MMB 257 mRNA Processing and Metabolism, Methods and Protocols

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1、METHODSINMOLECULARBIOLOGYMETHODSINMOLECULARBIOLOGYTMTMVolume257mRNAProcessingmRNAProcessingandMetabolismandMetabolismMethodsandProtocolsMethodsandProtocolsEditedbyDanielR.SchoenbergDanielR.SchoenbergChromatinImmunoprecipitation11UsingChromatinImmunoprecipitationtoMapCotranscriptionalmRNAProc

2、essinginSaccharomycescerevisiaeMichael-ChristopherKeoghandStephenBuratowskiSummaryThechromatinimmunoprecipitation(ChIP)techniquehasbeenusedtodeterminewhereandunderwhatconditionsDNAbindingproteinsassociatewithspecificDNAsequences.Pro-teinsarecrosslinkedinvivowithformaldehyde,andchromatinisthe

3、nisolatedandsheared.TheproteinofinterestisthenimmunoprecipitatedandtheassociatedDNAsequencesidenti-fiedviaPCR.AlthoughthistechniquewasoriginallydesignedtoassayDNAbindingproteins,itcanalsobeusedtomonitormRNAprocessingfactorsassociatedwithtranscriptioncom-plexes.KeyWordsChromatinimmunoprecipit

4、ation;epitopetagging;polymerasechainreaction;tandemaffin-itypurification(TAP)tag.1.IntroductionSynthesisofmRNAbyRNApolymeraseII(RNApII)isacomplexprocessinvolvingthetransientassociationoflargeproteincomplexeswithDNA(1,2).Muchworkinthefieldhasconcentratedoninvitroreconstitution,examiningtherol

5、eofindividualproteinsorcomplexesatdifferentstepsofthetranscrip-tioncycle.However,studyofthisprocessinitsnaturalchromosomalenviron-mentisrequiredformorecompleteunderstanding.Thischapterdescribeschromatinimmunoprecipitation(ChIP),amethodusedtodeterminewhereandwhenaparticularproteinislocatednea

6、rspecificDNAsequences(3–6).Chromatinimmunoprecipitationhasbeenusedexten-sivelyinthebuddingyeastSaccharomycescerevisiae,butthetechniquehasFrom:MethodsinMolecularBiology,vol.257:mRNAProcessingandMetabolismEditedby:D.R.Schoenberg©HumanaPressInc.,Totowa,NJ12KeoghandBuratowskiFig.1.Chromatinimmun

7、oprecipitationschematic.ProteinXislocalizedintheregionofthepromoter(TATA)duringtranscription,butnotthroughouttheopenread-ingframe(ORF)oratthe3'UTR(AATAAA).Followingformaldehydecrosslinking,thecellsarelysedandthechromatinisolatedandshearedtosmallerf