Table Of Content

Preface Acentralchallengeofthepost-genomiceraistounderstandhowthe30,000to 40,000uniquegenesinthehumangenomeareselectivelyexpressedorsilenced tocoordinatecellulargrowthanddifferentiation.Thepackagingofeukaryotic genomes in a complex of DNA, histones, and nonhistone proteins called chromatinprovidesasurprisinglysophisticatedsystemthatplaysacriticalrole 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 domainsofcovalentmodificationsofDNAandhistones,canbeinheritedfrom onegenerationtothenext.Theheritabilityofchromatinstatesintheabsence ofDNAmutationhascontributedgreatlytothecurrentexcitementinthefield ofepigenetics. Thepast5yearshavewitnessedanexplosionofnewresearchonchroma- tinbiologyandbiochemistry.Chromatinstructureandfunctionarenowwidely recognizedasbeingcriticaltoregulatinggeneexpression,maintaininggenomic stability, and ensuring faithful chromosome transmission. Moreover, links be- tweenchromatinmetabolismanddiseasearebeginningtoemerge.Theidenti- fication of altered DNA methylation and histone acetylase activity in human cancers,theuseofhistonedeacetylaseinhibitorsinthetreatmentofleukemia, and the tumor suppressor activities of ATP-dependent chromatin remodeling enzymesareexamplesthatlikelyrepresentjustthetipoftheiceberg. Assuch,thefieldisattractingnewinvestigatorswhoenterwithlittlefirst 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)andVolume304(Chromatin).Whilemanyoftheclassicproto- colsinthosevolumesremainastimelynowaswhentheywerewritten,itisour hopethepresentserieswillfillinthegapsforthenextseveralyears. This3-volumesetofMethodsinEnzymologyprovidesnearlyonehundred procedurescoveringthefullrangeoftools—bioinformatics,structuralbiology, biophysics, biochemistry, genetics, and cell biology—employed in chromatin research.Volume375includesahistonedatabase,methodsforpreparationof xv xvi preface histones,histonevariants,modifiedhistonesanddefinedchromatinsegments, protocolsfornucleosomereconstitutionandanalysis,andcytologicalmethods forimagingchromatinfunctionsinvivo.Volume376includeselectronmicro- scopy and biophysical protocols for visualizing chromatin and detecting chromatin 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 fortranscriptionandDNArepaironchromatintemplates.Weareexceedingly gratefultotheverylargenumberofcolleaguesrepresentingthefield’sleading laboratories, who have taken the time and effort to make their technical expertiseavailableinthisseries. Finally, we wish to take the opportunity to remember Vincent Allfrey, AndreiMirzabekov,HaroldWeintraub,AbrahamWorcel,andespeciallyAlan Wolffe,co-editorofVolume304(Chromatin).Alloftheseindividualshadkey rolesinshapingthechromatinfieldintowhatitistoday. 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,SankarL.AdhyaandSusanGarges,Editors. METHODS IN ENZYMOLOGY EDITORS-IN-CHIEF John N. Abelson Melvin I. Simon DIVISIONOFBIOLOGY CALIFORNIAINSTITUTEOFTECHNOLOGY PASADENA,CALIFORNIA FOUNDINGEDITORS Sidney P. Colowick and Nathan O. Kaplan Contributors to Volume 377 Articlenumbersareinparenthesesandfollowingthenamesofcontributors. Affiliationslistedarecurrent. WoojinAn(30),LaboratoryofBiochemis- Brad Cairns (20), University of Utah try and Molecular Biology, The Rocke- SchoolofMedicine,DepartmentofOnco- feller University, New York, New York logicalSciences,HowardHughesMedical 10021 Institute and HuntsmanCancerInstitute, SaltLakeCity,Utah84112 Jennifer A. Armstrong (4), Department of Molecular, Cell and Developmental Yuh-Long Chang (16), Institute of Mo- Biology, UniversityofCalifornia, Santa lecularBiology,AcademiaSinica,Taiwan Cruz,SantaCruz,California95064* 115,RepublicofChina Orr G. Barak (25), The Wistar Institute, GillianE.Chalkley(28),GeneRegula- Philadelphia,Pennsylvania19104 tion Laboratory, Center for Biomedical Genetics, Department of Molecular BrianC.Beard(32),DepartmentofBio- and Cell Biology, Leiden University chemistryandBiophysics,SchoolofMo- Medical Center, 2300 RA Leiden, The lecular Biosciences, Washington State Netherlands University, Pullman, Washington 99164 –4660 Nadine Collins (24), Chromatin Lab, Peter B. Becker (21), Adolf-Butenandt- Marie Curie Research Institute, Surrey RH80TL,UnitedKingdom(cid:1) Institut, Molekularbiologie, Mu¨nchen D-80336,Germany Davide F. V. Corona (4), Departmentof Shelley L. Berger (7), The Wistar Insti- Molecular,CellandDevelopmentalBiol- ogy,UniversityofCalifornia,SantaCruz, tute,Philadelphia,Pennsylvania19104 SantaCruz,California95064 Tiziana Bonaldi (6), Protein Analysis Unit, Adolf-Butenandt Institut, Ludwig JacquesCo¨te´(8),LavalUniversityCancer MaximilliansUniversita¨t,Mu¨nchen,80336 Research Center, Quebec, GIR 2J6 Mu¨nchen,Germany Canada LudmilaBozhenok(24),ChromatinLab, Tianhuai Chi (18), Howard Hughes Marie Curie Research Institute, Surrey Medical Institute, Stanford University, RH80TL,UnitedKingdom Stanford,California94305` Eli Canaani (15), Department of Mole- Carlo M. Croce (15), Kimmel Cancer cular Cell Biology, Weizmann Institute Center, Thomas Jefferson University, ofScience,Rehovot76100,Israel Philadelphia,Pennsylvania19107 *Current Affiliation: Joint Science Department, W. M. Keck Sceince Center, The Claremont Colleges,Claremont,California91711 (cid:1)Current Affiliation: Cellular Pathology, Royal Surrey County Hospital, Guildford, United Kingdom `CurrentAffiliation:SectionofImmunology,YaleUniversitySchoolofMedicine,NewHaven, Connecticut06520 ix x contributors to volume 377 Franck Dequiedt (10), Molecular and Karien Hamer (17), Swammerdam Insti- CellularBiologyUnit,FacultyofAgron- tute for Life Sciences, University of omy,GemblouxB-5030,Belgium Amsterdam, 1018 TV Amsterdam, The Netherlands Jim Dover (13), Department of Genetics, Washington University School of Medi- Ali Hamiche (22), Institut Andre Lwoff, cine,St.Louis,Missouri63110 94800Villejuif,France Yannick Doyon (8), Laval University Shu He (31), Johnson Research Founda- Cancer Research Center, Quebec, GIR tion, Department of Biochemistry and 2J6Canada Biophysics, University of Pennsylvania School of Medicine, Philadelphia, AntonEberharter(21),Adolf-Butenandt- Pennsylvania19104–6059 Institut, Molekularbiologie, Mu¨nchen D-80336,Germany Karl W. Henry (7), The Wistar Institute, Philadelphia,Pennsylvania19104 Stuart Elgar (23), Emory University School of Medicine, Department of DerHwa-Huang(16),InstituteofMolecu- Pathology and Laboratory Medicine, lar Biology, Academia Sinica, Taiwan Atlanta,Georgia30322 115,RepublicofChina YuhongFan(5),DepartmentofCellBiol- Axel Imhof (6), Histone Modifications ogy,AlbertEinsteinCollegeofMedicine, Group,Adolf-ButenandtInstitut,Ludwig Bronx,NewYork10461 Maximillians Universita¨t, Mu¨nchen, 80336Mu¨nchen,Germany JiaFang(12),LinebergerComprehensive CancerCenter,DepartmentofBiochem- Sandra J. Jacobson (1), Department of istryandBiophysics,UniversityofNorth Biology, University of California, San Carolina at Chapel Hill, Chapel Hill, Diego,LaJolla,California92093–0347 NorthCarolina27599–7295 Mark Johnston (13), Department of Wolfgang Fischle (10), Laboratory of Genetics,WashingtonUniversitySchool ChromatinBiology,TheRockefellerUni- ofMedicine,St.Louis,Missouri63110 versity,NewYork,NewYork10021 Rohinton T. Kamakaka (14), National RoyFrye (10),VA MedicalCenter, Pitts- InstituteofChildHealthandHumanDe- burgh,Pennsylvania15240 velopment,UnitonChromatinandTran- SunilGangadharan(14),NationalInsti- scription,Bethesda,Maryland20892 tute of Child Health and Human Mikhail Kashlev (29), National Cancer Development, Unit on Chromatin and InstituteCenterforCancerResearch,Na- Transcription,Bethesda,Maryland20892 tionalCancerInstitute-FrederickCancer SonjaGhidelli(14),NationalInstituteof ResearchandDevelopmentCenter,Fred- ChildHealthandHumanDevelopment, erick,Maryland21702 Unit on Chromatin and Transcription, JamesA.Kennison(3),LaboratoryofMo- Bethesda,Maryland20892§ lecular Genetics, National Institute of PatrickA.Grant(8),UniversityofVirgi- Child Health and Human Development, na School of Medicine, Charlottesville, National Institutes of Health, Bethesda, Virginia22908 Marlyland,20892–2785 §CurrentAffiliation:CellzomeAG,69117Heidelberg,Germany contributors to volume377 xi Roger D. Kornberg (19), Department of Alxander Mazo (15), Kimmel Cancer Structural Biology, Stanford University Center,DepartmentofMicrobiologyand SchoolofMedicine,Stanford,California Immunology, Thomas Jefferson Univer- 94305 sity,Philadelphia,Pennsylvania19107 Wladyslaw Krajewski (15), Kimmel StaceyMcMahon(8),UniversityofVirgi- CancerCenter,ThomasJeffersonUniver- na School of Medicine, Charlottesville, sity,Philadelphia,Pennsylvania19107{ Virginia22908 TedH.J.Kwaks(17),SwammerdamInsti- DeweyG.McCafferty(31),JohnsonRe- tute for Life Sciences, University of search Foundation, Department of Bio- Amsterdam, 1018 TV Amsterdam, The chemistry and Biophysics, University Netherlands of Pennsylvania School of Medicine, Philadelphia,Pennsylvania19104–6059 Gernot La¨ngst (21), Adolf-Butenandt- Institut,Molekularbiologie,Mu¨nchenD- TatsuyaNakamura(15),KimmelCancer 80336,Germany Center,DepartmentofMicrobiologyand Immunology,ThomasJeffersonUniver- Patricia M. Laurenson (1), Department sity,Philadelphia,Pennsylvania19107 ofBiology,UniversityofCalifornia,San Diego,LaJolla,California92093–0347 Brian North (10), Gladstone Institute of VirologyandImmunology,Universityof Hong Liu (27), Laboratory ofMolecular California,SanFrancisco,SanFrancisco, Immunology,NationalInstitutesofHealth, California94103 Bethesda,Maryland20892–1674 SantaekOh(31),HowardHughesMedical Wan-Sheng Lo (7), The Wistar Institute, Institute, Division of Nucleic Acids En- Philadelphia,Pennsylvania19104 zymology, Department of Biochemistry, LorrainePillus(1),DepartmentofBiol- University of Medicine and Dentistry of ogy,UniversityofCalifornia,SanDiego, NewJersey,RobertWoodJohnsonMed- LaJolla,California92093–0347 ical School, Piscataway, New Jersey 08854–5635 Yahli Lorch (19), Department of Struc- turalBiology,StanfordUniversitySchool Erin K. O’Shea (2), Howard Hughes ofMedicine,Stanford,California94305 Medical Institute, University of Califor- nia,SanFrancisco,Department.ofBio- RomainLoury(11),InstitutdeGeńe´tique chemistry and Biophysics, San etdeBiologieMoleculaireetCellulaire, Francisco,California94143–2240 67404Illkirch,Strasbourg,France ArieP.Otte(17),SwammerdamInstitute AlejandraLoyola(31),HowardHughes forLife Sciences, UniversityofAmster- Medical Institute, Division of Nucleic dam,1018TVAmsterdam,TheNether- AcidsEnzymology,DepartmentofBio- lands chemistry, University of Medicine and Dentistry of New Jersey, Robert Wood MatthewB.Palmer(23),EmoryUniver- Johnson Medical School, Piscataway, sity School of Medicine, Department of NewJersey08854–5635 Pathology and Laboratory Medicine, Atlanta,Georgia30322 BrettMarshall(10),GladstoneInstitute ofVirologyandImmunology,University Svetlana Petruk (15), Kimmel Cancer of California, San Francisco, San Center, Thomas Jefferson University, Francisco,California94103 Philadelphia,Pennsylvania19107 {CurrentAffiliation:InstituteofDevelopmentalBiology,Moscow117808,Russia xii contributors to volume 377 Raymond Poot (24), Chromatin Lab, RichardG.A.B.Sewalt(17),Swammer- Marie Curie Research Institute, Surrey damInstituteforLifeSciences,University RH80TL,UnitedKingdom ofAmsterdam,1018TVAmsterdam,The Netherlands Danny Reinberg (31), Howard Hughes Medical Institute, Division of Nucleic Xuetong Shen (26), Department of Car- AcidsEnzymology,DepartmentofBio- cinogenesis, University of Texas, M.D. chemistry, University of Medicine and Anderson Cancer Center, Science Park Dentistry of New Jersey, Robert Wood Research Division, Smithville, Texas Johnson Medical School, Piscataway, 78957 NewJersey08854–5635 RaminShiekhattar(25),GeneExpression Jo¨rgT.Regula(6),ProteinAnalysisUnit, and Regulation Program, The Wistar Adolf-ButenandtInstitut,LudwigMaxi- Institute, Philadelphia, Pennsylvania millians Universita¨t, Mu¨nchen, 80336 19104 Mu¨nchen,Germany AliShilatifard(13),SaintLouisUniver- NatalieRezai-Zadeh(9),H.LeeMoffitt sity School of Medicine, Department of Cancer Center and Research Institute, Biochemistry,St.Louis,Missouri63104 University of South Florida, Tampa, Florida33612 Arthur I. Skoultchi (5), Department of RobertRoeder(30),Head,Laboratoryof CellBiology,AlbertEinsteinCollegeof Medicine,Bronx,NewYork10461 BiochemistryandMolecularBiology,The Rockefeller University, New York, New Mick Smerdon (32), Department of Bio- York10021 chemistryandBiophysics,SchoolofMo- Anjanabha Saha (20), UniversityofUtah lecular Biosciences, Washington State University,Pullman,Washington99164– SchoolofMedicine,DepartmentofOnco- 4660 logicalSciences,HowardHughesMedical Instituteand HuntsmanCancerInstitute, Sheryl T. Smith (15), Kimmel Cancer SaltLakeCity,Utah84112 Center, Thomas Jefferson University, PaoloSassone-Corsi(11),InstitutdeGe´- Philadelphia,Pennsylvania19107 ne´tiqueetdeBiologieMoleculaireetCel- DavidJ.Steger(2),HowardHughesMed- lulaire,67404Illkirch,Strasbourg,France icalInstitute,UniversityofCalifornia,San Jessica Schneider (13), Saint Louis Uni- Francisco, Department of Biochemistry versity School of Medicine, Department and Biophysics, San Francisco, ofBiochemistry,St.Louis,Missouri63104 California94143–2240 Marc F. Schwartz (7), The Wistar Insti- VassilyM.Studitsky(29),Departmentof tute,Philadelphia,Pennsylvania19104 Biochemistry and Molecular Biology YuriiSedkov(15),KimmelCancerCenter, WayneStateUniversitySchoolofMedi- Thomas Jefferson University, Phila- cine,Detroit,Michigan4820** delphia,Pennsylvania19107 JohnW.Tamkun(4),DepartmentofMo- Edward Seto (9), H. Lee Moffitt Cancer lecular,CellandDevelopmentalBiology, CenterandResearchInstitute,University University of California, Santa Cruz, ofSouthFlorida,Tampa,Florida33612 SantaCruz,California95064 **CurrentAffiliation:DepartmentofPharmacology,UniversityofMedicineandDentistryof NewJersey,RobertWoodJohnsonMedicalSchool,Piscataway,NewJersey08854 contributors to volume377 xiii Shih-ChangTsai(9),H.LeeMoffittCancer JacquelineWittmeyer(20),Universityof CenterandResearchInstitute,University UtahSchoolofMedicine,Departmentof ofSouthFlorida,Tampa,Florida33612 Oncological Sciences, Howard Hughes MedicalInstituteandHuntsmanCancer Patrick Varga-Weisz (24), Chromatin Institute,SaltLakeCity,Utah84112 Lab, Marie Curie Research Institute, SurreyRH80TL,UnitedKingdom Hua Xiao (22), Laboratory of Molecular Cell Biology, National Institute of Eric Verdin (10), Gladstone Institute of Health,Bethesda,Maryland20892–4255 VirologyandImmunology,Universityof California,SanFrancisco,SanFrancisco, YutongXue(18),LaboratoryofGenetics, California94103 NationalInstituteonAging,NationalIn- stitute of Health, Baltimore, Maryland C.PeterVerrijzer(28),GeneRegulation 21224 Laboratory,CenterforBiomedicalGen- etics, Department of Molecular and Cell ZhijiangYan(18),LaboratoryofGenet- Biology, Leiden University Medical ics,NationalInstituteonAging,National Center,2300RALeiden,TheNetherlands InstituteofHealth,Baltimore,Maryland 21224 Paul A. Wade (23), Emory University SchoolofMedicine,DepartmentofPath- Wen-Ming Yang (9), H. Lee Moffitt ologyandLaboratoryMedicine,Atlanta, Cancer Center and Research Institute, Georgia30322 University of South Florida, Tampa, Florida33612 WendyWalter(29),CenterforMolecular MedicineandGenetics,WayneStateUni- Ya-Li Yao (9), H. Lee Moffitt Cancer versity School of Medicine, Detroit, CenterandResearchInstitute,University Michigan48201 ofSouthFlorida,Tampa,Florida33612 HengbinWang(12),LinebergerCompre- YiZhang(12),LinebergerComprehensive hensive Cancer Center, Department of CancerCenter,DepartmentofBiochem- BiochemistryandBiophysics,University istryandBiophysics,UniversityofNorth ofNorthCarolinaatChapelHill,Chapel Carolina at Chapel Hill, Chapel Hill, Hill,NorthCarolina27599–7295 NorthCarolina27599–7295 Wei-DongWang(18),LaboratoryofGen- Keji Zhao (27), Laboratory of Molecular etics, National Institute on Aging, Na- Immunology,NationalInstitutesofHealth, tional Institute of Health, Baltimore, Bethesda,Maryland20892–1674 Maryland21224 Yu-Der Wen (9), H. Lee Moffitt Cancer CenterandResearchInstitute,University ofSouthFlorida,Tampa,Florida33612 [1] functional analysesof chromatin inyeast 3 [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,andubiquitin-conjugatingenzymesthatarehighlyconservedfrom yeasttohumans.Histonemodifyingenzymesoftenresideinmulti-protein complexes whose subunitstargetand/or regulate therespective enzymatic activity.Anumberofthesecomplexeshavenowbeenbiochemicallypuri- fiedandanalyzed.Inthebuddingyeast,Saccharomycescerevisiae,biochemical approaches are readily combined with genetic analyses to coordinate understandingofhistonemodifyingcomplexes,theirinvivosubstratespeci- ficity, their target genetic loci, and the functional consequences of their activity. HerewepresentprinciplesandmechanicsofusingS.cerevisiaetoana- lyze the function of histones and histone modifiers. We depict the well- studied, posttranslational modifications of yeast histone residues (see Fig.1)andthehistonegenes(seeFig.2),andoutlinetheenzymesrespon- 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- sourcescurrentlyavailableinyeast(seeTableVIII;TableI)andhowthese maybe used to complement more traditionalgeneticapproaches. Copyright2004,ElsevierInc. Allrightsreserved. METHODSINENZYMOLOGY,VOL.377 0076-6879/04$35.00 4 chromatin modification and remodeling [1] Histone Genetics in S. cerevisiae Nucleosome Structure An underlying theme in considering chromatin modifications is that theyprovidemechanismsfordynamicregulationofgeneexpression.Such dynamism,whichcorrelateswithepigeneticaspectsofregulation,iscritical 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 arereadilyaccessible inyeast.Experimentaldissectionofchromatinfunc- tion has gained momentum with the availability of high-resolution structural 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 withDNA.1,2Thisimageofthenucleosomeshowedaminoacidsthatwere 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 posttranslational modifications of N-terminal histone tails. These solvent ex- posed tails are modified at discrete sites through the covalent addition of acetyl,methyl,phosphateorubiquitingroups(seeFig.1).Themarkshave significant effects on chromatin structure and function where they may alter nucleosome structure or inter-nucleosomal interactions and regulate binding ofchromatin-associatedproteins. The role of chromatin modifications in the process of DNA transcription has been studied in detail, particularly that of acetylation which impacts basal transcription levels and reversible activation of genes (reviewed in Kurdistani and Grunstein3). Genome-wide screening of histone 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 1K.Luger,A.W.Mader,R.K.Richmond,D.F.Sargent,andT.J.Richmond,Nature389, 251(1997). 2C.L.White,R.K.Suto,andK.Luger,EMBOJ.20,5207(2001). 3S.K.KurdistaniandM.Grunstein,Nat.Rev.Mol.Cell.Biol.4,276(2003). 4M.Vogelauer,J.Wu,N.Suka,andM.Grunstein,Nature408,495(2000).