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INTRODUCTION Incellsandtissues,thehistoneproteinsthatconstitutethenucleosomescanpresentmultiplepost-translationalmodifications,suchaslysineacetylation,lysineandargininemethylation,serinephosphorylation,andlysineubiquitination.Ontheirown,orincombination,thesecovalentmodificationsonthecorehistonesarethoughttoplayessentialrolesinchromatinorganizationandgeneexpressionineukaryotes.Importantly,patternsofhistonemodificationsmaybesomaticallyconservedandcan,thereby,maintainlocus-specificrepression/activityindefinedlineages,orthroughoutdevelopment.Indirectimmunofluorescencestudiesonculturedcellshavebeenpivotalinunravelingtherolesofhistonemodifications.However,toaddressindetailwhathappensatspecificsitesinvivo,chromatinimmunoprecipitation(ChIP)isthemethodofchoice.Here,wedescribehowChIPcanbeperformedonnon-fixedchromatinfromanimalcellsortissues(freshorfrozen)toanalyzehistonemodificationsatspecificchromosomalsites.Theseprotocolsaresuitableonlyforanalyzinghistonesandtheirmodifications.Forotherapplications,chromatinimmunoprecipitationshouldbeperformedoncross-linkedchromatin. RELATEDINFORMATION ThisChIPprotocolwasderivedfrommethodologiesoriginallydescribedbyO’NeillandTurner(1995).ItwaspublishedearlierontheWebsiteoftheEuropeanNetworkofExcellence"EPIGENOME"(www.epigenome-noe.net),andwasadaptedfromUmlaufetal.(2003).AnoverviewoftheprocedureisprovidedinFigure1.FollowingChIP,weusedifferentPCR-basedmethodsthatallowonetoanalyzealocusofinterestintheprecipitatedchromatin(seePCR-basedAnalysisofImmunoprecipitatedChromatinfordetails). MATERIALS Reagents 100-bpDNAsizeladder(Promega) Agarose Antisera(affinity-purified) Theseshouldberaisedagainsthistonepeptideswithmono-,di-,ortrimethylationoracetylationataspecificlysine/arginineresidueofinterest.Inaddition,includeacontrolprecipitationwithan(IgG)antiserumthatisnotdirectedagainstchromatinproteins. Cellculturefromwhichnucleiaretobeextracted(1x107to1x108cellsarerequired;seeSteps9-16) Cellculturemedium,appropriateforcellsofinterest(seeStep10) Ethanol(70%,v/v) Glycogensolution(20mg/ml)(Roche) Micrococcalnuclease(MNase;10units/µlin50%[v/v]glycerol)(AmershamBioscience) Storein10-to20-µlaliquotsat–20°C.Eachaliquotshouldbeusedonlyoncetoensureequalenzymeactivityindifferentchromatinpreparations. ProteinA(e.g.,CL-4BSepharosefromAmershamBioscience)orproteinGSepharose ProteinAandproteinGarebacterialcellwallproteinsthatbindtotheFcregionofantibodies.TheseproteinsarecovalentlycoupledtoSepharose.ThechoicebetweenproteinAorproteinGSepharosedependsonthenatureoftheantibodyusedforChIP.Ingeneral,proteinAworksbestforrabbitpolyclonalantiseraandformousemonoclonalantibodiesfromtheIgG2a,IgG2b,andIgG3subclasses.ProteinGSepharoseispreferredformouseIgG1monoclonalantibodiesandforpolyclonalantiserafrommouse,rat,sheep,andgoat. Sodiumbutyrate(optional;foranalyzinghistoneacetylationonly) Toanalyzehistoneacetylation,werecommendaddingsodiumbutyrate(toafinalconcentrationof5mM)tothesolutionsusedforthepurificationofnucleiandforthepreparationofinputchromatin.Sodiumbutyratepreventslossofhistoneacetylationviathenonspecificactionofendogenoushistonedeacetylases. Tissuesamples(freshorfrozen)fromwhichnucleiaretobeextracted(seeSteps1-8) Equipment Centrifuges: Dialysistubing(0.5-mmthick,10-kDaporewidth)(VWRinternational) Homogenizer,prechilledonice Weuseatissuegrinder/homogenizer(fromBDH)thathasaglassmortar(tube)andapestlewithahardplastichead.Theclearancebetweenpestleandmortaris0.15-0.25mm. Horizontalgelelectrophoresistankforagarosegels Ice Magneticstirrer(seeStep32) Microcentrifugetubes(1.5mland2.0ml) Microscope,invertedlight(optional;seeStep8) Mortarandpestle,prechilledinliquidnitrogen(forfrozentissueonly;seeStep1) Muslincheesecloth PreparethecheeseclothbyrinsingwithH2Oandthenautoclaving. Pasteurpipettes Polypropylenetubes,14ml(e.g.,17x100-mmFalcontubes)and15ml(e.g.,17x120-mmFalconconicaltubes) Rotatingwheelsat4°Candroomtemperature Spectrophotometer Trayforstaininggels(seeStep41) Universaltubingclamps(5mm)(SpectrumLaboratories) UVlamp Vortexmixer Waterbathsetat37°C METHOD NucleiPreparationfromTissuesandCells Steps1-8describethepurificationofnucleifromtissue,whileSteps9-16describethepurificationofnucleifromculturedcells.Topreventchromatindegradation,allstepsofthenucleipurificationprocedureshouldbeperformedonice,orat4°C(e.g.,precoolthecentrifugerotors).Inaddition,onesetofmicropipettesshouldbededicatedonlytothepreparationofnuclei,chromatin,andChIPanalysis,toavoidDNAcontamination.Wearglovesthroughoutallprocedures,andrespectthesafetyrules,especiallywhenhandlingphenol. PurificationofNucleifromTissue(2h) NucleiPreparationfromCulturedCells(2h) MicrococcalNuclease(MNase)FractionationandPurificationofChromatin PreparationofDialysisTubing(2hincludingcoolingtime) MNaseFractionation(30min) RecoveryofSolubleChromatinFractions(16h) QualityControlofChromatin(3h) ChromatinImmunoprecipitation IncubationofChromatinwithAntiserum(16h) PreparationofProteinA(G)Sepharose(45min) ExtractionofImmunoprecipitatedChromatinwithProteinA(G)Sepharose(6-7h) DNAExtractionfromPrecipitatedChromatin(3h) AssessmentofPrecipitatedChromatin TROUBLESHOOTING Problem:Howmuchtissue(andwhichkind)mustbeusedtopurifyenoughnucleiforaChIPexperiment? [Step1] Solution:Thisprotocolworkswellontissues,suchasliver,brain,lung,andplacenta,andalsoonearlymammalianembryos.Inourlaboratory,forinstance,wehaveperformedstudieson8.5-9.5d.p.c.mouseembryosandplacentas(Umlaufetal.2004).Some60dissectedembryoswereusedforeachChIPexperiment.Nomorethan~0.2goftissueshouldbeusedforthevolumesandtubesizesindicatedintheprotocol. Problem:Howcantheyieldofintactnucleibemaximized? [Steps4and12] Solution:AtStep4(or12)ofthenucleipurificationprocedure,itiscriticalnottoextendtheincubationinnucleipreparationbufferIIfor>10minutes.Forthatreason,Step5shouldbeinitiatedafter5minutesofincubationinordertocommencecentrifugation(Step6)atexactly10minutesaftertheadditionofnucleipreparationbufferIIinStep4.Longerincubationscangreatlyreducetheyieldofintactnuclei.Formanytissues(liver,kidney,placenta),afinalconcentrationof0.2%ofthenonionicdetergentIGEPALCA-630(innucleipreparationbufferII)willbeenoughtolysethecellularmembranesduringthe10-minuteincubation.However,werecommendtesting0.4%IGEPALCA-630forothertissues.Forinstance,thishigherconcentrationofdetergentslightlyimprovestheyieldofnucleifrombrainandmuscletissues. Problem:Isitaproblemifthetoplayer(containingtheIGEPALCA-630)comesintocontactwiththenucleipellet? [Steps7and15] Solution:AtStep7(or15),itisessentialthatnotracesofthetoplayer(containingtheIGEPALCA-630)comeintocontactwiththenucleipellet(evensmalltracesofIGEPALCA-630mayaberrantlyaffectthesubsequentdigestionofchromatinbyMNase).Usually,weremovethetoplayerandthesucrosecushionfromthetubebyusingPasteurpipettes.Thisisdonebyaspiratingfromthesurfaceofthesolution,whilechangingthePasteurpipetteveryoften.Ifthetoplayerneverthelesscomesincontactwiththenucleipellet,thepelletshouldbegentlyrinsedoncewith1mlofnucleipreparationbufferIIIbeforeproceedingwithStep8(or16). Problem:Whatcanbedoneif,aftertheMNasedigestion,thechromatinappearstobedigestedtoomuchortoolittle? [Step43] Solution:FractionationofchromatindependsonthebatchofMNaseused,theconcentrationofthenucleiinthetube,thetimeofincubation,andthetissuetypefromwhichthenucleiwerepurified.Ifoneobservestoomuchdigestionofthechromatin(i.e.,almostallchromatinisdigestedtomono-anddinucleosomefragments),alowerconcentrationofMNaseshouldbeused.Inversely,incaselittlematerialisobtainedintheS1fraction,theamountofenzymeshouldbeincreased. Problem:WhatcanbedonetoimproveChIPwhenusingchickenantiserathatdonotbindwelltoeitherproteinAorproteinG? [Step47] Solution:Werecommendadding5µgofarabbitanti-chickenantiserumdirectlyafterStep47forasecondprecipitationof3-4hours,beforeproceedingwiththeextractionoftheantibody-boundchromatin. Problem:Whenanantiserumisusedforthefirsttime,howdoesoneverifythatthehistonemodificationitisdirectedagainsthasbecomeenrichedintheantibody-boundfraction? [Step69] Solution:Thiscanbedonebypurifyingthehistoneproteinsfromtheantibody-boundfraction(fromStep69),followedbyelectrophoresisthroughacid-urea-Tritongels.Afterelectrophoresis,proteinsareWestern-blottedtonylonfilters,whichareimmunostainedwiththeantiserumfollowingstandardprocedures(Gregoryetal.2001). DISCUSSION Therearedifferentwaystoobtaininputchromatin.Severalgroupsinthefieldprepare"cross-linkedchromatin,"forexample,bychemicallycross-linkingproteinsandDNAwithspecificsubstancessuchasformaldehyde.However,usuallyonlyasmallfractionofthechromatinisprecipitated,andthismethodreliesonrandomshearingaftercross-linking,whichdoesnotalwaysproducesmall-enoughchromatinfragmentsattheregionsofinterest.Forthisreason,andtobeabletoconductexperimentsonfreshandfrozentissues,weandothershavepreferredtomakeuseof"nativechromatin."Inourprotocol(Fig.1),thechromatinisfractionatedbyincubationofpurifiednucleiwithmicrococcalnuclease(MNase),anenzymethatcleavespreferentiallyatthelinkerDNAbetweenthenucleosomes.ByperformingpartialdigestionswithMNase,itispossibletoobtainnativechromatinfragmentsof,onaverage,onetofivenucleosomesinlength(Fig.2).Theseoligo-nucleosomefragmentsarepurifiedfromthenucleiandarethenusedtoperformChIP.ThechoiceofnativechromatinastheinputmaterialforChIPisadvantageousbecausetheepitopes,recognizedbytheantibody,remainintactduringthechromatinpreparation.Asaconsequence,nativechromatintendstogivehigherlevelsofprecipitationforaspecifichistonemodificationthanformaldehydecross-linkedchromatin.Becausefractionationoccursbetweenthenucleosomes,ratherthanrandomly,precipitatednativeoligo-nucleosomefragmentsarealsoparticularlysuitabletobeusedfor"ChIPonchip."InarecentChIPonchipstudy(Bernsteinetal.2006),bothnativeandformaldehyde-cross-linkedchromatinwereprecipitatedwithantiseraagainsthistonemodifications.DNAsamplesextractedfromtheprecipitatedchromatinwereusedasprobestohybridizeDNAtilingarrayscoveringmanylargechromosomalregio,ns.Inthislarge-throughputstudy,resultsobtainedwithnativechromatinwereverysimilartothoseobtainedwithcross-linkedchromatin.Forlocus-specific,smaller-scalestudies,amplificationbythepolymerasechainreaction(PCR)remainsthemethodofchoice.DifferentPCR-basedapproachescanbeusedtodeterminehowmuchDNAisprecipitatedatasiteofinterest(seePCR-basedAnalysisofImmunoprecipitatedChromatin). AlthoughChIPispresentlythebestmethodologytoanalyzehistonemodificationsatspecificchromosomalloci,ithasseverallimitations.First,unlikeDNAmethylationstudies,ChIPdoesnotallowanalysisofhistonemodificationsinindividualcellsoronindividualchromosomes.ChIPstudiesarealwaysperformedonpopulationsof(cultured)cellsorontissuesamplescomprisingmanycells.Moreover,althoughsequentialprecipitationswithdifferentantiseracanbedone(Bernsteinetal.2006),orantiseraagainstcombinationsofdifferenthistonemodificationscanbeused,itisnoteasytodeterminewhethertherearespecificcombinationsofcovalentmodificationsonindividualhistonesatagivenlocus.Again,thisisbecausemanycellsareusedforchromatinpurificationandChIP,andchromatinisusuallyfractionatedintofragmentsthatcomprisemultiplenucleosomes.Last,itshouldbenotedthatquantificationofthelevelsofhistonemodificationsatspecificchromosomallociisdifficulttoobtainbyChIP,becauselevelsofprecipitationdonotdependsolelyonthelocalabundanceofthemodificationstudied.Theyalsodependonthequalityofthepreparedchromatin,onthespecificityandconcentrationoftheantiserumused,andontheglobalabundanceofthehistonemodificationthatisbeingstudied.Factorsthatcaninfluencetheoutcomeoftheexperimentare(1)thedistributionofthehistonemodificationsonthechromosomes,(2)theamountofantiserumused,and(3)the"strength"oftheantibodies(i.e.,theaffinityfortheirepitope).Ontheotherhand,theefficiencyofprecipitationofmodifiedhistonesatalocusofinterestgreatlydependsonwhetherthemodificationiscommonorrareinthegenome.Forinstance,araremodification(e.g.,H3-K4methylation)givesusuallygoodprecipitationatthesitewhereitispresent.Thiscanbeexplainedbythefactthat,intheChIP,thequantityofantibodyaddedtothetubeishighenoughtoprecipitateallthechromatinthatcarriesthatspecificmodification.However,foramodificationthatisabundantinthegenome,theindicatedamountofantibody(5-10µg)sometimesdoesnotprecipitateallthechromatinthathasthemodification.Thesedifferentfactorsshouldbetakenintoaccountwhencomparingdifferentchromatinimmunoprecipitationexperiments. ACKNOWLEDGMENTS WethankRichardGregoryandDavidUmlauffordesignofmethodologies,andBryanM.TurnerandLauraP.O’Neill(Birmingham,UK)forintroducingustoChIPonunfixedchromatin.TheCNRS,theAssociationpourlaRecherchesurleCancer(ARC),andtheESFEuroCORESProgrammeEuroSTELLSareacknowledgedforgrantsupport. 
Viewlargerversion(16K):Figure1.Flowchartoftheproceduresusedtoinvestigatesite-specificcovalentmodifications(i.e.,methylationandacetylation)onhistones.Insummary,nucleiarepurifiedfromfresh/frozentissuesorfromcells,andthechromatin,afterfractionationwithmicrococcalnuclease(MNase),ispurifiedfromthenuclei.This"inputchromatin,"madeupoffragmentsofuptosevennucleosomesinlength,isincubatedwithanantiserumdirectedagainstthehistonemodificationofinterest.Theantibody-boundfractionisseparatedfromtheunboundfractionand,afterextractionofgenomicDNAfromtheboundandunboundfractions,PCRtechnologiesareappliedtospecificallyanalyzethegeneorchromosomalregionofinterest.PrecipitatedDNAscanbeusedasprobestohybridizeDNAtilingarrays(ChIPonchip)aswell. 
ChIPelutionbufferChIPincubationbufferDialysis-lysisbufferDNAloadingbuffer(6X)Ethidiumbromidesolution(20mg/mlinH2O)IsopropanolLiquidnitrogen(forfrozentissueonly;seeStep1)MNasedigestionbufferMNasestopsolutionNaCl(5M)NucleipreparationbufferI,prechilledoniceNucleipreparationbufferII,prechilledoniceNucleipreparationbufferIII,prechilledonicePhenol:chloroform:isoamylalcohol25:24:1(v:v:v)ForextractionofgenomicDNA,thephenolshouldbesaturatedbeforehandwith100mMTris-Cl(pH7.5)andstoredat4°Cunder10mMTris-Cl(pH7.5).Phosphate-bufferedsaline(PBS),coldProteinaseK(10mg/ml)(optional;seeStep70)Sodiumdodecylsulfate(SDS;10%,w/v)TBEbuffer(1X)TEbuffer(1X,pH7.5)Trypsinsolution(0.05%[w/v])(Sigma)TubingpreparationsolutionITubingpreparationsolutionIIWashingbufferAWashingbufferBWashingbufferCChromatinimmunoprecipitationsandincubationswithProteinA(G)Sepharose(Steps44-69)areperformedinmicrocentrifugetubes.Thesetubesmaybesiliconizedbeforehand(e.g.,witha2%[v/v]dichloromethylsilanesolution)inordertopreventnonspecificassociationofchromatinandantibodiestotheinnerwallsofthetubes.Inourlaboratory,wehaveobtainedcomparableresultswithnonsiliconizedandsiliconizedmicrocentrifugetubes.
SeeTroubleshooting.
Viewlargerversion(32K):Figure2.Photographofnativechromatinpreparationwithfragmentsof,onaverage,onetofivenucleosomesinlength.Forthisexperiment,nucleiwerepurifiedfromprimaryfibroblastsandincubatedwithMNasefor6,9,12,and15minutes(lanes1-4,respectively).TheS1fractionswereobtaineddirectlyafterMNasedigestion,whereastheS2fractionswererecoveredbyovernightdialysis.Bandscorrespondingtochromatinfragmentsofonenucleosome(mono)tofivenucleosomes(penta)inlengthareindicated(1.2%agarosegel).Fractions1and2ofS1werecombinedwithfractions3and4ofS2forsubsequentChIP.
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