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1、MCPPapersinPress.PublishedonDecember29,2015asManuscriptM115.053207CharacterisationoftwodistinctNucleosomeRemodellingandDeacetylaseComplex(NuRD)assembliesinembryonicstemcells.1121*1DanielBode,LuYu,PeriTate,MercedesPardo,JyotiChoudhary1ProteomicMassSpectromet
2、ry,WellcomeTrustSangerInstitute,WellcomeTrustGenomeCampus,HinxtonCB101SA,UK.2StemCellEngineering,WellcomeTrustSangerInstitute,WellcomeTrustGenomeCampus,HinxtonCB101SA,UK.*Correspondingauthor:mp3@sanger.ac.ukRunningtitle:TwodistinctNuRDassembliesinembryonics
3、temcells1Copyright2015byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc.ABBREVIATIONSAP–affinitypurificationBAC–bacterialartificialchromosomeBN-PAGE–bluenativepolyacrylamidegelelectrophoresisCBB–CoomassieBrilliantBlueG-250ChIP-seq–chromatinimmunopr
4、ecipitationfollowedbydeepsequencingESC–embryonicstemcellsiBAQ–intensitybasedabsolutequantificationLC–liquidchromatographyMS/MS–tandemmassspectrometryNuRD-NucleosomeRemodellingandDeacetylasecomplexPCP–proteincorrelationprofilingPES–polyethersulfonePRC2–Polyc
5、ombrepressivecomplex2PVDF–polyvinylidenedifluorideSEC–sizeexclusionchromatography2SUMMARYPluripotencyandself-renewal,thedefiningpropertiesofembryonicstemcells,arebroughtaboutbytranscriptionalprogramsinvolvinganintricatenetworkoftranscriptionfactorsandchroma
6、tinremodellingcomplexes.TheNucleosomeRemodellingandDeacetylase(NuRD)complexplaysacrucialanddynamicroleintheregulationofstemnessanddifferentiation.SeveralNuRD-associatedfactorshavebeenreportedbuthowtheyareorganisedhasnotbeeninvestigatedindetail.Here,wehaveco
7、mbinedaffinitypurificationandbluenativepolyacrylamidegelelectrophoresisfollowedbyproteinidentificationbymassspectrometryandproteincorrelationprofilingtocharacterisethetopologyoftheNuRDcomplex.OurdatashowthatinmouseembryonicstemcellstheNuRDcomplexispresentas
8、twodistinctassembliesofdifferingtopologywithdifferentbindingpartners.CellcycleregulatorCdk2ap1andtranscriptionfactorSall4associateonlywiththehighermassNuRDassembly.WefurtherestablishthatonlyisoformSall
