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 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 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 375 Articlenumbersareinparenthesesandfollowingthenamesofcontributors. Affiliationslistedarecurrent. Chad Alexander (3), The University of Hinrich Boeger (11), Department of Tennessee-Oak Ridge Graduate School Structural Biology, Stanford University of Genome Science and Technology, SchoolofMedicine,Stanford,California Oak Ridge National Laboratory, Life 94305 SciencesDivision,OakRidge,Tennessee 37831-8080 William M. Bonner (5), Laboratory of Molecular Pharmacology, National Genevie`ve Almouzni (8), Institut Curie, Cancer Institute, Bethesda, Maryland Section de Recherche, F-75248, Paris 20892 Cedex05,France Michael Bruno (14), Division of Gene Satoshi Ando (18), Department of Mo- Regulation and Expression, The Well- lecularLifeScience,SchoolofMedicine, come Trust Biocentre, Department of Tokai University, Kanagawa 259-1193, Biochemistry, University of Dundee, Japan Dundee, DD1 5EH, Scotland, United YunheBao(2),DepartmentofBiochemis- Kingdom. try and Molecular Biology, Colorado GerardJ.Bunick(3),LifeSciencesDiv- State University, FortCollins, Colorado ision, Oak Ridge National Laboratory, 80523-1870 OakRidge,Tennessee37831-8080 Blaine Bartholomew (13), Department of Biochemistry & Molecular Biology, MichaelBustin(21),NationalCancerIn- Southern Illinois University School of stitute, National Institutes of Health, Medicine, Carbondale, Illinois Bethesda,Maryland20892 62901-4413 AnneE.Carpenter(23),WhiteheadInsti- David P. Bazett-Jones (28), Programme tuteforBiomedicalResearch,Cambridge, in Cell Biology, Hospital for Sick Massachusetts02142 Children, Toronto, Ontario M5G 1X8, Gustavo Carrero (26), Department of Canada Mathematical and Statistical Sciences, AndrewS.Belmont(23),Departmentof Faculty of Science, University of CellandStructuralBiology,Universityof Alberta, Edmonton, Alberta T6G 2E1, Illinois at Urbana-Champaign, Urbana, Canada Illinois61801 David Carter (29), Laboratory of Chro- LeiseBerven(16),Children’sMedicalRe- matin and Gene Expression, Babraham search Institute, Westmead, New South Institute, Cambridge CB2 4AT, United Wales2415,Australia Kingdom YehuditBirger(21),NationalCancerIn- Fre´de´ricCatez(21),NationalCancerIn- stitute, National Institutes of Health, stitute, National Institutes of Health, Bethesda,Maryland20892 Bethesda,Maryland20892 ix x contributors to volume 375 Lyubomira Chakalova (29), Laboratory Pamela N. Dyer (2), Department of Bio- ofChromatinandGeneExpression,Bab- chemistryandMolecularBiology,Color- raham Institute, Cambridge CB2 4AT, ado State University, Fort Collins, UnitedKingdom Colorado80523-1870 Srinivas Chakravarthy (2), Department Raji S. Edayathumangalam (2), Depart- ofBiochemistryandMolecularBiology, mentofBiochemistryandMolecularBiol- Colorado State University,Fort Collins, ogy, Colorado State University, Fort Colorado80523-1870 Collins,Colorado80523-1870 Lakshmi N. Changolkar (15), Depart- Thomas G. Fazzio (6), Fred Hutchinson mentofAnimalBiology,SchoolofVeter- Cancer Research Center, Seattle, Wash- inary Medicine, University of ington98109-1024 Pennsylvania,Philadelphia,Pennsylvania Andrew Flaus (14), Division of Gene 19104 Regulation and Expression, The Well- LisaAnnCirillo(9),DepartmentofCell comeTrustBiocentre,DepartmentofBio- Biology, Neurobiology, and Anatomy, chemistry,UniversityofDundee,Dundee, Medical College of Washington, DD15EH,Scotland,UnitedKingdom. Milwaukee,Wisconsin53149 PeterFraser(29),LaboratoryofChroma- PeterR.Cook(24),TheSirWilliamDunn tinandGeneExpression,BabrahamInsti- SchoolofPathology,UniversityofOxford, tute, Cambridge CB2 4AT, United OxfordOX13RE,UnitedKingdom Kingdom EllenCrawford(26),DepartmentofOn- SusanM.Gasser(22),DepartmentofMo- cology,FacultyofMedicine,Universityof lecular Biology, University of Geneva, Alberta and Cross Cancer Institute, 1211Geneva4,Switzerland Edmonton,AlbertaT6G2E1,Canada Stanislaw A. Gorski (25), National WouterdeLaat(30),DepartmentofCell Cancer Institute, National Institutes of Biology, ErasmusMC, 3015 GE Rotter- Health,Bethesda,Maryland20892 dam,TheNetherlands JoachimGriesenbeck(11),Departmentof GerdadeVries(26),DepartmentofMath- Structural Biology, Stanford University ematical and StatisticalSciences, Faculty SchoolofMedicine,Stanford,California ofScience,UniversityofAlberta,Edmon- 94305 ton,AlbertaT6G2E1,Canada Graham Dellaire (28), Programme in FrankGrosveld(30),DepartmentofCell Biology, ErasmusMC, 3015 GE Rotter- CellBiology,HospitalforSickChildren, dam,TheNetherlands Toronto,OntarioM5G1X8,Canada JohnD.Diller(10),DepartmentofBio- B.LeifHanson(3),TheUniversityofTen- chemistryandMolecularBiology,Center nessee-Oak Ridge Graduate School of for Gene Regulation, The Pennsylvania Genome Science and Technology, Life State University, University Park, Sciences Divison, Oak Ridge National Pennsylvania16802 Laboratory, Oak Ridge, Tennessee 37831-8080 Charles E. Ducker (10), Departmentof Biochemistry and Molecular Biology, Joel M. Harp (3), Department of Bio- CenterforGeneRegulation,ThePennsyl- chemistryandCenterforStructuralBiol- vania State University, University Park, ogy, Vanderbilt University, Nashville, Pennsylvania16802 Tennessee37232-8725 contributors to volume375 xi Keiji Hashimoto (17), Core Research for Karolin Luger (2), Department of Bio- Evolutional Science and Technology, chemistryandMolecularBiology,Color- Saitama332-0012,Japan ado State University, Fort Collins, Colorado80523-1870 JeffreyJ.Hayes(12),DepartmentofBio- chemistry and Biophysics, University of James G. McNally (27), Laboratory of Rochester Medical Center, Rochester, ReceptorBiologyandGeneExpression, NewYork14642 NationalCancerInstitute,NationalInsti- tutes of Health, Bethesda, Maryland Florence Hediger (22), Department of 20892 MolecularBiology,UniversityofGeneva, 1211Geneva4,Switzerland Tom Misteli (25) National Cancer Insti- tute, National Institutes of Health, MichaelJ.Hendzel(26),Departmentof Bethesda,Maryland20892 Oncology, University of Alberta and Cross Cancer Instutite, Edmonton, CraigA.Mizzen(19),DepartmentofCell AlbertaT6G1Z2,Canada & Structural Biology, University of MikiHieda(24),SirWilliamDunnSchool Illinois at Urbana-Champaign, Urbana, Illinois61801 of Pathology, University of Oxford, OxfordOX13RE,UnitedKingdom SetsuoMorishita(17),DepartmentofMo- Stefan R. Kassabov (13), Department of lecularBiology,SchoolofScience,Nagoya University,Nagoya464-8601,Japan Biochemistry & Molecular Biology, Southern Illinois University School of Uma M. Muthurajan (2), Department of Medicine, Carbondale, Illinois Biochemistry and Molecular Biology, 62901-4413 ColoradoState University, Fort Collins, HiroshiKimura(24),HorizontalMedical Colorado80523-1870 ResearchOrganization,SchoolofMedi- Frank R. Neumann (22), Department of cine,KyotoUniversity,Kyoto606-8510, MolecularBiology,UniversityofGeneva, Japan 1211Geneva4,Switzerland Roger D. Kornberg (11), Departmentof RozaliaNisman(28),ProgrammeinCell Structural Biology, Stanford University Biology, Hospital for Sick Children, SchoolofMedicine,Stanford,California Toronto,OntarioM5G1X8,Canada 94305 TomOwen-Hughes(14),DivisionofGene David Landsman (1) NationalCenterfor Regulation and Expression, The Well- BiotechnologyInformation,NationalLi- comeTrustBiocentre,DepartmentofBio- braryofMedicine,NationalInstitutesof chemistry,UniversityofDundee,Dundee, Health,Bethesda,Maryland20894 DD15EHScotland,UnitedKingdom. PaulJ.Laybourn(7),DepartmentofBio- John R. Pehrson (15), Department of chemistryandMolecularBiology,Color- Animal Biology, School of Veterinary ado State University, Fort Collins, Medicine, University of Pennsylvania, Colorado80523-1870 Philadelphia,Pennsylvania19104 Jae-HwanLim(21),NationalCancerInsti- CraigL.Peterson(4)UniversityofMas- tute, National Institutes of Health, sachusetts Medical School, Worchester, Bethesda,Maryland20892 Massachusetts01605 xii contributors to volume 375 RobertD.Phair(25),BioInformaticsSer- Erik Splinter (30), Department of Cell vices,Rockville,Maryland20854 Biology, ErasmusMC, 3015 GE Rotter- dam,TheNetherlands Duane R. Pilch (5), Laboratory of Mo- lecular Pharmacology, National Cancer Diana A. Stavreva (27), Laboratory of Institute,Bethesda,Maryland20892 ReceptorBiologyandGeneExpression, NationalCancerInstitute,NationalInsti- YuriV. Postnikov (21), National Cancer tutesofHealth,Bethesda,Maryland20892 Institute, National Institutes of Health, Bethesda,Maryland20892 J. Seth Strattan (11), Department of Structural Biology, Stanford University DannyRangasamy(16),TheJohnCurtin SchoolofMedicine,Stanford,California School of Medical Research, Australian 94305 NationalUniversity,Canberra,Australia CapitalTerritory2601,Australia Steven A. Sullivan (1), National Center forBiotechnologyInformation,National Dominique Ray-Gallet (8), Institut LibraryofMedicine,NationalInstitutes Curie, Section de Recherche, F-75248, ofHealth,Bethesda,Maryland20894 ParisCedex05,France Ulrica Svensson (16), The John Curtin ChristopheRedon(5),LaboratoryofMo- School of Medical Research, Australian lecular Pharmacology, National Cancer NationalUniversity,Canberra,Australian Institute,Bethesda,Maryland20892 CapitalTerritory2601,Australia RaymondReeves(20),SchoolofMolecu- AngelaTaddei(22),DepartmentofMo- larBiosciences,Biochemistry/Biophysics, lecular Biology, University of Geneva, Washington State University, Pullman, 1211Geneva4,Switzerland Washington99164-4660 John Th’ng (26), Northwestern Ontario Patricia Ridgway (16), The John Curtin Regional Cancer Centre, Thunder Bay, School of Medical Research, Australian OntarioP7A7T1,Canada National University, Canberra, Austra- David John Tremethick (16), The John lianCapitalTerritory2601,Australia CurtinSchoolofMedicalResearch,Aus- ChunRuan(10),DepartmentofBiochem- tralian National University, Canberra, istry and Molecular Biology, Center for Australian Capital Territory 2601, GeneRegulation,ThePennsylvaniaState Australia University,UniversityPark,Pennsylvania Toshio Tsukiyama (6), Fred Hutchinson 16802 Cancer Research Center, Seattle, Wash- Olga A. Sedelnikova (5), Laboratory ington98109-1024 of Molecular Pharmacology, National JayC.Vary,Jr.(6),MolecularandCellu- Cancer Institute, Bethesda, Maryland lar Biology Program, University of 20892 Washington,Seattle,Washington98195 MichaelA.Shogren-Knaak(4),Univer- Cindy L. White (2), Department of Bio- sity of Massachusetts Medical School, chemistryandMolecularBiology,Color- Worchester,Massachusetss01605 ado State University, Fort Collins, Colorado80523-1870 Robert T. Simpson (10), Department of Biochemistry and Molecular Biology, SriwanWongwisansri(7),Departmentof CenterforGeneRegulation,ThePennsyl- Biochemistry and Molecular Biology, vania State University, University Park, ColoradoState University, FortCollins, Pennsylvania16802 Colorado80523-1870 contributors to volume375 xiii KinyaYoda(17,18),BioscienceandBio- Chunyang Zheng (12), Department of technology Center, Nagoya University, BiochemistryandBiophysics,University Nagoya,464-8601,Japan ofRochesterMedicalCenter,Rochester, NewYork14642 Kenneth S. Zaret (9), Cell and Devel- opmental Biology Program, Fox Chase CancerCenter,Philadelphia,Pennsylva- nia19111 [1] mining corehistone sequencesfrompublic proteindatabases 3 [1] Mining Core Histone Sequences from Public Protein Databases By Steven A. Sullivan and David Landsman Introduction Constructinganonlinedatabaseofhistonesandhistonefold-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 inpublicsequencedatabases. Considerations Ourinitialgoalwastocollectasmanyreportedhistonesequencesaswe 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 GenBank6 and RefSeq,7 a curated, nonredundant set of sequences. 1S.Sullivan,D.W.Sink,K.L.Trout,I.Makalowska,P.M.Taylor,A.D.Baxevanis,and D.Landsman,NucleicAcidsRes.30,341(2002). 2S.A.SullivanandD.Landsman,Proteins52,454(2003). 3B.Boeckmann,A.Bairoch,R.Apweiler,M.C.Blatter,A.Estreicher,E.Gasteiger,M.J. Martin,K.Michoud,C.O’Donovan,I.Phan,S.Pilbout,andM.Schneider,NucleicAcids Res.31,365(2003). 4C. 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,andW.C.Barker,Nucleic AcidsRes.31,345(2003). 5J.Westbrook,Z.Feng,L.Chen,H.Yang,andH.M.Berman,NucleicAcidsRes.31,489 (2003). METHODSINENZYMOLOGY,VOL.375 0076-6879/04$35.00 4 histone bioinformatics [1] 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 6D.A.Benson,I.Karsch-Mizrachi,D.J.Lipman,J.Ostell,andD.L.Wheeler,NucleicAcids Res.31,23(2003). 7K.D.Pruitt,T.Tatusova,andD.R.Maglott,NucleicAcidsRes.31,34(2003). 8S. F. Altschul, T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman,NucleicAcidsRes.25,3389(1997). 9S.R.Eddy,Bioinformatics14,755(1998).