Forhumanstoproperlydevelop,thefertilizedeggmustdivideina mannerthateventuallyresultsinabodythathasallofthecorrectorgansandappendagesinall ofthecorrectplaces.Muchofthisisdeterminedbythepropertemporalandspatialregulation ofmasterregulatorygenesthatguideformationofthedifferentiatedcellsthatmakeuporgans andappendages.Akeyaspectofthisdevelopmentalprocessistheabilitytomaintainmaster regulatorygenesinarepressedstateincellswheretheirexpressionmightcauseinappropriate cellbehavior.Itisknownthatmisexpressionofevenasinglemasterregulatorygene,suchas thoseencodedintheHOXloci,canresultinacellbehavinginamannerincompatiblewithits bodylocationandtissuetype.ThePolycomb-??Group(PcG)groupofgenesislargelyresponsible formaintainingthisrepression,baseduponintensivestudyofthisgroupoverthepast70years. Thisapplicationdescribesthecontinuationofourworkonunderstandingthefunctionofthe proteinsencodedbythePcGgenes.Theseproteinsformseveralcomplexes.Thefocusofthis applicationisPolycombRepressiveComplex1(PRC1),themain?engine?ofrepressioninthe Polycomb-??Group.PRC1isknowntointeractwithchromatin,thestructurethatpackagesgenes inthenucleusofcells.ChromatinstructurecanrendertheenclosedDNAinaccessibleto activatingfactors.Aprominenthypothesisinthefieldhasbeenthatrepressioncanbe generatedbygeneratinghighlypackagedDNAthatisnolongerabletobetranscribedand expressed.WewillinvestigatetherolethatPRC1hasingeneratingpackagedchromatininthe firsttwoAims.Wewillinvestigatehowlongnon-??codingRNAsinterfacewithPRC1toregulate itsfunctioninthethirdAim.Specifically,inAim1wewillstudytheknownabilityofPRC1to interactwithnucleosomes,theprimarybuildingblockofchromatin,toformthemintoa compactedstructure.Wewilltestthehypothesisthatcompactionisessentialtofunctionby analyzingthedevelopmentalphenotypes,inmice,ofmutationsthatimpedetheabilityofPRC1 toformcompactedstructures.
In Aim2 wewillinvestigatetheabilityofPRC1togenerate higher-??orderstructures(onthescaleof100kbofDNA)thatarecompactedasvisualizedby superresolutionmicroscopyandbychromatinconformationcapture.
Aim3 characterizesthe abilityofPRC1complexestobindtolncRNAinvitro.Wewillalsodeveloptechnologiestomap thelocationoflncRNAbindingtochromatinincells.Wewillthencomparetheimpactof specificmutationsinlncRNAsandinPRC1onfunctiontotestthehypothesisthatspecific interactionsbetweenPRC1andlncRNAsareneededforproperfunctionofthePcGsystem.
Afundamentalquestioninbiologyconcernshowasinglefertilizedeggcandevelopinto acorrectlyformedorganism.ThisprocessrequiresthePolycomb-??Group,whichsilences genestomaintainpropercellidentityinmammalsandisinvolvedincancerandin developmentalsyndromes.Wewillcharacterizeproteinsandmolecularmechanisms thatgeneratecompactedchromatinstatesthatmightcausesilencing,andwilldetermine howlongnon-??codingRNAsmightregulatefunctionofthePolycomb-??Group.
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