ResearchReportChromatinimmunoprecipitationassaysrevealedCREBandserine133phospho-CREBbindingtotheCARTgeneproximalpromoterGeorgeA.Rogge,Li-LingShen,MichaelJ.Kuhar⁎YerkesNationalResearchPrimateCenterofEmoryUniversity,Atlanta,GA30329,USAARTICLEINFOABSTRACTArticlehistory:Accepted26April2010Availableonline6May2010BothoverexpressionofcyclicAMPresponseelementbindingprotein(CREB)inthenucleusaccumbens(NAc),andintra-accumbalinjectionofcocaine-andamphetamine-regulatedtranscript(CART)peptides,havebeenshowntodecreasecocainereward.Also,overexpressionofCREBintheratNAcincreasedCARTmRNAandpeptidelevels,butitisnotknownifthiswasduetoadirectactionofP-CREBontheCARTgenepromoter.ThegoalofthisstudywastotestifCREBandP-CREBbounddirectlytotheCREsiteintheCARTpromoter,usingchromatinimmunoprecipitation(ChIP)assays.ChIPassaywithanti-CREBantibodiesshowedanenrichmentoftheCARTpromoterfragmentcontainingtheCREregionoverIgGprecipitatedmaterial,anon-specificcontrol.Forskolin,whichwasknowntoincreaseCARTmRNAlevelsinGH3cells,wasutilizedtoshowthatthedrugincreasedlevelsofP-CREBproteinandP-CREBbindingtotheCARTpromoterCRE-containingregion.Aregionofthec-FospromotercontainingaCREcis-regulatoryelementwaspreviouslyshowntobindP-CREB,anditwasusedhereasapositivecontrol.ThesedatasuggestthattheeffectsofCREBoverexpressiononbluntingcocainerewardcouldbe,atleastinpart,attributedtotheincreasedexpressionoftheCARTgenebydirectinteractionofP-CREBwiththeCARTpromoterCREsite,ratherthanbysomeindirectaction.©2010ElsevierB.V.Allrightsreserved.Keywords:CARTCREBCocainelrewardChIP1.IntroductionCocaine-andamphetamine-regulatedtranscript(CART)wasdiscoveredasanmRNAthatwasincreasedintheventralstriatumofratsafteranacuteinjectionofpsychostimulants(DouglassandDaoud,1996;Douglassetal.,1995).Whiletheincreasewasnotalwaysfound(Hunteretal.,2005;Marie-Claireetal.,2003;Vrangetal.,2002;JonesandKuhar,2006),recentstudiesdemonstratedthatacutecocaineinjectionsdose-andtime-dependentlyincreasedc-Fosimmunoreactiv-ityinCART-containingcellsoftheratnucleusa...
