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 acces- sibletothetranscriptionmachinery,whileothergenesarereversiblysilenced. 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. As such, the field is attracting new investigators who enter with little firsthand experiencewith thestandard assays used to dissectchromatin struc- tureandfunction.Inaddition,evenseasonedveteransareoverwhelmedbythe 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 histones,histonevariants,modifiedhistonesanddefinedchromatinsegments, xv xvi preface 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 376 Articlenumbersareinparenthesesandfollowingthenamesofcontributors. Affiliationslistedarecurrent. Rhoda M. Alani (12), Department of Juliana Callaghan (10), Department of Oncology, Johns Hopkins University Biochemistry, University of Cambridge, SchoolofMedicine,Baltimore,Maryland CambridgeCB21GA,UnitedKingdom 21218 Marek Cebrat (12), Department of FranciscoAsturias(4),DepartmentofCell Pharmacology and Molecular Sciences, Biology, The Scripps Research Institute, Johns Hopkins University School of LaJolla,California92037 Medicine,Baltimore,Maryland21218 Andrew J. Bannister (18), Wellcome JulieChaumeil(27),MammalianDevelop- Trust/CancerResearch,UnitedKingdom mental Epigenetics Group, UMR Institute and Department of Pathology, 218-NuclearDynamicsandGenomePlas- University of Cambridge, Cambridge ticity, Curie Institute-Research Section, CB21QR,UnitedKingdom 75248Paris,Cedex05-France P.B.Becker(1),AdolfButenandtInstitut, DinaChaya(24),CellandDevelopmental Lehrstuhl fu¨r Molekularbiologie, Schil- Biology Program, Fox Chase Cancer lerstr.44,80336Munich,Germany Center,Philadelphia,Pennsylvania19111 MartinL.Bennink(6),BiophysicalTech- Peter Cheung (15), Department of Med- niques Group and MESAþ Research ical Biophysics, University of Toronto, Institute,DepartmentofScienceTechnol- Ontario Cancer Institute, Toronto, ogy, University of Twente, 7500 AE OntarioM5G2M9,Canada Enschede,TheNetherlands J. Chin (1), Department of Biochemistry, BradleyE.Bernstein(23),Departmentof NorthwesternUniversity,MolecularBio- ChemistryandChemicalBiology,Harvard logyandCellBiology,Evanston,Illinois University, Cambridge, Massachusetts 60208-3500 02138 David N. Ciccone (22), Department of MargieT.Borra(11),DepartmentofBio- Molecular Biology, Massachusetts Gen- chemistryandMolecularBiology,Oregon eral Hospital, Boston, Massachusetts HealthandScienceUniversity,Portland, 02114 Oregon97239 Philip A. Cole (12), Department of Brent Brower-Toland (5), Biology De- Pharmacology and Molecular Sciences, partment, Washington University in St. Johns Hopkins University School of Louis,St.Louis,Missouri63130 Medicine,Baltimore,Maryland21218 Michael Bustin (14), Protein Section, Carlos Cordon-Cardo (13), Division of NationalCancerInstitute,NationalInsti- Molecular Pathology, Memorial Sloan tutes of Health, Bethesda, Maryland Kettering Cancer Center, New York, 20892 NewYork10021 ix x contributors to volume 376 Carolyn A. Craig (25), Biology Depart- Jeffrey C. Hansen (2), Department of ment,WashingtonUniversityinSt.Louis, Biochemistry and Molecular Biology, St.Louis,Missouri63130 ColoradoState University, FortCollins, Colorado80523 John M. Denu (11), Department of Biochemistry and Molecular Biology, Edith Heard (27), Mammalian De- Oregon Health and Science University, velopmental Epigenetics Group, UMR Portland,Oregon97239 218-NuclearDynamicsandGenomePlas- ticity, Curie Institute-Research Section, Meghann K. Devlin (12), Department of 75248Paris,Cedex05,France Oncology, Johns Hopkins University SchoolofMedicine,Baltimore,Maryland Rachel A. Horowitz-Scherer (3), 21218 Department of Biology, University of Massachusetts, Amherst, Massachusetts Marija Drobnjak (13), Division of 01003 Molecular Pathology, Memorial Sloan Kettering Cancer Center, New York, Emily L. Humphrey (23), Department of NewYork10021 ChemistryandChemicalBiology,Harvard University, Cambridge, Massachusetts Brian Dynlacht (20), Department of 02138 Pathology,NewYorkUniversitySchool ofMedicine,NewYork,NewYork10016 StevenA.Jacobs(9),DepartmentofBio- Sarah C. R. Elgin (25), Biology De- chemistryandMolecularGenetics,Uni- versity of Virginia, Charlottesville, partment, Washington University in St. Virginia22908 Louis,St.Louis,Missouri63130 ChukwudiEzeokonkwo(4),Department ThomasJenuwein(16),ResearchInstitute of Molecular Pathology (IMP), The of Cell Biology, The Scripps Research Institute,LaJolla,California92037 ViennaBiocenter,Vienna,A-1030,Austria PeggyFarnham(21),McArdleLaboratory Monika Kauer (16), ResearchInstituteof MolecularPathology(IMP),TheVienna forCancerResearch,UniversityofWis- Biocenter,Vienna,A-1030,Austria consin,Madison,Wisconsin53706 Wolfgang Fischle (9), Department of W. Kevin Kelly (13), Genitourinary On- cologyServiceandDepartmentofMedi- Biochemistry and Molecular Genetics, cine, Memorial Sloan Kettering Cancer University of Virginia, Charlottesville, Center,NewYork,NewYork10021 Virginia22908 FredK.Friedman(14),LaboratoryofMe- Sepideh Khorasanizadeh (9), Depart- tabolism, National Cancer Institute, mentofBiochemistryandMolecularGen- National Institutes of Health, Bethesda, etics, University of Virginia, Maryland20892 Charlottesville,Virginia22908 Philippe T. Georgel (2), Department of Roger D. Kornberg (4), Department of BiologicalSciences,MarshallUniversity, Structural Biology, Stanford University Huntington,WestVirginia25755 SchoolofMedicine,Stanford,California 94305 Michael Grunstein (19), Department of BiologicalChemistry,SchoolofMedicine Tony Kouzarides (18), Wellcome Trust/ andMolecularBiologyInstitute,Univer- CancerResearch,UnitedKingdomInsti- sity of California, Los Angeles, Los tute,UniversityofCambridge,Cambridge Angeles,California90095 CB21QR,UnitedKingdom contributors to volume376 xi Siavash K. Kurdistani (19), Department ShirazMujtaba(8),DepartmentofPhysi- of Biological Chemistry, University of ologyandBiophysics,StructuralBiology California,LosAngelesSchoolofMedi- Program, Mt. Sinai School of Medicine, cineandMolecularBiologyInstitute,Los New York University, New York, New Angeles,California90095 York10029 G. La¨ngst (1), Adolf Butenandt Institut, AlexeyG.Murzin(10),MRCCentrefor Lehrstuhl fu¨r Molekularbiologie, Schil- Protein Engineering, Cambridge, CB2 lerstr.44,80336Munich,Germany 2QHUnitedKingdom Ernest Laue (10), Department of Bio- NataliaV.Murzina(10),Departmentof chemistry, University of Cambridge, Biochemistry, University of Cambridge, CambridgeCB21GA,UnitedKingdom CambridgeCB21GA,UnitedKingdom Sanford H. Leuba (6), Department of Peter R. Nielsen (10), Department of CellBiologyandPhysiology,University Biochemistry, University of Cambridge, of Pittsburgh School of Medicine, Hill- CambridgeCB21GA,UnitedKingdom man Cancer Center, UPCI Research Pavilion, Pittsburgh, Pennsylvania KenichiNishioka(17),DepartmentofDe- 15213-1863 velopmentalGenetics,NationalInstitute ofGenetics,Shizuoka,Japan,411-8540 Yuhong Li (25), University of Iowa, De- partment of Biochemistry, Iowa City, Matthew J. Oberley (21), McArdle Iowa52242 LaboratoryforCancerResearch,Univer- sity of Wisconsin, Madison, Wisconsin John Lis (26), Cornell University, Ithaca, 53706 NewYork14853 MarjorieA.Oettinger(22),Department ChihLongLiu(23),DepartmentofChem- of Molecular Biology, Massachusetts istryandChemicalBiology,HarvardUni- GeneralHospital,Boston,Massachusetts versity,Cambridge,Massachusetts02138 02114 YahliLorch(4),DepartmentofStructural IkuhiroOkamoto(27),MammalianDevel- Biology, Stanford University School of opmentalEpigeneticsGroup,UMR218– Medicine,Stanford,California94305 Nuclear Dynamics and Genome Plasti- Paul A. Marks (13), Cell Biology Pro- city, Curie Institute-Research Section, gram,MemorialSloan-KetteringCancer 75248Paris,Cedex05,France Center,NewYork,NewYork10021 SusanneOpravil(16),ResearchInstituteof RonenMarmorstein(7),StructuralBiol- MolecularPathology(IMP),TheVienna ogy Program, The Wistar Institute, Biocenter,Vienna,A-1030,Austria Philadelphia,Pennsylvania19104-4268 Barbara Panning (28), Department of KarlMechtler(16),ResearchInstituteof BiochemistryandBiophysics,University MolecularPathology(IMP),TheVienna of California, San Francisco, San Fran- Biocenter,Vienna,A-1030,Austria cisco,California94143-0448 KatrinaB.Morshead(22),Massachusetts LauraPerez-Burgos(16),ResearchInsti- General Hospital, Department of tuteofMolecularPathology(IMP),The MolecularBiology,Boston,Massachusetts Vienna Biocenter, Vienna, A-1030, 02114 Austria xii contributors to volume 376 Antoine H. F. M. Peters (16), Research Paul R. Thompson (12), Department of InstituteofMolecularPathology(IMP), Pharmacology and Molecular Sciences, The Vienna Biocenter, Vienna, A-1030 Johns Hopkins University School of Austria Medicine,Baltimore,Maryland21218 DannyReinberg(17),DepartmentofBiol- Julissa Tsao (21), Microarray Centre, ogy, Howard Hughes Medical Institute, University Health Network, Toronto, University of Medicine and Dentistry OntarioM5G2C4,Canada of New Jersey, Piscataway, NJ Lori L. Wallrath (25), Department of 08854-5635 Biochemistry, University of Iowa, Iowa BingRen(20),SanDiegoBranchandDe- City,Iowa52242 partment of Cellular and Molecular Michelle D. Wang (5), Department of Medicine, Ludwig Institute for Cancer Physics,LaboratoryofAtomicandSolid Research, University of California, San StatePhysics,CornellUniversity,Ithaca, Diego School of Medicine, La Jolla, NewYork14853 California92093-0653 VictoriaM.Richon(13),DiscoveryBiol- LingWang(12),DepartmentofPharma- ogy,AtonPharma,Inc.,Tarrytown,New cology and Molecular Sciences, Johns HopkinsUniversitySchoolofMedicine, York10591 Baltimore,Maryland21218 RichardC.Robinson(14),Laboratoryof Metabolism, National Institutes of JanisK.Werner(26),CornellUniversity, Health, National Cancer Institute, Ithaca,NewYork14853 Bethesda,Maryland20892 Jon Widom (1), Northwestern University, Daniel Robyr (19), Department of Biol- DepartmentofBiochemistry,Molecular ogicalChemistry,UniversityofCalifornia, Biology and Cell Biology, Evanston, Los Angeles, School of Medicine and Illinois60208-3500 MolecularBiologyInstitute,LosAngeles, ChristopherL.Woodcock(3),Department California90095 of Biology, University of Massachusetts, KavithaSarma(17),DepartmentofBiol- Amherst,Massachusetts01003 ogy, Howard Hughes Medical Institute, PatrickYau(21),MicroarrayCentre,Uni- University of Medicine and Dentistry versityHealthNetwork,Toronto,Ontario of New Jersey, Piscataway, NJ M5G2C4,Canada 08854-5635 Ken Zaret (24), CellandDevelopmental Stuart Schreiber (23), Department of BiologyProgram,W.W.SmithChairin ChemistryandChemicalBiology,Harvard Cancer Research, Fox Chase Cancer University, Cambridge, Massachusetts Center,Philadelphia,Pennsylvania19111 02138 BrianE.Schwartz(26),CornellUniversity, Yujun Zheng (12), Department of Pharmacology and Molecular Sciences, Ithaca,NewYork14853 Johns Hopkins University School of J. Paul Secrist (13), Discovery Biology, Medicine,Baltimore,Maryland21218 AtonPharma,Inc.,Tarrytown,NewYork Ming-Ming Zhou (8), Structural Biology 10591 Program,DepartmentofPhysiologyand Gena E. Stephens (25), Biology Depart- Biophysics,Mt.SinaiSchoolofMedicine, ment,WashingtonUniversityinSt.Louis, New York University, New York, New St.Louis,Missouri63130 York10029-6574 contributors to volume376 xiii XianboZhou(13),DiscoveryBiology,Aton Jordanka Zlatanova (6), Department of Pharma, Inc., Tarrytown, New York ChemicalandBiologicalSciencesandEn- 10591 gineering,PolytechnicUniversity,Brook- lyn,NewYork11201 [1] fluorescence anisotropyassays 3 [1] Fluorescence Anisotropy Assays for Analysis of ISWI-DNA and ISWI-Nucleosome Interactions By J. Chin, G. La¨ngst, P. B. Becker, and J. Widom Fluorescenceanisotropyisarapid,sensitive,andquantitativetechnique thatiswellsuitedtotheanalysisofprotein-proteinandprotein-DNAinter- actionsinsolution.Fluorescenceanisotropyisameasureofthedepolarization of emitted fluorescence intensity obtained after excitation by a polarized lightsource,anddependsdirectlyontherelativerateoffluorescenceemis- sion versus the rate of tumbling in solution. The concept is simple: if a fluorescentmolecule(or,moretypically,amoleculetowhichafluorescent probehasbeenattached)tumblesslowlyinsolutionrelativetothelifetime of fluorescence emission, then the light emitted in response to polarized excitation will remain highly polarized. However, if the molecules tumble rapidlyincomparisontotheemissionlifetime,then,priortoemitting,they willhavetumbledsufficientlysoastohave‘‘forgotten’’theirorientationat themomentofexcitation,thusdepolarizing(randomizingthepolarization of)the emitted light. Fluorescence anisotropy is applicable for analysis of macromolecular interactions because there is a good match between typical fluorescence lifetimes and typical macromolecular tumbling times. For approximately sphericalmolecules,thetumblingtimescalesasthemolecularvolume,that is, as the molecular weight. Thus, binding of an unlabeled macromolecule can make a significant change to the tumbling time of the molecule to whichthefluorescentprobeisattached,andhencetothemeasuredanisot- ropy.Forthestudiesdescribedinthefollowing,weutilizeDNAmolecules labeled at one end with the fluorescent dye fluorescein (these DNA mol- ecules may be ‘‘naked DNA’’ or they may be incorporated into nucleo- somes), and we use fluorescence anisotropy to monitor the binding of the Drosophila ISWI chromatin remodeling protein1–3 to the labeled DNA ornucleosomes. Fluorescenceanisotropyisespeciallyusefulbecauseofitshighinherent sensitivity.Dyessuchasfluoresceinallowquantitativeanalysisofemission polarization from sub-nanomolar concentrations. Since dissociation con- stants are typically nanomolar or greater, this allows experiments to be 1T.Tsukiyama,C.Daniel,J.Tamkun,andC.Wu,Cell83,1021(1995). 2P.D.Varga-Weiszetal.,Nature388,598(1997). 3G.La¨ngstandP.B.Becker,J.CellSci.114,2561(2001). Copyright2004,ElsevierInc. Allrightsreserved. METHODSINENZYMOLOGY,VOL.376 0076-6879/04$35.00 4 chromatin proteins [1] setupwiththeprobeconcentration(cid:1)K ;consequentlythefreeconcentra- d tion of the added macromolecule (ISWI, in our case), which is generally eitherdifficulttomeasureoriscompletelyunknown,willbeapproximately equal to the total concentration, which can be definitively measured, thus greatlysimplifyingtheanalysisofthebindingmeasurements.Anotherim- portant benefit of the sensitivity of the anisotropy measurement is that it preservespreciousreagents.Measurementscanbemadeinsmallvolumes, and samplescan be recovered and reused if desired. Finally,asdiscussedlater,theexperimentcanbecarriedoutusinginex- pensive conventional fluorometers such as are found at most biochemical or chemical research laboratories, or, alternatively, using an inexpensive instrument specialized for thefluorescenceanisotropy experiment. Investigators planning to carry out such studies should study two par- ticularly useful references, one on fluorescence theory and methodology ingeneral4andonefocusedonfluorescenceapproachestoanalysisofpro- tein-DNA interactions in particular.5 These references nicely define and explain the set of four fluorescence intensity measurements that go into a single measurement of fluorescence anisotropy; we will not duplicate this important topic here, but rather refer readersto these other sources. Fluorescein-Labeled DNA WeuseDNAsequenceslabeledwithfluoresceinattachedatthe50-end through a C6 linker. Relatively short sequences are purchased as a pair of complementary oligonucleotides, one containing 50-fluorescein. These are annealed, and the resulting duplex purified away from any remaining single strand by reverse-phase HPLC on a Zorbax-10 column using a gradient of 10–20% acetonitrille in 0.1 M triethanolamine-acetate, pH 7.0, 0.1 mM EDTA, developed over 10 min at 1 ml min(cid:3)1. When longer sequences (e.g., nucleosome-length DNAs) are required, direct synthesis is not practical. Instead we use preparative scale PCR, with one of the two primers again containing 50-fluorescein. The resulting PCR productispurifiedbygelelectrophoresisin1%agarosegelswithstandard TAE buffer, and extracted from the gel using Ultra-DA (Millipore) gel extraction kits. DNA concentrationsarequantified by UV absorbance. 4J. R. Lakowicz, ‘‘Principles of Fluorescence Spectroscopy,’’ 2nd Ed. Kluwer Academic/ PlenumPress,NewYork,1999. 5J.J.HillandC.A.Royer,Meth.Enzymol.278,390(1997).