chromatin and chromatin remodeling enzymes, part a

Preface A central challenge of the post-genomic era is to understand how the 30,000 to 40,000 unique genes in the human genome are selectively expressed or silenced to coordinate cellular growth and differentiation. The packaging of eukaryotic genomes in a complex of DNA, histones, and nonhistone proteins called chromatin provides a surprisingly sophisticated system that plays a critical role in controlling the flow of genetic information. This packaging system has evolved to index our genomes such that certain genes become readily access- ible to the transcription machinery, while other genes are reversibly silenced. Moreover, chromatin-based mechanisms of gene regulation, often involving domains of covalent modifications of DNA and histones, can be inherited from one generation to the next. The heritability of chromatin states in the absence of DNA mutation has contributed greatly to the current excitement in the field of epigenetics. The past 5 years have witnessed an explosion of new research on chroma- tin biology and biochemistry. Chromatin structure and function are now widely recognized as being critical to regulating gene expression, maintaining genomic stability, and ensuring faithful chromosome transmission. Moreover, links be- tween chromatin metabolism and disease are beginning to emerge. The identi- fication of altered DNA methylation and histone acetylase activity in human cancers, the use of histone deacetylase inhibitors in the treatment of leukemia, and the tumor suppressor activities of ATP-dependent chromatin remodeling enzymes are examples that likely represent just the tip of the iceberg. As such, the field is attracting new investigators who enter with little first hand experience with the standard assays used to dissect chromatin structure and function. In addition, even seasoned veterans are overwhelmed by the rapid introduction of new chromatin technologies. Accordingly, we sought to bring together a useful ‘‘go-to’’ set of chromatin-based methods that would update and complement two previous publications in this series, Volume 170 (Nucleosomes) and Volume 304 (Chromatin). While many of the classic proto- cols in those volumes remain as timely now as when they were written, it is our hope the present series will fill in the gaps for the next several years. This 3-volume set of Methods in Enzymology provides nearly one hundred procedures covering the full range of tools—bioinformatics, structural biology, biophysics, biochemistry, genetics, and cell biology—employed in chromatin research. Volume 375 includes a histone database, methods for preparation of xv histones, histone variants, modified histones and defined chromatin segments, protocols for nucleosome reconstitution and analysis, and cytological methods for imaging chromatin functions in vivo. Volume 376 includes electron micro- scopy and biophysical protocols for visualizing chromatin and detecting chro- matin interactions, enzymological assays for histone modifying enzymes, and immunochemical protocols for the in situ detection of histone modifications and chromatin proteins. Volume 377 includes genetic assays of histones and chromatin regulators, methods for the preparation and analysis of histone modifying and ATP-dependent chromatin remodeling enzymes, and assays for transcription and DNA repair on chromatin templates. We are exceedingly grateful to the very large number of colleagues representing the field’s leading laboratories, who have taken the time and effort to make their technical expertise available in this series. Finally, we wish to take the opportunity to remember Vincent Allfrey, Andrei Mirzabekov, Harold Weintraub, Abraham Worcel, and especially Alan Wolffe, co-editor of Volume 304 (Chromatin). All of these individuals had key roles in shaping the chromatin field into what it is today. C. David Allis Carl Wu Editors’ Note: Additional methods can be found in Methods in Enzymology, Vol. 371 (RNA Polymerases and Associated Factors, Part D) Section III Chromatin, Sankar L. Adhya and Susan Garges, Editors. xvi preface METHODS IN ENZYMOLOGY EDITORS-IN-CHIEF John N. Abelson Melvin I. Simon DIVISION OF BIOLOGY CALIFORNIA INSTITUTE OF TECHNOLOGY PASADENA, CALIFORNIA FOUNDING EDITORS Sidney P. Colowick and Nathan O. Kaplan Contributors to Volume 375 Article numbers are in parentheses and following the names of contributors. Affiliations listed are current. Chad Alexander (3), The University of Tennessee-Oak Ridge Graduate School of Genome Science and Technology, Oak Ridge National Laboratory, Life Sciences Division, Oak Ridge, Tennessee 37831-8080 Genevie ` ve Almouzni (8), Institut Curie, Section de Recherche, F-75248, Paris Cedex 05, France Satoshi Ando (18), Department of Mo- lecular Life Science, School of Medicine, Tokai University, Kanagawa 259-1193, Japan Yunhe Bao (2), Department of Biochemis- try and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870 Blaine Bartholomew (13), Department of Biochemistry & Molecular Biology, Southern Illinois University School of Medicine, Carbondale, Illinois 62901-4413 David P. Bazett-Jones (28), Programme in Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada Andrew S. Belmont (23), Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 Leise Berven (16), Children’s Medical Re- search Institute, Westmead, New South Wales 2415, Australia Yehudit Birger (21), National Cancer In- stitute, National Institutes of Health, Bethesda, Maryland 20892 Hinrich Boeger (11), Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305 William M. Bonner (5), Laboratory of Molecular Pharmacology, National Cancer Institute, Bethesda, Maryland 20892 Michael Bruno (14), Division of Gene Regulation and Expression, The Well- come Trust Biocentre, Department of Biochemistry, University of Dundee, Dundee, DD1 5EH, Scotland, United Kingdom. Gerard J. Bunick (3), Life Sciences Div- ision, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-8080 Michael Bustin (21), National Cancer In- stitute, National Institutes of Health, Bethesda, Maryland 20892 Anne E. Carpenter (23), Whitehead Insti- tute forBiomedical Research, Cambridge, Massachusetts 02142 Gustavo Carrero (26), Department of Mathematical and Statistical Sciences, Faculty of Science, University of Alberta, Edmonton, Alberta T6G 2E1, Canada David Carter (29), Laboratory of Chro- matin and Gene Expression, Babraham Institute, Cambridge CB2 4AT, United Kingdom Fre ´ de ´ ric Catez (21), National Cancer In- stitute, National Institutes of Health, Bethesda, Maryland 20892 ix Lyubomira Chakalova (29), Laboratory of Chromatin and Gene Expression, Bab- raham Institute, Cambridge CB2 4AT, United Kingdom Srinivas Chakravarthy (2), Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870 Lakshmi N. Changolkar (15), Depart- ment of Animal Biology, School of Veter- inary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Lisa Ann Cirillo (9), Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Washington, Milwaukee, Wisconsin 53149 Peter R. Cook (24), The Sir William Dunn SchoolofPathology,UniversityofOxford, Oxford OX1 3RE, United Kingdom Ellen Crawford (26), Department of On- cology, Faculty of Medicine, Universityof Alberta and Cross Cancer Institute, Edmonton, Alberta T6G 2E1, Canada Wouter de Laat (30), Department of Cell Biology, ErasmusMC, 3015 GE Rotter- dam, The Netherlands Gerda de Vries (26), Department of Math- ematical and Statistical Sciences, Faculty of Science, University of Alberta, Edmon- ton, Alberta T6G 2E1, Canada Graham Dellaire (28), Programme in Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada John D. Diller (10), Department of Bio- chemistry and Molecular Biology, Center for Gene Regulation, The Pennsylvania State University, University Park, Pennsylvania 16802 Charles E. Ducker (10), Department of Biochemistry and Molecular Biology, Center for Gene Regulation, The Pennsyl- vania State University, University Park, Pennsylvania 16802 Pamela N. Dyer (2), Department of Bio- chemistry and Molecular Biology, Color- ado State University, Fort Collins, Colorado 80523-1870 Raji S. Edayathumangalam (2), Depart- ment of Biochemistry and Molecular Biol- ogy, Colorado State University, Fort Collins, Colorado 80523-1870 Thomas G. Fazzio (6), Fred Hutchinson Cancer Research Center, Seattle, Wash- ington 98109-1024 Andrew Flaus (14), Division of Gene Regulation and Expression, The Well- come Trust Biocentre, Department of Bio- chemistry, University of Dundee, Dundee, DD1 5EH, Scotland, United Kingdom. Peter Fraser (29), Laboratory of Chroma- tin and Gene Expression, Babraham Insti- tute, Cambridge CB2 4AT, United Kingdom Susan M. Gasser (22), Department of Mo- lecular Biology, University of Geneva, 1211 Geneva 4, Switzerland Stanislaw A. Gorski (25), National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 Joachim Griesenbeck (11), Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305 Frank Grosveld (30), Department of Cell Biology, ErasmusMC, 3015 GE Rotter- dam, The Netherlands B. Leif Hanson (3), The University of Ten- nessee-Oak Ridge Graduate School of Genome Science and Technology, Life Sciences Divison, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-8080 Joel M. Harp (3), Department of Bio- chemistry and Center for Structural Biol- ogy, Vanderbilt University, Nashville, Tennessee 37232-8725 x contributors to volume 375 Keiji Hashimoto (17), Core Research for Evolutional Science and Technology, Saitama 332-0012, Japan Jeffrey J. Hayes (12), Department of Bio- chemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642 Florence Hediger (22), Department of Molecular Biology, University of Geneva, 1211 Geneva 4, Switzerland Michael J. Hendzel (26), Department of Oncology, University of Alberta and Cross Cancer Instutite, Edmonton, Alberta T6G 1Z2, Canada Miki Hieda (24), Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom Stefan R. Kassabov (13), Department of Biochemistry & Molecular Biology, Southern Illinois University School of Medicine, Carbondale, Illinois 62901-4413 Hiroshi Kimura (24), Horizontal Medical Research Organization, School of Medi- cine, Kyoto University, Kyoto 606-8510, Japan Roger D. Kornberg (11), Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305 David Landsman (1) National Center for Biotechnology Information, National Li- brary of Medicine, National Institutes of Health, Bethesda, Maryland 20894 Paul J. Laybourn (7), Department of Bio- chemistry and Molecular Biology, Color- ado State University, Fort Collins, Colorado 80523-1870 Jae-Hwan Lim (21), National Cancer Insti- tute, National Institutes of Health, Bethesda, Maryland 20892 Karolin Luger (2), Department of Bio- chemistry and Molecular Biology, Color- ado State University, Fort Collins, Colorado 80523-1870 James G. McNally (27), Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Insti- tutes of Health, Bethesda, Maryland 20892 Tom Misteli (25) National Cancer Insti- tute, National Institutes of Health, Bethesda, Maryland 20892 Craig A. Mizzen (19), Department of Cell & Structural Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 Setsuo Morishita (17), Department of Mo- lecularBiology, School of Science, Nagoya University, Nagoya 464-8601, Japan Uma M. Muthurajan (2), Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870 Frank R. Neumann (22), Department of Molecular Biology, University of Geneva, 1211 Geneva 4, Switzerland Rozalia Nisman (28), Programme in Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada Tom Owen-Hughes (14), Division of Gene Regulation and Expression, The Well- come Trust Biocentre, Department of Bio- chemistry, University of Dundee, Dundee, DD1 5EH Scotland, United Kingdom. John R. Pehrson (15), Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Craig L. Peterson (4) University of Mas- sachusetts Medical School, Worchester, Massachusetts 01605 contributors to volume 375 xi Robert D. Phair (25), BioInformatics Ser- vices, Rockville, Maryland 20854 Duane R. Pilch (5), Laboratory of Mo- lecular Pharmacology, National Cancer Institute, Bethesda, Maryland 20892 Yuri V. Postnikov (21), National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 Danny Rangasamy (16), The John Curtin School of Medical Research, Australian National University, Canberra, Australia Capital Territory 2601, Australia Dominique Ray-Gallet (8), Institut Curie, Section de Recherche, F-75248, Paris Cedex 05, France Christophe Redon (5), Laboratory of Mo- lecular Pharmacology, National Cancer Institute, Bethesda, Maryland 20892 Raymond Reeves (20), School of Molecu- lar Biosciences, Biochemistry/Biophysics, Washington State University, Pullman, Washington 99164-4660 Patricia Ridgway (16), The John Curtin School of Medical Research, Australian National University, Canberra, Austra- lian Capital Territory 2601, Australia Chun Ruan (10), Department of Biochem- istry and Molecular Biology, Center for Gene Regulation, The Pennsylvania State University, University Park, Pennsylvania 16802 Olga A. Sedelnikova (5), Laboratory of Molecular Pharmacology, National Cancer Institute, Bethesda, Maryland 20892 Michael A. Shogren-Knaak (4), Univer- sity of Massachusetts Medical School, Worchester, Massachusetss 01605 Robert T. Simpson (10), Department of Biochemistry and Molecular Biology, Center for Gene Regulation, The Pennsyl- vania State University, University Park, Pennsylvania 16802 Erik Splinter (30), Department of Cell Biology, ErasmusMC, 3015 GE Rotter- dam, The Netherlands Diana A. Stavreva (27), Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Insti- tutesofHealth,Bethesda,Maryland 20892 J. Seth Strattan (11), Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305 Steven A. Sullivan (1), National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894 Ulrica Svensson (16), The John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory 2601, Australia Angela Taddei (22), Department of Mo- lecular Biology, University of Geneva, 1211 Geneva 4, Switzerland John Th’ng (26), Northwestern Ontario Regional Cancer Centre, Thunder Bay, Ontario P7A 7T1, Canada David John Tremethick (16), The John Curtin School of Medical Research, Aus- tralian National University, Canberra, Australian Capital Territory 2601, Australia Toshio Tsukiyama (6), Fred Hutchinson Cancer Research Center, Seattle, Wash- ington 98109-1024 Jay C. Vary,Jr. (6), Molecular and Cellu- lar Biology Program, University of Washington, Seattle, Washington 98195 Cindy L. White (2), Department of Bio- chemistry and Molecular Biology, Color- ado State University, Fort Collins, Colorado 80523-1870 Sriwan Wongwisansri (7), Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870 xii contributors to volume 375 Kinya Yoda (17, 18), Bioscience and Bio- technology Center, Nagoya University, Nagoya, 464-8601, Japan Kenneth S. Zaret (9), Cell and Devel- opmental Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylva- nia 19111 Chunyang Zheng (12), Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642 contributors to volume 375 xiii [1] Mining Core Histone Sequences from Public Protein Databases By Steven A. Sullivan and David Landsman Introduction Constructing an online database of histones and histone fold-containing proteins has allowed our group to analyze histone sequence variation in some detail. 1,2 Here, we describe how we have inventoried core histone protein sequences as part of this project. The issues involved in such an undertaking are for the most part not unique to histone sequences. Our methods and observations should be broadly applicable to studies of protein families that are highly represented in public sequence databases. Considerations Our initial goal was to collect as many reported histone sequences as we could find. Among the considerations that came into play were the following. 1. Sourcing of sequences. Several excellent public sequence reposi- tories make protein sequences available to researchers. We relied on the protein database maintained by the National Center for Biotechnology Information (NCBI), which is updated frequently and has been compiled from worldwide sources, including Swiss-Prot, 3 the Protein Information Resource (PIR), 4 the Protein Research Foundation (PRF) (http:// www.prf.or.jp/en/), the Protein Data Bank (PDB), 5 and translations from annotated coding regions in GenBank 6 and RefSeq, 7 a curated, nonredundant set of sequences. 1 S. Sullivan, D. W. Sink, K. L. Trout, I. Makalowska, P. M. Taylor, A. D. Baxevanis, and D. Landsman, Nucleic Acids Res. 30, 341 (2002). 2 S. A. Sullivan and D. Landsman, Proteins 52, 454 (2003). 3 B. Boeckmann, A. Bairoch, R. Apweiler, M. C. Blatter, A. Estreicher, E. Gasteiger, M. J. Martin, K. Michoud, C. O’Donovan, I. Phan, S. Pilbout, and M. Schneider, Nucleic Acids Res. 31, 365 (2003). 4 C. H. Wu, L. S. Yeh, H. Huang, L. Arminski, J. Castro-Alvear, Y. Chen, Z. Hu, P. Kourtesis, R. S. Ledley, B. E. Suzek, C. R. Vinayaka, J. Zhang, and W. C. Barker, Nucleic Acids Res. 31, 345 (2003). 5 J. Westbrook, Z. Feng, L. Chen, H. Yang, and H. M. Berman, Nucleic Acids Res. 31, 489 (2003). [1] mining core histone sequences from public protein databases 3 METHODS IN ENZYMOLOGY, VOL. 375 0076-6879/04 $35.00 2. Sequence-harvesting tools. In general, a sequence database search is a similarity search of either the actual sequence data or its annotation. We find that both must be targeted in order to maximize the sequence harvest, because sequence-based searches alone can miss small or ambiguous sequence fragments that have been deposited in the public databases, and text-based searches can miss ‘‘cryptic’’ histones, that is, those with inadequate or incorrect annotation. For text-based searches of sequence annotation we used the Entrez search engine at the NCBI Web site (http://www.ncbi.nlm.nih.gov/Entrez). For sequence-based searching we used several varieties of the popular Basic Local Alignment Search Tool (BLAST) pairwise alignment algo- rithm. The most commonly used sequence similarity search tools find ‘‘hits’’ based on pairwise alignments of each sequence in the database to either the query sequence alone, for example, in the case of BLAST, or a query profile derived from a previously aligned set of similar sequences, for example, in the case of PSI-BLAST or HMMER. 8,9 The latter tools are better at finding highly divergent members of a protein family but can be expected to return false positives, requiring further filtering of results. PSI-BLAST is actually a hybrid tool that performs one round of standard BLAST, using a user-supplied query sequence, and then builds a profile from the alignment of the initial BLAST results, which becomes the query for the next round of BLAST. The process is reiterated until ‘‘conver- gence’’ is reached, that is, until no more new matches are found above the cutoff score. Ideally this should take fewer than 10 iterations, but con- vergence can be elusive when the query sequence matches a diverse and perhaps distantly related set of proteins. This was more difficult to interpret with searches for nonhistone proteins containing the histone fold than for harvesting core histone sequences. With the latter we found that seven iter- ations were sufficient to reach either convergence or the point at which all the ‘‘new’’ hits appeared by other criteria to be false positives. PSI-BLAST routinely returned a small number of true-positive matches to the query sequences that gapped protein BLAST (BLASTPGP) had missed. Reasonably fast BLASTPGP and PSI-BLAST servers are available at the NCBI Web site (http://www.ncbi.nlm.nih.gov/BLAST). One advantage of the NCBI Web site PSI-BLAST implementation over a command-line 6 D. A. Benson, I. Karsch-Mizrachi, D. J. Lipman, J. Ostell, and D. L. Wheeler, Nucleic Acids Res. 31, 23 (2003). 7 K. D. Pruitt, T. Tatusova, and D. R. Maglott, Nucleic Acids Res. 31, 34 (2003). 8 S. F. Altschul, T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman, Nucleic Acids Res. 25, 3389 (1997). 9 S. R. Eddy, Bioinformatics 14, 755 (1998). 4 histone bioinformatics [1] [...]... Nucleosome Core Particles from Recombinant Histones and DNA By Pamela N Dyer, Raji S Edayathumangalam, Cindy L White, Yunhe Bao, Srinivas Chakravarthy, Uma M Muthurajan, and Karolin Luger Introduction The ability to prepare nucleosome core particles (NCPs), or nucleosomal arrays, from recombinant histone proteins and defined-sequence DNA has become a requirement in many projects that address the role... Entrez ‘‘eukaryota[ORGN]’’ ‘‘H3’’ ‘‘histon’’ ‘‘eukaryota[ORGN] and H3’’ ‘‘eukaryota[ORGN] and histon’’ BLASTPGP BLASTPGP BLASTPGP BLASTPGP BLASTPGP BLASTPGP BLASTPGP BLASTPGP H3human H3humanþseg H3humanþeukgi H3humanþeukgiþseg H3yeast H3yeastþseg H3yeastþeukgi H3yeastþeukgiþseg PSIBLASTPGP PSIBLASTPGP PSIBLASTPGP PSIBLASTPGP PSIBLASTPGP PSIBLASTPGP PSIBLASTPGP PSIBLASTPGP H3human H3humanþseg H3humanþeukgi... evident in a true frameshift Several tools exist on the Web for doing such translations; we commonly use the one at the ExPASy (Expert Protein Analysis) Web site: http://us.expasy.org/tools/dna.html A translation tool is also available in the SEALS package Comparison of Search Strategies There are many available variations on the basic BLAST search protocol We investigated several parameters for their... protein databases 5 version is that the user can edit each set of aligned sequences before it is used to generate a profile This can redirect a diverging sequence search back toward convergence Unfortunately, however, it can also happen that a valid match from one iteration falls below the noise cutoff in the next, and in the WWW-based implementation, that match is lost Therefore we ran PSI-BLAST (and BLASTPGP)... fungal H 2A subclass clusters near the H 2A. X subclass, and also features a conserved SQ motif at its C terminus H 2A. F/Z sequences constitute another pan-eukaryotic subclass and are necessary but not sufficient for H 2A function in organisms tested Characteristic H 2A. F/Z residues in a C-terminal, H3-binding portion of the protein (positions 145–193 in Fig 1A) have been suggested to impart a specific, although... Nucleic Acids Res 25, 4876 (1997) [1] mining core histone sequences from public protein databases 7 To verify a frameshift, assuming access to the genomic DNA or cDNA record for the protein (which are often, but not always, available in public databases), one should translate the DNA in all frames and add those conceptual translations to the alignment; the correct frames will be visually evident in a true... For all core histone classes, sequence alignments show clear distinctions between metazoan, plant, fungal, and various basal eukaryote subclasses Distinct subclasses within the metazoan sequences are also common (e.g., insect or echinoderm sequences) Nomenclature is only occasionally helpful in classifying histone variants It is not standardized, and thus ‘‘H3.2’’ in one species may not be similar to... blue, basic; red, acidic (B) C-terminal section of macroH 2A subclasses (Fig 1A and B) H 2A. X is found in species spanning the eukaryotic spectrum and features a conserved serine four residues from the carboxyl terminus (part of an SQ motif, positions 208 and 209 in Fig 1A) that is phosphorylated in response to double-stranded DNA breaks, perhaps marking the site for repair (reviewed in Redon et al.16)... modifications, histone variants, or histone mutations in nucleosome and chromatin structure This approach offers many advantages, such as the ability to combine histone variants and tail deletion mutants, and the opportunity to study the effect of individual histone tail modifications on nucleosome structure and function We have previously described comprehensive protocols for the expression and purification... as given by the manufacturer Incubate at room temperature for at least 15 h, and check completion of ligation by PAGE Add more ligase if necessary 10 If necessary, purify ligated from unligated fragments by ionexchange chromatography on a TSK-DEAE column (or another ion-exchange column of similarly high resolution) This separation depends strongly on the DNA sequence and must be optimized individually . assays of histones and chromatin regulators, methods for the preparation and analysis of histone modifying and ATP-dependent chromatin remodeling enzymes, and assays for transcription and DNA. United Kingdom Fre ´ de ´ ric Catez (21), National Cancer In- stitute, National Institutes of Health, Bethesda, Maryland 20892 ix Lyubomira Chakalova (29), Laboratory of Chromatin and Gene Expression, Bab- raham Institute,. Rotter- dam, The Netherlands Gerda de Vries (26), Department of Math- ematical and Statistical Sciences, Faculty of Science, University of Alberta, Edmon- ton, Alberta T6G 2E1, Canada Graham Dellaire