IdentificationofunknowntargetgenesofhumantranscriptionfactorsusingchromatinimmunoprecipitationAmyS.WeinmannandPeggyJ.Farnham*McArdleLaboratoryforCancerResearch,UniversityofWisconsinMedicalSchool,1400UniversityAvenue,Madison,WI53706,USAAccepted15January2002AbstractThestandardchromatinimmunoprecipitation(ChIP)assayisusedtoexaminethespecificassociationoftranscriptionfactorswithDNAinthecontextoflivingcells.Herewereviewtwomodificationstothisprotocolwhicharedesignedtoidentifynoveltargetgenesoftranscriptionfactorsinmammaliancells.ThemainadvantagetobothoftheseapproachesisthatonlyDNAsequencesdirectlyboundbyafactorwithinthecontextofalivingcellwillbeidentified.Therefore,artifactsassociatedwithoverexpressionand/oralterationsinsignalingpathwaysareavoided.Thefirstmodificationwedescribe,aChIPcloningstrategy,canbeusedtoisolateanygenomicfragmentspecificallyassociatedwithaparticularfactor.Itrequiresnospecialequipmentorreagentsotherthanahigh-affinityantibodytobeusedforimmunoprecipitationofthefactorofinterest.However,itismostusefulfortheisolationofasmallnumberofgenomictargets.Incontrast,thesecondmodification,whichcombinesChIPwithspecializedCpGmicroarrays,isidealforamoreglobalanalysisoftargetgenes.Advantages,commonproblems,anddetailedprotocolsforthesetwoChIPtech-niquesarediscussed.�2002ElsevierScience(USA).Allrightsreserved.Keywords:Chromatinimmunoprecipitation;Transcriptionfactors;Targetgenes;CpGmicroarrays;Shotguncloning;E2F1.IntroductionAsthesequencingofthehumangenomenearscom-pletion,thechallengethatfacesthescientificcommunityistodeciphertheunderlyingmeaningbehindthesepreciselyorderednucleotides.Oneportionofthissequencewillprovidethegeneticinformationtocreatethelargenumberofproteinsrequiredformaintainingthecriticalfunctionsfordiversecelltypes.Inaddition,asignificantfractionofthegenomewillprovidetheinformationrequiredtodirecttheprecisetimingandpatternofexpressionfortheseproteins.Thepreciseregulationofgeneexpressiondeservesgreatattentionbecausetheinappropriateexpressionofasinglegen...
