ProgramDirector/PrincipalInvestigator(Last,First,Middle):Peterson,Craig,Lewis Theoverallobjectiveofourresearchistodeterminehowchromosomestructureinfluencesgene transcription,DNAreplicationandrepair,withspecialemphasisonidentifyingandcharacterizingthechromatin remodelingmachinesthatcontrolchromosomedynamics.Notably,geneticexperimentshaverevealedATP- dependentchromatinremodelingenzymesasessentialregulatorsofvirtuallyeverychromosomalprocess,and theirdysregulationleadstoavarietyofdiseases,includingcancer.Ourresearcheffortscanbeorganized intothreeinter-relatedareas:(1)MechanisticstudiesofATP-dependentchromatinremodelingenzymes;?(2) Roleofchromatindynamicsingenomestabilitypathways;?and(3)Assembly/functionofchromatinhigherorder structures.Amajorfocusofourmechanisticstudiesistocontinuetodissectthestructureand biochemicalmechanismsoftheINO80CandSWR1Cenzymes.Theseremodelingenzymescatalyze novel,ATP-dependenthistoneexchangeeventsthatcontrolthedepositionanddistributionoftheH2A.Z histonevariantwithinnucleosomesthatflankpromotersofgenestranscribedbyRNApolymeraseII,aswellas nucleosomesthatflankchromatinboundaryelements,centromeres,andreplicationorigins.Mammalian homologsofSWR1CandINO80C,includingthep400/Tip60andhINO80complexes,arekeyforproperstem cellfunction,genomestability,development,andgeneexpression.Duringthepastbudgetperiod,weidentified anovelregulatoryinteractionbetweenSWR1Candtheacetylationoflysine56ofhistoneH3(H3-K56Ac)that regulatesnucleosomedynamics,noncodingRNAexpression,andassemblyoflarge-scale,chromosome interactiondomains(CIDs)thatarerelatedtomammaliantopologically-associateddomains(TADs).These mechanisticstudieswillincludequantitative,fluorescence-basedassaystodefinestepsofthehistonedimer exchangereaction,aswellasthereconstitutionofthesemulti-subunitenzymeswithrecombinantsubunits. Studiesfromusandothersoverthepast10yearshavedemonstratedthatchromatindynamicsplayalarge roleinstabilizingthereplisomeandincontrollingvariousstepsinDNAdoublestrandbreakrepair.Ourrecent datasuggeststhatchangesinchromatindynamicscanalsoleadtodysregulationoftranscriptionwhich impactsgenomestabilitypathways.Ourresearchwilladdressseveralkeyunansweredquestions focusedongenomestabilitypathways:(1)DoRemodelersregulatethehomologysearchstepof homologousrecombinationanddotheyfunctioninconcertwithhistoneacetylases?(2)HowdoesINO80C stabilizethereplisomeandisthisroleduetotheregulationofncRNAexpression?(3)HowdoesINO80C preventncRNAexpressionfromintergenicregions?(4)DoesthehyperacetylationofH3-K56Acleadto formationofR-loopsthatdisruptreplisomefunction?(5)DoeshypoacetylationofH3-K56Acandtheresulting defectinnucleosomeassemblyalsoleadtoaberranttranscriptionduringSphasethatleadstogenome instability?Weplantocontinuetoexploitacombinationofinvitroandinvivoapproachestoaddresssuch questions. MechanisticstudiesofRemodelershaveprimarilyfocusedonsinglenucleosomesubstratesorsimple nucleosomalarrays.Invivo,theseenzymeslikelytargetnucleosomeswithinthecontextofcondensed chromatinfibers.Recently,weusedacombinationofsedimentationvelocityanalysesandAFMtodissectthe stoichiometryandsolutiondynamicsofasimpleformofyeastheterochromatinthatcontainstheprimary structuralcomponent,Sir3.Weplantoextendsuchstudieswithchromatinfibersreconstitutedwithacomplete complementofSirproteins(Sir2/3/4).Theoverallgoalwillbetocharacterizethestructureofyeast heterochromatinfibersaswellasunderstandinghowthesestructurescanbemodulatedby Remodelers.Asecondtypeofhigherorderchromosomestructureoccursthroughoutthegenome.CIDs containstronglyself-associatingnucleosomesthatspan~1-5genes,separatedbydistinctboundaryregions. LittleisknownaboutwhatcontrolsCIDorTADassemblyorwhatfunctionalrolestheyplay.Onepossibilityis thatdomainsofself-associatingnucleosomesassemblespontaneouslyadjacenttonucleosomedepleted regions(NDRs),andmutantsthatdisruptCIDseitheraffecttheefficiencyofNDRformationorincrease transcriptionthroughCIDs.Oneofourgoalsistouseagenome-wide,nucleosomereconstitutionsystem todirectlytestwhetherformationofpromoter-associated,nucleosome-depletedregionsissufficient forCIDassemblyinvitro.TheseinvitrostudieswillalsobecomplementedbygeneticanalysesofCID assembly,wherewewilleithereliminatekeyfactorsbygenedeletion,ormanipulatespecificCIDboundary regionsbyDNAalterations.Inthelongterm,understandinghowCIDsareassembledwillfacilitatestudiesto disruptthisprocessandinvestigatethefunctionalconsequencesonbothtranscriptionandgenomestability. OMBNo.0925-0001/0002(Rev.08/12ApprovedThrough8/31/2015) Page ContinuationFormatPage
Peterson,Craig,Lewis Chromosomestructureplaysacentralroleinregulatingthetheexpressionofgenes,therepairofDNA,and thefaithfulcopyingofDNAduringcelldivision.Thestudiesdescribedherewillinvestigatetheroleofkey enzymesthatremodelchromosomestructureandregulatetheseevents.Inmammals,thesefactorsare essentialfordevelopmentandstemcellidentity,andtheirinactivationcanleadtotumorogenesisandother diseases. PHS398/2590(Rev.06/09) Page ContinuationFormatPage
| Gioacchini, Nathan; Peterson, Craig L (2017) Chromatin remodeling: a complex affair. EMBO Rep 18:1673-1674 |
| Azmi, Ishara F; Watanabe, Shinya; Maloney, Michael F et al. (2017) Nucleosomes influence multiple steps during replication initiation. Elife 6: |
| Adkins, Nicholas L; Swygert, Sarah G; Kaur, Parminder et al. (2017) Nucleosome-like, Single-stranded DNA (ssDNA)-Histone Octamer Complexes and the Implication for DNA Double Strand Break Repair. J Biol Chem 292:5271-5281 |
| Xue, Yong; Pradhan, Suman K; Sun, Fei et al. (2017) Mot1, Ino80C, and NC2 Function Coordinately to Regulate Pervasive Transcription in Yeast and Mammals. Mol Cell 67:594-607.e4 |
