Chromosome Structural Analysis: A Practical Approach (The Practical Approach Series, 200)

Chromosome Structural Analysis

The Practical Approach Series SERIES EDITOR B. D. HAMES Department of Biochemistry and Molecular Biology University of Leeds, Leeds LS2 9JT, UK

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Chromosome Structural Analysis A Practical Approach Edited by

WENDY A. BICKMORE Cell Genetics Section, MRC Human Genetics Unit, Western General Hospital, Edinburgh

OXFORD UNIVERSITY PRESS

1999

OXFORD UNIVERSITY PRESS

Great Clarendon Street, Oxford OX2 6DP Oxford University Press is a department of the University of Oxford and furthers the University's aim of excellence in research, scholarship, and education by publishing worldwide in Oxford New York Athens Auckland Bangkok Bogota Buenos Aires Calcutta Cape Town Chennai Dar es Salaam Delhi Florence Hong Kong Istanbul Karachi Kuala Lumpur Madrid Melbourne Mexico City Mumbai Nairobi Paris Sao Paulo Singapore Taipei Tokyo Toronto Warsaw and associated companies in Berlin Ibadan Oxford is a registered trade mark of Oxford University Press Published in the United States by Oxford University Press Inc., New York © Oxford University Press 1999 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, without the prior permission in writing of Oxford University Press. Within the UK, exceptions are allowed in respect of any fair dealing for the purpose of research or private study, or criticism or review, as permitted under the Copyright, Designs and Patents Act, 1988, or in the case of reprographic reproduction in accordance with the terms of licenses issued by the Copyright Licensing Agency. Enquiries concerning reproduction outside those terms and in other countries should be sent to the Rights Department, Oxford University Press, at the address above. This book is sold subject to the condition that it shall not, by way of trade or otherwise, be lent, re-sold, hired out, or otherwise circulated without the publisher's prior consent in any form of binding or cover other than that in which it is published and without a similar condition including this condition being imposed on the subsequent purchaser Users of books in the Practical Approach Series are advised that prudent laboratory safety procedures should be followed at all times. Oxford University Press makes no representation, express or implied, in respect of the accuracy of the material set forth in books in this series and cannot accept any legal responsibility or liability for any errors or omissions that may be made. A catalogue record for this book is available from the British Library Library of Congress Cataloging in Publication Data (Data available) ISBN 0-19-963699-0 (Hbk) 0-19-963698-2 (Pbk) Typeset by Footnote Graphics, Warminster, Wilts Printed in Great Britain by Information Press, Ltd, Eynsham, Oxon.

Preface The transcription of genes and the duplication and segregation of the eukaryotic genome occurs within the context of chromatin and chromosomes. Chromosomes are macromolecular complexes of the primary DNA sequence complexed with protein, and perhaps also with RNA. The formation of chromatin occurs through a series of hierarchical interactions with proteins: starting with the interaction with the histone octamer to form nucleosomes and finishing with the chromosome in its fully condensed form ready for mitosis. By this stage a 10000-fold linear compaction of the DNA has been achieved. It is this very hierarchy of packaging that has made the analysis of chromatin and chromosome structure so difficult because of the problems in dissecting out any one particular layer of packaging without destroying or disrupting preceding levels. The history of research into the regulation of transcription well illustrates the progression of thinking about how DNA/protein interactions within the chromosome occur and what their roles are. Prokaryotic paradigms for transcriptional regulators are often single proteins, or multimers of a single protein, binding to a specific recognition sequence and influencing the activity of RNA polymerase directly. The study of single transcription factors binding to specific recognition motifs also has a long track record in studies of gene regulation in eukaryotes. Nucleosomes then became seen as a mere nuisance and hindrance to transcription factor-access to the DNA sequence. Chromatin became a bit more exciting when it was realized that modifications of histones, for example by acetylation, were important in regulating transcriptional potential and in propagating chromatin memory from cell to cell. The enzymes responsible for these modifications have now been found to be transcriptional regulators and to be part of large complexes of proteins that include other transcriptional activators or repressers. The complexes seem to be getting bigger and bigger. Large megadalton complexes are now known to be responsible for remodelling chromatin in the cell. In the most repressed parts of the genome (heterochromatin) it was soon apparent that both modifications of histones and the presence of large multiprotein complexes were at the heart of the genetic inertness of these regions of the genome. This extreme form of chromatin structure may also have an important role to play in maintaining the integrity of the chromosome itself. Advances in visual techniques for analysing the genome have added another layer of complexity to the problem. It is clear that in the interphase nucleus, chromosomes do not decondense into the tangled mess of spaghetti depicted in some textbook diagrams. Rather each chromosome maintains a distinctive identity (territory). Moreover, the silencing of some genes appears to be intimately linked with their spatial sequestration into discrete compartments

Preface of the nuclear volume—often those parts of the nucleus occupied by visible heterochromatin. To reflect this complexity of structure and control this Practical Approach volume seeks to bring together techniques from a variety of eukaryotes that all aim to study complex levels of chromatin and/or chromosome structure in vitro, ex vivo, and in vivo. The opening chapter from Donald MacLeod describes procedures that can be used to define specific sites of protein interaction (such as transcription factor binding sites or the position of nucleosomes) on DNA sequences within chromatin, both in vitro and in vivo, taking advantage of ligation-mediated PCR. Renato Paro and his colleagues and Janet Partridge and Karl Ekwall, have also explored ways of looking at specific interactions between chromatin proteins and DNA in vivo, by formaldehyde cross-linking of living cells (from Drosophila embryos or from fission yeast cells) and followed by chromatin immunoprecipitation, respectively. The chapter from Partridge and Ekwall also introduces us to visual analysis of chromosome structure using fluorescence in situ hybridization (FISH)—in this case in a yeast. Even in a eukaryote as small as Schizosaccharomyces pombe FISH has had a profound impact on our understanding of chromosome structure and biology. The chapter from Jeff Craig describes FISH in its more familiar form—as a tool to study the large and distinctive metaphase chromosomes of vertebrates. Special emphasis is placed here on the ways in which FISH can explore questions of chromosome structure, both by the nature of the probes that can be used and by the biochemical manipulation of the chromosomes to be used as hybridization substrates. Beth Sullivan develops this theme by showing how the combination of FISH to detect DNA sequence, immunofluorescence to detect specific proteins, and drugs to interfere with chromosome segregation and cytokinesis assist in our understanding of a key step in chromosome biology—the accurate segregation of the genetic material to daughter cells by mitosis. Analysis of mitotic chromosomes has been possible for so long because of each chromosome's compact structure and distinctive shape. Individual chromosomes in interphase cells cannot be distinguished by simple DNA stains alone. Hence to study interphase chromosome structure, individual chromosomes must be delineated with specific paints and probes. Joanna Bridger and Peter Lichter describe how FISH in combination with immunofluorescence can be used to analyse chromatin and chromosome structure in the nuclei of vertebrate cultured cells that have been treated to preserve as much as possible of their three-dimensional structure. Biochemical manipulation of the interphase nucleus prior to visual analysis is also described. Abby Dernburg takes us one step further to the real animal by describing ways in which FISH can be performed in whole-mount tissues of nonvertebrate animals. A delicate balance must be maintained here between the viii

Preface preservation of structure, the permeability of the cells/nuclei to probe, and the necessary denaturation of target chromosomal DNA. These last five chapters also all describe the benefits and limitations of the image acquisition and analysis systems that are necessary to do justice to the data produced from these fluorescence-based approaches. The preservation of subcellular and chromosomal architecture is a problem demanding compromises in FISH and immunofluorescence studies in whole cells and tissues. There are additional problems in studying complex underlying nuclear structures using classical biochemical approaches. Dean Jackson details the principal methods through which attempts have been made to study nuclear substructures using a variety of biochemical extraction procedures. Continuing the biochemical theme, Jason Swedlow presents techniques for generating and studying mitotic chromosomes in an in vitro system developed from Xenopus eggs. This system is proving its worth in identifying components important for higher order chromosome structure. Methods for the immunodepletion of specific factors from these extracts and also the immunolocalization of specific proteins on the resulting chromosomes are presented. The final chapter, from Christine Farr, describes a truly top-down approach to studying chromosome structure through the genetic manipulation and fragmentation of existing mammalian chromosomes. It is my belief, and I am sure that of the authors as well, that the methods presented here will be of continuing and increasing importance for studies of chromosome structure and function and gene regulation in eukaryotes, and that the combined forces of genetics, biochemistry, and cytology will augment and complement each other in this field to an increasing extent. Edinburgh May 1998

W.A.B.

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Contents List of contributors Abbreviations 1. Mapping protein/DNA interactions in vivo using ligation-mediated polymerase chain reaction Donald Macleod 1. Introduction Methods used in determining protein/DNA interactions in vitro Determining protein/DNA interactions in vivo

2. The ligation-mediated polymerase chain reaction (LMPCR) Applications of LMPCR

3. Mapping protein factor binding sites in vivo with LMPCR and DMS DNA modification by DMS in vitro DMS modification of DNA in vivo Amplification of DMS/piperidine-cleaved DNA by LMPCR Analysis of LMPCR reactions

4. Mapping nucleosomes using micrococcal nuclease and LMPCR Isolation of nuclei from cultured cells Preparation of DNA from nuclei treated with MNase Cleavage of genomic DNA with MNase

Acknowledgements References

2. Mapping DNA target sites of chromatinassociated proteins by formaldehyde cross-linking in Drosophila embryos Giacomo Cavalli, Valeria Orlando, and Renato Paro 1. Introduction 2. Outline of the method

1 1 1 2

3 4

5 5 1 7 9

13 13 14 16

18 18

21 21 22

Contents 3. Formaldehyde cross-linking in staged Drosophila embryos Preparation of fly cages and collection of staged embryos Optimizing cross-linking conditions

4. Immunoprecipitation of cross-linked embryonic chromatin and PCR amplification of the immunoprecipitated DNA 5. Analysing the enrichment of putative target sequences in the PCR-amplified DNA Slot-blot analysis of the enrichment of putative PC target sequences Mapping DNA target sites for Polycomb and GAGA factor in the Drosophila bithorax complex

6. Concluding remarks References

23 23 24

28 31 31 31

36 37

3. Fission yeast chromosome analysis: fluorescence in situ hybridization (FISH) and chromatin immunoprecipitation (CHIP) Karl Ekwall and Janet F. Partridge 1. Introduction 2. Fluorescence in situ hybridization (FISH) analysis of fission yeast Preparation of probes Cell fixation and cell-wall digestion

3. Chromatin immunoprecipitation from fission yeast Fixation of yeast cells to maintain protein localization Preparation of chromatin extract Immunoprecipitation of chromatin Analysis of immunoprecipitated DNA sequences

Acknowledgements References

39

39 40 40 43

48 49 50 51 55

56 56

4. Isolation of vertebrate metaphase chromosomes and their analysis by FISH

59

Jeff Craig 1. Introduction

59

2. General equipment required for FISH

59

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Contents 3. Production of metaphase chromosomes as substrates for FISH Production of fixed metaphase chromosome spreads Production of long prometaphase chromosomes Isolation of suspensions of unfixed metaphase chromosomes

60 60 62 63

4. Spreading fixed chromosomes

65

5. Pretreatments of slides Pretreatment of mitotic chromosome spreads Salt extraction of isolated metaphase chromosomes

66 66 67

6. Labelling DNA probes Choice of label Nick translation Random priming Labelling by PCR Quantifying label incorporation

69 69 69 70 70 71

7. Hybridization Preparation of probes and slides Hybridization

72 72 73

8. Detecting hybridized probe

75

9. Counterstaining and mounting Simple counterstaining Chromosome banding

77 77 77

Acknowledgements

78

References

78

5. Studying progression of vertebrate chromosomes through mitosis by immunofluorescence and FISH

81

Beth A. Sullivan and Peter E. Warburton 1. Introduction

81

2. Fundamental aspects of mitosis The mitotic spindle Chromosomes

81 81 82

3. Detecting centromere/kinetochore proteins on metaphase chromosomes

83

4. In situ hybridization following immunofluorescence

87

5. The use of anti-mitotic drugs Colcemid/colchicine Nocodazole

90 90 91

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Contents Vinblastine and other drugs Cytochalasin/dihydrocytochalasin B (DCB)

91 91

6. Immunofluorescence on anaphase and telophase cells/chromosomes C-anaphase: sister chromatid separation and anaphase in the presence of Colcemid Anaphase chromosomes visualized on the mitotic spindle Anaphase studies on cytokinesis-blocked cells References

92 92 94 96 100

6. Analysis of mammalian interphase chromosomes by FISH and imimmofluorescence

103

Joanna M. Bridger and Peter Lichter 1. Introduction

103

2. Preparation of sample material Adherent cells Suspension cells DNA halo preparations Fixation and permeabilization Improving probe penetration

105 105 106 106 107 109

3. Probes

111

Chromosomal painting probes Probe labelling

111 112

4. Fluorescence in situ hybridization

113

Denaturation Hybridization Washing Detection of reporter molecules

113 115 115 116

5. Immunofluorescence in combination with FISH Primary and secondary antibody incubations after FISH Primary antibody incubation predenaturation and secondary antibody incubation postdenaturation Primary and secondary antibody incubations prior to FISH denaturation Reporter-conjugated primary antibody incubated predenaturation

116 118 119 119 119

6. Mounting the slides

119

7. Analysis

120

References

121

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Contents

7. Fluorescence in situ hybridization in whole-mount tissues Abby F. Dernburg 1. Introduction 2. Probe synthesis and labelling

3. 4. 5. 6. 7.

125 125 127

General considerations Why use fluorescence-based detection? Choice of labelling and detection reagents Probe synthesis

127 127 128 131

Fixation methods for whole-mount FISH Hybridization methods Troubleshooting Microscopy and image analysis Future directions References Acknowledgement

133 136 141 143 144 144 144

8. Analysing the substructure of mammalian nuclei, in vitro Dean A. Jackson 1. Introduction 2. The nuclear matrix and nucleoskeleton 3. Methods used to analyse nuclear organization The nuclear matrix Nucleoids The nuclear scaffold The 'low-salt' nuclear matrix The nucleoskeleton

147 147 147 148 148 149 150 151 151

4. Studying the chromatin loops of different nuclear derivatives 153 Chromatin loops after hypertonic or hypotonic treatment Chromatin loops under 'physiological' conditions Technical tips on cutting and electroeluting chromatin The frequency and nature of attachment sites in different nuclear derivatives

5. The morphology of different nuclear derivatives 6. Assaying nuclear function and nuclear proteins in permeabilized cells Labelling sites of replication and transcription in vitro Technical tips on labelling sites of replication and transcription xv

153 154 156 157

159 160 160 161

Contents Studying protein distribution relative to sites of transcription or replication in permeabilized cells. A typical example

162 164

7. Conclusions

164

References

165

9. Chromosome assembly in vitro using Xenopus egg extracts

167

Jason R. Swedlow 1. Introduction

167

2. Chromosome structure and biochemistry

167

3. Preparation of Xenopus egg extracts for chromatin and chromosome assembly in vitro Xenopus egg maturation Xenopus egg extracts Chromosome assembly extracts—technical tips

168 168 168 169

4. Chromatin and chromosome assembly in vitro Assembly and isolation of chromatin and chromosomes—technical tips

175

5. Immunofluorescence of in vitro assembled chromosomes

178

6. Functional analysis of the role of specific proteins in chromatin and chromosome structure by irnmunodepletion

180

Acknowledgements

181

References

182

10. Chromosome fragmentation in vertebrate cell lines

175

183

Christine J. Farr 1. Introduction

183

2. Telomere-associated chromosome fragmentation

183

3. Experimentally induced de novo telomere formation Design of the telomere-seeding construct Transfection of the telomere-seeding construct Screening stable transfectants for de novo telomere formation

185 185 186 188

4. Targeted de novo telomere formation

191

5. Targeted truncation events in the recombination-proficient avian cell line DT40

192

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Contents 6. The characterization of chromosomes modified by de novo telomere formation and fragmentation Estimation of minichromosome size Assays for mitotic stability of minichromosomes

193 194 196

7. Concluding remarks

197

References

197

A1. List of suppliers

199

Index

207

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Contributors JOANNA M. BRIDGER

MRC Human Genetics Unit, Crewe Road, Edinburgh EH4 2XU, UK. GIACOMO CAVALLI

Zentrum fur Molekulare Biologie der Universita't Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany. JEFF CRAIG

The Murdoch Institute, Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia. ABBY F. DERNBURG

Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA. KARL EKWALL

Karolinska Institute, Department of Biosciences at Novum, SE-141 57 Huddinge, Stockholm, Sweden. CHRISTINE J. FARR

Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK. DEAN A. JACKSON

CRC Structure and Function Research Group, Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE. UK. PETER LICHTER

Abteilung Organisation Komplexer Genome, DKFZ, In Neuenheimer Feld 280, D-69120 Heidelberg, Germany. DONALD MACLEOD

Institute of Cell and Molecular Biology, University of Edinburgh, King's Buildings, West Mains Road, Edinburgh EH9 3JR, UK. VALERIO ORLANDO

DIBIT hSR Scientific Park, Via Olgettina 58, 20132 Milano, Italy. RENATO PARO

Zentrum fur Molekulare Biologie der Universita't Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany. JANET F. PARTRIDGE

MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK.

Contributors BETH A. SULLIVAN

MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK. JASON R. SWEDLOW

Department of Biochemistry, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, UK. PETER E. WARBURTON

Department of Human Genetics, Mount Sinai School of Medicine, New York, USA.

xx

Abbreviations Ab ActD AMCA ATCC BAC BCIP bp BrdU BSA CCD CENPs CHIP CHO CISS CLSM CREST

antibody actinomycin D 7-amino-4-methylcoumarin-3-acetic acid American Tissue Culture Company bacterial artificial chromosome 5-bromo-4-chloro-3-indolyl phosphate base pair 5-bromo-2'-deoxyuridine bovine serum albumin charged-coupled device centromeric proteins chromatin immunoprecipitation Chinese hamster ovary chromosomal in situ suppression confocal laser scanning microscopy calcinosis, Raynaud's phenomenon, (o)gsophogeal dysmotility, scleroderma, and telangectasia syndrome CSF cytostatic factor CytB cytochalasin B DABCO l,4-diazabicyclo-[2.2.2]-octane (triethylenediamine) DAPI 4, 6-diamidino-2-phenylindole DCB dihydrocytochalasin B dH2O double-distilled water dCTP deoxycytidine triphosphate DHS DNase hypersensitive site DIG digoxigenin DMS dimethylsulfate DMSO dimethylsulfoxide DNase deoxyribonuclease DNP dinitrophenol dNTP deoxyribonucleotide triphosphate(s) DTT dithiothreitol dTTP deoxythymidine triphosphate dUTP deoxyuridine triphosphate EDTA ethylenediaminetetraacetic acid, disodium salt EGS ethylene glycol-bis(succinimidylsuccinate) EGTA ethylene glycol-bis(b-aminoethyl ether-N,N,N',N''-tetraacetic acid EtBr ethidium bromide EtOH ethanol F farad (SI unit of capacitance)

Abbreviations FCS fetal calf serum FISH fluorescence in situ hybridization FITC fluorescein isothiocyanate GFP green fluorescent protein hCG human chorionic gonadotropin HPLC high-performance liquid chromatography HPRI human placental ribonuclease inhibitor HPRT hypoxanthine-guanine phosphoribosyl transferase IP immunoprecipitation IRS interspersed repetitive sequences kb kilobase LAS loop attachment sequences LCR locus control region LIS lithium 3,5-diiodosalicylic acid LMPCR ligation-mediated polymerase chain reaction LPS lipopolysaccharide mAb monoclonal antibody MARs matrix-associated regions MeOH methanol MI mitotic index MMCT microcell-mediated chromosome transfer MNase micrococcal nuclease MT microtubule MTX methotrexate NaOAc sodium acetate NBT nitroblue tetrazolium chloride NCS newborn calf serum NPG n-propyl gallate PAGE polyacrylamide gel electrophoresis PBL peripheral blood lymphocytes PBS phosphate-buffered saline PC Polycomb (chromatin-associated proteins in Drosophila) PCNA proliferating cell nuclear antigen PCR polymerase chain reaction pFA paraformaldehyde PFGE pulsed-field gel electrophoresis PHA phytohaemagglutinin PI propidium iodide PMSF phenylmethlysulfonyl fluoride PMSG pregnant mare serum gonadotropin PNA protein nucleic acid PPD p-phenylenediamine RBC red blood cells RNase ribonuclease xxii

Abbreviations SAAP SARs SDS SIR SSC TAE TBS TCA TdT TE TRF TRITC YAC

streptavidin-alkaline phosphatase conjugate scaffold-associated regions sodium dodecyl sulfate Silent Information Regulator standard saline citrate Tris-acetate, EDTA Tris-buffered saline trichloroacetic acid terminal deoxynucleotidyl transferase Tris-HCl, EDTA terminal restriction fragment tetramethylrhodamine isothiocyanate yeast artificial chromosome

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1

Mapping protein/DNA interactions in vivo using ligation-mediated polymerase chain reaction DONALD MACLEOD

1. Introduction This chapter describes the procedures currently used to identify sites of protein interaction on specific DNA sequences. One of these methods, ligationmediated PCR (LMPCR), can be used for in vivo footprinting analysis and is discussed in detail. Using the mouse aprt gene as an example, I show how this method can be used to build up a profile of gene structure in vivo, by mapping transcription factor binding sites and the position of nucleosomes.

1.1 Methods used in determining protein /DNA interactions in vitro There are a variety of methods used to examine protein interactions in vitro which are now standard protocols. Two methods are briefly described here: (a) Bandshift. This method uses polyacrylamide or agarose gel electrophoresis to determine whether a labelled DNA fragment contains binding sites for proteins in an in vitro assay. The labelled DNA probe is incubated with either a crude protein extract, usually prepared from nuclei, or with purified proteins. Binding of the protein to the DNA, in the presence of competitor, results in a 'bandshift' as the mobility of the probe is impaired during electrophoresis (1). (b) DNase I footprinting. This is in an extension of the previous procedure. Once bandshift conditions have been determined which optimize DNA/ protein binding, the specific sites of interaction on the DNA can be mapped using DNase I. This nuclease introduces random nicks into an end-labelled DNA probe to produce a ladder of bands resolvable by polyacrylamide gel electrophoresis (PAGE). The pattern of bands produced from DNase I cleavage of naked DNA is compared to that obtained when the probe has been preincubated with protein extract. Missing bands

Donald Macleod (footprints) indicate positions on the DNA which are not cleaved by DNase I due to masking by bound proteins. Some sites, generally at the edge of footprints, are often hypersensitive to DNase I compared to naked DNA (2).

1.2 Determining protein/DNA interactions in vivo In vitro analysis may not give a true picture of the DNA/protein interactions within the nucleus. Sites which bind protein factors in vitro may be unoccupied in vivo (3), and a different combination of factors may interact with a promoter to regulate the same gene in different cell types (4). Until the development of LMPCR it had been difficult to identify specific sites of protein binding in vivo due to the complexity of the genome. Most analyses have used DNase I and micrococcal nuclease (MNase) which are able to cleave exposed sites in DNA within nuclear chromatin. Cleavage sites in a specific DNA sequence can be mapped by indirect end-labelling (5): (a) DNase I hypersensitive sites. DNase I cleaves at preferred sites within chromatin. When these DNase I hypersensitive sites (DHS) are mapped, they are often found at gene promoters where polymerases and transcription factors interact with the DNA. However, they are also found at enhancers, silencers, replication origins, recombination sites, telomeres, and locus control regions (LCRs) (6). DHS are generally 50-100 bp stretches of DNA but 'domains' of sensitivity have also been identified which extend over much larger regions (7). DHS can be transient or persistent and are thought to indicate a localized decondensation of chromatin which allows access of proteins such as transcription factors and polymerases to the DNA. This open conformation can exist in the absence of factor binding (8). CpG islands, are CpG-rich, non-methylated regions of ~1 kb which are found at the 5' ends of many genes (9). Fractionation of CpG island chromatin has revealed a nucleosome-free fraction which is hypersensitive to nucleases such as DNase I (10). (b) MNase cleavage. MNase is a relatively non-specific nuclease, although it does prefer to cleave AT-rich DNA, and with prolonged digestion will eventually degrade DNA to small fragments and mononucleotides. When isolated nuclei are incubated with low concentrations of MNase there is preferential cleavage of linker DNA between nucleosomes. MNase will also preferentially cleave DNA at DHS. Limited digestion of nuclei with MNase in the presence of Ca2+ produces a nucleosomal ladder when fractionated by electrophoresis (11). The problem with in vivo analysis is in obtaining footprints at the same high level of resolution as can be achieved in vitro. This has been carried out by transferring fractionated genomic DNA from polyacrylamide gels to nylon membranes and probing with single-stranded probes labelled radioactively to

1: Mapping protein/DNA interactions high specific activity (12,13). Indirect end-labelling (5) and primer extension (14,15) have also been used. However, these methods are technically difficult due to the large amounts of DNA and long autoradiographic exposure times required, and the high signal to noise ratio (16). However, some analysis has been successful, especially when multiple copies of the target sequence are present in the genome (17).

2. The ligation-mediated polymerase chain reaction (LMPCR) LMPCR (16, 18) circumvents many of the technical problems of in-vivo analysis and is diagrammed in Figure 1. Partially cleaved DNA is denatured and a gene-specific primer is annealed and extended in a primer extension reaction to obtain blunt-ended fragments. These products are then ligated to

Figure 1. A flow diagram showing the LMPCR method. Step 1: partial cleavage of genomic DNA. Step 2: denaturation and annealing of gene-specific primer AP1. The primer will anneal to multiple fragments of different sizes—only one of which is shown in the diagram. Step 3: primer extension to produce blunt-ended fragments. Steps 4 and 5: ligation of linkers. Step 6: denaturation and annealing of second (nested) linker AP2. Step 7: primer extension in the first step of PCR which copies the lower strand (including the L1 sequence). Step 8: PCR amplification of molecules with AP2 and L1 primers.

Donald Macleod a blunt-ended, unidirectional linker molecule which consists of a 25mer (L1) and an llmer (L2) which have been preannealed together. The linker oligomers do not contain 5' phosphates so will not self-ligate. This will yield a mixture of fragments of different sizes each of which contain a linker molecule at one end which can then be amplified using a second (nested) gene-specific primer on the upper strand and the L1 primer on the lower strand.

2.1 Applications of LMPCR 2.1.1 Genomic sequencing Genomic sequencing is used to identify methylated cytosines in DNA. 5Methylcytosine is present in vertebrate genomic DNA where it is associated with transcriptional repression, and is also found in differentially methylated (imprinted) regions (19). Transcriptional repression may be directly caused by the methylated base excluding transcription factor interaction with its recognition sequence on the DNA, or, indirectly, by proteins which specifically bind to the methylated DNA (19). It is therefore of interest to accurately map the position of the methylated bases in the DNA sequence under study. However, as methylation is lost upon cloning into Escherichia coli, the sequence analysis has to be carried out directly on genomic DNA by Maxam and Gilbert chemical cleavage (20). Partial modification of the DNA by hydrazine or dimethylsulfate (DMS) and subsequent cleavage of the modified bases by piperidine is used to produce sequence ladders when the DNA is fractionated by PAGE. Methylated cytosine is not cleaved in the sequencing reaction and is identified as a 'missing' base in the sequence ladder. LMPCR considerably improves this analysis and has been used to amplify genomic DNA sequence from the 5' regions of the human PGK-1 and HPRT genes (16,21). 2.1.2 In vivo footprinting The same technology used in genomic sequencing is used for in vivo footprinting, and LMPCR is now widely used for this purpose (18). Cultured cells or isolated nuclei are treated with reagents which either directly cleave the DNA (e.g. DNase I, MNase) or modify specific DNA bases (e.g. DMS) which are subsequently cleaved after the DNA is isolated. (a) DNase I footprinting in vivo. DNase I will cleave exposed sites in DNA within the nucleus of cells permeabilized with lysolecithin (3,17). Cleaved DNA fragments from the gene of interest are specifically amplified by LMPCR, and the ladder of bands produced from in vitro modified DNA are compared to the in vivo LMPCR reactions after PAGE. As with in vitro DNase I footprinting, missing bands or 'footprints' of proteins which bind to a DNA sequence in vivo are revealed. In addition, hypersensitive cleavage sites can also be found, particularly at the edge of foot-

1: Mapping protein/DNA interactions prints, which are thought to be due to conformational changes in chromatin that makes these sites more accessible. (b) DM5 footprinting in vivo. DMS can be used to modify DNA at exposed sites in vivo which are then cleaved with piperidine when the DNA is isolated and amplified by LMPCR (18). This procedure is detailed in Section 3. 2.1.3 In vivo mapping of nucleosomes LMPCR has been used to map nucleosome-like structures on chromosomes in vivo using DNase I (3). In an analysis of the human PGK-1 gene on the inactive X chromosome two regions were cleaved by DNase I at 10 bp intervals, suggesting that the DNA is wrapped around nucleosomes. This periodicity is similar to that created by DNase I at exposed regions of the DNA helix, in isolated nucleosome core particles (22). MNase which preferentially cleaves nucleosome-free regions or linker DNA in vivo has also been used, but the results were not informative at the nucleotide level (3). A lowresolution method has been used to map positioned nucleosomes over the aprt gene using LMPCR and MNase cleavage (23) and this method is detailed in Section 4.

3. Mapping protein factor binding sites in vivo with LMPCR and DMS 3.1 DNA modification by DMS in vitro Conditions are determined which result in the incomplete cleavage of genomic DNA, whether in vitro or in vivo, to give a range of fragment sizes after cleavage by piperidine. Using DMS, a random selection of guanines will be methylated at the N7 position and subsequently cleaved by piperidine at the site of modification. Each of the fragments generated by this cleavage will terminate at a G in the sequence, and the DMS conditions must, therefore, be monitored to obtain a complete sequencing ladder where every G is represented. Fragments between 50 and 500 bp are optimal (16), and to achieve this, samples of DNA are incubated for various times with DMS, cleaved by piperidine, and subjected to denaturing gel electrophoresis to determine the average fragment size (Protocol 1). Protocol 1. DMS/piperidine treatment of naked DNA Caution: DMS and piperidine are hazardous chemicals. DMS waste should be deactivated by disposing into a waste bottle containing 5 M NaOH. The lyophilizer should be vented into a fume-hood to remove hazardous piperidine fumes. The use of the screw-capped microcentrifuge tube

Donald Macleod Protocol1. Continued provides an efficient seal when heating the samples at 90°C, an alternative is to seal the tube with Teflon tape, close the cap, and place a weight on top. Equipment and reagents • DMS (BDH) • TE buffer: 10 mM Tris-HCI pH 7.5, 1 mM EDTA . Chloroform/isoamyl alcohol (24:1) • 1:1 phenol (buffered with TE pH 8.0): chloroform/isoamyl alcohol (24:1) • 3 M sodium acetate (NaOAc), pH 5.0

. EtOH (absolute ethanol, stored at -20°C) • 10% piperidine (Sigma) • Alkaline gel electrophoresis buffer: 50 mM NaOH, 1 mM EDTA pH 14.0 . 5 M NaOH . Lyophilizer (Speedvac, Savant)

Method 1. Mix several samples of genomic DNA (10-50 mg) in 100 ml TE buffer with 11xmlDMS in a 1.5 ml microcentrifuge tube and incubate for various times (e.g. 0.5, 1, 1.5, or 2 min) at room temperature. 2. Stop the reactions by adding 100 ml 1.5 M NaOAc and 500 (ml chilled EtOH. Mix and chill the samples at -70°C for at least 1 h and centrifuge for 30 min in the cold at 15800 g in a microcentrifuge. Carefully remove the supernatant and gently wash the inside of the tube with 70% EtOH twice, then air-dry the DNA pellet. 3. To reduce the viscosity of the DNA sample, resuspend the DNA in 20 ml of an appropriate restriction enzyme buffer and incubate with a restriction enzyme whose site lies outside of the region of interest. 4. Extract the DNA with an equal volume of phenol:chloroform isoamyl alcohol, centrifuge for 5 min at 15800 g at room temperature, and remove the aqueous phase to a fresh tube. Repeat the extraction with an equal volume of chloroform:isoamyl alcohol and remove the aqueous phase to a screw-capped microcentrifuge tube. Add 1/10 volume 3 M NaOAc and 2.5 volumes of EtOH, then pellet the DNA by centrifugation as in step 2. Wash the pellet with 70% EtOH and air-dry. 5. Resuspend the samples in 100 ml 10% piperidine. Close the cap tightly and heat at 90°C for 30 min. Cool at room temperature for 5 min and spin down. Add 20 ul 3 M NaOAc and 500 ml EtOH. Chill at -70°C for at least 1 h and centrifuge for 30 min as in step 2. Gently wash the inside of the tube with 70% EtOH twice, and air-dry. 6. Resuspend in 50 ul dH2O (i.e. double-distilled), freeze on dry-ice, and lyophilize to remove traces of piperidine. Resuspend in 50 ml dH2O and repeat the lyophilization. Resuspend in 30 ml TE buffer. 7. Check a small amount of each sample on a 1.5% agarose, alkaline denaturing gel and run at 8V/cm. Stain the gel with ethidium bromide (EtBr) and determine the DNA size under UV light. Note: when the desired size range is achieved, the DNA can be used for LMPCR (Protocol 3).

1: Mapping protein/DNA interactions

3.2 DMS modification of DNA in vivo Since cell membranes are permeable to DMS, modification of DNA within the nucleus can be achieved by incubating cells for a short time in a culture media containing DMS. The DMS concentration, or the time that the cells are exposed, should be determined empirically. Protocol 2. Reagents

DMS treatment of culture cells

• . . .

Phosphate-buffered saline pH 7.3 (PBS) • Proteinase K buffer: 0.3 M NaCI, 10 mM Cells and culture medium EDTA, 10 mM Tris-HCI pH 7.5, 100 m-l/ml DMS (see Protocol 1) proteinase K 1 x trypsin-EDTA diluted with PBS from a • 20% sodium dodecyl sulfate (SDS) w/v in dH 10 x solution (Gibco/BRL) 2° • RNase A

Method 1. Grow several dishes (75 cm2) of culture cells until 60–80% confluent (approx. 1 x 107 cells). Remove the culture medium and replace with fresh medium containing 1% DMS (add just before use and use a control dish with no added DMS). Treat each dish for different time intervals (e.g. 0.5, 1, 1.5, and 2 min one dish for each); remove the medium/DMS and wash the cells twice with PBS. 2. Detach the cells from the dishes using 2 ml 1 x trypsin-EDTA for 2-5 min and centrifuge them at 219 g in 15 ml tubes. Resuspend the pellets in 10 ml aliquots of ice-cold PBS. Repeat the centrifugation and PBS wash to remove traces of DMS. 3. Extract DNA from the cells either by preparing nuclei (Section 4.1) or by resuspending the cells in 10 ml proteinase K buffer. Add SDS to 1% and incubate at 37°C for 3 h, then add 10 m.g RNase A/ml and continue the incubation at 37°C for a further 1 h. 4. Extract the DNA (Protocol 1, step 4), resuspend in 50 ml TE buffer, and assay DNA concentration by spectrophotometry. Note: The in-vivo modified DNA is then cleaved with piperidine to produce a range of fragment sizes that are assayed by denaturing gel electrophoresis (Protocol 1}. When the desired range of fragments is obtained, the DNA samples can be used in LMPCR reactions (Protocol 3).

3.3 Amplification of DMS-/piperidine-cleaved DNA by LMPCR DNA sequence ladders can be obtained by amplifying the partially cleaved molecules using gene-specific primers and linker molecules.

Donald Macleod Protocol 3. Ligation-mediated PCR amplification of cleaved DNA Equipment and reagents • . . • . . .

Linkers and gene specific primers • T4 DNA ligase (NBL) 250 mM Tris-HCI, pH 7.7 . DMS ligation mix: 50 mM Tris pH 7.5, 2 mM 0.1 M MgCI2 ATP 0.1 M dithiothreitol (DTT) • MNase ligation mix: 84 mM Tris pH 7.5, 2 mMATP Thermocycler (e.g. Techne) Sequenase T7 DNA polymerase (USB) * 3 M NaOAc 5 X Seq. buffer: 250 mM NaCI, 200 mM •tRNA Tris-HCI pH 7.7 • Dimethylsulfoxide (DMSO) (Sigma) . 10 mM dNTP (10 mM each of dATP, dGTP, • Red Hot Taq polymerase and supplied dCTP, and dTTP) buffer IV (Advanced Biotech.) . TE buffer (Protocol 7)

Method 1. Prepare annealed linkers. I have used the HPLC-purified oligomers described in ref. 18— LI: 5'-GCG GTG ACC CGG GAG ATC TGA ATT C3' and L2: 5'-GAA TTC AGA TC-3'. Anneal oligomers together at 20 pM/ml of each in 250 mM Tris-HCI pH 7.7. Denature at 95°C for 5 min. Place in a water-bath at 70°C and then cool to room temperature. Store at -20°C and thaw on ice before use. 2. Anneal 0.5 pM of a gene-specific primer to 1-3 mg of cleaved genomic DNA in a 15 ml reaction mix containing 1 x Seq buffer. Denature at 95°C for 2 min and anneal primer at 55°C for 30 min. Chill on ice. 3. Primer extension is carried out using Sequenase. Add 3 ml 5 x Seq. buffer, 3 ml 0.1 M MgCI2, 3 ml H20,1ml10 mM dNTP, 3 ml of 0.1 M DTT and mix. Then add 1.5 ml of Sequenase (diluted 1:8 in ice-cold TE immediately before use). Incubate at 45°C for 15 min and inactivate at 68°C for 15 min. Chill on ice and centrifuge briefly. 4. Ligate the linkers to the DNA. As the linkers are in 250 mM Tris-HCI pH 7.7, it is important that the ligation buffer is adjusted so that the final concentration of Tris is optimal for the ligase. For DMS reactions, add 30 ml ice-cold DMS ligation mix and 5 ml linkers (100 pM). For MNasetreated DNA samples add 30 ml MNase ligation mix and 1 ml linkers (40 pM). In each case, add 200 units (u) T4 DNA ligase and incubate at 18°C overnight. 5. Precipitate DNA by adding 1/10 volume 3 M NaOAc, 10 mg tRNA, and 2 vol. EtOH (prechilled at -20°C). Centrifuge for 30 min at 15800 g in the cold. Wash pellet carefully with 70% EtOH and air-dry. 6. Amplify the ligated DNA by PCR using the L1 primer and a second gene-specific primer that is nested with respect to that used in the annealing reaction (step 1). Resuspend the DNA pellet in 100 ml of a standard PCR reaction mix. The type of thermostable polymerase used

8

1: Mapping protein/DNA interactions is largely a matter of choice, currently I use Red Hot Taq polymerase with the supplied buffer. The final reaction mix contains 20 mM (NH4)3SO4, 75 mM Tris-HCI pH 9.0, 0.01% Tween-20, 1.5 mM MgCI2, 250 mM dNTP, 10 pM of gene-specific and L1 primers, and 1 U Taq polymerase. For GC-rich sequences include 10% DMSO in the reaction mixture. Denature samples at 94°C for 5 min, cycle 20 times (94°C x 30 sec, 55°C X 30 sec, 72°C x 2 min) and finally extend at 72°C for 10 min.

3.4 Analysis of LMPCR reactions The products of LMPCR reactions can be analysed by agarose gel electrophoresis, but if high resolution is required, as in footprinting of protein factors, PAGE is used. The most convenient method is to include another PCR step using an end-labelled third nested gene-specific primer. This allows the products to be directly visualized by autoradiography after PAGE (Protocol 4). 3.4.1 LMPCR analysis using an end-labelled primer Protocol 4. LMPCR analysis by PCR with an end-labelled primer Equipment and reagents • [-y-32P]ATP 10 mCi/ml 5000 Ci/mmol (Amer- • 5 M NH4OAc (ammonium acetate), pH 7.0 sharn) • Red Hot Taq polymerase (Protocol 3) • T4 polynucleotide kinase (NBL) . Thermocycler (Protocol 3) • Tris-HCI pH 7.6 . 6% acrylamide sequencing gel and equip• Labelling mixture: 70 mM Tris-HCI pH 7.6, ment 10 mM MgCI2, 5 mM DTT, 50 mCi [y- . 10 x TBE buffer: 890 mM Tris, 890 mM 32 P]ATP, 50 pM primer, 10 U T4 polynuboric acid, 20 mM EDTA pH 8.0 cleotide kinase . Gel loading mix: 80% formamide, 20 mM . Labelled primer mix: 20 mM (NH4)3SO4, 75 EDTA, 0.05% Bromophenol blue, 0.05% mM Tris-HCI pH 9.0, 0.01% Tween-20, 1.5 xylene cyanol blue mMMgcl2,1mMdNTP' 2 • TE buffer (Protocol • tRNA

PM

of labelled • 3MM chromato9raPhY PaPer (Whatman) • XAR-5 autoradiographic film (Kodak) 1) ,_ . • Vacuum gel drier (Savant)

Method 1. End-label a third primer, nested to that used in the LMPCR reaction, using 10ml labelling mixture. Incubate the reaction mix at 37°C for 1 h and heat inactivate the enzyme at 65°C for 15 min. Precipitate the labelled primer by adding 40 ml TE, 40 ml 5 M NH4OAc, 1 ml tRNA, and 270 ml ice-cold EtOH. Mix and centrifuge for 10 min at 15800 g in the cold. Wash the pellet with 70% EtOH and air-dry. Resuspend in 50 ml TE buffer. 2. To the PCR reactions from Protocol 3, add 5 ml labelled primer mix and a further 1 U Red Hot Taq polymerase/reaction. Cycle twice (94°C x 30

9

Donald Macleod Protocol 4.

Continued

sec, 55°C x 30 sec, 72°C X 2 min) and then extend at 72°C for 10 min. Chill the samples and add 10 ml 3 M NaOAc, 1 ml tRNA, and 250 |xl EtOH; precipitate DNA by centrifugation as in step 1. Wash the pellet in 70% EtOH and air-dry. 3. Resuspend the samples in 10 ml gel loading mix. Denature 2-5 ml of the sample (the rest can be stored at -20°C to repeat the electrophoresis) at 90°C for 2 min and quench on ice. 4. Load the samples on a 6% acrylamide sequencing gel and electrophorese in 1 x TBE buffer. Include a set of DNA sequence reactions as a marker. A DNA clone which contains the sequence analysed can be used with dideoxy-terminator sequencing using established methods (e.g. Sequenase, USB). Preferably the sequencing reactions should be carried out using the same primer as in step 1 since this can be directly compared with the G ladder produced by the LMPCR reactions. 5. After electrophoresis, transfer the gel to 3MM paper, cover with Saran wrap and place on a vacuum drier. Dry down at 80°C, under vacuum, and expose to autoradiographic film. An alternative to this procedure is to transfer the DNA from the gel to a nylon membrane by electroblotting (Protocol 5) and then to hybridize the membrane to a gene-specific probe.

For low-resolution analysis, such as in nucleosome mapping, agarose gel electrophoresis can be used. DNA fragments from the agarose gels can be

Figure 2. Electroblotting apparatus. A drawing of a workshop-made apparatus as described in ref. 16. It consists of a Perspex box containing the blotting apparatus. The electrodes are two stainless-steel plates connected to the power supply through holes in the box lid. A safety mechanism should be incorporated to interrupt the power supply when the lid is removed. The lower plate sits on top of Perspex blocks and on top of this are 10 pieces of Whatman filter paper (cross-hatched) soaked in buffer and rollered to remove air bubbles. On top of this is the gel, then the nylon membrane, another stack of filter paper soaked in buffer, the top electrode plate, and finally a 2 kg weight. The buffer level is adjusted to halfway up the bottom filter paper stack.

10

2: Mapping protein/DNA interactions transferred to nylon membranes by conventional Southern blotting and hybridized to gene-specific probes. 3.4.2 Analysis of LMPCR reactions by electroblotting from acrylamide gels DNA can be transferred from the acrylamide gel by electroblotting using the method described in ref. 24. Equipment is commercially available, but I have used an apparatus made in our workshops as described in ref. 16 and shown in Figure 2. Protocol 5. Electroblotting from acrylamide gels Equipment and reagents • • • •

3 M NaOAc Loading dye mix (Protocol 4) PAGE equipment and reagents Genescreen Plus nylon membrane (NEN)

. TBE buffer (Protocol 4} • Electroblotting apparatus • Whatman 17 filter paper

Method 1. Chill LMPCR samples from Protocol 4, add 1/10 volume 3 M NaOAc and 2.5 volumes of EtOH precipitate the DNA by centrifugation for 30 min at 15800 g in the cold, wash the pellet with 70% EtOH and air-dry. 2. Resuspend in 10 ul loading dye mix and fractionate the DNA by PAGE. 3. After electrophoresis, transfer the gel from the glass plate to Saran wrap. Overlay with 10 layers of filter paper, presoaked in TBE, and squeezed out to remove trapped air bubbles. Invert, so the gel is to the top, remove Saran wrap after rolling out any bubbles, and transfer to the buffer chamber on top of the lower electrode. Lay the prewetted nylon membrane on top (again take care not to trap air bubbles) followed by a second layer of ten presoaked filter papers, the top electrode, and the 2 kg weight. Electroblot onto the membrane at 30V (1.6 A) for 1-2 h. Remove the membrane, air-dry for 15–30 min, and bake in a vacuum oven at 80°C. UV cross-link the DNA. The membranes can then be hybridized using a gene-specific probe.

After hybridization, LMPCR samples showing the most complete sequence ladders from the naked DNA or in vivo modified DNA reactions can be rerun alongside each other on a new gel so that the intensities of the bands can be directly compared. Figure 3 shows a region of the aprt gene promoter which interacts with the transcription factor Spl. This analysis was done with primers which amplify either the coding or the non-coding strand. In each case a footprint is identified at the three Sp1 binding sites by weak or missing G bands in the in vivo samples. Hyper-reactive G bands (relative to the naked

11

Donald Macleod

Figure 3. In vivo footprints at the aprt gene promoter. Autoradiographs showing footprints on lower strand [tracks B and C) and upper strand (tracks E and F) of the mouse aprt DNA sequence from ref. 23. Tracks A and B are LMPCR reactions produced from DMS-modified naked DNA and tracks B, C, D and E from in vivo modified DNA. Samples B and C were treated for 2 min with DMS and samples in D and E for 5 min. Each of the bands in the tracks terminate at a G residue in the aprt sequence. Weak or missing bands in the in vivo samples are indicated by open circles, hyper-reactive bands by filled circles. Numbered brackets refer to the positions of the Sp1 sites and the consensus sequence motifs are shown at the bottom of the figure.

12

1: Mapping protein/DNA interactions DNA sample) are also seen in the in vivo samples. The DNA sequence motifs have been shown to interact with Sp1 in vitro by DNase I footprinting (25).

4. Mapping nucleosomes using micrococcal nuclease and LMPCR MNase-cleaved DNA obtained from nuclei, or an in vitro reaction, can also be amplified using LMPCR.

4.1 Isolation of nuclei from cultured cells This procedure is based on the method described in ref. 26. Cells are treated with weak detergent that ruptures the cell membrane but not the nuclear membrane. The nuclei are purified from cell debris by pelleting through a sucrose cushion, resuspended in a buffer containing glycerol, and stored at -70 °C until use. Protocol 6.

Preparation of nuclei from tissue culture cells

Equipment and reagents • PBS pH 7.3 • Nuclei storage buffer: 50 mM Tris-HCI pH . Buffer R: 10 mM Tris-HCI pH 7.5, 10 mM 7.5, 5 mM MgCI2, 0.1 mM EDTA NaCI, 3 mM MgCI2, 0.1 mM PMSF, 0.25 M . Glycerol sucrose . Centrifuge with swing-out rotor (e.g. Beck• NP-40 man J2-21, JS13 rotor) • Sucrose • 30 ml Corex tubes . 1% SDS

Method 1. Culture cells (approx. 2 x 107) and detach from flasks as in Protocol 2. 2. Wash the cell pellet twice with PBS and resuspend in 10 ml ice-cold buffer R. Add 0.2 ml 10% NP-40 in buffer R and agitate cells by repeated pipetting back and forth using a 10 ml glass pipette (alternatively, use a glass Dounce homogenizer) to lyse cell walls and release nuclei. Check the degree of lysis by removing a drop of the mixture and examining under a microscope using phase contrast. 3. Prepare an ice-cold 5 ml cushion of 1.1 M sucrose in buffer R in a 30 ml glass Corex tube and overlay with 10 ml lysed cells. Centrifuge at 3900 g in a prechilled, swing-out rotor for 3 min at 0°C to pellet the nuclei. 4. Remove the supernatant and gently resuspend the nuclear pellet in 5 ml buffer R and repeat the centrifugation through a sucrose cushion (Step 3). 5. Resuspend the nuclei in 1 ml nuclei storage buffer and examine a drop under the microscope to check that the nuclei are intact and that there is no contaminating cell debris. If contamination is a problem, repeat procedure perhaps with an additional step 3. 13

Donald Macleod Protocol 6.

Continued

6. Remove a small aliquot (10 ml) into a fresh tube and add 1 ml of 1% SDS. Mix to lyse the nuclei and check the DNA concentration using a spectrophotometer. 7. Add glycerol to the nuclei suspension to 40%, aliquot into microcentrifuge tubes, and store at -70 °C.

The amount of NP–40 solution used can be varied depending on the cell type. However, it is important to treat the nuclei gently so that they do not lyse or break open. Checking the DNA concentration before freezing determines how much of the nuclei suspension to aliquot into each tube (generally this is equivalent to 1 mg of DNA/tube). 4.2 Preparation of DNA from nuclei treated with MNase MNase preferentially cleaves the chromatin between nucleosomes to give a nucleosomal ladder when the DNA is purified and fractionated by electrophoresis. For LMPCR, a range of DNA samples should be produced which are only partially digested with the nuclease, as overdigestion will result in preferential amplification of the smaller fragments. Protocol 7. Micrococcal nuclease (MNase) digestion of nuclei Reagents • Buffer M: 50 mM Tris-HCI pH 7.4, 60 mM KCI, 3 mM CaCI2, 0.34 M sucrose • MNase (Worthington) resuspended at 17 U/ml in 10 mM Tris-HCI pH 7.5, 0.5 mM EDTA, 0.5 mM DTT, and 50% glycerol • Proteinase K stop mix: 0.6 M NaCI, 20 mM EDTA, 20 mM Tris-HCI pH 7.5, 1% SDS, 200 m,g/ml proteinase K . 10 mg/ml RNase A (Sigma); heat treat before use (27)

Phenol:chloroform (1:1) (Protocol 1) Chloroform:isoamyl alcohol (24:1) (Protocol 7) 3 M NaOAc (Protocol 7) TE buffer (Protocol 7) T4 polynucleotide kinase buffer: 70 mM Tris-HCI pH 7.6 10 mM Mgcl2, 5 mM DTT' 2 mM ATP •4 polynucleotide kinase (NBL)

Method 1. Thaw a frozen aliquot of nuclei on ice and centrifuge at 720 g in a microcentrifuge for 3 min. Carefully remove the supernatant and resuspend the nuclear pellet in 600 ml ice-cold buffer M. 2. Remove 100 ml aliquots of nuclei into fresh microcentrifuge tubes containing variable amounts of MNase. The amount used must be determined empirically as enzyme activity varies from batch to batch. As an example, 1-15mlof a 1/500 dilution of the stock solution in buffer M was used to produce the range of digestion products shown in Figure 2. 3. Incubate the samples for 4 min at 37°C then stop the reaction with an

14

1: Mapping protein/DNA interactions

4.

5. 6.

7.

equal volume of proteinase K stop mix. Mix and continue the incubation at 37°C for 3 h. Add 10 mg RNase A and incubate at 37°C for a further 1 h, Extract DNA with an equal volume of 1:1 phenol:chloroform and again with chlorofom:isoamyl alcohol (24:1) at room temp. Precipitate the DNA by adding an equal volume of isopropanol and pellet by centrifugation for 30 min at 15800 g at room temp. Wash the DNA pellet with 70% EtOH and air-dry, Resuspend in 100 ml TE buffer. Determine the DNA concentration by UV spectrophotometry and analyse a small sample (e.g. 5 ug) by agarose gel electrophoresis. Resuspend 5 mg MNase-digested DNA in 20 ml T4 polynucleotide kinase buffer and add 5 U T4 polynucleotide kinase. Incubate at 37°C for 1 h. Inactivate the enzyme at 65°C for 15 min, then add 2 ml 3 M NaOAc, 50 ml ice-cold EtOH and precipitate the DNA by centrifugation for 30 min at 15800 g in the cold. Wash the pellet in 70% EtOH and air-dry. Resuspend in 10 ml TE buffer and use for LMPCR (Protocol 5).

8 As MNase cleavage does not produce a 5' phosphate for the ligation step of the LMPCR the DNA is phosphorylated by T4 poiynucleotide kinase.

Figure 4 shows EtBr-stained DNA isolated from nuclei that had been digested with increasing amounts (0, 1, 2, 5, 10, and 15 ml) of a 1/500 dilution

Figure 4. Agarose gel fractionation of micrococcal digested DNA. (A) shows samples of naked DNA incubated with increasing amounts of micrococcal nuclease. Track 0: no enzyme added; tracks 1, 2, 5, and 10 correspond to 1, 2, 5, and 10 ml.1 of a 1/1000 dilution of stock enzyme, (B) shows samples of DNA extracted from nuclei digested with MNase. As above, the numbering of the tracks refers to the amount of enzyme added in each sample (0, incubated without enzyme). DNA markers are 1 kb (m1) and 123 bp (m2) ladders IGibco/BRL).

15

Donald Macleod of MNase stock and run on a 1.5% agarose gel. As the amount of MNase is increased a ladder of nucleosomal bands is detectable.

4.3 Cleavage of genomic DNA with MNase As a control, a set of naked DNA samples cleaved with MNase is also prepared to run alongside the in vivo samples. This will allow preferentially cleaved sites to be mapped on the naked DNA which can be directly compared with sites cleaved in vivo. Protocol 8.

Micrococcal nuclease cleavage of DNA

Equipment and reagents • see Protocol 7

Method 1. Prepare several tubes of genomic DNA resuspended in buffer M and add increasing amounts of MNase. This needs to be determined empirically (e.g. 0, 1, 2, 5, 10, and 15 |xl of a 1/1000 dilution of MNase stock). 2. Incubate for 4 min at 37°C then stop with an equal volume of phenokchloroform mix and extract the DNA (Protocol 1, step 4). 3. Resuspend the DNA in 50 ml TE buffer and analyse fragment size by agarose gel electrophoresis. 4. Phosphorylate DNA ends (Protocol 7, step 6) and use in the LMPCR method (Protocol 3).

Figure 4 also shows an analysis of naked DNA cleaved by MNase. As with in vivo cleaved DNA a range of fragments is produced, but a smear of DNA is evident rather than a nucleosomal ladder. Figure 5 shows the results of in vitro and in vivo treated mouse F9 cell DNA samples which were subjected to LMPCR using primers from the aprt gene, fractionated on a 1.6% agarose gel, and hybridized to a DNA probe which lies downstream of the primer sequences used for LMPCR. Figure 5 clearly shows that MNase cleaves the naked DNA and nuclear DNA at hypersensitive sites. Some are cleaved in naked DNA but not nuclei (e.g. sites D1 and D2), and others are hypersensitive in both samples (e.g. site 1). Site 5 is weakly cleaved in nuclei but becomes more prominent with higher concentrations of MNase. Hypersensitive sites 2, 3, and 4 map to the promoter region where Sp1 interacts with the DNA. The other in vivo sites map at —200 bp intervals on either side and were mapped to the DNA sequence (see Figure 6). A suggested array of positioned nucleosomes over the gene is indicated in 16

1:Mapping protein/DNA interactions

Figure 5. Micrococcal cleavage of aprt DNA amplified by LMPCR. Autoradiographs of Southern blots of agarose gel-fractionated LMPCR samples probed with the mouse aprt gene Isee Figure 6), The LMPCR samples are produced from the same DNA samples shown in Figure 3. Each track refers to the amount of MNase used to cleave the DNA [see Figure 4) (0: incubated without enzyme). The DNA marker is a 1 kb ladder (Gibco/BRL) end-labelled with [32P)dNTP (track M). The arrows indicate prominent MNase cleavage sites in vivo (1-8) and sites which are hypersensitive only in vitro (D1 and D2) and are referred to in Figure 6.

Figures. Sites of in vivo protein/DNA interactions in the 5' region of the aprt gene. Numbers refer to base pairs, and short vertical lines show the positions of the CpG nucleotides in the gene sequence (the bracket indicates the boundaries of the CpG island). The position of the first two exons (open rectangles) and three Sp1 motifs are shown under the CpG plot. Below this, the mapped sites for MNase cleavage in vivo are indicated by arrows (see Figure 5) and the inferred positions of nucleosomes as hatched rectangles. The positions of the Ap2 primer and probe are also shown.

17

Donald Macleod Figure 6. The in vivo cleavage sites were confirmed using other primer sets (23); outside this region nucleosomes could not be mapped, suggesting that they may be randomly positioned in these regions or that the linker DNA is protected from MNase cleavage (23). A similar analysis, using a different method, has been undertaken on another CpG island gene, O6-methylguanine DNA methyltransferase, and shows a very similar in vivo organization (28).

Acknowledgements I would like to thank G.P. Pfeifer et al. (Molecular Biology Section, Beckman Research Institute of the City of Hope, Department of Biology, Duarte, California 91010) and P.R. Mueller and B. Wold (Division of Biology, California Institute of Technology, 156–29 Pasadena, CA 91125) for their detailed protocols on the LMPCR method. I thank Cold Spring Harbor Laboratory Press and the editors of Genes and Development for permission to include data from published work (23). I would also like to thank Adrian Bird for his helpful discussions and The Wellcome Trust, the Imperial Cancer Research Fund, and the Howard Hughes Medical Institute for their support.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Strauss, E. and Varshavsky, A. (1984). Cell, 37,889. Galas, D. and Schmitz, A. (1978). Nucl. Acids Res., 5, 3157. Pfeifer, G.P. and Riggs, A.D. (1991). Genes Dev., 5,1102. Somma, M.P., Gambino, I., and Lavia, P. (1991). Nucl. Acids Res., 19, 4451. Wu, C., Wong, Y-C., and Elgin, S.C.R. (1979). Cell, 16, 807. Gross, M. and Garrard, W.T. (1988). Annu. Rev. Biochem., 57, 159. Bonifer, C., Vidal, M., Grosveld, F, and Sippel, A.E. (1990). EMBO J., 9, 2843. Weintraub, H. (1985). Cell, 42, 705. Bird, A.P. (1986). Nature, 321, 209. Tazi, J. and Bird, A. (1990). Cell, 60, 909. Kornberg, R. (1977). Annu. Rev. Biochem., 46, 931. Church, G.M. and Gilbert, W. (1984). Proc. NatlAcad. Sci. USA, 81,1991. Becker, P.B., Ruppert, S., and Schutz, G. (1987). Cell, 51, 435. Saluz, H.P. and Jost, J.P. (1989). Proc. NatlAcad. Sci. USA, 86, 2602. Mueller, P.R., Salser, S.J., and Wold, B. (1988). Genes Dev., 2, 412. Pfeifer, G.P., Steigerwald, S.D., Mueller, P.R., Wold, B., and Riggs, A.D. (1989). Science, 246, 810. Zhang, L. and Gralla, J.D. (1989). Genes Dev., 3,1814. Mueller, P.R. and Wold, B. (1989). Science, 246, 780. Tate, P. and Bird, A.P. (1993). Curr. Opin. Genet. Dev., 3, 226. Maxam, A.M. and Gilbert, W. (1980). In Methods in enzymology, Vol. 65 (ed. L. Grossman and K. Moldave) p. 499. Academic Press, New York. Hornstra, I.K and Yang, T.F. (1994). Mol. Cell. Biol., 14,1419. Noll, M. (1974). Nature, 251, 249. 18

1: Mapping protein/DNA interactions 23. Macleod, D., Charlton, J., Mullins, J., and Bird, A.P. (1994). Genes Dev., 8, 2282. 24. Saluz, H.P. and Jost, J.P. (1987). A laboratory guide to genomic sequencing. Birkhauser, Boston. 25. Dush, M.K., Briggs, M.R., Royce, M.E., Schaff, D.A., Khan, S.A., Tischfield, J.A., and Sambrook, P.J. (1988). Nucl. Acids Res., 16, 8509. 26. Shimada, T., Inokuchi, K., and Nienhuis, A.W. (1986). J. Biol. Chem., 261,1445. 27. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular cloning. A laboratory manual. Cold Spring Harbor Press, NY. 28. Patel, S.A., Graunke, D.M., and Pieper, R.O. (1997). Mol. Cell. Biol., 17, 5813.

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2

Mapping DNA target sites of chromatin-associated proteins by formaldehyde cross-linking in Drosophila embryos GIACOMO CAVALLI, VALERIC ORLANDO, and RENATO PARO

1. Introduction The method described here allows the mapping of protein-DNA interactions through the ability of formaldehyde to cross-link proteins and nucleic acids in living cells. Formaldehyde is a very reactive dipolar compound which reacts with the amino groups of proteins and amino acids (1, 2). It shows no reactivity, however, towards free double-stranded DNA, and thus does not cause the extensive DNA damage seen after prolonged exposure to other crosslinking reagents such as UV. Each formaldehyde molecule has the capacity to interact with two amino groups. Therefore DNA-protein, protein-protein, and RNA-protein cross-links are rapidly formed after formaldehyde treatment, creating a stable structure which prevents the redistribution of cellular components. Furthermore, a simple heat treatment is sufficient to reverse the reaction equilibrium, and to allow the isolation of pure DNA for further analysis (3). Formaldehyde cross-linking combined with chromatin immunoprecipitation (IP) is a way of mapping the in vivo distribution of chromatinassociated proteins. As such, this technique is of great value in the analysis of protein-DNA interactions, even more so when studying proteins which do not show specific DNA binding activities in vitro. This has recently been demonstrated for several chromatin-associated proteins, such as Polycomb (PC) in Drosophila (4) and the Silent Information Regulators (SIR) proteins in budding yeast (5). Additionally, analysis of the cross-linking pattern can not only allow mapping of sites of protein-DNA interaction, but can also give an estimation of the relative binding affinity to different sequences across a large genomic region. The method presented here is based on a previously described method of

Giacomo Cavalli, Valeria Orlando, and Renato Paro formaldehyde cross-linking in Drosophlla cultured Schneider SL-2 cells (4). This method was recently improved with a modification of the PCR amplification step, allowing a more accurate quantification of relative binding affinities for adjacent sequences with a resolution in the order of 500 bp (6). Here we present the adaptation of this methodology to the analysis of chromatin from Drosophila embryos. Although SL-2 cells have been successfully used as a model system for the study of various cellular processes, the analysis of embryonic chromatin permits additional functional studies. For example, competitive protein binding to overlapping target sites during development might be investigated at endogenous sites or in transgenic constructs. Moreover, the wealth of genetic mutants of Drosophila allows DNA binding of multimeric protein complexes to be studied in the absence of single components. In this chapter, we will discuss the different steps involved in the mapping of in vivo DNA binding sites by formaldehyde cross-linking, focusing in particular on the steps of preparation and cross-linking of the embryos. As an example of the application of the method, we will show the analysis of the binding profile of two proteins. The first is PC, a protein involved in the maintenance of the repressed state of homeotic genes from mid-embryogenesis to the adult state (7). The second is GAGA factor, which counteracts PCmediated silencing, maintaining the spatially restricted activation pattern of homeotic genes (8).

2. Outline of the method The analysis of in vivo DNA-protein interactions by formaldehyde crosslinking involves the following steps: (a) preparation of embryos for cross-linking; (b) formaldehyde cross-linking and purification of soluble cross-linked chromatin; (c) IP of purified cross-linked chromatin, reversal of the cross-links, and DNA purification; (d) PCR-amplification of the immunoprecipitated DNA; (e) analysis of the immunoprecipitated DNA, which can be done in two ways: (i) using probes to analyse the enrichment of specific DNA fragments in slot-blots, these fragments may represent putative target sites of the protein under study; or (ii) using PCR-amplified DNA as a radioactively labelled probe to hybridize a Southern blot of DNA from chromosome walk of a genomic region of interest. 22

2: Mapping DNA target sites of chromatin-associated proteins

3. Formaldehyde cross-linking in staged Drosophila embryos 3.1 Preparation of fly cages and collection of staged embryos For analysing cross-linked chromatin of 11-16-h-old (after egg lay) embryos, Drosophila embryo collection was performed at 25 °C in cylindrical fly cages of 30 cm diameter and 35 cm depth containing 7500 to 15 000 flies per cage. Flies were 5-12 days old. We used two fly cages for wild-type (Oregon R) flies, while four cages were necessary for the transgenic line 5F24 25,2, which lays significantly less eggs then the wild-type flies. For collection, six apple-juice agar plates, 14 cm in diameter, distributed on two levels in the cage were used. Under these conditions about 1 g wet weight of 11-16-h-old embryos could be collected (in our experience, a minimum of 0.5 g is required to obtain enough DNA for PCR amplification). For staging, a prelay collection of 2 h with plates streaked with 45% acetic acid and fresh yeast paste was first made. Collection was made from 5 p.m. to 10 p.m. and the plates were incubated overnight at 25 °C. At 9 a.m., embryos were collected in embryo wash buffer and further processed as described in Protocol 1. Essentially, embryos were dechorionated, washed, and cross-linked. Cross-linked embryos were extensively washed and then sonicated to produce soluble chromatin, which was used for further analysis (see below). Many chromatin proteins may change their target size distribution at different developmental stages, therefore a careful staging is essential in these experiments. In order to monitor staging, nuclei from a small aliquot of the cross-linked embryos were stained with the DNA staining dye Hoechst 33258. Stained embryos were analysed by fluorescence- and light microscopy to assess the average stage of development. We found that preparations containing a large proportion of embryos older then 16 h resulted in a high fraction of uncross-linked chromatin, most likely due to the poor permeability of these embryos to formaldehyde. On the other hand, cross-linking of embryos during early development can be easily performed, but it must be noted that the same weight equivalent contains a smaller number of nuclei, i.e. of chromatin. This is particularly important when studying early stages, such as preblastoderm embryos, where up to 5-10 g wet weight might be required to obtain DNA amounts sufficient to represent the whole genome complexity after IP. With this set-up, up to four embryo collections could be obtained from wild-type fly cages on alternate days. On the days when no cross-linking was performed, flies were fed in the morning and in the evening with three applejuice agar plates/cage, streaked with about 20 g of fly medium and 2 g of yeast paste. When transgenic flies were used, only two to three embryo collections per cage could be performed. 23

Giacomo Cavalli, Valeria Orlando, and Renata Paro

3.2 Optimizing cross-linking conditions Permcabili/alion of the embryos was obtained by adding three volumes of n-heptane directly to the cross-linking solution (Protocol 1), and cross-linking with vigorous shaking. This was sufficient to permeabilLze embryos until the late developmental stages, but it seems to be insufficient for cross-linking

Figure 1. Analysis of cross-linking efficiency with different concentrations of formaldehyde. Cross-linking of embryonic chromatin was performed with 1.8% (A, C), or 1% (B, D) formaldehyde. (A) and (B): Analysis of the DNA from each fraction collected from a CsCI gradient of 1.8% and 1% formaldehyde cross-linked chromatin, respectively. Fraction numbers are indicated at the top of each gel. The size of the DNA (Mr) markers (Boehringer) is shown in bp on the right of each panel. In the 1.8% sample, the majority of the DNA elutes with a density of 1.36 to 1.42 g/cm3, corresponding to protein-DNA complexes; while in the 1% sample, a large amount of the DNA elutes in the bottom of the gradient. This has a density of about 1.60 g/cm3, corresponding to free, uncrosslinked DNA. (C) and (D): The gels from (A) and (B), respectively, were photographed, and the intensity of the DNA signal was estimated by scanning each lane using the NIH Image software package. The relative intensity of the signal (after background subtraction, calculated from an empty lane) is plotted against the fraction number. The density of fractions which were pooled for chromatin analysis of the 1.8% sample is indicated.

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2: Mapping DNA target sites of chromatin-associated proteins embryos at the very end of embryogenesis or upon hatching, as judged by the high loss of material observed from preparations containing a high fraction of 16–20-h–old embryos. These embryos were also refractory to Hoechst staining. Thus, more severe methods of fixation should be tested for these late stages. The concentration of formaldehyde required for optimal cross-linking has been tested extensively using different concentrations of formaldehyde. The optimal range was found to be between 1.8% and 3.7%. The effect of undercross-linking is shown in Figure 1. Using 1% formaldehyde, a large fraction of the embryonic chromatin is not cross-linked, resulting in the fractionation of a large amount of free DNA towards the bottom of the gradient (Figures 1B and ID). Sometimes, this was also observed using 1.4% formaldehyde. On the other hand, a concentration of 1.8% resulted in a reproducible cross-linking of about 80% of the material, eluting in a fraction with the density characteristic of protein-DNA complexes ( = 1.39, see Figures 1A and 1C). Concentrations of 2.8% and 3.7% gave a pattern indistinguishable from that observed with 1.8% formaldehyde, while higher concentrations resulted in the loss of a large fraction of the material and to DNA of a high molecular weight (Mr), probably since highly cross-linked material is refractory to shearing by sonication.

Protocol 1. Formaldehyde cross-linking of Drosophila embryos Equipment and reagents • Sonifier apparatus (Branson Ultrasonics Corporation, Sonifier Model 250) equipped with a Microtip 3/16 inch (cat. no. 101–148–069) . 0.1 mm diameter glass beads •Wash . 3% NaOCl . Cross-linking solution: 1.8% formaldehyde, 50 mM Hepes, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCI, pH 8.0. Add formaldehyde immediately before use from a 37% stock solution stabilized with 10% methanol. . n-heptane • Glycine • Phosphate buffered saline pH 7.4 (PBS) • Triton X-100

• 100% glycerol . Staining dye: Hoechst 33258 (Sigma) . Embryo wash buffer (EWB): 0.03% Triton X100, 0.4%NaCI solution A: 10 mM Hepes pH 7.6, 10 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0, 0.25% Triton X-100 . Wash solution B: 10 mM Hepes pH 7.6, 200 mM NaCI, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0, 0.01% Triton X-100 . Sonication buffer: 10 mM Hepes pH 7.6, 1 mM EDTA pH 8.0, 0.5 mM EGTA, pH 8.0 • 10% N-lauroylsarcosine • CsCI (optical grade) • 15 ml and 50 ml Falcon tubes

Method 1. Dechorionate approximately 1 g embryos in 3% NaOCI in EWB for 2-3 min at room temperature (r.t). Wash extensively with EWB. Transfer the embryos to a 50 ml Falcon tube and wash once in 0.01% Triton X100 made up in PBS.

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Giacomo Cavalli, Valeria Orlando, and Renato Paro Protocol 1. Continued 2. Cross-link with 10 ml of cross-linking solution for 15 min in the presence of 30 ml n-heptane. Shake vigorously. 3. Spin embryos for 1 min in a tabletop centrifuge at maximum speed. Stop cross-linking by washing with 50 ml of PBS, 0.125 M glycine, 0.01% Triton X-100. Let embryos sediment without centrifuging. 4. DNA stain a small aliquot of embryos to check embryo staging as follows: (a) Wash embryos with 0.5 ml PBS. (b) Add 250 ml 1 mg/ml Hoechst 33258 in PBS, stain for 4 min in the dark, allow embryos to sediment. (c) Wash twice with 0.4 ml PBS. (d) Resuspend embryos in 25 ml PBS (at this stage the embryos can be stored overnight at 4°C in the dark). (e) Add 25 ml of 100% glycerol and mix well. Mount on a slide and coverslip. (f)

Analyse on a fluorescence microscope with a suitable filter and score 100-200 embryos to control staging.

5. Resuspend embryos in 10 ml of wash solution A. Transfer them to a 15 ml Falcon tube and wash them for 10 min on a roller. Repeat with wash solution B. Resuspend in 5.5 ml (final volume) sonication buffer. Up to this stage, embryos should still be intact. This can be checked under a microscope. 6. Sonicate with the Branson Model 250 sonifier equipped with a microtip. Adjust sonication empirically and keep the sample on ice throughout. We recommend that the tip be immersed about 2 cm deep in the solution. Perform four sonication cycles of 30 sec each at constant power in the presence of 0.1 mm diameter glass beads. Gradually increase the power up the maximum level possible, but avoid foaming. It should be possible to reach level 6 to 8 in the output control scale of the Branson Model 250 sonifier. Pause for 90 sec between each cycle. At the end, inspect a 10 ml aliquot under phase-contrast microscopy to ensure that all nuclei are lysed (no large particles should be left). 7. Adjust to 0.5% N-lauroylsarcosine and rotate for 10 min. Spin the debris at high speed (in microcentrifuge tubes for 5 min). The supernatant can be further processed by CsCI-gradient purification (Protocol 2) immediately, or frozen in liquid N2 and stored for several days before further purification. This is particularly useful when working with fly lines that lay poorly, so that several cross-linking preparations can be pooled together.

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2: Mapping DNA target sites of chromatin-associated proteins Protocol 2. Purification of soluble cross-linked chromatin Equipment and reagents • Supernatant from Protocol 7 • 1 mg/ml RNase A (DNase-free) . 10% W-lauroylsarcosine . 10 mg/ml proteinase K (in TE buffer, stored • Sonication buffer (Protocol 1) at -20°C) •Peristaltic pump with 0.114 cm (0.045 . 3 M sodium acetate (NaOAc) pH 5.2 inches) internal diameter tubing .Dialysis buffer: 4% glycerol, 10 mM •20% SDS/v Tris-HCI pH 8.0, 1 mM EDTA pH 8.0, 0.5 • 100 % ethanol (on ice) mM EGTA pH 8.0 . Phenol/chloroform 1:1 v/v . Dialysis bags: microcollodion bags (Sarto- •Gel loading buffer rius, cat. no. 13202) 1% agarose gel and electrophoresis equip. TE buffer: 10 mM Tris-HCI 1 mM EDTA, pH 8.0

Method 1. To the supernatant from Protocol 1 add sonication buffer (Protocol 1) containing 0.5% N-Mauroylsarcosine to a volume of 8 ml. Add 5.68 g CsCI (i.e. adjust to a density of 1.42g/ml) and make up to 10 ml final volume with sonication buffer containing 0.5% N-lauroylsarcosine. 2. Divide the sample between two 5 ml tubes to avoid overloading the gradient (after centrifugation a broad sarkosyl/lipid/protein aggregate is present at the top of the gradient, and overloading can result in poor resolution of the gradient, and thus poor quality chromatin). Spin the CsCI gradient at 195000 g (40000 r.p.m. in a Beckman rotor SW55Ti) for 72 hat 20 °C. 3. Elute 12 x 400 ml fractions per gradient with a peristaltic pump, fitted with 0.114 cm (0.045 inches) internal diameter tubing, at a speed of about 1 ml/min. 4. Check the density profile of the fractions (refraction index should be in the range of 1.390 to 1.362). 5. Dialyse fractions at 4°C in microcollodion bags against 2 litre dialysis buffer. After 2 h change the buffer and continue the dialysis overnight. 6. After the buffer change in step 5, remove about 1/10 vol. (40 ml) to a microcentrifuge tube to check the size and quantity of DNA in each gradient fraction (see Figure 1) as follows: (a) Add RNase A to 50 mg/ml and incubate for 30 min at 37°C. (b) Add proteinase K to 500 ng/ml and SDS to 1%. (c) Incubate at 56°C for 1 h to partially reverse the cross-links. (d) Purify DNA once by phenol/chloroform extraction, then ethanol precipitate and resuspend in 15 ml 1 x loading buffer for agarose gel electrophoresis. (e) Check DNA samples on a 1% agarose gel.

27

Giacomo Cavalli, Valeria Orlando, and Renato Paro Protocol 2. Continued 7. Pool appropriate dialysed chromatin fractions together (usually good fractions have densities of between 1.350 and 1.450 g/ml and, depending on the efficiency of the sonication, DNA is from 0.2 kb to 20 kb in size with an average size of 1.0 kb). Freeze 500 ml aliquots in liquid N2 and store at -80°C. These aliquots should contain 30–60 mg DNA. To analyse DNA of the pooled fractions, take 50 ml of the final pool and repeat the RNase treatment, proteinase K/SDS, and DNA purification of step 6. Estimate the DNA concentration by measuring the optical density at 260 nm and run an agarose gel to check DNA size-distribution. Frozen chromatin aliquots are suitable for IP and should be stable for several months.

4. Immunoprecipitation of cross-linked embryonic chromatin and PCR amplification of the immunoprecipitated DNA IP of the cross-linked purified chromatin, reversal of the cross-links, and DNA purification are performed as previously described (4, 9) and detailed in Protocol 3. Recently, an improvement of the PCR amplification step has been introduced in order to allow a more reliable quantification of the relative enrichment of neighbouring DNA fragments (6). The purified DNA is directly ligated to a blunt-ended linker and PCR-amplified. This assumes that: (a) chromatin DNA fragments which are not blunt-ended are not ligated to the linker; and (b) among the ligated molecules, short fragments, in the range of 200 to 500 bp, are amplified more efficiently than longer ones. Thus, a fraction of the cross-linked DNA molecules is lost during the procedure. Since this loss is random throughout the genome, it does not compromise the quantification as long as the whole genomic complexity is represented in the samples before PCR amplification. Amplification artefacts may be observed under non-optimal sonication conditions, since too large a proportion of very long fragments results in a correspondingly high loss of DNA molecules during PCR. This might result in the loss of entire regions of genomic DNA in the PCR-amplified samples. Such artefacts can be identified by analysing the hybridization profile of a PCR-amplified, radioactively labelled, sample from a mock IP to a restriction digest of a large genomic walk. Since no antibody is used in the mock IP, the probe obtained from this sample should hybridize uniformly to all fragments of the digest in the absence of artefactual loss of genomic complexity (see Protocol 4 and also Figures 3A, B, and C, mock IP samples). Finally, it should be noted that the preferential amplification of shorter DNA fragments sets the resolution of the technique to about 500 bp. 28

2: Mapping DNA target sites of chromatin-associated proteins Protocol 3. Chromatin immunoprecipitation Reagents • RIPA buffer: 140 mM NaCI, 10 mM Tris-HCI pH 8.0, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, 1 mM PMSF (on ice); add PMSF immediately before use from a 100 mM stock in isopropanol • Protein A Sepharose CL4B (PAS, Sigma), equilibrate in RIPA buffer by mixing at 4°C for 30–60 min. (100 mg PAS equilibrated in 1 ml RIPA buffer results in a 50% v/v suspension. After equilibration, PAS is stable for up to 1 week at 4°C.) . TE buffer: 10 mM Tris-HCI pH 8.0, 1 mM EDTA • RNase A • Proteinase K • 100 mM PMSF in isopropanol

Stock solutions for adjusting chromatin samples to RIPA conditions: 10% Triton X100, 1% SDS, 1% sodium deoxycholate, 1.4 M NaCI . LiCI buffer: 250 mM LiCI, 10 mM Tris-HCI pH 8.0, 1 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate (on ice) > 1:1 v/v phenol:chloroform/isoamyl alcohol (24:1) . 50 mM Tris-HCI pH 8.0 > 20 mg/ml glycogen > 3 M sodium acetate pH 5.2 > 5 mg/ml RNase A (DNase-free) 10% SDS • 10 mg/ml proteinase K in TE buffer, store at -20°C

Method 1. Thaw a 500 ml aliquot of chromatin and adjust to RIPA buffer conditions by the sequential addition of 100 ml 10% Triton X-100, 100 ml 1% sodium deoxycholate, 100 ml 1% SDS, and 100 ml 1.4 M NaCI. Allow 2 min gentle mixing between additions for equilibration of the chromatin into the new conditions. Finally add 10 ml 100 mM PMSF. Always include one additional sample to be mock-treated as a negative control. 2. Add 30-40 ml of the 50% (v/v) PAS suspension to the chromatin sample. Incubate for 1 h at 4°C, then remove the PAS by centrifuging in a microcentrifuge at top speed for 30 sec. This acts as a preclearing step to reduce non-specific binding to the protein A Sepharose. 3. Remove the chromatin sample to a new tube, and add 2-5 mg of appropriate antibody. Incubate overnight at 4°C, with gentle mixing. The optimal amount of antibody may need to be determined empirically, and a control (mock) IP without antibody should be carried out in parallel. Mock IP isolates DNA non-specifically, but specific antibodybound DNA fragments should be several-fold enriched in the antibody IP. 4. Purify immunocomplexes by adding 30-40 ul 50% (v/v) PAS, and incubating for 3 h at 4°C, with gentle mixing. 5. Wash PAS-antibody-chromatin complexes 5 times for 10 min each in RIPA buffer, once in LiCI buffer, and twice in TE buffer. Carry out all wash steps at 4°C using 1 ml wash buffer, and between washes centrifuge at full speed for 20 sec to pellet the PAS before removing the supernatant. 29

Giacomo Cavalli, Valeria Orlando, and Renato Paro Protocol 3. Continued 6. Resuspend the PAS complexes in 100 ml TE buffer, add RNase A to 50 mg/ml, and incubate for 30 min at 37°C. 7. Adjust the samples to 0.5% SDS, 0.5 mg/ml proteinase K and incubate overnight at 37°C, followed by 6 h at 65°C. 8. Phenol-chloroform extract the sample, and back-extract the lower phenol phase by adding an equal volume of 50 mM Tris-HCI pH 8.0, mixing, and centrifuging. Combine the aqueous phases from the phenol extraction and the back-extraction, and extract the combined phases with chloroform/isoamyl alcohol. 9. Precipitate by adding 1 ml 20 mg/ml glycogen (as carrier), 1/10 volume 3 M sodium acetate pH 5.2, and 2 volumes ice-cold 100% ethanol. Store on ice for 30 min, before centrifuging at 4°C for a further 30 min. Wash the DNA pellet in 70% ethanol, air-dry and resuspend in 20 ul sterile dH20. Store at–20°C. To minimize the risk of contamination during the PCR, we would recommend the use of aerosol-free pipette tips, and the storage of nucleotides, linkers, primers, etc. in small aliquots to prevent contamination of valuable reagents (in addition to preventing frequent freeze-thawing which may cause the destabilization/inactivation of buffers). Protocol 4.

PCR amplification of immunoprecipitated DNA

Equipment and reagents . • Sample from Protocol 3 • 1 uM linker DNA: two oligonucleotides • annealed: (a) a 24mer of sequence 5'-AGA • AGC TTG AAT TCG AGC AGT CAG (phosphorylated at the 5'-end); (b) a 20mer of • sequence 5'-CTG CTC GAA TTC AAG CTT CT. Store in small aliquots at–20°C. . 10 x ligation buffer: 0.5 M Tris-HCI pH 7.6, 125 mM MgCI2, 250 mM DTT, 12.5 mM • ATP. Store at–20°C in small aliquots. . 10 mM dGTP, dATP, dCTP, dTTP: dilute • from commercially available stocks in 10 • mM Tris-HCI pH 8.0, and store in small aliquots at–20°C

T4 DNA ligase, 4 U/ml Taq polymerase and buffer (Boehringer) PCR primer: linker oligonucleotide (b) at 100 MM, store in small aliquots at–20°C Phenol–chloroform (Protocol 3), chloroform/isoamyl alcohol (24:1), 3 M NaOAc pH 5.2, 100% ethanol (20 mg/ml glycogen (Boehringer), 70% ethanol Hindlll restriction endonuclease and corresponding buffer PCR purification columns (e.g. Qiagen) 1% agarose gel, and electrophoresis equipment and reagents

Method 1. Ligate the linker to 7 ul of the sample from Protocol 3 by adding 1 ul 10 x ligation buffer, 1 ul 1 uM linker, and 1 ul (4 U) T4 DNA ligase. Incubate overnight at 4°C. 2. Carry out the amplification directly, without purifying the ligated DNA. Make the volume of the sample up to 78.5 ul with dH2O, and add 10 ml 10 x Taq polymerase buffer, 2.5 ul each 10 mM dNTP, 1 ml 100 mM 30

2: Mapping DNA target sites of chromatin-associated proteins primer and 0.5 ml Taq polymerase. Use the following amplification scheme: 1 cycle of 94°C for 2 min; 35 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 3 min; 1 cycle of 94°C for 1 min, 55°C for 1 min, 72°C for 10 min. 3. Check 5 ul of the amplified samples on a 1% agarose gel; the product should be a smear ranging between 200 and 500 bp. Obvious bands in the smear may be a result of contamination. 4. Purify the amplified DNA by phenol-chloroform extraction and ethanol precipitation. Remove linker DNA sequences by digesting with 10 U HinDIII. Purify the amplification products from linker DNA using Qiagen PCR purification columns, according to the manufacturer's conditions. The expected yield from the PCR reaction is approximately 5 ug, and amplification of DNA from the mock IP should be as efficient as that of the antibody IP.

5. Analysing the enrichment of putative target sequences in the PCR-amplified DNA 5.1 Slot-blot analysis of the enrichment of putative PC target sequences When putative target sequences of the chromatin protein under study are known, it is possible to test whether they are enriched in the complex DNA sample (obtained by IP from the cross-linked chromatin) using slot-blot analysis: 100 ng of mock IP and antibody IP DNAs are immobilized on the membrane and hybridized with a radioactive probe from the DNA fragment to be tested (Protocol 5A). Relative enrichments are calculated as a ratio between antibody and mock IPs. Figure 2A shows the relative enrichment for a known PC target site, a 6.0 kb EcoRI fragment from the bithorax complex (BX-C), named MCP (10,11). A strong enrichment of this sequence in the PC IP compared to the mock IP was seen in two independent Drosophila lines, as previously reported for Drosophila cultured SL-2 cells (4,6). Only specific PC target sites are enriched by this procedure (Figure 25). Heat-shock sequences, which are known not to be targets of PC-binding, are not enriched. It should be noted that, although this method is very valuable for quantitative measurements of relative enrichments, it is inconvenient for scanning binding to extended chromosomal domains.

5.2 Mapping DNA target sites for Polycomb and GAGA factor in the Drosophila bithorax complex In order to scan binding to a large genomic region, or to compare relative binding affinity to neighbouring DNA fragments from regions with a size of several kb, the PCR-amplified DNA can be used as a complex radioactive 31

B Figure 2. The relative enrichment of the MCP sequence by PC immunoprecipitation in wild-type and transgenic fly lines. Cross-linked Chromatin from two Drosophita lines was analysed: wild type, OregonR flies (wt) and the 5F24 25,2 transgentc line, which contains a P element-derived transgene inserted in the X chromosome (11), The enrichment factor of PC IP versus Mock IP Chromatin, calculated by Phosphorimager quantitation of the hybridization signals, is reported to the right of each panel. (A): Slot-blot enrichment of the 6.0 kb Ecor MCP element from the BX-C was assayed by hybridization to 100 ng of crosslinked, PCR-amplified DNA obtained from mock or PC IP. A substantial enrichment factor is observed in both lines. (B) The same DNAs were hybridized to a 10 kb FcoRI DMA fragment from the heat-shock 87 °C genomic locus (4). In this case, no significant enrichment is detected. Figure 3. Hybridization of control, PC, and GAGA immune precipitated DNA to putative target sites in the BX-C. From the previously published analysis of PC and GAGA factor binding to the BX-C, three fragments sutacloned into Bfuescript KS+ (6) were analysed in order to characterize the binding of PC and GAGA at higher resolution. The three fragments are (i) a 3384 bp EcoRI fragment, from coordinate 218 241 to 221 625 of the published BX-C sequence (15). This fragment contains 3 PC and GAGA factor target site, named the BXD PRE (12). (ii) a 7652 bp FcoRI fragment (coordinates 123 772-131 424), a PC and GAGA target site named Peak C (6). (iii) a 5989 bp EcoRI fragment (coordinates 109 688-115 677) containing the MCP element, a known PC target (6, 10). (A-C): DNAs were digested, run on a 1% agarose gel, blotted and hybridized to PCR-amplified mock IP (panels A, B, C, left), GAGA IP (panel A, right), or PC IP (panels B and C, right). The size of the DNA fragments (in bp) most strongly bound by PC and GAGA factor is shown on the right side of each panel. Panels A and B, lanes 1 and 4; BXD PRE digested by Kpnl (K) and Ps (P). Panels A and B, lanes 2 and 5: Peak C digested by Pstl, BamHI (B), and Xhol (Xh), Panels A and B, lanes 3 and 6: MCP digested by Acc\ (A) and Pstl. In order to analyse PC binding to the two largest MCP fragments of 2208 bp and 1556 bp, the 2208 bp fragment was gel-elmed and digested with Rsall (R) and Xmnl (X) (C, lanes 8, 10), while the gel-eluted 1556 bp fragment was digested by Pvull (Pv) (C, lanes 7, 9). (D-F): Calculated relative enrichments for each DNA fragment. For BXD PRE and Peak C (D, F, respectively!, enrichments for PC and GAGA factor are shown, whilst only PC enrichments are shown for MCP (E) since no GAGA factor binding could be detected to any fragments of the MCP region (panel A, compare lane 3 with 6). The plots show the DNA fragments of each DNA element to scale in the proximal-distal orientation along the BX-C.

32

2: Mapping DNA target sites of chromatin-assaciated proteins probe to hybridize a membrane obtained by Southern blotting an agarose gel containing a restriction digest of DNA from the corresponding genomic region. When large regions are analysed, genomic walks of severa) lambda phage or P1 clones are digested and loaded side by side on the gel. The restriction digests can be set to obtain DNA fragments as small as 500 bp, the maximal resolution of the methodology. Figure 3 shows an analysis of PC- and

33

Giacomo Cavalli, Valeria Orlando, and Renato Paro GAGA factor-binding to three regions from the BX-C. The first is the 6.0 kb MCP fragment mentioned previously. The second is another PC target, a 3.4 kb EcoRl fragment containing the so-called BXD Polycomb response element (PRE) (12). The third sequence is a 7.6 kb fragment, named Peak C, which was identified during the analysis of the whole 340 kb BX-C in SL-2 cultured cells as a specific target for both PC and GAGA binding (6). Specific enrichments were observed for the binding of both proteins to the same DNA subfragments in Drosophila embryos as previously described for SL-2 cells. These enrichments can be plotted to display relative binding affinities to neighbouring regions better (see Figure 3D to F). Importantly, the binding profile of the two proteins is qualitatively different. PC, a chromatin-associated protein which associates to large regions of its target genes, shows rather broad peaks. On the other hand GAGA factor, a DNA binding protein which recognizes GA repeats on the DNA both in vitro and in vivo, binds only to DNA fragments containing its target sites. Therefore, the methodology presented here is suited to mapping the binding of both broadly distributed proteins as well as sequence-specific binding proteins.

Protocol 5. Analysis of the enrichment of specific DNA sequences in PCR-amplified DNA Equipment and reagents • Standard materials for agarose gel electro• Standard materials for random primed DNA phoresis and Southern blotting onto posilabelling with (a-32P)dATP (Specific activity tively charged nylon membrane (e.g. Gene10 mCi/ml 3000 Ci/mmol, Amersham) screen Plus, NEN) . Hybridization buffer: 0.5 M NaHP04 pH 7.2, •Slot-blot apparatus minifoldll (Schleicher 7%SDS, 1 mM EDTA pH 8.0, 1% BSA (a 1 M and Schuell, SRC 072/0) NaHP04 pH 7.2 stock is 0.5 M Na2HP04 con. Phosphorimager apparatus taining 4 ml orthophosphoric acid per litre) . Denaturation buffer: 0.5 M NaOH, 1 M NaCI • Wash buffer 1: 40 mM NaHPO4 pH 7.2, 5% . Dilution buffer: 0.1 x SSC solution, 0.125 M SDS, 1 mM EDTA PH8.0.0.5%BSA NaOH • Wash buffer 2: 40 mM NaHPO4 pH 7.2, 1% SDS 1 mM EDTA . Neutralization buffer: 0.5 M Tris-HCI PH 7.5, ' PH8.0,0.5%BSA 0.5 M NaCI

A. Analysis by slot-blot of enrichment of specific DNA fragments 1. Denature 100 ng DNA of the mock IP and antibody IP samples made up to 15 ml with dH2O by adding 15 ml of the denaturation buffer and incubating for 10 min at room temperature. 2. Add 270 ml of dilution buffer and transfer on ice. Load samples into the slots of the minifold apparatus containing the hybridization membrane and let the liquid flow through the membrane for 30 min. Connect the minifold to a vacuum pump until no liquid is left in the slot. Uncast the slot-blot and neutralize the membrane for 1-2 min in neutralization buffer. Fix the DNA to the membrane by baking at 80°C for 1 h.

34

2: Mapping DNA target sites of chromatin-associated proteins 3. Prehybridize for 2-4 h at 65°C in hybridization buffer (hybridization is carried out essentially as described in ref. 13). 4. Label 25 ng of the specific DNA fragment to be tested for specific enrichment by random priming with 50 ml (a-32P)dATP. 5. Denature the probe by boiling it for 5 min. Cool in ice, then add to 5-10 ml hybridization buffer and incubate overnight at 65°C. 6. Wash the filter twice for 10 min at 65°C in wash solution A, and 3-4 times for 5 min each at 65°C in wash solution B. Expose the filter overnight to a Phosphorimager screen. Quantify the signals of the mock- and the antibody-IP samples to calculate the specific enrichment due to IP of the cross-linked chromatin for the sequence used as probe. 7. After exposure, strip the probe from the membrane according to manufacturer's instructions, and reprobe with 25 ng of labelled Drosophila genomic DNA. This gives a precise estimate of the amount of DNA loaded onto the membrane, to use as a correction factor for standardizing the calculated specific enrichments. B. Searching target DNA binding sites by analysis of the Southern hybridization profile to large genomic walks or subcloned DNA fragments 1. Prepare a Southern filter of a genomic walk which is a potential target of the immunoprecipitated protein, or with the subcloned putative target DNA fragments digested by appropriate restriction enzymes. Prehybridize for 2-4 h at 65°C in hybridization buffer. 2. Random prime label 50–100 ng of amplified DNA with (a-32P)dATP. 3. Hybridize as described above (see part A, steps 3-6), and expose the filter (overnight or several days) to a Phosphorimager screen. 4. Quantify the signal intensity of each band using the software package of the Phosphorimager, and calculate the enrichment of each fragment taking into account the following points: (a) Intensity is proportional to Mr and the resulting values should be normalized with respect to M, if the relative enrichments of different DNA fragments are being compared. The amount of signal per kb of DNA in each fragment may be calculated and plotted against a map of the genomic region (as shown for PC and GAGA factor in Figures 3D to F). (b) The mock IP probe should hybridize approximately uniformly to all fragments (dependent on Mr). As the method is only semiquantitative some sequence-specific differences in amplification may occur, but amplification of different fragments generally varies by no more than 50% from the mean. This degree of error

35

Giacomo Cavalli, Valerio Orlando, and Renato Paro Protocol 5. Continued must therefore be assumed for all experiments. If only a few random restriction fragments of a genomic walk hybridize to the mock IP probe, it is likely that too little input DNA was added to the ligation reaction, or that ligation occurred at low efficiency. (c) Repetitive elements are always strongly enriched in immunoprecipitations and therefore hybridize strongly to all IP DNA probes. These elements can be identified by their strong hybridization to genomic DNA. (d) When analysing large genomic walks, antibody IP probes will hybridize to all fragments of a genomic region to some extent (although enriched fragments hybridize much more strongly). Slot-blot analysis may be used to determine the background level accurately. Typically 100-200 ng of DNA from mock and antibody IPs are immobilized on nylon membrane by slot-blot and hybridized to a number of probes derived from the target DNA of interest. The resulting signals are quantitated and the actual enrichment accurately determined. Comparison between a number of fragments allows the setting of a 'background' level, and only hybridization signals above this level are considered to be enriched.

6. Concluding remarks The formaldehyde cross-linking method presented here allows in-vivo mapping of the DNA target sites of chromatin-associated proteins and site-specific DNA binding proteins in Drosophila embryos. This has several applications in the molecular dissection of the regulation of gene expression for developmentally relevant genes. For example, whenever the potential target sites of a given regulatory factor are known, it can be determined whether or not they are actually bound; in addition, the developmental timing of DNA binding can also be analysed. Moreover, competition between regulatory proteins within the nucleus can be analysed, either at natural target sites or in transgenic constructs. In several regulatory sequences of developmental genes, it has been found that DNA binding sites for regulatory transcription factors are overlapping. With cross-linking analysis, it might be possible to investigate whether binding of a given regulator physically displaces a second from the DNA. Many chromatin-associated proteins show no site-specific DNA binding in vitro. In this case, formaldehyde cross-linking appears to be the method of choice for the analysis of binding to the regulatory regions of potential target genes. Moreover, this method is also applicable to the analysis of histone 36

2: Mapping DNA target sites of chromatin-associated proteins modifications in the chromatin template in vivo. Formaldehyde cross-linking has already been successfully applied in the study of changes in histone acetylation in the mating type loci and telomeres of yeast chromatin (14). Additionally other chromosomal processes, e.g. DNA replication, recombination, and repair, can now be subjected to an in-vivo analysis of participating components.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

McGhee, J.D. and von Hippel, P.H. (1975). Biochemistry, 14, 1281. McGhee, J.D. and von Hippel, P.H. (1975). Biochemistry, 14,1297. Solomon, M.J. and Varshavsky, A. (1985). Proc. Natl Acad. Sci. USA, 82, 6470. Orlando, V. and Paro, R. (1993). Cell, 75,1187. Hecht, A., Strahl-Bolsinger, S., and Grunstein, M. (1996). Nature, 383, 92. Strutt, H, Cavalli, G., and Paro, R. (1997). EMBO J., 16, 3621. Paro, R. (1995). Trends Genet., 11, 295. Farkas, G., et al. (1994). Nature, 371, 806. Orlando, V., Strutt, H., and Paro, R. (1997). Methods. A companion to methods in enzymology, Vol. 11 (ed. K. Zaret), pp. 205-14. Academic Press, New York. Busturia, A. and Bienz, M. (1993). EMBO J., 12, 1415. Zink, D. and Paro, R. (1995). EMBO J., 14, 5660. Chan, C.S., Rastelli, L., and Pirrotta, V. (1994). EMBO J., 13, 2553. Church, G.M. and Gilbert, W. (1984). Proc. Natl Acad. Sci. USA, 81,1991. Braunstein, M., et al. (1993). Genes Dev., 7, 592. Martin, C.H., et al. (1995). Proc. Natl Acad. Sci. USA, 92, 8398.

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3

Fission yeast chromosome analysis: fluorescence in situ hybridization (FISH) and chromatin immunoprecipitation (CHIP) KARL EKWALL and JANET F. PARTRIDGE

1. Introduction The fission yeast (Schizosaccharomyces pombe/S. pombe) is a unicellular ascomycete, that is a well-studied model organism for cell biology and molecular genetics. Some of the main advantages of using fission yeast as a model eukaryote are: • well-defined classical and molecular genetics; • distinct growth properties for cell-cycle analysis; • approximately 50% of the 15 Mb genome has been sequenced; (http://www.sanger.ac.uk/); • immunofluorescence- and electron-microscopy techniques are well developed. Here we describe two recently developed methods for chromosome analysis in fission yeast: fluorescence in situ hybridization (FISH) and chromatin immunoprecipitation (CHIP). FISH allows the detection of any chromosomal DNA sequences (>30 kb) in the nuclei of fixed yeast cells, using fluorescence microscopy after hybridization to DNA probes labelled either directly or indirectly with fluorochromes. In addition, FISH in combination with immunofluorescence, can be used to demonstrate the co-localization of specific proteins to their associated DNA sequences during all stages of the cell cycle (for example co-localization of proteins to centromere DNA). CHIP makes it possible to isolate and precisely map the DNA sequences associated with chromosomal proteins (see Chapter 2). This is achieved by the chemical cross-linking (fixation) of cells and the preparation of soluble chromatin extracts from which specific chromatin fragments are then precipitated using

Karl Ekwall and Janet F. Partridge antibodies to chromatin proteins. Both FISH and CHIP have already contributed significantly to our understanding of chromosomal proteins in many areas of biology. It is our belief that these two methods will be of increasing importance for studies of fission yeast chromosomal proteins and their associated DNA sequences, augmenting and complementing conventional biochemical and genetic approaches. Here we describe detailed protocols for FISH and CHIP in fission yeast, with the emphasis on problems that are peculiar to studies in yeast and that are not encountered in other eukaryotes.

2. Fluorescence in situ hybridization (FISH) analysis of fission yeast The FISH method for fission yeast described in this chapter was originally developed by Uzawa and Yanagida (1). The same laboratory has also reported some modifications to the original protocol (2). Fission yeast FISH involves five steps: (a) (b) (c) (d) (e)

preparation of probes; fixation of cells; denaturation of cellular DNA by alkali treatment; in situ hybridization of the probe to cellular DNA; and fluorescence microscopy.

The main improvement to the FISH protocol described in this chapter and outlined below is the optimization of probe preparation (3). The cell fixation conditions used in the FISH protocol are based on the aldehyde fixation method described by Hagan and Hyams (4). Recently two-colour FISH has been reported (5), enabling the simultaneous detection and co-localization of different DNA sequences, for example centromeres and telomeres, by direct labelling probe DNAs with FITC-dUTP (green) and Cy3-dUTP (red), respectively. The protocols developed for FISH analysis of budding yeast (S. cerevisiae) are quite different from the procedures outlined below for fission yeast. For guidelines on the budding yeast procedure see Guacci et al. (6).

2.1 Preparation of probes Two parameters have to be considered: • the size of the target sequence; and • the length of the probe fragments. As with all FISH experiments the larger the target sequence, the stronger the resulting hybridization signal. In practice this means that, for example, detection of ribosomal DNA is relatively easy, because the target is large and 40

3: Fission yeast chromosome analysis repetitive. The smallest low copy number targets for FISH reported in fission yeast are the mating type loci, probed using three plasmids with a combined target size of ~20 kb (7). Probe cos212 utilized in this chapter contains a 30-kb insert of telomere adjacent sequence and detects telomere regions of chromosome / and // (2). 2.1.1 Probe labelling It is very important to use hybridization probes of denned fragment length. Probe fragments that are too large are unable to penetrate yeast cells and stick to the cell wall, causing high background. Probe fragments that are too short tend to hybridize inefficiently (Figure 1). At least three methods of probe labelling for FISH in fission yeast have been reported: random priming (1); nick translation (3); and direct labelling using terminal deoxynucleotidyl transferase (5). Here we describe the method for nick translation (Protocol 1), as the use of calibrated amounts of DNase I gives control over the length of probe fragments produced. The amount of DNase I has to be titrated and the resulting probe fragment lengths checked (Protocol 2, Figure 1A). Once calibrated, the same batch of DNase I can be used for years (enzyme stock is stable in glycerol at -20 °C). Protocol 1. Labelling of cosmidorplasmid DNA probes by nick translation Equipment and reagents • Cosmid or plasmid DNA of good quality (e.g. supercoiled CsCI preparation) . Water-bath at 15°C .• 0.5 DTT, 500 mg/ml BSA, 100 mM MgS04, 500 alkali stable' (DIGdUTP) (Boehringer, cat. no. 1093088) . dNTPs •7'5

• 10U/mlDNase I (Gibco BRL) . •10 u/ml DNA polymerase 1 (Gibco BRL) MEDTA pH 8.0 • Sephadex 17004201)

G-50 (Pharmacia, cat.

no.

MNH Ac

'

Method 1. To 1.0 M-g DNA add 4 ml each of: 10 XNTS buffer, 2 mM dATP, 2 mM dCTP, 2 mM dGTP, and 1 mM DIG-dUTP; 2 ml of 0.5 mM dTTP, 1 ml (10 Units) DNA polymerase 1 and 1 ml DNase I freshly diluted,b dependent on the batch from 1:15 to 1:500 in sterile dH20. Bring the volume up to 40 ml with dH2O. 2. Incubate for 90 min at 15°C. Stop the reaction by adding 1.2 ml 0.5 M EDTA and place on ice. 3. Purify the labelled DNA by either step (a) or (b): (a) Purify the labelled DNA by filtering, using a G-50 Sephadex spin column as described in ref. 8. 41

Karl Ekwall and Janet F. Partridge Protocol 1. Continued (i)

Plug an empty 1 ml syringe barrel with glass-wool. Fill with Sephadex G-50 swollen in TE buffer pH 8.0 and pack the column to 0.9 ml by centrifuging at 3000 fir for 5 min at room temp. (ii) Add 40 ml of TE buffer pH 8.0 and check that the same volume is recovered by centrifuging at 3000 g for 5 min at room temp. (iii) Add 40 ml of the nick translation reaction from step 2 to the column and elute the purified probe by centrifuging at 3000 g for 5 min at room temp. (b) Purify the labelled fragments by removing proteins by precipitation (step (i)) and then precipitate DNA from free dNTPs (steps (ii) and (iii)). (i)

Add 20 ml 7.5 M NH4Ac to the nick translation reaction, and centrifuge for 5 min at 15000 g at room temp, (ii) Transfer the supernatant to afresh tube. Add 150 ml EtOH, mix and centrifuge for 15 min at 15000 g at room temp. Wash the DNA pellet with 150 ml 80% EtOH. (iii) Centrifuge for 5 min as in (ii). Dry the pellet for 10 min under vacuum then resuspend the probe in 40 ml TE buffer 8.0. 'It is important to use alkali-stable rather than labile DIG-UTP, because NaOH is used during the denaturation step (Protocol 4). b See Section 2.1.1 and Figure 1A for calibration of DNase 1. We recommend a titration procedure for each batch.

2.1.2 Assessing probe fragment length Probe fragments are only able to diffuse through the permeabilized yeast cell wall if they are of sufficiently small size. However, if probe fragments are too short they will not hybridize efficiently to the target sequence. The desired mean probe fragment size is 0.2 kb (range between 0.1 and 0.5 kb) (see Figure 1). The nick translation reaction described in Protocol 1 uses the single-strand nicking activity of DNase I in combination with the polymerization activity of DNA polymerase 1 to incorporate DIG-dUTP into double-stranded DNA. The length of the resulting fragments is measured using a strand-separating gel system such as alkaline agarose gel electrophoresis (8). The efficiency of DIG incorporation into probe fragments can be assessed by Southern blotting of the alkaline gel, followed by detection of the labelled fragments using an anti-DIG alkaline phosphatase assay (Protocol 2). Protocol 2. Checking probe fragment length and efficiency of DIG labelling This protocol is based on ref. 9 and the manufacturer's instructions for the Vectastain kit (Vector)

42

3: Fission yeast chromosome analysis Equipment and reagents . DIG-labelled molecular weight marker VI (Boehringer, cat. no. 1 218 611) . 5 M NaOH . 0.5 M EDTA (pH 8.0) . 6 x alkaline loading buffer (ALB): 300 mM NaOH, 6 mM EDTA, 18% Ficoll (w/v), 0.15% Bromocresol green (wM) » Alkaline gel running buffer: 30 mM NaOH, 1 mM EDTA pH 8.0 • • Midigel (for example Pharmacia GNA 100 apparatus, 10 cm)

. 1MTris-HCI pH 7.6, 1.5 M NaCI . Buffer 1: 0.1 M Tris-HCI pH 7.5, 1.5 M NaCI . 20 X SSC buffer: 0.3 M sodium citrate pH 7.0, 3 M NaCI • BSA fraction V .• 0.1 M Tris-HCI pH 9.5 . Anti-DIG-alkaline phosphatase (anti-DIGAP, Boehringer) > Vectastain kit IV BCIP/NBT (Vector SK5400) or equivalent

Method 1. Make up fresh gel running buffer and place in a cold room. 2. Melt 1% agarose in H2O and cool to 60 °C. 3. To the molten agarose add 5 M NaOH to 50 mM and 0.5 M EDTA (pH8.0) to1 mM. 4. Pour a midigel (for example Pharmacia GNA 100 apparatus 10 cm) and place in the cold room. 5. To 10–15 ml probe sample, add 0.5 M EDTA (pH 8.0) to 10 mM and 0.2 volumes of 6 x ALB. 6. Load the gel dry and layer agarose on top of the wells. 7. Electrophorese in alkaline gel running buffer in the cold room at 75 V for 3-4 h (should give Vectashield mounting medium (Vector H1000)

• DAPI • Polylysine (Sigma, cat. no. P1274) • No.1 22 X 22 mm coverslips (Chance Propper Ltd. )

Method 1. Resuspend fixed cells from Protocol 3 in 0.5 ml 1 x PEMBAL buffer with 0.2 mg/ml RNase A, and incubate for 2 h at 37°C. 2. Boil the DIG-labelled probe (Protocol 1) for 5 min and then add 50–100 ng (2-4 ml) to 100 ml of the Hybridization mix prewarmed to 65°C. Incubate at 65°C for 15 min. 3. Spin down the cells (see footnote a in Protocol 3) and add 100ml,l0.1 M NaOH to each cell pellet containing 0.5-1 x 107 cells, resuspend and wait for 2 min (including the spin time), before removing the NaOH by centrifugation and adding 100 ml of the probe solution. Resuspend the cells by pipetting and incubate at 37°C overnight. 4. Next day, wash out the non-specifically bound probe by adding 200 ml 2 X SSC + 0.1% sodium azide. Spin down the cells as in Protocol 3. 5. Wash the cells three times for 30 min each time at 37°C with 2 x SSC + 0.1% sodium azide. 6. Pellet the washed cells, resuspend in 100 ml PBS-BAG, and incubate for 30 min at room temperature. 7. Pellet the cells and resuspend in 150 ml PBS-BAG containing a 1/100 dilution of FITC-conjugated anti-DIG antibodies (raised in sheep). 8. Incubate, covered in aluminium foil to exclude light, for at least 12 h on a rotary wheel at room temperature. 9. Pellet the cells and wash out secondary antibodies once with 150 ml PBS-BAG and twice with PBS + 0.1% sodium azide. 10. Resuspend the cells in 50 ml of PBS + 0.1% sodium azide with DAPI (0.5 (mg/ml). 11. Mount 5 ml of the cell suspension on polylysine-coated clean coverslips3 and allow to dry in the dark. 12. Mount the coverslip onto a slide using one drop of Vectashield, gently press using Kleenex tissue to remove excess Vectashield. a Coverslips are usually greasy and need to be washed extensively using hot water and detergent, then rinsed several times with water and finally with acetone. Coating with polylysine is carried out by spreading 1 mg/ml solution (w/v) on the clean coverslips, wiping off excess with filter paper, and allowing to dry.

47

Karl Ekwall and Janet F. Partridge

3. Chromatin immunoprecipitation from fission yeast The technique of chromatin immunoprecipitation (CHIP), as used to study histone occupation of transcribed promoters (10), and localization of regulatory chromatin factors in Drosophila (11,12 and Chapter 2) and S. cerevisiae (13, 14), has recently been modified to analyse chromatin components in fission yeast (15,16). The technique relies upon the rapid fixation of large multimeric complexes within whole cells, prior to chromatin extraction and analysis of recognition sites for particular proteins by immunoprecipitation (IP). We describe methods developed for the analysis of histone isoform distribution, and for the isolation of DNA sequences associated with the Swi6 chromodomain protein (Figure 2). Formaldehyde Fix cells

i i Sonicate chromatin

Prepare chromatin extracts

to 0.5 - 1 kb

.

Immunoprecipitate chromatin protein Wash immunoprecipitates Recover DNA Quantitate DNA by competitive PCR

Figure 2. Schematic diagram of chromatin immunoprecipitation procedure. Cells are fixed (Protocol 5) and soluble chromatin extracts, with chromatin sheared to a few nucleosomes in size, are prepared (Protocol &. Protein-DNA complexes of interest are immunoprecipitated by adding antibodies and Sepharose beads (Protocol 7). DNA is recovered by reversing the cross-linking (Protocol 8), and analysed by PCR (Section 3.4).

48

3: Fission yeast chromosome analysis The CHIP protocol consists of four steps: (a) fixation of cells to maintain localization of proteins of interest; (b) preparation of chromatin extract, with DNA of 500–1000 bp target length; (c) IP of chromatin extract, to specifically isolate target sequences; and (d) evaluation of the IP by PCR or slot-blot analysis.

3.1 Fixation of yeast cells to maintain protein localization For histones and other abundant components of chromatin, a good starting point for fixation is to use the conditions outlined in Protocol 5A. For less abundant antigens, base your trial experiments on the conditions you have determined for immunofluorescence microscopy. We rapidly screened a wide range of pFA fixation parameters using fluorescence microscopy of cells bearing a functional green fluorescent protein (GFP) Swi6 fusion, which is expressed at levels similar to the endogenous Swi6p (A.L. Pidoux unpublished observation). Our assay was the detection of bright punctate spots of fluorescence in the nucleus. Conditions tested were: fixing cells in the absence/presence of 1.2 M sorbitol; varying pFA concentration from 0.5 to 4%; and using either a 15- or 30-min fixation time. In parallel, we performed a more limited series of experiments on wild-type yeast, performing standard immunocytochemistry to visualize endogenous Swi6p staining. These parameters must be evaluated in conjunction with the preparation of a chromatin extract (Section 3.2). For instance, we found that 3% pFA fixation for 15 min in the presence of sorbitol generated chromatin that was too highly cross-linked to be efficiently extracted, and resulted in no detectable signal for our target DNA in the crude lysate, even before IP. In the absence of sorbitol, the fixation time could be increased to 30 min with 3% pFA to yield good cytology and extractable chromatin. Protocol 5.

Fixation of cells for CHIP

Equipment and reagents • Shaking incubator(s) at 32°C and 18°C, 65°C water-bath in fume hood . 2 ml screw-top Sarstedt tubes for the mini beadbeater (ref. no. 72693005) • 2.5 M glycine • 50 ml centrifuge tubes

• Paraformaldehyde (pFA) : Fisons AR 37% stock (F/1500/PB17); or for part B, paraformaldehyde powder (Sigma P6148) . Tris-buffered saline (TBS): 8 g NaCI, 0.2 g KCI, 3 g Tris base in 1 litre, pH to 7.4 with HCI

A. For histones and other abundant chromatin proteins 1. Grow 20 ml of yeast culture overnight to 5 X 106/ml. 2. Add pFA to 1%, from a 37% stock (Fisons).

49

Karl Ekwall and Janet F. Partridge Protocol 5.

Continued

3. Fix cells for 30 min at room temperature with shaking. 4. Stop the fixation by adding 2.5 M glycine to 0.125 M, with shaking for 5 min at room temperature. 5. Transfer the cells to a 50 ml centrifuge tube, and spin at 3000 g for 5 min at 4°C, dispose of the supernatant (containing pFA) in a fumehood sink. 6. Gently resuspend the pelleted cells in 20 ml ice-cold TBS. 7. Repeat step 5. 8. Resuspend the cells in 1 ml TBS, and transfer to a 2 ml screw-cap Sarstedt tube. Microcentrifuge the tube at 15000 g for 30 sec and remove the supernatant. Cells can be stored on ice at this point for up to2h. B. For less abundant chromatin proteins 1. Grow 50 ml of yeast overnight to 5 x 106 /ml.alf sorbitol is to be used, add 50 ml of 2.4 M sorbitol in growth medium to the culture and incubate for 5 min prior to fixation. 2. Prepare 50% pFA (w/v) in growth media. To dissolve the pFA, add 5 M NaOH to 0.25 M, and heat at 65°C. Allow the pFA to cool to room temperature, and add to the yeast culture while swirling to the desired percentage. 3. Fix the cells for 15-30 min at room temperature. 4. Follow Steps 4 to 8, part A. * If antigen localization is improved at low temperature, incubate the culture at 18°C for 2 h prior to fixation.

3.2 Preparation of chromatin extract Efficient cell lysis can be achieved by bead-beating cells in the presence of glass beads. As heat is generated, this lysis should be performed in pulses at 4°C. The chromatin is then sheared by sonication to generate fragments of a few nucleosomes in length (~600 bp). It is important to determine the parameters required for good fixation in parallel with the conditions for the generation of sheared chromatin. Material that is overfixed can not be efficiently sheared, and will be pelleted during the clarification steps at the end of Protocol 6. Time courses of sonication should be performed, and the size of the recovered DNA (Protocol 8) evaluated by agarose gel electrophoresis. 50

3: Fission yeast chromosome analysis Protocol 6.

Preparation of chromatin extract

Equipment and reagents • Mini beadbeater (Biospec) • Lysis buffer: 50 mM Hapes-KOH pH 7.5, 140 . Soniprep 150 MSE sonicator or equivalent MM NaCI, 1 mM EDTA, 1% (v/v) Triton Xthat can be used to sonicate material in a 100. 0-1% (w/v) sodium deoxycholate 1.5 ml tube • Protease inhibitors: 'complete' protease . Acid-washed glass beads (425-600 inhibitor cocktail (Boehringer, cat. no. microns; Sigma, cat. no. G8772) 16997498), 1 mM PMSF (Sigma, cat. no. . 15 ml round-bottomed Falcon tubes (cat. P7626) no. 2006) • 19-gauge needle • 1.5 ml and 15 ml centrifuge tubes

Method 1. Resuspend the fixed cell pellet from Protocol 5 in 400 ml ice-cold lysis buffer with protease inhibitors added. 2. Add approximately 400 ml of cold glass beads to the resuspended cells. 3. Lyse the cells with 4 x 40-sec pulses on the bead beater at 4°C, set at maximum power. 4. Check for >95% cell lysis under a microscope. 5. Cut the lid off a 1.5 ml centrifuge tube, and place it inside a 15ml centrifuge tube on ice. 6. Puncture the base of the 2 ml screw-cap Sarstedt tube with a 19gauge needle, and place it on top of the 1.5 ml tube. 7. Centrifuge the 15 ml tube containing both tubes at 3000 g for 5 min, at 4°C, to spin the cell lysate, but not the glass beads, into the rnicrocentrifuge tube. 8. Place the lysate on ice, and resuspend it gently with a 1 ml Gilson pipette blue tip. 9. Sonicate the lysate on ice to shear the chromatin to —600 bp, e.g. 1 x 10-sec pulse at 22 m amplitude.9 10. Spin out the cell debris at 15000 g. 4°C, for 5 min. 11. Transfer the supernatant to a fresh tube, and spin at 15000 g for a further 15 min, at 4°C. 12. Transfer the supernatant (crude lysate) to a fresh 1.5 ml tube on ice. "The sonication time required will vary dependent on the fixation conditions, and the sonicator used.

3.3 Immunoprecipitation of chromatin The parameters used for IP depend to a large extent on the abundance of the antigen. For histones, follow the procedure outlined in Protocol 7A, and for less abundant antigens, Protocol 7B. The crude lysate generated in Protocol 6 51

Karl Ekwall and Janet F. Partridge will suffice for six reactions for abundant antigens, and for one IP of a nonabundant chromatin protein. In general, the cleanest results will be generated by using the most specific reagents possible. For IP of centromeric heterochromatin to which Swi6p is bound, we used affinity-purified polyclonal antibodies (Ab) either crosslinked to protein A-Sepharose (Protocol 7), or we added protein ASepharose to cell lysates after incubating with Ab to collect the antigen-bound chromatin. We also found that it was necessary to preclear extracts with protein A-Sepharose beads before adding antibodies, to reduce non-specific interactions with the beads (Protocol 7B). In addition, high backgrounds resulted from overnight incubations, and these were reduced by shortening the incubation times of Ab with lysate to a maximum of 3 h (Protocol 7B). For histone antibodies, crude sera coupled to protein A-Sepharose gave good results, preclearing was not necessary, and the time of IP was not critical (Protocol 7A and ref. 16). Protein A-Sepharose can be used to bind polyclonal Abs and monoclonal mouse Abs of classes IgG2a, IgG2p, or IgG3. For epitopes for which there are only mouse monoclonal antibodies (mAb) of class IgG1 available, protein G-Sepharose should be substituted. Before use, protein A-Sepharose beads should be swollen in TBS and thoroughly washed twice by resuspending them in 10 volumes of TBS, and centrifuging at 3000 g for 5 min. The beads should finally be resuspended to 50% (v/v) in TBS, sodium azide added to 0.01%, and stored at 4°C. Protein G-Sepharose is sold as a slurry in ethanol; again these beads need to be thoroughly washed and resuspended to 50% (v/v) in TBS before use. Note that there is some batch-to-batch variation in the efficiency of coupling of protein A in the protein A-Sepharose available from suppliers. It is well worth spending the time to properly wash away residual non-covalently coupled protein A from the beads before use, as the efficiency of cross-linking or immunoprecipitating Ab onto beads will be greatly reduced by competition with free protein A. Protocol 7 gives details on CHIP for antibodies which can be bound by protein A. For mouse IgG1, protein A-Sepharose should be substituted by protein G-Sepharose. Protocol 7. Immunoprecipitation of yeast chromatin Equipment and reagents • Rocking platform at 4°C • Antibody-conjugated Sepharose beads (see Protocol 9) or protein A-Sepharose (Sigma, cat. no. P3391)

• Refrigerated microcentrifuge • Duck-bill pipette tips (Sorenson Bioscience Ltd, cat. no. 13760)

A. Immunoprecipitation of abundant chromatin antigens 1. Store 50 ml of the crude lysate from Protocol 6 as the control non-IP chromatin.

52

3: Fission yeast chromosome analysis 2. Add 25 ml of antibody-conjugated Sepharose beads (50% v/v, Protocol 5) to 50 ml of the crude lysate. 3. Incubate the bead/lysate mixture with rocking at 4°C for at least 2 h to overnight. B. Immunoprecipitation of non-abundant chromatin antigens 1. Preclear the crude lysate from Protocol 6, by adding 60 (ml 50% (v/v) protein A-Sepharose beads to 400 ml of the crude lysate. Incubate the bead/lysate mixture with rocking for 1 h at 4°C. 2. Centrifuge at 15000 g for 5 min at 4°C. Carefully remove the supernatant to a clean tube using a duck-bill tip, taking care to leave the beads behind. Save 10% of this material at -20°C as the control nonimmunoprecipitated chromatin sample. 3. To the remainder of the sample, add 60 ml of antibody-conjugated beads (Protocol 9), or antibody.8 4. Bind the antibody-conjugated beads with the lysate for 3 hb with gentle rocking at 4°C. 'Antibody-conjugated beads can be replaced at this stage by the addition of Ab alone. We use 1010mlof affinity-purified polyclonal sera/350 ml precleared lysate; this sera is used at 1/600 for Westerns, and at 1/30 for immunofluorescence. b Antibody incubation with lysate is performed for 1 h prior to the addition of 60 ml (50% (v/v)) protein A-Sepharose beads, and a further 2 h incubation at 4°C.

Protocol 8 gives methods for washing the IPs and reversal of cross-linking for the recovery of DNA. See Protocol 9 for methods for the covalent linking of antibodies to Sepharose beads.

Protocol 8. Washing immunoprecipitations, and reversal of cross-linking Equipment and reagents • • • •

Duck-bill tips {Protocol 7) . Wash buffer: 10 mM Tris-HCI pH 8, 0.25 M LiCI, 0.5% NP–40, 0.5% sodium deoxyRotating wheel cholate, 1 mM EDTA 65 °C water-bath . • TE buffer: 10 mM Tris-HCI, 1 mM EDTA pH 2 ml centrifuge tubes (e.g. Anachem, cat. 8.0 no. HT-20-NG/1000) » 10 mg/ml proteinase K (Boehringer • Lysis buffer (see Protocol & Mannheim, cat. no. 1000144) « Lysis buffer, 500 mM NaCI: replace 140 mM • Phenol:chloroform 1:1 v/v NaCI in lysis buffer (Protocol 6) with 500 . 3 M NaOAc mM NaCI • Glycogen (Sigma, cat. no. G1508) . TES: 50 mM Tris-HCI pH 8.0, 10 mM EDTA, • RNase A (DNase free) 1% SDS

53

Karl Ekwall and Janet F. Partridge Protocol 8.

Continued

Method 1. Collect the beads from Protocol 7 by centrifuging at room temperature for 2 min at 15000 g. Use a duck-bill tip to discard the supernatant, being careful to retain all beads in the tube. 2. Wash the beads successively with 1 ml of the following buffers. Wash the beads for 5 min at room temperature on a rotating wheel, then collect the beads by centrifuging for 2 min at 15000 g, and discard the supernatant: • 1 ml lysis buffer twice • 1 ml lysis buffer, 0.5 M NaCI • 1 ml wash buffer • 1 ml TE buffer 3. Carefully remove all the supernatant. Add 50 ml TES to the beads and incubate at 65°C for 10 min. 4. Centrifuge the beads at 15000 g for 1 min at room temperature, and transfer the supernatant to a fresh 1.5 ml tube. 5. Wash the beads with 200 ml TES, centrifuge as in step 4, and pool the supernatants. 6. Add 200 ml TES to the control non-immunoprecipitated chromatin from Protocol 7A, step 1, or Protocol 76, step 2. 7. Incubate the control and IP chromatin samples at 65°C for at least 6 h to reverse the cross-linking. 8. Cool the samples to room temperature. Add 25 ml 10 mg/ml proteinase K and 250 ml TE buffer to each tube. Incubate at 37°C for 2 h. 9. Phenol:chloroform-extract the DNA, and transfer the supernatant to a 2 ml microcentrifuge tube. 10. Ethanol-precipitate the DNA by adding 1/10 volume of 3 M NaOAc, 2.5 volumes of 96% ethanol, and 2 ml 10 mg/ml glycogen. Freeze the mixture on dry ice for 30 min, and collect the DNA by centrifuging at 15000 g for 30 min at 4°C. 11. Vacuum-dry the pellet, resuspend in 40 ml TE + 10 ml RNase A and incubate for 30 min at 37°C.

54

3: Fission yeast chromosome analysis Protocol 9. Covalent coupling of antibodies to protein Sepharose Equipment and reagents • Protein A-Sepharose or Protein G-Sepharose"'Sigma, cat. no. P3391 or P4691) • 0.2 M borax pH 9.0 (sodium tetraborate; Sigma, cat. no. B9876) « TBS (Protocol 75), 0.01% sodium azide

• Dimethyl pimelimidate (Sigma, cat. no. D8388) • 0.2 M ethanolamine pH 8.0 (Sigma, cat. no. E9508)

Method (based on ref. 17) 1. Incubate Ab with protein-Sepharose for 1 h with rocking at room temperature. We use a 1:10 ratio of affinity-purified antisera to 50% (v/v) protein-Sepharose. This sera is used at 1/600 for Western blotting. Perform a control preparation of placebo-coupled beads for the preclearing steps (Protocol 76). 2. Wash the beads twice with 10 vol. of 0.2 M sodium tetraborate pH 9.0 by spinning at 3000 g for 5 min.b 3. Dissolve dimethylpimelimidate in 0.2 M sodium tetraborate pH 9.0 to a final concentration of 20 mM, and incubate the Sepharose beads in 10 vol. of this solution for no longer than 30 min, including step 4. 4. Rapidly remove the coupling mixture by centrifuging at 3000 g for 5 min. 5. Wash the beads twice with 10 vol. of 0.2 M ethanolamine pH 8.0. 6. Resuspend the beads in 10 vol. of 0.2 M ethanolamine, and incubate with rocking for 2 h at room temperature.6 7. Wash the beads by centrifuging with 10 vol. TBS three times. 8. Resuspend the beads to 50% (v/v) in TBS, 0.01% sodium azide, and store at 4°C. ' Protein-Sepharose of appropriate type for the Ab should be chosen (Section 3.3), and should be washed and resuspended to 50% (v/v) in TBS before use. b Coupling efficiency of Ab to beads can be estimated by taking samples of beads at these points, boiling them in Laemmli buffer (17), and comparing the amount of heavy chain (55 kDa) stained by Coomassie blue after SDS-PAGE of these samples before and after coupling.

3.4 Analysis of immunoprecipitated DNA sequences Several methods have been described in the literature for the analysis of immunoprecipitated chromatin sequences. Analyses by slot blotting, Southern blotting, and by PCR are described in Chapter 2. Here we describe a strategy (competitive PCR) that takes advantage of the ease of genetic manipulation in fission yeast and that gives an accurate measurement of the relative enrichment of a particular locus compared with an internal control within the 55

Karl Ekwall and Janet F. Partridge same genome (Figure 2, ref. 16). We use 5. pombe strains bearing a ura4+ marker gene inserted at particular sites within the telomeres, centromeres, or mating type loci. In addition, these strains carry a ura4 minigene, called DS/E, with a 268-bp deletion, at the endogenous ura4 locus (18). Primers were designed which span the region deleted in the minigene, and give rise to products of 426 and 694 bp from the minigene and ura4+, respectively. These primers can be used in competitive PCR reactions (19) to determine accurately the ratio of ura4+ to minigene in the input DNA sample, and the ratio of ura4+ to minigene in the IP sample. The fold enrichment of the IP is calculated by dividing the IP DNA ratio by the non-IP ratio for each strain. A further control can be built into these calculations, by determining the ratios for a strain with a non-heterochromatic ura4+ locus at the same time. The potential disadvantages of this method are that strains need to be constructed, and that the technique relies on the spreading of the protein of interest across the ura4+ chromatin. For these experiments, as only a small proportion of the total input DNA is immunoprecipitated, we use 3 ml of a 1/100 dilution of the input material, and 3 ml of the IP material in a 40-ml PCR reaction. For quantification of the relative ratios of ura4+ and minigene, we include 5 ml [a-32P]dCTP (10mCi/ml) (3000 Ci/mmol) in each ml of PCR mixture. PCR-amplified products are resolved on 4% polyacrylamide gels, dried onto Whatman 3MM paper, exposed to a phosphorimage screen, and quantified using Molecular Dynamics software. IP can be fickle, it is therefore essential to reproduce results several times, and to build appropriate controls (e.g. IP with placebo beads, or unrelated antibodies) into the experimental design.

Acknowledgements FISH technology was transferred to the Allshire laboratory with the generous support of Prof. Mitsuhiro Yanagida. We would like to thank the following for advice, help, and encouragement: 'Chromosome Biology', 'Photography', W. Bickmore, I. Hagan, J.P. Javerzat, A. Pidoux, S. Strahl-Bolsinger, and B. Turner. Special thanks to Robin Allshire for his continued support and enthusiasm. KE is supported by the MFR (K97-13P-1182), and our work is supported by a core grant to R. Allshire from the Medical Research Council

References 1. Uzawa, S. and Yanagida, M. (1992). J. Cell Sci., 101, 267. 2. Funabiki, H., Hagan, I., Uzawa, S., and Yanagida, M. (1993). J. Cell Biol., 121, 961. 3. Ekwall, K., Nimmo, E. R., Javerzat, J-P., Borgstrom, B., Egel, R., Cranston, G., and Allshire, R. (1996). J. Cell Sci., 109,2637. 56

3: Fission yeast chromosome analysis 4. Hagan, I. M. and Hyams, J. S. (1988). J. Cell Set., 89,343. 5. Chikashige, Y., Ding, D-Q., Imai, Y., Yamamoto, M., Haraguchi, T., and Hiraoka, Y. (1997). EMBOJ., 16, 193. 6. Guacci, V., Hogan, E., and Koshland, D. (1994) J. Cell Biol., 125, 517. 7. Ekwall, K., Javerzat, J-P, Lorentz, A., Schmidt, H., and Allshire, R. (1995). Science, 269,1429. 8. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1989). In Molecular cloning—A laboratory manual (1st edn). Cold Spring Harbor Laboratory Press, New York. 9. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) In Molecular cloning—A laboratory manual (2nd edn). Cold Spring Harbor Laboratory Press, New York. 10. Solomon, M.J., Larsen, P.L., and Varshavsky, A. (1988). Cell, 53, 937. 11. Orlando, V. and Paro, R. (1993). Cell, 75,1187. 12. Strutt, H., Cavalli, G., and Paro, R. (1997). EMBO J., 16, 3621. 13. Hecht, A., Strahl-Bolsinger, S., and Grunstein, M. (1996). Nature, 383, 92. 14. Strahl-Bolsinger, S., Hecht, A., Luo, K., and Grunstein, M. (1997). Genes Dev., 11, 83. 15. Saitoh, S., Takahashi, K., and Yanagida, M. (1997). Cell, 90,131. 16. Ekwall, K., Olsson, T., Turner, B.M., Cranston, G., and Allshire, R.C. (1997). Cell, 91,1021. 17. Harlow, E. and Lane, D. (1988). In Antibodies a laboratory manual. Cold Spring Harbor Laboratory Press, New York. 18. Allshire, R.C., Nimmo, E.R., Ekwall, K., Javerzat, J-P., and Cranston, G. (1995). Genes Dev., 9,218. 19. Gilliland, G., Perrin, S., and Bunn, H.F. (1990). In PCR protocols (ed. M.A. Innis, D.H. Gelfand, J.J. Sninsky, and, T.J. White), p. 60. Academic Press, San Diego, California.

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4

Isolation of vertebrate metaphase chromosomes and their analysis by FISH JEFF CRAIG

1. Introduction This chapter concentrates on the in situ hybridization of DNA probes to metaphase chromosomes isolated from vertebrate cells. Fluorescence in situ hybridization (FISH) has a number of applications within the field of chromosome structure, ranging from the simple mapping of specific genes or markers (1-3), mapping transgene integration sites (4-7), through to analysis of the distribution of particular classes of DNA sequence across the genome, e.g. repeat sequence families (8-10), CpG islands (11), fractions of defined base composition (12), and scaffold or matrix-associated sites (13). Whole chromosomes can also be highlighted by chromosome painting probes (14, 15, and Chapter 6), and interspecies hybridization can highlight regions of conserved synteny (16, 17). There are also other novel applications of FISH in studies of chromosome structure, these either analyse karyotype changes or explore the higher order organization of chromosomes (18-24). The protocols detailed in this chapter have been developed from those used by two different research groups (25, 26), after various workshop discussions—such as those set up by Oncor Appligene, and from the research of this author and colleagues.

2. General equipment required for FISH Some basic equipment is needed for all the different FISH techniques. This includes: • two or three water-baths; • a hot-block capable of reaching 75 °C; • frosted slides;

Jeff Craig • coverslips of various sizes (the most commonly used being 22 X 22 mm, 22 X 32 mm, and 22 X 50 mm and between 0.1 and 0.2 mm thick); • rubber cement; • a phase-contrast microscope; • a small lightproof slide incubation box; • slide forceps; and • a rotating platform. Also essential are 50 ml glass Coplin jars; these can hold up to 10 slides backto-back or 9 slides in a zigzag pattern, the latter being better for buffer circulation. If more slides are needed this can be done in 200 ml slide troughs (20 slides). Equipment for FISH can be bought from many large suppliers. Optional, but useful, equipment includes a shaking hot-block and a rotating vacuum dryer. Many of the methods in this chapter contain slide washing steps. Solutions and Coplin jars are prewarmed in water-baths; washing is performed by placing the Coplin jars on a rotating platform, preferably within a small Perspex incubation hood.

3. Production of metaphase chromosomes as substrates for FISH Described below are methods for obtaining fixed metaphase and prometaphase chromosome spreads, as well as techniques for obtaining suspensions of isolated, unfixed metaphase chromosomes.

3.1 Production of fixed metaphase chromosome spreads The technique outlined below describes the production of chromosome spreads from peripheral blood lymphocyte (PBL) cultures and other cell cultures. Subconfluent cell cultures contain a proportion of cells undergoing cell division at any given time. Human PBLs are stimulated to divide with the plant lectin phytohaemagglutinin (PHA), resulting in a wave of cell division after 2-3 days. Similarly, cells isolated from mouse spleens can be stimulated to divide in culture by the addition of lipopolysaccharide (LPS). Mitotic cells are accumulated using a spindle inhibitor, most commonly colchicine, or its derivative demecolcine (Colcemid). A longer incubation time with these agents not only increases the percentage of mitotic cells within a population (the mitotic index, MI), but also increases the degree of chromosome condensation. If absolute numbers of mitotic cells are important, as in Protocol 3, cells can be incubated with Colcemid for many hours and they will have high MIs (50–100%) but extremely condensed chromosomes. On the other hand, cultures released from other cell-cycle blocks and harvested without spindle 60

4: Isolation of vertebrate metaphase chromosomes inhibitors can produce a low, but workable, MI (0.5-5%) of long prometaphase chromosome spreads (Protocol 2). Protocol 1 uses a 40-min incubation with Colcemid. Cells are then harvested and swollen in a hypotonic solution before being fixed with 3:1 methanol:acetic acid. It is important, especially when working with PBLs, to take care with these two steps—especially the fixation—as lysed red blood cells (RBCs) can precipitate if the 'fix' is added too quickly. The number of RBCs can be reduced significantly by culturing only the 'buffy coat' (the whitish interface between the RBCs and plasma after centrifugation of whole blood) that is enriched in white blood cells. The fixative should be freshly made with absolute methanol (HPLC grade if possible) and glacial (>99% pure) acetic acid just before it is needed, since it absorbs water from the atmosphere. Protocol 1. Production of metaphase chromosomes from human peripheral blood Caution: Treat all blood samples with extreme care as 'Biological hazard' material, and take all recommended precautions. Equipment and reagents Venipuncture equipment Sodium heparin (Sigma) RPMI 1640 medium, 200 mM L-glutamine (Gibco BRL) Fetal calf serum (PCS, Sigma) Penicillin-streptomycin (Sigma P0781) PHA (PHA-L, Sigma) 75 cm3 tissue culture flasks

• 10 mg/ml Colcemid (Sigma); store at–20°C in 10 ml aliquots • 5% CO2 incubator . Hypotonic 0.075 M KCI • 37 °C water-bath • 3:1 fixative (HPLC grade methanol:glacial acetic acid) (Sigma)

Method 1. Collect peripheral blooda and transfer immediately to tubes containing preservative-free sodium-heparin (e.g. 50 ul of 5000 U/ml Naheparin for 10 ml blood). Centrifuge in 15 ml tubes at 200 g for 7 min at room temp. 2. Remove 2 ml of the buffy coat.b Alternatively, use heparinized whole blood. 3. Add 2 ml of the buffy coat or 3.5 ml whole blood to 50 ml prewarmed RPMI 1640 medium containing 15% FCS, 100 U/ml penicillin, 100 mg/ ml streptomycin, 2 mM L-glutamine, and 1% PHA. 4. Mix well, transfer to a 75 cm2 tissue culture flask and incubate at 37°C with 5% CO2 for 69-75 h. 5. Add 500 ul 10 mg/ml Colcemid for 40 min. Alternatively, synchronize the cultures by arresting the cells in S-phase with methotrexate (Protocol 2). 61

Jeff Craig Protocol 1.

Continued

6. Centrifuge at 200 g for 7 min at room temp, remove the supernatant down to 5 ml and mix with the pellet using a vortex mixer on a low setting. 7. While mixing as in step 6, add 5 ml prewarmed (to 37°C) hypotonic KCI dropwise, then top up to 50 ml. 8. Incubate in a 37°C water-bath for 15-20 min, then add 5 ml freshly made fixative at room temperature. 9. Repeat step 6. 10. Add 45 ml fixative, adding the first 10 ml dropwise while vortexing on a low setting. 11. Repeat steps 9 and 10. 12. Repeat step 6. (The pellet should now be white). Add 10 ml fix, transfer to a 10-15 ml tube, centrifuge again at 200 g for 7 min, and remove the supernatant. 13. Repeat step 12 twice more, and finally resuspend the pellet in 5 ml fix, mixing well. The chromosome preparation is now ready to spread on slidesc or it can be stored at -20°C. * It is advisable to test the peripheral blood from a number of individuals, as there can be some variability in the quantity and quality of chromosome preparations produced. b The buffy coat is not always obvious; aim to remove approximately equal amounts of plasma and RBCs. This step increases the proportion of white blood cells, making the culture easier to harvest and fix. c It is advisable to spread as soon after fixation as possible, as preparations deteriorate slowly during storage.

Protocol 1 is also applicable for cultured cell lines. After the required length of exposure to Colcemid, cells are harvested, washed in PBS, and processed as in Protocol 1 (from step 7). The concentration of cells in hypotonic KCI should be 3-fold though the nuclear volume is almost unaffected. 2. Add Triton X-100 to 0.5% and break the cells using either 10-20 strokes of a tight-fitting Dounce homogenizer or 3-10 passes through a 22-gauge syringe needle; nuclei prepared using a syringe generally have less cytoplasmic material (8). Use a light microscope to inspect the sample at appropriate intervals and stop when the cells are 95-100% broken, but before nuclear/chromatin fragments appear. 3. Collect nuclei by centrifuging at 300 g for 5 min at 4°C, wash the pellet 2 x in TM buffer (mix without pipetting at this stage to avoid damage that will cause the nuclei to aggregate) and resuspend at ~5 x 107 nuclei/ml. 4. Add MgCI2 to a final concentration of 2.5 mM and DNase I at ~30 U/ml, incubate for 30 min at 4°C. 5. Mix gently, while adding an equal volume of 4 M NaCI, then add TM (0.2) buffer to give ~107 nuclei/ml. Collect nuclear matrices by centrifuging at 750 g for 15 min at 4°C and resuspend the pellet in TM 0.2.

3.2 Nucleoids For some purposes it is necessary to use nuclear derivatives that retain all nuclear DNA. This can be achieved using Protocol 1 but omitting step 4. However, such preparations tend to be unstable and will usually aggregate on manipulation. If cells are lysed directly, as described in Protocol 2, the resulting 'nucleoids' are stabilized by cytoskeletal elements that collapse onto the nuclear lamina during lysis (9). Nucleoids have been used to investigate the organization of sites of DNA replication, transcription, and repair (4).

Protocol 2.

Nucleoid preparations

Equipment and reagents • PBS (Protocol 1) . 30% (w/v) sucrose in 10 mM Tris-HCI pH . Lysis mix: 2.7 mM Tris-HCI pH 8.0, 133 mM 8.0,1.95 M NaCI, 1 mM EDTA EDTA, 2.6 M NaCI, 0.67% Triton X-100 » 10 mM Tris pH 8.0 . 15% (w/v) sucrose in 10 mM Tris-HCI pH • Beckman SW 28 rotor (or similar) 8.0, 1.95 M NaCI, 1 mM EDTA • Siliconized, wide-bore Pasteur pipettes

Method 1. Harvest 108 tissue culture cells, wash once in PBS, and resuspend in 3 ml PBS. Lyse the cells by adding 9 ml of the lysis mix. 149

Dean A. Jackson Protocol 2.

Continued

2. Layer the mixture directly onto two 25 ml step gradients of 15% (w/v) sucrose floating on 5 ml 30% (w/v) sucrose. Incubate on ice for 10 min. 3. Centrifuge at 7500 gmax (5500 r.p.m. in the Beckman SW 28 rotor) for 25 min at 4°C.a 4. Use a siliconized, wide-bore Pasteur pipette to recover the fluffy white aggregate of nucleoids from the surface of the sucrose cushion. Dilute the nucleoids with 9 vol. 10 mM Tris, pH 8.0 to a working concentration of ~5 x 106 nucleoids/ml. a If nucleoids from a particular cell type do not sediment under these conditions, use trial and error to establish the centrifugation conditions that should be used.

3.3 The nuclear scaffold The possibility that structures isolated following hypertonic extraction might not reflect in-vivo organization, stimulated the development of procedures that gave stable nuclear derivatives without the need for extraction with buffers containing high concentrations of salt. The two 'mild' treatments used most often extract chromatin with either lithium 3,5-diiodosalicylic acid (LIS; (10)) or 0.25M (NH4)2SO4 (11). While nascent replication products maintain a tight association with the nuclear scaffold, the detergent treatment used destroys transcription sites so that nascent transcripts and active genes are not preferentially associated with this structure (10-12). Protocol 3.

Preparing nuclear scaffolds

Equipment and reagents • Dounce homogenizer . Isolation buffer: 3.75 mM Tris-HCI pH 7.4, 0.05 mM spermine, 0.125 mM spermidine, 0.5 mM EDTA/KOH pH 7.4, 1% thiodiglycol, 20 mM KCI, 0.2 mM PMSF • Low-salt extraction buffer: 5 mM Hepes/ NaOH pH 7.4, 0.25 mM spermidine, 2 mM EDTA/KOH pH 7.4, 2 mM KCI, 0.1% digitonin, 25 mM LIS (Sigma)a

• 0.5 mM CuS04 . Digestion buffer: 20 mM Tris-HCI pH 7.4, 0.05 mM spermine, 0.125 mM spermidine, 20 mM KCI, 70 mM NaCI, 5 mM MgCI2, 0.1% digitonin, 0.2 mM PMSF

Method 1. Prepare nuclei as described in Protocol 1 steps 1-3 using isolation buffer and 0.1% digitonin instead of Triton X-100. Release nuclei by Dounce homogenization, wash 2 x, and resuspend in isolation buffer without EDTA but with 0.1% digitonin at ~5 x 107 nuclei/ml. 2. To 'stabilize' nuclei, either incubate at 37°C for 1 h or add 0.5 mM CuSO 4 for 10 min at O°C.

150

8: Analysing the substructure of mammalian nuclei, in vitro 3. Slowly add 50 vol. of the low-salt extraction buffer. Incubate at room temperature for 10 min. 4. Recover the histone-depleted nuclei by centrifuging at 750 g for 20 min at room temperature, wash 3 x in 10 ml digestion buffer and resuspend in digestion buffer at ~5 x 106 nuclei/ml. s

ln some circumstances it may be better to USe a lower concentration, e.g. E mM.

3.4 The'low-salt'nuclear matrix A 'low-salt' nuclear matrix can be prepared using Protocol I, steps 1-5, by extracting with 0.4-0.5 M (NH4)2SO4 in place of 4 M NaCl at step 5. This extraction is commonly used for morphological studies (8,11).

3.5 The nucleoskeleton While the different preparations described so far suggest that eukaryotic nuclei are highly organized, their impact is compromised by the possibility that the extraction conditions used generate structures that do not always resemble those existing in vivo. To avoid such extreme treatments, nuclei and derivatives can be prepared under isotonic conditions, but they then tend to lyse and aggregate upon manipulation making analysis difficult. However, if cells are first encapsulated in agarose microbeads (13) as in Protocol 4, this physically isolates cells and protects the fragile structures that result when cells are treated under conditions resembling those existing in vivo. Protocol 4 describes a simple procedure for encapsulating living cells in microbeads (Figure /). Cells in PBS supplemented with molten agarose are

Figure 1. Cells encapsulated in agarose microbeads. HeLa cells were encapsulated in agarose as described in Protocol 4. Beads were incubated in medium and visualized by phase microscopy either before (A) or after (B) permeabilizing with Triton X-100 in a physiological buffer. Bar = 100 mm.

151

Dean A. Jackson homogenized with an immiscible phase of liquid paraffin to form an emulsion. When this is cooled, suspended agarose droplets containing cells gel to form microbeads. Subsequently, cells can be grown to give microcolonies or can be permeabilized in solutions containing a 'physiological' concentration of salts. Such cells are permeable to triphosphates and continue to replicate and transcribe the chromatin template at rates close to those found in vivo (14–16). Protocol 4 can be used for any cell culture but is most appropriate for use with cells that are generally grown in suspension. Protocol 4. Encapsulating cells in agarose microbeads Equipment and reagents • . . . •

100 ml round-bottomed flask Flask-shaker (BDH) PBS DEAE-cellulose (Whatman) 50 ml Falcon tubes

•Low-gelling temperature agarose (Sigma Type VII) • Liquid paraffin (Boots the chemist; BDH cat. cat. no. 7162) no.29436:Merck

Method 1. Dissolve 5 g low-gelling temperature agarose in 200 ml PBS and mix with 100 ml fined DEAE-cellulose (pre-equilibrated with PBS) at 50°C for 15 min.a 2. Pour the slurry into 50 ml Falcon tubes and pellet the DEAE-cellulose using a bench centrifuge at 1000 g for 5 min at 37 °C. Decant the molten agarose and store the purified agarose in 10 ml aliquots at -20 °C. 3. For most purposes, encapsulate mammalian cells at cell densities ranging from 106-107 cells/ml beads. Harvest the cells and resuspend to give 5 ml of the desired cell density in 4 ml PBS. Warm the cells to 37°C in a 100 ml round-bottomed flask. 4. Melt 2.5% agarose in PBS, cool to 37°C, add 1 ml to the cell suspension and mix thoroughly. 5. Pour 10 ml liquid paraffin at 37°C into the flask, cover with Parafilm and shake at room temperature at about 800 cycles/min in a flaskshaker until a creamy emulsion forms (—10 see).b Plunge the flask into ice-cold water and leave for ~10 min, until the agarose sets. 6. Recover the beads by mixing the cooled slurry with 35 ml ice-cold PBS, then transfer to a 50 ml Falcon tube and pellet the beads using a bench centrifuge at 750 g for 2.5 min. If some beads remain at the PBS/paraffin interface—remove most of the paraffin, mix and respin.

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8: Analysing the substructure of mammalian nuclei, in vitro 7. Aspirate the PBS and any remaining paraffin, resuspend the encapsulated cells in fresh medium and incubate under growth conditions for at least 2 h.c a Different batches of agarose contain variable amounts of contaminants that inhibit restriction enzymes and ligases. Lower gelling types have fewer sulfate groups and, for work with DNA, tend to have fewer inhibitors. However, for nuclease digestions, it is advisable to further remove impurities by extracting agarose with DEAE-cellulose. b If a flask shaker is not available shake by hand, as fast as possible for 10 sec. Bead size is controlled by shaking speed, temperature, viscosity, and the interfacial tension between water and the immiscible liquid used. Step 5 gives spherical beads, 50-100 mm in diameter; beads prepared by hand are often non-spherical and more varied in size. Larger beads can be removed by filtering dilute solutions through monofilament nylon filters (R. Cadisch and Sons) in a Swinnex filter (Millipore). Filtration through a 150 mm mesh should remove few beads and leave a filtrate that passes freely through yellow pipette tips. c The cell cycle of encapsulated cells continues normally for one or two cycles, cells then slow and eventually stop growing.

Though almost any cell type can be encapsulated in this way some are more sensitive than others to the encapsulation process. For example, HeLa cells grown in suspension are robust and will survive much more vigorous shaking than freshly isolated lymphocytes, that are rather fragile. Optimal conditions for each cell type should be established by trial and error.

4. Studying the chromatin loops of different nuclear derivatives 4.1 Chromatin loops after hypertonic or hypotonic treatment DNA inside mammalian cells is constrained at intervals so that it forms topologically isolated DNA 'loops' when the histones are removed. These were first described using 'nucleoid' preparations that, unlike early nuclear matrix preparations, retained supercoiled, undamaged, DNA (9). Subsequently, a number of studies demonstrated that particular classes of sequence were associated preferentially with the nuclear cage (4), nuclear matrix (5, 17), and nuclear scaffold (10,12), in vitro. By convention, DNA sequences that remain bound to these structures after removing most chromatin/DNA by nuclease digestion are called nuclear cage-associated sequences, nuclear matrix-associated regions (MARs), and nuclear scaffold-associated regions (SARs), respectively. Nuclear derivatives extracted using either high-salt or LIS have few residual histones. The naked DNA remaining in these preparations provides an excellent substrate for restriction endonucleases that can be used to analyse the arrangement of sequences around the DNA loops, as described in Protocol 5. 153

Dean A. Jackson Protocol 5. Fractionating chromatin loops from histone-depleted nuclear derivatives Reagents • EDTA • Restriction endonucleases

• Gel electrophoresis equipment and reagents • Southern blotting equipment and reagents

Method 1. Prepare derivatives of choice using Protocols 1-3 and dilute them in a suitable digestion buffer3 at a density of ~5 x 106 nuclei/ml. Add the desired restriction endonuclease(s) at an appropriate concentration.b Incubate at 33°C for 30 min, mixing intermittently. 2. Add EDTA to 5 mM to stop the reaction and separate the soluble (released or loop) DNA from the insoluble, derivative-associated DNA by centrifuging at 5-10000 g for 15 min at 4°C. 3. Recover the supernatants (loop) and pellets, prepare DNA,C and analyse by gel electrophoresis and Southern blotting, using standard procedures (see Figure 2). a Most restriction endonucleases work over a wide salt range. Isolation buffers with -100 mM NaCI and 1-2 mM MgCI2 should be OK. Proprietary buffers can also be used. b The concentration required should be established by trial and error; usually in the range 100-1000 U/ml. c To quantitate DNA, it is better to prelabel DNA as described in Protocol 6 and establish the amounts in the pellet and supernatant fractions by scintillation counting. Crude estimates can be obtained from UV scans of the purified material.

4.2 Chromatin loops under 'physiological' conditions If cells are encapsulated in agarose microbeads as in Protocol 4, they can subsequently be lysed using detergents and the chromatin examined under an appropriate environment without difficulty (18, 19). The precise 'physiological' conditions inside mammalian nuclei are not known. Nevertheless, it is clear that a buffer system such as that described in Protocol 6 offers advantages for the preservation of nuclear structure and function. Protocol 6. Fractionation of chromatin loops from the nucleoskeleton under 'physiological' conditions Equipment and reagents • • . .

10 ml clear polycarbonate tubes Vertical gel electrophoresis apparatus Southern blotting apparatus and reagents Peristaltic pump

• Glass-fibre filters • Scintillation counter and Ecolite Scintillation cocktail (ICN) • 200 ™M KH2PO4

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8: Analysing the substructure of mammalian nuclei, in vitro PBS (Protocol 1) . 40mMEGTA 10 ml clear polycarbonate tubes • |3H)thymidine (0.1 mCi/ml; -50 Ci/mmol, Amersham) 1% SDS • Physiological buffer (PB): 100 mM potas5% trichloroacetic acid (TCA) sium acetate, 30 mM KCI, 10 mM Na2HPO4, Diethylether 1 mM MgCI2, 1 mM Na2ATP (Sigma Type I), Sarkosyl 1 mM dithiothreitol, 0.2 mM PMSF in HPLC pure water RNase Proteinase K . Triton X-100 . TAE buffer: 40 mM Tris, 20 mM NaAl, 2 mMPhenol and phenol/chloroform (1:1 v/v) EDTA pH 8.3

Method 1. To allow quantitation of DNA, grow cells for 24 h in medium supplemented with [3H]thymidine (0.05-0.25 ml (Ci/ml). 2. Adjust the pH of the PB to 7.4 by adding 90

Farr, unpublished

The assay used involves the transfection of cells with linearized (TTAGGG)n-constructs based on various selectable markers. Genomic DNAs have been analysed by Southern blotting and BAL 31 nuclease digestion. Percentage estimates are derived from the number of telomeric integration events identified.

of the cis-acting requirements for telomere formation in the HeLa cell line by de Lange and colleagues demonstrated that the process is a highly sequencespecific interaction, with heterologous telomeric DNAs (such as (TTAGGC)n and (TTTAGGG)n) seeding new telomeres only very inefficiently, if at all (6). Another crucial aspect affecting the frequency with which new chromosome ends are created is the recipient cell line, and although a variety of established vertebrate cell lines will seed new telomeres efficiently in this assay, some do not (see Table 1). The factors affecting telomere seeding have not been fully established, but may include telomerase activity, the presence of other telomere-associated proteins, levels of nuclease activity, and the cell's DNA damage checkpoints and repair pathways. Although following random integration many of the telomere-seeding events observed by Barnett et al. in the HT1080 line and mouse embryonic stem-cell line EFC-1 appeared to occur in the vicinity of existing telomeres (3), the application of powerful selection schemes and targeting of telomere-seeding events to specific chromosomal loci by homologous recombination has resulted in the removal of 184

10: Chromosome fragmentation in vertebrate cell lines whole arms from non-essential chromosomes (usually human chromosomes maintained on a rodent background) (7, 8). These observations underlie the use of the (TTAGGG)n repeat array as a tool for dissecting mammalian chromosomes and creating reagents for studies into chromosome structure and function relationships.

3. Experimentally induced de novo telomere formation 3.1 Design of the telomere-seeding construct A typical construct for de novo telomere formation is shown in Figure la. The critical components are: • a dominant selectable marker for use in mammalian cell lines (such as the bacterial hygromycin B kinase gene placed under the SV40 early promoter); • a few hundred base pairs (bp) of (TTAGGG)n DNA (the lower limit has not been established and may vary depending on the recipient cell line; in general, arrays of around 500-1000 bp have been used); and • a unique restriction enzyme site immediately distal to the repeat array. This allows the plasmid to be linearized prior to transfection. It appears that for optimal telomere seeding this restriction enzyme site should lie no more than 100 bp distal to the start of the terminal repeat array, although this may be partly dependent on the recipient cell line and mode of transfection. Long stretches (more than a few hundred bp) of non-terminal repeat DNA distal to the (TTAGGG)n array appear to diminish seeding efficiencies. In early versions of telomere-seeding constructs additional 'tag' DNA sequences were included. These were 5-10 kb stretches of human genomic DNA designed simply to increase the total size of the introduced DNA molecule in order to allow direct visualization of the integration sites by fluorescence in situ hybridization (FISH) (2). Today, however, with improved tyramide signal-amplification systems, the inclusion of additional DNA sequences is no longer essential (9). If there is a requirement to characterize the endogenous DNA at the integration site this can be achieved by plasmid-rescue of the flanking DNA. This approach requires the plasmid backbone (i.e. the bacterial antibiotic resistance marker and origin of replication) to be positioned at the opposite end of the ingoing linear DNA molecule from the (TTAGGG)n repeat array (as shown in Figure la). At least one (and ideally a choice of) restriction enzyme sites should be available for releasing the plasmid DNA and the sequences immediately adjacent (see the EcoR1 site in pHyTMl). This DNA flank can then be recovered by recircularizing the released genomic DNA fragment and transforming it into a bacterial host, such as DH5a. 185

BamHITRF >8.9 kb Figure 1. Telomere seeding constructs, (a) A (TTAGGG)n-construct, pHyTMl, designed to integrate and seed new telomeres randomly in the recipient genome. It is based on a pSV2 plasmid backbone into which has been inserted 1.6 kb of the vertebrate terminal repeat sequence (TTAGGG)n, a dominant selectable marker and 4.5 kb of 'tag' DNA sequences. A unique Not1 site immediately distal to the (TTAGGG)n repeat array has been used to linearize the plasmid. The DNA probe suitable for detecting the SamH1 terminal restriction fragment (TRF) is indicated, (b) A generic targeting-breakage construct designed to seed a new telomere at a specific chromosomal locus. The positions of the endogenous centromere and telomere are indicated. The predicted product of homologous recombination between the construct and the target locus is shown.

3.2 Transfection of the telomere-seeding construct The linearized (TTAGGG)n-construct can be introduced into the recipient cell line using a variety of transfection techniques. The most commonly used are lipofection and electroporation. Electroporation can be used with virtually any cell type and is particularly good at generating cell lines which have integrated only one, or a few, copies of the ingoing plasmid DNA; this technique is discussed in more detail below. Electroporation uses an electric field to open up pores in the cell membrane through which the DNA can diffuse into the cell. Optimal electroporation conditions have to be established empirically for each cell line, varying in turn the voltage and capacitance used. Detailed electroporation protocols have been collated by Bio-Rad. In general for human cells, conditions which result in a long time constant (e.g. capacitance 960 mF and voltage 550 V giving a time constant of about 10 msec) are selected, while for rodent cells conditions resulting in a very short time pulse seem optimal (e.g. capacitance 25 mF and voltage 1.2 kV giving a time constant of 0.3 msec) (Protocol 1). For the avian DT40 cell line electroporation conditions of 25 mF and 550 V are effective. Protocol 1. Electroporation of an established vertebrate cell line with a telomere-seeding construct Equipment and reagents • (TTAGGG)n-construct • 175 cm3 tissue culture flask

Plasmid DNA • Haemocytometer

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10: Chromosome fragmentation in vertebrate cell lines • Appropriate restriction enzyme and buffer as recommended by manufacturer • 0.4 mm electroporation cuvettes (e.g. BioRad) • TE buffer pH 7.4: 10 mM Tns-HCI pH 7.4, 1 mM EDTA pH 8.0 • Multiwell dishes

• Capacitance discharge device/ electroporater (e.g. Bio-Rad Gene Pulsar) • Phosphate-buffered saline without Mg2+ or Ca2+ (PBSA) pH 7.2 • Non-selective tissue culture medium . Appropriate selection reagents

Method 1. Linearize the (TTAGGG)n-construct with the appropriate restriction enzyme (allow 20-30 jj-g of plasmid DNA per ml of recipient cells). Heat (or otherwise) inactivate the restriction enzyme. Precipitate the linear DNA and resuspend at 1 M-g/n-l in sterile TE buffer. 2. Harvest exponentially growing recipient cells for transfection (replate adherent cells, or refeed suspension cells, 24-48 h prior to transfection. Estimate the cell number (e.g. using a haemocytometer). 3. Wash the cells once with PBSA then resuspend in PBSA at an appropriate cell concentration. In general, for electroporation, the cell concentration should be between 1 x 106/ml and 2 x 107/ml depending on the transfectability and plating efficiency of the particular cell line.9 4. Aliquot 1 ml of cells into a sterile 0.4 mm electroporation cuvette and add 20-30 |xg of plasmid DNA. Mix gently and leave at room temperature for 5 min. 5. Subject the DNA/cell suspension to an electric pulse using a capacitance discharge device. 6. After a further 5 min at room temperature transfer the cells to 25 ml of non-selective medium in a 175 cm3 tissue culture flask and incubate at 37°Cfor18-24h. 7. Add selection to the cells the next day, e.g. for isolating pSV2 Hygro (Figure 1). CHO transfectants use a hygromycin B concentration of 500 jig l^r1 (equivalent to 500 u,/ml,Calbiochem). For adherent cells the selective medium is added directly to the flask. For cell lines which grow in suspension the transfected cells should be pelleted (1000 grfor 5 min), resuspended in selective medium, and plated into multiwell dishes, (e.g. for DT40 cells 2 x 107 transfected cells should be divided among 4 x 96-well microtitre dishes). For adherent cells dead cells and cellular debris can be removed after 3-4 days and fresh selective medium applied. Drug-resistant colonies should be visible after 2-3 weeks. 8. Pick off colonies and expand into cell lines. Suspension colonies should be transferred initially to 24-well plates. Adherent colonies can be picked from the 175 cm3 flask using bent Pasteur pipettes and transferred into 25 cm3 flasks. "For example, for Chinese hamster ovary-derived cell lines (CHO) the number of stable transfectants generated is in the range of 102to 103per 10" cells electroporated; while the numbers generated for the human cell line HT1080 are significantly less (10-100 per 106).

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Christine J. Fair

3.3 Screening stable transfectants for de novo telomere formation In brief, the criteria for de novo telomere formation by the introduced construct are the following: • A heterogeneous terminal restriction fragment (TRF) must be detected on a Southern blot. • For mapping purposes the telomere can be regarded as a universal restriction enzyme site. Therefore in any cell line in which the construct DNA is providing a functional telomere a restriction analysis can be used to detect a series of heterogeneous DNA smears of increasing size. • Terminal DNA sequences must be sensitive to BAL 31 nuclease (Protocol 2). In contrast, a chromosome-internal sequence will be resistant to such degradation. • A terminal signal must be detected when the construct DNA is used as a probe for in situ hybridization on metaphase chromosomes. 3.3.1 Molecular analysis The terminal restriction fragments of a chromosome are heterogeneous in length. This is caused by variability in the precise amount of (TTAGGG)n present at any particular chromosome end across a population of cells. Therefore, as an initial screen for de novo telomere formation, 20-30 stable transfectants are picked and expanded into cell lines. Genomic DNA is extracted from these cell lines using a standard procedure and is analysed on a Southern blot. A 20 ug aliquot of genomic DNA should be digested for 6-16 h with a restriction enzyme that will define a TRF if the construct has seeded a new chromosome end (e.g. in the plasmid illustrated in Figure la the BamHl site would be suitable). The digested genomic DNA is size-fractionated on a 0.5% agarose gel, transferred to a nylon membrane, and hybridized with a diagnostic probe (Figure la). Transfectants in which the construct has integrated interstitially without seeding a new chromosome end will display one, or a few (if more than one copy of the plasmid has become incorporated into the genome) discrete hybridization bands of variable size (due to the random nature of the integration events). The ease with which a TRF can be detected varies depending on the quality of the blot and the particular probe DNA used. With probes giving a higher level of background hybridization, the diffuse signal associated with a TRF can be difficult to detect. An example of the hybridization signals generated by a series of CHO-derived cell lines transfected with the pHyTMl telomere-seeding construct is shown in Figure 2a. Examples of both diffuse hybridization smears, characteristic of TRFs, and of discrete bands characteristic of interstitial integration events are illustrated. Note that both events are sometimes present within the same clonal cell line. 188

10: Chromosome fragmentation in vertebrate cell lines Although a heterogeneous hybridization signal is characteristic of TRFs derived from a functional telomere, the definitive test for the terminality of any DNA is its sensitivity to digestion by the nuclease BAL 31 as described in Protocol 2. BAL 31 is predominantly a 3' exonuclease that removes mononucleotides from the 3' termini of linear DNA molecules. It also has an endonuclease activity which removes the single-stranded DNA generated by the 3' exonuclease activity. Thus BAL 31 degrades both the 5' and 3' termini resulting in a controlled shortening of the DNA, and so a fragment of DNA present at a naturally occurring DNA end (a telomere) will be reduced in size before a more internal one. Degradation is absolutely dependent on the presence of Ca2+, and so the reaction can be stopped at different stages by the addition of the chelating agent EGTA. Protocol 2.

BAL 31 sensitivity assay

Equipment and reagents • 30°C water-bath • 5 x BAL 31 reaction buffer 100 mM Tris-HCI pH 8.0, 50 mM CaCI2, 50 mM MgClz, 3 M NaCI, 5 mM EDTA pH 8.0 . 0.1 M EGTA

• BAL 31 nuclease (New England BioLabs) • phenol:chloroform:octan-2-ol (25:24:1 v/v) . chloroform:octan-2-ol 24:1 v/v . TE buffer (Protocol 1)

Method 1. Aliquot out 200 ug of intact genomic DNA from cell lines which appear, from the initial Southern analysis, to be potential single-copy integrants in which the construct provides a functional telomere. This provides sufficient DNA for the analysis of five time points.a 2. Make up to 500 ul in 1 x BAL 31 reaction buffer. Preincubate the DNA at 30°C for 2 h in an uncovered water-bath. 3. Prelabel a series of Eppendorf tubes with various time points; a suitable range would be 0, 10, 20, 40, and 80 min exposure to BAL 31 nuclease activity. Aliquot 20 ul 0.1 M EGTA per Eppendorf tube (this will give a final concentration of between 15 and 20 mM with the addition of 100 ul DNA BAL 31 nuclease solution). 4. Add 20 U BAL 31 nuclease to the genomic DNA; mix by inversion and incubate the open tube at 30°C. Immediately remove 100 u1 and stop the reaction by adding EGTA. 5. Continue the incubation and remove further samples at the appropriate time intervals. 6. When the incubation step is complete remove the nuclease by a phenol/ chloroform:octan-2-ol extraction followed by a chloroform:octan-2ol extraction step. Ethanol-precipitate the DNA and resuspend it in sterile TE buffer. 189

Christine J. Farr Protocol 2. Continued 7. Digest the nuclease-treated DNA with a restriction endonuclease suitable for TRF identification and analyse the DNA by Southern blotting and hybridization.b a BAL 31 nuclease is sensitive to RNA contamination, so 'good' quality DNA preparations are required. b Some restriction enzymes are inhibitad by EGTA.

An example of BAL 31 digestion of DNA from a cell line with a functioning (TTAGGG)n construct is illustrated in Figure 2b. BAL 31 degrades AT-rich DNA sequences significantly more rapidly than it degrades GC-rich DNA. This may be responsible for the end products of some BAL 31 digestions appearing as a discrete hybridi/ation band at late time points (see Figure 2b). 3.3.2 Cytogenetic analysis Finally, the location of the exogenous DNA can be examined cytogenetically by fluorescence in situ hybridization using the introduced telomere- see ding

Figure 2. Molecular analysis of transfectants. (a) Southern blot analysis of a series of CHO-derived cell lines transfected with Not1-linearized pHyTM1, Genomic DNAs have been digested with BamH1 and probed with the DNA fragment indicated in Figure 1a. (b) BAL 31 sensitivity of DMA from one transfectant in which the (TTAGGG)n-construct has seeded a new telomere.

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10: Chromosome fragmentation in vertebrate cell lines construct, minus the (TTAGGG)n repeat array itself, as the probe. Appropriate cytogenetic techniques for the preparation of fixed metaphase chromosomes from Colcemid-blocked cells are described in Chapter 4. The exogenous DNA is detected by FISH using the (TTAGGG)n-containing construct as the probe, minus the terminal repeat (since this would result in the probe DNA hybridizing to all endogenous chromosome ends). It should be noted that even if transfectants are scored cytogenetically as having integrated the construct at apparently terminal locations this is not definitive, and the molecular analyses described in Section 3.3.1 must also be performed. A region of DNA may appear cytogenetically to be terminal, but in fact may lie several megabases away from the actual chromosome end; conversely, DNA shown by the molecular analyses to be at the very end of the chromosome and sensitive to the action of BAL 31 nuclease may appear subtelomeric on metaphase chromosomes. The reason for the latter observation is unclear, but it has been speculated that it may reflect loops of DNA extending out and beyond the telomeres, resulting in the appearance of DNA beyond the site of hybridization. Visualization of the integration site by FISH therefore provides supportive data, but is not in itself proof of terminality.

4. Targeted de novo telomere formation The (TTAGGG)n-construct shown in Figure la is designed to seed telomeres randomly in the recipient genome. Two strategies have been used to obtain truncation events on a particular chromosome: • the use of a randomly integrating (TTAGGG)n-construct combined with a powerful selection scheme to enrich for truncation events of interest; and • the use of homologous recombination to target the (TTAGGG)n-construct to a particular chromosomal location. The first approach is reliant on the presence of two selectable markers on the chromosome of interest: a negative or bidirectional selectable marker (e.g. HSV-Tk, or HPRT) distally located on the chromosome arm to be truncated; and a dominant selectable marker (e.g. hygror, neor, or hisDr) located on the segment of chromosome which will be retained. Using this 'PushmiPullyu' counterselection system a panel of somatic cell hybrids, in which the long arm of the human X chromosome is truncated to various extents, has been created (7). Selection against the human HPRT gene at Xq26 was used to isolate cells in which the introduced hygromycin-based (TTAGGG)nconstruct had replaced the long-arm telomere. Out of 85 cell lines isolated, 42 displayed apparently random terminal truncations of Xq, extending from qter to the a-satellite array at the centromere. Presumably, the stringent biochemical selection permits the isolation of the desired truncation events from a much larger number of truncations generated by the random integration of 191

Christine J. Farr the (TTAGGG)n-construct. Although a powerful approach, its application is strictly limited due to the absolute requirement for strategically placed selectable markers. Since there are relatively few endogenous biochemically selectable markers in the mammalian genome, this necessitates retrofitting the chromosome of interest using homologous recombination. The feasibility of creating defined truncation events was first demonstrated by Itzhaki et al. who seeded a de novo telomere within the interferoninducible 6-16 gene located on the distal tip of chromosome Ip in the HT1080 cell line (10). In addition to targeting unique loci, (TTAGGG)n-constructs have also been targeted to the repetitive a-satellite DNA at the centromere of the human Y chromosome, resulting in the creation of both long-arm and short-arm derivatives (8). In both instances, the basic (TTAGGG)n-construct is modified to include a stretch of genomic DNA homologous to the locus being targeted. When linearized, the targeting (TTAGGG)M-construct has the vertebrate terminal repeat array at one end and a stretch of DNA homologous to the locus of interest at the other (Figure Ib). This approach requires either that the orientation of the locus being targeted relative to the centromere and telomere is known (so that the (TTAGGG)n-array can be orientated correctly relative to it) or, alternatively, the use of two targeting constructs with the region of homology in both possible orientations. Targeted chromosome breakage has been used extensively in the mapping and characterization of both natural and artificial chromosomes in yeast, where the integration of exogenous DNA by homologous recombination is very efficient. The major limitation to the use of this approach for the functional analysis and modification of mammalian chromosomes has been the very low levels of homologous recombination observed in mammalian somatic cell lines.

5. Targeted truncation events in the recombinationproficient avian cell line DT40 The chicken pre-B cell line DT40 is unusual for a vertebrate cell line in that it exhibits high levels of homologous recombination, the reasons for which are as yet unclear (11). Human chromosomes can be transferred into DT40 cells using microcell-mediated chromosome transfer (MMCT) and appear to segregate in a relatively stable manner. Moreover, it has recently been demonstrated that the transferred chromosomes can also be efficiently modified by homologous recombination in the DT40 hybrids (12, 13). Systematic manipulation of human chromosomes in the DT40 background can therefore be undertaken, with subsequent transfer of the modified human chromosome back into a mammalian cell line by MMCT for further analysis. To construct microcell hybrids, donor cells are subjected to a prolonged mitotic block, which induces micronucleation (partitioning of individual or a 192

10: Chromosome fragmentation in vertebrate cell lines few chromosomes into subnuclear packets). The micronuclei are extruded from the cells by centrifugation in the presence of cytochalasin B, forming microcells (micronuclei surrounded by a plasma membrane) and fused to intact recipient cells. Hybrids are selected using a metabolic or transgenic marker located on the desired chromosome. A detailed protocol for the production of DT40-human hybrids by MMCT is provided by Dieken and Fournier (14). Microcell fusion is technically demanding, temperamental, and requires proficiency in tissue culture techniques. Moreover, the transfer of human chromosomes into DT40 cells is probably an order of magnitude more difficult than fusions between mammalian donor and recipient cell lines. Success is dependent upon careful organization and monitoring of each step, and on patience and perseverance (15). Fortunately, laboratories highly proficient at microcell hybrid production have already generated several DT40-human hybrids and, although a full panel representing all human chromosomes is not yet available, several hybrids have been described in the literature (e.g. DT40-human microcell hybrids carrying chromosomes 1, 2, 3, and 11) (12, 13). The frequency of transfer of the modified human chromosome back into a mammalian cell line for further study is much higher than in the reciprocal cross. However, after each transfer step the chromosome under investigation will need to be thoroughly examined, both at the molecular and cytogenetic level, to determine whether or not it has been transferred intact, or, as frequently happens during MMCT, has undergone rearrangement. Since homologous recombination is very efficient in DT40 cells and hybrids, vector construction is straightforward, with no requirement for elaborate schemes to enrich, or screen, for targeting events. A simple targeting construct with a single block of homology at one end and screening of a small number of transfectants by Southern blotting is generally sufficient, although this may depend on the type of locus being targeted (e.g. unique or repetitive, euchromatic or heterochromatic) and on the nature of the targeted product (e.g. targeting a functional chromosomal element may affect stability, which will in turn affect the number of targeting events isolated).

6. The characterization of chromosomes modified by de novo telomere formation and fragmentation Characterization of the modified chromosome product will involve both molecular and cytogenetic analyses. FISH is essential for confirming that the integrated (TTAGGG)n-construct is in the predicted location and for determining the effect of the de novo seeding event on the overall morphology of the targeted chromosome, including whether it still exists as an unrearranged, independently segregating entity. If the target chromosome has been fragmented within the a-satellite DNA sequences, which are localized at the 193

Christine J. Farr primary constriction of all naturally occurring human centromeres, pulsedfield gel electrophoresis (PFGE) will be required to estimate the amount of a-satellite DNA remaining and its integrity compared with the parental chromosome. In addition, some measure of the functional status of the centromere can be obtained by determining the presence of centromere-binding proteins associated with activity, such as CENP-C and CENP-E, by immunofluorescence (Chapter 5).

6.1 Estimation of minichromosome size If the target chromosome has been subjected to several rounds of telomereassociated chromosome fragmentation, in order to create minichromosomes based on human centromeric sequences, the total size of the minichromosome (if it has a linear structure) can be investigated by PFGE of undigested, intact, genomic DNA (circular structures are thought not to resolve under these conditions) as in Protocol 3. To resolve linear DNA molecules in the 2-10 Mbp size range, long runs (5 days or longer) and long pulse times are required on a suitable apparatus (e.g. BioMetra's Rotaphor system). Size markers covering the Mbp range are available commercially (Bio-Rad and BioMetra); chromosomes in S. pombe are approximately 3.5, 4.6, and 5.7 Mbp in size, while the Hansenula wingei genome provides markers in the 1 to 3.1 Mbp range, overlapping with chromosomes in 5. cerevisiae (0.2-2.2 Mbp). Following electrophoresis, the DNA is transferred to a nylon membrane and hybridized with a repeat probe, such as the appropriate chromosome-specific a-satellite DNA. A DNA probe which hybridizes to a high copy-number repeat is required since only a very small proportion of the DNA molecules migrate into the agarose. Estimation of minichromosome size by PFGE is much easier if the human-derived minichromosome is in a non-human background, since the presence of other human chromosomes will give rise to some level of background hybridization from inevitable degradation arising during block preparation and electrophoresis. Protocol 3. Pulsed-field gel electrophoresis of large DNA Caution: PMSF must be treated with extreme caution. It is highly toxic and volatile and should always be handled in a fume hood. It is unstable and must be freshly prepared. Equipment and reagents • Pulsed-field block formers (Bio-Rad, BioMetra) • Water-baths at 48, 50, and 65°C • Pulsed-field gel electrophoresis apparatus (e.g. Bio-Rad CHEF DRII or BioMetra Rotaphor system) • Stratalinker UV crosslinker (Stratagene)

• Pulsed-field grade and chromosomal grade agarose (Bio-Rad) « SE: 75 mM NaCI, 25 mM EDTA, pH 8.0 • PBSA (Protocol 7) . 50 ml Falcon tubes . ESP: 0.5 M EDTA pH 8.0, 1% sodium lauryl sarcosinate, 2 mg/ml proteinase K

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10: Chromosome fragmentation in vertebrate cell lines • 0.5 M EDTA pH 8.0 • TE buffer (Protocol 1) . Phenylmethylsulfonyl (Sigma)

fluoride

(PMSF)

• 10 mg/ml ethidium bromide • Equipment and reagents for Southern blotting, hybridization, and autoradiography

Method 1. Prepare 1% pulsed-field grade agarose in SE. Place at 65 °C and transfer to a 48°C water-bath 10-15 min before use. Clean and dry pulsed field block formers. 2. Harvest exponentially growing cells and wash once with PBSA. Check the cell number (e.g. with a haemocytometer) and resuspend in PBSA or SE at 2 x 107 cells/ml. 3. Add 1 ml of cooled agarose per ml of cell suspension. Quickly pipette up and down to mix and immediately aliquot into a block-forming mould. Leave to solidify. (If embedding several ml of the cell suspension do so in 1 ml aliquots to prevent the cell/agarose mix from solidifying during the process.) 4. Transfer the blocks to a 50 ml Falcon tube and incubate in 20 ml of ESP for 48 h at 50°C; swirl occasionally during this period. 5. For long-term storage of blocks (for up to 6-12 months) at 4°C replace the ESP with 0.5 M EDTA pH 8.0. 6. Prior to use, wash the blocks thoroughly with multiple (at least 10) changes of TE buffer. (TE supplemented with 1 mM PMSF can be used to inactivate and remove all traces of proteinase K. However, PMSF must be treated with extreme caution. It is highly toxic and volatile and should always be handled in a fume hood. It is unstable and must be freshly prepared.) 7. Load the rinsed and pre-equilibrated (in running buffer, see step 8) sample blocks and suitable size markers, into a 0.6% chromosomal grade agarose gel using a sterile spatula. Precool the gel in the buffer chamber before starting electrophoresis. 8. Run for a 5-10 day period using the conditions recommended by the manufacturer, e.g. on the Bio-Rad CHEF DRII apparatus, which has a fixed 120° angle, reasonable resolution in the 2-6 Mbp range can be obtained using: 75 V, 600-2700 sec pulse, 120 h run at 8°C in 0.5 x TBE. For the BioMetra Rotaphor system 5- and 10-day run conditions are detailed in the user manual. 9. After electrophoresis is complete stain the DNA with ethidium bromide for 30-60 min and photograph the gel. UV-treat the gel to nick the DNA (e.g. deliver 960 mJ of energy using a Stratalinker UV crosslinker). Denature, neutralize, and transfer the gel as usual for Southern blotting. 10. Hybridize the filter with a suitable DNA repeat probe and subject to autoradiography.

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Christine J. Fan

6.2 Assays for mitotic stability of minichromosomes The assays currently available for estimating mammalian chromosome stability are limited and cumbersome. However, if properly undertaken they do give some information as to the stability of the modified chromosome compared with intact human chromosomes, in the context of a somatic cell hybrid background. To determine whether modified chromosomes and minichromosomes are mitotically stable the cell lines should be grown for prolonged periods both in the presence and absence of selection (1-6 months), with frequent passaging of the cells to ensure that the cultures are kept in an actively proliferating growth state. At monthly time-intervals some measure of stability can be obtained by plating-efficiency assays. Plate 1-2 X 103 cells per 75 cm3 flask (in triplicate) into media both with and without the selective agent. After 5-10 days stain the colonies with Crystal violet and count. This assay is only suitable for adherent cell lines and, although it reveals whether or not the selectable marker being assayed is present/absent, it does not provide any information about copy number or the physical status of the chromosome being monitored. A more informative assay is to analyse the cells by FISH, screening 100-200 metaphases at each time-point off selection and comparing this with stability on selection. The presence or absence of autonomous minichromosomes should be determined using a highly efficient probe, such as a centromeric repeat DNA. If the chromosome displays instability this can be investigated using FISH to anaphase and early telophase cells (8,16). Exponentially growing cells are assayed without prior Colcemid treatment (Protocol 4). The presence of anaphase bridges between separating daughter cells and lagging chromosomes would indicate abnormal mitotic behaviour (see also Chapter 5). Protocol 4. Anaphase analysis for adherent cells Equipment and reagents • • • . •

Sterile coverslips (e.g. see Chapter 6) PBSA (Protocol 1) EDTA MgCI2 Triton X-100

• 1 mg/ml NaBH4 in H2O . 5-10 % goat serum in PBSA • Ethylene glycol-bis(succinimidylsuccinate) (EGS) (Pierce) • 2 x SSC: 0.3 M NaHCI, 0.03 M trisodium

citrate pH 7 . Paraformaldehyde or glutaraldehyde '° . Permeabilization and fixation solution: 10 * 10° ^9/ml RNase A in 2 x SSC mM EDTA, 2 mM MgCI2, 0.1% Triton X-100, • 0.2 M NaOH 3.7% paraformaldehyde (or 0.5% glu- • Equipment and reagents for hybridization taraldehyde) in PBSA and standard FISH analysis

Method 1. Plate cells onto coverslips and grow for 24-48 h until 80% confluent. 2. Simultaneously permeabilize and fix the cells by incubating them for 30 min in the permeabilization and fixation solution.

196

10: Chromosome fragmentation in vertebrate cell lines 3. Rinse the cells on the coverslip 3 x with PBSA. 4. Incubate 2 x with 1 mg/ml NaBH4 in dH20 for 10 min. 5. Wash 2 x with PBSA and incubate for 30 min in 5-10 % goat serum in PBS A at 37 °C. 6. Wash briefly 5 x in PBSA and incubate with the cross-linking agent EGS for 30 min at 37°C (to preserve the microtubule morphology). 7. Wash briefly 5 x in PBSA and treat the coverslip with 100 jig/ml RNase A in 2 x SSC for 30-60 min at 37°C. 8. Wash 3 x in PBSA and denature the DNA in 0.2 M NaOH at room temperature for 4 min. 9. Hybridize in situ with biotinylated probe DNA and visualize with FITC-avidin detection as for standard FISH analysis.

7. Concluding remarks The physical manipulation of mammalian chromosomes and the characterization of their phenotypes demands expertise in a wide range of molecular biology, tissue culture, and cytogenetic methodologies. In this brief chapter it has only been possible to outline current developments in this field; for more detailed protocols the reader is referred to the reference list below.

References 1. Harrington, J.J., van Bokkelen, G., Mays, R.W., Gustashaw, K., and Willard, H.F. (1997). Nature Genet., 15, 345. 2. Farr, C., Fantes, J., Goodfellow, P., and Cooke, H. (1991). Proc. Natl Acad. Sci. USA, 88,7006. 3. Barnett, M.A., Buckle, V.J., Evans, E.P., Porter, A.C.G., Rout, D., Smith, A.G., and Brown, W.R.A. (1993). Nucl. Acid Res., 21,27. 4. Farr, C.J., Bayne, R.A.L., Kipling, D., Mills, W., Critcher, R., and Cooke, H.J. (1995). EMBO J., 14,5444. 5. Heller, R., Brown, K.E., Burgtorf, C., and Brown, W.R.A. (1996). Proc. Natl Acad. Sci. USA, 93,7125. 6. Hanish, J.P., Yanowitz, J.L., and de Lange, T. (1994). Proc. Natl Acad. Sci. USA, 91, 8861. 7. Farr, C.J., Stevanovic, M., Thomson, E.J., Goodfellow, P.N., and Cooke, H.J. (1992). Nature Genet., 2, 275. 8. Brown, K.E., Barnett, M.A., Burgtorf, C., Shaw, P., Buckle, V., and Brown, W.R.A. (1994). Hum. Mol. Genet, 3,1227. 9. Raap, A.K., van de Corput, M.P.C., Vervenne, R.A.W., van Gijlswijk, R.P.M., Tanke, H.J., and Wiegant, J. (1995). Hum. Mol. Genet., 4,529. 10. Itzhaki, J.E., Barnett, M.A., MacCarthy, A.B., Buckle, V.J., Brown, W.R.A., and Porter, A.C.G. (1992). Nature Genet., 2, 283. 197

Christine J. Farr 11. Buerstedde, J.-M. and Takeda, S. (1991). Cell, 67,179. 12. Dieken, E.S., Epner, E.M., Fiering, S., Fournier, R.E.K., and Groudine, M. (1996). Nature Genet., 12,174. 13. Koi, M., Lamb, P.W., Filatov, L., Feinberg, A.P., and Barrett, J.C. (1997). Cytogenet. Cell Genet., 76,72. 14. Dieken, E.S. and Fournier, R.E.K. (1996). In Methods (ed. R.E.K. Fournier), Vol. 9, p.56. Academic Press, New York. 15. Schafer, AJ. and Farr, C.J. (1998). In The ICRF handbook of genome analysis (ed. N. Spurr), Chapter 14. Blackwell Science. 16. Larin, Z., Fricker, M.D., and Tyler-Smith, C. (1994). Hum. Mol. Genet, 3, 689.

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List of suppliers Advanced Biotechnologies Ltd. Advanced Biotechnologies Ltd., Units B1-B2, Longmead Business Centre, Blenheim Road, Epsom, Surrey KT19 9QQ, UK. Advanced Biotechnologies Ltd., Wendenstrasse 23,20097 Hamburg, Germany. Aladdin Enterprises, 1255 23rd St, San Francisco, CA 94122, USA. Amersham Amersham International pic, Lincoln Place, Green End, Aylesbury, Buckinghamshire HP20 2TP, UK. Amersham Corporation, 2636 South Clearbrook Drive, Arlington Heights, IL 60005, USA. Anachem, Anachem Ltd., Anachem House, Charles Street, Luton, Beds LU2 OEB, UK Andennan and Co. Ltd., 145 London Road, Kingston-Upon-Thames, Surrey KT17 7NH, UK. K. R. Anderson Company Inc., 2800 Bowers Ave, Santa Clara, CA 95051, USA. AndPak-EMA, 1560 Dobbin Drive, San Jose, CA 95133, USA. Applied Imaging International Ltd., Hylton Park, Wessington Way, Sunderland, SR5 3HD, UK. Applied Precision Inc., 1040 12th Ave, Northwest, Issaquam, Washington 98027, USA. Applied Spectral Imaging Ltd., P.O. Box 101, Migdal Haemek, 10551, Israel. AutoQuant Imaging, 877 25th St, Watervliet, NY 12189, USA. Bayer Diagnostics pic, Bayer House, Strawberry Hill, Newbury RG14 1JA, UK. BDH BDH Chemicals UdJMerck Ltd., Merck House, Poole, Dorset BH15 1TD, UK. BDH, Merck Ltd., Hunter Boulevard, Magna Park, Latterworth, Leicester, LE17 4XN, UK. Beckman Instruments Beckman Instruments UK Ltd., Progress Road, Sands Industrial Estate, High Wycombe, Buckinghamshire HP12 4JL, UK.

List of suppliers Beckman Instruments Inc., P.O. Box 3100,2500 Harbor Boulevard, Fullerton, CA 92634, USA. Becton Dickinson Becton Dickinson and Co., Between Towns Road, Cowley, Oxford OX4 SLY, UK. Becton Dickinson and Co., 2 Bridgewater Lane, Lincoln Park, NJ 07035, USA. Bio Bio 101 Inc., do Statech Scientific Ltd., 61-63 Dudley Street, Luton, Bedfordshire LU2 OHP, UK. Bio 101 Inc., P.O. Box 2284, La Jolla, CA 92038-2284, USA. BioMetra Biometra Ltd., Whatman House, St Leonard's Road, 20/20 Maidstone, Kent ME 16 OLS, UK. Biometra Inc., 550 N. Reo St. #101, Tampa, FL 33609, USA. Bio-Rad Laboratories Bio-Rad Laboratories Ltd., Bio-Rad House, Maylands Avenue, Hemel Hempstead HP2 7TD, UK. Bio-Rad Laboratories, Division Headquarters, 3300 Regatta Boulevard, Richmond, CA 94804, USA. Biospec Products Inc., P.O. Box 722, Bartlesville, OK 74005, USA. Biovation, Crombie Lodge, Aberdeen Science and Technology Park, Balgownie Drive, Aberdeen AB22 8GU, UK Boehringer Mannheim Boehringer Mannheim UK (Diagnostics and Biochemicals) Ltd., Bell Lane, Lewes, East Sussex BN17 1LG, UK. Boehringer Mannheim Corporation, Biochemical Products, 9115 Hague Road, P.O. Box 504 Indianopolis, IN 46250-0414, USA. Boehringer Mannheim Biochemica, GmbH, Sandhofer Str. 116, Postfach 310120 D-6800 Ma 31, Germany. Branson Ultrasonic Corp., 41 Eagle Rd., Danbury, CT 06813, USA. British Drug Houses (BDH) Ltd., Poole, Dorset, UK. R. Cadisch and Sons, Arcadia Avenue, Finchley, London, N3 2JW, UK. Calbiochem Calbiochem-Novabiochem, Boulevard Industrial Park, Padge Rd, Beeston, Nottingham NG9 2JR, UK. Calbiochem, Inc., P.O. Box 12087, San Diego, CA 92112-4180, USA. Cambio, 34 Newnham Road, Cambridge, CB3 9EY, UK. Cambridge Bioscience (Molecular Probes), 24-25, Signet Court, Newmarket Road, Cambridge, CB5 8LA, UK. Chance Propper Ltd., West Midlands, B66 1NZ, UK. Chemicon Chemicon International, 2 Bonnersfield Lane, Harrow HA1 2JR, UK. Chemicon International, 28835 Single Oak Dr.,Temecula, CA 92590, USA. 200

List of suppliers Chroma Technology Corporation, 72 Cotton Mill Hill Unit A-9, Brattleboro, VT 05301, US A. Citifluo, Connaught Buildings, City University, Northampton Square, London EC1N OH13, UK. Cytocell Ltd, Somerville Court, Banbury Business Park, Adderbury, Banbury, Oxon OX17 3SN, UK Difco Laboratories Difco Laboratories Ltd., P.O. Box 14B, Central Avenue, West Molesey, Surrey KT8 2SE, UK. Difco Laboratories, P.O. Box 331058, Detroit, MI 48232-7058, USA. Du Pont Dupont (UK) Ltd., Industrial Products Division, Wedgwood Way, Stevenage, Herts, SGI 4Q, UK. Du Pont Co. (Biotechnology Systems Division), P.O. Box 80024, Wilmington, DE 19880-002, USA. Electron Microscopy Sciences, PO Box 251, 321 Morris Road, Fort Washington, PA 19034, USA. European Collection of Animal Cell Culture, Division of Biologies, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG, UK. Falcon (Falcon is a registered trademark of Becton Dickinson and Co.) Fisher Scientific Co., 711 Forbest Avenue, Pittsburgh, PA 15219-4785, USA. Flow Laboratories, Woodcock Hill, Harefield Road, Rickmansworth, Hertfordshire WD3 1PQ, UK. Fluka Fluka-Chemie AG, CH-9470, Buchs, Switzerland. Fluka Chemicals Ltd., The Old Brickyard, New Road, Gillingham, Dorset SP8 4JL, UK. Gibco BRL Gibco BRL (Life Technologies Ltd.), Trident House, Renfrew Road, Paisley PA3 4EF, UK. Gibco BRL (Life Technologies Inc.), 3175 Staler Road, Grand Island, NY 14072-0068, USA. Growing Point, Toyobo Co Ltd, 2-8 Dojima Hama Z Chome, Kita-Ku, Osaka 530 8230, Japan. Heraeus Equipment Ltd., 9 Wates Way, Brentwood, Essex CM15 9TB, UK. Arnold R. Horwell, 73 Maygrove Road, West Hampstead, London NW6 2BP, UK. Hybaid Hybaid Ltd., 111-113 Waldegrave Road, Teddington, Middlesex TW11 8LL, UK. Hybaid, National Labnet Corporation, P.O. Box 841, Woodbridge, NJ 07095, USA. 201

List of suppliers HyClone Laboratories, 1725 South HyClone Road, Logan, UT 84321, USA. ICN ICN Pharmaceuticals Inc., 3300 Hyland Avenue Costa Mesa, CA 92226, USA. ICN Biochemicals Ltd., Unit 18, Wenman Rd, Thame, Oxfordshire OX9 3XA, UK. International Biotechnologies Inc., 25 Science Park, New Haven, CT 06535, USA. International Equipment Company (IEC), Needham Heights, MA 02194, USA. Improvision, Viscount Centre 11, University of Warwick Science Park, Millburn Hill Road, Coventry, CV4 7HS, UK. Invitrogen Corporation Invitrogen Corporation, 3985 B Sorrenton Valley Building, San Diego, CA 92121, USA. Invitrogen Corporation, do British Biotechnology Products Ltd., 4-10 The Quadrant, Barton Lane, Abingdon, OX14 3YS, UK. Jackson ImmunoResearch Laboratories Inc., P.O. Box 9, 872 West Baltimore Pike, West Grove, PA 19390, USA. Kodak: Eastman Fine Chemicals, 343 State Street, Rochester, NY, USA. Leica Inc. Leica Inc., Ill Deer lake Road, Deerfield, IL 60015, USA. Leica AG, CH-9435 Heerbrugg, Switzerland Leinco Technologies Inc. Leinco Technologies Inc., 14730 Manchester Rd, Ballwin, MO 63011, USA. Universal Biologicals Ltd., 30 Merton Road, London SW18 1QY, UK. Life Sciences International (Shandon UK), Unit 5, The Ringway Centre, Edison Rd, Basingstoke, Hampshire RG21 6YH, UK. Life Sciences Resources, Abberley House, Granham's Road, Great Shelford, Cambridge CB2 5LQ, UK. Life Technologies Inc., 8451 Helgerman Court, Gaithersburg, MD 20877, USA. Merck Merck Industries Inc., 5 Skyline Drive, Nawthorne, NY 10532, USA. Merck, Frankfurter Strasse, 250, Postfach 4119, D-64293, Germany. Millipore Millipore (UK) Ltd., The Boulevard, Blackmoor Lane, Watford, Hertfordshire WD1 8YW, UK. Millipore CorpJBiosearch, P.O. Box 255, 80 Ashby Road, Bedford, MA 01730, USA. Molecular Dynamics Ltd, 5 Beech House, Chiltern Court, Asheridge Road, Chesham, Bucks HP5 2PX, UK. Molecular Probes Inc. Molecular Probes Inc., P.O. Box 22010, Eugene, OR 97402-0469, USA. 202

List of suppliers Cambridge Bioscience (Molecular Probes), 24-25, Signet Court, Newmarket Road, Cambridge, CBS SLA, UK. MSE Scientific Instruments, Manor Royal, Crawley, West Sussex RH10 2QQ, UK. NBL Gene Sciences Ltd., South Nelson Industrial Estate, Cramlington, Northumberland NE23 9HL, UK. New England Biolabs (NBL) New England Biolabs (NBL), 32 Tozer Road, Beverley, MA 01915-5510, USA. New England Biolabs (NBL), c/o CP Labs Ltd., P.O. Box 22, Bishops Stortford, Hertfordshire CM23 3DH, UK. New England Nuclear Du Pont (UK) Ltd., Biotechnology Systems Division, Wedgewood Way, Stevenage, Hertfordshire SGI 4QN, UK. NEN Life Science Products, 549 Albany Street, Boston, MA 02118, USA. Nikon Corporation, Fuji Building, 2-3 Marunouchi 3-chome, Chiyoda-ku, Tokyo, Japan. Olympus America Inc., Two Corporate Center Drive, Melville, NY 11747-3157, USA. Omega Optical Inc., 3 Grove St, P.O. Box 573, Brattleboro, VT 05302-0573, USA. Oncor Appligene Inc. Appligene Oncor, Pinetree Centre, Durham Rd, Birtley, Chester-le-St, Co. Durham DH3 2TD, UK. Oncor Appligene Inc., 209 Perry Parkway, Gaithersburg, MD 20877, USA. Perkin-Elmer Perkin-Elmer Ltd., Maxwell Road, Beaconsfield, Buckinghamshire HP9 1QA, UK. Perkin Elmer Ltd., Post Office Lane, Beaconsfield, Buckinghamshire HP9 1QA, UK. Perkin Elmer-Cetus (The Perkin-Elmer Corporation), 761 Main Avenue, Norwalk, CT 0689, USA. Pharmacia Biosystems Pharmacia Biosystems Ltd., (Biotechnology Division), Davy Avenue, Knowlhill, Milton Keynes MK5 8PH, UK. Pharmacia LKB Biotechnology AB, Bjorngatan 30, S-75182 Uppsala, Sweden. Pharmacia Biotech Europe, Procordia EuroCentre, Rue de la Fuse-e 62, B-1130 Brussels, Belgium. Philip Harris Scientific, Lynn Lane, Shenstone, Lichfield, Staffordshire WS14 9BR, UK. Pierce Pierce and Warriner (UK) Ltd., 44 Upper Northgate St, Chester CHI 4EF, UK. 203

List of suppliers Pierce, 3747 N. Meridian Rd, P.O. Box 117, Rockford IL 61105, USA. Polysciences, 400 Valley Road, Wallington, PA 18976, USA. Princeton Instruments, 3660 Quakerbridge Road, Trenton, NJ, USA. Promega Promega Ltd., Delta House, Enterprise Road, Chilworth Research Centre, Southampton, UK. Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711-5399, USA. Qiagen Qiagen Inc., do Hybaid, 111-113 Waldegrave Road, Teddington, Middlesex, TW11 8LL, UK. Qiagen Inc., 9259 Eton Avenue, Chatsworth, CA 91311, USA. Sartorius AG., 37070 Gottingen, Germany. Savant Instruments Inc. 110-103 Bi-county Boulevard, Farmingdale NY11735, USA. Scanalytic Inc., 8550 Lee Highway, Suite 400, Fairfax, Virginia 22031-1515, USA. Schleicher and Schuell Schleicher and Schuell Inc., do Andermann and Company Ltd. Schleicher and Schuell Inc., Keene, NH 03431A, USA. Schleicher and Schuell Inc., D-3354 Dassel, Germany. Scientific Volume Imaging, Alexanderlaan 14, 1213 XS, Hilversum, Netherlands. Shandon Scientific Ltd., Chadwick Road, Astmoor, Runcorn, Cheshire WA7 1PR, UK. Sigma Chemical Company Sigma Chemical Company (UK), Fancy Road, Poole, Dorset BH17 7NH, UK. Sigma Chemical Company, 3050 Spruce Street, P.O. Box 14508, St Louis, MO 63178-9916, USA. Sorenson Bioscience Inc., 6507 South 400 West, Salt Lake City, UT 84107, USA. Sorvall DuPont Company, Biotechnology Division, P.O. Box 80022, Wilmington, DE 19880-0022, USA. Stratagene Stratagene Ltd., Unit 140, Cambridge Innovation Centre, Milton Road, Cambridge CB4 4FG, UK. Stratagene Inc., 11011 North Torrey Pines Road, La Jolla, CA 92037, USA. Stratech Scientific Ltd (Jackson ImmunoResearch), 61-63, Dudley St, Luton, Bedfordshire, LU2 ONP, UK. Techne Techne (Cambridge) Ltd., Duxford, Cambridge CB2 4PZ, UK. Techne Incorporated, 3700 Brunswick Pike, Princeton, New Jersey 08540-6192, USA. Ted Pella, Inc., P.O. Box 492477, Reading, CA 96049-2477, USA. 204

List of suppliers Thomas Scientific, 99 High Hill Road, PO Box 99, Swedesboro, NJ 08085, USA. Tip Top Stahlgruber, D-81675, Muenchen, Germany. United States Biochemical, P.O. Box 22400, Cleveland, OH 44122, USA. VayTek Inc., 305 West Lowe Ave, Suite 109, Fairfield, IA 52556, USA. Vector Vector Laboratories Ltd., 16 Wulfric Square, Bretton, Peterborough PE3 8RF, UK. Vector Laboratories Inc. 30 Ingold Rd, Burlingame, CA 94010, USA. Vysis Vysis (UK) Ltd., Rosedale House, Rosedale Road, Richmond, Surrey TW9 2SZ, UK. Vysis Inc., 3100 Woocreek Drive, Downers Grove, IL 60515-5400, USA. Wellcome Reagents, Langley Court, Beckenham, Kent BR3 3BS, UK. Whatman International Ltd., St Leonard's Road, 20/20 Maidstone, Kent ME16 OLS, UK. Worthington, 7 Tavistock Estate, Ruscombe Business Park, Ruscombe Lane, Twyford, Reading RG10 9NJ, UK. Xillix Technologies Corporation, #300-13775 Commercial Parkway, Richmond, BC V6V 2V4, Canada. Zeiss Optical Systems Inc., One Zeiss Drive, Thornwood, NY 10594, USA.

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Index acetone, fixation with 108-9,111 actinomycin D 62-3,77-8,161 agarose beads 151-5,159-60 alkaline agarose gel electrophoresis 42-3,45 alpha satellite 82,89-90,93^t, 100,191-3 AMCA 117,120,130 a-amanatin 161 anaphase 82-3,91-6,98,100,196 antibodies conjugated 119 coupling to protein Sepharose 52,55 antifade agents, see mountants artificial chromosomes 183 ATP 9,155-6,173,176-7 Bal 3l nuclease 184,188-91 bandshift 1 biotin, labelling with 69-70,112,127-8,161 biotin, detection of 712,75,104,116,129-30, 138,141,162-3,165 bithorax complex, Drosophila 31-4 BrdU 62,77,160 Caenorhabditis elegans 126,134 cell cycle stage 104,116 cell wall, digestion of 45-6 centromere 82-3,90,93,108,191-2,194 centromere proteins (CENPs) 82-3,90,194 CHIP (chromatin immunoprecipitation), see immunoprecipitation, chromatin chromosomal proteins 175-6 chromosome assembly in vitro 169,172-3, 175-8 chromosome banding 77-8,158,164 chromosome condensation 60-2,103,134-5, 167,169,174-6,180-1 chromosome paints 59,69,103,105,111-12 chromosome scaffold 67 chromosome stability 93-4,100,196 chromosome territories 103-4,117,164 CISS (chromosomal in situ suppression), see suppression of hybridization from repeats Colcemid 60-1,63-4, 84-5,90,92-^t colony picking 187 competitive PCR 48,55-6 confocal laser scanning microscopy (CLSM) 120-1,128,143,164 Cot-1 DNA 73,112 counterselection 191 coverslips, see slides CpG island 2,17 creatine kinase 177

CREST 82 CsCl gradients 24-8 CSF, see cytostatic factor cyanine dyes 40,69,75,120,129-30,137,163 cyclins 168,174 cytocentrifugation 83-7,94-5,106-7 cytochalasinB and D 912,96-9,170-4,193 cytokinesis 82,91-2,96,99 cytosol, Xenopus egg extract clarified 168-9, 172,176 cytostatic factor (CSF) 168 DAPI, see 4,6-diamidino-2-phenylindole deconvolution 120-1,128,134,143 depurination 66,109-10 detection of hybridized probes 47,75-6,89, 138,141 DHS, see DNase I hypersensitive sites 4,6-diamidino-2-phenylindole 47,77,86,120, 130,138,141,163 dicentric chromosomes 90,94,100 digitonin, permeabilization of cell membrane with 63-5,150 digoxigenin, labelling with 41-2,69-70,112, 127-8,161 digoxigenin, detection of 42-3,47,71-2,75, 129-30,138,141,162 dimethylsulfate, modification of DNA by 4-7 DMS, see dimethylsulfate DNA staining with fluorescent dyes 23,26,47, 77,129-30,163,165 DNase 11,2,4-5,41-2,45,69-70,147,149 DNase I hypersensitive sites (DHS) 2,4 Drosophila embryos, collection and staging of 23 cross-linking of 23-6 preparation of chromatin from 23,27-8 DT40 cells 192-3 EGS, fixation and cross-linking with 196-7 electroblotting 10-11 electroelution 154-8 electron microscopy 159-60,162 electroporation 186-7 end-labelling of DNA 3,9,12,127 ethanol, fixation with 108,135 FISH, see fluorescence in situ hybridization FITC, see fluoroscein isothiocyanate fluorescence in situ hybridization 39,46-7,59, 69,72-7,81,84,87-90,99,113-15,125, 134,137-41,157,185,188,190,193,196-7

Index fluorochromes 69,129,144 flouroscein isothiocyanate 40,75,97,100,120, 130,163 footprinting DNase I 1,4,13 dimethylsulfate 5,12,16-17 formaldehyde, cross-linking and fixation with 21-6,36-7,43,45-6,48-50,53,86-8,96-8, 108-9, 111, 133,135,163,179,196 freeze-fracture 109-10 GAGA factor 22,31-4 genomic sequencing 4 GFP, see green fluorescent protein glutaraldehyde, fixation with 45-6,133,196 green fluorescent protein 49,109 Hl kinase 168,173-4 halos 106-7,157 histones, and modifications of 36-7,49,51, 153,167 Hoechst dyes 26,130 homologous recombination 191-3 HPRT, see hypoxanthine-guanine phosphoribosyl transferase hydrazine, modification of DNA by 4 hygromycin 185,191 hypotonic, swelling of cells with 61-2,64,83-5, 94-5,99,107,148 hypoxanthine-guanine phosphoribosyl transferase 191 immunodepletion 173,180 immunofluorescence 39,49,81,83-7,92,94, 96,116,118,164,178-80 after FISH 117-19,133 before FISH 43,87,90,116,119 immunoprecipitation, of chromatin (CHIP) 21,28-9,31-3,36,39,48-9,51-5 interphase extract, see Xenopus egg extract activated interphase interspersed repetitive sequences (IRS) 59,112 IP, see immunoprecipitation kinasing 9,14-17 kinetochore 81-3,169 LCR, see locus control regions ligation of DNA 3,8,30 ligation-mediated polymerase chain reaction (LMPCR) 1,3-4,8-13,17 lipofection 186 LIS, 3,5-diiodosalicylic acid 68,107,150,153 LMPCR, see ligation-mediated polymerase chain reaction

locus control regions (LCRs) 2 loop size 157-8 low gelling temperature agarose 152-3 matrix-associated regions (MARs) 59,106, 153,157 Maxam and Gilbert chemical cleavage 4 meiosis 126,168-9 metaphase chromosomes extraction of 66-8 preparation from blood 60-3 preparation from cultured vertebrate cells 62-4,84-5 preparation from in vitro assembly reactions 173,175-8 spreading of 65-6,83-5 structure of 67,83,169 treatment with RNase 66-7 methanol, fixation with 94-5,108-9, 111, 133-4 methanol:acetic acid, fixation with 61-2,65-6, 87-8,94,98-9,108,133 methotrexate (MTX) 62-3 methylcytosine 4 microcell-mediated chromosome transfer 192-3 micrococcal nuclease (MNase) 2,5,13-17 microtubules (MTs) 81-3,91,94-7 inhibitors of polymerization, see Colcemid; nocodazole; vinblastine mitotic extract, see Xenopus egg extracts, CSF(mitotic) mitotic index (MI) 60,63-4,84,94 mitotic shake-off 64,94-5 mitotic spindle 60,81-3,94 MNase, see micrococcal nuclease 78 mountants 47,78,86,119-20,137 nick translation 41,69 nocodazole 91,94-5,97-9 nuclear envelope/lamina or periphery 82,108, 117,133,159,172 nuclear matrix 67,106-7,147-9,153,157, 165 nuclear scaffold 148,150,153,157,159,165 nuclear speckles 164-5 nuclease, see DNase I; micrococcal nuclease 2 nuclease hypersensitive sites 2,4,16-17 nuclei, isolation of 13-14,142,149 nuclei, preservation of structure in 103-8,110, 125,133-5,147,151,154 nucleoids 149,153,157 nucleolus 118-19,159,165 nucleoskeleton 147-8,151,157,165 nucleosome ladder 2,14-16, nucleotides, conjugated 41,69,112,127-9, 161-2 optical sectioning microscopy 128,134,165

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Index paraformaldehyde, see formaldehyde PC, see Polycomb PCR, see polymerase chain reaction pepsin, digestion with 111 peptide nuceic acid (PNA) probes 144 permeabilizing cells nuclei and tissues 24-5, 45-6,107,109,133,151,160-1,163,196 phenylmethylsulfonyl fluoride (PMSF) 29,67, 148,150,194-5 phosphocreatine 173,177 phosphorimaging 56 piperidine, cleavage of DNA with 4-6 plasmid-rescue 185 PMSF, see phenylmethylsulfonyl fluoride polyacrylamide gel electrophoresis (PAGE) 55,164 Polycomb 21,31-4 polylysine, coating of slides or coverslips with 47,106,136,179 polymerase chain reaction 22,28,31—2,48, 70-1 see also ligation mediated polymerase chain reaction; competitive PCR probe denaturation 47,74,89,99,113,138,140 probe labelling by end-labelling 127,131-2 by nick translation 41-2,45,69,112,127 by PCR 70,112,127 by random priming 70,127 probe size for FISH 40-4,69-70,112,127 propidium iodide 77,129-30,138 protein A Sepharose 29,52-3,55 protein G Sepharose 52-3 proteinase K digestion 7,14-15,27-9,53-4, 156,194-5 pulsed-field gel ecetrophoresis 194-5 radiolabelling DNA in vivo 155,162 replication, sites of 148-50,159-62 rhodamine 69,100,120,130 RN A polymerase 160-2 RNase, treatment of slides with 66,137,110, 140

denaturation of 72-4, 87-8,99,113-15,138, 140,142 freezing 66,109-10,136,142 growing and attaching cells on 105-6,135-6, 139,163 hybridizing probes to 73-4,89,99,115,140, 142 incubation of antibodies with 75-7,86,89,140 mounting tissue onto 136,138-9 washing of 75-6,87,89,115,140 somatic cell hybrids 87,111-12,193-4,196 sonication 25-6,28,48,50-1 Southern hybridization 2,11,17,22,31-6,55, 154,156,184,188,190,195 SpI transcription factor 11-13,16-17 spindle pole 82,91 sucrose step gradient 64-5,149-50 suppression of hybridization from repeats 73, 112 Swi6 protein 48-9,52 synchronization of cells 62-3 telomeres, detection of 188-91 telomere formation 184-7,191 telomere structure 183,188 telophase 82-3,92-3,100,196 tetramethylirhodamine isothiocyanate (TRITC) 69 Texas Red 69,97,117,120 topoisomerase II 157 transcription, sites of 148-50,157,159-62, 164-5 transfections, see lipofection; electroporation TRITC, see tetramethylirhodamine isothiocyanate tubulin, see microtubules tyramide 185 vinblastine 91

S phase 62-3,104,116 salt extraction of chromosomes or nuclei 67-9, 106-7,147-50,153,159-60 scaffold-associated regions (SARs) 59,153, 157 scintillation counting 154,156 Sephadex G5O 41,69 Silent Information Regulators 21 SIR, see Silent Information Regulators slides and coverslips cleaning 65,84,105,179 counterstaining 77,86,89,141 dehydration of 66,74,88,99,107,142

whole-mount tissues detection of fluorescence in 130,143 penetration into 128,131,133,135 preparation of for FISH 125,133,135-7, 139-40,143 Xenopus egg extracts activated (interphase) 168,173-5 CSF(mitotic) 168-75 Xenopus eggs, collection of 168-70 Xenopus sperm nuclei 168,172,175-7 yeast cell lysis 50-1

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