangement is associated with loss of the

other TEL allele. We initially observed in a small number of patients that TEL LOH (deletion) accompanied the TEL/AML1 fusion. Raynaud and colleagues subsequently demonstrated in a FISH-based study that the majority of TEL/AML1-positive patients also have loss of the other (nontranslocated) TEL allele (Raynaud et al., 1996b), and frequently have cytogenetic evident abnormalities of chromosome 12p (Kobayashi et al., 1996; O’Connor HE, 1998; Raimondi et al., 1997) (Fig. 24.6). One hypothesis to explain the consistent loss of the other allele of TEL is that native TEL can interfere with the transforming properties of the TEL/AML1 fusion protein. Consistent with this hypothesis, TEL is capable of heterodimerizing with TEL/AML1 in vitro, as shown in Figure 24.7 (McLean et al., 1996). Although the biological consequence of TEL/AML1 heterodimerization with TEL is not understood, it is possible that this interaction abrogates the transforming potential of unopposed TEL/ AML1 homodimers. Such heterodimerization

Figure 24.7. Coimmunoprecipitation of TEL/AML1 and TEL. Both proteins were translated in vitro with 35S methionine. Pre indicates the proteins prior to immunoprecipitation. Post indicates the proteins recovered after immunoprecipitation with anti-AML1 antibody. TEL co-IPs with TEL/AML1 when translated together, but is not recovered by the AML1 antibody when translated alone. This experiment demonstrates that TEL/AML1 and TEL physically interact. Similar experiments demonstrated that TEL/AML1 forms homodimers in vitro.

TEL/AML1 GENE REARRANGEMENT IN PEDIATRIC ALL

would be eliminated by deletion of the other TEL allele. Since Tel null mice have embryonic lethal phenotypes, it will be necessary to generate Tel conditional mice that will allow for tissue-specific excision of Tel to determine whether TEL/AML1 expression in a Tel-deficient background can cause leukemia. Second, the deletion of the residual TEL allele is also accompanied by deletion of the neighboring p27Kip1 gene (CDNK2), (Cave et al., 1997; Filatov et al., 1996; Kim et al., 1996; Nishimura et al., 1999; Raynaud et al., 1996a, b; Stegmaier et al., 1995; Stegmaier et al., 1996; Wlodarska et al., 1996). It was recently demonstrated that mice hemizygous for p27Kip have increased susceptibility to cancer, a phenomenon referred to as haploinsufficient tumor suppression (Fero et al., 1998). It is thus plausible that loss of one allele of p27Kip1 contributes to pathogenesis of leukemia in TEL/ AML1-positive patients. Finally, a proportion of TEL/AML1-positive patients also have p16 deletions, suggesting that p16 deficiency may contribute to the TEL/AML1-mediated leukemogenesis (Anguita et al., 1997). Additional experimentation will be required to determine whether expression of TEL/AML1 in these genetic backgrounds can recapitulate the disease phenotype.

THE CLINICAL SIGNIFICANCE OF THE TEL/AML1 GENE REARRANGEMENT IN PEDIATRIC ALL To place the TEL/AML1 fusion in an appropriate clinical context, it is worthwhile to provide a historical perspective on the treatment of childhood ALL. Acute lymphoblastic leukemia (ALL) is the most common malignancy of child-

435

hood, with approximately 2,000 new cases diagnosed annually in the United States. Treatment advances over the past four decades have resulted in a tremendous improvement in rates of cure for this disease (Kersey, 1998; Pui, 1999; Rubnitz, 1998; Rubnitz et al., 1997a, 1997b). Prior to the development of modern chemotherapy, childhood ALL was uniformly and rapidly fatal. As shown in Figure 24.8, single-agent chemotherapy used in the early 1960s resulted in a measurable remission induction rate, but the patients invariably relapsed. With the development of multiagent chemotherapy programs and specific treatment of the central nervous system over the past 25 years, disease-free survival has dramatically improved (Kersey, 1998). Currently, approximately 75% of children with ALL can be expected to be cured of their disease (Kersey, 1998). Unfortunately, the remainder either fail to achieve remission or relapse, suggesting that current treatment for those individual patients is inadequate. With few exceptions, such as Philadelphia chromosome positivity, there are no molecular genetic markers that can identify patients at diagnosis who have a high likely of relapse and therefore may benefit from more intensive treatment such as allogeneic bone marrow transplantation. In addition, the fact that the majority of children with ALL can now be cured of their leukemia has resulted in new challenges in the field. Specifically, it is now becoming clear that long-term side effects of chemotherapy and cranial radiation are common in childhood ALL survivors. Studies done at the Dana-Farber Cancer Institute and elsewhere have demonstrated, for example, that cardiac dysfunction, endocrine abnormalities, neuropsychiatric problems, and the development of second malignant neoplasms are all potential consequences of intensive treatment of childhood leukemia.

Figure 24.8. Relapse-free survival of childhood ALL over the past four decades.

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Figure 24.9. Recurring chromosomal translocations in childhood ALL. The t(12;21) TEL/AML1 translocation accounts for 25—30% of all cases. The next most frequent translocation is the t(1;19), accounting for 5—6% of cases.

In contrast to the 1960s, therefore, a new series of challenges face the treatment of childhood ALL. New efforts are required to minimize the long-term side effects of children who can be cured of their leukemia, while increasing the likelihood of cure of those patients who are at a high risk of relapse. Successful preservation (if not improvement) of cure rates for childhood ALL accompanied by a diminution of long-term toxicities of treatment will require better risk group stratification and more novel, highly specific approaches to therapy based upon the molecular pathogenesis of the disease.

TEL/AML1 Is the Most Common Gene Rearrangement in Childhood ALL Although many questions remain about the molecular mechanisms of transformation mediated by TEL/AML1, clinically useful insights have emerged from analysis of the TEL/AML1 gene rearrangement in pediatric ALL patients (Borkhardt et al., 1999; Rubnitz et al., 1999). As noted above, the t(12;21) translocation is cryptic by standard cytogenetics. To determine the frequency of the t(12;21) in pediatric ALL, we performed a retrospective analysis of 98 childhood ALL patients treated on Dana-Farber Cancer Institute (DFCI) childhood ALL protocols between 1980 and 1991 (McLean et al., 1996). Patients were chosen based on the availability of frozen bone marrow samples, but were not selected with regard to cytogenetic analysis, age, risk category, or outcome. RNA of sufficient quality could be extracted from 81 of the 98 samples identified. RT-PCR demonstrated that a TEL/AML1 fusion was present in

22/81 (27%) patients. As shown below, 16 of the patients generated a PCR product of the expected size for the previously reported TEL/ AML1 fusion, but 6 of the patients generated a PCR product that was 39 base pairs smaller in size. Sequence analysis showed that this variant corresponded to a TEL/AML1 fusion that lacked AML1 exon 2. None of the patients in this study for whom bone marrow karyotypes were available had cytogenetic evidence of a t(12;21) translocation. It is likely that the t(12;21) is cryptic because of the similar banding patterns of the small portions of chromosomes 12 and 21 involved in the rearrangement. A similarly high frequency of TEL/AML1 fusion has also recently been noted in retrospective analyses performed by other investigators using RT-PCR, Southern blotting, and FISH (Amor et al., 1998; Borkhardt et al., 1997; EguchiIshimae et al., 1998; Fears et al., 1996; Liang et al., 1996; Romana et al., 1995b; Takahashi et al., 1998). Although TEL/AML1 frequency in pediatric ALL has been approximately 20—25% in several studies in both Caucasian and Asian populations, there is a suggestion that in some pediatric populations the incidence may be less than 10% (Garcia-Sanz et al., 1999; Inamdar et al., 1998). The TEL/AML1 gene rearrangement is rare in adult ALL and AML (Aguiar et al., 1996; Hoshino et al., 1997; Kwong, 1997; Loh et al., 1998; McLean et al., 1996; Raynaud et al., 1996a; Shih et al., 1996), in infant ALL (Silverman et al., 1997), in lymphoid blast crisis of CML (Hoshino et al., 1997), and in ALL associated with Down’s syndrome (Lanza et al., 1997). The TEL/AML1 gene rearrangement thus appears to be restricted to pediatric ALL,

TEL/AML1 GENE REARRANGEMENT IN PEDIATRIC ALL

437

Figure 24.10. Kaplan-Meier curves demonstrating difference in probability of being relapse-free. TEL/AML1positive patients were more likely to be free of relapse compared to TEL/AML1-negative patients (p : 0.004).

and indeed is the most common gene rearrangement of any childhood cancer (Figure 24.9). TEL/AML1 Is Associated with a Favorable Outcome. We performed a retrospective study of ALL patients treated on DFCI consortium protocols. Although the patients in this study spanned four consecutive DFCI protocols, there was no association between TEL/AML1 status and treatment protocol. Similarly, there was no significant association between TEL/AML1 status and peripheral white blood cell count, sex, or central nervous system disease. In contrast, the presence of the TEL/AML1 fusion was significantly associated with age (p : 0.00007) and immunophenotype (p : 0.02). All but one of the TEL/AML1-positive patients were 2 to 9 years of age. TEL/AML1-positive patients also had a consistent cell surface immunophenotype, consistent with B-lineage ALL (CD10>, CD19>, HLA Class II>, CD2\, CD33\). None of the 13 patients with T-cell disease was TEL/ AML1 positive. Of particular interest was the role of the TEL/AML1 fusion as a predictor of relapse. Five children did not achieve a complete remission following induction chemotherapy and were excluded from the analysis of relapse. All five patients lacked the TEL/AML1 fusion. Of the 76 patients who achieved a complete remission, 16 have relapsed. All relapses occurred among the 54 patients who lacked the TEL/ AML1 fusion (16/54, 30%); none of the 22

TEL/AML1-positive patients relapsed (0/22, 0%). The association between TEL/AML1 status and rate of relapse was highly significant (Fisher exact test, p : 0.004). The Kaplan-Meier curve for time to relapse in the 76 patients analyzed is shown below. Median follow-up from diagnosis of surviving patients was 8.3 years, with a range of 2.8—15.2 years (McLean et al., 1996) (Fig. 24.10). When the 13 T-cell patients were excluded from the analysis, the association between TEL/ AML1 positivity and freedom from relapse remained highly significant. In addition, even among the patients within the favorable age range of 2 to 9 years at diagnosis, there was a strong suggestion that TEL/AML1-positive patients were less likely to relapse compared to those who were TEL/AML1 negative (p : 0.07). A similarly favorable outcome has been observed in retrospective analyses of TEL/AML1positive patients treated at several different centers (Ayigad et al., 1999; Maloney et al., 1999; McLean et al., 1996; Rubnitz et al., 1997a, 1997b; Rubnitz et al., 1999; Rubnitz et al., 1999a, 1999b; Shurtleff et al., 1995; Takahashi et al., 1998; Zuna et al., 1999a, 1999b).

The Favorable Prognosis of TEL/AML1Positive Leukemia Is Controversial In contrast with the favorable prognosis of TEL/AML1 reported by our center and others, two studies from Europe have reported an incidence of TEL/AML1 at relapse that approxi-

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TABLE 24.2. Comparison of TEL/AML1 Rearrangement in Reports of Relapsed Patients

Immunophenotype B-cell only B-cell and t(9;22) negative

Study

TEL/AML Positive

TEL/AML1 Negative

Loh 98 Seeger 98 Loh 98 Seeger 98

1 9 1 32

30 26 27 101

mates the incidence of TEL/AML1 at diagnosis. These latter findings would suggest that TEL/ AML1 positivity does not confer a favorable prognosis. Harbott and colleagues reported on a cohort of patients initially treated on BFM-86, BFM-90, or Co-ALL-05-92. Nine of 36 (25%) patients with relapsed B-progenitor ALL were identified as having TEL/AML1-positive disease. Five of the TEL/AML1-positive patients were diagnosed at initial relapse and the other four were diagnosed at second relapse. Seeger and co-workers reported on a cohort of patients treated on a relapse protocol, and 32 of 133 (24%) patients with relapsed B-progenitor ALL were TEL/AML1-positive. Thus, the frequency of TEL/AML1 positive patients at relapse (25% and 24%, respectively) is similar to the incidence of TEL/AML1 positivity at diagnosis, suggesting that the rearrangement does not confer a favorable prognosis (Harbott et al., 1997; Seeger et al., 1998, 1999a, 1999b). In addition, some TEL/AML1-positive cases demonstrated late relapses in these studies, emphasizing the necessity of long-term follow-up studies to determine prognostic significance (Chow et al., 1999; Seeger et al., 1998). Since our initial study had not focused on relapsed patients, we determined the incidence of TEL/AML1 in the relapsed population initially treated on DFCI-ALL consortium trials conducted between 1981 and 1995. Samples were chosen based on the following criteria: patients had to have experienced a relapse of ALL, age 18 years at diagnosis, treatment on one of four consecutive DFCI protocols, and diagnostic bone marrow or peripheral blood availability in the DFCI cell bank. Samples were selected without prior knowledge of age, sex,

p-Value

0.01 0.02

race, immunophenotype, or cytogenetics. Additionally, paired samples from initial diagnosis and relapse diagnosis were analyzed if available. Thirty-two patients with a total of 50 cryopreserved bone marrow samples yielded RNA of sufficient quality for analysis of TEL/AML1 gene rearrangement (Loh et al., 1998a). In striking contrast to the data from the BFM studies, and consistent with our previous report, only 1/32 (3%) patients were TEL/AML1 positive at relapse. The difference in outcome between these reports is statistically significant (Table 24.2). There are several possible explanations for the different relapse rates of TEL/AML1-positive patients between the DFCI-ALL consortium and the BFM consortium. First, it is possible that there was retrospective sample bias in one of more of the studies. Second, since most of the patients from the BFM reports had only relapse samples tested, it is conceivable that the increased incidence of TEL/AML1 positivity could reflect the emergence of a therapy-related TEL/AML1 ; ALL. Although this possibility seems unlikely, BFM protocols incorporate the use of agents that have been reported to increase the risk of therapy-associated-AML such as epipodophyllotoxins. A third possibility is that the low incidence of TEL/AML1 in the DFCI group reflects differences in the efficacy of the initial therapy. Historically, the DFCI and BFM treatment programs have achieved similar outcome results in children with newly diagnosed ALL, but with different treatment strategies. It is plausible that TEL/AML1-positive patients represent a biologically distinct subset of patients whose leukemia is more effectively treated by the agents used more intensively by the DFCI group, such as asparaginase.

REFERENCES

It is critical to clarify this controversy. It has been suggested that if TEL/AML1 confers a favorable prognosis, it may be possible to consider decrements in therapy for this subgroup to decrease long-term morbidity. It is plausible that differences in therapy or in host immune response to the unique TEL/AML1 fusion peptide could account for a favorable outcome in this subgroup of pediatric ALL (Yotnda et al., 1998; Yun et al., 1999). However, if prospective analysis does not confirm a favorable prognosis, or if it is necessary to treat this subgroup with the same dose intensity to achieve a cure, then a decrease in therapy intensity would compromise disease-free survival in a cohort of patients with curable disease. These data also emphasize the importance of developing animal models for this disease to understand the differences in outcome reported by different centers. For example, an animal model would allow for testing of sensitivity of TEL/AML1-positive leukemic cells to specific chemotherapeutic agents and for exploring the possibility that host response plays a role in the development of TEL/AML1 leukemia.

Minimal Residual Disease Detection in TEL/AML1-Positive ALL Patients In addition to determining the prognostic significance of TEL/AML1 positivity at diagnosis, it will be critical to identify those TEL/AML1positive individuals who are destined to relapse. One approach has been to monitor minimal residual disease (MRD) using RT-PCR of the unique TEL/AML1 fusion (Nakao et al., 1996; Pallisgaard et al., 1999; Rubnitz et al., 1999a, b; Satake et al., 1997; Scurto et al., 1998; Viehmann et al., 1999). For example, Cayeula et al. assessed 8 TEL/AML1-positive patients and demonstrated that 4 of the 8 showed persistence of detectable MRD after induction therapy using RT-PCR. One patient continued to have detectable TEL/AML1 transcripts 242 days into therapy, but remained in continuous remission at the measured time points (Cayuela et al., 1996). Although follow-up was short in this study, it was suggested that the RT-PCR assay might identify those patients at high risk for relapse. In support of this hypothesis, another group studied 7 TEL/AML1-positive patients, and 1

439

patient was identified with persistence of detectable TEL/AML1 transcripts followed by subsequent relapse (Satake et al., 1997). Prospective analyses will be necessary to determine the value of quantitative MRD detection in TEL/AML1positive leukemias.

CONCLUSION Although many questions remain about the molecular mechanisms of transformation, clinically useful insights have been gained from analysis of hematologic malignancies that harbor TEL gene rearrangements. Analysis of the TEL-tyrosine kinase fusions has demonstrated that at a minimum the kinase is an appropriate and tenable target for therapeutic intervention, and that some downstream effectors of transformation such as Stat family members may also be potential targets for drug development. In addition, analysis of the prognostic significance of the TEL/AML1 fusion should determine whether there are true differences in outcome in this subgroup of patients treated on different protocols. If true differences are identified, attention can be focused on differences in therapy that might affect outcome in the subgroup of TEL/AML1-positive patients. In the interim, efforts to identify the molecular determinants required for transformation in animal models of TEL/AML1 leukemia may allow for design and testing of novel therapeutic approaches to this disease.

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further evidence for lineage specificity of TEL/ AML1. Leuk. Res. 22, 461—464. Lutterbach, B., Westendorf, J. J., Linggi, B., Patten, A., Moniwa, M., Davie, J. R., Huynh, K. D., Bardwell, V. J., Lavinsky, R. M., Rosenfeld, M. G., Glass, C., Seto, E., and Hiebert, S. W. (1998). ETO, a target of t(8;21) in acute leukemia, interacts with the N-CoR and mSin3 corepressors. Mol. Cell. Biol. 18, 7176—7184. Maloney, K., McGavran, L., Murphy, J., Odom, L., Stork, L., Wei, Q., and Hunger, S. (1999). TELAML1 fusion identifies a subset of children with standard risk acute lymphoblastic leukemia who have an excellent prognosis when treated with therapy that includes single delayed intensification. Leukemia 13, 1708—1712. McLean, T. W., Ringold, S., Stegmaier, K., Neuberg, D., Tantravahi, R., Ritz, J., Koeffler, H. P., Takeuchi, S., Janssen, J. W., Seriu, T., Bartram, C. R., Sallan, S. E., Gilliland, D. G., and Golub, T. R. (1996a). TEL-AML1 dimerizes and is associated with a favorable outcome in childhood acute lymphoblastic leukemia. Blood 88, 4252—4258. Meyers, S., Lenny, N., Sun, W., and Hiebert, S. W. (1996). AML-2 is a potential target for transcriptional regulation by the t(8;21) and t(12;21) fusion proteins in acute leukemia. Oncogene 13, 303—312. Nakao, M., Yokota, S., Horiike, S., Taniwaki, M., Kashima, K., Sonoda, Y., Koizumi, S., Takaue, Y., Matsushita, T., Fujimoto, T., and Misawa, S. (1996). Detection and quantification of TEL/ AML1 fusion transcripts by polymerase chain reaction in childhood acute lymphoblastic leukemia. Leukemia 10, 1463—1470. Nishimura, R., Niida, Y., Saikawa, Y., Goto, Y., Noguchi, T., Ichihara, T., and Koizumi, S. (1999). Microsatellite analysis of childhood leukemia: correlation of 9p and 12p chromosome abnormalities with expression of related genes. Pediatr. Int. 41, 346—352. O’Connor, H. E., Butler, T. A., Clark, R., Swanton, S., Harrison, C. J., Secker-Walker, L. M., and Foroni, L. (1998). Abnormalities of the ETV6 gene occur in the majority of patients with aberrations of the short arm of chromosome 12: a combined PCR and Southern blotting analysis. Leukemia 12, 1099—1106. Odero, M. D., Carlson, K., Calasanz, M. J., Chinwalla, V., and Rowley, J. D. (1999). Identification of eight new translocations involving TEL/ETV6 in hematological malignancies using FISH and spectral karyotyping. Blood 94, (suppl. 1), 495a. Okuda, K., Golub, T. R., Gilliland, D. G., and Griffin, J. D. (1996). p210BCR/ABL, p190BCR/ABL and TEL/ABL activate similar signal transduction pathways in hematopoeitic cell lines. Oncogene 13, 1147—1152.

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resulting from a cryptic t(12;21) in adult B lineage acute lymphoblastic leukemia in Taiwan. Leukemia 10, 1456—1458. Shurtleff, S., Buijs, A., Behm, F., Rubnitz, J., Raimondi, S., Hancock, M., Chan, G., Pui, C., Grosveld, G., and Downing, J. (1995). TEL-AML1 fusion resulting from a cryptic t(12;21) is the most common genetic lesion in pediatric ALL and defines a subgroup of patinets with an excellent prognosis. Leukemia 9, 1985—1989. Silverman, L. B., McLean, T. W., Gelber, R. D., Donnelly, M. J., Gilliland, D. G., Tarbell, N. J., and Sallan, S. E. (1997). Intensified therapy for infants with acute lymphoblastic leukemia: results from the Dana-Farber Cancer Institute Consortium. Cancer 80, 2285—2295. Slupsky, C. M., Gentile, L. N., Donaldson, L. W., Mackereth, C. D., Seidel, J. J., Graves, B. J., and McIntosh, L. P. (1998). Structure of the Ets-1 pointed domain and mitogen-activated protein kinase phosphorylation site. Proc. Natl. Acad. Sci. USA 95, 12,129—12,134. Song, H., Kim, J. H., Rho, J. K., Park, S. Y., Kim, C. G., and Choe, S. Y. (1999). Functional characterization of TEL/AML1 fusion protein in the regulation of human CR1 gene promoter. Mol. Cells 9, 560—563. Speck, N. A., Stacy, T., Wang, Q., North, T., Gu, T. L., Miller, J., Binder, M., and Marin-Padilla, M. (1999). Core-binding factor: a central player in hematopoiesis and leukemia. Cancer Res. 59 (suppl. 7), 1789s—1793s. Stegmaier, K., Pendse, S., Barker, G. F., Bray-Ward, P., Ward, D. C., Montgomery, K. T., Krauter, K. S., Reynolds, C., Sklar, J., Donnelly, M., Bohlander, S. K., Rowley, J. D., Sallan, S. E., Gilliland, D. G., and Golub, T. R. (1995). Frequent loss of heterozygosity at the TEL gene locus in acute lymphoblastic leukemia of childhood. Blood 86, 38—44. Stegmaier, K., Takeuchi, S., Golub, T. R., Bohlander, S. K., Bartram, C. R., Koeffler, H. P., and Gilliland, D. G. (1996). Mutational analysis of the candidate tumor suppressor genes TEL and KIP1 in childhood acute lymphoblastic leukemia. Cancer Res. 56, 1413—1417. Sternberg, D. W., Tomasson, M. H., Carroll, M., Kazlauskas, A., and Gilliland, D. G. (1999). Phosphorylation sites in the TEL/PDGFR fusion protein that are required for STAT5 activation are essential for a lethal myeloproliferative dsease in a murine bone marrow transplant model. Blood 94 (suppl. 1), 389a. Takahashi, Y., Horibe, K., Kiyoi, H., Miyashita, Y., Fukuda, M., Mori, H., Nozaki, C., Hasegawa, S.,

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Kawabe, T., Kato, K., Kojima, S., Matuyama, T., and Naoe, T. (1998). Prognostic significance of TEL/AML1 fusion transcript in childhood B-precursor acute lymphoblastic leukemia. J. Pediatr. Hematol. Oncol. 20, 190—195. Thandla, S. P., Ploski, J. E., Raza-Egilmez, S. Z., Chhalliyil, P. P., Block, A. W., de Jong, P. J., Aplan, P. D. (1999). ETV6-AML1 translocation breakpoints cluster near a purine/pyrimidine repeat region in the ETV6 gene. Blood 93, 293—299. Tomasson, M. H., Williams, I. R., Hasserjian, R., Udomsakdi, C., McGrath, S. M., Schwaller, J., Druker, B., and Gilliland, D. G. (1999). TEL/ PDGFR induces hematologic malignancy in mice that responds to the tyrosine kinase inhibitor CGP57148. Blood 93, 1707—1714. Tomasson, M. H., Sternberg, D. W., Williams, I. R., Carroll, M., Dain, D., Aster, J. C., Ilaria, R. L., Van Etten, R. A., and Gilliland, D. G. (2000). Fatal myeloproliferation, induced in mice by TEL/ PDGFR expression, depends on PDGFR tyrosines 579/581. J. Clin. Invest. 105, 423—432. Uchida, H., Downing, J. R., Miyazaki, Y., Frank, R., Zhang, J., and Nimer, S. D. (1999). Three distinct domains in TEL-AML1 are required for transcriptional repression of the IL-3 promoter. Oncogene 18, 1015—1022. Viehmann, S., Borkhardt, A., Lampert, F., and Harbott, J. (1999). Multiplex PCR — a rapid screening method for detection of gene rearrangements in childhood acute lymphoblastic leukemia. Ann. Hematol. 78, 157—162. Wang, L. C., Kuo, F., Fujiwara, Y., Gilliland, D. G., Golub, T. R., and Orkin, S. H. (1997). Yolk sac angiogenic defect and intra-embryonic apoptosis in mice lacking the Ets-related factor TEL. EMBO J. 16, 4374—4383. Wang, L. C., Swat, W., Fujiwara, Y., Davidson, L., Visvader, J., Kuo, F., Alt, F. W., Gilliland, D. G., Golub, T. R., and Orkin, S. H. (1998). The TEL/ ETV6 gene is required specifically for hematopoiesis in the bone marrow. Genes Dev. 12, 2392— 2402. Wang, Q., Stacy, T., Binder, M., Marin-Padilla, M., Sharpe, A. H., and Speck, N. A. (1996a). Disruption of the Cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis. Proc. Natl. Acad. Sci. USA 93, 3444—3449. Wang, Q., Stacy, T., Miller, J. D., Lewis, A. F., Gu, T. L., Huang, X., Bushweller, J. H., Bories, J. C., Alt, F. W., Ryan, G., Liu, P. P., Wynshaw-Boris, A., Binder, M., Marin-Padilla, M., Sharpe, A. H., and Speck, N. A. (1996b). The CBF beta subunit is essential for CBF alpha2 (AML1) function in vivo. Cell 87, 697—708.

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CHAPTER 25


MLL IN NORMAL AND MALIGNANT HEMATOPOIESIS PAUL M. AYTON AND MICHAEL L. CLEARY Department of Pathology, Stanford University School of Medicine

INTRODUCTION Alterations of the mixed lineage leukemia (MLL) gene (also termed ALL-1 or HRX), located at chromosome band 11q23, define a distinct group of acute leukemias with unique clinical and biological features. Although mutations of MLL account for only about 5% of acute leukemias, they are particularly prevalent in leukemias of infants under 1 year of age, accounting for at least 70% of cases (Mitelman, 1994). Chromosomal translocations involving MLL are also prevalent in secondary leukemias associated with prior use of drugs that inhibit topoisomerase II (Bower et al., 1994; Hunger et al., 1993). MLL mutations occur in leukemias of either lymphoid or myeloid lineages, and the transformed lineage generally correlates with the specific partner gene that participates in the reciprocal translocation. Regardless of age at presentation, these leukemias are associated with an extremely poor prognostic outcome, even after intensive combination chemotherapy (Chen et al., 1993).

For recent guides to the clinical aspects of MLL leukemogenesis, the reader is referred to the detailed series of reports from the European Workshop on 11q23 (Secker-Walker, 1998). The cloning of the MLL gene and its cytogenetics have been previously reviewed and therefore are not covered here (Bernard and Berger, 1995; Canaani, et al. 1995; Waring and Cleary, 1997). This review focuses exclusively on aspects of normal MLL function during development, its disruption by various types of leukemogenic mutations, and how these data can be utilized to formulate models of MLL-mediated leukemogenesis. DOMAIN STRUCTURE AND PUTATIVE FUNCTIONS OF MLL MLL homologues have now been sequenced, wholly or partially, from human, mouse, chick, pufferfish and Drosophila. Taken together, the sequences predict a consensus domain structure that has been conserved throughout evolution (Caldas et al., 1998; Gu et al., 1992; Ma et al., 1993; Mazo et al., 1990; Schofield et al., 1999;


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Figure 25.1. Wild-type and mutant MLL proteins. Schematic representations are shown for the domain structures of the wild-type MLL protein (above) and three classes of oncogenic mutants (below). Various homology domains are shown as colored boxes and labeled as follows: AT1-3, AT hook DNA-binding motifs 1—3; SNL 1 and 2, speckled nuclear localization signals 1 and 2; CxxC, cysteine-rich motif homologous to DNA methyltransferase and MBD1; PHD1-3, PHD fingers 1—3; TA, transactivation domain; SET, SET domain. Domains conserved with Drosophila trx are indicated by the (trx) underlabel.

Tillib et al., 1995; Tkachuk et al., 1992). The predicted MLL protein is very large and basic, consisting of 3969 amino acids that predict a molecular weight of 431 kD (Tkachuk et al., 1992). The domains of MLL that are shared with other proteins of known function are summarized below and their potential relevance to MLL function is highlighted.

STRUCTURAL SIMILARITIES BETWEEN MLL AND DROSOPHILA trx MLL shares significant, albeit limited, homology with Drosophila trithorax (trx), the founding member of a genetically distinctive group of genes that functions to maintain the expression of the homeotic selector genes (HOM-C) (Ingham, 1985). Early during Drosophila embryogenesis, homeotic gene expression is transiently initiated by the gap and pair rule genes. Members of the Trithorax (TrxG) and

Polycomb (PcG) gene families subsequently impose a cellular memory system that maintains the stable, heritable positive or negative regulation of homeotic gene expression throughout embryonic and adult life (Simon, 1995). Recent data have suggested that the developmental functions of TrxG and PcG proteins have been conserved in mammals (reviewed Gould, 1997; Schumacher and Magnusan, 1997). While TrxG and PcG proteins are thought to influence target gene expression via epigenetic regulation of chromatin accessibility within critical regulatory regions, the molecular mechanisms that mediate such activity are unknown. Three regions of significant homology shared by trx and MLL include subnuclear localization signals, central PHD finger domains, and the carboxy-terminal SET domain (Fig. 25.1). Genetic evidence from Drosophila indicates that the PHD and SET domains are absolutely required for trx function. Hypomorphic alleles of trx that arrest fly development at the pupal stage, such

DOMAINS NOT CONSERVED WITH trx

as trx and trx, harbor point mutations in the PHD and SET domains, respectively (Stassen et al., 1995). The highest degree of identity between MLL and trx lies within the extreme carboxy terminal 200 residues that encode the SET domain (for review, see Jenuwein et al., 1998). This highly conserved motif has been identified in members of both the trxG and PcG families of epigenetic transcriptional regulators and may participate in molecular interactions with other proteins. In support of this proposal, interactions between the SET domains of MLL/trx and SNF-5, as well as trx and the SET domain of ASH1 (another TrxG protein), have recently been reported. These observations suggest that the SET domain mediates formation of a higher-order MLL complex that contains at least two components previously defined functionally as members of the TrxG family (Rozenblatt-Rosen et al., 1999; Rozovskaia et al., 1999). It is not yet clear whether such interactions are mutually exclusive or components of a single complex that regulates chromatin accessibility. The central region of MLL contains three tandem copies of a modified zinc-finger structure known as C HC PHD domains. These are followed by a second region that forms an imperfect PHD domain (Aasland et al., 1995). The PHD domain has been identified in a broad range of proteins from arthropods, plants, and mammals, although it has yet to be assigned a functional role. The genetic properties and nuclear localization for many of these proteins raises the possibility that the PHD domain may participate in chromatin-mediated transcriptional regulation (Aasland et al., 1995). Of note, the region of the MLL gene encoding its three PHD fingers coincides with the translocation breakpoint cluster region (BCR) that is consistently targeted for mutation in leukemias. Short, conserved sequences in the amino terminus of MLL direct its localization to discrete subnuclear domains (Yano et al., 1997). Two regions, termed SNL1 and SNL2, mediate speckled subnuclear localization. Both regions are highly conserved with Drosophila trx. This suggests that the ability of MLL/trx to localize to discrete subnuclear sites has been conserved through evolution and may play a critical role in regulating MLL/trx function. Furthermore, these results suggest that at least some trx

449

downstream target genes/signaling pathways may also be conserved in mammals.

DOMAINS NOT CONSERVED WITH trx The amino terminus of MLL contains three short AT hook motifs thought to mediate specific binding to the minor groove of AT-rich DNA (Reeves and Nissen, 1990; Tkachuk et al., 1992). The AT hook motif consists of three components as recently determined by the solution structure of HMGI/Y in complex with an in vivo binding site in the interferon -enhancer (Huth et al., 1997). Interactions between the AT hook and the minor DNA groove are mediated by a central RGR motif. Flanking this central core, the amino terminal KR dipeptide and a carboxy terminal array of nine residues mediate extensive hydrophobic and polar contacts. The three AT hooks present in the amino terminus of MLL lack the carboxy-terminal residues required for extensive phosphodiester backbone contacts and are therefore predicted to exhibit reduced affinity for the minor groove relative to the motifs present in HMGI/Y (Huth et al., 1997). The AT hook motifs may therefore allow MLL to indirectly stabilize protein-DNA interactions by inducing conformational changes, such as bends in DNA of the target-binding site, which may in turn facilitate the binding of specific trans-acting factors that regulate target gene transcription. Alternatively, upon binding to the minor groove, MLL may mediate protein-protein interactions that allow distinct factors to efficiently interact with each other and the basal transcription machinery. The AT hook motifs of HMG-I/Y are phosphorylated both in vitro by cdc2 kinase and on the same residue during M phase in vivo (Nissen et al., 1991; Reeves et al., 1991). This induces a substantial reduction in DNA-binding activity. A consensus cdc2 kinase site is also present in the most amino-terminal AT hook of MLL, suggesting that it also may be modulated by cdc2 kinase during the cell cycle. MLL contains a second cysteine-rich region, termed the CxxC motif, which exhibits sequence homology with two proteins implicated in the epigenetic regulation of transcription via methylation. One of these is mammalian DNA methytransferase (DMT) and the other is methyl-bind-

450


ing domain protein 1 (MBD1) (Cross et al., 1997; Domer et al., 1993; Hendrich et al., 1998). The CxxC motif is unlikely to endow MLL with the ability to recognize and bind methylated target promoters, since proteins such as MBD1 utilize an alternative motif, the conserved methyl-binding domain, for this purpose. Rather, the CxxC motif may constitute a protein-protein interaction interface that contributes to the transcriptional effector properties of these proteins. In support of this possibility, transcriptional repression activity has been localized to the CxxC motif of MLL (Prasad et al., 1995; Zeleznik-Le et al., 1994). MLL also contains a potent transcriptional activation domain in its carboxy terminal portion, between its PHD and SET domains. This is lost and replaced by partner protein sequences following chromosomal translocations in leukemias (Prasad et al., 1995; Zeleznik-Le et al., 1994). The minimal transactivation domain utilizes central hydrophobic and acidic residues for its activity (Prasad et al., 1995).

THE ALL-1 RELATED (ALR) PROTEIN A recent search for human genes that encode proteins related to MLL has identified a close structural homologue named ALL-1 related (ALR) (Prasad et al., 1997). The predicted ALR protein is larger than MLL, containing 5262 residues with a predicted mass of 562 kD. ALR contains two clusters of PHD fingers, containing two and three fingers respectively, that are highly conserved with both MLL and trx. A third cysteine-rich cluster of 105 residues, located at the carboxy terminus of ALR, is highly similar to the central imperfect PHD domain present in MLL. The extensive similarity of putative functional domains between MLL, ALR, and trx suggests that ALR may represent another member of the trxG family of proteins. Notably, ALR does not contain AT hook motifs implicated in binding to the minor groove of DNA. Furthermore, a putative null allele of the TRR gene, the recently identified fly homologue of ALR, did not result in homeotic transformations, suggesting that, in contrast to MLL and trx, TRR and ALR may not regulate the expression of HOX genes (Sedkov et al., 1999).

ALTERNATIVELY SPLICED FORMS OF MLL Several variant forms of the MLL transcript appear to result from alternative splicing. These do not appear to be associated with tissuespecific patterns of expression. Seven regions of the MLL transcript are modified by alternative splicing (Domer et al., 1993; Ma et al., 1993; Nam et al., 1996) and the various alternatively spliced forms of MLL expressed within the same cell have the combinatorial potential to encode 128 variant MLL proteins. The amino acid sequences encoded by alternatively spliced MLL exons and the possible effect of their inclusion/ loss on the function of the MLL protein are depicted in Figure 25.2.

ROLE OF MLL DURING MAMMALIAN DEVELOPMENT The contributions of MLL during mouse development have been investigated by two groups using gene-targeting techniques, although neither generated a complete MLL null allele. In the first case, a large amino-terminal portion of MLL, including its DNA-binding AT hook motifs, was fused in frame to -galactosidase (Yu et al., 1995). Mice carrying a single copy of the MLL -galactosidase fusion allele displayed defects in growth and fertility as well as homeotic transformations of the vertebrae along the length of the axial skeleton. Embryos homozygous for this fusion allele died around E10.5 with multiple patterning defects to neural crest— derived structures of the branchial arches, cranial nerves, and ganglia, but also loss of segmental boundaries for both somites and spinal ganglia along the trunk of the embryo (Yu et al., 1995, 1998). Although expression of Hox a7 and c8 was properly initiated in homozygous embryos at E8.5, it was not maintained at later stages of development (Yu et al., 1998). These results suggest that MLL functions as a mammalian counterpart of Drosophila trx, which positively maintains the expression of multiple Hox genes during development. Interestingly, this particular line of MLL mutant mice displayed phenotypic defects in anterior regions of the embryo that are not patterned by class I Hox genes, implying that MLL may regulate

ROLE OF MLL DURING MAMMALIAN DEVELOPMENT

451

Figure 25.2. Hypothetical variant MLL proteins generated by alternative splicing. A schematic representation of the wild-type MLL protein is shown at the top. The effects of alternative RNA splicing on predicted MLL proteins are shown below. Solid lines indicate preserved MLL sequences. Amino acid insertions are denoted by triangles, deletions by brackets, and open-reading frame truncations by Xs. Possible functional consequences are indicated to the right.

other developmentally important genes in addition to Hox. Further studies of these MLL mutant mice revealed hematopoietic abnormalities (Hess et al., 1997). Definitive hematopoietic colony assays of MLL nullizygous yolk sac progenitors at E10.5 generated CFU-M and CFU-GEMM colonies that were consistently smaller, were fewer in number, and exhibited a slower growth rate than those from wild-type littermates. Mice homozygous for another mutant MLL allele, truncated further 3 at the BCR (Yagi et al., 1998), also displayed embryonic lethality, but at a slightly later stage of development between E12.5 and E13.5. Homozygous mutant embryos from this line exhibited edema and bleeding, somewhat reminiscent of the block in definitive hematopoiesis associated with AML-1 deficiency (Okuda et al., 1996). Examination of fetal liver and yolk sac hematopoietic progenitors from embryos homozygous for this MLL mutation revealed reduced numbers and size of myeloid colonies after 7 days of culture. However, with further in vitro culture, colony numbers increased to almost normal, although their size remained reduced, suggesting delayed kinetics of colony formation. Furthermore, the ex-

pression of a number of Hox genes (Hox-a7, -a9, -a10, and -c4) was greatly reduced in the MLL mutant fetal liver (Yagi et al., 1998). Despite the reduction in size and number of myeloid colonies in progenitor assays of hematopoietic cells from embryos homozygous for either MLL mutant allele, all mature cell types were observed. This suggests that MLL is not absolutely required for terminal myeloid differentiation, but influences the proliferation and/or survival of multipotent progenitors. No defects were observed in erythroid colony formation, consistent with the lack of MLL expression in the erythroid compartment (Hess et al., 1997). The available data suggest that the contributions of MLL to normal hematopoietic development are most similar to those of GATA-2, a previously characterized regulator of multipotential progenitor cell development (Tsai et al., 1994). It is notable that GATA-2 is expressed in MLL homozygous mutant embryos, suggesting that MLL lies either downstream or on a parallel pathway to GATA-2 (Hess et al., 1997). The early embryonic lethality of homozygous MLL mutants has thus far prevented analysis of the role of MLL during lymphoid development.

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MLL MUTATIONS ASSOCIATED WITH ACUTE LEUKEMIAS MLL Fusion Proteins Generated from Chromosomal Translocations MLL fusion proteins constitute the most prevalent class of MLL mutations associated with acute leukemia. Recent studies have led to the molecular cloning of 20 of the estimated 30 partner proteins that participate with MLL in reciprocal chromosomal translocations in leukemias. The studies were initiated to identify a common theme regarding the structure and function shared between the MLL partner proteins and thereby provide mechanistic insights into the ability of MLL fusion proteins to transform hematopoietic progenitors. These results have not yet yielded a consistent explanation for transformation by MLL fusion proteins. Some general comments, however, can be made regarding the MLL partner proteins identified so far. Most importantly, all MLL fusions with various partner proteins maintain a productive open-reading frame, suggesting that this feature is strongly selected for and therefore essential for the transformation properties of the fusion protein. In one instance, where MLL lies in opposite transcriptional orientation to the AF10 partner gene, complex inversion rather than translocation events were required to maintain the open-reading frame encoded by the resulting chimeric transcript (Beverloo et al., 1995). Cytogenetic analysis of complex threeway MLL translocations identified the absolute conservation of the derivative chromosome 11 product encoding the amino-terminal 1400 residues of MLL fused to the variable portions of the partner gene (Rowley, 1992). The derivative chromosome 11 product encodes amino-terminal sequences of MLL including AT hook, SNL 1 and 2 motifs, and the CxxC domain. Partner protein sequences consistently replace the PHD, transactivation and SET domains of MLL. The typical structure of the MLL fusion protein is depicted in Figure 25.1. The genes for all partner proteins are widely expressed in a variety of adult tissues and cells, including the hematopoietic cell types associated with MLL mutations in leukemias. This suggests that the partner proteins may normally perform general rather than cell-type specific

functions. Currently, five pairs of MLL partner proteins (ENL/AF9, CBP/p300, AF10/AF17, AF-X/AF6q21, hCDCrel-1/MSF) exhibit considerable homology within functional domains and can, therefore, be regarded as members of novel protein families. It is anticipated that future cloning of the remaining unidentified partner genes may reveal further homologies among the MLL fusion partners. At this point, MLL partner proteins fall into two broad categories: signaling molecules that normally localize to the cytoplasm/cell junctions or nuclear factors implicated in various aspects of transcriptional regulation. The current list of MLL partner proteins, including their putative functional domains, close structural homologues, and potential regions necessary for transforming activity, are summarized in Tables 25.1 and 25.2.

MLL Partial Amino-Terminal Duplication Mutants Associated with AML A second type of MLL mutation has been identified as a recurrent event in AML. These are characterized by partial internal tandem duplications (Caligiuiri et al., 1996; Schichman et al., 1994) and are typically associated with myeloid leukemias containing trisomy 11 or lacking cytogenetic evidence of 11q23 translocation (Caligiuiri et al., 1994). Interestingly, these cases display a more immature AML M1/M2 phenotype instead of the characteristic AML M4/M5a subtypes associated with MLL translocations. The internal tandem duplications maintain the MLL open-reading frame, suggesting that expression of the internally duplicated allele is necessary for malignant transformation. Most tandem duplications encompass sequences encoding the AT hook, SNL 1 and 2, and CxxC domains (Caligiuri et al., 1996) and are schematically represented in Figure 25.1. However, one AML-M1 patient was reported to contain a shorter duplication confined to sequences encoding the SNL-2 and CxxC domains of MLL, possibly defining the minimal region of duplication necessary for myeloid transformation. Allelotype analysis revealed that internal tandem MLL duplications invariably affect only one allele of the MLL gene in myeloid leukemias (Caligiuri et al., 1997). These results

453

t(4;11)(q21;q23)

t(9;11)(p22;q23) t(11;19)(q23;p13.3)

t(10;11)(p12;q23) t(11;17)(q23;q21)

t(11;16)(q23;p13.3) t(11;22)(q23;q13)

t(11;19)(q23;p13.1)

t(11;X)(q23;q13) t(6;11)(q21;q23)

AF9 ENL

AF10 AF17

CBP P300

ELL

AFX AF6q21

Translocation

AF4

Partner Protein

T-ALL AML M4/M5a

AML M4/M5a

AML M4/M5a AML M4/M5a

AML M4/M5a AML M4/M5a

AML M4/M5a AML M4/M5a Pro B ALL.

Pro B ALL

Leukemia Classification

TABLE 25.1. The Nuclear MLL Partner Proteins

Both contain a forkhead DNA-binding domain and are transcriptional activators in vitro. Genetics suggests proapoptotic functions in vivo

Both contain PHD, AT hook, and leucine zipper motifs, suggesting chromatin association. Transcriptional coactivators with histone acetyltransferase (HAT) activity. Transcriptional elongation factor inhibits transcription initiation.

Transcriptional adaptor in vitro (ENL).

Transcriptional activator in vitro.

Putative Function of Wild-Type Partner Protein Residues 391—1211. Includes transactivation domain. Residues 478—568 of AF9 and 371—559 of ENL. Carboxy terminal transactivation domain predicted to form helical region. Residues 681—1027 of AF10 and 52—1093 of AF17. Includes leucine zipper region. Residues 1022—2442 of CBP and 940—2414 of p300. Includes HAT, E1A-binding and Q-rich regions. Residues 46—621. Includes transcriptional elongation activity and conserved carboxy terminus. Residues 148—501 of AFX and 208—673 of AF6q21. Carboxy terminal transactivation domain.

Minimal Region Required for MLL-Mediated Transformation

FKHR DAF-16

ELL2

None

CEZF

None

LAF4 FMR2

Structural Homologues

Borkhardt et al., 1997; Hillion et al., 1997

Thirman et al., 1994

Sobulo et al., 1997; Taki et al., 1997; Ida et al., 1997

Chaplin et al., 1995a, 1995b Prasad et al., 1994

Tkachuk et al., 1992; Nakamura et al., 1993; Yamamoto et al., 1993

Morrisey et al., 1993; Nakamura et al., 1993

References

454

t(6;11)(q27;q23)

t(1;11)(p32;q23)

t(11;19)(q23;p13)

t(11;22)(q23;q11.2) t(11;17)(q23;q25)

t(1;11)(q21;q23)

t(10;11)(p11.2;q23)

Fip/EPS15

EEN

hCDCrell MSF

AF1q

ABI-1

Translocation

AF6

Partner Protein

AML M4

AML M4/M5a

AML M1/2 AML M4/M5a

AML

AML M4/M5a

AML M4/M5a

Leukemia Classification

TABLE 25.2. The Cytoplasmic MLL Partner Proteins

SH3 domain. Negative regulator of v-abl transformation.

Unknown.

GTP-binding domain.

EH domain, coiled-coil and DPF motifs. Tyrosine phosphorylated target of EGF signaling. SH3 domain.

Ras and PDZ interaction domains.

Wild-Type Partner Protein Function

Residues 16—368. Includes SH3 domain. Residues 59—1089 of hDCrel and 8—568 of MSF. Includes the GTP-binding domain. Residues 1—90. Includes complete open-reading frame. Residues 95—447. Includes SH3 domain.

Residues 12 —896. All 3 domains retained.

Residues 36—1611. Includes Ras and PDZ interaction domains.

Minimal Region Required for MLL-Mediated Transformation

ABI-2

EEN-B1, EEN-B2 SH3p4, SH3p13 Carboxy terminus similar to septin family members such as CDC10. None

None

Ce AF6

Canoe

Structural Homologues

Taki et al., 1998

Tse et al., 1995

Megonigal et al., 1998; Osaka et al., 1999; Taki et al., 1999

So et al., 1997

Bernard et al., 1994

Prasad et al., 1993

References

MECHANISMS BY WHICH MLL FUSION PROTEINS TRANSFORM HEMATOPOIETIC PROGENITORS

strongly suggest that internal tandem duplications constitute dominant as opposed to recessive mutations of MLL. It is not yet clear whether these mutant MLL proteins function through positive gain-of-function or dominant negative mechanisms. However, a dominant negative mechanism seems unlikely, since it would require antagonism of MLL protein expressed from two wild-type alleles in leukemic cells with trisomy of chromosome 11. MLL PHD Finger 1 Deletion Mutants Associated with T-ALL A third type of infrequent structural alteration of the MLL gene has recently been reported. An interstitial deletion within the BCR of the MLL gene was identified in three cases of childhood T-ALL, resulting in the removal of exon 8 sequences from one allele of the MLL locus (Figure 25.1) (Lochner et al., 1996). MLL exon 8 sequences encode 33 residues including the first two conserved cysteine residues of PHD finger 1. Their deletion maintains the MLL open-reading frame, a common feature that is strongly selected for in the production of all three classes of leukemic MLL mutants. These same sequences derived from exon 8 are excised from the MLL precursor RNA by alternative splicing that gives rise to a minor transcript in a variety of cell lines (Figure 25.2) (Nam et al., 1996). Thus, it is possible that removal of exon 8 sequences, either by splicing or deletion events, may create different isotypes of MLL that differ in their functional properties mediated through the first PHD finger.

MECHANISMS BY WHICH MLL FUSION PROTEINS TRANSFORM HEMATOPOIETIC PROGENITORS MLL proteins arising from the three types of oncogenic mutations described above retain their amino-terminal AT hook motifs, indicating that they all share an ability to recognize putative MLL response elements within the regulatory regions of target genes. Therefore, a unifying mechanism by which these MLL mutants transform hematopoietic progenitors is likely to involve an MLL target gene—dependent pathway. However, due to the structural

455

diversity of the numerous MLL partner proteins, as well as leukemia-associated mutations that do not involve fusion to a heterologous partner protein, it has been controversial as to whether such an MLL-dependent mechanism involves loss or gain of MLL function. Two different approaches have clearly established that MLL fusion genes act as dominant alleles. First, chimeric mice generated from ES cells carrying a single MLL-AF9 knock-in allele succumbed to AML, whereas chimeric mice generated from ES cells heterozygous for an MLL truncation allele remained disease-free (Corral et al., 1996). Second, in vitro transformation of myeloid progenitors was observed following retroviral transduction of MLL-ENL, but not with mutant constructs containing amino-terminal MLL or carboxy terminal ENL protein fragments (Lavau et al., 1997). Subsequent structure/function analysis of MLL-ENL revealed a critical requirement for both the AT hook and CxxC amino-terminal motifs of MLL for in vitro myeloid transformation. A predicted helical region at the extreme carboxy terminus of ENL that mediates strong transcriptional transactivation potential was also necessary (Slany et al., 1998). Thus, the ENL moiety donates transcriptional effector properties to the MLL-ENL fusion protein and not simply enhanced protein stability. These results suggest that the mechanism by which MLL-ENL mediates transformation involves a dominant gain of MLL function, whereby MLL-ENL acts as a chimeric transactivator that constitutively activates MLL target genes. It remains to be determined how generalized this mechanism may be for the diverse MLL fusion proteins described to date. Final confirmation that MLL fusion proteins act via a dominant gain-of-function mechanism has been recently reported (Dobson et al., 1999). The MLL-AF9 knock-in allele has been transmitted through the germ-line to generate a mouse strain genetically predisposed to AML. Since MLL function is required for midgestation embryogenesis, the generation of germ-line MLL-AF9 mutant mice confirms that expression of the fusion protein does not compromise wild-type MLL function in vivo. If MLL fusion proteins acted as dominant negative inhibitors of MLL function, their activity should have blocked embryonic development at a stage when normal MLL function is required. Thus,

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the mechanism of transformation by MLL fusion proteins is quite distinct from the trans dominant activity of leukemogenic AML1/CBF fusion proteins that phenocopy AML1/CBF loss-offunction in vivo (Castilla et al., 1996; Okuda et al., 1996; Wang et al., 1996; Yergeau et al., 1997). Interestingly, MLL-AF9 mutant mice also exhibit rapid expansion of myeloid progenitors prior to disease onset, although it has not yet been reported whether such an expansion results from defective growth control, impaired differentiation, or enhanced survival. MLL loss of function, either by dominant negative inhibition, biallelic recessive mutations, or haploinsufficiency, has been proposed as a possible contributing mechanism for hematopoietic cell transformation. While MLL fusion proteins are unable to compromise MLL function during embryogenesis (Dobson et al., 1999), they could act as tissue-specific dominant negative inhibitors of MLL function during adult bone marrow—derived hematopoiesis. Interestingly, recent data have described a critical requirement for the leukemic transcription factor TEL during adult bone marrow derived definitive hematopoiesis but not yolk sac or fetal liver hematopoiesis (Wang et al., 1998). Mutation of both copies of MLL by translocation on one allele and complete deletion of the second allele has been reported in the ML-1 leukemic cell line, although such events have not been consistently observed (Strout et al., 1996). Loss of heterozygosity in a subset of leukemias that lack MLL translocations raises the possibility of a tumor suppressor role for MLL independent of its contributions to MLL fusion proteins (Webb et al., 1999). Genetic analyses demonstrate that mice that are chimeric or heterozygous for mutant MLL genes truncated at the BCR are not predisposed to development of acute leukemia over an 18month observation period (Ayton, unpublished observations; Corral et al., 1996). Moreover, MLL fusion proteins transform primary myeloid progenitors in the presence of two copies of the wild-type MLL gene (Lavau et al., 1997). It therefore seems unlikely that MLL haploinsufficiency plays a critical role in MLL-mediated leukemogenesis, although a subsidiary role cannot be excluded completely given the effects of haploinsufficiency on Hox expression and embryonic patterning.

The carboxy-terminal SET domain of MLL has been shown to interact with SNF 5 (Rozenblatt-Rosen et al., 1998), a component of the mammalian SWI/SNF chromatin remodeling complex. SNF5 is also targeted by mutations in a number of pediatric malignant rhabdoid tumors (Versteege et al., 1998) and is thus likely to have tumor suppressor properties. The association of a tumor suppressor protein with a region of MLL that is deleted from leukemic MLL fusion proteins raises the possibility that SNF5 function could be compromised in MLL leukemias. However, SNF5 is present in at least two distinct complexes with either MLL or SWI/SNF (Croce, 1999) and it is currently not clear whether MLL regulates the growth inhibitory properties of SNF5 in the hematopoietic lineages.

IS DISRUPTION OF PARTNER PROTEIN FUNCTION A CONTRIBUTORY FACTOR IN MLL FUSION PROTEIN-MEDIATED LEUKEMOGENESIS? The essential role of the partner protein in mediating the transforming activity of MLL fusion proteins via a dominant gain of MLL function is now well documented (Corral et al., 1996; Dobson et al., 1999; Lavau et al., 1997; Slany et al., 1998). However, it is not clear whether the sole transforming effect of MLL fusion proteins is due to a gain of MLL function. For instance, MLL fusion proteins may also exert effects on the function of the corresponding partner protein, resulting in a double genetic hit that strongly cooperates to promote leukemogenesis. Most of the MLL fusion partners are novel proteins and of unknown function including the three most common partners AF4, AF9, and ENL, respectively. Although the latter three have been defined as transcriptional activators in vitro, their in vivo functions are unknown (Ma and Staudt, 1996; Prasad et al., 1995; Rubinitz et al., 1994; Slany et al., 1998). Genetic and biochemical data implicate the cytoplasmic partner AF6 and the forkhead transcription factors AFX and AF6q21 to be components of mitogenic and survival signaling pathways, respectively (Brunet et al., 1999; Kops et al., 1999; Kuriyama et al., 1996; Miyamoto et al., 1995;


Ogg et al., 1997; Paradis and Ruvkun, 1998). Recent additions to the MLL partner protein family, identified from rare translocations, include examples of proteins with known function, such as CBP, its close homologue p300, and Abi-1 (Ida et al., 1997; Sobulo et al., 1997; Taki et al., 1997; Taki et al., 1998). Furthermore, two fusion partners, CBP and AF10, participate in reciprocal chromosomal translocations and subsequent fusions with genes other than MLL (Borrow et al., 1996b; Dreyling et al., 1996, 1998). Mechanistically, how could expression of an MLL fusion protein subvert the function of the native partner protein? First, partner protein function could be subject to haploinsufficiency after disruption of one allele by a translocation event. CBP exhibits haploinsufficiency associated either with multiple developmental defects of Rubinstein-Taybi syndrome in humans or the variably penetrant embryonic lethal phenotype of heterozygous mouse mutants (Petrij et al., 1995; Yao et al., 1998). However, we have found that three MLL fusion proteins (MLL-ENL, MLL-AF10, and MLL-ELL) transform primary myeloid progenitors in the presence of two copies of the respective partner genes, suggesting that partner protein haploinsufficiency may not play a crtical role in MLL fusion protein—mediated transformation (Di Martino et al., 2000; Lavau et al., 1997). Alternatively, MLL fusion proteins may also exert a dominant effect on partner protein function. Whether such a dominant effect results in a subsequent loss or gain of function may well be dependent upon the specific partner protein. For example, the variant forkhead transcription factor AF6q21/FKHRL1 is thought to control an evolutionarily conserved pathway that promotes apoptosis, by activating target genes such as Fas Ligand (Brunet et al., 1999). In its normal context, the proapoptotic activity of AF6q21 is negatively regulated by survival factor signaling, which results in the activation of PI-3 kinase. This in turn activates its downstream target Akt, which phosphorylates AF6q21, preventing its nuclear localization and inhibiting its ability to transactivate target gene reporter constructs (Brunet et al., 1999). Thus, it may be that an MLL-AF6q21 fusion protein behaves as a dominant negative inhibitor of AF6q21 function and results in enhanced cell survival and therefore

457

cooperates with an MLL gain of function to promote leukemogenesis. A similar case could be made for the MLLAbi1 fusion protein. Abi1 functions as a negative regulator of c-abl tyrosine kinase activity and v-abl transformation (Shi et al., 1995). Disruption of normal Abi1 function via dominant negative inhibition may generate a constitutively active and therefore oncogenic form of c-abl. Conversely, some MLL fusion proteins could act as dominant gain-of-function mutants for partner protein function. For example, genetic and biochemical data implicate mammalian AF6 and its fly homologue Canoe as novel downstream effectors of Ras and Notch signaling (Kuriyama et al., 1996; Matsuo et al., 1997; Miyamoto et al., 1995). Uncontrolled activation of AF6 targets along this mitogenic pathway could synergistically promote leukemogenesis in combination with MLL gain of function. These data suggest that deregulated partner protein function could contribute to development of the malignant phenotype via one of two models. First, the function of each partner protein and its downstream signaling components may be deregulated by the expression of the corresponding MLL fusion protein. Thus, each partner protein family would be predicted to control distinct pathways. In an alternative model, all MLL partner proteins could lie as various components along a common signaling pathway, such that cytoplasmic MLL partners are upstream regulators of the nuclear MLL partners. In such a model, activation of the single common pathway would be predicted to potently cooperate with an MLL gain-of-function mutation to promote leukemogenesis.

CONCLUSION AND FUTURE DIRECTIONS MLL promiscuously participates in multiple translocations, inversions, duplications, and deletions that generate three classes of mutant proteins in human leukemias. Current data suggest that at least one class of mutants, the translocation-derived MLL fusion proteins, transform myeloid progenitors via a dominant gain-of-function mechanism. However, a number of issues remain to be addressed. First, what is the mechanism of transformation utilized by other types of MLL

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mutants? Do they transform via dominant gain of MLL function? Second, the development of leukemias in mice, either by expression of an MLL-AF9 knock-in allele or transplantation of MLL-ENL—expressing bone marrow cells, requires a significant latency period, presumably due to the necessity for further secondary mutations. What is the nature of these cooperative genetic lesions? Third, for MLL fusion proteins, the partner protein seems to specify the lineage of the transformed hematopoietic cells. What is the mechanism for partner protein lineage selection? Perhaps ubiquitous partner proteins associate with lineage-specific cofactors to mediate transformation. Fourth, emerging evidence suggests that at least some native partner proteins function in mitogenic/survival pathways. Do MLL fusion proteins perturb these pathways in addition to altering MLL-dependent pathways to promote leukemogenesis? Finally, what are the critical downstream target genes that mediate the transforming potential of oncogenic MLL proteins? Attractive candidates include the Hox genes, some of which have been previously directly implicated in the pathogenesis myeloid leukemias (Borrow et al., 1996a; Nakamura et al., 1996a; 1996b; Thorsteindottir et al., 1997). Future studies designed to address these questions will allow a molecular dissection of MLL-mediated leukemogenesis and ultimately direct the design of newer therapeutic modalities for these malignancies that are refractory to current treatments. REFERENCES Aasland, R., Gibson, T. J., and Stewart, A. F. (1995). The PHD finger: implications for chromatin-mediated transcriptional regulation. Trends Biochem. Sci. 20, 56—59. Bernard, O. A., and Berger, R. (1995). Molecular basis of 11q23 rearrangements in hematopoietic malignant proliferations. Genes Chrom. Cancer 13, 75— 85. Bernard, O. A., Mauchauffe, M., Mecucci, C., Van den Berghe, H., and Berger, R. (1994). A novel gene, AF-1p, fused to HRX in t(1;11)(p32;q23), is not related to AF-4, AF-9 nor ENL. Oncogene 9, 1039—1045. Beverloo, H. B., Le Coniat, M., Wijsman, J., Lillington, D. M., Bernard, O., de Klein, A., van Wering,

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Waring, P. M., and Cleary, M. L. (1997). Disruption of a homolog of trithorax by 11q23 translocations: leukemogenic and transcriptional implications. Curr. Top. Microbiol. Immunol. 220, 1—23.

REFERENCES

Webb, J. C., Golovleva, I., Simpkins, A. H., Kempski, H., Reeves, B., Sturt, N., Chessells, J. M., and Brickell, P. M. (1999). Loss of heterozygosity and microsatellite instability at the MLL locus are common in childhood acute leukemia, but not in infant acute leukemia. Blood 94, 283—290. Yagi, H., Deguchi, K., Aono, A., Tani, Y., Kishimoto, T., and Komori, T. (1998). Growth disturbance in fetal liver hematopoiesis of Mll-mutant mice. Blood 92, 108—117. Yamamoto, K., Seto, M., Komatsu, H., Iida, S., Akao, Y., Kojima, S., Kodera, Y., Nakazawa, S., Ariyoshi, Y., Takahashi, T., et al. (1993). Two distinct portions of LTG19/ENL at 19p13 are involved in t(11;19) leukemia. Oncogene 8, 2617—2625. Yano, T., Nakamura, T., Blechman, J., Sorio, C., Dang, C. V., Geiger, B., and Canaani, E. (1997). Nuclear punctate distribution of ALL-1 is conferred by distinct elements at the N terminus of the protein. Proc. Natl. Acad. Sci. USA 94, 7286—7291. Yao, T. P., Oh, S. P., Fuchs, M., Zhou, N. D., Ch’ng, L. E., Newsome, D., Bronson, R. T., Li, E., Livingston, D. M., and Eckner, R. (1998). Gene dosage— dependent embryonic development and prolifer-

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ation defects in mice lacking the transcriptional integrator p300. Cell 93, 361—372. Yergeau, D. A., Hetherington, C. J., Wang, Q., Zhang, P., Sharpe, A. H., Binder, M., Marin-Padilla, M., Tenen, D. G., Speck, N. A., and Zhang, D. E. (1997). Embryonic lethality and impairment of haematopoiesis in mice heterozygous for an AML1-ETO fusion gene. Nat. Genet. 15, 303—306. Yu, B. D., Hess, J. L., Horning, S. E., Brown, G. A., and Korsmeyer, S. J. (1995). Altered Hox expression and segmental identity in Mll-mutant mice. Nature 378, 505—508. Yu, B. D., Hanson, R. D., Hess, J. L., Horning, S. E., and Korsmeyer, S. J. (1998). MLL, a mammalian trithorax—group gene, functions as a transcriptional maintenance factor in morphogenesis. Proc. Natl. Acad. Sci. USA 95, 10,632—10,636. Zeleznik-Le, N. J., Harden, A. M., and Rowley, J. D. (1994). 11q23 translocations split the ‘‘AT-hook’’ cruciform DNA-binding region and the transcriptional repression domain from the activation domain of the mixed-lineage leukemia (MLL) gene. Proc. Natl. Acad. Sci. USA 91, 10,610—10,614.

CHAPTER 26


COACTIVATORS AND LEUKEMIA: THE ACETYLATION CONNECTION WITH TRANSLOCATIONS INVOLVING CBP, p300, TIF2, MOZ, and MLL VANDANA CHINWALLA AND NANCY J. ZELEZNIK-LE Oncology Institute, Loyola University Medical Center, Chicago, IL

INTRODUCTION Transcriptional coactivators are molecules that act as bridges between the basal transcriptional machinery and transcription factors such as the cAMP response element binding protein (CREB) and nuclear hormone receptors. The activity of these transcription factors is enhanced by coactivators. One of the most well-studied coactivators is the CREB-binding protein (CBP), which was originally identified as a coactivator for CREB (reviewed in Shikama et al., 1997). CBP binds to the phosphorylated form of CREB and can also interact directly with TFIIB and with RNA polymerase II. Thus, it acts as a bridging factor between CREB and the basal transcription apparatus. It has subsequently been shown that some transcriptional coactivators do not just simply act as bridging molecules, but rather have enzymatic functions of their own. In particular, many of the coactivators have histone acetyltransferase (HAT) activity, which suggests that these proteins could also be directly involved in chromatin remodeling. Recently, the coactivators have also been

linked directly to human disease: chromosomal translocations involving CBP/p300 and TIF2 (which are described below in detail) are now implicated in causing particular types of acute myeloid leukemia. Here, we review the literature that demonstrates this astounding association between coactivators and leukemia, and hypothesize about potential mechanisms whereby the novel fusion proteins produced by coactivator gene translocations could ultimately produce leukemia.

CBP/p300 GENERAL COACTIVATORS CBP and p300 are global transcriptional coactivators that interact with many different DNAbinding transcription factors. CBP and p300 are proteins expressed from different genes, but they share extensive sequence similarity and very similar functions (Arany et al., 1994). Because they are considered as functional homologues, they are usually referred to as CBP/p300 (Lundblad et al., 1995). CBP was initially named for


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COACTIVATORS AND LEUKEMIA

its interaction with CREB, the cyclic AMP response element binding protein (Chrivia et al., 1993). p300 was identified as a 300 kDa protein that interacted with adenoviral protein E1A (Wang et al., 1993). CBP/p300 have subsequently been shown to bind to a variety of proteins including nuclear hormone receptors, viral proteins, transcription factors, the HAT P/CAF, and other nuclear receptor coactivators including P/CIP, SRC-1 and TIF2 (reviewed in Shikama et al., 1997; Torchia et al., 1997). It is thought that CBP/p300 functions, in part, by mediating the association of large multiprotein complexes that contain other cofactors. CBP and p300 also possess intrinsic HAT activity that is required for their coactivator function (Bannister and Kouzarides, 1996; Ogryzko et al., 1996). This finding led to the hypothesis that these proteins may contribute directly to transcriptional regulation by targeted acetylation of chromatin, thereby facilitating access of transcription factors to target gene promoter regions. Subsequent studies have demonstrated that additional substrates for CBP/p300 acetyltransferase activity include DNA-binding transcription factors, such as p53, GATA-1, and HMG-I(Y) (Boyes et al., 1998; Gu and Roeder, 1997; Munshi et al., 1998). Therefore, in addition to its potential role in altering chromatin structure, CBP/p300 could also act by changing the DNA-binding activities of transcription factors.

activation by NR requires additional factors that are present in limiting amounts in the cell. Thus, the search for these putative limiting factors led to the identification of TIF2 along with many other proteins belonging to this family of transcriptional coactivators (Onate et al., 1995; Voegel et al., 1996). Since then, several structurally distinct subclasses of NR coactivators have been identified including members of the p160 family (SRC-1/NCoA-1, TIF2/NcoA-2/ GRIP-1/SRC-2, pCIP/RAC3/ACTR/AIB1), the general coactivators CBP/p300 and their associated proteins, mammalian homologues of yeast SWI/SNF proteins, and the less-well-characterized E3 ubiquitin-protein ligase coactivators. TIF2 and SRC-1 share a high degree of homology and mediate the ligand-dependent transcriptional activation function of all different NRs such as estrogen receptor (ER), glucocorticoid receptor (GR), thyroid receptor (TR), retinoic acid receptor (RAR), and retinoid-X receptor (RXR) in different cell types (Onate et al., 1995; Voegel et al., 1996). The p160 group of coactivators is characterized by the presence of the signature motif LXXLL, which is required for interaction with NR (Heery et al., 1997; Montminy, 1997). The p160 coactivator group and CBP/p300 have intrinsic HAT activity, suggesting a role in chromatin remodeling (Chen et al., 1997; Ogryzko et. al., 1996; Spencer et al., 1997).

TIF2/NUCLEAR RECEPTOR COACTIVATORS

HISTONE ACETYLTRANSFERASES

TIF2, also known as NcoA-2/GRIP-1/SRC-2, belongs to the growing class of transcription intermediary factors or coactivators, which play a role in mediating the transcriptional activation function of a nuclear receptor (NR) (reviewed in Glass et al., 1997; Chen and Li, 1998; Freedman, 1999; Shibata et al., 1997). Nuclear receptors are a large family of ligand-inducible transcription factors that are important during development, growth, and homeostasis. They bind to small hydrophobic ligands such as steroid and thyroid hormones, vitamin D, and retinoids and activate transcription in response to their ligands via enhancer elements located in the promoters of target genes. Overexpression studies of NRs suggested that transcriptional

Histone acetyltransferases (HATs) are enzymes that acetylate the N-terminal histone tails. This process of acetylation is thought to be important in regulating the accessibility of nucleosomal templates to the transcriptional machinery (reviewed in Roth and Allis, 1996, and Brownell and Allis, 1996). There are two different types of HATs that are classified based on their subcellular location. HAT A proteins are nuclear HATs that act on histones already present in the nucleosomes, whereas HAT B proteins are cytoplasmic HATs that acetylate nascent histone H4 before it is transported to the nucleus. These two types of HATs have different substrate specificities and acetylate different lysine residues of histone tails. The im-

LEUKEMIA TRANSLOCATIONS INVOLVING COACTIVATORS

portance of such posttranslational modification of histone tails appears to be in regulating histone interaction with DNA and other nonhistone proteins. Histones contain globular core domains with highly charged, unstructured tails protruding out of the histone octamers present in nucleosomes. These highly charged tails are capable of interacting with DNA or other proteins. Acetylation of lysine residues present in these tails neutralizes charge and leads to reversal of these DNA-histone or protein-histone interactions, thus providing a level of regulation of such interactions. Many proteins such as the coactivators discussed here possess HAT A activity and therefore presumably can activate transcription from specific promoters by nucleosomal rearrangement at the promoter region. When the HAT A, Tetrahymena p55, was cloned, it was found to be a homologue of yeast Gcn5p, a well-studied yeast adapter/coactivator protein (Brownell et al., 1996). Gcn5p was known to be required for the full activity of some transcriptional activators. When the two were linked, it established a connection between histone acetylation and gene activation, and suggested that adapter/ coactivator complexes might influence gene activation through the direct modification of chromatin templates. It is intriguing that many of the HAT A enzymes described in the literature contain bromodomains. It has been proposed that one function of the bromodomain may be to tether A-type HATs to chromatin templates undergoing gene activation (Brownell et al., 1996).

LEUKEMIA TRANSLOCATIONS INVOLVING COACTIVATORS MOZ-CBP t(8;16) (p11;p13) Patients with the translocation t(8;16) generally are classified as having French-American-British (FAB) acute myeloid leukemia (AML)-M4 (myelomonocytic) or M5 (monocytic), with a pronounced erythrophagocytosis by the blast cells in the majority of cases. Both de novo and therapy-related cases have been reported in the literature. The experiments performed to demonstrate the involvement of CBP (CREB-binding protein) in this translocation were facilitated

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by studies done on a constitutional human disease mapped to chromosome band 16p13, Rubinstein-Taybi syndrome (RT) (Petrij et al., 1995; Tanaka, et al., 1997). Since the same band on chromosome 16 was involved in the t(8;16), yeast artificial chromosome (YAC) and cosmid contigs were generated across the CBP gene, and then probes devoid of repetitive sequences from this region were used to determine whether CBP was involved in the t(8;16) by Southern blot analysis (Borrow et al., 1996). Both on pulsed field gels and on standard Southern blots, the CBP gene is consistently rearranged in t(8;16) patients (Borrow et al., 1996; Giles et al., 1997). In fact, the genomic breakpoint cluster region in CBP seems to be localized to a relatively small 13 kb region near the 5 (centromeric) end of the gene (Giles et al., 1997). Once CBP was found to be rearranged at the genomic level, the chimeric junction was cloned from a cosmid library generated from t(8;16) patient material. This chimeric junction cosmid was subsequently used for exon trapping to identify transcribed sequences in this region, and to ultimately identify and clone the CBP fusion partner. This newly identified gene on chromosomal band 8p11, called MOZ, was rearranged in all patients with the t(8;16) as indicated by Southern blot analysis (Borrow et al., 1996). Surprisingly, the chimeric cDNA junction was amplified from patient material using reverse transcriptase—polymerase chain reaction (RTPCR) in only one case with the t(8;16) (Borrow et al., 1996). This is in contrast to the t(11;16) in which the MLL-CBP junction has been amplified by RT-PCR from all the patients analyzed to date (see below; Satake et al., 1997; Sobulo et al., 1997; Taki et al., 1997). Perhaps this fusion message is expressed at a very low level or is unstable and is, therefore, more difficult to detect in this manner. Another interesting feature of the MOZ-CBP cDNA junction that was cloned is that the breaks in the two genes occurred within exons. This is unusual for the majority of breakpoints that have been cloned from AML patients, but is certainly not unprecedented. Furthermore, two nontemplated nucleotides were added at the fusion junction to maintain the open-reading frame in the resulting fusion protein (Borrow et al., 1996). This could have implications in terms of understanding the mechanisms involved in generating the translocation at the genomic level.

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CBP seems to act as a tumor suppressor gene in RT syndrome (Miller and Rubinstein, 1995), but this does not seem to be the case for the t(8;16) in AML (Petrij et al., 1995). Although not as many cases have been studied in detail, it does not appear that the untranslocated CBP allele is deleted in these patients, at least at the level of detection of fluorescence in situ hybridization (FISH) with cosmid probes (Rachel Giles, unpublished data). Although there is still the possibility that small deletions or critical mutations of the remaining ‘‘normal’’ CBP allele exist, the present data do suggest that the CBP gene plays different roles in AML versus RT — in RT, acting as a classical tumor suppressor gene, and in the case of AML, acting as an oncogene created by expression of a novel fusion protein. MOZ and CBP proteins both contain multiple functional domains (Fig. 26.1). MOZ consists of 2004 amino acids and is predicted to be a 225 kDa nuclear protein (Borrow et al., 1996). Although at present its function is unknown, one can suggest some potential activities based on homology to other proteins. There are two potential nuclear localization signals (NLS) that presumably target MOZ to the nuclear compartment. It contains two C4HC3 domains, also known as plant homology domain (PHD) or leukemia-associated protein (LAP) domain, near the amino terminus (Aasland et al., 1995; Saha et al., 1995). The PHD domain is found in many proteins that have been implicated in chromatin modeling such as Trithorax (TRX), Polycomblike (pcl), and Mixed Lineage Leukemia (MLL) (see below). This domain is also found in coactivator proteins such as CBP and its functional homologue, p300, which are also both partner proteins of MLL (see below). PHD domains are present in two additional MLL partner genes, AF10 and AF17 (Saha et al., 1995). Although the function of the PHD domain is unclear, it is thought to be involved in protein—protein interactions. The MOZ C4HC3 fingers are most homologous to those encoded by the myeloid apoptosis gene Requiem and the neuro-D4 gene. It is interesting that several PHD domain—containing proteins are involved in chromosomal translocations. Another MOZ motif is the MYST domain present in the amino terminal half of the protein (Borrow et al., 1996). The MYST domain con-

sists of a single C2HC zinc finger as well as a region previously defined as an acetyltransferase signature (Kleff et al., 1995; Tanaka et al., 1989). Along with MOZ, the MYST domain was identified in two yeast proteins, SAS2 and YBF2, and in the human TIP protein, and was named after these four proteins (MOZ, YBF2, SAS2 and TIP). SAS2 and YBF2 have been studied and may give some insight into MOZ function. Both yeast genes are involved in yeast silencing, a process associated with acetylation. Disruptions of either of these genes affect transcriptional silencing, generating phenotypes similar to those seen with disruption of an N-acetyltransferase (Mullen et al., 1989; Reifsnyder et al., 1996). This suggests that MOZ likely has acetyltransferase activity, potentially with a chromatin-associated gene regulatory function. Very recently, it was experimentally confirmed that, indeed, MOZ does have histone acetyltransferase activity with the same substrate specificity as TIF60, another member of the MYST family (Julian Borrow, personal communication). They predominantly acetylate histones H2A and H4, and to a lesser extent histone H3. MOZ contains an extensive acidic domain in the middle of the protein, with 28% of residues being either glutamic acid or aspartic acid. The carboxy terminus of MOZ is methionine-rich (10%). The acidic domain of MOZ is unusually large and could interact with chromatin through contacts with exposed basic histone residues or it could act as a transcriptional activation domain. The methionine-rich domain and its importance is still uncharacterized, but it is highly conserved in ESTs T24773 and M28647, suggesting that it is important functionally (Borrow et al., 1996). CBP has been extensively studied in the past several years. Protein domains were identified with a combination of functional experiments and sequence similarity searches (Fig. 26.1). A direct nuclear receptor—binding function is localized to the amino (N)-terminal region of the protein, and indirect nuclear receptor binding is mediated through the SRC-1—binding/transactivation domain at the carboxy (C)-terminal region (Chakravarti et al., 1996; Kamei et al., 1996; Smith et al., 1996). The CREB-binding domain (also known as the KIX domain) near the N-terminus is important for binding to a


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Figure 26.1. Diagramatic representation of the MOZ, MLL, CBP/p300, and TIF2 proteins. The locations of breaks in different translocations are indicated by arrows. AT-H, AT-hook region; AD, activation domain; BCR, breakpoint cluster region; bromodomain, a highly conserved motif predicted to be important in chromatin remodeling; CID, CBP interaction domain; HAT, histone acetyltransferase domain; M-rich, methionine-rich domain; MYST, encompasses both the C2HC finger and the HAT domain; NID, nuclear receptor interaction domain; PAS/bHLH, domain involved in DNA binding and protein-protein interactions; PHD/LAP, C4HC3 zinc fingers believed to be important for protein-protein interaction; RD, repression domain; SET, a highly conserved motif of unknown function. The PHD domain of CBP/p300 is embedded in the HAT domain. The regions of CBP proteins involved in binding to various different proteins are indicated.

variety of transcription factors, similar to the E1A-binding domain near the C-terminus (Parker et al., 1996; and reviewed in Andrisani, 1999). CBP contains a PHD domain (discussed above with regard to the MOZ protein) and a bromodomain. The bromodomain is an approximately 110 amino acid module that is highly conserved evolutionarily (Jeanmougin et al., 1997). Although its function is not known, several bromodomain-containing proteins are involved in transcriptional regulation as mediators or coactivators, including SNF2-SWI2, CBP, TAF-250, and GCN5. Several of these have HAT activity and are present in large

multiprotein complexes. It has therefore been proposed that the bromodomain is important for protein-protein interactions and may influence the assembly or activity of these complexes (Brownell et al, 1996). Recently, the solution structure of the HAT coactivator P/CAF (p300/ CBP-associated factor) bromodomain revealed an unusual left-handed four-helix bundle (Dhalluin et al., 1999). Structure/function studies demonstrated that the bromodomain interacts specifically with acetylated lysine, in a manner similar to the interaction of acetyl-CoA with histone acetyltransferases (Dhalluin et al., 1999). Modification of chromatin by several of these

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Figure 26.2. Diagramatic representation of the fusion proteins formed by MOZ, MLL, CBP/p300, and TIF2 proteins. The symbols are the same as in Figure 26.1.

proteins also suggests that the bromodomain might be involved in chromatin interactions. CBP has intrinsic HAT activity, and the domain that mediates this function localizes to the central portion of CBP (Bannister and Kouzarides, 1996; Ogryzko et al., 1996). The HAT domain has also recently been shown to have acetyltransferase (AT) activity on substrates other than histones, including p53, GATA-1 and HMGI(Y) (Boyes et al., 1998; Gu and Roeder, 1997; Munshi et al., 1998; ). In the fusion protein that is produced as a result of the t(8;16), many of the functional domains of both MOZ and CBP would be retained (Fig. 26.2). The MOZ PHD domain, MYST/HAT domain, and acidic domain would be fused to CBP. The CBP CREB-binding domain, bromodomain, PHD domain, HAT domain, E1A-binding domain, and transactivation

domain (SRC-1/TIF2—binding domain) would all juxtapose the amino-terminal MOZ protein. The nuclear receptor—binding domain and a nuclear localization signal would be lost from CBP in the fusion, but the nuclear localization signal would presumably be replaced by nuclear localization signals from MOZ. MLL-CBP t(11;16) (q23;p13) The MLL gene (also called ALL-1, HRX, and Htrx—1) on chromosomal band 11q23 is involved in recurrent translocations in many leukemias, either acute lymphoblastic or myeloid/monocytic, and is involved in the majority of infant leukemias (reviewed in Rowley, 1993; and Bernard and Berger, 1995). MLL is also involved in translocations that occur secondary to therapy of a primary malignant disease with


drugs that target DNA topoisomerase II and result in therapy-related acute myeloid leukemia or acute lymphoblastic leukemia (t-AML or t-ALL) (Pedersen-Bjergaard and Rowley, 1994). MLL is involved in translocations with at least 40 different partner genes, 18 of which have been cloned so far. The rare but recurrent translocation t(11;16)(q23;p13) is unusual in that it has been almost exclusively associated with therapyrelated leukemia (Rowley et al., 1997; Sobulo et al., 1997). Of the 13 patients with this translocation of whom we are aware, only one is not therapy related (Rowley et al, 1997). This is in contrast with other MLL translocations where the majority of cases involving a particular partner gene are de novo, with a small percentage of cases arising secondary to therapy with drugs that target DNA topoisomerase II. Although the reasons for this are not known, it is thought that further studies of the structure of the genes involved may be informative. This translocation is also unusual in that many of the patients present with myelodysplastic syndrome (MDS), which is very unusual for MLL translocations (Rowley et al., 1997). Several groups have independently studied the t(11;16) and cloned CBP as a partner gene of MLL in this translocation (Rowley et al., 1997; Satake et al., 1997; Sobulo et al., 1997; Taki et al., 1997). Our laboratory initially cloned the genomic breakpoint from one patient with a t(11;16) (Sobulo et al., 1997). Using the sequence of the non-MLL portion of the genomic clone, we identified a P1 artificial chromosome (PAC) and a yeast artificial chromosome (YAC) that contained this sequence. The YAC had previously been mapped to near the CBP gene on chromosome 16, band p13. 3. We therefore used the candidate gene approach to determine whether CBP was the gene involved in this translocation with MLL. The candidate gene approach was also used by two other groups to identify CBP as the partner of MLL (Satake et al., 1997; Taki et al., 1997). In both cases, the researchers utilized recently published information that CBP was the partner gene of a newly identified gene, MOZ, in the t(8;16) (p11;p13) (see above). They reasoned that since CBP was located at the same chromosome band and was involved in a translocation that resulted in myeloid leukemia, it may be involved in another translocation with MLL that also resulted in

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myeloid leukemia. One of these groups first confirmed by Northern blot analysis using CBP as a probe that an abnormal transcript was present in patient material, although Southern blot analysis did not reveal a rearranged genomic band with the CBP probe (Taki et al., 1997). All groups were able to demonstrate that MLL formed a fusion product with CBP in patients with the t(11;16) using RT-PCR analysis. MLL codes for a very large protein, with a predicted molecular mass of 431 kDa (Djabali et al., 1992; Gu et al., 1992; Tkachuk et al., 1992). The protein contains several domains identified by homology to other proteins or by functional analysis (Fig. 26.1). Three AT-hook DNA-binding domains near the amino terminus also are found in the high-mobility-group proteins HMG-I(Y) (Reeves and Nissen, 1990). These domains bind AT-rich, cruciform, and scaffold attachment region (SAR) DNA, recognizing structure rather than a specific sequence. MLL contains a region of homology to mammalian DNA methyltransferases, has functional transcriptional activation and repression domains, as well as a cysteine-rich region that forms three PHD domains (Domer et al., 1993; Ma et al., 1993; Saha et al., 1995; Schindler et al., 1993; Zeleznik-Le et al., 1994). The PHD domain and the SET [Su(var)3-9, enhancer of zeste, and trithorax] domain at the carboxyl terminus are the regions most conserved with the Drosophila trithorax (trx) protein. The SET domain has recently been shown to interact with myotubularin-related proteins and with components of the SWI/SNF complex (Cui et. al., 1998; Rozenblatt-Rosen et. al., 1998) The trx gene is required to maintain the proper expression of homeotic genes of the Bithorax and Antennapaedia complexes in Drosophila. Mice with a single disrupted MLL gene created by homologous recombination display bidirectional homeotic transformations and those with homozygous deletions die at embryonic day 10. 5 (Yu et al., 1995). This is similar to changes observed in trx mutant Drosophila. It is thought that trx regulates homeotic gene expression at the level of chromatin organization by maintaining an ‘‘open’’ chromatin structure, but this remains to be proven experimentally. Of the five t(11;16) patient samples that have been analyzed by RT-PCR to date, all have

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breaks within the MLL BCR, and four have the same breakpoint at the cDNA level in the CBP gene (Satake et al., 1997; Sobulo et al., 1997; Taki et al., 1997). This would result in a fusion protein where the amino portion of MLL is fused to amino acid 267 of CBP (Fig. 26.2). MLL would contribute the AT hook DNAbinding domain, nuclear localization signals, and the DNA methyltransferase homology domain/transcriptional repression domain to the fusion. From CBP, only the region of CBP that binds nuclear hormone receptors would be lacking in the fusion. This is similar to the fusion produced in the t(8;16); however, unlike the MOZ-CBP fusion produced in the t(8;16), the breakpoints at the genomic level occur in introns, and no extra sequences are needed at the junctions to retain the open-reading frame in the fusion protein. The fusion junction that was cloned from another patient, however, would produce a protein with CBP amino acid 1021 fused to the amino portion of MLL (Sobulo et al., 1997; Fig. 26.2). This fusion is particularly informative because less of CBP is present in the fusion product that is necessary for leukemogenesis. In this case, only the region of CBP from the bromodomain on is brought into the fusion. Although it is unknown what CBP contributes to the fusion that facilitates abnormal growth of hematopoietic cells, we at least know that only about half of CBP is absolutely required for this process. This shorter fusion product is likely not an isolated aberrant incident either. Analysis of patients by FISH with our CBP PAC probe demonstrates that at least two other patients have a similar breakpoint at the genomic DNA level and would likely have a similar cDNA junction (Rowley et al., 1997). Furthermore, the MLL-p300 fusion has this similar smaller region of p300 juxtaposed to MLL (see t(11;22), below). MLL-p300 t(11;22) (q23;q13) With the identification of CBP as the partner gene of MOZ in the t(8;16) and of MLL in the t(11;16), it was hypothesized that p300, the functional homologue of CBP, might also be involved in chromosomal translocations that result in myeloid leukemia. Indeed, this was shown to be the case. MLL is fused to p300 in the leukemic cells from a t-AML patient with a

t(11;22) (q23;q13) (Ida et al, 1997). This is the single published case with this type of translocation to date; however, it is intriguing that it was observed in a t-AML patient treated with etoposide for his primary malignancy. It may be that this translocation is also primarily associated with t-AML in patients previously treated with DNA topoisomerase II—targeting drugs, similar to leukemias with a t(11;16) (see above). More patients with the t(11;22) need to be identified and characterized before this association can be made. FISH analysis of patient leukemia cells with p300 genomic clones was used to initially demonstrate that p300 was split by this translocation. Southern blot analysis of DNA with various p300 cDNA fragments as probes confirmed this and also localized the breakpoint to a region near the bromodomain of p300. An abnormally sized transcript was detected using either the p300 or an MLL probe. Subsequently, RT-PCR and sequence analysis confirmed the presence of the MLL-p300 fusion transcript and showed that amino acid 1445 of MLL, within the MLL BCR, was juxtaposed to p300 with an in-frame junction to amino acid 940 of p300, just upstream of the bromodomain (Fig. 26.2). In this patient sample the reciprocal RT-PCR product of p300-MLL was also detected. The functional domains of p300 that are retained in the MLL-p300 fusion protein are similar to the domains retained in the MLL-CBP fusion where less of CBP is brought into the translocation. MOZ-TIF2 Inversion (inv)(8) (p11q13) Translocations involving chromosome band 8p11 are associated with two distinct leukemia syndromes: either an acute myeloid leukemia (AML), classified as FAB M4 or M5, or a myeloproliferative disorder with features characteristic of both chronic myeloid leukemia and non-Hodgkin’s lymphoma (Inhorn et al., 1995; Still et al., 1997). The M4/M5 AML is characterized by pronounced erythrophagocytosis by the blast cells, young age at diagnosis, and poor outcome (Heim et al., 1987; Lai et. al. 1987). The recurrent translocation t(8;16)(p11;p13) discussed above is associated with the first type of syndrome and is found in approximately 2% of cases of M4 or M5 AML (Mitelman and Heim,


1995). Other rare cytogenetic rearrangements involving 8p11 with the same type of phenotype as that of the t(8;16) have also been reported, such as inv(8)(p11q13), t(8;22)(p11;q13) and t(8;19)(p11;q13) (Aguiar et al. 1997; Brizard et al., 1988; Lai et al., 1992). As noted, translocation (8;16)(p11;p13) involves MOZ and CBP (see above; Borrow et al., 1996). It was postulated that MOZ may be involved in other 8p11 rearrangements associated with AML with the M4 or M5 phenotype. CBP could presumably be replaced by analogous genes located on the other chromosomes (Borrow et al., 1996). Aguiar and co-workers. (1997) screened leukemic bone marrow cells with 8p11 abnormalities for evidence of MOZ involvement. This patient group included three cases with t(8;13)(p11;q12) and a single case with inv(8)(p11q13). The one patient with inv(8)(p11q13) had AML— M5 disease similar to that associated with the t(8;16)(p11;p13), whereas the other three patients with t(8;13)(p11;q12) abnormality suffered from the myeloproliferative disorder. MOZ was rearranged only in the patient with the inv(8)(p11q13) rearrangement, suggesting that the other 8p11 translocations associated with the myeloproliferative disorder may involve a different gene. This also provides an indication that patients with 8p11 involvement with AML M4/M5 disease may primarily involve the MOZ gene. Inv(8)(p11q13) is a rare abnormality and there are very few cases reported in the literature (Aguiar et. al., 1997; Coulthard et. al. 1998; Liang et. al., 1998). Recently, the inv(8)(p11q13) has been cloned by two separate groups (Carapeti et al., 1998; Liang et. al., 1998). Carapeti and colleagues (1998) cloned by screening a genomic library made from a patient sample with a 0. 9 kb cDNA probe from MOZ, which detects the breakpoints in MOZ. One clone that was isolated contained MOZ sequence plus 1. 1 kb of unique sequence. A probe derived from this unique sequence was used to obtain a PAC clone that mapped to 8q13. To clone the fusion junction between the two genes at the cDNA level, 3 rapid amplification of cDNA ends (RACE) was performed on RNA from a patient sample using MOZ forward primers. Along with PCR fragments containing normal MOZ, a PCR fragment in which MOZ was fused to unique sequence was obtained. A search of the

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Genbank database with this unique sequence showed that it derived from the TIF2 gene, a nuclear receptor transcriptional coactivator. Fluorescence in situ hybridization (FISH) using a PAC that contains TIF2 sequences confirmed that TIF2 is located at 8q13. In this fusion between MOZ and TIF2, amino acid 1117 of MOZ is fused to amino acid 870 of TIF2 (Fig. 26.2). This break occurs within the same exon of MOZ as in the t(8;16) but at a different sequence. RT-PCR using MOZ and TIF2 primers gave two products, which are the result of an alternatively spliced exon in TIF2. The reciprocal transcript with TIF2-MOZ was not detected by the RT-PCR. Liang and colleagues (1998) also described cloning of inv(8)(p11q13) by RT-PCR from a different patient. In this fusion the same amino acid of MOZ, 1117, is fused to amino acid 939 of TIF2. The fusion in TIF2 is at the splice acceptor site of an alternatively spliced exon. Unlike the first case where they report presence of both splice forms in the MOZ-TIF2 fusion, here they detect only the smaller fusion transcript without the alternatively spliced exon. It is not clear if the genomic break in MOZ in both patients is the same or not. This is the first time TIF2 has been implicated in the pathogenesis of a disease. Several sporadic leukemias have been described with translocations or other rearrangements that involve chromosome band 8q13 that could potentially involve TIF2. The other family member pCIP/ RAC3/ACTR/AIB1, located at chromosomal band 20q12, is amplified in breast and ovarian cancers (Anzick et al., 1997). The MOZ protein structure is discussed above (see MOZ-CBP). TIF2 belongs to the class of coactivators collectively called the p160 coactivators, which includes SRC-1/NCoA—1 and pCIP/RAC3/ ACTR/AIB1 (Chen and Li, 1998; Freedman, 1999; reviewed in Glass et al., 1997; Onate et al., 1995; Shibata et al., 1997; Voegel et al., 1996). They all act as general coactivators for the different nuclear receptors such as estrogen receptor (ER), glucocorticoid receptor (GR), thyroid receptor (TR), retinoid acid receptor (RAR), and retinoid-X receptor (RXR) in different cell types. TIF2 and SRC-1 have been found in the same complex (McKenna et al., 1998). All these proteins share a high degree of sequence

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similarity. Different motifs have been mapped in the TIF2 protein, partly based on its homology to the SRC—1 protein and partly by functional analysis (Fig. 26.1; Voegel et al., 1996). The predicted amino acid sequence of TIF2 contains N-terminal putative nuclear localization signals, one glutamine and three serine/ threonine-rich regions, and two charged clusters (Voegel et al. 1996). The N-terminal end of TIF2 contains the two nuclear localization signals and one of the charged clusters. This region of the protein is not homologous to the SRC-1 protein. All three proteins belonging to the p160 family contain a PAS/bHLH domain at their N-termini. The PAS/bHLH domain is believed to be involved in DNA binding and protein heterodimerization (Lindebro et al., 1995). SRC1 contains a C-terminal HAT domain (Spencer et al., 1997). TIF2 shares sequence homology with the HAT domain of SRC-1 and is therefore predicted to also have HAT activity. However, to date, the HAT activity of TIF2 has not been successfully demonstrated experimentally (Voegel et al., 1998). Voegel and co-workers (1998) performed detailed functional mapping of the TIF2 protein and identified an NR-interacting domain, activation domain 1 and 2 (AD1 and AD2). Both TIF2 and SRC-1 interact with CBP and are highly homologous in the CBP interaction domain (CID). CID in TIF2 maps exactly to the AD1, suggesting that the TIF2 AD1 activity may be a result of CBP recruitment. CID contains a leucine motif (LLXXLXXXL) and has been shown by mutational analysis to be necessary but not sufficient for interaction with CBP and its activation function. The nuclear receptor interacting domain (NID) of TIF2 was mapped to the central region of TIF2 (AA 624—869) by the GST pulldown assay using either ER, RAR or RXR . Sequence comparison of the NIDs of SRC-1 and TIF2 revealed the presence of three LXXLL motifs (motifs I, II, and III) in the NID, which have been described as a signature motif present on transcriptional coactivators. The LXXLL motif is necessary and sufficient for binding of these proteins to liganded NRs. Mutational analysis of LXXLL (also known as NR boxes) showed that motif II was always more detrimental in double mutants with respect to NR binding and stimulation of AF-2 activity than

mutations in motifs I and III in a GST affinity chromatography experiment. There have been conflicts in the literature as to whether there is preferential binding of individual NR boxes to specific NR (Leers et al., 1998; Voegel et al., 1998). The MOZ-TIF2 fusion transcripts cloned from both patient samples maintain an open reading frame giving rise to a novel fusion protein retaining the same domains from MOZ and TIF2 (Carapeti et al., 1998; Liang et. al., 1998). The fusion protein retains the nuclear localization signals, PHD, the MYST domain including the HAT domain and part of the large acidic domain of MOZ and CID, the two activation domains and the putative HAT domain of TIF2 (Figures 26.1 and 26.2).

HOW MIGHT THE FUSION COACTIVATORS FUNCTION? In order to formulate hypotheses about the function of fusion proteins formed by the translocation of coactivator genes, we need to understand how these coactivators may function under normal conditions (Fig. 26.3A). Activation of transcription of a target gene by nuclear receptors (NR) seems to be a multistep process triggered by binding of ligand to the ligandbinding domain. This is believed to cause a conformational change in the NR, which leads to dissociation of the NR-corepressor complex and allows for formation of the NR-coactivator complex. The NR-coactivator then binds to other proteins such as CBP or p300, which then makes a contact with the RNA Pol II (if these are not the coactivators themselves). SRC-1 has been shown to bind the proteins of the basal transcriptional machinery (Takeshita et al., 1996). The NRs themselves can bind the basal transcription factors, potentially making the bridging function of the coactivator redundant or less important (May et al., 1996; Mengus et al., 1997; Rochette-Egly et al., 1997). The coactivators may act by making the activation complex more stable and by providing a chromatin remodeling function to the activation complex. As more studies are published, the data all suggest that chromatin remodeling through histone acetylation plays a major role in transcrip-

HOW MIGHT THE FUSION COACTIVATORS FUNCTION?

475

Figure 26.3. Models of wild-type coactivators and their fusion proteins in target gene coactivator complex interactions. In the wild-type situation (A) TIF2 and CBP/p300 proteins bind to their normal transcriptional activator proteins. The example shown here is for transcriptional activation of NR, but similar scenarios could be envisioned for CREB, STAT-1, and other activators with which CBP/p300 interacts at its respective target sites on chromosomes. In these later complexes, CBP/p300 would bind the transcriptional activator proteins directly and may or may not include p/CAF or p/CIP. TIF2, CBP/p300, p/CAF, and p/CIP have HAT activity, which may lead to opening of the chromatin, making the target sequence accessible to the transcriptional activation complex. B, C, and D depict what may happen in case of coactivator fusion proteins. In MOZ-CBP, MOZ-TIF2, and MLL-CBP/p300, the HAT activity of CBP/p300 and its associated proteins may be targeted to new sites, presumably at the MOZ and MLL target sequences, respectively. It is not known how MOZ functions and whether it binds DNA directly or through interaction with other proteins. The PHD domain and nuclear localization signals of MOZ are retained in the fusion, and presumably MOZ target genes are preserved in case of the fusion proteins. It is not clear if MLL binds its target sequences directly or indirectly, but in this case the AT-H domain involved in DNA binding is also retained in the fusion. Hence the MLL fusion proteins may target MLL target genes, including the HOX genes. Novel HAT activity at unusual target sites may cause aberrant expression of certain genes in tissues where they are not usually expressed, leading to leukemia.

tion activation by nuclear receptors and general coactivators. CBP/p300 and p160 (SRC-1/ pCIP) coactivators each possess intrinsic HAT activity, and have been postulated to act together to remodel chromatin. Furthermore, p/ CAF, which interacts with CBP/p300 and with some p160 coactivators, is the mammalian

homologue of the yeast HAT GCN5, and is part of a multiprotein complex that contains TAFs (Ogryzko et al., 1998). Therefore, large chromatin-remodeling complexes that are comprised of CBP/p300, p160 coactivators, and p/CAF would contain multiple HAT activities that could be recruited to specific genomic regions

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via nuclear receptors (Fig. 26.3A). CBP/p300 is involved in translocations with both MOZ and MLL. The fusion proteins produced as a result of these translocations juxtapose CBP or p300 downstream of the aminoterminal portions of MLL or of MOZ. The MLL domains retained in the resultant fusion proteins include nuclear localization signals, the AT hook DNA-binding domains, the methyltransferase homology region, and the transcriptional repression domain. It is likely that this portion of MLL would provide signals to target the fusion protein to specific nuclear sites and to certain DNA target regions (Figure 3D). The AT hook region binds to DNA, recognizing structure rather than specific sequence. Specificity of binding may be determined by other domains in the amino portion of MLL or could also be influenced by the partner sequences contributed by the fusion. It is unknown at this point what determines target specificity. It is possible that MOZ could act in a similar manner to MLL (Fig. 26.3C). Little is known about the function of MOZ, but its homology to other MYST family members and the presence of a HAT domain suggests its involvement in chromatin remodeling. MOZ domains retained in the fusion proteins include the PHD and HAT/MYST domains, as well as nuclear localization signals. SAS2 and YBF2, yeast MYST family members, are involved in silencing mating-type loci in yeast. Histones at the silencing loci are generally hypoacetylated and the acetylation pattern is identical to the histones present in metazoan heterochromatin (Braunstein, et. al., 1996). The HAT domain of MYST family members is similar to a yeast HAT B enzyme and therefore may function in chromatin assembly, as newly synthesized histones are acetylated and then deposited on DNA (Brownell and Allis, 1996). SAS2 mutants show G arrest defects (Reifsnyder, et al., 1996). Also, distinct silenced loci may be critical for life-span control (Kennedy, et al., 1995). These data suggest that absence of normal silencing/ heterochromatinization might contribute to loss of cell cycle control and reprogramming of transcriptional activities. Thus, the fusion proteins may act by changing the normal heterochromatin structure around the target genes, which may lead to loss of cell cycle control. The MOZ-CBP and the MLL-CBP proteins may be

targeted to chromosomes by association with a DNA-binding protein via interaction through their PHD domains, or through interaction with a specific gene structural region as recognized, for example, by the MLL AT hook domain. The functional domains of CBP that are brought into the smaller MLL-CBP fusion (and the analogous MLL-p300 fusion) define the maximum domains required for the leukemia phenotype. These domains include the bromodomain, the HAT domain (which includes a PHD domain), the E1A-binding domain, and the transactivation domain. Just which of these functional domains are crucial for leukemogenesis remains to be determined, but one could conceive of many different scenarios. It is possible that the bromodomain brings in a critical interacting protein or protein complex that is not normally present in a complex with MLL. The same could be true of the E1A-binding domain and the transactivation domain: binding of a transcription factor such as GATA-1 or FOS that is not usually in a complex with MLL could aberrantly affect transcriptional activity of MLL. Furthermore, binding of TIF2, SRC-1, the steroid receptor coactivator protein, or P/ CAF to these domains brings in acetyltransferase activities that have not previously been shown to be associated with MLL function (Fig. 26.3D). Thus, proteins that are normally physically associated with CBP in the cell are now potentially associated with the amino-terminal portion of MLL. A function inherent to CBP could also be critical to the novel function of an MLL-CBP fusion protein. The HAT activity of CBP could function to alter the chromatin structure of genomic regions targeted by MLL. CBP is capable of acetylating all four core histones, regardless of whether they are formed into nucleosomes (Bannister et al., 1996; Ogryzko et al., 1996). This could have a dramatic effect on the structure of chromatin in a genomic region that is targeted by MLL (Fig. 26.3D). CBP was also recently shown to acetylate transcription factors such as p53, HMG-I(Y), and GATA-1. Therefore, it is possible that acetylation of some protein other than histones could have an effect on the downstream function of the MLL-CBP fusion. For example, the site of p53 that is acetylated is in a domain that is critical for the regulation of its DNA-binding activity. Acetyla-

REFERENCES

tion stimulates the sequence-specific DNA-binding activity of p53, presumably as a result of an induced conformational change (Gu and Roeder, 1997). In a similar manner, acetylation of GATA-1 increases the amount of GATA-1 that is bound to DNA and directly stimulates GATA-1—dependent transcription (Boyes et al., 1998). A combination of some or all of these functions could be critical to causing leukemia. In case of the inv(8)(p11q13), TIF2 is fused with MOZ, giving rise to a putative novel MOZ-TIF2 fusion protein. The other alleles of MOZ and TIF2 are presumably wild type. The mechanism by which the fusion protein causes leukemia is not clear, but several different mechanisms or combinations of them are possible. Physiological levels of TIF2 and/or MOZ could be critical for normal functioning of a cell. In transfection experiments where NRs were overexpressed, the activation of a promoter was inhibited by overexpression unless TIF2 or other coactivators were also overexpressed (Bocquel et al., 1989; Meyer et al., 1989; Tasset et al., 1990). On the other hand, SRC-1 null mice did not show any dramatic effect leading to the conclusion that there could be some functional redundancy between different family members (Xu et al., 1998). This suggests that the haploinsufficiency of TIF2 in case of inv(8)(p11q13) may not be important for causing leukemia. The MOZ-TIF2 fusion protein may be targeted to the, as yet unidentified, downstream targets of MOZ (Fig. 26.3B). These target promoters would now be affected by a novel HAT activity provided by CBP, p300, and pCAF brought in by TIF2, which is normally not present at these genes. As discussed before, based on its homology to yeast proteins SAS1 and SAS2 and the presence of a HAT domain, it has been suggested that MOZ may act by chromatin remodeling. The PHD domain of MOZ may interact with DNA-binding proteins drawing MOZ to its putative downstream targets. The fusion protein does retain nuclear localization signals, PHD, part of the activation domain, and the HAT domain of MOZ, which may still be capable of targeting the fusion protein to the normal MOZ targets. The fusion protein retains CID, the two activation domains, and the putative HAT domain of TIF2, and is therefore capable of still interacting with CBP and mediating transactivation. Also, as

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stated before, proteins such as GATA-1, FOS, and E1A may now be aberrantly directed to the MOZ target sites causing abnormal expression of the putative MOZ target genes. In addition, the two activation domains of TIF2, which are retained in the fusion protein, may stimulate the genes, which it does not normally activate. Although it is purely speculative at this point, general chromatin remodeling may be a common theme of all of these recently described translocations involving coactivators. By bringing in a novel chromatin remodeling function into target regions where they were not previously localized, or by qualitatively changing chromatin remodeling because of alternative HAT specificities, aberrant gene regulation and deregulated cell growth could result in the development of leukemia. It will be important to first determine the critical targets of the chimeric protein’s function. Once identified, assessing the chromatin structure of the genomic regions encompassing the targets in the presence or absence of the leukemogenic fusion proteins would provide evidence toward this model. Both inducible in vitro cell line systems and in vivo mouse models are currently being developed to answer these questions.

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CHAPTER 27


THE LMO2 MASTER GENE; ITS ROLE AS A TRANSCRIPTION REGULATOR DETERMINING CELL FATE IN LEUKEMOGENESIS AND IN HEMATOPOIESIS YOSHIHIRO YAMADA AND TERENCE H. RABBITTS Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology

CHROMOSOMAL TRANSLOCATIONS, TRANSCRIPTION, AND DEVELOPMENTAL REGULATION The cytogenetic analysis of tumors, particularly those of blood cell origin, revealed that reciprocal chromosomal translocations are recurring features of these tumors. Since 1990, many important genes have been identified from the breakpoints of chromosomal translocations in acute tumors (in this group, we include acute leukemias and sarcomas), providing a rich source on new transcription factors. There are two frequent outcomes of chromosomal translocations in human tumors (reviewed in Rabbitts, 1994). The first is confined to the lymphoid tumors in which the process of antigen receptor gene rearrangement occurs (immunoglobulin or T-cell receptor genes) and which occasionally mediates chromosomal translocation. This type of translocation causes enforced oncogene expression, resulting from the new chromosomal environment of the rearranged gene. In general, this means inappropriate gene expression. The second and probably the most common out-

come of chromosomal translocations is gene fusion in which exons from a gene on each of the involved chromosomes are linked after the chromosomal translocation, resulting in a fusion mRNA and protein. This type of event is found in many cases of hematopoietic tumors and in the sarcomas. T-cell acute leukemia (T-ALL) is one notable case in the first category of chromosomal translocations. The disease is clinically rather constant, yet individual cases contain one of more than a dozen possible chromosomal translocations. The analysis of these has led to the discovery of many different, novel genes that can contribute to T-cell tumorigenesis (Rabbitts, 1994). The LMO2 gene typifies the type of gene associated with chromosomal translocation breakpoints in acute leukemia (Rabbitts, 1998). This gene encodes a protein that displays features of a transcriptional regulator, a developmental regulator and a master gene regulator, as proposed previously for chromosomal translocation genes in acute tumors (Rabbitts, 1991). In this chapter we summarize data illustrating these points, which show the normal role of Lmo2 in


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hematopoiesis and vascular formation and the aberrant role in the development of T cells prior to tumor formation. THE Lmo2 GENE IS A PARADIGM FOR ACTIVATION OF A TRANSCRIPTION REGULATOR BY CHROMOSOMAL TRANSLOCATIONS The LMO Family of Genes The LMO family of genes comprises four known members (Fig. 27.1), of which two are found at the breakpoints of chromosomal translocations in T-cell acute leukemias. This family of genes was uncovered by the discovery of LMO1 (previously called RBTN1 or TTG1) at the junction of the chromosomal translocation t(11;14)(p15;q11) in a T-cell acute leukemia (TALL). The transcription unit was first observed in a T-cell line (Boehm et al., 1988a,b). The mRNA sequence was subsequently obtained from its cDNA sequence (Boehm et al., 1990b; McGuire et al., 1989) and shown to encode a protein essentially consisting of two zinc-binding LIM domains (Boehm et al., 1990a). Using an LMO1 probe, the two related genes LMO2 and LMO3 were isolated (previously called

RBTN2 or TTG2 and RBTN3, respectively) (Boehm et al., 1991; Foroni et al., 1992), of which LMO2 was also found at the junction of the chromosomal translocation t(11;14)(p13;q11) in T-ALL (Boehm et al., 1991; RoyerPokora et al., 1991). A fourth member of the LIM-only family, designated LMO4, was also described (Grutz et al., 1998b; Kenny et al., 1998; Racevskis et al., 1999; Sugihara et al., 1998). This gene is located on chromosome 1, band p22.3 (Tse et al., 1999), and no association of this gene with a specific chromosome breakpoint has yet been reported. The LMO Family of Genes Encode LIM-Domain-Only Proteins The unique feature of the LMO-derived protein sequences is that they are small proteins comprising two tandem LIM domains. The LIM domain is a zinc-binding finger-like structure (Archer et al., 1994), with a structural similarity to the DNA-binding GATA fingers (Perez-Alvarado et al., 1994; Sanchez-Garcia and Rabbitts, 1994). Each LIM domain comprises two LIM fingers, each having about 17 amino acids in a finger region held in place by a zinc atom coordinated by cysteine and histidine or aspar-

Figure 27.1. The LMO family of genes and T-cell oncogenes. The LMO gene family (LIM-Only, genes, previously called RBTN and TTG genes) has four known members. LMO1 (previously called RBTN1/TTG1) was identified first and then LMO2 (previously RBTN2/TTG2) and LMO3 (previously RBTN3). Subsequently, LMO4/Lmo4 was described. LMO1 and LMO2 are both located on the short arm of chromosome 11 and are both involved in independent chromosomal translocations in human T-cell acute leukemia. As yet, LMO3 and LMO4 have not been found associated with any chromosomal translocations. The chromosome locations of the LMO genes in humans and mice are shown, together with the chromosomal translocations at which LMO1 and LMO2 are found in leukemias. nk, none known.

THE NORMAL ROLE OF Lmo2 IN CELL FATE

tate residues. The characteristic feature of the LIM domain is the separation of the two LIM fingers by only two amino acids. A function was found for the LIM domain in protein interaction (Schmeichel and Beckerle, 1994; ValgeArcher et al., 1994; Wadman et al., 1994) and, as yet, no case of a direct, specific LIM—nucleic acid interaction has been reported, despite the similarity to the GATA DNA-binding zinc finger. The role of the LIM protein LMO2 in multiprotein complexes in hematopoiesis (see the following sections) provides a framework for understanding the function of this protein in development and leukemogenesis. Distinct t(11;14) Chromosomal Translocations Activate the LMO1 and LMO2 Genes The LMO1 and LMO2 genes are both involved in chromosomal translocations in T-cell acute leukemias, namely, t(11;14)(p15;q11) and t(11;14)(p13;q11) respectively (Rabbitts, 1994). Both of these distinct chromosomal translocations involve the T-cell receptor (TCR) chain gene at 14q11, and in addition LMO2 is rearranged with the TCR gene in the t(7;11)(q35;p13) in a subset of T-ALL (Sanchez-Garcia et al., 1991). LMO-associated chromosomal translocations appear to occurr by an error of the RAG-mediated V-D-J recombination process, as sequence analysis of the breakpoints on chromosome 11p13 and 11p15 detected recombinase signal-like sequences at the junctions (at least heptamers, if not nonamers) and because the joints on chromosome 14 in the TCR locus, or on chromosome 7 in the TCR locus, occur precisely at the end of TCR D or J-segments (Boehm et al., 1988a, 1988b; Sanchez-Garcia et al., 1991). This association with mistakes of VDJ recombinase seems pertinent to the stage of the T-cell differentiation at which initiation of T-cell tumors occurs (see later in this chapter). THE NORMAL ROLE OF Lmo2 IN CELL FATE DETERMINATION DURING DEVELOPMENT LMO2 Is Necessary for Both Primitive and Definitive Hematopoiesis In order to gain insights into the function of the LMO genes in tumorigenesis, an integrated ap-

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proach was adopted to understand normal function at the biological and molecular levels of LMO proteins and to utilize this information to explore the function in tumors. In mouse embryogenesis, hematopoiesis begins at about embryonic day 7.5 (E7.5) in yolk sac blood islands (yolk sac erythropoiesis). The first hematopoietic stem cell activity appears in the aortagonad-mesonephros (AGM) region at about E10.5 (definitive hematopoiesis). As a first step, gene targeting was used to introduce null mutations in mouse Lmo2 gene, which showed that Lmo2 is necessary for yolk sac erythropoiesis in mouse embryogenesis (Warren et al., 1994). The Lmo2 null mutant embryos die at about E9.5, due to the lack of yolk sac erythropoiesis. Furthermore, the use of ES cells with null mutations of both alleles of Lmo2 in chimeric mice showed that definitive hematopoiesis, including lymphopoiesis and myelopoiesis, fails completely in the absence of the Lmo2 gene (Yamada et al., 1998). Thus, Lmo2 must function in a cellautonomous way during the early stages of definitive hematopoiesis. This important function must be at the level of the pluripotent stem cell, at the level of the immediate multipotential progeny or even perhaps before this, when mesoderm gives rise to these precursors (Fig. 27.2 and the next section). The Lmo2 Gene Functions in Vascular Formation in Mouse Embryogenesis The earliest expression of Lmo2 protein is observed in yolk sac mesodermal cells and in posterior embryonic mesoderm at E7.5. During yolk sac hematopoiesis, Lmo2 is expressed in yolk sac blood islands. Using a lacZ knock-in of the endogenous Lmo2 gene to establish a reporter system for Lmo2 expression in embryogenesis (Yamada et al., 2000), we found that from E8.5, Lmo2 has a more specific intraembryonic expression pattern in endothelial cells and blood progenitor cells. At E10.5, when definitive hematopoiesis is thought to begin in the AGM region, Lmo2 is expressed in endothelial cells of the vascular system and multipotent blood progenitor cells (Delassus et al., 1999; Yamada et al., 2000) (Fig. 27.3). Additional sites of expression were observed in the limb buds and hippocampus. This expression pattern of Lmo2 in yolk sac and blood progenitor cells is consistent with its function in primitive and definitive

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Figure 27.2. Lmo2 is necessary for adult hematopoiesis in mice. Lmo2 null mutations introduced into the germ line of mice cause lethality at E9—10 due to a failure of yolk sac erythropoiesis (Warren et al., 1994). In addition, the contribution of embryonic stem cells with homozygous null mutation of Lmo2 to hematopoietic cell populations showed that definitive hematopoiesis in adult mice is dependent on Lmo2 (Yamada et al., 1998). Thus, the Lmo2 gene product is required for both embryonic and adult hematopoiesis in mice.

Figure 27.3. Whole-mount -galactosidase staining of Lmo-lacZ>\ heterozygous embryos shows Lmo2 expression in vascularization. A targeted ES clone with one null Lmo2 allele, in which the lacZ gene was knocked-in as a gene fusion with Lmo2, was injected into blastocysts, and germ-line transmission of the Lmo2 null allele was obtained. Heterozygous mice were crossed with C57/Bl6 mice and embryos obtained at E10.5 and E12.5. These embryos were stained with Xgal as a substrate for -galactosidase activity. Dark staining denotes areas of -galactosidase due to Lmo2—lacZ gene expression. A: E10.5 embryo. X-gal staining was seen on major blood vessel walls and capillaries of whole body. B: E12.5 embryo. In addition to -galactosidase staining of vasculature, prominent staining was found in the limb buds and the tip of tail. C: Histological section of an E12.5 embryo stained for -galactosidase and counterstained with eosin. Blood vessel endothelial cells can be observed to be positive in a background of eosin stained tissue. D: Histological section of the limb bud of an E12.5 embryo stained for -galactosidase and counterstained with eosin. The region beneath the apical ectodermal ridge of a limb bud is -galactosidase positive.

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Figure 27.4. Lmo2 is necessary for angiogenesis in mice. The formation of hematopoietic cells and vascular endothelium is thought to occur through a putative precursor cell, the hemangioblast, which derives from the posterior mesoderm. Formation of the capillary network de novo (vasculogenesis) precedes a remodeling of the existing endothelium (angiogenesis). Studies with the Lmo2-lacZ null ES cell derivatives in chimeric mice showed that the Lmo2 gene is necessary only for the latter process of angiogenesis. The failure of adult hematopoiesis may be due to the failure of angiogenesis in null Lmo2 cells, as hematopoietic cells may arise in large blood vessels that do not form in the absence of Lmo2. An alternative origin for hematopoietic cells is directly from hemangioblasts.

hematopoiesis. It is important to note in relation to development of T-cell tumors that Lmo2 is not expressed in mature T cells. The first intraembryonic hematopoietic stem cell activity is thought to appear in the AGM region at around E10.5, and it is believed that hematopoietic stem cells are derived from endothelium of large arteries, such as dorsal aorta (Medvinsky and Dzierzak, 1996; Mu¨ller et al., 1994). In mouse embryogenesis, hemangioblasts (putative common precursors of endothelium and blood cells) are proposed to arise from unspecified posterior mesoderm and thus the primary capillary network is made from these hemangioblasts. This first process of vasculature construction in which the capillary network is formed is called vasculogenesis. The more mature vascular system is made by the remodeling

of primary capillary network, in the process called angiogenesis (Hanahan and Folkman, 1996). The earlier role of Lmo2 before specification of hematopoietic stem cells, especially in the construction of the vascular system, was studied by following the fate of Lmo2 null ES cells in chimeric mice (Yamada et al., 2000). Lmo2-null ES cells can contribute to the capillary network in chimeric mice before E9. At about E10, however, a marked vascular disorganization is observed in chimeric mice, due to failure of Lmo2 null ES cell contribution in endothelium. After E11, there is no contribution of Lmo2 null ES cells to the endothelium of large arteries. These results indicate that Lmo2 is necessary for angiogenesis but not for vasculogenesis (Fig. 27.4). As we discussed before, Lmo2 is expressed in both endothelial cells and blood progenitor

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cells, and has dual functions both in hematopoiesis and angiogenesis. Considering the close relationship between endothelium and blood cell specification, the role of Lmo2 during this critical stage of mouse development is pivotal.

THE MOLECULAR FUNCTION OF THE LM02 PROTEIN The Lmo2 Protein Appears to Act as a Bridging Molecule in Multiprotein Complexes The broad function of Lmo2 in mouse hematopoiesis appears to be related to the ability of Lmo2 to form protein complexes through its LIM domain. A particularly relevant observation was that the T-cell oncogenes Lmo2 and Tal/Scl1 were coexpressed in erythrocytes (Warren et al., 1994), suggesting a possible synergy between the function of the two proteins. This turned out to be even more germane, since it was shown that the Lmo2 and Tal1 proteins could interact directly with each other (ValgeArcher et al., 1994; Wadman et al., 1994) through the LIM domains of Lmo2. The ability of the LIM domain to bind various proteins was shown for Lmo2, which can

bind to GATA-1 (Osada et al., 1995; Wadman et al., 1997) and to Ldb1/Nli1 protein (Agulnick et al., 1996; Bach et al., 1997; Jurata et al., 1996; Wadman et al., 1997). LMO1 was also shown to interact in vivo with LDB1 in T-ALL cells and neuroblastoma cell lines (Valge-Archer et al., 1998). This array of interactions led to the observation that Lmo2 can be found in an oligomeric complex in erythroid cells that involves Tal1, E47, Ldb1, and GATA-1 (Wadman et al., 1997). This complex is able to bind DNA through the GATA and bHLH components, thereby recognizing a unique bipartite DNA sequence comprising an E box (CANNTG) separated by about one helix turn from a GATA site, with Lmo2 and Ldb1 proteins seeming to bridge the bipartite DNA-binding complex (Fig. 27.5). These findings suggest that Lmo2 can be part of a transcription complex in hematopoiesis, and that this complex binds to and controls the expression of target genes during development. Variation in Multiprotein Transcription Factor Complexes in Hematopoietic Development It is possible that different Lmo2-containing complexes may exist in different hematopoietic

Figure 27.5. Lmo2 participates in complexes that can bind bipartite DNA sites. The LIM domains of Lmo2 function in protein interaction. The ability of the Lmo2 protein to interact with Tal1 and GATA-1 (in conjunction with Ldb1) appears to facilitate the formation of a complex in which two DNA-contacting regions comprise a Tal1—E47 dimer, binding an E box (CANNTG), and a GATA-1 molecule, binding a GATA site, as part of an erythroid complex. In model experiments (Wadman et al., 1997), the two parts of the bipartite DNA recognition sequence are separated by approximately one turn of the DNA helix. The function of this complex is presumably to bind to target genes and regulate their expression in erythropoiesis. If other complexes of this type, involving Lmo2 with different proteins, occur at other stages of hematopoiesis, these might bind and control distinct sets of target genes.

THE LMO2 PROTEIN IN T-CELL ACUTE LEUKEMIA

cell types, which may differ in the types of protein factors expressed and may control distinct sets of target genes. The involvement of Lmo2, Tal-1, and GATA proteins in common DNA-binding complexes during hematopoiesis suggests a role for the complex in regulation of downstream target genes, perhaps explaining why the null mutation of each of these genes leads to lack of primitive erythropoiesis (Pevny et al., 1991; Robb et al., 1995; Shivdasani et al., 1995; Warren et al., 1994). Conversely, there are differences in some specific aspects of hematopoiesis related to the individual null mutations, suggesting that each of the components can act in separate complexes at different stages of hematopoiesis. It is possible that Lmo2/Tal-1/GATA-1 complex plays an important role at a certain stage of erythroid development by regulating (positively and/or negatively) target genes. A function in other lineages (myeloid and lymphoid) is not readily explained by this complex. However, Gata2 is another transcription factor that can bind to Lmo2, and a broad arrest of blood cell development is observed in Gata-2 knockout mice. Therefore, an Lmo2/Tal-1/GATA-2 complex might play a role in progenitor cells before Lmo2/Tal-1/GATA-1. The more extensive hypothetical mechanism of protein complex transition during the progress of erythroid differentiation was presented by Sieweke and Graf in their cocktail party model (Sieweke and Graf, 1998). In this model, as differentiation proceeds, Gata2 of a Lmo2/Tal1/Gata2 complex, in an early hematopoietic progenitor, is replaced by Gata1 in erythroid/megakaryocytic progenitor cells. In a later stage, Lmo2-Tal1 could be replaced by FOG (Friend of GATA) (Tsang et al., 1997). Thus, the composition of the Lmo2containing complexes could be changed in a one-joins/one-leaves manner. This model explains the finding that overexpression of Lmo2 in the proerythroblastic cell line negatively regulates the erythroid differentiation (Visvader et al., 1997), possibly by preventing interaction of GATA-1 with FOG. A similar model could be applied to T-cell differentiation. The enforced expression of Lmo2 in CD2-Lmo2 transgenic mice causes T-cell differentiation block before overt tumor progression (Rabbitts, 1998).

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THE LMO2 PROTEIN IN T-CELL ACUTE LEUKEMIA Inhibition of Differentiation by Lmo2 Precedes T-cell Leukemia in Transgenic Mice The oncogenic activity of Lmo2 (also Lmo1) was studied in several transgenic mouse models. Emulation of the enforced LMO1 or LMO2 human T-cell expression after chromosomal translocation was achieved using transgenic mouse expression of Lmo1 (Aplan et al., 1997; Fisch et al., 1992; McGuire et al., 1992) and Lmo2 in the T-cell lineage (Fisch et al., 1992; Larson et al., 1994, 1995, 1996; Neale et al., 1995). Enforced Lmo2 expression results in clonal T-cell leukemia arising in the transgenic mice with a long latency. It indicates that the transgenes are necessary but not sufficient to cause tumors in these models, as is the case for many transgenic oncogene models (Adams and Cory, 1991), and that mutations in other oncogenes or tumor suppressor genes occur before development of overt disease. The long latency period before tumors arise in Lmo2 transgenics facilitated detailed studies of the possible effects of oncogene overexpression in the asymptomatic thymi of transgenic mice (Larson et al., 1994, 1995; Larson et al., 1996; Neale et al., 1995). An outline of normal T-cell differentiation is shown in Figure 27.6. A marked accumulation of immature CD4\, CD8\, CD25>, CD44> T cells (herein referred to as double negative or DN T cells) was observed in transgenic thymi compared to nontransgenic littermates, an effect that was exacerbated in mice transgenic for both Lmo2 and Tal1 (Larson et al., 1996). Thus, the role of the transgene products is to cause an inhibition in T-cell differentiation, which appears reversible, presumably by antigenic stimulation occurring after birth, since different transgenic mice exhibit different levels of DN cell accumulation. It is of note that the DN T-cell population is RAG VDJ recombinase positive. Chromosomal translocation breakpoint sequencing (discussed in a previous section) suggests that TCR rearrangement, mediated by recombinase, causes the formation of the aberrant chromosomes in humans, and in turn suggests that T-cell acute

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Figure 27.6. Inhibition of T-cell differentiation model for Lmo2 function. Bone marrow (BM) produces pre-T cells (designated as TN because these cells do not yet express typical T-cell markers CD3, CD4, and CD8), which become DN cells (having a CD4\, CD8\, CD25>, CD44> marker phenotype). These do not yet express functional T-cell receptors (TCR) but begin to express the RAG recombinase proteins for performing VDJ recombination. Further differentiation of this immature DN T-cell subset results after TCR rearrangement, giving mature functional TCR-bearing T cells. The Lmo2 transgenic mice and double Lmo2-Tal1 transgenics (Larson et al., 1996) accumulate the DN cells, apparently as a result of differentiation inhibition caused by the transgene(s). Because the LMO1- and LMO2-associated chromosomal translocations seem to result from RAG-mediated recombinase errors, we proposed that this same target population of DN T cells is affected in humans with the chromosomal translocation (Rabbitts, 1998). This would be the population from which the overt tumors arise.

leukemia precursors in humans acquire the LMO2 chromosomal translocations within the DN T-cell population. If so, this would produce a cell with inhibited differentiation, analogous to those of the transgenic mice (Fig. 27.6), providing the precursors for overt tumor to arise. Models Involving Protein Interaction Explain the Role of LMO2 in T-cell Acute Leukemia The consistent clinical phenotype of T-cell acute leukemia, irrespective of type of chromosomal translocation and activated oncogene, could be partly explained by interaction between T-cell oncogenic proteins. The putative role of Lmo2 function in T-cell development is probably in the very early stage of T-cell specification. A putative transcription factor complex including Lmo2 might regulate cell fate after the enforced expression of LMO2, either by chromosomal

translocation or by transgenesis. In a search for evidence of such Lmo2-containing complexes, T-cell lines derived from CD2-Lmo2 transgenic mice were used as a source of Lmo2 protein complexes (Grutz et al., 1998a). This work resulted in the detection of a Lmo2 complex, which, like its analogue in erythroid cells, binds to a bipartite recognition site, but in this case within T-cell lineage. The complex recognizes a dual E-box motif, in which the two E-box sequences are separated by about one DNA helical turn (Grutz et al., 1998a) rather than the E-box—GATA motif previously detected in erythroid cells (Wadman et al., 1997). Analysis of the components of this complex showed that E47-Tal1 bHLH heterodimeric elements were present as well as Lmo2 and the Ldb1 proteins (Fig. 27.7A). By analogy with arguments proposed for the normal erythroid E-box—GATAbinding complex, a possible role for the E-box— E-box—binding T-cell complex may be to regu-

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Figure 27.7. Molecular models for the effect of enforced LMO2 expression in T cells. A: An Lmo2-containing multiprotein complex that binds to a bipartite E-box—E-box sequence has been found in T cells of Lmo2expressing transgenic mice (Grutz et al., 1998a). This novel complex is formed that can recognize a dual E-box motif (CANNTG-CANNTG), apparently involving bHLH heterodimers linked by Lmo2 and Ldb1 proteins. Like the distinct complex detected in erythroid cells, this Lmo2-containing complex may bind to dual E-box motifs near target genes and accordingly control their expression. B: The sequestration mode of Lmo2 function in T-ALL (Rabbitts, 1998). The Lmo2-containing multiprotein complex serves not necessarily to bind target gene sequences but to sequester proteins from their natural functions. In the example shown, Ldb1 is limited by its being removed into the Lmo2-containing complex, and thus by mass action the amount of Ldb1-Y complex is limited. In turn, concentrations of putative Y-Z, free Y and Z, as well as other possible interactions, may be altered. In such a model, enforced Lmo2 expression causes biochemical alterations to the cell.

late expression of specific sets of target genes, which, based on the difference in DNA-binding site, would differ from those putative genes controlled by the Lmo2 multimeric complex in erythroid cells. Alternatively, LMO2 may work in T-ALL by a similar mechanism to which overexpression of Lmo2 in erythroid lineage suppresses the differentiation of that lineage. Thus, LMO2 may have natural binding partners in T-cell development, but the enforced expression may influence protein interaction equilibria by simple mass action. In this sequestration model (Rabbitts, 1998), the interaction of Lmo2 with protein X (e.g., Ldb1 as illustrated in Fig. 27.7B) causes a shift in a putative equilibrium of protein interactions, po-

tentially resulting in the aberrant function of members of this equilibrium. In the context of this model, it may be that the Lmo2 multimeric complex identified in T-ALL (Grutz et al., 1998a) does not itself influence target gene expression but rather does so indirectly in a mass action effect. In support of such a model is the finding that the proportion of transgenic mice expressing Lmo1 or Lmo2 that develop tumors is related to the transgene copy number (Fisch et al., 1992; McGuire et al., 1992); the more transgenic copies, the more tumors occur, suggesting a titration of protein components. Furthermore, the Drosophila Lmo gene homologue (Boehm et al., 1990a; Zhu et al., 1995), when mutated, causes wing defects that can be modi-

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fied by the dosage of the Drosophila gene Chip (equivalent to Ldb1) (Fernandez-Funez et al., 1998; Milań et al., 1998; Shoresh et al., 1998; Zeng et al., 1998), suggesting that Chip/Ldb can work by titrating binding partners.

CONCLUSION A distinction has been drawn between the general types of gene activated by chromosomal translocations in chronic leukemias and acute leukemias (Rabbitts, 1991). This in turn has led to the master gene model of chromosomal translocations in acute leukemia, in which it was proposed that master gene regulators are activated (by enforced expression) or created as chimeric proteins, and these gene products are at the head of transcription cascades of target genes (Rabbitts, 1991), controlling cell fate and thus involved in developmental processes. Many examples of genes fitting this general criterion have been published (reviewed in Rabbitts, 1994). LMO2 is an oncogenic transcription regulator that serves as a paradigm of such a master regulator. The gene is involved in T-ALL after chromosomal translocations, but normally is important as a regulator of yolk sac erythropoiesis and definitive hematopoiesis. The gene is also specifically necessary for angiogenesis in vascular formation. Lmo2 overexpression in erythroid cells and T cells leads to differentiation arrest, which, in the case of T cells, is the prerequisite for T-cell tumor development. Lmo2 overexpression can result in aberrant complexes that recognize different DNA sequences, underlining the importance of LIM domain protein and its protein complex throughout the critical stages of tumor development. Thus, it is perhaps an ironic twist of fate that the normal role of LMO2 in angiogenesis may provide a novel target for inhibition of tumor growth.

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ACKNOWLEDGMENTS

Boehm, T., Foroni, L., Kennedy, M., and Rabbitts, T. H. (1990a). The rhombotin gene belongs to a class of transcriptional regulators with a potential novel protein dimerisation motif. Oncogene 5, 1103— 1105. Boehm, T., Greenberg, J. M., Buluwela, L., Lavenir, I., Forster, A., and Rabbitts, T. H. (1990b). An unusual structure of a putative T cell oncogene which allows production of similar proteins from distinct mRNAs. EMBO J. 9, 857—868.

Our own work cited in this review was supported by the Medical Research Council and by grants from the Leukaemia Research Fund (UK) and the National Foundation for Cancer Research (USA).

Boehm, T., Foroni, L., Kaneko, Y., Perutz, M. P., and Rabbitts, T. H. (1991). The rhombotin family of cysteine-rich LIM-domain oncogenes: distinct members are involved in T-cell translocations to human chromosomes 11p15 and 11p13. Proc. Natl. Acad. Sci. USA 88, 4367—4371.

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Zhu, T.-H., Bodem, J., Keppel, E., Paro, R., and Royer-Pokora, B. (1995). A single ancestral gene of the human LIM domain oncogene famly LMO in Drosophila: characterisation of the Drosophila Dlmo gene. Oncogene 11, 1283—1290.

CHAPTER 28


THE ACETYLTRANSFERASES CBP AND p300: MOLECULAR INTEGRATORS OF HEMATOPOIETIC TRANSCRIPTION INVOLVED IN CHROMOSOMAL TRANSLOCATIONS GERD A. BLOBEL Department of Pediatrics, Division of Hematology, Children’s Hospital of Philadelphia University of Pennsylvania School of Medicine, Philadelphia, PA 19104

INTRODUCTION Differentiation of pluripotent hematopoietic stem cells into mature circulating blood cells is coordinated by a complex series of transcriptional events. Numerous transcription factors have been identified whose expression is highly lineage restricted within the hematopoietic system. However, the control of tissue-specific and developmentally correct gene expression is not achieved by a single transcription factor. Rather, unique combinations of restricted and widely expressed nuclear factors account for the enormous specificity and diversity in gene expression profiles. Recently, two highly related and widely expressed molecules, CREB-binding protein (CBP) and p300, have emerged as important cofactors for a broad number of transcription factors both within and outside the hematopoietic system. The complexity of CBP/p300mediated gene regulation is underscored by the observation that CBP/p300 play a role in both transcriptional stimulation and repression. Moreover, CBP/p300 support cellular differenti-

ation, but can also cooperate with gene products that interfere with it. Thus, the precise functions of p300/CBP depend on promotor context and cellular environment. A series of recent reviews (Eckner, 1996; Giles et al., 1998; Shikama et al., 1997) serve as excellent guides through the large number of factors interacting with CBP/p300. This chapter focuses on the role of CBP/p300 in the transcriptional control of hematopoietic cell differentiation. Naturally, the survey of a burgeoning field such as this might not do justice to all contributions. Therefore, I apologize to those whose work is not represented here. Following an initial general overview of CBP/ p300, the hematopoietic transcription factors regulated by CBP/p300 are described in a systematic fashion. Subsequently, leukemia-associated chromosomal translocations involving the CBP/p300 genes are discussed briefly (See Chapter 26 in this book). This is followed by an attempt to conceptualize our knowledge by discussing mechanistic aspects of CBP/p300 function.


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THE ACETYLTRANSFERASES CBP AND p300

OVERVIEW; CBP/p300 STRUCTURE AND FUNCTION CBP was originally discovered based on its ability to interact with cAMP response elementbinding protein (CREB) (Chrivia et al., 1993), and p300 was isolated as a cellular target of the adenoviral oncoprotein E1A (Eckner et al., 1994). While E1A binds to various cellular proteins, including the Rb family of tumor suppressor proteins, its ability to block cell differentiation and to induce cell cycle progression in many cell types depends at least in part on its interaction with CBP/p300 (see also below). The functions of CBP and p300 appear interchangeable in many published reports, yet both molecules also fulfill unique roles as revealed by gene inactivation studies (Kawasaki et al., 1998; Yao et al., 1998; see below). Here I refer to them as CBP/p300 unless the study under review involved only one of the two molecules. Since the mid-1990s, numerous transcriptional regulators have been found to interact

with CBP/p300 (see Fig 28.1 for examples; for review, see Shikama et al., 1997). CBP/p300 are widely expressed and are believed to regulate gene expression in most cell types. Consistent with a function in a wide range of tissues, CBP-1, a Caenorhabditis elegans factor closely related to CBP/p300, acts at an early stage in development and is essential for all nonneuronal differentiation pathways (Shi and Mello, 1997). In mammals the situation is more complex because of the existence of at least two such molecules, CBP and p300. The complexity of protein-protein interactions surrounding CBP/ p300 has led to their description as molecular integrators. The ability of CBP/p300 to integrate multiple transcriptional signals is illustrated by the observation that many nuclear factors that depend on CBP/p300 can synergize with each other when bound to the same promoter in cis. On the other hand, mutual transcriptional inhibition between these factors might occur when they are bound to different promoters. Inhibition has been proposed to result at least in some cases from competition of

Figure 28.1. Structure of CBP adapted from Shikama and co-workers (Shikama et al., 1997). Not all known CBP-interacting proteins are shown. Amino acid numbers indicated on the top are approximate. HAT, histone acetyltransferase domain; CH, cysteine/histidine-rich region; BROMO, bromodomain. Bromodomains are found in most histone acetyltransferases and in many chromatin-associated factors. Bromodomains specifically bind to acetylated lysine (Dhalluin et al., 1999). CBP and p300 interact with tissue-specific (e.g., MyoD), broadly expressed (e.g., nuclear receptors), and general (e.g., TFIIB and TBP) transcription factors. In addition, CBP and p300 interact with oncoproteins, including c-Jun and c-Fos, and tumor-suppressor proteins such as p53. CBP and p300 also interact with other HAT-containing molecules, such as p/CAF, SRC-1, and ACTR. Finally, CBP and p300 regulate the activity of signal-dependent transcriptional activators such as CREB and the STATs.

ROLES OF CBP/p300 IN HEMATOPOIESIS

these factors for limiting amounts of CBP/p300 in the nucleus (Horvai et al., 1997; Kamei et al., 1996). Genetic evidence for the idea that CBP/ p300 is limiting stems from the discovery that patients who lack one allele of CBP suffer from Rubinstein-Taybi syndrome (RTS), a disease characterized by mental retardation, craniofacial abnormalities, broad toes and thumbs, and an increased risk for tumors (Petrij et al., 1995; see also below). Similarly, mice lacking one allele of CBP or p300 have reduced viability, and, in the case of CBP, display a phenotype reminiscent of RTS (Tanaka et al., 1997; Yao et al., 1998). Finally, normal development of Drosophila embryos is highly dependent on CBP gene dosage (Akimaru et al., 1997a, 1997b). Of note, some but not all of the CBP/p300 interactions are regulated by cellular signals. For example, phosphorylation regulates the interaction of CBP/p300 with the transcription factors CREB, p65 NFkB, c-Jun, IRF3, and Smad3, and hormones such as estrogens, glucocorticoids, and retinoic acid stimulate CBP/p300 binding to nuclear hormone receptors. To add to the complexity, CBP/p300 can stimulate both the activating and repressive functions of certain nuclear factors. For example, while CBP/p300 increase p53 activity on certain p53-dependent promoters (Avantaggiati et al., 1997; Gu et al., 1997; Lill et al., 1997), CBP/p300 can also augment p53-mediated transcriptional repression on others (Lee et al., 1999). Thus, promoter and cellular context are critical determinants of transcriptional output. A breakthrough in the understanding of CBP/ p300 function was the discovery that they act not only in a stoichiometric fashion, as is the case for most transcriptional cofactors, but that they also possess enzymatic activity. The laboratories of Kouzarides and Nakatani (Bannister and Kouzarides, 1996; Ogryzko et al., 1996) found that CBP/p300 possess intrinsic histone acetyl transferase (HAT) activity. Acetylation of histones is associated with a relaxed chromatin configuration, which is thought to facilitate transcription factor access to DNA. For example, work by Hebbes and collegues demonstrated a strong correlation between the presence of acetylated core histones and DNase I sensitivity at the chicken -globin locus (Hebbes

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et al., 1994). DNase I sensitivity occurs before transcription is initiated and might reflect a state poised for transcriptional activation. The importance of a balance between the acetylated and nonacetylated state of histones in transcriptional regulation is underscored by the discovery that certain transcriptional repressors are associated with histone deacetylases (for review see (Pazin and Kadonaga, 1997)). More recently, CBP/p300 have been shown to acetylate nonhistone nuclear proteins, including the tumor suppressor protein p53 (Gu and Roeder, 1997; Liu et al., 1999; Sakaguchi et al., 1998), dTCF (Waltzer and Bienz, 1998), EKLF (Zhang and Bieker, 1998), GATA-1 (Boyes et al., 1998a; Hung et al., 1999), NF-Y (Li et al., 1998), the basal transcription factors TFIIE and TFIIF (Imhof et al., 1997; Wong et al., 1998), and the architectural transcription factor HMG I(Y) (Munshi et al., 1998). In the case of p53, acetylation strongly increases DNA binding in vitro, providing a potential mechanism for CBP/p300-mediated transcriptional control (Gu and Roeder, 1997; Liu et al., 1999; Sakaguchi et al., 1998). Given the large number of factors regulated by CBP/p300 it is likely that more will be found to be acetylated by CBP/p300 or by the CBP/p300-associated HATs, such as p/CAF, SRC-1 and ACTR (Chen, H., et al., 1997; Spencer et al., 1997; Yang et al., 1996). Additional mechanisms by which CBP/p300 might operate are discussed below.

ROLES OF CBP/p300 IN HEMATOPOIESIS The viral oncoprotein E1A has been an invaluable tool to test the requirements of CBP/p300 in gene expression and differentiation in various cell types. E1A binds to CBP/p300 via defined domains and blocks their functions (Bayley and Mymryk, 1994; Moran, 1993). Indeed, in numerous studies the first clues suggesting a requirement for CBP/p300 during gene regulation derived from experiments showing that forced expression of E1A, but not mutant forms of E1A defective for CBP/p300 binding, interfered with expression of certain myeloid, erythroid, and B-lymphocytic genes. The following recurring themes are found in many of the studies summarized below. First, the activity of most transcription factors that

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Figure 28.2. Structure of CBP indicating docking sites for hematopoietic transcription factors. Details as in Figure 28.1.

interact with CBP/p300 is sensitive to coexpressed E1A. Inhibition by E1A can occur even if the transcription factor interaction occurs outside the E1A-binding domain of CBP/p300, suggesting that simple competition for CBP/ p300 binding cannot account for all of E1A’s effects. Second, stimulation of transcription factor activity by overexpressed CBP/p300 usually ranges between 2-fold and 10-fold in transient transfection assays, indicating that CBP/p300 are limiting under these conditions. Third, varying combinations of nuclear factors regulated by CBP/p300 display synergy during their activation of hematopoietic gene promoters. The following section is divided according to classes of CBP/p300-regulated hematopoietic transcription factors (summarized in Fig. 28.2) rather than according to hematopoietic cell lineages, since most transcription factors are expressed in multiple cell types. Introduction to these factors is kept to a minimum since they are discussed in detail in other chapters. c-Myb c-Myb is among the first hematopoietic transcription factors found to be regulated by CBP. c-Myb is the cellular counterpart of the v-Myb oncoprotein identified in the avian myeloblastosis virus (AMV). c-Myb expression is highest in progenitor cells of the myeloid, erythroid and

lymphoid lineages and is downregulated during maturation/differentiation of these cells (for review, see Weston, 1990). Forced expression of c-Myb blocks differentiation of erythroid, and myeloid cell lines (Clarke et al., 1988; McClinton et al., 1990; McMahon et al., 1988; Todokoro et al., 1988; Yanagisawa et al., 1991). Expression of a dominant interfering form of c-Myb results in enhanced erythroid differentiation (Weber et al., 1990) while treatment with antisense oligonucleotides directed against the c-Myb mRNA reduces proliferation of immature cells of the erythroid, myeloid, and T-lymphoid lineages (Anfossi et al., 1989; Gewirtz et al., 1989; Gewirtz and Calabretta, 1988). Disruption of the c-Myb gene in mice leads to lethal anemia during fetal liver hematopoiesis (Mucenski et al., 1991). Together with the leukemogenic potential of c-Myb, the above studies suggest that c-Myb functions in maintaining hematopoietic precursor cells in a proliferative state. CBP was found to stimulate both c-Myb and v-Myb transcriptional activity in transient transfection experiments (Dai et al., 1995; Oelgeschlger et al., 1996). c-Myb binds CBP in vivo and in vitro in a phosphorylation-independent manner at a site that overlaps with the CREBbinding domain of CBP. Expression of E1A, of antisense CBP RNA, or of dominant-negative CBP interferes with c-Myb-dependent transac-


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Figure 28.3. Expression of E1A blocks DMSO-induced differentiation of MEL cells. MEL cells expressing a conditional, estradiol-dependent form of E1A were left untreated (U), or were treated with DMSO (D), estradiol (E), or both (D/E). Cells were stained with benzidine, which stains hemoglobin (dark), and counterstained with May-Grunwald. Note the absence of benzidine-positive cells following estradiol-induced E1A activation (D/E). For details, see Blobel et al., 1998.

tivation (Dai et al., 1995; Oelgeschla¨ger et al., 1996). While CBP moderately enhances c-Myb activity (about 3-fold), the presence of another CBP-regulated DNA-binding protein such as NF-M strongly increases the effects of CBP in a synergistic fashion (Oelgeschla¨ger et al., 1996). Forced expression of E1A leads to a differentiation arrest of various cell types, indicating a requirement for CBP/p300 function during cellular differentiation. Therefore, it seems paradoxical that CBP also cooperates with gene products, such as c-Myb or v-Myb which block differentiation. A possible explanation is that factors inducing differentiation and those stimulating proliferation compete for the action of CBP, depending on their expression levels during cellular maturation (see also below), or depending on cellular signals that regulate their interaction with CBP/p300.

The Proteins Encoded by the E2A Gene Work from more than a decade ago demonstrated that E1A can repress the activity of the immunoglobulin 2b heavy-chain (IgH) and the kappa light-chain genes in lymphoid cells (Bergman and Shavit, 1988; Hen et al., 1985). However, at that time, the identity of transcription factors inhibited by E1A was unknown. Recent studies suggest that the basic helix-loop-helix (bHLH) proteins E47 and E12 might present critical targets for inhibition by E1A. E12 and E47, which are both encoded by the E2A gene (not to be confused with E1A), are essential regulators of B-cell gene expression (see Chapter 16 in this book). In most cell types, E12 and E47 proteins bind to DNA and regulate transcription as heterodimers with tissue-specific bHLH proteins, such as the hematopoietic transcrip-

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tion factor tal-1/SCL or the muscle-determining factors of the MyoD family. Remarkably, despite its broad distribution, only in B cells can E47 bind DNA and activate gene expression as a homodimer (Shen and Kadesch, 1995). Targeted disruption of the E2A gene in mice leads to perinatal death and a selective ablation of mature B cells (Bain et al., 1994; Zhuang et al., 1994). The cause of death is uncertain, but surprisingly there are no obvious abnormalities present in other hematopoietic and nonhematopoietic tissues (Bain et al., 1994; Zhuang et al., 1992, 1994). Work by Eckner and colleagues demonstrated that p300 forms a stable complex with E47 bound to its cognate DNA element (Eckner et al., 1996). In addition, p300 stimulates E47 activity in transient transfection experiments using a reporter gene driven by an intact IgH enhancer or by isolated E47-binding sites. p300 also interacts with bHLH proteins involved in myogenesis (Eckner et al., 1996), suggesting that it has the capacity to target various members of bHLH protein superfamily, which might include those involved in hematopoiesis. Together with the findings outlined below, this suggests a role for p300 in B-lymphoid gene expression. GATA-1 GATA-1, one of the best studied hematopoietic transcription factors, is a zinc finger protein involved in the regulation of virtually all erythroid-specific genes. GATA-1 is required for survival and maturation of primitive and definitive erythroid precursor cells (Fujiwara et al., 1996; Pevny et al., 1991, 1995; Weiss et al., 1994; Weiss and Orkin, 1995). In addition, GATA-1 plays a critical role during megakaryocytic proliferation and differentiation (Pevny et al., 1995; Shivdasani et al., 1997). GATA-1 can trigger terminal differentiation and cell cycle arrest when reintroduced into a GATA-1—deficient immortalized proerythroblastic cell line (Weiss et al., 1997). Among the multitude of genes regulated by GATA-1 are the globin genes, which are in turn under the influence of the locus control regions (LCRs). The LCRs, which contain multiple functionally important GATA-binding sites, are thought to act in part by regulating the chromatin structure at the globin gene loci (see

Chapter 1 in this book). Given that CBP has histone acetyltransferase activity, it is noteworthy that GATA-1 interacts with CBP in vivo and in vitro (Blobel et al., 1998). Interaction involves the zinc-finger region of GATA-1 and the E1A-binding domain of CBP. CBP strongly augments GATA-1 activity in transient expression assays (Blobel et al., 1998). Expression of a conditional form of E1A in the erythroid cell line MEL leads to a complete block in differentiation and to reduced expression of GATA-1—dependent genes, including the - and -globin genes (Fig. 28.3) (Blobel et al., 1998). These findings are consistent with a mechanism by which CBP/p300 mediate at least some of GATA-1’s functions in intact erythroid cells. In addition, other GATA factors including GATA-2 and GATA-3, which have distinct expression patterns, are also stimulated by CBP/ p300 (G. A. Blobel, unpublished). GATA-2 levels are high in progenitor cells and decline during erythroid maturation (Leonard et al., 1993; Sposi et al., 1992). In contrast, GATA-1 levels increase as cells mature (Leonard et al., 1993; Sposi et al., 1992). Thus, it is possible that as its levels rise, GATA-1 recruits CBP/p300 away from factors required for proliferation of precursor cells such as GATA-2 (Tsai et al., 1994) and c-Myb (Mucenski et al., 1991), employing them for the activation of differentiation-specific genes. One mechanism by which CBP regulates GATA-1 activity appears to involve direct acetylation of GATA-1 itself. Two reports showed that CBP and p300 acetylate GATA-1 at two highly conserved lysine-rich motifs near the zinc fingers (Boyes et al., 1998; Hung et al., 1999). In addition, CBP stimulates acetylation of GATA-1 in vivo at the same sites acetylated by CBP in vitro (Hung et al., 1999). In vivo acetylation of GATA-1 by CBP is inhibited by E1A but not by mutant E1A defective for CBP/ p300 binding (Hung et al., 1999), establishing a correlation between acetylation of GATA-1 and its transcriptional activity. While Boyes and colleagues (Boyes et al., 1998) reported that acetylation by p300 stimulates DNA binding of chicken GATA-1 to a single GATA site in vitro, no change in DNA binding upon acetylation was observed by Hung and co-workers (Hung et al., 1999). This discrepancy may be the result of using chicken GATA-1 and p300 versus murine


GATA-1 and CBP, respectively. However, several lines of evidence suggest that changes in DNA binding might not be the mechanism by which acetylation regulates GATA-1 activity in vivo. First, mutations in the acetylation sites do not affect DNA binding of mammalian expressed GATA-1 molecules but do affect the response to CBP/p300 (Boyes et al., 1998; Hung et al., 1999). Second, although CBP/p300 stimulate GATA-1 activity in transient transfection assays, no evidence exists showing that this stimulation is associated with an increase in DNA binding of GATA-1. Third, when assayed in the context of differentiating erythroid cells, mutations in either of the two acetylation motifs impair the ability of murine GATA-1 to trigger erythroid differentiation without affecting its ability to bind DNA (Hung et al., 1999). This indicates that the biological activity of the acetylation sites can be uncoupled from their putative role in DNA binding. While acetylation of GATA-1 does not affect its interaction with FOG, CBP, or with GATA-1 itself (Hung et al., 1999), it is possible that acetylation leads to changes in the conformation of GATA-1 or affects interaction with other as yet unidentified GATA-1 cofactors. NF-E2 Given the large number of CBP-interacting proteins, it is likely that the strong inhibitory effects of E1A on erythroid cell differentiation (see above) involves multiple CBP-interacting factors. Consistent with this notion, two other erythroid transcription factors, p45 NF-E2 and EKLF (see below), have been reported to interact with CBP. NF-E2 is composed of a hematopoietic-restricted p45 subunit and a widely expressed p18 subunit, which is a member of the maf family of proteins (Andrews et al., 1993a, 1993b; Igarashi et al., 1994; for review, see Blank and Andrews, 1997). Other p45-related molecules capable of interacting with maf family members include Nrf1, Nrf2, Nrf3, Bach 1, and Bach 2 (for references, see Kobayashi et al., 1999). Multiple functionally important NF-E2— binding sites are present in the - and -globin LCRs. Loss of a functional p45 gene leads to a pronounced defect in platelet formation, while globin gene expression and erythroid development are largely unaffected. This suggests that

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other members of the p45 family might substitute for p45 function in erythroid cells (see Chapter 2 in this book). In vitro binding studies showed that the p45 subunit of NF-E2 binds directly to CBP (Cheng et al., 1997). The study by Cheng and coworkers (Cheng et al., 1997) further suggests that CBP might participate in mediating the ligand-dependent stimulation of the thyroid hormone receptor by p45. This is of biological interest given the role of thyroid hormone during erythropoiesis (Braverman and Utiger, 1991). Although the functional and molecular consequences of the p45-CBP interaction have not been studied in detail, it is conceivable that NF-E2 cooperates with GATA-1 and EKLF in the formation at the LCR of a high-molecularweight transcription factor complex (enhanceosome; see below) surrounding CBP/p300. It is important to point out that NF-E2 activity on chromatinized templates cannot be attributed solely to the recruitment of histone acetyltransferases. A report by Armstrong and Emerson demonstrated that NF-E2 can disrupt chromatin structure on templates containing regulatory regions of the -globin locus, and that the NF-E2—associated chromatin-modifying activity is ATP dependent (Armstrong and Emerson, 1996). EKLF Another transcription factor regulated by CBP is the zinc finger—containing erythroid Kru¨ppellike factor EKLF (Miller and Bieker, 1993). EKLF is specifically required for the expression of adult - but not -globin genes, and loss of EKLF function leads to lethal thalassemia (Nuez et al., 1995; Perkins et al., 1995). Moreover, EKLF\\ mice carrying a human globin gene locus display an incomplete - to -globin switch, which occurs at the onset of adult bone marrow erythropoiesis (Perkins et al., 1996; Wijgerde et al., 1996). Interestingly, absence of EKLF also results in a loss of DNase 1 hypersensitive site formation at both the transgenic and endogenous -globin promoters (Wijgerde et al., 1996), consistent with a role of EKLF in remodeling chromatin at these promoters (see Chapter 5 in this book). EKLF can interact with both CBP/p300 and the CBP/p300-associated acetyltransferase p/

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Figure 28.4. Hypothetical model in which CBP (or p300) is recruited to the LCR by NF-E2, GATA-1, and EKLF, leading to acetylation of histones and transcription factors. It is possible that this high-molecular-weight complex also connects to the promoters of the globin genes through a looping mechanism.

CAF in transfected cells. However CBP and p300, but not p/CAF, acetylate EKLF in vitro (Zhang and Bieker, 1998). Acetylation most likely occurs at two residues (Lys-279 and Lys288) that are part of an inhibitory domain adjacent to the zinc-finger region. Metabolic labeling experiments using [H]acetate further suggest that EKLF is acetylated in vivo (Zhang and Bieker, 1998). CBP/p300, but not p/CAF, stimulate EKLF transcriptional activity in transient transfection experiments using the erythroleukemia cell line K562 (Zhang and Bieker, 1998). It will be interesting to determine whether acetyltransferase activity of CBP/p300 is required for stimulation of EKLF activity. Acetylation did not affect DNA binding of EKLF and the molecular consequences of acetylation are not yet known (Zhang and Bieker, 1998). Together, the above reports suggest that erythroid transcription factors controlling globin gene expression at the promoter regions and at the LCRs might act together in the formation of a high-molecular-weight complex in which GATA-1, NF-E2, and EKLF are linked via CBP/p300 (Fig. 28.4). Consistent with such a model is the observed synergy between GATA-1 and EKLF in transactivation experiments (Merika and Orkin, 1995).

C/EBP CCAAT-box/enhancer—binding proteins (C/ EBPs) belong to the basic region/leucine zipper class of transcription factors and play a role in the differentiation of a broad range of tissues (see Chapter 8 in this book). In the hematopoietic system C/EBP family members are expressed mostly in the myelomonocyctic lineage and participate in the regulation of macrophage- and granulocyte-restricted genes, such as the M-CSF receptor, G-CSF receptor, and GMCSF receptor genes among others (for review, see Lekstrom-Himes and Xanthopoulos, 1998; Yamanaka et al., 1998). Targeted disruption of the C/EBP, C/EBP, or C/EBP genes resulted in defects predominantly affecting the granulocytic lineage (Screpanti et al., 1995; Tanaka et al., 1995; Yamanaka et al., 1997; Zhang et al., 1997), while other hematopoietic lineages remained intact. C/EBP transcription factors are also critical mediators of inflammatory and native immune functions (for review, see Poli, 1998). Studies by Mink and colleagues (Mink et al., 1997) showed that C/EBP-dependent transcription is E1A sensitive and that overexpressed p300 stimulates C/EBP activity on the


macrophage/granulocyte-specific mim-1 promoter and, importantly, also on an endogenous C/EBP-regulated gene, called 126. Moreover, p300 increases the synergy between c-Myb and C/EBP. C/EBP binds to the E1A-binding region of p300 via its N-terminus. Overexpression of the minimal C/EBP-binding domain of p300 region reduced the activity of C/EBP presumably by interfering with the C/EBP/ p300 interaction (Mink et al., 1997). The Nterminus of C/EBP contains stretches of amino acids conserved among C/EBP family members, suggesting that other C/EBP molecules might also be regulated by CBP/p300 (Mink et al., 1997). Together, these results imply CBP/p300 during granulocytic gene expression.

Ets The Ets family of transcription factors is a diverse group of approximately 30 proteins that share a conserved DNA-binding domain (see Chapter 7 in this book). Ets-1 is expressed predominantly in lymphoid cells and is thought to regulate a number of lymphocyte-specific genes. Gene knockout studies demonstrated a role for Ets-1 in T-cell proliferation and survival (Bories et al., 1995; Muthusamy et al., 1995). Effects on B-cell differentiation were also observed (Bories et al., 1995; Muthusamy et al., 1995). Ets-1 and some of its relatives synergize with a number of transcriptonal regulators known to interact with CBP/p300, such as AP-1 (Wasylyk et al., 1990), and Myb (Dudek et al., 1992; Postigo et al., 1997; Ratajczak et al., 1998; Sharpiro, 1995). Especially striking is the frequently observed cooperativity between Ets-like factors and GATA-1 during the expression of several megakaryocyte-restricted genes, including the GPIIB (Lemarchandel et al., 1993), GPIX (Bastian et al., 1996), GP1b- (Hashimoto and Ware, 1995), the thrombopoietin receptor (c-mpl) (Deveaux et al., 1996), and PF4 genes (Minami et al., 1998). However, in some cases, it remains to be determined which of the many Ets-like factors cooperate with GATA-1 in vivo to control gene expression. The synergy of Ets proteins with CBP/p300-regulated factors led to the hypothesis that they too are regulated by CBP. Indeed, Yang and co-workers (Yang et al., 1998) showed that the activity of the Myb-

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and Ets-dependent promoter of the myeloidexpressed gene CD13/APN is sensitive to the expression of E1A but not of mutant E1A defective for CBP/p300 binding. Ets-1 activity is stimulated by coexpressed CBP, and Ets-1 associates with CBP in nuclear extracts. In vitro, the N-terminus of Ets-1 can form two contacts with CBP involving CBP’s CH1 and CH3 domains. In support of the functional importance of the physical interaction between Ets-1 and CBP, the authors demonstrated a good correlation between binding of Ets-1 to the the CH1 region and its ability to transactivate. In addition, Ets-1 coprecipitates with histone acetyltransferase activity, consistent with its association with CBP/p300 and/or other HATs in vivo (Yang et al., 1998). It is not yet known whether CBP acetylates members of the Ets family of proteins.

AML1 Another potentially leukemogenic transcription factor controlled by p300 is AML1 (Kitabayashi et al., 1998). The AML1 gene is rearranged in several distinct chromosomal translocations associated with acute myeloid leukemia (AML; t(8;21)), acute lymphatic leukemia (ALL; t(12;21)), and myelodysplastic syndrome (t3;21) (for review, see Look, 1997). The AML1 gene is the most frequent target in chromosomal translocations in human leukemias. AML1 constitutes a family of at least three factors derived from the same gene by alternative splicing (see Chapter 6 in this book). The AML1 gene products bind to DNA as heterodimeric complexes with CBF. Of note, the CBF gene itself is involved in a chromosomal rearrangement found in cases of AML (Look, 1997). Consistent with its broad expression pattern and the presence of functionally important AML1-binding sites in the promoters and enhancers of myeloid and lymphoid expressed genes, knockout studies revealed that both AML1 and CBF genes are essential for the formation of all definitive blood lineages (Niki et al., 1997; Okuda et al., 1996; Sasaki et al., 1996; Wang et al., 1996a, 1996b). AML1b and p300 associate in vivo and in Far Western blots, and p300 stimulates AML1b activity on the myeloperoxidase promoter in

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transient transfection experiments (Kitabayashi et al., 1998). Overexpression of the (t8;21) translocation product AML1-ETO, also called AML1MTG8, in the IL-3—dependent myeloid cell line L-G interferes with G-CSF—induced differentiation along the neutrophilic lineage. Forced expression of wild-type AML1b, one of the AML1 isoforms that contains an activation domain, can overcome the effects of AML1-ETO and restore differentiation (Kitabayashi et al., 1998). In contrast, AML1a, which lacks this activation domain, is inactive in this assay. The differentiation-inducing ability of AML1b constructs is further enhanced by coexpression of p300 and correlates well with their ability to interact with p300 (Kitabayashi et al., 1998). This indicates that p300 plays a role in myeloid cell differentiation and suggests that the rearranged AML genes found in chromosomal translocations act as dominant negative alleles. The latter notion is consistent with the recent finding that AML-ETO associates with a transcriptional repressor complex containing histone deacetylases and that this deactylase complex is required for blocking differentiation of myeloid cells (Gelmetti et al., 1998; Lutterbach et al., 1998; Wang et al., 1998) (see Chapter 23 in this book). This raises the interesting possibility that the intrinsic (or associated) acetyltransferase activity of p300 might be required to overcome the repressive effects of AML-ETO on differentiation. Indeed, a truncated form of p300 lacking the acetyltransferase domain was impaired in its ability to synergize with AML-1b. However, a more detailed mutagenesis of p300 will be required to establish a correlation between its HAT activity and its ability to cooperate with AML1b. Finally, AML-1 synergizes with c-Myb and C/EBP on myeloid and lymphoid promoters (Britos-Bray and Friedman, 1997; HernandezMunain and Krangel, 1994; Zhang et al., 1996). This synergy is apparently not the result of cooperative DNA binding (Hernandez-Munain and Krangel, 1995; Zhang et al., 1996), suggesting that it is instead mediated through recruitment of a common cofactor such as CBP/p300, similar to what has been proposed for other CBP/p300-regulated factors.

CBP/P300 IN LEUKEMIA-ASSOCIATED CHROMOSOMAL TRANSLOCATIONS Both CBP and p300 bind the viral oncoproteins E1A and SV40 T antigen. This raised the possibility that alterations in the functions of CBP/ p300 might play a role in the development of malignancies in humans. This suspicion was supported by the finding that one copy of the CBP gene is inactivated in the rare disease Rubinstein-Taybi syndrome (Petrij et al., 1995), which is manifested by an increased propensity for tumors (mostly of the nervous system), craniofacial malformations, and mental retardation (Miller and Rubinstein, 1995; Rubinstein and Taybi, 1963). The involvement of CBP/p300 in hematological malignancies was realized through the discovery of leukemia-associated chromosomal translocations involving the CBP and p300 genes (Borrow et al., 1996; Giles et al., 1997; Ida et al., 1997; Rowley et al., 1997; Satake et al., 1997; Sobulo et al., 1997; Taki et al., 1997) (see Chapter 26 in this book). These translocations, which fuse the CBP and p300 genes to the MOZ or MLL genes, generally result in fusion proteins that preserve most of the CBP and p300 molecules, suggesting that the disease mechanism does not simply involve loss of function of CBP, as is the case of Rubinstein-Taybi syndrome. Instead, they suggest altered function (dominant positive or dominant negative) through fusion to another molecule. In general, any translocation event could lead to gain or loss of function of either fusion partner, or to the formation of dominant interfering alleles. Fusion of CBP to a given transcription factor might result in aberrant recruitment of CBP to certain promoters, leaving less free CBP available for other transcription factors involved in balancing proliferation and differentiation. In addition, it is possible that misguided or deregulated acetyltransferase activity by CBP/p300 fusion products causes the changes in gene expression that contribute to the transformed state. An interesting example of the importance of balancing histone acetylation and deacetylation comes from studies of acute promyelocytic leukemia (APL)—associated translocations that fuse the retinoid acid recep-

MECHANISMS OF CBP/p300 FUNCTION

tor (RAR) to PLZF or PML (see Chapter 20 in this book). PML-RAR and PLZF-RAR fusion proteins have a high affinity for a transcriptional repressor complex containing histone deacetylases. While normal RAR responds to retinoic acid (RA) by shedding the deacetylase complex, followed by association with an acetyltransferase complex, the PMLRAR responds only to very high concentrations of RA, and PLZF-RAR is RA resistant (Grignani et al., 1998; He et al., 1998; Lin et al., 1998). The ability of leukemic cells to differentiate upon RA treatment correlates with the ability of their translocation fusion proteins to displace the repressor complex in response to RA. In fact, patients with PML-RAR APL achieve remission upon treatment with high doses of RA, while PLZF-RAR APL patients do not. Together, these findings underscore the importance of CBP/p300 function and protein acetylation in balancing growth and differentiation of hematopoietic cells.

MECHANISMS OF CBP/p300 FUNCTION Clues From Studies of Intact Animals Some unexpected insights into the function of CBP/p300 came from gene knockout studies. The CBP and p300 null mice display similar phenotypes (Yao et al., 1998). The p300\\ mice die between days 9 and 11.5 of embryonic development. Their main defects are severe developmental retardation, reduced size, failure to close the neural tube, and altered trabeculation in the cardiac ventricles. A fraction of the p300>\ mice die early, displaying neural tube closure defects similar to the p300\\ mice, indicating a requirement for full p300 dosage during neural development. Mice heterozygous for CBP deficiency suffer from skeletal abnormalities and growth retardation (Tanaka et al., 1997). CBP/ p300 compound heterozygous mice die early and display a phenotype very similar to the individual homozygous knockouts (Yao et al., 1998). Unexpectely, fibroblasts derived from p300\\ embryos grow more slowly than their wild-type

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counterparts, suggesting that p300 might exert a positive effect on cell proliferation. This finding is especially surprising in light of the positive effects of E1A expression on cell proliferation. Despite their many similarities, p300 and CBP also perform distinct functions in vivo. For example, cells deficient for p300 have a compromised response to signaling by nuclear hormone receptors but not by cAMP/CREB. Nonoverlapping functions of CBP and p300 were also observed upon their ribozyme-mediated inactivation in embryonal carcinoma F9 cells (Kawasaki et al., 1998). The abnormalities observed in p300>\ mice indicate that a full CBP/p300 gene dosage is essential for normal development. This is consistent with the observation that CBP haploinsufficiency causes Rubinstein-Taybi syndrome in humans and leads to a phenotype somewhat similar to this disease in mice (Petrij et al., 1995; Tanaka et al., 1997). However, since most tissues form normally, this suggests that different cell types display variable sensitivity to CBP/p300 protein levels. The effects of CBP or p300 deficiency on the various cell lineages of the hematopoietic system have not yet been characterized in detail. A recent report describes the consequences of a CBP gene disruption in mice where a truncated form of CBP is generated that retains the Nterminal 1084 amino acids (out of 2441) but lacks the HAT domain (Oike et al., 1999). Mice homozygous for this defect show a phenotype similar to that observed in the p300 knockouts. They appear anemic and their yolk sacs contain fewer erythroid cells and display a defective vascular network. While the number of yolk sac—derived erythroid colony-forming units is reduced, mature erythroid cells are found, suggesting that the requirement of CBP for erythroid maturation is not absolute, and that p300 might be able to compensate for the CBP defect. To examine definitive hematopoiesis in the CBP-defective mice, organ culture was performed from E9.5 embryos using tissue from the aorta-gonad-mesonephros (AGM) region, followed by colony-forming assays. These experiments showed that homozygous loss of CBP leads to dramatically reduced numbers of definitive erythroid progenitors and to a significant

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reduction in granulocyte/macrophage colonyforming units. Organ cultures from homozygous embryos also revealed a strong reduction in vasculoangiogenesis. Whether the presence of the truncated CBP molecule retains any biological activity in these mice remains to be determined. In any case, these results suggest a role for CBP in hematopoiesis and vasculoangiogenesis. A more thorough analysis of the mice null for CBP and p300 is required to determine potential defects in proliferation or differentiation of various hematopoietic lineages. It is possible that defects in gene expression resulting from CBP or p300 deficiency might be limited to certain subsets of genes in select cell types. Thus, more detailed profiling of gene expression in various tissues is necessary to assess the requirement of CBP or p300 in gene regulation. Alternatively, loss of CBP might be compensated for by p300 and vice versa. Clearly, more detailed examination of gene expression in different tissues as well as the study of chimeric animals will give new and important insights. Strength in Numbers CBP/p300 interact with numerous transcription factors. Many of these interactions might take place simultaneously since they are mediated by dedicated domains. This could account for the observed synergy between factors regulated by CBP (see above for examples). Thus, CBP might provide a platform for the assembly of highmolecular-weight-complexes (enhanceosomes; for review, see Carey, 1998) containing multiple DNA-binding proteins that position the complex in a sterically correct fashion at promoters/ enhancers. This complex could serve to form multiple contacts with components of the basal transcription machinery via CBP or via other components of the complex (see below). Since this complex is likely to include non-DNAbinding proteins such as p/CAF, ACTR, or SRC-1, which also possess acetyltransferase activity, it would constitute a powerful regulator of chromatin structure. For example, a highmolecular-weight complex centered around CBP/p300 could form at the LCR, which participates in regulating chromatin structure at the -globin locus (Fig. 28.4). The LCR contains binding sites for GATA-1, EKLF, and NF-E2,

all of which bind to CBP/p300 (Blobel et al., 1998; Cheng et al., 1997; Hung et al., 1999; Zhang and Bieker, 1998). Thus, CBP could serve to integrate signals from multiple transcriptional regulators and perhaps even present a target for global regulators of gene expression such as signaling cascades activated by growth/ differentiation factors. The latter notion is supported by the observation that CBP/p300 is acetylated and phosphorylated. CBP/p300 are also thought to mediate negative cross-talk between transcription factors. Competition for limiting amounts of CBP/p300 has been invoked to account for mutual inhibition of CBP/p300-regulated transcription factors when bound to separate DNA templates (Kamei et al., 1996). This might explain the inhibition of GATA factors by ligand-activated nuclear hormone receptors (NR) (Blobel and Orkin, 1996; Blobel et al., 1995; Chang et al., 1993). The observations that overexpression of CBP alleviates NR-mediated repression of GATA-1, and that ligand-bound NR reduces the stimulation of GATA-1 activity by CBP (G. A. Blobel, unpublished), are consistent with such a model. Together, these findings support a role of CBP/p300 as molecular integrators of positive and negative transcriptional signals that govern hematopoietic gene expression.

Building a Bridge The large number and diversity of genes and transcription factors regulated by CBP/p300 could be explained if CBP/p300 were components of the basal transcription apparatus. In support of such a model, CBP/p300 have been found to interact with TFIIB (Kwok et al., 1994), TBP (Abraham et al., 1993; Dallas et al., 1997; Sang et al., 1997; Swope et al., 1996), and RNA polymerase II (Cho et al., 1998; Kee et al., 1996; Nakajima et al., 1997a, 1997b; Neish et al., 1998). Thus, recruitment of CBP by a DNAbound transcription factor could facilitate the formation of a preinitiation complex at relevant promoters (Fig. 28.5). Such a mechanism would imply that CBP/p300 act in a stoichiometric fashion. While this mechanism might hold for some promoters, additional evidence suggests that CBP/p300 also act catalytically (see next paragraph).

MECHANISMS OF CBP/p300 FUNCTION

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Figure 28.5. Hypothetical model in which CBP (or p300) links DNA-bound nuclear factors to components of the basal transcription machinery. GTFs, general transcription factors; TBP, TATA-binding protein, Pol II, RNA polymerase II.

Action by Catalysis The observation that CBP/p300 and some of its associated factors possess acetyltransferase activity suggests an enzymatic mechanism of gene regulation. Targeting of CBP/p300 to the appropriate sites could lead to local increases in histone acetylation followed by rearrangement of chromatin structure (Fig. 28.4). This in turn could favor access of other transciptional regulators. Again, the LCR provides an example where such a mechanism could be operating. As mentioned above, histone acetylation and open chromatin correlate well at the chicken -globin gene locus (Hebbes et al., 1994). However, depending on transcription factor/promoter context, CBP/p300 can also act in HATindependent fashion (Korzus et al., 1998). If certain nuclear factors function by recruiting a histone-modifying enzyme to trigger chromatin opening, how do they find access to DNA in the first place? One possibility is that other transcription factors might pave their way by facilitating chromatin opening in an acetylation-independent fashion. An example for such a scenario is provided by the observation that NF-E2 disrupts chromatin structure in an ATPdependent manner on a chromatinized template containing DNase 1 hypersensitive site 2 of the globin LCR (Armstrong and Emerson, 1996). This leads to increased access of GATA-1 to adjacent GATA-binding sites (Armstrong and Emerson, 1996). Alternatively, GATA-1 might find access to chromatin without the assistance of other factors. A recent report (Boyes et al., 1998) demon-

strated that chicken GATA-1 or a peptide comprising just its DNA-binding domain can bind DNA when packaged into a nucleosome. This leads to a reversible breakage of histone/DNA contacts, thus, perturbing nucleosome stucture (Boyes et al., 1998). Once bound to DNA, the GATA-1—associated acetyltransferase complex might modify adjacent histones, thus paving the way for access of other transcription factors to DNA. It is important to keep in mind that modification of chromatin is not restricted to acetylation and that numerous regulated chromatinmodifiying complexes have been identified (for review, see Kadonaga, 1998). For example, an elegant study by Armstrong and co-workers (Armstrong et al., 1998) demonstrated that EKLF interacts with a complex, called E-RC1, which contains components of the mammalian SWI/SNF complex, an ATP-dependent chromatin remodeling machine (Kadonaga, 1998). ERC1 stimulates EKLF-dependent transcription on a chromatinized -globin gene template and is required for the formation of a DNase 1 hypersensitive site. Interestingly, this activity is ATP dependent, and E-RC1 does not appear to contain histone acetyltransferases (Beverly Emerson, personal communication). Similarly, as pointed out above, NF-E2 requires an ATPdependent chromatin remodeling activity for the formation of a DNase 1 hypersensitive site on in vitro assembled chromatin (Armstrong and Emerson, 1996). Acetylation of nonhistone proteins, including transcription factors, might turn out to be of equal importance. For example, acetylation of

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p53 leads to a direct increase in DNA-binding activity (Gu and Roeder, 1997; Liu et al., 1999; Sakaguchi et al., 1998). It is likely that acetylation of other nuclear factors will lead to altered gene expression through a variety of mechanisms. For example, acetylation has been shown to regulate protein-protein interactions. In the case of the transcription factor dTCF, acetylation by CBP decreased its affinity for its cofactor -catenin/Armadillo (Waltzer and Bienz, 1998). An interesting variation of this mechanism is the finding that acetylation of HMG I(Y) by CBP leads to destabilization of an enhanceosome complex at the interferon -gene promoter, resulting in termination of transcription (Munshi et al., 1998). These examples demonstrate that CBP can negatively regulate transcription via its acetyltransferase activity. It is conceivable that acetylation might be a widely used mechanism to trigger allosteric changes in proteins, thereby regulating proteinprotein and protein-DNA interactions, similar to what has been observed upon protein phosphorylation. In both cases, the modification results in a change of charge: addition of a negative charge in the case of phosphorylation and neutralization of a positive charge in the case of acetylation. Moreover, acetylation changes the size of the lysine side chain, which could be equally important in protein folding.

CONCLUSION CBP/p300 are extraordinarily versatile molecules that can exert positive and negative effects on transcription and cell differentiation. It is likely that more factors will be identified that interact with CBP/p300, and that a subset of these will be regulated by acetylation. The challenge that lies ahead will be to determine the significance of such interactions in physiologically relevant settings. Given that CBP and p300 share many functions, this will not be an easy task, especially since it has not been possible so far to generate CBP/p300 double knockout cell lines. The mechanisms by which CBP/p300 act likely depend on promoter and cellular context as well as the chromatin configuration in which a given target gene is embedded. One approach that would allow for dissection of CBP/p300 functions in a physio-

logical context would be to knock-in mutant CBP/p300 alleles bearing alterations in domains associated with specific functions, such as the HAT domain or important protein-docking sites. The early lethality of homozygous p300 and CBP knockout mice likely obscures critical functions of these molecules at later stages of development. Analysis of tissue-directed knockout mice and chimeric mice is likely to yield important insights. Given the broad variety of CBP/p300-regulated factors, an important and challenging task will be the identification of the relevant genes that mediate their function in vivo. Subtractive hybridization and microarray technologies might be useful approaches to identify genes most sensitive to changes in CBP/p300 levels. Although CBP/p300 are expressed in most tissues, their importance in regulating gene expression and differentiation in hematopoietic cells is illustrated by their involvement in leukemia-associated chromosomal translocations. It remains to be determined why these chromosomal translocations result in leukemias mostly of the myeloid/monocytic lineage. Since CBP/p300 have intrinsic and associated acetylase activity, they might present targets for pharmacological intervention. It can be envisioned that novel drugs might be developed that alter specific activity or substrate specificity, thereby allowing for modulation of gene expression and cell differentiation. For example, trichostatin A, a deacetylase inhibitor, has been successfully used to activate silenced transgenes in mice carrying gene-delivery vectors designed for use in gene therapy (Chen et al., 1997). Furthermore, drugs targeting histone deacetylases have been used against malaria and toxoplasmosis (Darkin-Rattray et al., 1996). Thus, a detailed understanding of the role of protein acetylation might reveal new approaches to controling gene expression and treating human diseases.

ACKNOWLEDGMENTS I want to thank Margaret Chou, Merlin Crossley, Richard Eckner, Stuart Orkin, Morty Poncz, and Mitchell Weiss for helpful suggestions and critical reading of the manuscript.

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Yang, X.-J., Ogryzko, V. V., Nishikawa, J., Howard, B. H., and Nakatani, Y. (1996). A p300/CBP-associated factor that competes with the adenoviral oncoprotein E1A. Nature 382, 319—324. Yao, T.-P., Oh, S. P., Fuchs, M., Zhou, N.-D., Ch’ng, L.-E., Newsome, D., Bronson, R. T., Li, E., Livingston, D. M., and Eckner, R. (1998). Gene dosagedependent embryonic development and proliferation defects in mice lacking the transcriptional integrator p300. Cell 93, 361—372. Zhang, D. E., Hetherington, C. J., Meyers, S., Rhoades, K. L., Larson, C. J., Chen, H. M., Hiebert, S. W., and Tenen, D. G. (1996). CCAAT enhancer-binding protein (C/EBP) and AML1 (CBF alpha2) synergistically activate the macrophage colony-stimulating factor receptor promoter. Mol. Cell. Biol. 16, 1231—1240. Zhang, D. E., Zhang, P., Wang, N. D., Hetherington, C. J., Darlington, G. J., and Tenen, D. G. (1997). Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding protein alpha-deficient mice. Proc. Natl. Acad. Sci. USA 94, 569—574. Zhang, W., and Bieker, J. J. (1998). Acetylation and modulation of erythroid Kru¨ppel-like factor (EKLF) activity by interaction with histone acetyltransferases. Proc. Natl. Acad. Sci. USA 95, 9855— 9860. Zhuang, Y., Kim, C., Bartelmez, S., Cheng, P.-F., Groudine, M., and Weintraub, H. (1992). Helixloop-helix transcription factors E12 and E47 are not essential for skeletal or cardiac myogenesis, erythropoiesis, chondrogenesis, or neurogenesis. Proc. Natl. Acad. Sci. USA 89, 12,132—12,136. Zhuang, Y., Soriano, P., and Weintraub, H. (1994). The helix-loop-helix gene E2A is required for B cell formation. Cell 79, 875—884.

PART V


ONCOGENESIS AND HEMATOPOIESIS

CHAPTER 29


THE ROLES OF THE c-myc AND c-myb ONCOGENES IN HEMATOPOIESIS AND LEUKEMOGENESIS MARCELLO ARSURA AND GAIL E. SONENSHEIN Department of Biochemistry, Boston University School of Medicine

INTRODUCTION Throughout evolution, acutely transforming retroviruses have acquired oncogenes, which are altered forms of normal cellular genes or protooncogenes. Structural analysis of these viral genomes revealed that as a result of integration, oncogenes have suffered multiple deletions and/ or point mutations of essential regulatory domains, which led to deregulation of gene function. These structural alterations are thought to be responsible for the high tumorigenicity of the retrovirus. In the past 20 years, the study of the v-myb and v-myc viral oncogenes has greatly increased our understanding of the molecular mechanisms leading to neoplastic transformation of hematopoietic cells. The v-myb and v-myc oncogenes were first identified in several naturally occurring retroviruses that cause myeloid leukemias and bursal lymphomas in birds, respectively (Bishop et al., 1982; Roussel et al., 1979). Aberrant activity of the v-myb and v-myc oncogenes is essential for the transformed phenotype of infected hemato-

poietic cells. Their cellular counterparts, the cmyb and c-myc proto-oncogenes, have been shown to regulate transformation, proliferation, differentiation, and survival of hematopoietic cells. Both genes have been shown to encode labile nuclear phosphoproteins and are members of multigene families. These two oncoproteins are structurally and functionally distinct. Furthermore, c-myc has been found to be expressed in all proliferating cells, while c-myb displays a much more restricted pattern of expression — that is predominantly hematopoietic cells. This review dissects the roles of the c-myb and c-myc genes in the regulation of hematopoiesis and leukemogenesis. Due to the extensive literature for the material covered here, we apologize at the outset for the failure to cite all of the original literature and have in a few places instead referred readers to several outstanding reviews.

THE c-myc ONCOGENE The c-myc gene was discovered as the cellular homologue of the transforming gene of the avi-


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THE ROLES OF THE c-myc AND c-myb ONCOGENES IN HEMATOPOIESIS AND LEUKEMOGENESIS

an myelocytomatosis virus, v-myc (Bishop, 1982). The v-myc oncogene was first implicated in tumor formation in birds infected with acutetransforming viruses such as MC-29, OK-10, and MH-2 (reviewed in Alitalo et al., 1987). Insertion mutagenesis by slow transforming retroviruses has further implicated the cellular homologue c-myc in bursal lymphomas in chickens, T-cell lymphomas in mice, as well as T-cell leukemias in cats (Alitalo et al., 1987). However, a dramatic surge of interest in this oncogene developed following the observation that the c-myc gene, normally located on chromosome 15, was at the breakpoint of t(12;15) chromosomal translocations characteristic of mouse plasmacytomas (Shen-Ong et al., 1982). As a result of the gene rearrangement, only a portion of exon 1 and exons 2 and 3 of the translocated c-myc gene were present, and these were now in a head-to-head configuration with an immunoglobulin (Ig) heavy constant region. Thus, the upstream regulatory regions and normal promoters were lost. This finding was rapidly confirmed in other murine B-cell tumors, and followed by similar observations in human Burkitt’s lymphomas

(BLs). Direct demonstration that such a truncated c-myc gene could result in B-cell tumors in transgenic mouse models was made by Adams and co-workers (1985). Overexpression of c-myc has now been found to typify many cancers (reviewed in Dang and Lee, 1995). In this section of the review, we first discuss the structure and function of the normal c-myc oncogene and then describe mutations and other genetic alterations that lead to its activation in tumor cells. In particular, we focus on the human and mouse c-myc genes, since these are the two most wellcharacterized systems. We then describe the genetic events linking c-myc to tumors of the hematopoietic lineage, including translocations, point mutations, and amplification. Finally, the studies with transgenic mice demonstrating a causal role for the c-myc gene are described. Studies on the posttranscriptional events, including mRNA stability and posttranslational modifications, and on other members of the myc gene family (N-myc and L-myc), have been covered extensively in other recent review articles (Cole, 1986; DePinho et al., 1991; Marcu et al., 1992), and are, for the most part, beyond the scope of this review.

Figure 29.1. Schematic representations of the c-myc gene and c-Myc protein structures. A: c-myc gene. The two major (P1 and P2) and two minor (P0 and P3) promoter regions of the human c-myc gene are indicated. The positions of the start sites of translation in exon 1 (CUG), leading to synthesis of c-Myc1, and in exon 2 (AUG), leading to synthesis of c-Myc2 and c-MycS, proteins are shown. Open area indicates the coding regions in exons 1, 2, and 3. B: c-Myc2 protein structure. Structural and functional domains of the human c-Myc protein are indicated: MB I, Myc homology box I; MB II, Myc homology box II; NLS, nuclear localization sequence; B, basic region; HLH, helix-loop-helix region; LZ, leucine zipper region. P indicates the positions of potential sites of phosphorylation at amino acids thr 58 and ser 62.

THE c-myc ONCOGENE

c-myc Gene Structure and Transcripts The normal mammalian c-myc gene is composed of three exons with large 5 and 3 untranslated regions within exons 1 and 3, respectively (Fig. 29.1A). In the human gene, these exons are approximately 550, 770, and 870 base pairs (bp) (Battey et al., 1983). In both the human and mouse c-myc genes there are two major promoters, P1 and P2, separated by approximately 160 bp (Bernard et al., 1983, Stanton et al., 1984) (Fig. 29.1A). Both the P1 and P2 promoters contain canonical TATA and CAAT eukaryotic basal promoter elements. The sizes of the P1- and P2-driven transcripts are 2.4 and 2.2 kb, respectively. The normal half-lives of these c-myc transcripts are relatively short, approximately 20—40 minutes (reviewed in Spencer and Groudine, 1990). In most cell types, transcription from the P2 promoter predominates, giving rise to 75—90% of steady-state c-myc mRNAs; however, under certain conditions and in some tumors, there is a switch in promoter usage (Bentley and Groudine, 1986b; Chang et al, 1991; Spencer et al, 1990; Stewart et al., 1984a; Taub et al., 1984). There is also a minor promoter P3, located near the 3 end of intron 1 (Ray and Robert-Lezenes, 1989) (Fig. 29.1A). Furthermore, the human gene contains a fourth, and minor, promoter P0, located significantly upstream of the P1 promoter at 9550 to 9650 bp (Bentley and Groudine, 1986a). The 3.1 and 2.3 kb transcripts from the P0 and P3 promoters account for only about 5% of total c-myc steady-state RNA in normal cells (Ray and Robert-Lezenges, 1989; Spencer and Groudine, 1991). As discussed below, P3 promoter usage dramatically increases in tumor cells in which the c-myc gene has translocated with concomitant loss of normal upstream promoter regions.

c-Myc Protein Products Two c-Myc proteins have been identified in mammalian cells (Hann et al., 1988; Hann and Eisenman, 1984; Ramsey et al., 1984) (Fig. 29.1A). The major translation start site was localized to an AUG codon at the 5 end of exon 2; initiation at this site leads to synthesis of the major 439 amino acid c-Myc protein, c-Myc2. A

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nonconsensus initiation codon (CUG) was identified at the 3 end of exon 1, leading to synthesis of the second protein, c-Myc1. These sites are separated by approximately 42 nucleotides in the RNA, and the protein products are in frame but differ at their amino termini, with c-Myc1 having an additional 14 amino acids. More recently, additional, shorter c-Myc protein products have been identified, which result from a leaky scanning mechanism (Spotts et al., 1997). These polypeptides initiate at two closely spaced downstream AUG residues giving rise to two shorter c-Myc proteins termed c-MycS, which lack approximately 100 amino acids at the amino terminal domain (Fig. 29.1A). All of the c-Myc proteins localize to the nucleus and are extremely labile phosphoproteins (reviewed in Lemaitre et al., 1996; Spotts et al., 1997). The carboxy termini of all of the c-Myc proteins contain a basic region (B), and helix-loophelix/leucine zipper (HLH/LZ) domains, necessary for DNA binding and heterodimerization with the binding partner Max (Fig. 29.1B). Max is also characterized by the presence of b/HLH/ LZ motifs. The HLH and LZ motifs together mediate specific protein:protein interactions of Max with c-Myc proteins, and the basic region facilitates DNA binding of the dimerized factors to the E-box c-Myc sites (EMS) CACGTG DNA sequence (Amati et al., 1992; Berberich et al., 1992; Blackwood and Eisenman, 1991; Halazonetis and Kandil, 1991; Kato et al., 1992; Prendergast et al., 1991; Reddy et al., 1992). In addition, Max can bind several antagonists of c-Myc function, including Mad1, Mxi1 (or Mad2), Mad3, and Mad4, which recruit histone deacetylases that remodel the structure of the chromatin repressing transcription (reviewed in Grandori and Eisenman, 1997). Other factors have been found to interact with carboxy terminal domains of c-Myc, including YY1, TFII-I, AP-2, BRCA1, Miz-1, and Nmi (reviewed in Sakamuro and Prendergast, 1999). The amino termini of c-Myc have two highly conserved regions, termed Myc box (MB) I and II (Fig 29.1B). These two regions and surrounding sequences have been implicated in transactivation. As expected, the c-MycS proteins, which lack the MB I, are unable to transactivate and function as dominant negative inhibitors of full-length c-Myc transactivation (Spotts et al., 1997). Additional proteins have

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been found to interact with c-Myc via its amino terminal domain, including the transcription factor TBP (Maheswaran et al., 1994), implicated in both positive and negative transcriptional regulation, TRRAP, a component of the transcriptional adaptor complex SAGA, and the Rb pocket protein p107 (reviewed in Cole and McMahon, 1999; Sakamuro and Prendergast, 1999). In addition to this positive mechanism of regulation, c-Myc has also been found to repress transcription of several genes (see below). A direct role for c-Myc in repression of transcription mediated by an initiator (Inr) element was elucidated by Roy and co-workers (1993a). Inr elements were originally defined from a loose consensus site, 5-YYCAYYYYY-3, found at the transcription initiation sites of genes containing upstream TATA elements (Grosschedl and Birnstiel, 1980; Smale and Baltimore, 1989). Subsequently, a number of genes lacking a TATA box were found to have Inr elements (Smale and Baltimore, 1989). Formation of a preinitiation complex is facilitated through the Inr element located at the start site of transcription (Smale and Baltimore, 1989; Roy et al., 1993b). Using cell-free transcription analyses, Roy and co-workers (1993a) demonstrated that c-Myc can repress Inr-mediated transactivation. In vivo inhibition of Inr mediated transcription has been shown by several laboratories (reviewed in Dang, 1999; Li et al. 1994; Mai and Martensson, 1995; see below). Repression appears to require AA 106—143, encompassing MB II, (Li et al., 1994), and has been shown to be facilitated through interaction with the transcription factor YY1 (reviewed in Dang, 1999). Thus, c-Myc can function in both a positive and a negative pathway of transactivation. Interestingly, we have identified a naturally occurring splice variant of Max, termed dMax, which lacks the basic region and helix 1 of the HLH domain; dMax interacts with c-Myc, and inhibits E-box Myc site—driven transcription in transient transfection assays (Arsura et al., 1995). Furthermore, more recently we have shown that dMax has no effect on c-Myc repression through Inr elements (FitzGerald et al., 1999). Thus, since it is widely expressed, dMax has the ability to differentially regulate c-Myc function.

Role of the c-myc Gene in Proliferation, Differentiation, and Apoptosis c-Myc has been implicated positively in control of proliferation and neoplastic transformation; conversely, c-myc gene expression often prevents differentiation. Thus, in cell culture, c-myc RNA levels are low in quiescent untransformed cells, including B and T lymphocytes, and increase early during the G0 to G1 transition following mitogen stimulation (Campisi et al., 1984; Kelly et al., 1983). Furthermore, RNA levels remain elevated in proliferating cells (Dean et al., 1986). Conversely, a drop in expression of c-myc was detected in proliferating murine WEHI 231 B cells induced to growth arrest at the G1/S phase transition (Maheswaran et al., 1991; McCormack et al., 1984), and in mouse erythroleukemia (MEL) cells and human HL-60 promyelocytic cells induced to differentiate (Lachman and Skoultchi, 1984; Reitsma et al., 1983). Importantly, the role of the drop in c-myc expression was illustrated when constitutive ectopic expression was shown to prevent the growth arrest (Wu et al., 1996a) and terminal differentiation (Coppola and Cole, 1986; Dmitrovsky et al., 1986), respectively. Furthermore, selective reduction in c-myc function upon either addition of an antisense oligonucleotide or expression of an antagonist such as Mad1 was found to promote differentiation directly (Cultraro et al, 1997; Holt et al., 1988; Prochownik et al., 1988). In general, removal of required growth factors in untransformed cells leads to a drop in c-myc levels (Dean et al., 1986), while overexpression of c-myc in growing cells leads to reduced growth factor requirements and a shortening of the time to pass through G1 (Karn et al., 1989). Further indicating a role in proliferation, c-myc knockout mice displayed early embryonal lethality (between day 9.5 and 10.5) with multiple pathologic disorders including of the heart, pericardium, and neural tube (Davis et al., 1993). However, difficulties in establishing cells from these mice hindered further insights with this model. More recently, a somatic cell knockout of the c-myc gene has been made in Rat1 fibroblasts (Mateyak et al., 1997). These c-myc null Rat1 fibroblasts proliferate with a greatly increased time for progression through

THE c-myc ONCOGENE

the G1 and G2 phases of the cycle. Consistent with these findings, targeted expression of c-myc in hematopoietic cells in several mouse transgenic models has now been shown to lead to arrest of differentiation (Thompson et al., 1996), and to enhanced proliferation and neoplastic transformation (see below). The c-myc gene has also been implicated in control of apoptosis and can both stimulate and inhibit cell death. Somewhat paradoxically with its role in promoting proliferation and neoplastic transformation, c-myc overexpression was found to enhance the rate of apoptosis of cells upon growth-limiting conditions. For example, increased levels of cell death were seen in myeloid or fibroblast cells overexpressing c-myc upon IL-3 or serum deprivation, respectively (Askew et al., 1991; Evan et al., 1992). In contrast, there are many experimental systems in which treatments that cause apoptosis result in c-myc downregulation, and ectopic c-myc expression decreases the extent of cellular death (reviewed in Thompson, 1998). For example, we showed that a drop in c-myc expression in immature B lymphocytes following either B-cell receptor (BCR) engagement or TGF-1 treatment leads to a drop in c-Myc that induces apoptosis (Arsura et al., 1996; Wu et al., 1996b). Furthermore, similar findings were seen upon withdrawal of growth factors from Ba/F3 hematopoietic cells (Besancon et al., 1998) or treatment of human lymphoblastic leukemia cells with glucocorticoids (Thulasi et al., 1993). In vivo experiments have also shown similar protection for glucocorticoid treatment of activated immature T cells by c-myc (Wang et al., 1999). Thus, c-myc can either promote survival or apoptosis of cells depending upon the treatment conditions. Different models have been proposed to account for the ability of c-Myc to either sensitize to or protect from apoptosis (Thompson, 1998). One model suggests apoptosis results from a mismatch in GO/STOP signals for advancing through the cell cycle. Another model argues that c-Myc itself is responsible for specific signals that both advance the cell cycle and regulate apoptotic death. Experimental evidence exists supporting both models, and future studies are necessary to resolve this apparently paradoxical feature of c-Myc function.

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c-Myc Function Multiple genes have been identified as having one or more E-box c-Myc sites (EMS), and many of these have been reported to be transactivated by c-Myc. These include ornithine decarboxylase (ODC), -prothymosin, cad, p53, cyclin A, cyclin D1, cdc25A, DHFR, eIF-2, eIF4E, LDH-A, Rcl, telomerase, and thymidine kinase (reviewed in Cole and McMahon, 1999; Dang, 1999; Grandori and Eisenman, 1997). Some of these genes are expressed in a growthrelated fashion and would presumably control cell cycle progression, while others would appear to be involved in metabolism or immortality. Surprisingly, however, in Rat1 cells null for c-Myc, normal expression was observed for almost all of the EMS-containing genes tested, with the exception of cad, which encodes carbamoyl phosphate synthase/aspartate carbamoyltransferase/dihydro-orotase (Bush et al., 1998). This observation raises the intriguing question of whether c-Myc acts via direct binding or indirectly to regulate transcription of most EMS-containing genes. This issue has been critically evaluated in a recent review by Cole and McMahon (1999). In addition, multiple genes have been found to be repressed by c-Myc proteins. Many, but not all, of these genes have promoters driven by an Inr element. Genes identified that are repressed by c-Myc include those encoding gadd45, involved in control of proliferation, and C/EBP, terminal transferase, rag-1, albumin, collagen 1 and 2(I), Ig chain, thrombospondin, LFA-I, and MHC class I, involved in mediating differentiation phenotype, adhesion, metastasis, or immune surveillance (reviewed in Claassen and Hann, 1999; Dang, 1999). In addition, c-Myc represses transcription of its own promoter, in an autoregulatory fashion (Cory, 1986; Claassen and Hann, 1999). Interestingly, overexpression of gadd45 was seen in the Rat1 c-myc null cells, consistent with a loss of repression (Bush et al., 1998). Recently, we have found that transcription of the cyclin-dependent kinase (cdk) inhibitor protein p27Kip1 gene is repressed by c-Myc (Yang et al., unpublished findings). This gene has been found to control the G1 to S phase transition, suggesting another mechanism for c-myc involvement in cell cycle pro-

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gression, as well as differentiation. Increased levels of p27 mRNA and protein have also been detected in the Rat1 c-myc null cells (Mateyak et al., 1999). Interestingly, we find that Max can facilitate binding of c-Myc to the Inr element of the p27 promoter, suggesting involvement of this central binding partner in repression (Yang et al., unpublished findings). c-myc Gene Involvement in Neoplastic Transformation Abundant examples of genetic alterations that lead to increased levels of c-myc gene expression, constitutive synthesis resulting from the loss of normal regulation, or altered function have indirectly implicated the c-myc oncogene in leukemias, lymphomas, and other tumors of the hematopoietic lineage. Translocations. While it had been known that the nonproductively arranged Ig heavy-chain locus in mouse plasmacytomas had undergone gene rearrangement, it was the work of Michael Cole and his co-workers that elucidated the critical nature of this structural alteration. In particular, they discovered that the c-myc oncogene, which had been implicated in several retrovirally induced B-cell tumors in birds, was at the chromosomal breakpoint in mouse plasmacytomas (Shen-Ong et al., 1982). Furthermore, they showed that this led to the synthesis of an altered c-myc mRNA. This observation was quickly confirmed, with c-myc-Ig gene translocations detected in various mouse myelomas and Burkitt’s lymphomas (BLs) (reviewed in Alitalo et al., 1987; Cole, 1986; Cory, 1986; Kelly and Siebenlist, 1986; Klein, 1983; Spencer and Groudine, 1991). In the case of the murine plasmacytomas, which were often induced by mineral oil injection, the large majority of the rearrangements involved the Ig heavy-chain locus, in particular, the -constant region (C). These translocations most often occurred within exon 1 of the c-myc gene, truncating the normal gene, removing key upstream and internal regulatory elements, as well as the P0, P1, and P2 promoters; moreover, the two genes were placed in a head-to-head configuration. Resulting transcripts have been mapped to start at multiple sites near the P3 promoter, within intron 1 (Fig 29.1A). Further-

more, the unrearranged c-myc gene was transcriptionally silenced (reviewed in Cory, 1986). In BL, which predominantly affects children, c-myc was again implicated in the neoplastic transformation of B cells. In this case, however, reciprocal translocations were seen with the human Ig heavy chain, the kappa () light chain, or the lambda () light chain located at chromosome locations 14q32, 2p11, or 22q11, respectively (reviewed in Cole, 1986; Cory, 1986; Varmus, 1984). The most common rearrangement observed was a t(8;14) occurring within c-myc exon 1 or intron 1 at chromosome 8q24 and the switch region of the heavy-chain locus (S), again placing the genes in a head-to-head configuration. The translocation resulted in loss of normal promoter P1 and P2, and transcription of the truncated c-myc genes initiated from cryptic promoters within intron 1 (Hayday et al., 1984). Loss of normal c-myc regulation was detected in somatic BL cell hybrids, as was the silencing of the unrearranged c-myc allele (reviewed in Croce and Nowell, 1985). In the less frequent ‘‘variant’’ translocations, t(2;8) and t(8;22), involving the and light chains, respectively, the rearrangement of the c-myc gene can even occur within sequences downstream of the transcribed region (Denny et al., 1985; Henglein et al., 1989; Showe and Croce, 1986). While the exact molecular mechanisms by which the rearranged c-myc gene is activated has not been delineated in every case, it is likely that the activation of c-myc plays a fundamental role in the neoplastic transformation of these cells. This was confirmed in a transgenic mouse model where a truncated c-myc gene, when placed under the control of the heavy-chain enhancer region (E), was found to cause B-cell tumors (Adams et al., 1985). Furthermore, the observation that these tumors were either of monoclonal or oligoclonal origin led to the hypothesis that activation of c-myc is an initial phase in a multistep transformation process. Subsequently, c-myc proviral inserts were observed in some T lymphomas, and an analogous translocation of a c-myc allele was seen in T-cell cancers (Alitalo et al., 1987; reviewed in Showe and Croce, 1986). In the case of T-cell leukemias (T-ALL), the most frequent alteration is a t(8;14) translocation, involving the chain of the T-cell receptor (TcR-) and the 3 end of the

THE c-myc ONCOGENE

c-myc gene (Lewis et al., 1985; Mathieu-Mahul et al., 1985). This rearrangement, therefore, resembles the translocations in the variant BLs. In addition to the loss of normal regulatory and promoter regions and the concomitant use of alternate promoter discussed above, the new position often places c-myc closer to an active Bor T-cell—specific enhancer region, for example, in the case of the typical BL or plasmacytoma, c-myc is now moved closer to either the enhancer or 3 C enhancer, respectively. Furthermore these types of rearrangements have been found to lead to activation of c-myc via additional mechanisms, including enhanced mRNA stability or translation efficiency. For example, the resulting alternative c-myc transcript forms have been found to have longer than normal half-lives, of the order of 45—60 minutes (Eick et al., 1985; Piechaczyk et al., 1985; Rabbitts et al., 1985). This result implies that 5 sequences of the c-Myc transcript regulate mRNA stability. However, simply adding these sequences to another transcript does not affect the mRNA half-life of the resulting chimera (Pei and Calame, 1988). Other work has implicated sequences within the 3 end of the c-myc transcript, in part to an AU-rich element (reviewed in Spencer and Groudine, 1991). Regardless of the precise mechanism, extending the half-life of the c-myc mRNA would likely result in higher levels of polypeptide produced per mRNA, promoting enhanced production of cMyc proteins. Mutations Associated with Cancer. Point mutations have been detected in both the coding and noncoding regions of the rearranged c-myc genes in BLs (Rabbitts et al., 1984; Showe et al., 1985). Within the coding region, a preponderance of BLs have been found to have missense mutations that localize to Mb I (reviewed in Bhatia et al., 1993; Yano et al., 1993). One site of particular interest surrounds threonine 58 (Thr-58) (Fig. 29.1B). Interestingly, three avian myc retroviruses have mutations at the same threonine residue (Papas and Lautenberger, 1985). The regions surrounding Thr-58, as well as serine 62 (Ser-62), are highly conserved over evolution, and these residues are in vivo sites of phosphorylation; furthermore, evidence suggests that phosphorylation at one position affects the other (Gupta et al., 1993; Lutterbach and Hann,

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1994). In vitro, these sites are targets of several kinases, including glycogen synthase kinase 3 (Lutterbach and Hann, 1994), ERK MAP kinases (Alvarez et al., 1991), cdk1 (p34) (Lutterbach and Hann, 1994; Seth et al, 1991), and a p107-cyclinA-cdk2 complex (Hoang et al., 1995). Transformation by c-Myc is negatively affected by phosphorylation on Thr-58 and positively enhanced upon phosphorylation of Ser-62 (Henriksson et al., 1993; Pulverer et al., 1994). Meanwhile, the role of this phosphorylation in transactivation is controversial. Gupta and colleagues (1993) found that mutation of either site reduced transactivation by c-Myc, whereas more recently Lutterbach and Hann (1994) found it unaffected. While these results suggest that neoplastic transformation may depend only partially on transactivation, the situation may be more complex, since this same site has more recently been implicated in direct interaction of c-Myc with the Rb pocket protein p107 (Beijersbergen et al., 1994; Gu et al., 1994; Hoang et al., 1995). In these studies, c-Myc with a mutation in Thr-58 was resistant to suppression of activation by p107. Raffeld and co-workers have mapped another frequent site of c-Myc mutation to leucine 115 (Leu-115), resulting in a substitution of phenylalanine in proteins encoded by 4 of 45 lymphoma-derived c-myc genes (Clark et al., 1994; Hoang et al., 1995; Yano et al., 1993). Analysis of this mutation by Dang and coworkers (Lee et al., 1996) showed that this alteration indeed increases c-myc transformation ability, as well as transcriptional repression, without affecting transactivation capacity. These results indicate that repression by c-Myc plays an important role in neoplastic transformation, which is consistent with studies on c-MycS that showed that loss of the transactivation domain did not ablate its ability to transform cells. Amplification. Overexpression of c-myc has been linked to amplification of c-myc genes in a number of human tumors. This has most often been observed at late stages of progression and is generally associated with aggressively malignant phenotype. Tumors displaying elevated cmyc expression due to amplification include plasma cell leukemia (Sumegi et al., 1985), promyelocytic leukemia (Collins and Groudine, 1982; DallaFavera et al., 1982), granulocytic

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leukemia (McCarthy et al., 1984), acute myelogenous leukemia (Alitalo et al., 1985; Fugazza et al., 1997), chronic myeloid leukemia (Jennings and Mills, 1998), and T-cell lymphomas (Silva et al., 1988). Transformation is a Multistep Process. Some of the key tests of the functional role of the c-myc gene rearrangements in neoplastic transformation of hematopoietic lineage cells came from studies with transgenic mouse models. Initial constructs utilized the Ig E heavy- or light-chain enhancer with either wild-type or translocated versions of c-myc (Adams et al., 1985). Dramatically, all of the mice expressing the translocated allele driven by the highly potent E enhancer developed pre-Band B-cell tumors, demonstrating a causal role for c-myc in neoplastic transformation. Interestingly, the transgenic mice carried an enlarged pool of pre-B cells, which were more actively proliferating, compared to normal mice (Langdon et al., 1986, 1989). These findings suggest a pre-neoplastic state. Also of note, transgenic mice carrying the normal c-myc allele, similarly driven by the E enhancer, displayed much reduced levels of tumor formation. Work with other promoters that led to expression in a wide variety of cells confirmed the oncogenic capacity of c-myc (reviewed in Cole et al., 1986; Meichle et al., 1992). Within the hematopoietic lineage, Skoda and colleagues (1995) have more recently shown that c-myc under the control of GATA-1 regulatory sequences developed either early- or late-onset erythroleukemia. Cell lines established from these mice displayed proerythroblast morphology and limited potential to differentiate in response to erythropoietin. Another striking observation made with the E-c-myc mice was that, in most cases, tumor onset was variable among littermates. Furthermore, analysis of the immunoglobulin protein produced by the tumors indicated that they were of monoclonal or oligoclonal origin. These findings suggested that an additional rarer event(s), beyond the activation of c-myc, was required for full malignancy. Similar results had been seen with transgenic mice using the mouse mammary tumor virus (MMTV) long-terminal repeat driving c-myc expression (Stewart et al., 1984b). Mammary adenocarcinomas were found to develop at high frequency, but only in one

mammary gland per animal, even though the transgene was expressed in the other mammary glands. Harris and co-workers (1988) confirmed a second event in E-c-myc mice with the demonstration that activation of the N-ras oncogene had occurred by somatic mutation in some of the oligoclonal tumors. Thus, additional, rarer genetic changes appear to be required for cells to become fully malignant. The hypothesis of a multistep transformation process was confirmed using double transgenic mouse models. Mice carrying both activated c-myc and ras transgenes developed tumors after a significantly shorter latency period in comparison with either single transgenic mouse (Sinn et al., 1987). Furthermore, infection of the E-c-myc mice with helper free retrovirus containing either ras or raf genes similarly resulted in significantly reduced latency periods compared to infected normal mice or uninfected transgenic animals (Alexander et al., 1989). More recently, several genes have been found to decrease the latency time for tumors to appear in E-c-myc mice, for example, bcl-2 (Strasser et al., 1990) and cyclin D1 (Bodrug et al., 1994), although neither bcl-2 or cyclin D1 alone resulted in tumor formation. Mice expressing the casein kinase II catalytic subunit were found to develop lymphomas at an accelerated rate in c-myc bitransgenic animals (Seldin and Leder, 1995). Use of murine leukemia virus (MuLV) as an insertional mutagen is another strategy that has been successfully used to identify genes that can cooperate with c-myc in neoplastic transformation: neonatal mice expressing c-myc were infected with the virus and insertions that activated a cooperating gene via introduction of the viral promoter were isolated using the MuLV DNA sequences as tags. Four loci were identified: Bmi-1, Pim-1, pal-1, and bla-1 (Haupt et al., 1991; Van Lohuizen et al., 1989; 1991a, 1991b). Bmi-1 is a RING-finger nuclear protein that is a member of the polycomb group of transcriptional repressors and has recently been implicated in entry into S phase (Jacobs et al., 1999). Bi-transgenic mice directly demonstrated the ability of c-myc and Bmi-1 to cooperate, as well as the role of the RING finger in this cooperation (Alkema et al., 1997). Pim-1 encodes a ubiquitously expressed serine/threonine protein kinase and is a member of a multigene

CONCLUSION

family. Its ability to cooperate with c-myc was established directly in bi-transgenic analysis (Van Lohuizen et al., 1989). More recently, Pim-2, which can compensate for Pim-1, has similarly been found to strongly collaborate with c-myc as manifested by the appearance of pre-B cell leukemias in neonate bitransgenic mice (Allen et al., 1997). Interestingly, Pim-1 has now been shown to be downstream of the MAP kinase signaling pathway (Nagata and Todokoro, 1995), indicating that ras, raf, and Pim-1 genes may all be part of a common signaling pathway. The genes within the pal-1 or bla-1 insertional sites remain to be identified.

CONCLUSION The c-myc gene, which is highly regulated and expressed in virtually all proliferating mammalian cells, has been implicated at several levels in hematopoiesis: c-myc expression promotes proliferation and inhibits differentiation. Furthermore, the c-myc gene has been found to play a causal role in tumor formation of cells of the hematopoietic lineage in humans, as well as in most mammalian species, including mouse, rat, and rabbits. Importantly, the avian myelocytomatosis virus has further implicated myc in tumor formation in birds. Multiple alterations that lead to activation or deregulated expression of c-myc have been delineated, including gene translocation and amplification, as well as point mutation. However, the exact mechanism by which c-myc controls all of these events, as well as the identification of critical regulatory genes, is an area of intense investigation. Recent work has focused on the ability of c-Myc to repress gene transcription, and we have identified a new target of repression, the p27Kip1 cdk inhibitor. Interestingly, decreased expression of p27 has been found to represent a marker of poor prognosis in many tumors recently (reviewed in Lloyd et al., 1999), suggesting one plausible mechanism for c-Myc—mediated loss of normal control of growth and differentiation.

THE c-myb ONCOGENE The c-myb proto-oncogene was first identified as the cellular counterpart of the v-myb oncogene

529

found in two acutely leukemogenic chicken retroviruses AMV and E26 (Roussel et al., 1979). Upon injection in young chicks, the AMV strain caused a strong and fatal myeloid leukemia while the E26 strain gave rise to an erythroid leukemia (reviewed by Introna et al., 1994). The populations of transformed myeloid cells were in both cases oligoclonal suggesting that the short latency of the AMV and E26 viruses was sufficient to transform hematopoietic cells in the absence of somatic mutations. Furthermore, both retroviruses integrated randomly in the genome of the host cell and transformed bone marrow cells in vitro (Radke et al., 1982). The phenotype of the transformed bone marrow cells was that of immature monoblast in the case of AMV and that of myeloblast in the case of E26. Furthermore, infection of hematopoietic cells with E26 transformed cells of either the erythroid or the myeloid lineage (Radke et al., 1982). Intriguingly, mutant and chimera analysis indicated that the ability of E26 to transform myeloid cells was due to the v-myb oncogene, while the v-ets oncogene, also present in the viral genome, was found to be responsible for the erythroid transformation (Beug et al., 1984; Golay et al., 1988; Metz et al., 1991). Also, AMV temperature-sensitive mutants within the v-myb DNA-binding domain gave rise to distinct cellular phenotypes (Introna et al., 1990), suggesting that the v-myb oncogene is indeed responsible for the transformed phenotype. Sequence analysis revealed that the v-myb oncogene of both AMV and E26 viruses are truncated versions of the c-myb gene, with deletions at both the Nand C-termini as well as additional point mutations. Since then, a large body of work has analyzed the structural alterations within the c-myb gene that could lead to its oncogenic activation and to induction of leukemogenesis. These studies are summarized below. c-myb Gene and Protein Structure The human and mouse c-myb genes have been localized on chromosomes 6 and 10, respectively (reviewed in Introna et al., 1994). The genome of chicken, murine, and human c-myb is organized in at least 15 exons, which span over 15—50 kbp (Fig. 29.2A). The promoter region of c-myb lacks a TATA box and transcription is initiated within a GC-rich region. The resulting 3.4—4

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Figure 29.2. Schematic representations of the c-myb gene and c- and v-Myb protein structures. A: c-myb cDNA. Exons 1 through 15 are represented by numbered white boxes. The alternatively spliced exon 9A is represented by a striped box. The positions of the start and the stop signals of translation (ATG and TGA, respectively) are indicated. Nt: nucleotide. B: c- and v-Myb proteins. The locations of the DNA-binding domain, the transcriptional activation domain (TA), and the negative regulatory domain (NRD), which includes the leucine zipper (LZ) and the EVES (E) domains, are indicated. R1, R2, and R3 represent the three imperfect tryptophan repeats contained in the DNA-binding domain. The potential phosphorylation sites for casein kinase II (CKII) and for mitogenactivated protein kinase (mapk) are labeled P and indicated by black arrowheads. The locations of PEST sequences, 1 through 3, are shown. The single-letter amino acid sequence of the EVES domain is depicted and the serine phosphoacceptor is underlined. The regions of the c-Myb protein that interact with other proteins involved in transcriptional cooperation, including Cyp40, p100, CREB-binding factor (CBP), and p160/p67, are indicated. The size of the truncated v-Myb proteins of the AMV and E26 retroviruses in relation to c-Myb are schematized.

kb c-myb mRNA transcript is found predominantly in hematopoietic tissues with expression decreasing during differentiation (reviewed in Introna et al., 1994). As seen with other nuclear oncogenes, the c-myb mRNA is unstable. The c-myb cDNA encodes for a protein of approximately 640 amino acids, which is highly conserved in humans, mice, and chickens (about 95% aa identity). The c-myb and v-myb gene products have been shown to be nuclear phosphoproteins of 80

and 45 kDa, respectively, able to bind the specific consensus sequence PYACC(G/T)G, termed Myb-binding sequence or MBS (Biedenkapp et al., 1988; Klempnauer and Sippel, 1987). The alignment of myb protein sequences revealed the existence of functional domains conserved in Myb proteins throughout evolution. The most conserved domain is the DNA-binding domain located at the N-terminal third of the protein (Fig. 29.2B). This domain is comprised of three tandem imperfect repeats of 51—52 amino acids

CONCLUSION

(termed R1, R2, R3) (Klempnauer and Sippel, 1987). Each repeat contains three regularly spaced tryptophan residues required for maintaining an active DNA-binding activity (Anton et al., 1988). Mutant analysis revealed that only the last two tandem repeats, R2 and R3, are necessary and sufficient for DNA binding (Gabrielsen et al., 1991). NMR analysis of the R2/R3 domain bound to DNA has shown that each of the myb repeats forms a helix-loop-helix—like structure similar to that of eukaryotic homeodomain (Ogata et al., 1994). Two other members of the myb gene family of transcription factors, the A-myb and B-myb genes, were isolated based on high homology with the region encoding the DNA-binding domain (Nomura et al., 1988). These two latter genes have been the subject of several recent reviews (Golay et al., 1997; Introna et al., 1994; Lipsick et al., 1996; Lyon et al., 1994). Two potential casein kinase II (CKII) phosphorylation sites are present in the N-terminus of c-Myb at Ser-11 and Ser-12 (Fig. 29.2B). Phosphorylation of these sites by CKII has been shown to downregulate the DNA-binding activity to the MBS sequence (reviewed in Luscher and Eisenman, 1990). The transactivation properties of Myb proteins are mediated through AA residues 275—325, termed the transactivation domain (TA; see Fig. 29.2B). A third region of homology between Mybrelated proteins is localized to the C-terminus. Deletion of this region was found to increase the transcriptional activity of c-Myb (Dubendorff et al., 1992; Hu et al., 1991; Ibanez and Lipsick, 1990; Kalkbrenner et al., 1990) and was termed the negative regulatory domain (NRD; see Fig. 29.2B). The NRD is characterized by the presence of a LZ, as well as putative MAP kinase phosphorylation sites. The NRD also mediates intramolecular interaction with the DNA-binding domain through a sequence termed EVES, in the single-letter amino acid code (E in Fig. 29.2B).

Oncogenic Alterations in c-Myb Structural Alterations of c-Myb DNA-Binding Domain and CKII Phosphorylation Sites. Analysis of v-Myb mutants indicated that the DNA-binding domain is rel-

531

evant to the oncogenicity of Myb proteins, although nuclear localization or DNA-binding activity in the absence of transcriptional activity is not sufficient for transformation of hematopoietic cells (Ibanez and Lipsick, 1988). For example, single-point mutations within the DNA-binding region of v-Myb, which affected the DNA-binding activity of the v-myb gene, were sufficient for the AMV virus to transform monoblasts and promyelocytes (Introna et al., 1990). Interestingly, the mim-1 gene, which is a related neutrophil chemotactic factor, was induced by the v-Myb of E26 and by AMV v-Myb point-mutated within the DNA-binding domain but not by the wild-type v-Myb (Introna et al., 1990). Furthermore, expression of the chicken myelomonocytic growth factor (cMGF) was induced by the homeobox gene GBX2, which was shown to be a target of AMV-Myb but not of E26-Myb (Kowenz-Leutz et al., 1997). In accordance, ectopic expression of the GBX2 gene in E26 transformed cells led to a monocytic phenotype resembling that of AMV-Myb transformed cells, and their cell growth was independent from cMGF (Kowenz-Leutz et al., 1997). The differences in biological activity between these Myb proteins can potentially be explained by the recent finding that the cyclosporin-A—binding protein cyclophilin Cyp-40 binds through its tetratricopeptide motif to E26-Myb and to cMyb, but not to the AMV-Myb protein, probably due to point mutations in the domain (see Fig. 29.2B) (Leverson et al., 1998). Interestingly, Cyp40 was found to inhibit the DNA-binding activity of E26-Myb and c-Myb, and this could be overridden by cyclosporin A treatment (Leverson et al., 1998). Thus, these results suggest the possibility that Myb-mediated transformation of hematopoietic cells can also be regulated by conformational changes of Myb proteins at the level of the DNA-binding domain due to interaction with lineage-specific cofactors. The two Ser-11 and Ser-12 at the N-terminal that are phosphorylated by CKII are frequently lost in oncogenically activated Myb proteins upon insertion of retroviruses that cause murine myeloid leukemias and chicken bursal lymphomas (Gonda et al., 1987; Press et al., 1995; Kanter et al., 1988; Shen-Ong et al., 1984). The loss of this CKII-dependent negative regulatory domain could explain the higher DNA-binding

532


affinity of v-myb or of other oncogenically activated Myb proteins relative to that of c-myb. Also, it has been shown that mutations of both Ser-11 and Ser-12 can affect transcriptional cooperativity between c-myb and the NF-M factor during transactivation of an MBS-driven element and the neutrophil elastase promoter (Oelgeschlager et al., 1995). Transcriptional Activation Domain. The acidic and highly hydrophilic domain downstream of the DNA-binding domain comprised within residues 275—325 has been shown to be required for transcriptional activation by c-Myb and vMyb proteins (Kalkbrenner et al., 1990; Klempnauer et al., 1989; Weston and Bishop 1989) (TA, see Fig. 29.2B). This transactivator domain is conserved in A-Myb but not in B-Myb (Nomura et al., 1988), and probably reflects the distinct transactivating properties of these two proteins (reviewed in Introna et al., 1994). Both the transcriptional activation and DNA-binding domain are sufficient to stimulate promoters containing MBS elements (Weston and Bishop, 1989). Furthermore, mutant analysis has demonstrated that that the transcriptional activation domain is necessary for transformation of avian bone marrow cells by v-myb (Lane et al., 1990). Interestingly, none of the acidic residues in this domain is essential for transcriptional activation by v-Myb in animal cells, whereas a deletion of a portion of this domain (234—295 aa) inactivates the oncogenic potential of v-Myb (Chen et al., 1995). Thus, transcriptional activation by v-Myb is not sufficient for oncogenic transformation. Intriguingly, transactivation by c-Myb of the HSP70 promoter, which is DNAbinding domain independent, requires the presence of an intact transcriptional activation domain in addition to C-terminal downstream sequences (Foos et al., 1993). This observation is consistent with the findings that c-Myb associates through its activation domain with coactivators, such as CBP (Dai et al., 1996), during activation of certain promoters (see below). To date, no structural alterations at the level of the transactivation domain of Myb proteins has been reported in tumors. Negative Regulatory Domain. As discussed above, deletion of the NRD increases transcriptional activation of c-Myb. Interestingly, as

shown in Figure 29.2B, v-Myb proteins of both AMV and E26 are truncated for the NRD (Klempnauer et al., 1982). Thus, it was proposed that the loss of the NRD, in concert with the loss of the N-terminus CKII phosphorylation site, contributes to the oncogenic activation of the c-Myb protein. Accordingly, overexpression of truncated forms of c-Myb results in a more efficient transformation of primary hematopoietic cells (Gonda et al., 1989; Hu et al., 1991; Kanei-Ishii et al., 1992) relative to wildtype c-Myb protein (Ferrao et al., 1995; Grasser et al., 1992). More recently, Lipsick and coworkers were able to transform avian myelomonocytic yolk sac cells through high-efficiency infection of a full-length c-Myb. These transformed cells were morphologically heterogeneous with phenotypes characteristic of both macrophage and granulocyte lineage, but distinct from that of cells transformed with v-myb— harboring retroviruses (Fu and Lipsick, 1997). Overall, these findings indicate that C-terminal deletions increase the transforming potential of Myb proteins in a cell lineage—specific manner. In addition to C-terminal truncations due to integration in the viral genome, c-Myb is activated upon viral insertional mutagenesis in several murine leukemias. For example, induction of promonocytic tumors upon injection with pristane and intravenous inoculation of murine leukemia viruses, such as Moloney or Friend-Mulv, leads to the rearrangement of the c-myb gene as a consequence of viral integration (Wolff et al., 1991). Although this rearrangement occurs preferentially at the N-terminus in exons 2 and 3 (see below), resulting in viral LTRdriven expression of N-terminally truncated Myb proteins (Gonda et al., 1987; Shen-Ong et al., 1984), cases have been reported in which murine nondefective retroviruses can integrate within the regions encoding the NRD (reviewed in Wolff et al., 1996). However, it appears that the disruption of negative regulatory elements of c-myb in this system might not be sufficient for induction of leukemias, since the growth in vitro of hematopoietic cells transformed by these Myb proteins requires growth factors (NasonBurchenal and Wolff, 1993). Thus, a multistep process appears to be required for leukemogenesis by oncogenic Myb proteins. One potential mechanism leading to repression of the c-myb gene through the NRD ap-

CONCLUSION

pears to be mediated by the leucine zipper domain (Fig. 29.2B), which is located just upstream of the C-terminal conserved sequence (Hu et al., 1991; Kanei-Ishii et al., 1992). Deletions or point mutations within this region have been shown to induce transactivation and transformation by c-myb, suggesting that an inhibitor that binds to the leucine zipper can suppress transactivation by c-Myb (Kaneii-Ishii et al., 1992). Accordingly, two nuclear proteins, p67 and p160, have been shown to interact with the leucine zipper domain of c-Myb in a tissuespecific fashion (Favier et al., 1994) (Fig. 29.2). Furthermore, p67, the N-terminal portion of p160, was shown to repress transactivation by c-Myb (Tavner et al., 1998). Alternatively, it has been proposed that the leucine zipper mediates the formation of c-Myb homodimers which ultimately are unable to bind DNA (Nomura et al., 1993). Intriguingly, as shown in Fig. 29.2A, an alternative spliced variant of c-myb, which contains an additional exon, termed exon 9A, has been shown to contain a disrupted leucine zipper domain (Woo et al., 1998). This structural alteration is believed to enhance the transcriptional activation by c-Myb. However, no direct correlation between the increased transcriptional activation with a higher-level transforming potential of the variant Myb protein was noted (Woo et al., 1998). It has also been shown that p42 mitogenactivated protein kinase (MAPK) phosphorylates the NRD at Ser-528, a conserved serine residue, leading to repression of c-Myb activity (Aziz et al., 1995; Vorbrueggen et al., 1996). Conversely, mutation of this serine residue to alanine leads to an increase of c-Myb— mediated transactivation of the CD34 promoter but not of the c-myc and mim-1 promoters (Miglarese et al., 1996). Interestingly, sequence analysis revealed that Ser-528 is localized within a conserved C-terminal 10 amino acid long region. This region displays the characteristic structural motif of a PEST sequence (PEST 3; see Fig. 29.2B), being rich in proline, glutamic acid, serine, and threonine and contains the EVES sequence responsible for c-Myb intramolecular regulation (Dash et al., 1996), discussed above. Since PEST regions have been shown to mediate the ubiquitin-26S proteasome degradation pathway of other short-lived proteins, it has been proposed that c-Myb protein

533

turnover might be regulated by its C-terminal sequence (Bies et al., 1997). In agreement with this hypothesis, a myeloid-specific C-terminal truncated version of the c-Myb protein, which has lost the PEST sequence, displayed longer half-life relative to the normal full length c-Myb protein (Bies et al., 1997). Interestingly, the c-Myb protein contains two other PEST sequences, located upstream of the transactivation domain and of the leucine zipper (PEST 1 and PEST 2, respectively, in Fig. 29.2B), but only the C-terminal PEST is conserved among higher vertebrate c-Myb proteins. Thus, it is tempting to speculate that upon deletion of the C-terminal PEST due to retroviral insertion, the loss of this stability determinant sequence leads to a more stable c-Myb protein with increased oncogenic potential. Taken all together, these observations indicate that structural alterations can oncogenically activate the c-Myb protein. These include an amino- and a carboxyl-terminal truncation, leading to the loss of negative regulatory domains. In addition, disruption of the negative regulatory regions can occur by integration within the c-myb genome of nondefective murine retroviruses or by alternative splicing. Aberrant Transcriptional Activation of the c-myb Gene. The c-myb gene is normally expressed at high levels in immature precursors of hematopoietic cells (reviewed in Introna et al., 1994). During differentiation, c-myb mRNA levels are rapidly downregulated at the transcriptional level. This downregulation is thought to be mediated by a conditional arrest of transcriptional elongation that occurs within the first intron of the gene (Bender et al., 1987; Castellano et al., 1992; Watson, 1988a, 1988b). This transcriptional termination mechanism has also been implicated in the regulation of mRNA levels during proliferation and differentiation of hematopoietic cells (Bender et al., 1987). For example, during differentiation of myeloid and erythroid cells, the block in mRNA elongation becomes virtually complete, leading to c-myb mRNA downregulation (Watson, 1988b). Thus, it has been proposed that the disruption of c-myb transcriptional attenuation might play an important role during induction of leukemogenesis. Accordingly, proviral integration in intron 1, which occurs mostly in chicken B-cell lym-

534


phomas (Pizer et al., 1992), has been shown to bypass the transcriptional elongation block of the c-myb gene. In addition to the elongation release, the retroviral insertion of LTR elements within the 5 regulatory sequence of c-myb might represent a potential mechanism of c-myb transcriptional deregulation. For example, Wolff and coworkers identified a common proviral integration site 20—25 kb upstream of the c-myb locus in MuLV-induced promonocytic leukemias (Koller et al., 1996). In these leukemias c-myb is not rearranged. Thus, it is tempting to speculate that upstream proviral insertion might influence the transcriptional regulation of the c-myb gene. However, no direct evidence of c-myb overexpression was found in these promonocytic leukemias. Interaction of Myb with Other Factors. Two other mechanisms potentially relevant to Mybinduced transformation of hematopoietic cells include transcriptional cooperation of Myb with other transcription factors and transcriptional repression. It is now well established that c-Myb cooperates with several factors during transcriptional regulation of a distinct set of genes. For example, transactivation of the chicken mim-1 gene and lysozyme genes by c-Myb requires the cooperation with the NF-M transcription factor, which is the homologue of mammalian C/EBP (Burk et al., 1993; Ness et al., 1993; Mink, et al. 1996). Also, c-Myb and v-Myb have been shown to cooperate with the ETS factors during activation of the mim-1 (Dudek et al., 1992), CD34 (Melotti and Calabretta, 1994), CD13/APN (Shapiro, 1995), and lck (McCracken et al., 1994) promoters. Similarly, c-Myb was found to synergistically interact with the Epstein-Barr virus BZLF1 transactivator in lymphoid cells (Kenney et al., 1992). In addition, the CREB-binding protein CBP binds to the activation domain of c-Myb and functions as a coactivator of Myb transcriptional activity (Dai et al., 1996). This effect can be blocked by ectopic expression of E1A protein, suggesting that E1A-mediated transformation could depend in part on the modulation of c-Myb activity. Furthermore, c-Myb and the core-binding factor cooperate during regulation of the T-cell receptor enhancer (Hernandez-Munain et al., 1994). Another ubiquitous coactivator, p100,

binds to the DNA-binding domain of c-Myb acting as a competitor of intramolecular association between the DNA-binding domain and the C-terminal EVES motif (Dash et al., 1996) (Fig. 29.2B). More recently, it has been shown that the serine/threonine kinase Pim-1 phosphorylates p100, affecting c-Myb activity in a Ras-dependent manner (Leverson et al., 1998). Additional cooperativity has been observed between Myb and C/EBP and PU.1 for transcriptional activation of the neutrophil elastase promoter (Oelgeschlager et al., 1996), and between Myb and C/EBP for the monocytic/ myeloic gene MRP14 (Klempt et al., 1998). The c-myb protein has also been shown to repress artificial and natural promoters containing MBS elements. In particular, c-myb was found to repress transcription of the c-erbB-2 promoter due to competition for binding with the transcription factor TFIID (Mizuguchi et al., 1995). In accordance with this finding, c-myb expression was found to be inversely correlated with c-erbB-2 overexpression in non inflammatory breast cancer (Guerin et al., 1990). Similarly, v-Myb was found to be a potent inhibitor of the N-ras promoter in transient transfection experiments (Ganter et al., 1997). More recently, inhibition of the early granulocytic gene CD13/ APN by c-Maf transcription factor through physical association with c-Myb was reported (Hedge et al., 1998). Thus, direct or indirect Myb-mediated repression constitutes a novel mechanism potentially relevant to transformation and tumorigenicity of hematopoietic cells. Roles of the c-myb Gene in Hematopoietic Cell Proliferation, Differentiation, and Apoptosis The c-myb gene is expressed predominantly, although not exclusively, in hematopoietic cells. Mounting evidence indicates a direct correlation between c-myb expression and proliferation, differentiation, and death of hematopoietic cells. Experiments aimed at defining the role of c-myb in the regulation of these processes relevant to hematopoiesis and leukemogenesis are discussed below. Proliferation. A number of studies have demonstrated a direct role for c-myb in the regulation of proliferation of mature and immature

CONCLUSION

535

TABLE 29.1. Summary of Myb-regulated genes involved in proliferation, differentiation, or apoptosis of hematopoietic cells. Effector Myb

(;) Transactivation (9) Repression Reference

Promoter

Gene Function Neutrophil chemotactic factor Microbicidal Transcription factor Transcription factor Cyclin-dependent kinase Cell cycle Growth factor Growth factor receptor GTPase Stem cell receptor Microbicidal

; ; ; ; ; ; ; 9 9 ; ;

Ness et al., 89; Foos 92 Introna et al., 90 Zobel et al., 92 Nicolaides, 91 Ku et al., 93 Brandt et al., 97 Miglarese et al., 97 Mizuguchi et al., 95 Ganter et al., 97 Melotti et al., 94 Oelgeschlager et al., 96

c-myb c-myb c-myb v-myb c-myb c-myb c-myb c-myb/v-myb

mim-1 Lysozyme c-myc c-myb p34cdc2 DNA topoII FGF-2 c-erbB-2 N-ras CD34 Neutrophil elastase c-kit CD13/APN MRP14 rem-1 lck ADA TCR bcl-2

Tyrosine kinase receptor Metallopeptidase Calcium-binding protein Calcium binding protein Protein tyrosine kinase Deaminase T-cell receptor (enhancer) Antiapoptotic

; ; ; ; ; ; ; ;

v-myb/c-myb

GBX2

Homeobox

;

v-myb v-myb

tom-1 A2b

? Adenosine receptor

; ;

Hogg et al., 97 Shapiro et al., 95 Klempt et al., 98 Kraut et al., 95 McCracken et al., 94 Ess et al., 95 Hernandez et al., 94 Frampton-Munain et al., 96 Kowenz-Leutz et al., 97 Burk et al., 97 Worpenberg et al., 97

v-myb/c-myb v-myb v-myb/c-myb c-myb c-myb c-myb c-myb c-myb v-myb c-myb c-myb

blood cells. These include experiments with antisense c-myb oligonucleotides or constructs, which are able to block cell proliferation of myeloid, erythroid, and lymphoid cells (Anfossi et al., 1989; Arsura et al., 1992; Gewirtz et al., 1988). In particular, Calabretta and co-workers demonstrated the requirement of c-myb for proliferation of erythroid and myeloid precursors during normal human hematopoiesis (Caracciolo et al., 1990). Furthermore, c-myb expression levels were dramatically induced in G1/S phase upon mitogenic stimulation of mature quiescent B and T cells (Golay et al., 1991; Stern and Smith, 1986; Torelli et al., 1985). T-cell proliferation upon PHA or anti-CD3 treatment is dramatically inhibited in transgenic mice ectopically expressing a dominant interfering allele of c-myb (Badiani et al., 1994). Infection with a recombinant Myb murine retrovirus rendered T-cell lines proliferation IL-2 independent (Rose and Reddy, 1992). Furthermore, ectopic express-

ion of the c-myb gene rescued M1 myeloid leukemia from TGF-1—mediated cell growth arrest (Selvakumaran et al., 1994). Previous work has identified numerous Myb target genes that have been involved in cell cycle regulation. One of these genes is the c-myc proto-oncogene (Zobel et al., 1992) (Table 29.1). The c-myc gene contains 15 MBS elements, situated in two clusters (Cogswell et al., 1993). However, the physiological relevance of these elements remains obscure as the levels of c-myc RNA do not vary during the G to S-phase progression in BALB/c 3T3 fibroblasts (Dean et al., 1986), commensurate with the increase in c-myb expression. Other genes targeted by c-myb essential for cell proliferation in both lymphoid and myeloid cell lines include the p34cdc2 (Ku et al., 1993), which is activated at the G /S interphase in hematopoietic cells, but whose function is primarily in G M. Also targeted are fibroblast

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growth factor 2 (FGF-2) (Miglarese et al., 1997) and the DNA topoisomerase IIa gene (Brandt et al., 1997). This latter finding is particularly relevant since the DNA topoisomerase IIa gene, which is essential for cell proliferation, is a target for a number of antitumor drugs (Table 29.1). Moreover, c-Myb has been shown to positively or negatively autoregulate itself in fibroblast and myeloid cells through MBS elements located within the 5 flanking region (Guerra et al., 1995; Nicolaides et al., 1991). Intriguingly, myeloid precursor cells lacking cmyb are able to proliferate normally during yolk sac erythropoiesis, suggesting that either c-Myb can be complemented by other Myb proteins or its function is temporally regulated throughout development. Accordingly, primary murine fetal hematopoietic cells ectopically expressing a Cterminal truncated version of c-Myb protein did not display c-myc and p34cdc2 regulation by c-myb (Hogg et al., 1997). Thus, Myb-mediated regulation of genes involved in proliferation appears to be cell-type specific, probably due to tissue-specific expression of distinct coactivators, as discussed above. Differentiation. Several studies have shown that the c-myb gene is a key regulator of differentiation of hematopoietic cells. Expression of cmyb is downregulated during differentiation of hematopoietic cells (Sheiness and Gardinier, 1984). Moreover, ectopic c-myb expression is sufficient to block differentiation of erythroid and myeloid cell lines as judged by cell morphology and differentiation markers (Bies et al., 1995; Clarke et al. 1988; Selvakumaran et al., 1992). Thus, based on these studies, the downregulation of c-Myb appears to be necessary to erythroid and myeloid terminal differentiation. Interestingly, c-myb null mice displayed an impaired fetal hepatic erythropoiesis, which occurred at day 15 of gestation and shortly thereafter died of anoxia (Mucenski et al., 1991). Morphologic analysis of c-myb\\ littermates revealed that both the erythroid and the myeloid compartments, with the exception of the megakaryocyte lineage, were severely affected with a significant decrease in total cell number. Surprisingly, the mutant mice did not display anomalies during the yolk sac erythropoiesis, which occurs at days 7—12 of gestation, suggesting that c-myb regulates hematopoiesis in a

temporal manner during murine development. Thus, it appears that c-Myb expression is necessary for late-stage hematopoiesis likely due to c-myb—dependent proliferation of erythroid and myeloid precursors (Caracciolo et al., 1990). In contrast, downmodulation of c-myb expression appears to be required for terminal differentiation of erythroid and myeloid cell lines. Myb target genes related to specific stages of myeloid differentiation include the early hematopoietic stem cell receptor CD34 (Melotti et al., 1994), the tyrosine kinase receptor c-kit (Hogg et al., 1997; Ratajczak et al., 1998), the microbicidal neutrophil elastase protein (Oelgeschlager et al., 1996), the metallopeptidase CD13/ aminopeptidase (Shapiro, 1995), the macrophage migration inhibitory MRP14 (Klempt et al., 1998), and the related calcium-binding protein rem-1 (Kraut et al., 1995) (Table 29.1). In addition, c-myb appears to interact with a 5 regulatory element of genes involved in lymphoid cell differentiation. These include the promoter of the lymphoid-specific tyrosine kinase lck (McCracken et al., 1994), the promoter of the CD4 gene (Siu et al., 1992), the enhancer within the locus-control region of the human adenosine deaminase gene in thymocytic T cells (Ess et al., 1995), and the T-cell—specific enhancer of the T-cell receptor (Hernandez-Munain et al., 1994). This latter finding is supported by the observation that an intact Myb-binding site is essential for the activation of VDJ recombination at the T-cell receptor locus (HernandezMunain et al., 1994). The important role of c-myb during T-cell development has been further analyzed by the generation of transgenic mice ectopically expressing interfering alleles of c-myb. These animals displayed a partial block in thymopoiesis and a severely impaired proliferation of T cells upon mitogenic stimulation (Badiani et al., 1994). Conversely, T-cell—specific expression of v-Myb protein in mice led to an increase of the total number of thymocytes and to a late onset of T-cell lymphomas (Badiani et al., 1996). Apoptosis. Recent evidence has also demonstrated a role for the c-myb gene in positive as well as negative regulation of apoptosis. On the one hand, c-myb gene expression accelerated TGF--1—induced apoptosis of the murine M1 myeloid leukemia cell line (Selvakumaran et al.,

CONCLUSION

1994), and, p53 when co-expressed with stimulated apoptosis of both the murine promyelocytic 32D and the human osteosarcoma SAOS2 cell lines (Sala et al., 1996). On the other hand, Lipsick and co-workers found that ectopic expression of c-myb rendered chicken monoblasts more resistant to TPA-induced differentiation and cell death (Smarda et al., 1994). Similarly, a Gag-Myb-Ets fusion product enhanced cell survival of the IL-3—dependent granulocytic FDCP2 cells when these latter were transferred from IL-3 to erythropoietin-containing media (Athanasiou et al., 1996). Moreover, Frampton and colleagues (1996) found that the v-myb oncogene contained in the E26 retrovirus acts as a survival factor in infected chicken myeloblasts by directly transactivating the antiapoptotic gene Bcl2. In agreement with this finding, ectopic c-myb expression could rescue the IL-2—dependent cytotoxic T-cell line CTLL-2 from cell death induced by IL-2 deprivation or dexamethasone via induction of Bcl2 gene expression (Salomoni et al., 1997). In addition, a dominant negative c-Myb protein was found to induce programmed cell death when introduced in the thymoma cell line EL4 through selective downregulation of Bcl2 gene expression (Taylor et al., 1996). Thus, c-myb may cause cell death in a cell-type—specific fashion. Taken together, these data indicate that c-myb regulates genes involved in proliferation, differentiation, and cell survival. Although a more extensive identification and analysis of downstream target genes by Myb proteins is needed, it is tempting to speculate that the deregulation of one or all of these downstream targets following Myb oncogenic activation could significantly contribute to induction of leukemogenesis. Amplification, Rearrangement, and Expression of the c-myb Gene in Human Leukemias The human c-myb gene has been localized to the q22—24 region of chromosome 6 (Harper et al., 1983; Janssen et al., 1986). Deletions in this region have been reported in acute lymphoblastic leukemias (Oshimura et al., 1977), chronic myelogenous leukemias (Mitelman et al., 1983), and non-Hodgkin’s lymphomas (Bloomfield et al., 1983). Often the cell lines that have suffered 6q deletions express higher levels of c-myb

537

mRNA than those expressed by non 6q\ at a similar stage of development and lineage (Barletta et al., 1987; Castaneda et al., 1991; Okada et al., 1990). In particular, four 6q\ acute myelogenous leukemia cell lines, ML-1, ML-2, ML-3, and HL-60, displayed amplification of the c-myb locus (Pelicci et al., 1984). Taken together, these observations suggested a possible involvement of c-myb during induction of leukemogenesis in hematopoietic cells with 6q deletions. In the past, several laboratories have tried to map possible rearrangements and deletion of the c-myb locus in human leukemias. Despite numerous efforts, in most of the cases the c-myb gene itself has not been found to be rearranged or deleted (Okada et al., 1990; Park et al., 1992). In only the case of the acute lymphoblastic leukemia CCRF-CEM, which is not 6q\, the c-myb promoter region was found to be rearranged due to a submicroscopic deletion in chromosome 6 (Jacobs et al., 1994). This evidence, together with the finding that 6q\ T-cell leukemias such as MOLT-4 and PEER do not show amplification of c-myb (Ohyashiki et al., 1988), suggests that the elevated levels of c-myb gene expression are likely to be related to lineage development rather than to 6q\ abnormalities. Very recently, the chronic myelogenous leukemia TK-6 cell line has been found to express a C-terminal truncated c-Myb protein (Tomita et al., 1998). This shorter cMyb displayed higher transactivating activity than the full-length c-Myb (Tomita et al., 1998). However, it is unlikely that this truncated form of the c-myb gene contributed to the induction of leukemogenesis, since its expression was detected only after the T-cell blast crisis. The level of c-myb expression has also been investigated in a panel of B-cell neoplasia representing the entire spectrum of differentiation of the B-lymphoid compartment. Unlike A-myb, whose expression appears to be restricted to a subset of B-cell neoplasia such as Burkitt’s lymphomas and sIg> B acute lymphoblastic leukemias (ALLs), c-myb was found to be expressed in most of the cell lines studied, although with some heterogeneity (Golay et al., (1996)). In particular, c-myb was found to be expressed in most of the fresh cases of ALL studied but in only 3 out of the 20 chronic lymphocytic leukemias (CLLs), probably due to the lower mitotic index of CLLs (Golay et al.,

538


(1996)). Remarkably, c-myb expression, although present at lower levels than in the ALLs, was detected in 4 out of 5 myelomas analyzed, suggesting that c-myb might play a role in B-cell neoplasia at later stages of differentiation. In all these cases, however, no structural alterations of the c-myb gene were detected. In acute myelogenous leukemias, Gopal and co-workers (1992) observed the level of c-myb expression correlated with poor prognosis in response to therapy. Conclusion The c-myb gene, which is expressed predominantly in hematopoietic cells, has been found to play a critical role in development of the hematopoietic lineage. In particular, c-myb promotes cell proliferation and affects differentiation. This function probably reflects the ability of the oncogene v-myb of AMV and E26 to transform erythroid and myeloid cells in birds. In this system the oncogenic potential of v-myb appears to be dominant over that of v-myc. In fact, the phenotype of doubly v-myb and v-myc transformed chick myelomonocytic cells was found to be indistinguishable from that of cells transformed with v-myb alone (Ness et al., 1987). Ironically, there is little solid evidence to date implicating c-myb in neoplastic transformation of hematopoietic tissues in humans. In fact, the role of c-myb in leukemias has most clearly been delineated in birds and mice. In these models the c-myb genes have been found to be activated by deletion or point mutation of negative regulatory regions, rearrangements, and aberrant transcriptional activation. Taken together, these findings do not exclude the possibility that point mutations or microscopic rearrangements could also be involved in c-myb deregulation during human leukemogenesis. However, a more systematic analysis of the c-myb gene structure in these tumors is needed. Overall, much still needs to be learned about the mechanism of action of both c-myc and c-myb. In particular, recent work suggests that these genes function through multiple mechanisms in a tissue and cell-type—specific manner. Thus, the role of transactivation and/or repression, and the identification of the critical genes controlled by c-myc and c-myb that mediate their effects on proliferation, differentiation, and neoplastic transformation, remain to be elucidated.

ACKNOWLEDGMENTS Josee Golay is gratefully acknowledged for helpful discussions. We thank Darin Sloneker for assistance in preparation of this manuscript. This work was supported by grants from the Cure for Lymphoma Foundation (MA), the Charlotte Geyer Foundation (MA), and National Institutes of Health grant CA36355 (GES).

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CHAPTER 30


NF-B IN CELL LIFE AND DEATH WINNIE F. TAM Rosenstiel Basic Medical Sciences Research Center and Department of Biology, Brandeis University

JYOTI SEN Dana-Farber Cancer Institute, Department of Pediatrics, Children’s Hospital and Harvard Medical School

RANJAN SEN Rosenstiel Basic Medical Sciences Research Center and Department of Biology, Brandeis University

INTRODUCTION The NF-B/Rel family of transcription factors in mammalian cells regulates inducible transcription of a large number of genes in response to diverse stimuli. NF-B regulates expression of genes encoding cytokines, chemokines, growth factors, cytokine receptors, and cell adhesion molecules. It is activated in response to various stimulants such as cytokines, oxygen-free radicals, UV light, and viral gene products. Recent observations suggest that a key feature of NFB—dependent gene expression is the regulation of cell proliferation and cell death; therefore, both aspects are likely to be important in the

contribution of NF-B proteins to normal development, as well as to the role of NF-B in disease. Aberrant NF-B activation has been linked to several forms of human cancers, including Hodgkin’s lymphoma, breast cancer, and B-cell leukemias. Furthermore, ectopic expression of v-Rel in the mouse thymus leads to thymic lymphomas. NF-B activation has also been noted in inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, and autoimmune encephalitis. Therefore, rational manipulation of NF-B function is likely to be beneficial in diverse circumstances. In this chapter we review recent studies that address the mechanisms that link Rel proteins to the control of cell proliferation and cell death.


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THE NF-B FAMILY NF-B was identified as a DNA-binding protein that recognized a sequence within the immunoglobulin light-chain gene enhancer. This and other related sequences are referred to as B elements. The NF-B/Rel family consists of five proteins: p50 and p52, which are expressed as larger precursors of approximately 100 kD, RelA (p65), c-Rel, and RelB (Gerondakis et al., 1998; Ghosh et al., 1998; Sha, 1998). Rel family members form homo- or heterodimers, generating multiple B-binding activities within cells. Among these many B-binding proteins, NF-B refers to the p50/p65 (RelA) heterodimer. It also follows that a simple DNA-binding assay, such as electrophoretic mobility shift assays, cannot distinguish which specific B-binding proteins generate the observed nucleoprotein complex observed in vitro. Additional experiments such as ‘‘supershifting’’ with antibodies is usually required to determine the composition of the DNA-binding activity. The Rel proteins are characterized by the presence of a 300 amino acid domain, known as the Rel homology domain (RHD), which is also found in the v-rel oncogene of the avian acute transforming retrovirus RevT. The RHD is sufficient for DNA binding, nuclear localization, homo- and heterodimerization among Rel family members, and association with I-B, the inhibitor of NFB. X-ray crystallographic structures of the RHDs of p50 and RelA, with and without complexed I-B, were recently determined (Baeuerle, 1998; Chen, 1998; Cramer et al., 1997; Cramer and Muller, 1999; Ghosh et al., 1995; Huang et al., 1997; Huxford et al., 1998; Jacobs and Harrison, 1998; Muller and Harrison, 1995; Muller et al., 1995; Sengchanthalangsy et al., 1999). Unlike most sequence-specific DNA-binding proteins that use -helices to bind DNA in the major groove, the RHDs make DNA contacts using loops that project from the ends of more defined secondary structures. Contacts are made primarily in the major groove of DNA and result in high-affinity protein/DNA interaction. The five family members can be further classified according to their domain structure. p50 and p52 consist largely of RHDs (Fig. 30.1); they do not contain classical transcription activation domains and have been considered to be transcriptional repressors. Indeed, in cotransfec-

tion assays, overexpression of p50 suppresses NF-B—dependent transcription, presumably by displacing a transcriptionally active Rel heterodimer. However, the suppressive role of p50 and p52 should be taken with caution because it is possible that in the appropriate promoter context these proteins may act as transcription activators by recruiting other factors to the DNA. For example, the ankyrin domain—containing protein Bcl-3 contains a transcription activation domain but no DNA-binding domain. Bcl-3 is known to associate with p50 and may provide transactivation function to DNAbound p50. Furthermore, the Drosophila melanogaster Rel protein, dorsal, has been shown to activate or repress transcription depending on the promoter context. These observations suggest that the properties of Rel proteins may be modified by association with other factors. p65 (RelA) and c-Rel are closely related proteins that contain N-terminal RHDs and Cterminal transcription activation domains (Fig. 30.1). RelA is the more potent transcription activator of the two when analyzed by cotransfection with NF-B—dependent promoter constructs. These proteins can homodimerize or heterodimerize with each other, p50, or p52 to form functional units. Both c-Rel and RelA bind IB proteins (May and Ghosh, 1997; Miyamoto and Verma, 1995; Whiteside and Israel, 1997), whereas p50 and p52 do not. Rel/I-B complexes do not bind DNA, and I-B was shown to disrupt Rel/DNA complexes in vitro (Zabel and Baeuerle, 1990). Association with I-B also sequesters Rel proteins in the cell cytoplasm, leading to the generation of a cytosolic pool of Rel proteins that can be mobilized rapidly to respond to cell activation. Recent crystallographic analyses of Rel/I-B complexes provide molecular explanations for both these properties. The first and second ankyrin repeats of I-B interact closely with the nuclear localization sequence (NLS) of p65/RelA located at the carboxy end of the RHD, thereby blocking the NLS from interacting with nuclear importins. Furthermore, the N-terminal half of the RHD, which contains most of the DNA contact residues, is folded away in the Rel/I-B complex, thereby precluding DNA binding. RelB falls in a category by itself (Fig. 30.1); it can heterodimerize with p50/p52, but RelB-containing complexes are weakly inhibited by I-Bs.

THE I-B FAMILY

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Figure 30.1. Domain organization of NF-B/Rel and I-B proteins. A: Rel proteins in mammalian cells. The signature Rel homology domain (RHD) is shaded dark; the nuclear localization sequence located toward the C-terminus of the RHD is indicated as N. TD refers to transcription activation domains present in c-Rel, p65/RelA, and RelB. The p50 and p52 subunits of NF-B can be produced as larger polypeptides of 105 and 100 kD, respectively. The C-termini of the larger proteins contain six ankyrin domains. A glycine-rich hinge, indicated as G, separates the N-terminal RHD from the C-terminal ankyrin domains of these proteins, and the approximate termini of p50 and p52 are indicated by inverted black triangles. B: I-B proteins in mammalian cells. These proteins are characterized by the presence of multiple ankyrin domains indicated as shaded boxes. At the C-termini are PEST-rich sequences and several type II casein kinase phosphorylation sites (not shown in the figure). SS indicates the position of serine residues that undergo signal-induced phosphorylation by the I-B kinases. The K indicates a lysine residue that is ubiquitinated as a consequence of signal-induced phosphorylation, which targets the molecule for proteasome-mediated degradation. Bcl-3 is unique in containing a transcription activation domain (TD) at the C-terminus.

THE I- B FAMILY IB protein family is characterized by the presence of a small (37—40 amino acid) structural feature known as an ankyrin domain (Fig. 30.1). IB, , and contain six ankyrin repeats and are the most closely related. The ankyrin domains, which are clustered in the middle of the protein, are flanked by N- and C-terminal domains that are important for I-B function. The

N-terminal domain contains two crucial serine residues whose signal-induced phosphorylation targets I-B for ubiquitination and proteasomemediated degradation. The site of ubiquitination is also located in this domain. The C-terminal domain contains PEST sequences that contribute to protein turnover times and sites for phosphorylation by type II casein kinase. Degradation of IBs releases the associated Rel proteins to translocate to the nucleus and activate

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gene expression. This mechanism forms the basis of the classical posttranslational pathway of NF-B induction. The precursors of p50 and p52 also contain six ankyrin repeats at the C-terminus that serve I-B—like function. First, the full-length p105 or p100 proteins do not bind DNA well until the carboxy termini are deleted, suggesting that the ankyrin repeats intramolecularly inhibit DNA binding (Blank et al., 1991; Dobrzanski et al., 1995; Hatada et al., 1993; Mellits et al., 1993; Mercurio et al., 1992; Naumann et al., 1993a, 1993b; Rice et al., 1992). Second, the p105/p100 molecules can sequester other Rel proteins, such as c-Rel and RelA, in the cytoplasm to provide another pool from which posttranslational induction can take place (Harhaj et al., 1996; Naumann et al., 1993a, 1993b; Scheinman et al., 1993). However, it is not known if a p100/Rel heterodimer is targeted to release DNA-binding NF-B protein (Belich et al., 1999; Boland and O’Neill, 1998; Fujimoto et al., 1995; Harhaj et al., 1996; Lin et al., 1998; MacKichan et al., 1996). Recent studies indicate the HTLV-1 transactivator-mediated NF-B activation may proceed, in part, by induced degradation of p100/p110 (Munoz and Israel, 1995; Murakami et al., 1995; Rousset et al., 1996). Finally, the proto-oncogene bcl-3 contains seven ankyrin repeats and has been proposed to be an IB (Franzoso et al., 1992; Hatada et al., 1992; Inoue et al., 1993; Kerr et al., 1992; Lenardo and Siebenlist, 1994; Nolan et al., 1993; Zhang et al., 1994). Early studies showed that bcl-3 proteins inhibited DNA binding by p50 homodimers in vitro, as well as contained a transcription activation domain (Dechend et al., 1999; Fujita et al., 1993; Na et al., 1998). These apparently contradictory observations, plus the largely nuclear location of bcl-3, suggest that bcl-3 is not a typical IB. The posttranslational mechanism of NF-B induction has been substantiated in a wide variety of cell types in response to diverse stimuli. Indeed, the generality of this mode of induction has obscured more subtle levels of NF-B regulation, which nevertheless may be of functional significance. For example, c-Rel has been shown to be regulated at transcriptional and posttranscriptional levels. The regulation of c-Rel in B and T cells is different (Venkataraman et al., 1995; Wang et al., 1997). In T cells, c-Rel is

required to activate interleukin-2 (IL-2) gene expression, and this cytokine then serves as a growth factor to induce cell proliferation. However, signals that are sufficient to induce IL-2 gene expression do not induce posttranslational nuclear translocation of preexisting cytoplasmic c-Rel in T cells (Venkataraman et al., 1996). Instead, these signals induce de novo c-Rel synthesis by activating c-Rel gene transcription and translation. The newly synthesized protein translocates directly to the nucleus. In B cells, antigen receptor signals activate cytoplasmic c-Rel by the classical posttranslational pathway (Venkataraman et al., 1996). Overall, this example serves to illustrate that regulation of Rel family members may vary depending on the cell type and the nature of the signal. NF- B FUNCTION As transcriptional regulators, Rel family members function by altering gene expression. In the majority of hematopoeitic cells, NF-B is located in the cytoplasm in an inactive form, and transported to the nucleus as a result of cell stimulation. Rapid response of NF-B is due to I-B degradation and transport of the pre-existing protein to the nucleus; the response at late times is controlled by de-novo expression of Rel proteins. Therefore the composition of DNA binding Rel proteins in the nucleus may be different at early or late stages of cell stimulation. Mature B cells are an exception in that nuclear NF-B is present in unstimulated cells. Even in these cells, however, NF-B can be further induced and used to regulate inducible genes. The best characterized NF-B-dependent genes are those encoding cytokines, cytokine receptors, cell adhesion molecules and antigen receptors. In addition, several ubiquitously-distributed intracellular molecules such as c-Myc (Ji et al., 1994; Lee et al., 1995a, 1995b; Qin et al., 1999; Siebelt et al., 1997) and p53 (Hellin et al., 1998; Kirch et al., 1999; Qin et al., 1999; Wu and Lozano, 1994) have also been implicated as NF-B targets. The regulation of many NF-B— dependent genes has been intensively studied. Most studies start with the identification of NF-B binding sites in promoters and the demonstration that mutation of these sites reduces

LESSONS FROM THE ANALYSIS OF Rel GENE KNOCKOUTS

gene expression. Mechanistic studies of an inducible NF-B dependent promoter are best exemplified by the analysis of the -interferon gene promoter studied by Maniatis and colleagues (Algarte et al., 1999; Kim and Maniatis, 1997; Kirchhoff et al., 1999; Sica et al., 1997; Wathelet et al., 1998). In several instances, the proposed requirement for NF-B was confirmed by the analysis of gene knockout mice. For example, the IL-2 gene had been proposed to be an NF-B target based on promoter analysis; of the several Rel-knockout mice analyzed, no IL-2 production was seen in c-Rel\\ mice, confirming the role of this NF-B family member in IL-2 gene expression. However, all putative NF-B target genes were not inactivated in mouse strains deleted in single Rel family member genes. This observation indicates a degree of functional redundancy among Rel proteins. It is expected that these genes will be found to be inactive in mice deficient in two or more Rel genes. Future issues in the area of NF-B—mediated gene transcription will relate to the mechanisms by which Rel proteins, in concert with other transcription factors, regulate gene expression. For example, it is important to understand why some promoters are promiscuous and can be activated by several Rel proteins, whereas others, such as IL-2, are regulated by specific family members. It will also be important to understand how Rel proteins interact with other transcription factors in the context of specific promoters.

LESSONS FROM THE ANALYSIS OF Rel GENE KNOCKOUTS Analysis of mice deficient in Rel genes have been surveyed in excellent reviews (Attar et al., 1997; Baeuerle and Baltimore, 1996; Gerondakis et al., 1999). Three general observations can be gleaned from these studies. First, mice deficient in each of the Rel genes have a distinct phenotype. For example, lymphocytes from p50\\ mice proliferate poorly, while activated T lymphocytes from mice deficient in the related p52 gene produce higher levels of IL-2 and GMCSF. RelA\\ mice die at embryonic day 15 due to hepatocyte apoptosis, whereas deletion of the related c-Rel gene results in viable, though im-

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munocompromised, mice. RelA\\ lymphocytes proliferate well in response to mitogens, whereas c-Rel\\ lymphocytes do not proliferate even to strong signals such as lipopolysaccharide (B cells) or phorbol ester and calcium ionophore (T cells). These observations suggest that even the most closely related Rel proteins perform distinct functions in vivo. The specificity in function is difficult to explain in molecular terms because the DNA-binding specificity and the domain structure of these proteins are quite similar. One possibility is that these proteins are differentially expressed in different tissues; it is also possible that Rel proteins may interact with different partners in different cell types, thereby leading to the differences in the phenotypes of the knockouts. To accurately determine whether a particular Rel gene can functionally substitute for another, it must be ‘‘knocked-in’’ to the other locus so as to be appropriately regulated in each tissue. Second, no developmental defects are apparent in any of the hematopoeitic lineages in the single gene knockouts. This is based on the analyses of cell populations by flow cytometry using limited sets of markers, and it is possible that more sensitive analyses (such as arraybased RNA analysis) will reveal subtle maturational differences. However, when both p50 and p65/RelA genes are deleted, B- and T-lymphocyte development is blocked. Because p65\\ mice die at embryonic day 15, the hematopoeitic reconstitution potential of p50/p65 doubly deficient cells was tested by transferring fetal liver cells to lethally irradiated mice (Horwitz et al., 1997). Long-term survival of irradiated mice indicated successful reconstitution of erythroid and myeloid lineages; however, B and T cells were severely depleted while the numbers of neutrophils were enhanced. Impaired lymphocyte development was not a cell-autonomous defect and could be rescued by cotransplanted wild-type fetal liver cells. These observations suggest that NF-B is required for the synthesis of an extracellular factor that is necessary for lymphopoiesis. The known function of NF-B as a regulator of cytokine and cell-surface receptor genes is consistent with the idea of a missing factor. Third, most of the defects identified in the Rel knockouts pertain to aspects of cell cycle progression and cell viability. c-Rel\\ and p50\\

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TABLE 30.1. NF- B and the Cell Cycle NF-B as Pro-proliferative 1. NIH 3T3 cells ; serum NF-B!, proliferation (Baldwin et al., 1991) 2. SMC and IB ; proliferation (Bellas et al., 1995) 3. Regenerating liver ; NF-B! (Taub, 1996) 4. RS cells ; IB ; proliferation (Bargou et al., 1997) 5. p50\\, c-Rel\\ ; primary lymphocyte proliferation (Grumont et al., 1998) 6. HeLa cells ; IB ; proliferation (Kaltschmidt et al., 1999) 7. Myoblast proliferation ; NF-B (Guttridge et al., 1999) 8. Ras connection (Mayo et al., 1997)

NF-B as Antiproliferative 1. Growth-arrested Abelson virus transformed cells ; NF-B (Klug et al., 1994) 2. Jurkat cells ; p21 ; NF-B! (Perkins et al., 1977) 3. HeLa cells ; c-Rel ; G1 block (Bash et al., 1997) 4. Epidermal epithelial cells ; IB ; hyperplasia (Seitz et al., 1998) 5. Pro-B cells ; p65/RelA ; G1 block (Sheehy and Schlissel, 1999) 6. INK4 suppress p65-dependent transcription (Wolff and Naumann, 1999)

SMC, smooth muscle cells; RS, Reed-Sternberg cells.

lymphocytes show reduced proliferation and viability in vitro, while RelB\\ and p50 !2 !2 (deletion of the ankyrin domain—containing Cterminus of p105) have enhanced inflammatory responses in vivo. It is possible that the developmental defect in p50/p65\\ double knockouts is also a reflection of reduced viability of one (or more) precursor cell populations. In this regard, it was recently proposed that NF-B plays a role in the survival of human CD34> hematopoeitic precursor cells in the bone marrow (Pyatt et al., 1999). Because regulated cell proliferation and cell death are essential for appropriate development, the lack of significant developmental defects is surprising. One explanation is that multiple parallel pathways are probably involved in making life/death decisions during differentiation; Rel proteins constitute one such pathway, but their importance relative to others may vary among different cell types. Furthermore, functional overlaps between Rel family members may compensate for the loss of single Rel genes, thereby precluding the detection of developmental defects. Analyses of mice deleted in Rel genes underscore the importance of NF-B in the regulation of cell proliferation and cell viability. In the following sections, we discuss the mechanisms of NF-B function as it pertains to these two emerging areas.

NF- B AND THE CELL CYCLE The transforming potential of v-Rel, the oncogene of the acute transforming retrovirus RevT, first suggested a relationship of NF-B to the cell cycle. Subsequently, serum stimulation of quiescent BALB/C 3T3 cells was shown to induce NF-B and cell cycle progression (Baldwin et al., 1991). Since that study, several additional examples have strengthened the connection between NF-B and the cell cycle (Table 30.1). These studies show that Rel proteins can block or facilitate cell cycle progression. An analysis of these observations is presented below, though no unifying conclusions can be drawn at present. NF-B and Cell Cycle Inhibition Ectopic expression of c-Rel arrests HeLa cells in the G1/S stage of the cell cycle. However, overexpression of I-B, which should block NF-B function, also arrests HeLa cells at the same stage. If one discounts the trivial possibility that HeLa cell transfectants were coincidentally selected for other unrelated changes, this contradiction may be resolved by taking into account that c-Rel induces I-B expression in these cells (Bash et al., 1997). The results of ectopically expressing c-Rel in HeLa cells paral-

NF-B AND THE CELL CYCLE

lels the observation that RelA/p65 expression in a pro-B cell line also inhibits the cell cycle at the G1/S boundary (Sheehy and Schlissel, 1999). Like the c-Rel expression in HeLa cells, these studies also used an inducible expression system, which minimizes the possibility of secondary alterations during clone selection. One of the striking features of the latter study was that overexpressing RelA in a mature B cell line did not induce cell cycle arrest. Overall, these studies indicate that sustained NF-B activity inhibits cell cycle progression in G1. Additional evidence for a growth-inhibitory role for NF-B comes from a study of transgenic mice expressing nondegradable I-B in epidermal epithelial cells, which induced hyperplasia (Seitz et al., 1998). This suggested that inhibition of NF-B by I-B enhanced cell proliferation. In the same study, ectopic expression of p50 resulted in growth inhibition. The cyclin-dependent kinase (Cdk) inhibitors p21 and p27 have been shown to enhance NF-B activity in transient transfection assays (Perkins et al., 1997; Wolff and Naumann, 1999). The mechanism of transactivation by NF-B is not known; however, p65 association with CBP/ p300 and Cdk2 suggests that enhanced transcriptional activity may be due to posttranslation modification (such as phosphorylation) of CBP/p300, a known coactivator of p65. Because high p21/p27 activity (and low Cdk2 activity) is found in G1-arrested cells, these observations suggest that NF-B transcriptional activity can be enhanced in G1-arrested cells. This idea is further corroborated by studies of pre-B cell lines transformed with a temperature-sensitive v-abl oncogene. In these cells, when the v-abl kinase is active, the cells grow rapidly and express low levels of nuclear NF-B. Inactivation of kinase activity at the restrictive temperature leads to growth arrest and induction of NF-B activity (Klug et al., 1994). In contrast to HeLa or pro-B cell lines, where Rel proteins caused cell cycle arrest, these observations suggest that NF-B activity is increased when cells accumulate in G1. Finally, it was shown that the ankyrin-domain—containing Cdk inhibitor, p16/INK4, blocks NF-B—dependent transcription by directly interacting with p65/RelA (Wolff and Naumann, 1999). Therefore, NF-B activity can be either raised or lowered by Cdk inhibitors. Taken together, these observations show that

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NF-B activity can be modulated in G1-arrested cells; however, sustained NF-B—dependent gene expression prevents further cell cycle progress. NF- B and Cell Cycle Progression A requirement for Rel proteins for cell proliferation was noted in transformed as well as primary cells (Table 30.1). Many of these studies have involved assaying the effect of IB (or a nondegradable form of IB) expression in different cell types. With this assay, IB was shown to block proliferation of HeLa cells (Kaltschmidt et al., 1999), Hodgkin Reed-Sternberg cells (Bargou et al., 1997), smooth muscle cells, and hepatocytes (Autieri et al., 1995; Bellas et al., 1995; Bellas and Sonenshein, 1999; Bretschneider et al., 1997; Iimuro et al., 1998; Nakajima et al., 1994; Selzman et al., 1999a, 1999b; Taub, 1996; Xu et al., 1998). The block in proliferation is seen primarily in the transition of cells from the G1 to the S phase of the cell cycle. Successful negotiation of the G1/S checkpoint involves rescue of cells from a preprogrammed apoptotic pathway that is activated when cells reach the G1/S boundary without activating appropriate antiapoptotic genes (Evan and Littlewood, 1998). Therefore, NF-B can affect proliferation as a progression factor, an antiapoptotic factor, or both. In this section, we consider the evidence that NF-B can directly influence cell cycle progression. The clearest evidence for a role for Rel proteins in proliferation comes from the analysis of c-rel—deficient mice (Gerondakis et al., 1996; Ko¨ntgen et al., 1995; Tumang et al., 1998). c-rel\\ B lymphocytes do not proliferate in response to B-cell mitogens such as anti-immunoglobulin, anti-CD40, and bacterial lipopolysaccharide (LPS). These cells also undergo enhanced apoptosis in response to activation by cross-linking the antigen receptor with anti-Ig antibody. Provision of a transgenic bcl-2 gene to c-rel\\ B cells prevents anti-Ig—induced apoptosis, but does not rescue the proliferative defect (Grumont et al., 1998). Tumang and colleagues (Tumang et al., 1998) reached similar conclusions using a different assay. They showed that apoptosis of c-rel\\ B cells induced by in vitro culture could be rescued by CD40 or LPS; however, neither regimen induced proliferation.

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These results show that the antiapoptotic function of c-Rel can be separated from its requirement for cell cycle progression. p50-deficient B cells also show markedly reduced proliferation in response to LPS and CD40 cross-linking, but do not undergo apoptosis. In both c-Rel and p50-deficient animals, B cell cycle progression is blocked in early G1, showing that both these proteins are required for G1 progression. A similar distinction between cell proliferation and antiapoptosis was noted in cells derived from Hodgkin’s disease patients (Bargou et al., 1997). Analysis of primary lymph node cells, or cell lines, from Hodgkin’s patients showed elevated levels of active RelA. Upon expression of dominant negative IB, which should decrease functional NF-B levels, the growth of these cells was significantly impaired and they accumulated in the G1 phase. However, the cells did not die, again indicating that decreased NF-B expression blocked cell cycle progression without affecting viability. When these cells were induced to undergo apoptosis by serum starvation, the IB transfected cells underwent apoptosis more rapidly than the untransfected parental cells. These observations suggest that NF-B also functions as an antiapoptotic agent in these cells. The effects of NF-B on proliferation and cell viability, however, were clearly distinguishable. One mechanism by which NF-B may play a direct role in cell cycle progression is suggested by the observation that the cyclin D1 gene required for G1 progression is a target of NF-B (Guttridge et al., 1999; Hinz et al., 1999). In summary, Rel proteins can affect the cell cycle directly by activating genes required for progression, or indirectly by activating antiapoptotic genes, or by both mechanisms. The relative importance of each pathway will vary between cell types and needs to be experimentally evaluated in each situation. Given the central role of proliferation and quiescence during cell differentiation, it is likely that the growth regulatory properties of NF-B will be important during normal hematopoeisis. NF- B AND CELL VIABILITY The first demonstration of the role of NF-B in cell viability was provided by the hepatocyte

apoptosis observed in p65/RelA-deficient embryos (Beg and Baltimore, 1996). Hepatocyte cell death could have resulted from cell intrinsic defects due to the absence of p65, or from the absence of a hepatocyte survival signal/factor produced by another cell. Evidence for a direct role of p65/RelA in cell survival was provided by several in vitro studies: p65-deficient embryonic fibroblasts were more sensitive to TNFinduced death and a nondegradable form of I-B-enhanced HeLa cell death induced by TNF and DNA-damaging agents (Beg and Baltimore, 1996; Van Antwerp et al., 1996; Wang et al., 1996). Further evidence was provided by the observation that expression of NF-B prevented death of WEHI-231 cells induced by cross-linking surface immunoglobulin receptors (Lee et al., 1995a, 1995b; Schauer et al., 1996, 1998; Wu et al., 1996). Since these initial studies, dominant negative IB was shown to accentuate cell death in many cell types by downregulating NF-B levels and activity (Feig et al., 1999; Paillard, 1999; Sugiyama et al., 1999; Sumitomo et al., 1999). The diversity of death-inducing signals that are suppressed by NF-B, and the variety of cells in which this occurs, strongly suggests that most cells can utilize this pathway to enhance cell viability; it must be noted, however, that many of these studies have used transformed cells. Because the mechanism of transformation may affect the outcome of such experiments, parallel analysis of primary cells and cell lines will be required to determine the circumstances in which NF-B provides antiapoptotic functions in a particular cell type. Different Rel family members may affect cell survival in different circumstances. Because IB inhibits induction of both c-Rel— and p65— containing homo- or heterodimers, experiments using nondegradable I-B do not distinguish between effects mediated by p65/RelA or c-Rel, or both. One of the clearest examples of the differential effects of Rel proteins on cell viability comes from a comparison of B lymphocytes from mice in which Rel proteins have been selectively deregulated. In c-rel\\ animals, B cells develop normally and have normal turnover times in vivo (Grumont et al., 1998). In contrast, p50-deficient B cells turn over more rapidly in vivo, suggesting that a p50-containing NF-B protein is required to maintain B-cell

ANTIAPOPTOTIC GENES REGULATED BY NF-B

lifetime in vivo. Furthermore, B-cell numbers are reduced in transgenic mice that express nondegradable I-B in these cells (Bendall et al., 1999). A possible explanation is that I-B expression reduces the levels of functional NFB proteins that are required to maintain cell viability in vivo. Additional evidence in favor of this idea is the observation that low constitutive NF-B expression in xid/CBA-N mice, which are defective in signaling via Bruton’s tyrosine kinase, renders B cells particularly susceptible to death (Woodland et al., 1996). Because reducing NF-B function by I-B reveals a more extreme reduction in B-cell numbers compared to a single gene deficiency in p50\\ animals, these observations suggest that multiple Rel proteins (other than c-Rel) are involved in the maintenance of mature B cells. While p50 is required for viability of resting B cells, c-Rel is required to prevent apoptosis in activated B cells. c-Rel\\ B cells, but not p50\\ B cells, undergo apoptosis when the antigen receptor is cross-linked in vitro. Cell death in c-Rel\\ B cells can be prevented by ectopic expression of Bcl-2, indicating that c-Rel is required to maintain B-cell viability after activation. Taken together with studies discussed in the preceding sections, these observations suggest that c-Rel provides two critical functions in activated B cells via independent signaling pathways: one maintains cell viability and the other induces cell proliferation. These studies indicate that different Rel family members are utilized to provide maintenance signals for B cells in vivo, inhibit activation-induced cell death, and mediate activation-induced proliferation.

ANTIAPOPTOTIC GENES REGULATED BY NF- B NF-B manifests antiapoptotic activity by regulating the expression of several key genes. c-Myc was first identified as an antiapoptotic gene and a NF-B target in the immature B lymphoma WEHI-231 (Soneshein, 1997). Cross-linking of surface immunoglobulin on these cells transiently induces nuclear NF-B and its target gene c-myc. Cell death ensues soon after the drop in c-myc levels. If NF-B is maintained at high levels by cross-linking the cell surface receptor

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CD40, c-myc levels remain high and the cells survive. Ectopic c-myc expression in WEHI-231 cells rescues cells from apoptosis even after NF-B levels fall, suggesting that it is a downstream mediator of antiapoptotic functions. A close parallel to these results was observed in primary CD4>CD8> thymocytes. Freshly isolated CD4>CD8> thymocytes contain nuclear NF-B and c-myc proteins (Wang, W., et al., 1999). The observation that NF-B and c-myc were downregulated prior to glucocorticoid-mediated death suggested a role for these proteins in cell survival in vivo. Accordingly, ectopic expression of transgenic c-myc in these cells partially protected against apoptosis, suggesting that viability of CD4> CD8> thymocytes is maintained by a NF-B/c-myc—dependent pathway (Wang, W., et al., 1999). Taken together, the studies in WEHI-231 and primary thymocytes indicate that a NF-B/c-myc—dependent survival pathway may play an important role in lymphocyte development. NF-B—dependent activation of the inhibitor of the apoptosis (IAP) family of genes was implicated in the survival of primary human endothelial cells (ECs) (Stehlik et al., 1998a, 1998b). These cells are not susceptible to death when activated with TNF; however, expression of a dominant negative form of IB (that inhibits NF-B activation) in these cells sensitizes them to death by blocking expression of three IAP genes. NF-B—dependent activation of IAP genes was also observed in HeLa cells and Jurkat (T-lymphoblastoid) cells (Chu et al., 1997; Wang et al., 1998). It is interesting to note that reexpression of only one of three IAP genes by viral gene transfer is sufficient to rescue I-B transfected ECs from cell death. In addition, it was shown that human IAP-1 and IAP-2 can both induce NF-B, indicating the possibility of a feedback loop where IAPs and NF-B may reinforce each other (Chu et al., 1997; Wang et al., 1998). It is likely that enhanced NF-B activation by IAPs is a consequence of its association with TRAF2, an NF-B inducer downstream of TNF receptors 1 and 2. The broadspectrum antiapoptotic properties of IAPs and their close relationship to NF-B suggest that this pathway may play an important role in tumor formation (LaCasse et al., 1998). The expression of certain Bcl-2—like antiapoptotic proteins is also regulated by NF-B.

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Susceptibility of c-rel\\ B lymphocytes to apoptosis in response to sIg cross-linking was shown to be due to a defect in induction of Bfl-1/A1 gene. Ectopic expression of Bfl-1/A1 in a c-rel\\ B cell line restored cell viability after antiIg treatment, indicating that it is a critical c-Rel regulated antiapoptotic gene. In parallel studies, a subtractive cDNA hybridization approach also identified Bfl-1/A1 as a putative c-Rel target gene in HeLa cells (Zong et al., 1999), and transfection of Bfl-1/A1 protected HeLa cells from TNF-mediated death. Interestingly, in HeLa cells, p65/RelA can induce Bfl-1/A1, whereas in primary B lymphocytes this gene apparently cannot be activated by p65/ RelA, which is present and inducible in c-rel\\ cells. This difference could be because of the transformed phenotype of HeLa cells or tissuespecific gene regulation by c-Rel in primary cells. The involvement of the Rel/Bfl-1 pathway in the survival of other cell types remains to be established. Other genes implicated in mediating NF-B— dependent cell survival are the A20 zinc-finger— containing protein (Jaattela et al., 1996; Krikos et al., 1992), superoxide dismutase (Jones et al., 1997; Xu et al., 1999), and the immediate early gene IEX-IL (Wu et al., 1998). Therefore, NFB regulates a variety of genes that provide antiapoptotic function in different circumstances. It is likely that the relative importance of a particular NF-B—dependent survival pathway will vary between cell types. Indeed, even closely related cell types may utilize very different means of survival. For example, as described above, the NF-B/c-myc pathway was proposed to be important for the survival of CD4>CD8> thymocytes. Neither their developmental precursors, the CD4\CD8\ thymocytes, nor the more mature CD4 or CD8 single positive thymocytes appear to utilize this pathway. PROAPOPTOTIC ACTIVITY OF NF- B There are several examples where NF-B/Rel proteins were shown to promote cell death. For example, glutamate-induced toxicity of neuronal cells (Grilli and Memo, 1999) and the apoptotic death of serum-starved 293 (human embryonic kidney) cells (Grimm et al., 1996) coincide with increased NF-B activity. A recent study in

rodents has strengthened the idea that NF-B can be cytopathic under some circumstances (Schneider et al., 1999). Induction of transient ischemia in mice led to enhanced NF-B activation as measured by DNA-binding assays, or by activation of a transgenic B-dependent reporter gene. Use of p50\\ mice in this model resulted in reduced ischemic damage, suggesting that NF-B was required for cell death. These observations substantiate several earlier studies where NF-B activation was correlated with stimuli that induced neuronal cell death (Carter et al., 1996; Clemens et al., 1997, 1998; Hunot et al., 1997; Kaltschmidt et al., 1997). The mechanism by which NF-B induces cell death in these studies is not known. It was recently shown that both the Fas (receptor) and the Fas ligand (FasL) gene promoters are regulated by NF-B (Chan et al., 1999; Kasibhatla et al., 1999; Matsui et al., 1998). Because Fas/FasL interaction is a major mediator of cell death in lymphocytes, these observations provide a mechanism by which NF-B may induce cell death in susceptible cells. The Fas receptor is expressed at low levels on both B and T lymphocytes. Activation via CD40 on the surface of B cells induces Fas expression, presumably via NF-B. Fas-expressing cells are sensitive to FasL-dependent killing, and in this situation induced NF-B can be construed to have a proapoptotic effect. However, CD40 activation also protects WEHI-231 cells from autonomous cell death induced by anti-Ig cross-linking. Because CD40 activation induces NF-B, and NF-B is sufficient to rescue WEHI-231 cells from anti-Ig—induced death, these observations suggest that CD40induced NF-B plays an antiapoptotic role in these cells. Therefore, CD40-induced NF-B can be proapoptotic or antiapoptotic depending on the nature of the signals that the cells receive. Similarly, in T cells, NF-B activation is required to induce FasL expression, which is essential for the classical form of activationinduced cell death. In this context, the NF-B signal is proapoptotic rather than antiapoptotic. In the same cells, NF-B serves an antiapoptotic function to protect against TNF-induced death. Thus, the role of NF-B in cell death is context dependent. It will be determined by the nature of the NF-B—inducing stimulus — that is, is the activation transient or persistent, what

CONCLUSION

Rel family members are induced, and what other effects does the stimulus have on the cell. NF- B AND CANCER Given the relationship of Rel proteins to the cell cycle and the observation that v-rel is an acute transforming oncogene, it is not surprising that these proteins have been implicated in human cancers (Rayet and Gelinas, 1999). A recurrent theme has emerged, which is that nuclear NFB levels are often increased in tumors of both hematopoietic and nonhematopoietic origin. For example, elevated NF-B levels were reported in lymphoid tumors such as T-cell leukemias (Mori et al., 1999), multiple myelomas (Feinman et al., 1999), and Hodgkin’s lymphoma (Bargou et al., 1996). The mechanism of NF-B induction is understood best in the cases of cancer-inducing viruses such as type I human T-cell leukemia virus (HTLV-I) and EpsteinBarr virus (EBV), where viral genes that activate NF-B have been identified. The HTLV-I tax gene product is an intracellular protein that directly activates IB-kinases (IKKs) (Chu et al., 1998; Geleziunas et al., 1998; Li et al., 1999), which in turn leads to the phosphorylation and degradation of IB and the release of DNAbinding NF-B. EBV encodes a transmembrane protein, LMP-1, which induces constitutive NF-B activation that is critical for immortalization of human B lymphocytes (Farrell, 1998; Sylla et al., 1998). These examples demonstrate that the NF-B activation pathway can be usurped in different ways to deregulate NF-B— dependent gene expression. Deregulation of the Rel network may also occur by chromosomal translocations (Migliazza et al., 1994; Neri et al., 1991). (t14; 19) translocations, which involve the IB-like bcl-3 gene, have been observed in a small proportion of chronic (B) lymphocytic leukemias (Michaux et al., 1997; McKeithan et al., 1997: Ohno et al., 1993). These translocations result in higher levels of bcl-3 expression, and it is believed that other mutations in the protein are not required for its role in oncogenesis. Interestingly, transgenic overexpression of bcl-3 in murine thymocytes leads to increased p50 homodimer expression, but no other obvious consequences (Caamano et al., 1996; Caamano et al., 1998). In

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contrast, transgenic bcl-3 expression in B lymphocytes leads to the accumulation of B cells in the lymph nodes and spleen, though lymphoid tumors were not detected (Ong et al., 1998). These studies demonstrate that bcl-3 overexpression is likely to be one step in a multistep process of leukemia induction, and that its effects are cell-type specific. Constitutive NF-B expression may not only be important in the induction of tumors, but may impact cancer therapy. NF-B—expressing tumor cell lines were shown to be resistant to apoptosis-inducing therapeutic regimens such as radiation and chemotherapy (Beg and Baltimore, 1996; Paillard, 1999; Van Antwerp et al., 1996; Wang et al., 1996). Reduction of NF-B activity by expression of a nondegradable IB sensitized such tumor cells to undergo apoptosis and consequent tumor regression (Duffey et al., 1999; Wang, C. Y. et al., 1999).

CONCLUSION In summary, Rel proteins protect from cell death, induce cell death, and regulate cell proliferation depending on the cellular conditions. This is very likely due to the large variety of genes whose expression is regulated by Rel proteins. Protection from cell death is mediated by targets such as c-Myc, IAPs, and bcl-2—like proteins; induction of cell death results from expression of genes such as Fas and FasL; cell cycle regulation is the result of NF-B—dependent modulation of G1 progression. The observations that provide the connection of NF-B to the G1 phase of the cell cycle are as follows: (1) The G1-specific cyclin D1 gene is an NF-B target; (2) overexpression of RelA or c-Rel arrests cell cycle progression at the G1 stage; (3) the G1-specific cell cycle inhibitors, p21 and p27, enhance NF-B—dependent transcription; (4) c-Rel—deficient B lymphocytes are blocked at the G1 stage when activated by mitogenic signals. These apparently contradictory observations suggest that NF-B blocks or facilitates progression at different stages of the G1 phase. A model illustrating the role of NF-B in the G1 phase of the cell cycle is outlined below. We suggest that NF-B activity may be transiently induced during G1 progression (Fig. 30.2). This induction may be in response to

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Figure 30.2. A model linking cell cycle and apoptosis regulation by NF-B. The crux of this model is that NF-B activity is transiently induced during G1 progression as indicated by small and large circles labeled NF-B. Note that the term NF-B is used to indicate that different Rel family members could be involved depending on the type of cell or the inducing stimulus. The increase in NF-B may be induced by signals from the cell surface and is required for G1 progression by activating genes such as cyclin D1. It is also likely that the induced NF-B activates antiapoptotic genes, such as c-myc, IAP, and bcl-2 family members, which are required for successful G1/S transition. As G1 progresses, a decrease in NF-B is invoked to account for the observation that sustained p65 or c-Rel, expression leads to G1 arrest; thus, reduced NF-B appears to be necessary for G1/S transition. This sequence of events can be modulated at several steps. For example, p16/INK4 molecules, induced by growthinhibitory molecules such as TGF, may suppress NF-B activity in early G1. Conversely, p21 and p27 Cdk inhibitors induced in response to stress (jagged arrow) enhance NF-B activity and prevent downregulation of NF-B, which we propose to be necessary for G1/S transition. Therefore, the cell cycle is blocked in late G1. The bold arrow, marked apoptosis, indicates that many forms of apoptosis, including FasL-dependent death of activated T cells, occurs from a point in late G1. NF-B—induced antiapoptotic genes may also be required for successful negotiation of this checkpoint.

growth stimulatory signals at the cell surface, such as those initiated by serum or by the antigen receptors on lymphocytes. The role of NF-B may be to induce G1 progression genes such as cyclin D1, to activate antiapoptotic genes to enable the cell to transit through the G1/S boundary, and to induce growth-inducing genes such as c-myc. We envisage that the induction must be transient to account for the observation that ectopic expression of RelA (or c-Rel) leads to G1 arrest in two cell lines, which suggests that sustained NF-B

activation is detrimental to progression out of G1. NF-B activity during G1 can be decreased by the action of p16/INK4 protein. Alternatively, NF-B may be activated by p21/p27 in response to cellular stress via a pathway that involves p53. The combined action of the Cdk inhibitors plus sustained NF-B activity will halt cells in G1, as well as (presumably) induce survival genes. The cell cycle resumes when p21/p27 levels, and consequently NF-B activity, are reduced below a threshold.

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PART VI

SUMMARY


CHAPTER 31


TRANSCRIPTION FACTORS IMPLICATED IN HEMATOPOIESIS: IN VIVO STUDIES YULIA KALUZHNY AND KATYA RAVID Department of Biochemistry, Boston University School of Medicine, Boston, MA

The following chapters include a summary of in-vivo studies focusing on the role of transcription factors in hematopoiesis and in mouse development (chapter 31) as well as a compilation of chromosomal rearrangements in hematological malignancies which affect transcriptional regulators (chapter 32). The first table includes a synopsis of all the transcription factors surveyed in this paper, their protein family, consensus binding sites and expression pattern. It also

summarizes studies, described up to now, involving the use of knock out mice to explore the role of these transcription factors in normal and malignant development of different blood cell lineages. The data were collected from the literature and from chapters in this book (we apologize for potentially missing some details or data that have appeared after the publication of this book).


573

574

Zinc finger

Zinc finger

Zinc finger

bZIP

GATA-2

GATA-3

p45 NF-E2

Protein Family

GATA-1

Transcription Factor

Expression persists in Meg and mast cells but is shut off in erythroid maturation; HSC, immature multipotential progenitors, endothelial cells, undifferentiated ES cells. Widespread during embryogenesis; high level in developing kidney and nervous system; restricted to thymocytes, T lymphocytes (Th2, CD4 cells) and nervous system in adults.

Hematopoietic specific; multipotential hematopoietic progenitors, erythroid and mast cells, Meg.

(A/T)GATA(A/G)

(T/C)GCTGA(G/C)TCA (T/C)

AGATAG

Multipotential progenitors, erythroid, megakaryocytic, mast cell lineages, eosinophils; testis.

Expression Pattern

(A/T)GATA(A/G)

Consensus-binding Site

Homozygous mutant embryos die by day 12 p.c. and display massive internal bleeding, marked growth retardation, severe deformities of the brain and spinal cord, and gross aberrations in fetal liver hematopoiesis. Required for development of the T-cell lineage in chimeras generated with null ES cells. Mild blood cell abnormalities; complete absence of circulating platelets, mice die shortly after birth from extensive hemorrhage.

No mature RBC; megakaryocytes are abnormally abundant in chimeric fetal livers; no interference with hematopoiesis of other lineages and tissues. Marked thrombocytopenia in megakaryocyte-specific gene KO. Mice die at E10—11 with anemia due to lack of effective primitive erythropoiesis; all hematopoietic lineages are profoundly affected.

Comments on Knockout Phenotype in Mice

Andrews et al., 1993a; Ney et al., 1990; Shivdasani and Orkin, 1995; Shivdasani et al., 1995b

George et al., 1994; Ho et al., 1991; Pandolfi et al., 1995; Ting et al., 1996; Zhang, D. H. et al., 1997b

Dorfman et al., 1992; Tsai et al., 1994

Orkin, 1990; Pevny et al., 1991; Shivdasani et al., 1997; Tsai et al., 1989; Yamamoto et al., 1990

References

575

bZIP

bHLH

Kru¨ppel-like

Kru¨ppel-like

CREB/ATF

Runt related

Small-Maf proteins (p18) MafK, MafF, MafG

Tal-1/SCL

EKLF

PLZF

CREB

AML1 (Runx1)

TG(T/C)GGT

TGACGTCA

GT(A/C)(A/C)AGT

CCACACCCT

CAGTTG

T-MARE: TGCTGACTCAGCA; C-MARE: TGCTGACGTCAGCA

Myeloid cells, T-cells, hematopoietic progenitors; hemogenic endothelial cells.

Early hematopoietic progenitors, limb buds, segments of the CNS. Ubiquitous.

Early hematopoietic progenitors, erythroid precursors, Meg, mast cells; presumptive hemangioblast; endothelial cells; brain. Restricted to normal sites of hematopoiesis; mouse bone marrow, spleen; within erythroid and mast cell lines.

Widely expressed with varying levels and distinct spatial and temporal patterns; MafG and MafK display complementary expression patterns in early embryos.

Homozygous null mice die of anemia around day E14.5 during fetal liver erythropoiesis and show the molecular and hematological features of -globin deficiency. Homozygous null mice have aberrant limb development and T-cell lymphopenia CREB null mice are smaller than their littermates and die immediately after birth from respiratory distress; impaired fetal T-cell development. Block in development of all definitive hematopoietic lineages; death by E12.5 accompanied by perivascular edema and extensive hemorrhages in the CNS.

MafK null mice are viable, fertile, and normal; MafG null mice exhibit impaired thrombopoiesis with late differentiation block in megakaryocyte development; in addition, mice display behavioral abnormalities. Mice die in utero as a result of the absence of blood cell formation; essential for angiogenic remodelling.

Bae et al., 1993; Miyoshi et al., 1991; North et al., 1999; Okuda et al., 1996; Wang et al., 1996a

Avantaggiato et al., 1995; Hawe et al., 1996; Reid et al., 1995 Rudolph et al., 1998; Zauli et al., 1998

Begley et al., 1989; Mouthon et al., 1993; Porcher et al., 1996; Shivdasani et al., 1995a; Visvader et al., 1998 Miller and Bieker, 1993; Nuez et al., 1995; Perkins et al., 1995

Andrews et al., 1993b; Fujiwara et al., 1993; Kotkow and Orkin, 1996; Shavit et al., 1998

576 T lymphocytes.

HMG box

TCF-1

(A/T)(A/T)CAAAG

Novel

EBF

B lymphocytes, lymph node, spleen, adipocytes, olfactory neurons.

Endothelial cells, T cells, Meg.

(G/C)(A/C)GGAAGT

Ets

Ets-1

ATTCCCNNGGGAAT

Hematopoietic progenitors; monocytes/macrophages, B cells, granulocytes, low levels in erythroid cells.

GAGGAA

Ets

Ubiquitously expressed.

Expression Pattern

PU.1


Non-DNA-binding subunit of CBF

Protein Family

CBF

Transcription Factor Same spectrum of abnormalities as CBF2-deficient mice: embryos show normal morphogenesis and yolk sac—derived erythropoiesis, but lack fetal liver hematopoiesis and die around E12.5. KO{1: Mice die in utero at day E16; absence of monocytes, granulocytes, T and B cells; KO{2: Animals could be viable at birth; no monocytes and mature B cells; can produce B-cell progenitors. Abnormal pool of resting lymphocytes; T- and B-cell proliferation defect. Lack differentiated B cells with rearranged immunoglobulin DJH segments, contain progenitor B cells. Nonlymphoid tissues expressing EBF are apparently normal. Defects in thymocytes differentiation and expansion; adult KO mice are immunocompetent, contain competent T cells that may have arisen from the early wave of development.


Oosterwegel et al., 1991; Schilham et al., 1998

Hagman et al., 1993; Lin and Grosschedl, 1995; Travis et al., 1993

Barton et al., 1998; Watson et al., 1985

Klemsz et al., 1990; Scott et al., 1994

Ogawa et al., 1993; Sacchi et al., 1996; Wang et al., 1996b; Wang et al., 1993b

References

577

Sox

bZIP

bZIP

bZIP

Sox-4

C/EBP

C/EBP (NF-IL6)

C/EBP

ATTGCGCAAT

ATTGCGCAAT

ATTGCGCAAT

AACAAAG

Granulocytes and lymphoid cells

Liver, adipose tissue; myeloid lineages.

Hepatic and adipose tissue; specifically expressed in proliferating myelomonocytic cell lines and not in erythroid, B- or T-cell lines.

T and B lymphocytes; murine thymus.

Null embryos succumb to circulatory failure at day E14 due to impaired development of the endocardial ridges. Lethally irradiated mice reconstituted with SOX-4\\ fetal liver cells have a specific block in pro-B-cell development. Mutant mice die within the first few hours after birth of impaired glucose metabolism. Null mice display selective block in differentiation of neutrophils: mature neutrophils and eosinophils are absent in the blood or fetal liver of mutant animals, but all other lineages are quantitatively unaffected. Mutant mice develop marked splenomegaly, peripheral lymphadenopathy, and enhanced hemopoiesis; humoral, innate, and cellular immunity are profoundly distorted; defective activation of splenic macrophages in relation to bacterial killing and tumor cytotoxicity. Essential for terminal differentiation and functional maturation of committed granulocyte progenitor cells: opportunistic infections and tissue destruction lead to death by 3—5 months of age. Antonson et al., 1996; Lekstrom-Himes and Xanthopoulos, 1999; Yamanaka et al., 1997

Cao et al., 1991; Screpanti et al., 1995; Tanaka et al., 1995

Landschultz et al., 1988; Wang et al., 1995; Zhang, X. E. et al., 1997a

Schiham et al., 1996; van de Wetering et al., 1993

578

Homeobox

Homeobox

Zinc finger

Paired domain

HoxA10

Ikaros

BSAP (Pax-5)

Protein Family

HoxA9


Early hematopoietic progenitors; lymphoid lineages.

Developing central nervous system, fetal liver, bone marrow, spleen lymph node, testis; restricted to the B-lymphoid lineage within the hematopoietic system; pro-B, pre-B, and mature B cells.

(G/A)G(C/A)AT(G/C) A(A/T)GCG(T/G) (G/A)(A/C)(C/A)

Highest expression in CD34>-positive bone marrow cells; myeloid cells.

CD34> bone marrow cells; developing lymphocytes.

Expression Pattern

GGGAAT

NTGATNNAT

NTGATNNAT

Consensus-binding Site Homozygous null mice have significantly smaller spleens and thymuses; display reduction in peripheral leukocyte and granulocyte count; significantly reduced number of committed myeloid, erythroid, and B-cell progenitors. Knockout mice are viable but have defects in both male and female fertility — males have a severe defect in spermatogenesis and increasing sterility with age; females are sterile because of death of embryos between E2.5 and E3.5. Mice homozygous for a germ-line mutation in the DNA-binding domain lack T and B lymphocytes, NK cells, and their earliest defined progenitors. Mice with a mutation in a single copy of the gene develop a lethal lymphoproliferative disease. Homozygous mutant mice have growth retardation and most died within 3 weeks; about 5% of mutants survive to adulthood, are fertile, but have a complete block at the pro-B cell stage of B-cell differentiation as well as an altered pattern of the posterior midbrain.


Adams et al., 1992; Czerny et al., 1993; Nutt et al., 1997; Urbanek et al., 1994

Georgopoulos et al., 1992, 1994; Wang et al., 1996a; Winandy et al., 1995

Lowney et al., 1991; Satokata et al., 1995

Lawrence et al., 1997; Sauvageau et al., 1994

References

579

Nondefinable TTCC(G:C)GGAA

TTCC(GC)GGAA

TTCC(AT)GGAA

STAT2 STAT3

STAT4

STAT5A

GAS-binding sites:

TTCC(GC)GGAA

Src homology

STAT1

STAT

Ubiquitous with different level of expression in various tissues. Deficient mice display no gross developmental defects, but revealed a complete lack of responsiveness to either IFN or INF; highly susceptible to infection by microbial pathogens and viruses. Early embryonic lethal Mutant embryos die before gastrulation: show a rapid degeneration between E6.5 and E7.5. The mice are viable and fertile, with no detectable defects in hematopoiesis; however, all IL-12 functions tested are disrupted, including the induction of IFN-, mitogenesis, enhancement of natural killer cytolytic function and Th1 differentiation; mutant mice demonstrate a tendency toward the increased development of Th2 cells. Mutant mice develop normally; however, mammary lobuloalveolar outgrowth during pregnancy is severely reduced, and females fail to lactate because of a failure of terminal differentiation. Liu et al., 1997

Kaplan et al., 1996; Thierfelder et al., 1996

Darnell, 1997 Takeda et al., 1997

Durbin et al., 1996; Meraz et al., 1996

Review in Darnell, 1997; Ihle, 1996

580

E2A

bHLH

E box: CANNTG

TTCC(AT,N)GGAA

STAT6


TTCC(AT)GGAA

Protein Family

STAT5B


Ubiquitous, level of expression varies considerably.

Expression Pattern Mutant male mice grow slowly and have serum levels of liver-produced proteins that are characteristic of female mice: do not maintain sexual dimorphism of body growth rates and liver gene expression; mice display characteristics of Laron-type dwarfism, a disease associated with a defective GH receptor: dwarfism, elevated plasma GH, low plasma IGF, development of obesity. IL-4 mediated biological responses are mainly abolished, including expression of CD23 and MHC class II in resting B cells; B- and T-cell proliferative responses; production of Th2 cytokines from T cells, as well as IgE and IgG1 responses after nematode infection. Homozygous mutant mice develop to full term without apparent abnormalities, but then display a high rate of postnatal death; the surviving mice show retarded postnatal growth and fail to generate mature B cells due to early block in differentiation; have T-cell abnormality and develop lymphomas.


Bain et al., 1994, 1997; Murre et al., 1989a, 1989b; Zhuang et al., 1994

Takeda et al., 1996

Udy et al., 1997

References

581

POZ/Zinc finger

POU homeobox

bHLH-ZIP

myb

NF-B/Rel

BCL6

Oct-2

c-Myc

c-Myb

p50 NFB

B site: GGGRNNYYCC

(T/C)AAC(G/T)C

CACGTG

Upregulated in various proliferating cells, including those of hematopoietic origin; downregulated in differentiating myeloid cell lines. Progenitor cells of erythroid, myeloid, and lymphoid lineages; neural tissue of embryonic, fetal, and adult brain; ES cells; human malignancies of neuroectodermal and hematopoietic origin. Ubiquitously.

(T/A)NCTTTCNAGG Ubiquitously detected in (A/G)AT or human tissues; upregulated TTTNNNGNNATNCTTT in KC and sperm at their terminal stage of differentiation; high level in germinal centers, in B cells and CD34> T cells, but not in mature B cells. Octamer: B-lymphocytes. ATGCAAAT Mice deficient in BCL-6 display normal B-cell, T-cell and lymphoid-organ development but have a selective defect in T-cell—dependent antibody responses; abnormal Th2 response. Mutant mice develop normally but die within hours of birth from undetermined reasons; mutants contain normal numbers of B-cell precursors but are partially deficient in IgM> B cells with defect in their capacity to secrete immunoglobulin upon mitogenic stimulation in vitro. c-Myc protein is necessary for embryonic survival beyond 10.5 days of gestation. Pathologic abnormalities include heart, pericardium, and neural tube development. Mutant mice die by day 15 of gestation from severe anemia; adult-type erythropoiesis is greatly diminished; granulocytic and monocytic lineages are similarly affected; Meg are spared. Mice show no developmental abnormalities, but exhibit multifocal defects in immune responses involving B lymphocytes and nonspecific responses to infection. Ghosh et al., 1990; Sha et al., 1995

Biedenkapp et al., 1988; Boyle et al., 1986; Gewitz and Calabretta, 1988; Mucenski et al., 1991; Slamon et al., 1986

Amin et al., 1993; Davis et al., 1993; Dorn et al., 1994; Thompson et al., 1996

Corcoran et al., 1993; Kemler and Schaffner, 1990

Baron et al., 1995; Dent et al., 1997; Fukuda et al., 1997; Kawamata et al., 1994; Ye et al., 1997

582

NF-B/Rel

Nuclear hormone receptors

RelB

RAR*

RAR

RAR

RAR

NF-B/Rel

Protein Family

p65/ReIA


Wide variety of tissues.

RNA transcripts are very low or undetectable in early and late embryos; the level rapidly increases after birth; in the adult mouse, mainly restricted to thymus, spleen, lymph node, and intestine.

B site: GGGRNNYYCC

RARE: AGGTCAN AGGTCA \

B cells, T lymphomas.

Expression Pattern

B site: GGGRNNYYCC


Mutant mice display decreased viability, growth deficiency, male sterility (degeneration of seminiferous epithelium) and congenital malformations including webbed digits, homeotic transformations. Mice homozygous for the mutation are viable and fertile with no externally apparent abnormalities. Mutant mice display decreased viability, growth deficiency, male sterility (squamous metaplasia of seminal vesicle

Disruption of the relA locus leads to embryonic lethality at 15—16 days of gestation, concomitant with a massive degeneration of the liver by programmed cell death or necrosis. Mutant animals have multifocal, mixed inflammatory cell infiltration in several organs; myeloid hyperplasia, splenomegaly due to extramedullary hematopoiesis; a reduced population of antigen-presenting dendritic cells in the thymus; an impaired cellular immunity.


Lohnes et al., 1993

Luo et al., 1995

Lohnes et al., 1994; Lufkin et al., 1993

Reviewed in Kastner et al., 1995

Burkly et al., 1995; Carrasco et al., 1993; Weih et al., 1995

Beg et al., 1995; Nolan et al., 1991; Weih et al., 1994

References

583

RXR

RXR

RXR

RXR?

Nuclear hormone receptors

RARE: AGGTCAN AGGTCA \

Wide variety of tissues.

Reviewed in Kastner et al., 1995 Null mutants die between Dyson et al., 1995; E10.5 and E17.5 from Kastner et al., 1994; cardiac failure: the major Sucov et al., 1994 defect is grossly thinned ventricular wall with concurrent defects in ventricular septation; also display ocular malformations. Approximately 50% of the Kastner et al., 1996 homozygous mutants die before or at birth, but those that survived appear normal except that the males were sterile, owing to oligoastheno-teratozoospermia; failure of spermatid release occurred within the germinal epithelium and preceded by the progressive accumulation of lipids within the mutant Sertoli cells. Homozygous mutant mice Krezel et al., 1996 develop normally and are indistinguishable from their RXR>\ or wild-type littermates with respect to growth, fertility, viability, and apparent behavior in the animal facility.

and prostate gland epithelia); congenital malformations include webbed digits, homeotic transformations, and malformations of cervical vertebrate, fusion of trachea rings, agenesis of Harderian glands.

584

LIM domain

Zinc finger

HAT

LMO2

FOG@

CBP@

Cofactor, DNA binding not shown

Cofactor, DNA binding not shown

Does not bind DNA directly

VDRE: AGGTCA(N )AGGTCA


Ubiquitous.

Multipotential progenitors, high level in lineages of primitive and definitive erythropoiesis, fetal liver; CNS (hippocampus), thymus, limb buds (embryo). Coexpressed with GATA-1 during embryonic development and in erythroid and megakaryocytic cells.

Skin, testis, pancreas, colon, muscle, breast, prostate, thymus, and bone marrow.

Expression Pattern

Boehm et al., 1991; Warren et al., 1994; Yamada et al., 1998, 2000

Review in Freedman and Luisi, 1993; Yoshizawa et al., 1997

References

Mice die during midembryonic Tsang et al., 1997, 1998 development with severe anemia; FOG null erythroid cells display marked, but partial, arrest in development at the proerythroblast stage; loss of FOG leads to specific ablation of the megakaryocytic lineage. Embryos die between E9.5 and Blobel et al., 1998; E10.5; exhibit a defect in Kwok et al., 1994; neural tube closure, primitive Oike et al., 1999 hematopoiesis, and vasculoangiogenesis.

No defects in development and growth are observed before weaning in null mutant mice; in postweaning stage mutants failed to thrive, display alopecia, hypocalcemia, infertility with uterine hypoplasia, and severely impaired bone formation; most of the mutants died within first 15 weeks after birth. Embryonic lethality around E10.5; failure of yolk sac erythropoiesis. Essential for definitive hematopoiesis and embryonic angiogenesis.


?Ligand-dependent transcription factors. @Cofactor. bHLH, basic helix-loop-helix; bZIP, basic leucine zipper; HAT, histone acetyl transferase; RBC, red blood cells; WBC, white blood cells, Meg, megakaryocytes; ES cells, embryonic stem cells; CNS, central nervous system; PBP, platelet basic protein; TCR, T-cell receptor; MARE, Maf recognition elements; CBP, CREB-binding protein; NK, natural killer cells; Th2, T-helper 2 cells; KC, keratinocytes; GH, growth hormone; IGF, insulin-like growth factor; INF, interferon; HMG box, high-mobility-group box; Th1, T helper 1 cells; HSC, hematopoietic stem cells.

Nuclear hormone receptor

Protein Family

VDR?


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CHAPTER 32


CHROMOSOMAL TRANSLOCATIONS ASSOCIATED WITH DISRUPTION OF TRANSCRIPTIONAL REGULATORS IN LEUKEMIA AND LYMPHOMA JONATHAN D. LICHT Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, NY

The table in chapter 32 was derived by abstracting the chapters of this book as well as internet resources listed at the end of the table. It is safe to say that the fusion genes that most frequently occur in myeloid and lymphoid malignancy have been cloned and the functions of the wildtype and fusion genes have been or are on their way to being well characterized. The most common translocations may account for over 40% of AML and ALL (Look, 1997). Rarer translocations reported in a handful of patients might still highlight important genes for normal blood development and cell growth control. It is becoming increasingly apparent that some transcription factors may be deleted or silenced in hematological malignancy, consistent with roles as tumor suppressors. These genes may also be affected so as to generate dominant negative or gain of function proteins that may interfere with the normal transcription factor function and

lead to a block of differentiation or apoptosis and enhancement of cell growth. Hence, it is possible that genes rarely affected by chromosomal translocation may still be important in the pathogenesis of leukemia and lymphoma. While the tabulation below is not complete as it focuses only on transcriptional regulators and their partners, the reader may still appreciate the breadth of genes affected. In addition, upon examination of the table it becomes apparent that several genes such as those of the CBF/ RUNX complex and the retinoic acid receptor are repeatedly affected. It is hoped that this table might focus some of its readers on new avenues of investigation and discovery. Look, A.T. (1997) Oncogenic transcription factors in the human acute leukemias. Science. 278, 1059—1064.


593

594

T-ALL

AML

Pre-B ALL

AML

MDS/AML

B-NHL

ALL/AML

APL

AML

AML

t(1;11)(q23;p15)

t(1;19)(q23;p13.3)

t(2;11)(q31;p15)

inv(3)(q21;26) t(3;3)(q21;q26) ins(3;3)(q26;q21q26) t(3;7)(q27;q22) t(3;21)(q26;q22)

t(3;14)(q27;q32) t(2;3)(p12;q27) t(3;22)(q27;q11) t(3;13)(q27;q14) t(3;4)(q27;p13) and others

t(4;11)(q21;q23)

t(5;17)

t(6;9)(p23;q34)

t(6;11)(q21;q23)

Disease

t(1;14)(p32;q11) t(1;14)(p34;q11) t(1;3)(p34;p21) t(1;5)(p32;q31) t(1;7)(p32;q35)

Translocation

AF6q21 OMIM 602681

DEK OMIM 125264

NPM OMIM 164040

AF4 OMIM 159557

BCL6 OMIM 109565

EVI1 OMIM 165215

HOXD13

PBX1 OMIM176310

PMX1

SCL/TAL1 OMIM 187040

Transcription Factor (Online Mendelian Inheritance in Man Reference Number)

Forkhead

Novel DNA-binding domain

Cofactor?

Transcription factor?

Zinc finger

Zinc finger

Homeodomain

POU-homeodomain

Homeodomain

Helix-loop-helix

Type of Factor

6q21

6023

5q35

4q21

3q27

3q26

2q31-q32

1q23

Iq23

1p32

Chromosomal Location

MLL fusion

DEK-CAN (OMIM114350) fusion

NPM-RAR NPM-MLF1 (OMIM 601402)

AF4-MLL fusion

Fusion to -light-chain promoter activates expression Point mutation in BCL6 promoter upregulates expression

TEL-EVI1 fusion AML1-EVI1 fusion

Inversion leads to deregulated expression of EVI1

Nup98-HOXD13 fusion

E2A-PBX fusion protein

Nup98-PMX1 fusion

Activation of expression of TAL1 by rearrangement with TCR promoter 5 deletion of TAL1 puts SCL under control of the SIL (OMIM 181590) promoter

Fusions Produced

595

T-ALL

APL

t(11;14)(p15;q11)

t(11;17)(q13;q21)

NuMA OMIM 164009

LMO1 OMIM 186921

LMO2 OMIM 180385

Nuclear matrix

LIM domain

LIM domain

Zinc finger and leucine zipper

11q13

11p15

11p13

10p12

NuMA-RAR fusion

Activation by rearrangement with TCR

Activation by rearrangement with TCR

MLL fusion

Activation by rearrangement with TCR or

T-ALL

Fusion with MLL

t(11;14)(p13;q11) t(7;11)(q35;p13)

AF10 OMIM 602409

10q24

10q24

10p12

AF9-MLL fusion

AML

Homeodomain

Rel domain

Zinc-finger and homeodomain

9p22

E-mu enhancer of the IgH activates expression of pax 5

t(10;11)(p12;q23)

Hox 11 OMIM 186770

NFB2 OMIN 164012

AF10 OMIM 602409

Coactivator? SWI/SNF interaction

9p13

Ig promoter/enhancer activates expression of c-myc

T-ALL

AML

t(10;11)(p12;q23)

AF9 OMIM 159558

Paired box

8q24.12-q24.13

MOZ-TIF2 fusion

t(10;14)(q24;q11) t(7;10)(q35;q24)

AML

t(9;11)(p21;q23)

Pax5 OMIM 167414

Helix-loop-helix

8

NUP98-HoxA9 fusion

IgA constant-region promoter activates expression

Small lymphocytic lymphoma Large cell lymphoma

t(9;14)(pl3;q32)

c-myc OMIM 190080

Coactivator

7p15-p14.2

Expression of dominant negative forms of the protein is found in MLL fusion—associated leukemia

B cell low grade NHL

NHL-Burkitt’s

t(8;14)(q24;q32) t(2;8)(p12;q24) t(8;22)(q24;q11)

TIF2 OMIM 601993

Homeodomain

7p13-p11.1

t(10;14)(q24;q32)

MLL

inv(8)(p11q13)

Hox A9 OMIM 142956

Zinc finger

AF10 fusion with clathrin assembly protein (CALM) OMIM 603025

AML M2 or M4

t(7;11)(p15;p15)

Ikaros OMIM 603023

t(10;11)(p13;q14)

ALL

Various

596

APL

AML ALL MLL

MDS/AML

CMML

ALL CML/ALL AML/ALL ALL MDS/AML

Stem cell leukemia

APL

AML

MDS/AML

AML

APL

Multiple

t(3;12)(q26;p13)

t(5;12)(q33;p13)

t(6;12)(q23;p13) t(9;12)(p24;p13) t(9;12)(q34;p13) t(12;21)(p13;q22) t(12;22)(p13;q11)

t(8;13)(p11;q11-12)

t(15;17)(q22;q21)

inv(16)(p13;q22)

t(11;16)(q23;p13)

t(8;16)(p11;p13)

t(15;17)(q22;q21)

Disease

t(11;17)(q23;q21)

Translocation

RARA OMIM 180240

CBP OMIM 600140 MOZ OMIM 601408

CBF OMIM 121360

PML OMIM 102578

ZNF198 OMIM 602221

Tel OMIM 600618

MLL OMIM 159555

PLZF OMIM 176797

Transcription Factor (Online Mendelian Inheritance in Man Reference Number)

Nuclear receptor

Coactivator histone acetylase Coactivator histone acetylase

Non-DNA-binding partner of AML

RING finger cofactor?

Zinc finger

ETS domain

Probable cofactor

Zinc finger

Type of Factor

17q21

16p13.3

16q22

15q21

13q11-q12

12p13

11q23

11q23

Chromosomal Location

N-RAR fusion

MOZ-CBP fusion

MLL-CBP fusion

Fusion with MYH11 (OMIM 160745) smooth muscle myosin heavy chain

PML-RAR fusion

FGFR1 (OMIM 136350)-ZNF198 fusion protein

Tel-PDGFR fusion (OMIM 173410) Tel-STL fusion (OMIM 602532) Tel-JAK2 fusion (OMIM 147796) Tel-Abl fusion (OMIM 189980) Tel-AML1 fusion Tel MN1 fusion (OMIM 156100)

Tel-Evi1 fusion

Fusion with multiple partners

PLZF-RAR fusion RAR-PLZF fusion

Fusions Produced

597

ALL

AML

AMI

ALL

B-CLL

ALL

AML

CML-BC ALL

AML

AML

t(17;19)(q21;p13) t(1;19)(q23;p13)

t(11;19)(q23;p13)

t(11;19)(q23;p13.1)

t(7;19)

t(14;19)(q32;13.1)

t(7;9)(q34;q32)

t(8;21)(q22;q22)

t(3;21)(q26;q22) t(12;21)(p12;q22)

t(11;22)(q23;q13)

t(X;11)(q13;q23)

AFX OMM 300033

p300 OMIM 602700

AML1 OMIM 151385 ETO OMIM 133435

TAL2

BCL3 OMIM 109560

LYL1 OMIM 151440

ELL

ENL OMIM 159556

HLF OMIM 142385 E2A OMIM 147141

AF17 OMIM 600328

Forkhead

Coactivator/HAT

Runt corepressor

b-HLH

Inhibitor of Rel/NFkB

b-HLH

Pole II elongation factor

Coactivator? SWI/SNF interacting?

Basic leucine zipper Helix-loop-helix

Zinc finger and leucine zipper

Xq13.1

22q13

21q22.3 8q22

19q31

19q13

19p13.2-p13.1

19p13.1

19p13.3

17q21 19p13.3

17q21

Information on all of these translocations can be found at the following Web sites: 1. Online Mendelian inheritance in man http://www3.ncbi.nlm.nih.gov/Omim/ 2. Atlas of Genetics and Cytogenetics in Oncology and Haematology http://www.infobiogen.fr/services/chromcancer/index.html 3. Breakpoint Map of Recurrent Chromosome Aberrations http://www.ncbi.nim.nih.gov/CCAP/mitelsum.cgi

AML

t(11;17)(q23;q21)

AFX-MLL fusion

p300-MLL fusion

AML1-EVI1 Tel-AML1

AML1-ETO fusion

TCR activates expression of TAL2

IgA constant-region promoter activates expression of BCL3

TCR activates expression of LYL1

ELL-MLL

ENL-MLL fusion

HLF-E2A fusion E2A-PBX fusion

MLL fusion

Transcription Factors: Normal and Malignant Development of Blood Cells

Transcription Factors: Normal and Malignant Development of Blood Cells

Transcription Factors

Eukaryotic Transcription Factors,

A Handbook of Transcription Factors

Eukaryotic Transcription Factors, Fourth Edition

Adhesive Interactions in Normal and Transformed Cells

Transcription Factors: A Practical Approach, 2nd edition

Transcription Factors: A Practical Approach, 2nd edition

Transcription Factors: Methods and Protocols (Methods in Molecular Biology)

Myb Transcription Factors: Their Role in Growth, Differentiation and Disease

Forkhead Transcription Factors: Vital Elements in Biology and Medicine

Pathology of Malignant Mesothelioma

Transcription Factors in the Nervous System: Development, Brain Function, and Diseases

Apoptosis In Normal Development And Cancer

Blood Cells: A Practical Guide

Textbook of Malignant Hematology

Outsourcing Training and Development: Factors for Success

Neural stem cells: development and transplantation

Malignant Sadness

Neural Development and Stem Cells (Contemporary Neuroscience)

Stem Cells in Development and Disease

Malignant Mesothelioma

Malignant Lymphoma

Risk and Protective Factors in the Development of Psychopathology

TNM Classification of Malignant Tumours

The Biology of Schwann Cells: Development, Differentiation and Immunomodulation

Transcription (Cell and Biomolecular Sciences)

In Vitro Transcription and Translation

Transcription Factors: Normal and Malignant Development of Blood Cells