PROJECTSUMMARY DNAmethylationisanepigeneticmodificationthatplaysakeyroleinregulatingstemcells, developmentandmanydiseases.AbnormalDNAmethylationhasbeenobservedincancerformore thantwodecades,withmanyinvestigationsfocusingonpromoterhypermethylation,whichsilences tumorsuppressorgenes.Additionally,DNMT3A,oneofdenovoDNAmethyltransferases,is frequentlymutatedinaspectrumofhematologicalmalignancies.Ourlabhasdemonstratedthat Dnmt3alossimpairshematopoieticstemcell(HSC)differentiation,whileexpandingHSCnumbersin bonemarrow,suggestingDNMT3Amayhavearoleintumorigenesisandstemcellregulation. However,DNAmethylationprofilingofleukemiapatientsamplesshowsDNAmethylationcorrelates poorlywithgeneexpressionacrossthegenome,highlightingourlimitedunderstandingofthespecific functionsofDNAmethylation.RecentstudiesusingamurinemodelofthemostfrequentDNMT3A mutationinhematologicalmalignancies,DNMT3AR882,demonstratedthatDNMT3AR882cooperates withFLT3-ITDandNPM1cmutationstocontributetoleukemictransformation.Nevertheless,our knowledgeofwhichremainingDNMT3Amutationsleadtoleukemogenesisandthemechanismsby whichtheycontributetocancerformationremainslacking.Therefore,thelong-termgoalofthe proposedresearchistounderstandhowDNMT3Aaffectsgeneregulationincancer,andhow DNMT3Amutantspredisposestemcellexpansion.InAim1,IestablishedanovelDNAepigenome editingtool(dCas9-SunTag-DNMT3Asystem)toinvestigatethecausalrelationshipbetweenDNA methylationandgeneexpression.Usingpan-canceranalysisofgenome-wideprofiles,wehave identifiedDNAhypermethylationoccurringinthegene-bodyregionsofcanyons(broadand undermethylatedregions)withactivationofcorrespondinggeneexpression.InAim2,usinga Dnmt3amutantmurinemodelIdeveloped,IwillelucidatetheroleofoneDnmt3amutantinpriming stemcellexpansion.ThefindingsfromthisproposedresearchwillshedthelightonabnormalDNA methylationincancerandmolecularmechanismsofDNMT3A-associatedmalignancies.Littleis knownabouthowmutationsinepigeneticmodifiersaffectthe3Dgenomicstructureincancer. Therefore,inAim3,Iplantousemypostdoctoralstudiestounderstandhowepigeneticmodifiers shapethegenomiclandscapeincancerandtheirunderlyingmechanism.Thistrainingprogramis tailoredtogivemeacomprehensiveeducationinbasicscienceresearchthatwillbeextremelyuseful inachievingmylong-termcareergoalofbecominganindependentcancerresearcher.