Cardiomyocytesrequireprecisegeneregulationprogramstopreventpathophysiology.Upregulationofcardiac remodelinggenesresultsincardiomyocytehypertrophyanddecreasedheartfunction.Whilemuchisknown aboutthetranscriptionfactorsthatdrivecardiomyocytespecificgeneexpression,wehavealimited understandingofhowhistoneposttranslationalmodificationsregulatecardiomyocytedevelopmentand maintenance.PolycombRepressiveComplex2(PRC2)isahistonemodifyingcomplexthatsilencesgene expressionbytri-methylatinglysine27onhistoneH3.Conditionalknockoutstudiesshowtheabsenceof PRC2,duringmouseheartdevelopment,resultsinupregulationofcardiomyocytespecifictranscriptionfactors andcardiachypertrophy.However,thereisalackofmechanisticunderstandingregardinghowPRC2 regulatescardiomyocytespecificgenes.EmergingevidencedemonstratesthathumanPRC2isrecruitedto chromatinthroughaninteractionwithasetofaccessoryproteinsknownasAEBP2andPCLproteins.This proposalwillutilizePRC2separation-of-functionmutationstotestthehypothesisthattheinteractionbetween PRC2andtheseaccessoryproteinsisnecessaryforregulatingcardiomyocytespecificgenesandfor preventingcardiomyocytehypertrophy.High-throughputsequencingandimmunocytochemistryapproaches willbeusedtodeterminewhetherAEBP2orPCLproteinsis/areresponsibleformaintainingthetranscriptional profileandphenotypeofcardiomyocytesinaPRC2dependentmanner.Furthermore,live-cellsingle-molecule imagingwillbeusedtodefinehowAEBP2andPCLproteinsaffectthechromatin-bindingdynamicsofPRC2 throughoutcardiomyocytedifferentiation.Theexperimentsinthisproposalrepresentacriticalsteptoward uncoveringthemechanismofPRC2mediatedgene-silencingincardiomyocytes,amechanismthatiscentral tonormalheartdevelopmentandfunction. TrainingPlanSummary:TheproposedresearchwillbecompletedintheUniversityofColoradoBoulder BiochemistryDepartment,locatedinthemultidisciplinaryBioFrontiersBiotechnologybuilding.Theapplicant willdrawfromlocalexpertiseinthefieldsofcardiomyocytebiology,computationalbiology,chromatinbiology, andtranscriptionalregulationtodevelopintoanindependentphysician-scientistthatiswellversedin mechanismsofgeneregulation.Theapplicant?strainingplanincludestakingcoursework,receivingindividual mentorship,attendingconferences,givingoralpresentations,andpreparing/submittingmanuscripts.
ThisproposalwillstudyhowPolycombRepressiveComplex2regulatescardiomyocytespecificgene expression.Findingsgeneratedfromtheproposedaimswillexpandourunderstandingofamechanismthat governsproperheartdevelopment,function,andcouldprovidepathwaysforfuturecardiomyopathytherapies.
