chromatin and chromatin remodeling enzymes, part c

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 377 Article numbers are in parentheses and following the names of contributors. Affiliations listed are current. Woojin An (30), Laboratory of Biochemis- try and Molecular Biology, The Rocke- feller University, New York, New York 10021 Jennifer A. Armstrong (4), Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, California 95064 * Orr G. Barak (25), The Wistar Institute, Philadelphia, Pennsylvania 19104 Brian C. Beard (32), Department of Bio- chemistry and Biophysics, School of Mo- lecular Biosciences, Washington State University, Pullman, Washington 99164 –4660 Peter B. Becker (21), Adolf-Butenandt- Institut, Molekularbiologie, Mu ¨ nchen D-80336, Germany Shelley L. Berger (7), The Wistar Insti- tute, Philadelphia, Pennsylvania 19104 Tiziana Bonaldi (6), Protein Analysis Unit, Adolf-Butenandt Institut, Ludwig Maximillians Universita ¨ t, Mu ¨ nchen,80336 Mu ¨ nchen, Germany Ludmila Bozhenok (24), Chromatin Lab, Marie Curie Research Institute, Surrey RH8 0TL, United Kingdom Eli Canaani (15), Department of Mole- cular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel Brad Cairns (20), University of Utah School of Medicine, Department of Onco- logical Sciences, Howard Hughes Medical Institute and Huntsman Cancer Institute, Salt Lake City, Utah 84112 Yuh-Long Chang (16), Institute of Mo- lecular Biology, Academia Sinica, Taiwan 115, Republic of China Gillian E. Chalkley (28), Gene Regula- tion Laboratory, Center for Biomedical Genetics, Department of Molecular and Cell Biology, Leiden University Medical Center, 2300 RA Leiden, The Netherlands Nadine Collins (24), Chromatin Lab, Marie Curie Research Institute, Surrey RH8 0TL, United Kingdom À Davide F. V. Corona (4), Department of Molecular, Cell and Developmental Biol- ogy, University of California, Santa Cruz, Santa Cruz, California 95064 Jacques Co ¨ te ´ (8), Laval University Cancer Research Center, Quebec, GIR 2J6 Canada Tianhuai Chi (18), Howard Hughes Medical Institute, Stanford University, Stanford, California 94305 ` Carlo M. Croce (15), Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 *Current Affiliation: Joint Science Department, W. M. Keck Sceince Center, The Claremont Colleges, Claremont, California 91711 À Current Affiliation: Cellular Pathology, Royal Surrey County Hospital, Guildford, United Kingdom ` Current Affiliation: Section of Immunology, Yale University School of Medicine, New Haven, Connecticut 06520 ix Franck Dequiedt (10), Molecular and Cellular Biology Unit, Faculty of Agron- omy, Gembloux B-5030, Belgium Jim Dover (13), Department of Genetics, Washington University School of Medi- cine, St. Louis, Missouri 63110 Yannick Doyon (8), Laval University Cancer Research Center, Quebec, GIR 2J6 Canada Anton Eberharter (21), Adolf-Butenandt- Institut, Molekularbiologie, Mu ¨ nchen D-80336, Germany Stuart Elgar (23), Emory University School of Medicine, Department of Pathology and Laboratory Medicine, Atlanta, Georgia 30322 Yuhong Fan (5), Department of Cell Biol- ogy, Albert Einstein College of Medicine, Bronx, New York 10461 Jia Fang (12), Lineberger Comprehensive Cancer Center, Department of Biochem- istry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599–7295 Wolfgang Fischle (10), Laboratory of Chromatin Biology, The Rockefeller Uni- versity, New York, New York 10021 Roy Frye (10), VA Medical Center, Pitts- burgh, Pennsylvania 15240 Sunil Gangadharan (14), National Insti- tute of Child Health and Human Development, Unit on Chromatin and Transcription, Bethesda, Maryland 20892 Sonja Ghidelli (14), National Institute of Child Health and Human Development, Unit on Chromatin and Transcription, Bethesda, Maryland 20892 § Patrick A. Grant (8), University of Virgi- na School of Medicine, Charlottesville, Virginia 22908 Karien Hamer (17), Swammerdam Insti- tute for Life Sciences, University of Amsterdam, 1018 TV Amsterdam, The Netherlands Ali Hamiche (22), Institut Andre Lwoff, 94800 Villejuif, France Shu He (31), Johnson Research Founda- tion, Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104–6059 Karl W. Henry (7), The Wistar Institute, Philadelphia, Pennsylvania 19104 Der Hwa-Huang (16), Institute of Molecu- lar Biology, Academia Sinica, Taiwan 115, Republic of China Axel Imhof (6), Histone Modifications Group, Adolf-Butenandt Institut, Ludwig Maximillians Universita ¨ t, Mu ¨ nchen, 80336 Mu ¨ nchen, Germany Sandra J. Jacobson (1), Department of Biology, University of California, San Diego, La Jolla, California 92093–0347 Mark Johnston (13), Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110 Rohinton T. Kamakaka (14), National Institute of Child Health and Human De- velopment, Unit on Chromatin and Tran- scription, Bethesda, Maryland 20892 Mikhail Kashlev (29), National Cancer Institute Center for Cancer Research, Na- tional Cancer Institute-Frederick Cancer Research and Development Center, Fred- erick, Maryland 21702 James A. Kennison (3), Laboratory of Mo- lecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Marlyland, 20892–2785 § Current Affiliation: Cellzome AG, 69117 Heidelberg, Germany x contributors to volume 377 Roger D. Kornberg (19), Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305 Wladyslaw Krajewski (15), Kimmel Cancer Center,Thomas Jefferson Univer- sity, Philadelphia, Pennsylvania 19107 { Ted H. J. Kwaks (17), Swammerdam Insti- tute for Life Sciences, University of Amsterdam, 1018 TV Amsterdam, The Netherlands Gernot La ¨ ngst (21), Adolf-Butenandt- Institut, Molekularbiologie, Mu ¨ nchen D- 80336, Germany Patricia M. Laurenson (1), Department of Biology, University of California, San Diego, La Jolla, California 92093–0347 Hong Liu (27), Laboratory of Molecular Immunology,NationalInstitutesofHealth, Bethesda, Maryland 20892–1674 Wan-Sheng Lo (7), The Wistar Institute, Philadelphia, Pennsylvania 19104 Lorraine Pillus (1), Department of Biol- ogy, University of California, San Diego, La Jolla, California 92093–0347 Yahli Lorch (19), Department of Struc- tural Biology, Stanford University School of Medicine, Stanford, California 94305 Romain Loury (11), Institut de Ge ´ ne ´ tique et de Biologie Moleculaire et Cellulaire, 67404 Illkirch, Strasbourg, France Alejandra Loyola (31), Howard Hughes Medical Institute, Division of Nucleic Acids Enzymology, Department of Bio- chemistry, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854–5635 Brett Marshall (10), Gladstone Institute of Virology and Immunology, University of California, San Francisco, San Francisco, California 94103 Alxander Mazo (15), Kimmel Cancer Center, Department of Microbiology and Immunology, Thomas Jefferson Univer- sity, Philadelphia, Pennsylvania 19107 Stacey McMahon (8), University of Virgi- na School of Medicine, Charlottesville, Virginia 22908 Dewey G. McCafferty (31), Johnson Re- search Foundation, Department of Bio- chemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104–6059 Tatsuya Nakamura (15), Kimmel Cancer Center, Department of Microbiology and Immunology, Thomas Jefferson Univer- sity, Philadelphia, Pennsylvania 19107 Brian North (10), Gladstone Institute of Virology and Immunology, University of California, San Francisco, San Francisco, California 94103 Santaek Oh (31), Howard Hughes Medical Institute, Division of Nucleic Acids En- zymology, Department of Biochemistry, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Med- ical School, Piscataway, New Jersey 08854–5635 Erin K. O’Shea (2), Howard Hughes Medical Institute, University of Califor- nia, San Francisco, Department. of Bio- chemistry and Biophysics, San Francisco, California 94143–2240 Arie P. Otte (17), Swammerdam Institute for Life Sciences, University of Amster- dam, 1018 TV Amsterdam, The Nether- lands Matthew B. Palmer (23), Emory Univer- sity School of Medicine, Department of Pathology and Laboratory Medicine, Atlanta, Georgia 30322 Svetlana Petruk (15), Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 { Current Affiliation: Institute of Developmental Biology, Moscow 117808, Russia contributors to volume 377 xi Raymond Poot (24), Chromatin Lab, Marie Curie Research Institute, Surrey RH8 0TL, United Kingdom Danny Reinberg (31), Howard Hughes Medical Institute, Division of Nucleic Acids Enzymology, Department of Bio- chemistry, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854–5635 Jo ¨ rg T. Regula (6), Protein Analysis Unit, Adolf-Butenandt Institut, Ludwig Maxi- millians Universita ¨ t, Mu ¨ nchen, 80336 Mu ¨ nchen, Germany Natalie Rezai-Zadeh (9), H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612 Robert Roeder (30), Head, Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, New York 10021 Anjanabha Saha (20), University of Utah School of Medicine, Department of Onco- logical Sciences, Howard Hughes Medical Institute and Huntsman Cancer Institute, Salt Lake City, Utah 84112 Paolo Sassone-Corsi (11), Institut de Ge ´ - ne ´ tique et de Biologie Moleculaire et Cel- lulaire, 67404 Illkirch,Strasbourg, France Jessica Schneider (13), Saint Louis Uni- versity School of Medicine, Department of Biochemistry, St. Louis, Missouri 63104 Marc F. Schwartz (7), The Wistar Insti- tute, Philadelphia, Pennsylvania 19104 Yurii Sedkov (15), Kimmel Cancer Center, Thomas Jefferson University, Phila- delphia, Pennsylvania 19107 Edward Seto (9), H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612 Richard G. A. B. Sewalt (17), Swammer- dam Institute for Life Sciences, University of Amsterdam, 1018 TV Amsterdam, The Netherlands Xuetong Shen (26), Department of Car- cinogenesis, University of Texas, M.D. Anderson Cancer Center, Science Park Research Division, Smithville, Texas 78957 Ramin Shiekhattar (25), Gene Expression and Regulation Program, The Wistar Institute, Philadelphia, Pennsylvania 19104 Ali Shilatifard (13), Saint Louis Univer- sity School of Medicine, Department of Biochemistry, St. Louis, Missouri 63104 Arthur I. Skoultchi (5), Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461 Mick Smerdon (32), Department of Bio- chemistry and Biophysics, School of Mo- lecular Biosciences, Washington State University, Pullman, Washington 99164– 4660 Sheryl T. Smith (15), Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 David J. Steger (2), Howard Hughes Med- ical Institute, University of California, San Francisco, Department of Biochemistry and Biophysics, San Francisco, California 94143–2240 Vassily M. Studitsky (29), Department of Biochemistry and Molecular Biology Wayne State University School of Medi- cine, Detroit, Michigan 4820 ** John W. Tamkun (4), Department of Mo- lecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, California 95064 ** Current Affiliation: Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 xii contributors to volume 377 Shih-Chang Tsai (9), H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612 Patrick Varga-Weisz (24), Chromatin Lab, Marie Curie Research Institute, Surrey RH8 0TL, United Kingdom Eric Verdin (10), Gladstone Institute of Virology and Immunology, University of California, San Francisco, San Francisco, California 94103 C. Peter Verrijzer (28), Gene Regulation Laboratory, Center for Biomedical Gen- etics, Department of Molecular and Cell Biology, Leiden University Medical Center, 2300 RA Leiden, The Netherlands Paul A. Wade (23), Emory University School of Medicine, Department of Path- ology and Laboratory Medicine, Atlanta, Georgia 30322 Wendy Walter (29), Center for Molecular Medicine and Genetics, Wayne State Uni- versity School of Medicine, Detroit, Michigan 48201 Hengbin Wang (12), Lineberger Compre- hensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599–7295 Wei-Dong Wang (18), Laboratory of Gen- etics, National Institute on Aging, Na- tional Institute of Health, Baltimore, Maryland 21224 Yu-Der Wen (9), H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612 Jacqueline Wittmeyer (20), University of Utah School of Medicine, Department of Oncological Sciences, Howard Hughes Medical Institute and Huntsman Cancer Institute, Salt Lake City, Utah 84112 Hua Xiao (22), Laboratory of Molecular Cell Biology, National Institute of Health, Bethesda, Maryland 20892–4255 Yutong Xue (18), Laboratory of Genetics, National Institute on Aging, National In- stitute of Health, Baltimore, Maryland 21224 Zhijiang Yan (18), Laboratory of Genet- ics, National Institute on Aging, National Institute of Health, Baltimore, Maryland 21224 Wen-Ming Yang (9), H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612 Ya-Li Yao (9), H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612 Yi Zhang (12), Lineberger Comprehensive Cancer Center, Department of Biochem- istry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599–7295 Keji Zhao (27), Laboratory of Molecular Immunology,NationalInstitutesofHealth, Bethesda, Maryland 20892–1674 contributors to volume 377 xiii [1] Functional Analyses of Chromatin Modifications in Yeast By Sandra J. Jacobson,Patricia M. Laurenson,and Lorraine Pillus Site-specific modification of histones is fundamental to chromatin function. The enzymes that perform these modifications include protein acetyltransferases and deacetylases, methyltransferases, kinases and phos- phatases, and ubiquitin-conjugating enzymes that are highly conserved from yeast to humans. Histone modifying enzymes often reside in multi-protein complexes whose subunits target and/or regulate the respective enzymatic activity. A number of these complexes have now been biochemically puri- fied and analyzed. In the budding yeast, Saccharomyces cerevisiae, biochem- ical approaches are readily combined with genetic analyses to coordinate understanding of histone modifying complexes, their in vivo substrate speci- ficity, their target genetic loci, and the functional consequences of their activity. Here we present principles and mechanics of using S. cerevisiae to ana- lyze the function of histones and histone modifiers. We depict the well- studied, posttranslational modifications of yeast histone residues (see Fig. 1) and the histone genes (see Fig. 2), and outline the enzymes respon- sible for histone modifications and their known cellular functions (see Table II). We present experimental strategies for studying chromatin modifiers and histone mutants (see Fig. 3; Table III) with a case study (see Table IV) and examples from the literature. This is accompanied by methods for studying chromatin-related functions, including chromatin- related assays (see Table V) and silencing assays (see Table VI, Fig. 4). Beyond these studies, S. cerevisiae is valuable for examining chromatin- related functions of a favorite protein from multicellular eukaryotes. We consider briefly human chromatin modifier genes associated with disease (see Table VII) and methods for analyzing their functions in yeast (see Figs. 5 and 6). Finally, we include a discussion of genomics tools and re- sources currently available in yeast (see Table VIII; Table I) and how these may be used to complement more traditional genetic approaches. [1] functional analyses of chromatin in yeast 3 Copyright 2004, Elsevier Inc. All rights reserved. METHODS IN ENZYMOLOGY, VOL. 377 0076-6879/04 $35.00 Histone Genetics in S. cerevisiae Nucleosome Structure An underlying theme in considering chromatin modifications is that they provide mechanisms for dynamic regulation of gene expression. Such dynamism, which correlates with epigenetic aspects of regulation, is critical because it constitutes a framework for developmental switches and envi- ronmental responses without changes in primary DNA sequence. Under- standing how histone modifications contribute to biological regulation ultimately relies on coordinated biochemical and genetic approaches that are readily accessible in yeast. Experimental dissection of chromatin func- tion has gained momentum with the availability of high-resolution struc- tural data of chromatin proteins and their modifiers, which help guide the construction and interpretation of mutants. The X-ray crystallographic structure of the nucleosome core particle at 2.8A resolution provided key details of the precise spatial orientation of histones with each other and with DNA. 1,2 This image of the nucleosome showed amino acids that were poised for post-translational modification as well as those that were likely to support the structural integrity of the nucleosome. It has become the bench-side companion of investigators designing and interpreting chromatin-related experiments. Many studies have focused on understanding the significance of post- translational modifications of N-terminal histone tails. These solvent ex- posed tails are modified at discrete sites through the covalent addition of acetyl, methyl, phosphate or ubiquitin groups (see Fig. 1). The marks have significant effects on chromatin structure and function where they may alter nucleosome structure or inter-nucleosomal interactions and regulate binding of chromatin-associated proteins. The role of chromatin modifications in the process of DNA transcrip- tion has been studied in detail, particularly that of acetylation which impacts basal transcription levels and reversible activation of genes (reviewed in Kurdistani and Grunstein 3 ). Genome-wide screening of his- tone acetylation and RNA transcript profiles in acetylase and deacetylase mutants has revealed that histone acetylation can exert long range effects to create chromosomal domains. 4–9 In other cases, acetylation may affect only several neighboring nucleosomes to facilitate binding of regulatory 1 K. Luger, A. W. Mader, R. K. Richmond, D. F. Sargent, and T. J. Richmond, Nature 389, 251 (1997). 2 C. L. White, R. K. Suto, and K. Luger, EMBO J. 20, 5207 (2001). 3 S. K. Kurdistani and M. Grunstein, Nat. Rev. Mol. Cell. Biol. 4, 276 (2003). 4 M. Vogelauer, J. Wu, N. Suka, and M. Grunstein, Nature 408, 495 (2000). 4 chromatin modification and remodeling [1] [...]... pleiotropic effects in yeast,31 including slow growth, transcriptional silencing defects, and transcriptional activation defects (see Table II and references therein) A mechanistic explanation for these phenotypes is now emerging from converging biochemical and genetic approaches Biochemical tools, including epitope-tagged proteins, highly speci c antisera, chromatin immunoprecipitation (ChlP) experiments, and. .. transcriptional activation defect transcriptional repression defectf transcriptional repression defectf transcriptional repression defectf 6-AU sensitivity of sas3 Á/spt1-Á922 synthetic lethality with gcn5 Á transcriptional activation defect transcriptional activation and/ or elongation defect transcriptional repression defectg telomeric silencing defect G2/M cell cycle block, nucleolar disruption, transcriptional... IV GCN5: A Case Study in Principles of Histone Modification and Function Chromatin- modifier characteristic Gcn5p example 1 Substrate specificity of chromatinmodifying enzyme may vary Gcn5p in vitro substrate specificity depends on the source of enzyme used (recombinant or purified as a complex from cell lysates) and whether free histones, nucleosomes or synthetic peptides are used as substratesa Gcn5p... function a Ref.a Assay description Repair, homologous recombination, gene conversion, non-homologous end joining or cell cycle arrest after HO endonuclease cleavage Homologous recombination and double-strand break repair Oxidative DNA damage/repair Mutation spectra assays Integrated lac operator tandem array phenotypic analysis Microscopic examination Cell cycle phenotypes Mass spectroscopy to characterize... Haimberger, M W McIntosh, and D E Gottschling, Genetics 161, 995 (2002) (57) M Bucholc, Y Park, and A J Lustig, Mol Cell Biol 21, 6559 (2001) 30 chromatin modification and remodeling [1] in vitro Furthermore, hta1-S129E mutants that mimic the constitutively phosphorylated form of H2A exhibited subtle defects in chromatin structure Both plasmid superhelical density and micrococcal nuclease digestion... sequence-specific DNA-binding protein Swi5p, followed by the chromatin- remodeling complex SWI/SNF, then the Gcn5p-containing SAGA complexf Gcn5p resides in at least three distinct yeast HAT complexes: SAGAg, Adah, and SLIK/SALSAi,j 2 Chromatin modifier may exert short-range gene-specific effects and long-range effects on genome-wide chromatin structure 3 Histone modification may be part of a temporal process... or nucleosomes60,62 and ESA1 mutants also have a telomeric silencing defect (Clarke and Pillus, personal communication) The H2A C- terminal region also contributes to transcriptional regulation Deletion of this region likewise causes a telomeric silencing defect90 and transcriptional phenotypes characteristic of SWI/SNF-dependent gene activation defects.84 C- terminal mutants exhibited a significant loss... spectroscopy to characterize protein complexes and protein modifications Chromatin immunoprecipitations for presence of trans-acting factors and for post-translational modifications of histones at a specific locus Chromatin immunoprecipitations on a microchip Flow cytometry De novo telomere addition Telomere length Telomere protection Telomeric tract rapid deletion and movement (39,40) (40) (41) (42)... Modificationb Enzyme H3 K4 Me Set 1 K9, K14 Ac Gcn5e K9/14 deAc Rpd3 K9, 14, 18, 23, 27 K14 deAc Hda1 deAc Sir2 Sas3 S10 Snf1 K36 H4 P Me Set2 K79 K5,8,12,16 Me Ac Dot1 Esa1h K5, 12f deAc Rpd3 K12 Ac Hat1 K16 Ac Sas2 K16 deAc Sir2 Ref c Phenotype of mutant slow growth, rDNA silencing defect, telomeric silencing and/ or telomeric length defect transcriptional activation and/ or elongation defect transcriptional... enzymatic activity using histone substrates Correlate enzymatic activity with amino acid modification in vivo Correlate enzymatic activity with cellular function Method In vitro chemical transfer reaction Isolate modified substrates for mass spectrometrya To identify histone substrate in vivo: mutate candidate histone modified amino acid and look for change in histone modification in the cell by Western, ChIP . 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. 20892 Mikhail Kashlev (29), National Cancer Institute Center for Cancer Research, Na- tional Cancer Institute-Frederick Cancer Research and Development Center, Fred- erick, Maryland 21702 James A. Kennison. 19107 *Current Affiliation: Joint Science Department, W. M. Keck Sceince Center, The Claremont Colleges, Claremont, California 91711 À Current Affiliation: Cellular Pathology, Royal Surrey County