stronglyios11

ARTICLEReceived9Mar2015|Accepted7Feb2016|Published23Mar2016AcetylationofC/EBPainhibitsitsgranulopoieticfunctionDeepakBararia1,2,3,4,5,6,*,HuiSiKwok1,7,*,RobertS.
Welner8,9,AkihikoNumata1,MenyhartB.
Sarosi10,HenryYang1,SheenaWee11,SebastianTschuri3,4,5,6,DebleenaRay12,OliverWeigert3,4,5,6,ElenaLevantini2,8,13,AlexanderK.
Ebralidze2,8,JayanthaGunaratne11,14&DanielG.
Tenen1,2,8CCAAT/enhancer-bindingproteinalpha(C/EBPa)isanessentialtranscriptionfactorformyeloidlineagecommitment.
HerewedemonstratethatacetylationofC/EBPaatlysineresiduesK298andK302,mediatedatleastinpartbygeneralcontrolnon-derepressible5(GCN5),impairsC/EBPaDNA-bindingabilityandmodulatesC/EBPatranscriptionalactivity.
AcetylatedC/EBPaisenrichedinhumanmyeloidleukaemiacelllinesandacutemyeloidleukaemia(AML)samples,anddownregulatedupongranulocyte-colonystimulatingfactor(G-CSF)-mediatedgranulocyticdifferentiationof32Dcl3cells.
C/EBPamutantsthatmimicacetylationfailedtoinducegranulocyticdifferentiationinC/EBPa-dependentassays,inbothcelllinesandinprimaryhematopoieticcells.
OurdatauncoverGCN5asanegativeregulatorofC/EBPaanddemonstratetheimportanceofC/EBPaacetylationinmyeloiddifferentiation.
DOI:10.
1038/ncomms10968OPEN1CancerScienceInstitute,NationalUniversityofSingapore,Singapore117599,Singapore.
2DanaFarber/HarvardCancerCenter,Boston,Massachusetts02215,USA.
3DepartmentofInternalMedicineIII,UniversityHospitaloftheLudwig-Maximilians-UniversityMunich,MunichD-81377,Germany.
4ExperimentelleLeuka¨mie-undLymphomforschung(ELLF),LMU,MunichD-81377,Germany.
5GermanCancerConsortium(DKTK),HeidelbergD-69120,Germany.
6GermanCancerResearchCenter(DKFZ),HeidelbergD-69120,Germany.
7NationalUniversityofSingaporeGraduateSchoolforIntegrativeSciencesandEngineering,Singapore117456,Singapore.
8HarvardStemCellInstitute,HarvardMedicalSchool,Boston,Massachusetts02215,USA.
9UniversityofAlabamaatBirmingham,DepartmentofMedicine,DivisionofHematology/Oncology,Birmingham,Alabama35294,USA.
10InstituteofInorganicChemistry,FacultyofChemistryandMineralogy,Universita¨tLeipzig,LeipzigD-04103,Germany.
11TranslationalBiomedicalProteomicsLaboratory,InstituteofMolecularandCellBiology,AgencyforScience,TechnologyandResearch,Singapore138673,Singapore.
12PrograminCancerandStemCellBiology,Duke-NUSGraduateMedicalSchool,Singapore169857,Singapore.
13InstituteofBiomedicalTechnologies,NationalResearchCouncil(CNR),Pisa56124,Italy.
14DepartmentofAnatomy,YongLooLinSchoolofMedicine,NationalUniversityofSingapore,Singapore117594,Singapore.
*Theseauthorscontributedequallytothiswork.
CorrespondenceandrequestsformaterialsshouldbeaddressedtoD.
B.
(email:deepak.
bararia@med.
uni-muenchen.
de)ortoD.
G.
T.
(email:daniel.
tenen@nus.
edu.
sg).
NATURECOMMUNICATIONS|7:10968|DOI:10.
1038/ncomms10968|www.
nature.
com/naturecommunications1Hematopoiesisisapreciselycontrolledprocessinvolvingdifferentiationofmultipotentialhematopoieticstemcells(HSCs)intospeciclineages,regulatedbytranscriptionfactors.
CCAAT/enhancer-bindingproteinalpha(C/EBPa)isoneofthetranscriptionfactorsthatiscrucialforbothmyeloiddifferentiationandmaintenanceofquiescenceinadultHSCs.
TheroleofC/EBPaingranulopoiesisisdemonstratedthroughMx1-Cre-drivenconditionaldisruptionofC/EBPainadultmice,resultinginadifferentiationblockduringthetransitionfromcommonmyeloidprogenitorstogranulocytemonocyteprogenitorsandincreasedHSCself-renewal1,2.
Moreover,expressionofC/EBPainbothleukaemiccelllinesandinhumanCD34-enrichedHSCsleadtogranulocyticdifferentiation3–5.
ChangesinC/EBPaexpressionandfunctionareassociatedwithimpairedmyeloidlineagefatedecisionswhiledysregulationofC/EBPafunctionisfrequentlyassociatedwithleukaemogenesis6–10.
Acetylationcatalysedbylysineacetyl-transferases(KATs)isanimportantpost-translationalmodication(PTM),andKATsplaypivotalrolesinnumerouscellularprocesses.
Moreimportantly,histonesarenottheonlyphysiologicalacetylationtargets11.
C/EBPainvolvementwithKATshasbeenpreviouslyreported12–15,buttheroleofacetylationinmodulatingC/EBPafunctionhasnotbeenaddressed.
Inthisstudy,weidentifytheabilityofKATGCN5tointeractandacetylateC/EBPaonatleastlysineK298andK302inthebasicDNA-bindingdomain(DBD),whichresultsinimpairedDNA-bindingactivity.
Moreover,wild-type(WT)GCN5butnotthemutant,catalyticallyinactiveGCN5repressesC/EBPatransactivationandshorthairpinRNA(shRNA)knockdownofGCN5resultsinupregulationofC/EBPaactivity.
C/EBPaacetylationisdetectableinmyeloidcelllines,primaryleukaemicsamplesanddropsdramaticallyduringgranulocyticdifferentiationof32Dcl3cellsongranulocyte-colonystimulatingfactor(G-CSF)treatment.
AnacetylationmimeticmutantofC/EBPashowslossofDNAbindingresultinginthelossoftranscriptionalactivityaspredictedbymoleculardynamics(MD)simulations.
OurstudyisthersttoprovideanunderstandingofhowC/EBPaactivityisregulatedbyKATatthepost-translationallevel.
ResultsC/EBPaisacetylatedinitsC-terminalregionbyGCN5.
GivenC/EBPaexpressionnormallytriggersdifferentiationofimmaturemyeloblastsintomaturegranulocytes,weinvestigatedwhysomeleukaemiccelllinesremainundifferentiateddespitehighlevelsofexpressionofnon-mutatedC/EBPa.
OnepossibilityistheinactivationofWTC/EBPaproteinbyPTM.
Therefore,weinvestigatedwhetheracetylationisanegativeregulatorofC/EBPaactivity.
Usingapan-acetyl-lysineantibody,acetylatedC/EBPawasdetectedinleukaemiccelllinesHL-60andMolm-14(Fig.
1a).
Next,wesoughttoinvestigatewhichacetyltransferase/srepressesC/EBPaactivity.
AC/EBPa-dependentpromoter-luciferasereporterconstructwasusedtomeasurethetranscriptionalactivityofC/EBPa.
Co-transfectionofvariousacetyltransferaseswithaC/EBPaexpressionplasmidwasperformedin293TcellslackingendogenousC/EBPa.
Amongtheacetyltransferasestested,GCN5wasfoundtorepressC/EBPatranscriptionalactivity(Fig.
1b).
Co-transfectionofp300andC/EBParesultedinanincreaseintheC/EBPatransactivationactivityaspreviouslyreported(Supple-mentaryFig.
1a)14.
Co-transfectionofGCN5antagonizedtheabilityofC/EBPatoactivatethereporterplasmidinadose-dependentmanner,withoutaffectingC/EBPaproteinlevels(Fig.
1b,SupplementaryFig.
1a–c).
GCN5alonehadnoeffectonluciferaseactivity,indicatingthatGCN5specicallymediatesdownregulationofthereporterthroughinteractionwithC/EBPa(Fig.
1b).
ExpressionoftheseacetyltransferasesandC/EBPawereconrmedbyusingwesternblot(SupplementaryFig.
1b).
Aco-immunoprecipitationexperimentshowedbothGCN5andPCAF(p300/CBPassociatedfactor,aGCN5homologue)interactwithC/EBPa(SupplementaryFig.
1d).
ToaddressthequestionwhetheracetyltransferaseactivityofGCN5isrequiredtorepressC/EBPatranscriptionalactivity,anacetyltransferase-defectivemutationofGCN5(-HAT,Y621A/P622A)wasusedinplaceofWTGCN5(ref.
16).
Indeed,themutatedHAT-decientformfailedtorepressC/EBPatranscriptionalactivity,indicatingthatacetyltransferaseactivityisessentialforrepression(Fig.
1c,SupplementaryFig.
1c).
Inareciprocalexperiment,C/EBPatranscriptionalactivitydoubledwhenendogenousGCN5wasefcientlyknockeddown(shGCN5#3;Fig.
1d,e).
Takentogether,ourdataindicatethatGCN5inhibitsC/EBPatransactivationinanacetyltransferase-dependentmanner,althoughtheroleofotherHATsinthisprocesscannotbeexcluded.
Subsequently,weperformedaninvitroacetyltransferaseassayusingGCN5andC/EBPapeptides.
K298,K302andK326wereidentiedasthesitesofacetylationbyGCN5(Fig.
1f,SupplementaryFig.
1e–gandSupplementaryTable1).
Theselysineresidueshavehighdegreeofevolutionaryconservationacrossdifferentspecies,suggestingcrucialroleforC/EBPafunction(SupplementaryFig.
1h).
K298andK302areexposedonthebasicDBD,whereasK326residesintheleucinezipperdimerizationdomain(Fig.
1g)17,18.
TofurtherinvestigatetheproteindomainsinvolvedintheC/EBPa–GCN5interaction,weperformedco-immunoprecipita-tionassaysin293Tcells(SupplementaryFig.
1i).
WhileimmunoblotanalysisusingFLAGandV5antibodiesrevealedthatGCN5interactswithC/EBPaWT,C/EBPa1-207,andC/EBPap30/120-358,itfailedtointeractwithC/EBPa204-358(SupplementaryFig.
1j).
Byperformingpull-downassayswithFLAGantibodyforC/EBPa-TAD1(Transactivationdomain1),andC/EBPa-DBDseparately,wewereunabletodetectanyinteractionbetweenGCN5andTAD1orDBDdomainofC/EBPa(SupplementaryFig.
1k).
Collectively,theseobservationssuggestthattheGCN5interactiondomaininC/EBPaliesintheN-terminalregionofC/EBPa(SupplementaryFig.
1l).
Therelevantlysineresidues(K298,K302andK326)weresubstitutedwithargininetogeneratenon-acetylatedmimeticformsofC/EBPa(referredtoasK3R).
Wefurthertestedwhetherapan-acetylantibodyisabletodetectacetylationdifferencesbetweenC/EBPaWTandK3RorC/EBPa-DBDandC/EBPa-DBDK3R(SupplementaryFig.
1m).
Immunoprecipi-tatedC/EBPaWTorK3Rmutantshowednodifferenceinacetylationusingapan-acetylantibody,bothwith(lanes4and5)andwithout(lanes2and3)GCN5co-transfection.
Inaddition,co-transfectionwithDBDorDBDK3Rdidnotshowanyacetylationsignalusingapan-acetylantibody(lanes6and7).
ImmunoprecipitatedWT,K3R,DBD,andDBDK3RweredetectedbyusingV5antibody.
Theseresultsareinaccordancewithourdomain-mappingdata,suggestingthattheC/EBPaDBDdomaindoesnotinteractwithGCN5,andthereforenoacetylationsignalisobservedfromeitherDBDorDBDK3Rwhenco-transfectedwithGCN5(SupplementaryFig.
1l).
TodetectacetylationofC/EBPaincellsatK298,K302andK326,site-specicanti-acetyl-C/EBPaantibodiesweregeneratedusingsyntheticallyacetylatedpeptides.
Theacetylatedandnon-acetylatedformsofthesepeptideswererstconrmedbymassspectrometry.
OurantibodieswereabletoreadilyrecognizeacetylatedC/EBPaatK298,K302andK326.
Whenanon-acetylatedmimeticformofC/EBPa,thatis,K3Rwasused,ARTICLENATURECOMMUNICATIONS|DOI:10.
1038/ncomms109682NATURECOMMUNICATIONS|7:10968|DOI:10.
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com/naturecommunicationsnosignalwasdetected,conrmingthattheantibodieswegeneratedarecapableofspecicallydetectingacetylatedC/EBPa(SupplementaryFig.
1n).
Consistently,westernblottingwiththesesite-specicacetylationantibodiesshowedanincreaseinacetylatedC/EBPawhenGCN5andC/EBPawereco-expressedin293Tcells(SupplementaryFig.
1o).
WealsoexaminedwhetherK298,K302andK326wereacetylatedinHL-60andMolm-14,andtheresultsareconsistentwhenprobedwithsite-specicantibodies(Fig.
1h).
Thesedataindicatethatouracetylation-specicantibodieswereabletodetectC/EBPaacetylationintheDBDofC/EBPa.
LossofC/EBPaacetylationonmyeloiddifferentiation.
WelookedatwhetherendogenousC/EBPaisacetylatedatK298,302and326invivoandiftheacetylationstatusofC/EBPachangeswithrespecttomyeloiddifferentiation.
Withinthehematopoieticsystem,expressionofC/EBPaisdetectableinearlymyeloidprecursorsanditsexpressionisnecessaryandsufcientforneutrophilicdifferentiation5,19,20.
Weusednon-leukaemic32Dcl3cellstoassessC/EBPaacetylationstatusondifferentiation.
Murine32Dcl3cellsaredependentoninterleukin-3(IL-3)forsurvivalandproliferation,andreadilydifferentiateintomaturegranulocytesonremovalofIL-3andadditionofG-CSF21,22.
gK302K298DNAC/EBPαCtrlAbC/EBPαC/EBPαAcK298AcK302AcK326CtrlAb42kD42kD42kD42kDBlot:IP:HL-60Molm-14hK326aCtrlAb42kD42kDHL-60Panacetyl-lysineC/EBPαBlot:C/EBPαC/EBPαCtrlAbIP:Molm-14C/EBPαGCN54*GCSFRC/EBPsite-PTKRLU***bC/EBPαGCN5(-HAT)RLUc––504030201003020100––––––––4*GCSFRC/EBPsite-PTKRLUC/EBPα4*GCSFRC/EBPsite-PTK–NTCshGCN5#1shGCN5#2shGCN5#3***6040200**dfC/EBPαpeptides****1.
5*1043HincorporationGCN5GAPDH90kD37kDBlot:eNTCshGCN5#1shGCN5#2shGCN5#3Histones81–92aa154–165aa253–264aa266–276aa277–288aa294–306aa303–315aa323–333aa343–354aaGCN5alone162–173aa1*1045*103Figure1|GCN5-mediatedacetylationofC/EBPaislinkedtolossoftranscriptionalactivity.
(a)Westernblotanalysiswithanti-pan-acetyl-lysineantibodyfollowingimmunoprecipitation(IP)ofC/EBPawithrabbitanti-C/EBPainHL-60andMolm-14humanmyeloidcelllysates.
Rabbitanti-GFPantibodywasusedasIPcontrol.
(b,c)GCN5decreasestheabilityofC/EBPatotransactivateaminimalp(CEBP)4TKpromoterinadose-dependentmannerandisdependentontheGCN5histoneacetyltransferase(HAT)domain.
293Tcellsweretransientlytransfectedwithp(CEBP)4TK,pRL-null,andpcDNA6expressionplasmidsforC/EBPaandwithGCN5orGCN5(-HAT),respectively.
ProteinexpressionofcorrespondingconstructswereshowninSupplementaryFig.
1c.
Luciferaseactivitywasmeasuredinduplicateforeachexperimentanddataareshownasmean±s.
d.
(N3).
(d)KnockdownofendogenousGCN5resultsinanincreaseinC/EBPatransactivationpotential.
GCN5knockdownin293TcellsenhancedC/EBPatransactivationcapacityonaminimalp(CEBP)4TKpromoterwithpRL-nullandpcDNA6C/EBPaplasmids.
Fireyluciferasereadingswerenormalizedagainstinternalcontrolrenillaluciferase.
Luciferaseactivitywasmeasuredinduplicateforeachexperimentanddataareshownasmean±s.
d.
(N3).
(e)WesternblotdemonstratingknockdownefcienciesofendogenousGCN5by3differentshRNAsin293Tcellsusedind.
(f)GCN5acetylatesC/EBPaatK298,K302andK326.
C/EBPapeptideswereincubatedwiththeGST-HATdomainofGCN5inthepresenceof3H-labeledacetyl-CoA.
TritiumincorporationbyC/EBPapeptideswasmeasuredbyscintillationcounting.
Errorbarsrepresentmean±s.
e.
m.
Experimentswereperformedtwiceinduplicate.
(g)StructureoftheC/EBPabasicregion-leucinezipperdomainboundtoDNA.
Arrowsindicatethelocationoftheacetylatedlysineresidues.
(h)EndogenousC/EBPaisacetylatedinthebasicleucinezipperregioninHL-60andMolm-14cells.
C/EBPaproteinwasimmunoprecipitatedusingarabbitantibodyrecognizingtotalC/EBPaprotein,followedbyimmunoblottingagainstacetylatedC/EBPaantibodies(K298,K302,andK326).
Rabbitanti-GFPantibodywasusedasacontrolforIP.
**Po0.
01and***Po0.
001;Student'sunpairedt-test(b,dandf).
NATURECOMMUNICATIONS|DOI:10.
1038/ncomms10968ARTICLENATURECOMMUNICATIONS|7:10968|DOI:10.
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nature.
com/naturecommunications3FACSandGiemsa-stainedcytospinsanalysisrevealedanefciencyofZ80%ingranulocyticdifferentiationof32Dcl3cellsonday4onG-CSFinduction(SupplementaryFig.
2a).
WesternblotresultsshowedenrichmentofacetylatedC/EBPaforK298andK302in32Dcl3cellsbeforedifferentiationbyG-CSFisinitiated(Fig.
2a).
OnG-CSFinduction,therewasareductioninacetylationlevelsforK298andK302,whichcoincideswiththelossofGCN5proteinexpression.
Wethenusedacetylation-specicantibodiestodeterminetheendogenouslevelsofC/EBPaacetylationinprimaryacutemyeloidleukaemia(AML)samplesinwhichC/EBPalevelscouldbedetectedbywesternblot.
UndifferentiatedhumanCD34cellsexpressedsuchlowlevelsofC/EBPaproteinthatwewereunabletocomparetheirC/EBPaacetylationsignalwithAML.
Therefore,weusedG-CSFtreatedCD34cellsonday7,inwhichC/EBPaproteinexpressionwasdetectedbywesternblot.
C/EBPawasacetylatedatK298andK302,andGCN5washigherinAMLsamplesthaninpartiallydifferentiatedhumanCD34cells(day7;Fig.
2b,SupplementaryFig.
2c).
WedidnotobserveastrongeracetylatedsignalatK302inAMLsamples#3,#4and#5ascomparedwithotherAMLsamples(Fig.
2b).
WewerealsounabletodetectanysignalsusingacetylatedK326-specicantibodyinAMLsamplesandinG-CSF-induceddifferentiated32Dcl3cells(SupplementaryFig.
2b).
OurAMLsamplesizewasinsufcienttodistinguishthedifferencebetweenK302acetyla-tion,andlackofK326acetylation(SupplementaryTable2).
OurdataindicatelossofC/EBPaacetylationatK298andK302onG-CSF-mediateddifferentiationofmurine32Dcl3cells.
Inaddition,C/EBPaacetylationiseasilydetectableinAMLpatientscomparedwithpartiallydifferentiatedhumanCD34cells.
Interestingly,GCN5isexpressedathigherlevelsinAMLpatientsthaninnormalCD34cells(SupplementaryFig.
2d).
PredictinglossofDNAbindingforacetylatedC/EBPa.
Onacetylation,lysinesidechainsarechangedinthattheacetylgroupneutralizesthepositivecharge.
Substitutionoflysinewithglutamine(neutralsidechain)mimicstheacetylatedform11.
ToexaminewhetherglutaminesubstitutionwasconsistentwithlysineacetylationandtoexaminetheimpactofC/EBPaacetylationonitsstructure(Fig.
3a),weconductedMDsimulationsonfourmodelsystems.
InadditiontotheWTDNAandacetylationmimic(K2Q-DNA)systems,twoacetylatedmodelswerealsoincludedinthisstudy.
TheH-N-COdihedralangleoftheacetylatedlysineresidueswassetto0°and180°inthetwoacetylatedmodels(hereaftertermedasK2Ac_a-DNAandK2Ac_b-DNA,respectively,Fig.
3b;seeComputationalmethodssectionfordetails).
ThebackboneatomsofthefourmodelsshowedsimilaructuationsduringMDsimulations(SupplementaryFig.
3).
AscomparedwiththeC/EBPaWT–DNAcomplex,lysineacetylation(K2Ac_a-DNAandK2Ac_b-DNA)andacetylationmimic(K2Q-DNA)raisedthecalculatedprotein–DNA-bindingfreeenergiessubstantially(Table1).
ThisindicatesadecreaseinDNA-bindingpotencyofthemodels(K2Ac_a-DNA,K2Ac_b-DNAandK2Q-DNA).
AccordingtothecrystalstructureofC/EBPaWTDNA,oneofthekeyinteractionsisformedbetweenK298andA–5(ref.
18).
DuringMDsimulations,thethreeammoniumhydrogenatomsofK302formedhydrogenbondswiththephosphateoxygenatomsofT–4(Fig.
3c,SupplementaryTable3).
TheelectrostaticinteractionenergybetweenK298andK302,andDNAresiduesT–4andA–5isamajorcomponentofthetotalprotein–DNA-bindingenergy.
BothacetylatedlysineandglutamineformedsignicantlylesshydrogenbondswithDNAduringthe3nsMDsimulations(SupplementaryTable3).
Furthermore,theelectrostaticstabilizationbetweentheC/EBPaproteinandDNAresiduesweresignicantlyloweredinthemutantsystems(SupplementaryTable4).
AcetylationmightalsodestabilizetheC/EBPastructure.
15msMDsimulationshaveshownhigherconformationaluctuationsandlossofenzymaticactivitybothduringacetylationandmutationofK104toglutamineinRAS23.
SimilarstructuraldestabilizationmightalsoberesponsibleforthelossofC/EBPatranscriptionalactivityonacetylation.
Acetylationmimeticmutantsimpairsmyeloiddifferentiation.
Toinvestigatetheconsequence/sofacetylationonC/EBPafunction,wegeneratedandtestedsingleandvariouscombina-tionsofK298,K302andK326sitesinwhichlysinewasreplacedwitheitherarginine(hereaftertermedasnon-acetylatedmimetic)orglutamine(acetylationmimetic)inC/EBPa-dependentaC/EBPαAcK298AcK302GCN5β-actinHL-60+HDACiHL-6032D+IL-332D+G-CSF42kD42kD42kD90kD40kDbC/EBPα(N-19)AcK298AcK302#3#4#5AMLHumanCD34+d-7#2#6#8AMLHumanCD34+d-7293T293T+K3Rα293T+WTα+GCN5293T293T+K3Rα293T+WTα+GCN5GCN5β-actinBlot:42kD42kD42kD90kD40kDFigure2|LossofC/EBPaacetylationongranulocyticdifferentiationanddetectioninprimaryAMLsamples.
(a)32Dcl3murineIL-3dependentcellsaredifferentiatedintogranulocyteswithadditionofG-CSFfor4days.
WesternblotanalysisshowednuclearextractsusingantiacetylK298,K302,C/EBPa(D56F10),GCN5,andb-actinantibodies.
HL-60cellsandHL-60treatedwithhistonedeacetylaseinhibitors(HDACi:400nMTrichostatinacid(TSA),20mMnicotinamide(NA)and5mMsodiumbutyrate(NB)for12-16hbeforeharvest.
)wereusedasapositivecontrolforC/EBPaspecicacetylatedantibodies.
(b)EnrichmentofC/EBPaacetylationatK298andK302inhumanAMLcellscomparedtopartiallydifferentiated(day7)humanCD34cells.
Whole-celllysateswerepreparedandblottedwithanti-acetylK298,K302,C/EBPa,GCN5,andb-actinantibodies,respectively.
Non-acetylatedmimeticC/EBPaK3RwasusedasanegativecontrolwhileC/EBPaco-transfectedwithGCN5wasusedasapositivecontrolforacetylationattherespectiveresidues.
ARTICLENATURECOMMUNICATIONS|DOI:10.
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SingleacetylationmimeticsofallthreeresiduesdidnothaveanymajoreffectonC/EBPatransactivation(SupplementaryFig.
4a).
Interestingly,K298andK302Q(C/EBPa-K2Q)andK298,K302andK326Q(C/EBPa-K3Q)acetylationmimeticmutationcombinationscompletelyabolishedC/EBPatransactivation(SupplementaryFig.
4a,b).
WesternblotsoftheC/EBPamutationsconrmedsimilarexpressionlevels,indicatingthatthelossinreporteractivityisnotduetoreductioninC/EBPaproteinlevels(SupplementaryFig.
4a,b).
Allcorrespondinglysine-to-argininemutationshadluciferaseactivitysimilartothatofC/EBPa-WT,substantiatingthatmimickingabHTE-ITE-IIBRLZ1TE-III709712720028830634735871K298RK302RK326RK3RK2RK298QK302QK326QK2QK3QAcetyl-deficientmutantAcetyl-mimeticmutantLYS298A–5I)LYS302T–4GLN302GLN298A–5T–4II)III)A–5ACK302ACK298T–4IV)T–4ACK302A–5ACK298cNNHHOOH-N-C=O=180°H-N-C=O=0°Figure3|MoleculardynamicssimulationofacetylationatK298andK302.
(a)DiagramoftheC/EBPaprotein.
Thepositionsofthethreetransactivationelements(TEs),basicregion-leucinezipper(BR-LZ)andputativeacetylationsitesforGCN5areindicated.
(b)Thetwoconformationsoftheacetylatedlysine(ACK)sidechainbuiltintothestartingcoordinatesoftheacetylatedmodelsandpreservedduringtheMDsimulations.
(c)MDsnapshotsduring3nssimulationshowingselectedprotein–DNAinteractions:I)C/EBPaWT-DNA;II)K2Q-DNA;III)K2Ac_a-DNA;andIV)K2Ac_b-DNA.
Hydrogenbondsareshownasdashedlines.
Table1|C/EBPaandDNAinteractionenergy.
Averages.
d.
s.
e.
m.
WT-DNA84.
515.
70.
9K2Q-DNA43.
515.
10.
9K2Ac_a-DNA68.
913.
30.
8K2Ac_b-DNA56.
610.
90.
6s.
d.
,standarddeviation;s.
e.
m.
,standarderrorofthemean;WT,wildtype.
CalculatedMM/PBSAprotein–DNA-bindingfreeenergies(kcalmol1)basedon3nsMDtrajectories.
NATURECOMMUNICATIONS|DOI:10.
1038/ncomms10968ARTICLENATURECOMMUNICATIONS|7:10968|DOI:10.
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com/naturecommunications5acetylationresultsintheinactivationofC/EBPafunction.
Toeliminatethepossibilityofalteredconformationandaffectedtransactivationbyclustersoflysine-to-glutaminemutations,anothermutatedform(C/EBPa-KIIIQ)wasgeneratedtomimicacetylationofaclusterofthreelysineresidues(K275,K277andK278)locatedclosetotheDBD.
Thesethreelysineresiduesweresubstitutedwithglutamine.
Althoughbothmutantsrepresentedacetylationmimeticforms,unliketheC/EBPa-K3Qform,C/EBPa-KIIIQindeedretainedtranscriptionalactivity(SupplementaryFig.
4b).
Thisstronglyarguesthatretainingthepositivechargeisonlycrucialfortheresiduesinthebasicregion,andsubstitutinglysineresiduesforglutaminedoesnotalwaysresultinlossoffunction.
TheC/EBPa-decienthumancelllineK562wasengineeredtoconditionallyexpressb-estradiol-inducibleconstructstostudyhowvariousC/EBPamutantsaffectdifferentiation4.
Withoutinduction,fusionproteinsareconstitutivelyexpressedinthecytoplasm,wheretheyareinactive.
Oninductionwithb-estradiol,thefusionproteintranslocatestothenucleus,whereitisactive.
ConsistentwithresultsfromMDsimulationandluciferaseassays,cellsexpressingC/EBPaWT-oestrogenreceptor(ER),C/EBPaK3R-ERandC/EBPaK2R-ERdifferentiated,whereascellsexpressingERonly,C/EBPaK3Q-ERandC/EBPaK2Q-ER,remainedinanundifferentiatedstateasassessedbysurfaceexpressionofthemyeloidmarkerCD11b,cellmorphologyandneutrophilicenzymaticactivity(Fig.
4a,b,SupplementaryFig.
4c–f).
ExpressionlevelsoftheindividualfusionproteininK562stablecloneswereassessedbywesternblots(SupplementaryFig.
4g).
Furthermore,ourresultsshowedthatK562C/EBPa-WT-ERwasabletoinhibittheincreaseincellnumberswithin5daysofculture,ascomparedwithvector-aloneandvehicle(ethanol)control(SupplementaryFig.
4h).
TheK562C/EBPa-K2Qmutantcelllineshowedanintermediateinhibitionincellnumber,whichlackedstatisticallysignicantdifference(Pvalue0.
148,N6withtwo-wayanalysisofvariancetest;SupplementaryFig.
4h).
C/EBPaWT-ERwasabletodownregulateendogenousc-MycaspreviouslyreportedbyourgroupwhereasC/EBPa-K3QandC/EBPaK2Q-ERmutantsshowedpartialdownregulationofc-Myc(SupplementaryFig.
4i)24.
Usingamurinemodel,wefurtherexploredwhethertheacetylationmimeticformisunabletodifferentiateprogenitorsintomaturegranulocyticcells.
AsgranulocytedifferentiationisimpairedinCebpa/Mx1-cremice,wechosethismodeltoanalysethedifferentiationpotentialofthevariousC/EBPamutants.
Ten-totwelve-week-oldCebpa/Mx1-cremiceandcontrol(Cebpa/Mx1-cre-)miceweretreatedwithpoly(I:C)1,2,20.
Bonemarrowwasharvested17–21dayslater,andexcisionoftheCebpagenewasconrmedbyPCR.
MultipotentprogenitorLinSca-1c-Kit(LSK)cellsfromCebpaD/DmicewereFACS-puriedandcultured.
Cellsweretransducedwithretroviruses-expressinggreenuorescentprotein(pMSCVIRESGFP,MIGemptyvector),WT(C/EBPa-WT),ormutatedformsofC/EBPa(C/EBPa-K3QandC/EBPa-K3R).
ExpressionlevelsofMIGC/EBPa-WTandmutantswereassessedbywesternblot(SupplementaryFig.
4j).
After7daysofculture,thepercentageofgranulocyticdifferentiationwasmeasuredbyGr-1andMac-1surfacestainingandbyGiemsastainformorphologicalanalysis(Fig.
4c,d).
TheC/EBPa-K3Qacetylationmimeticmutationwasnotablyunabletoformmaturegranulocytes.
TheseresultssuggestthatacetylationatK298,K302andK326antagonizesC/EBPagranulocyticdifferentiationpotential.
Inaddition,acetylationofK298andK302aloneissufcienttoimpededifferentiation.
FurthertestingofthepossibilityofGCN5involvementinmyeloiddifferentiationwasdonebyusing10–12weeksoldGcn5D/DVav-iCreandcontrolmice(Gcn5/)25,26.
LSKcellswereFACS-puriedandplacedinmyeloidcultureconditionscontainingG-CSF.
LSKcellsfromGcn5conditionalknockout(Gcn5D/D)produceda1.
7-foldincreaseinthepercentageofGr-1Mac-1cellscomparedwithcontrolmice(Gcn5/;Fig.
4e,f;Pvalue0.
0137,N3,withunpairedtwo-tailedStudent'st-test).
Inconclusion,G-CSFinducedinvitromyeloiddifferentiationisenhancedintheabsenceofGCN5.
AcetylationatK298andK302reducesDNA-bindingability.
TounderstandhowacetylationofK298andK302leadstoC/EBPainactivation,weassessedchangesinhomodimerization,cellularsublocalizationanddominantnegativefunctionofthemutantformsoverC/EBPaWT.
NodifferenceinhomodimerizationorlocalizationbetweenC/EBPaWTandmutatedformswasdetected(SupplementaryFig.
5a,b).
InaC/EBPa-dependentpromoter-luciferasereporterassay,co-transfectionoftheC/EBPa-K2QmutanttogetherwithC/EBPaWTdidnotshowanydominantnegativeeffectbytheK2Qmutant,unliketheC/EBPap30form(SupplementaryFig.
5c)27.
ExpressionofC/EBPa,C/EBPap30form,andC/EBPa-K2Qmutantwereconrmedusingwesternblot(SupplementaryFig.
5d).
ToinvestigatewhetheracetylationbyGCN5altersC/EBPaDNAbindinginvitro,weperformedelectrophoreticmobilityshiftassays(EMSA).
C/EBPawasacetylatedbyaninvitroacetyltransferaseassayusingGCN5HATdomainprotein.
EMSAanalysisshowedthatacetylationofinvitro-translatedC/EBPareducesitsabilitytobindtotheC/EBPabindingsite(Fig.
5a).
Nobindingwasobservedinthecontrollysate.
WesternblotanalysisofthereactionusedforEMSAconrmedC/EBPaacetylationatK298andK302residues(Fig.
5b).
MDsimulationspredictedadramaticreductionintheabilityofC/EBPatobindtoitstargetDNAsequencesbytheacetylationmimeticC/EBPa-K2Q(Fig.
3c,Table1).
TheDNA-bindingabilitiesofWTandmutatedformsofC/EBPawerealsoassessedbyEMSA,usingnuclearextractsfromK562linesexpressingERemptyvector,C/EBPaWT-ER,C/EBPaK2Q-ERandC/EBPaK2R-ER.
Asexpected,C/EBPaK2Q-ERextractsdemonstratedweakenedDNAbindingascomparedwithC/EBPaWT-ERandC/EBPaK2R-ER.
Usinganti-C/EBPaantibodyforsupershift,wedemonstratedspecicityoftheC/EBPa-dependentbindinginEMSA(Fig.
5candSupplementaryFig.
5f).
EMSAperformedusing293TcellstransfectedwithC/EBPaK2QwithouttheER-tagshowedlossofDNAbindingsimilartoK2Q-ER(SupplementaryFig.
5e,g).
TheG-CSFreceptorisanimportantdownstreamtargetgeneofC/EBPa,anditspromoterregioncontainsC/EBPa-bindingsites28.
MinimalrecruitmentofC/EBPaK2Q-ERtotheG-CSFreceptorpromoterbychromatinimmunoprecipitation(ChIP)wasobserved(Fig.
5d).
Insummary,theseresultssupportthehypothesisthatacetylationofthesetworesiduesleadstothelossofDNA-bindingactivityandsubsequentlossofinductionofgranulocyticdifferentiation.
DiscussionInthisstudy,wedemonstratedthatacetylationonC/EBPaleadstothelossofdifferentiationpotential.
Weidentiedthreelysineresidues(K298,K302andK326)astheacetylationtargetsitesinC/EBPabyGCN5.
Withacetylationsite-specicantibodies,wedetectedacetylationatthesethreesitesinleukaemiccelllinesexpressingendogenousC/EBPa.
WeprovidedevidencethatC/EBPaacetylationlevelsandGCN5expressionarereducedongranulocyticdifferentiation.
Furthermore,wedemonstratedC/EBPaacetylationinprimaryleukaemicsamples.
UsingC/EBPa-dependentmurineandcellularmodels,wedemonstratedARTICLENATURECOMMUNICATIONS|DOI:10.
1038/ncomms109686NATURECOMMUNICATIONS|7:10968|DOI:10.
1038/ncomms10968|www.
nature.
com/naturecommunicationsabcdEVWTK3RK3QC/EBPα***%CD11bERWTC/EBPα-ERK3RK3Q100806040200***C/EBPα-ER%CD11b100806040200**********%Gr-1+-Mac-1+cells(gatedonGFP+)EVWTC/EBPαK3RK3QCebpaΔ/ΔLSKcells***151050eGcn5fl/flGcn5Δ/Δf%Gr-1+Mac-1+cellsLSKcells+G-CSFGcn5fl/flGcn5Δ/Δ0102030*ERWTK2QK2RK326QK326RFigure4|LossofdifferentiationpotentialinacetylationmimeticmutationsofC/EBPa.
(a,b)C/EBPaK3Q-ERandC/EBPaK2Q-ERfailtoupregulatedifferentiationmarkerCD11binK562cells.
K562cellswerestablytransfectedwithEV-ER,C/EBPaWT-ER,K3Q-ER,K3R-ER,K2R-ER,K2Q-ER,K326R-ER,orK326Q-ER.
InductionofC/EBPanuclearlocalizationwasperformedwithadditionof5mMb-estradiol.
CellswereanalysedwithFACSforsurfacemarkerCD11bfollowing4daysofculture.
BargraphshowsthepercentageofCD11bcells,whichisindicativeofgranulocyticdifferentiation.
Dataaremean±s.
d.
fromtwoindependentclonesforeachconstruct.
***Po0.
001;Student'sunpairedt-testN6(a)andN9(b).
(c)C/EBPa-K3Qfailstodifferentiatehematopoieticearlyprogenitorcells.
LinSca-1c-Kit(LSK)cellsfrombonemarrowofCebpaD/DmiceweretransducedwithretroviralexpressionvectorspMIGEV,C/EBPa-WT,C/EBPa-K3R,andC/EBPa-K3Qtoinducedifferentiationintogranulocytes.
CellsgatedontheGFPfractionwereanalysedonday7forsurfacemarkersGr-1andMac-1,indicativeofgranulocyticdifferentiation.
Dataaremean±s.
d.
***Po0.
001;Student'sunpairedt-testN3.
(d)LackofmaturegranulocytesinC/EBPa-K3QtransducedLSKcells.
CytospinswerestainedwithWright-Giemsa.
Originalmagnicationx100,scalebarsindicate10mm.
Thearrowsindicategranulocyteswiththeirpolymorphonuclearmorphology.
(e)IncreaseinmyeloidcellsondeletionofGcn5.
LSKcellsfromGcn5D/DVav-iCreandcontrol(Gcn5/)micewereculturedinG-CSFsupplementedmedia.
After7days,viablecellswereassessedforrelativeexpressionofGr-1andMac-1usingowcytometry.
Datashownarerepresentativefromfourindependentexperimentsdoneintriplicate.
Dataaremean±s.
d.
*Po0.
05;Student'sunpairedt-test.
(f)MaturegranulocytesmorphologyfromG-CSFinducedinvitroculture.
CellsweresubjectedtoWright-Giemsastainingandpictureswereacquiredatanoriginalmagnication40.
Scalebarsindicate10mm.
Thearrowsindicategranulocyteswiththeirpolymorphonuclearmorphology.
NATURECOMMUNICATIONS|DOI:10.
1038/ncomms10968ARTICLENATURECOMMUNICATIONS|7:10968|DOI:10.
1038/ncomms10968|www.
nature.
com/naturecommunications7aAcK298C/EBPαAcK302GCN542kD42kD42kD75kDbcInvitrotranslationGCN5(2)Probe+++–––+–+Anti-C/EBPα––––+––+––++–SSShiftFreeprobe1234567GCN5(1)+–C/EBPαEVProbeK562NEAnti-C/EBPα++++ERK2QWTK2RShiftSSXFreeprobe–123456789edC/EBPαAcG-CSFR…LowactivityGCN5C/EBPαG-CSFR…GCN5Advantageforleukemia/diseasestateNormal/stagespecificGCSFRpromoterregionfoldenrichmentC/EBPα-ER*100806040200C/EBPαC/EBPα+GCN5(1)C/EBPα+GCN5(2)ERWTK2QK2RK326QK326RFigure5|AcetylationinthebasicregionimpairsDNAbindingability.
(a)GCN5attenuatesDNAbindingabilityofC/EBPainvitro.
EMSAwasperformedusingadouble-strandedC/EBPabindingsiteoligonucleotide.
Invitro-translatedC/EBPawasincubatedwithtwoseparatesourcesofrecombinantGCN5(HAT)domainproteins.
Co-incubationofivtC/EBPawithGCN5(lane6,7)resultedindecreaseinDNAbindingascomparedtoC/EBPaalone(lane4).
Nobindingwasobservedfromivt.
Emptyvector(EV)control(lane2)orfromEVincubatedwithGCN5(lane3).
SpecicityofC/EBPabindingtotheprobewasshownbysupershift(SS)usingC/EBPaantibody(lane5).
(b)Westernblotshowinginvitro-translatedC/EBPaacetylationatK298andK302byrecombinantGCN5(HATdomain)proteinusedinEMSAina.
(c)C/EBPaacetylationmimetic(K2Q-ER)showedreducedDNAbindingafnityinEMSA.
EqualamountsofnuclearextractsfromK562linesstablytransfectedwithEV-ER(lanes2,3),C/EBPaWT-ER(lanes4,5),C/EBPaK2Q-ER(lane6,7),andC/EBPaK2R-ER(lane8,9)wereused.
Cellsweretreatedwith5mMb-estradiolfor45min.
Lane1containedprobeonly.
Inlanes3,5,7and9,1mLofasupershiftingC/EBPaantibodywasadded.
SSindicatessupershiftedcomplex;ShiftindicatesC/EBPacomplex;andXreferstononspeciccomplexobservedwiththisprobe.
Therepresentativeexperimentoutofthreeisshownhere.
(d)AcetylationmimeticformK2QwasnotenrichedataC/EBPatargetgenelocusG-CSFR27.
ChIPanalysisofstimulated(45min)K562EV-ER,C/EBPaWT-ER,K2Q-ER,andK2R-ERcellsusingERantibody.
Foldenrichmentiscalculatedcomparedtobindingtocontrolgene,theinactiveinvolucrin(IVL)locus.
Dataaremean±s.
d.
(N3).
*Po0.
05;Student'sunpairedt-test.
PvaluebetweenC/EBPaWTandK2R,K326R,andK326Qisnotsignicant.
(e)ModelhypothesizingtheeffectofC/EBPaacetylationonitsfunctioninnormalhematopoiesisandleukaemia.
Non-acetylatedC/EBPaiscapableofinducinggranulocyticdifferentiation.
C/EBPaacetylationleadstolossinDNAbindingandlossofrecruitmenttoC/EBPatargetgenessuchastheG-CSFreceptor,therebyinhibitingdifferentiation.
ARTICLENATURECOMMUNICATIONS|DOI:10.
1038/ncomms109688NATURECOMMUNICATIONS|7:10968|DOI:10.
1038/ncomms10968|www.
nature.
com/naturecommunicationsthattheacetylationmimeticforms(K2QandK3Q)areincapableofdrivinggranulocyticdifferentiationininducibleC/EBPa-dependentassaysandinmurineprimarycellsasaresultoflossofDNAbindingability.
ThisistherstreportdemonstratinganacetyltransferasethathasrepressiveactivityonC/EBPafunction,consistentwiththereciprocalpatternofexpressionofGCN5andC/EBPainHSCs,inwhichGCN5isenriched,whileC/EBPaisbarelydetectable1,29.
PreviousstudieshavedescribedtheinvolvementofmultiplePTMsinmodulatingC/EBPa30–33.
Inparticular,phosphorylationofserine21hasbeenreportedtobeclinicallyrelevantduringmyeloiddifferentiation.
Hyper-phosphorylationatS21blocksthematurationofleukaemicblastsandneutrophilicdifferentiationofCD34progenitors9,10.
AlthoughbothacetylationandphosphorylationimpairtheabilityofC/EBPatodrivedifferentiation,themechanismsofinactivationappeartobedistinct.
AcetylationresultsinalossofDNA-bindingcapacity,whilephosphorylationaltersconformationofC/EBPadimers34.
ThedifferentialeffectsofthesePTMsonC/EBPaareattributestotheinherentdifferencesinthechemistryandsitesofthesemodications.
OurproteindomainmappingdataforC/EBPaindicatesthatGCN5dockinginteractionoccursattheNterminus,whereasacetylationsitesarelocatedattheCterminusintheDBD.
SuchobservationshavebeenreportedinC/EBPaandE2FinwhichdeletionoftheC/EBPaNterminusorthebasicregionmutationleadstolossofinhibitionofE2Factivity4.
SeveralstudieshavereportedanumberofC/EBPamutationslackingdifferentiationpotential,someofwhicharelocatedinthebasicregion.
TheBRM2(I294A,R297A)mutantproteinfailstodifferentiatecellsintogranulocyteandadipocytelineages.
UnlikeC/EBPa-K2Qdescribedinourstudy,BRM2doesnotabrogateDNAbinding17,35.
TheBR3(R297G,K298T,R300G,K302N)mutantcontainsfoursubstitutionsinthebasicregion.
SimilartoourresultsinC/EBPa-K2Qmutations,althoughBR3isalsoincapableofbindingDNA,proteinretainstheabilitytodimerize36.
However,BR3wasdesignedonastructuralbasis,inwhichclustersofaminoacidswithinthebasicregionexhibitanetpositivecharge.
ThisdiffersfromC/EBPa-K2Qmutations,whichwasselectedbasedontheacetylationsitescatalysedbyGCN5.
ThisisconsistentwiththeideathattheC/EBPa-K2R,whichretainsapositivechargeontheseresidues,isindistinguishablefromWTC/EBPainitsbehaviourinDNAbindingandtransactivation.
ThesedatashowthatthepositivechargeonK298andK302locatedwithinthebasicregionisrequiredforDNAbinding.
Infact,ourmolecularsimulationdatapredictedthatacetylationdisruptsthecapacitytobindDNAbyneutralizingthepositivechargeontheselysineresidues.
ItisimportanttomaintainapositivelychargedclusterofaminoacidsfortheinteractionwithDNA.
InagreementwiththesedataandbyusingEMSAandChIPassays,wedemonstratedthattheacetylationmimeticmutation(K2Q)issufcienttodecreaseDNAbinding.
TheC/EBPa-K2Qmutant(oracetylatedC/EBPa)lacksDNAbinding;however,itdoesnotdisplaydominantnegativefunctionoverC/EBPaWT.
IncomparisonwithC/EBPaWT,variousmutantsdifferintheabilitytodrivegranulocyticdifferentiation.
Wefocusedonthecompletelackoftransactivationand/orgranulocyticdifferentiationconsistentlyobservedinK2QandK3QmutantsofC/EBPa.
K326liesinthedimerizationdomainofC/EBPa,andsubstitutionwithglutamine(anacetylationmimetic)alsopreventeddifferentiationcomparedwithC/EBPaWTandK326R.
Suchobservationscouldalsobeduetothedifferencesinprotein–proteininteractions,otherthanthehomodimerization,whichisrequiredforefcientgranulocyticdifferentiation.
TranscriptionfactorFoxo1acetylationhasbeenlinkedtodecreasedabilitytobindDNA,andtobecomephosphorylated37.
ItwillbeintriguingtoexaminewhetherthereiscrosstalkbetweendifferentPTMsonC/EBPa.
Furthermore,substitutionoflysinewithargininecreatespotentialnewmethylationsitesresultinginaltereddifferentiationpotentialofmutantC/EBPa.
Attheveryleast,substitutionoflysinewithglutamine(acetylationmimetic)eliminatesthepotentialforsuchself-modicationsites.
ToinvestigatetheroleofGCN5inhematopoiesis,wecrossedGcn5/micewithhematopoietic-specicVav-iCre.
WedidnotobserveanysubstantialalterationinmyeloidlineagecommitmentanddifferentiationinGcn5excisedmice(Gcn5D/DVav-iCre).
However,bytestinginvitrogranulocyticterminalmaturationinamorerenedexperimentaimedatanalysingtheeffectofG-CSFonLSKcellsfromGcn5D/DVav-iCre,weobserveda1.
7-foldincreaseinGr-1Mac-1cells.
Thepossibilityexiststhatthismodel,inwhichGCN5wasdeletedattheearlieststagesofembryonichematopoieticdevelopment,doesnotrepresentanidealmodeltostudyC/EBPa-dependentmyeloidcommitmentinadulthematopoiesis.
WecannotruleoutthepossibilitythatC/EBPaacetylationmaybecompensatedbyotherHATs.
PCAFshareshighhomology(88%)withGCN5intheacetyltransferasedomain,andisreportedtoberedundantwithGCN5(refs38–40).
WhilewedidnotobservePCAF-mediatedrepressionofC/EBPatransactivationpotentialintransienttransfectionassays,giventhesepreviousreportsofredundancyofGCN5andPCAFinvivo,futurestudiesinvolvingGCN5andPCAFdoubleknockoutmicecouldhighlighttheroleofC/EBPaacetylationinhematopoieticcells.
GCN5hasbeenreportedtobehighlyexpressedinnon-smallcelllungcancer,whichcorrelateswithtumoursize41.
GCN5alsorepressesPGC-1aandbtranscriptionalactivityviaacetylationinculturedhepatocytesinmouselivertissuesandinprimaryskeletalmusclecells.
Thisacetylationhasbeenlinkedtochangesinglucoseconcentrationandcontrolsexpressionofmetabolicgenesinvolvedinfattyacidoxidation16,42.
C/EBPbisalsoreportedasatargetofGCN5-mediatedacetylationduringglucocorticoidstimulatedpreadipocytedifferentiationof3T3L1andNIH3T3cells43.
GiventheroleofC/EBPainlungdevelopment,glucosemetabolismandadipogenesis,itwouldbeofinteresttoinvestigatewhetherC/EBPaacetylationparticipatesinthesecellularprocessesinadditiontoitscriticalroleingranulocyticdifferentiation.
C/EBPaisinactivatedinleukaemiabydiversemolecularmechanisms.
Inthisstudy,wedemonstratedC/EBPaPTM(acetylation)inprimaryleukaemiacells,whichmayaffectitsactivity.
ItwillbeofgreatinteresttoinvestigatewhethertheadministrationofaGCN5inhibitorrepresentsanoveltherapeuticapproachforthetreatmentofleukaemias.
MethodsCellCulture.
HEK293T(ATCC,CCL11268)cellsweremaintainedinDMEM(Biowest)supplementedwith10%heat-inactivatedFBS(Biowest).
BOSC23(ATTC,CRL11270),HL-60(ATCC,CCL240),K562(ATCC,CCL246)andMolm-14(kindlyprovidedbyChngWeeJoo,CancerScienceInstitute,Singapore)weremaintainedinRPMI1640supplementedwith10%heat-inactivatedFBS(Biowest).
Forinhibitionoflysinedeacetylases,K562C/EBPa-ERstablecelllinesweremaintainedinphenolred-freeRPMI1640supplementedwith10%charcoal-strippedFBS(Invitrogen).
K562,HL-60andMolm-14identitieswereconrmedbySTRproling(GeneticaDNALaboratories,NC,USA).
AllcelllinesaretestednegativeformycoplasmacontaminationbyMycoAlertPLUSmycoplasmadetectionkit(Lonza).
32Dcl3cells(kindlyprovidedbyMotomiOsato,CancerScienceInstitute,Singapore)weremaintainedinRPMI1640supplementedwith10%FBSandIL-3(5ngml1;PeproTech).
InductionofgranulocyticdifferentiationwasperformedwithremovalofIL-3bythreewasheswithPBSandadditionofrecombinanthumanG-CSF(100ngml1;PeproTech).
Cellswereharvestedforwesternblotanalysis4dayspostinduction.
Bonemarrow-derivedCD34cells(1106cells)wereobtainedfromStemCellTechnologiesandculturedinIMDM(ATCC)supplementedwith10%FBS,FLT3ligand(50ngml1),SCF(50ngml1),IL-6(50ngml1)andG-CSFNATURECOMMUNICATIONS|DOI:10.
1038/ncomms10968ARTICLENATURECOMMUNICATIONS|7:10968|DOI:10.
1038/ncomms10968|www.
nature.
com/naturecommunications9(50ngml1;PeproTech)toinducegranulocyticdifferentiation.
IL-6wasreplacedwithIL-3(10ngml1)andG-CSFwasreducedto20ngml1intheculturemediafromday4onwards.
Freshmediawasreplenishedevery3daysandcellswereharvestedonday7forwesternblotanalysis.
PatientAMLsamples.
WeobtainedfrozenbonemarrowblastsofAMLfrompatientsattheNationalUniversityHospital,Singapore.
TheinstitutionalreviewboardoftheNationalUniversityofSingaporehasapprovedthestudy.
AllpatientsprovidedwritteninformedconsentinaccordancewiththeDeclarationofHelsinkiPrinciples.
AsummaryofleukaemiastatusofthepatientsamplesisdescribedinSupplementaryTable2.
Plasmidconstruction.
WT42-kDahumanC/EBPawasclonedintothepcDNA6V5-His-taggedplasmid.
Allmutationsfromlysinetoeitherglutamineorargininewerecreatedbysite-directedmutagenesis(Agilent).
AllmutantconstructscreatedbyPCRweresequenceveried.
ThefollowingC/EBPamutantexpressionconstructswerecreatedinthepcDNA6V5-Hisbackbone:C/EBPaK298Q,C/EBPaK302Q,C/EBPaK326Q,C/EBPaK298,302Q(K2Q),C/EBPaK298,302,326Q(K3Q),C/EBPaK275,277,278Q(KIIIQ),C/EBPaK326R,C/EBPaK298,302R(K2R)andC/EBPaK298,302,326R(K3R).
ForC/EBPa-ERreceptorfusionprotein,the42-kDa('wild-type')humanC/EBPaconstructandtherespectiveC/EBPamutantsweresubclonedfrompcDNA6plasmidsintothepBabe-ERplasmid(providedbyAlanFriedman,JohnsHopkinsUniversitySchoolofMedicine,Baltimore,USA)digestedwithBamHIandXhoI,suchthattheyareinframewiththeC-terminalERligand-bindingdomain.
LentiviralshRNAstargetingGCN5werepurchasedfromMISSIONshRNAatSigma-Aldrich.
ForgenerationofpMIG-C/EBPaWTandmutantexpressionconstructs,therespectiveC/EBPainsertsweresubclonedfrompcDNA6plasmidsintopMIGplasmiddigestedwithEcoRIandXhoI.
C/EBPadeletionexpressionconstructswerePCRampliedusingpcDNA6C/EBPaWTV5-HisastemplateandclonedintothepcDNA6V5-HisplasmidusingEcoRIandXhoI.
InsertsequenceswereconrmedbytheSangersequencingmethod.
Luciferasereporterassays.
ThereyluciferasereportergeneinthepXP2vectorisdrivenbyfourC/EBP-bindingsitesderivedfromG-CSFreceptorpromoter,startingfrom57to37bpupstreamofthemajortranscriptionstartsiteoftheG-CSFreceptorgene28.
Asaninternalcontrolplasmidforco-transfections,thepRL-nullconstructencodingaRenillaluciferasegene(Promega)wasused44.
FireyandRenillaluciferaseactivitiesweredetermined24hposttransfectionwiththedual-luciferasereporterassaysystem(Promega).
FireyluciferasereadingswerenormalizedagainstinternalcontrolRenillaluciferaseandcalculatedasfolddifferencesagainsttheactivityobtainedforthereporterplasmidwithoutC/EBPatransfection.
Luciferaseassaysweredonewithp(CEBP)4TK(200ng),pRL-null(10ng),pcDNA6expressionplasmidsforC/EBPa(5,10,20or30ng),GCN5orGCN5(-HAT)(20,50,100,200ng)andGCN5orp300(1,5,20or50ng),respectively.
ForGCN5knockdowneffectonC/EBPatransactivation,100,500pgand2.
5ngofpcDNA6C/EBPaplasmidconcentrationswereused.
C/EBPaacetylationmimeticandnon-mimeticmutantsluciferaseassayswereperformedusing500pg,1and5ngconcentrationsofrespectivepcDNA6expressionplasmids.
LuciferaseassayswherepcDNA6C/EBPaK2QwascomparedwithpcDNA6C/EBPaK298QorpcDNA6C/EBPaK302Q1,5and50ngplasmidconcentrationswereused(SupplementaryFig.
4a).
Invitroacetylationassay.
AGST-taggedGCN5HATdomainexpressioncon-structwasprovidedbyAxelImhof(LMU,Munich,Germany).
GST-taggedGCN5proteinwasproducedandpuriedfromEscherichiacoliBL21.
GST-taggedrecombinantGCN5(Catalogue#K311-381G)wasalsopurchasedfromSignalChem.
Invitro-translatedC/EBPaproteinwasproducedinacoupledtranscriptionandtranslationrabbitreticulocytelysate(Promega,Catalogue#L1170).
PeptidesweresynthesizedbyrstBase(Singapore;SupplementaryTable1).
Allpeptideswerepuriedbyhigh-performanceliquidchromatographytoobtain495%purity.
Substratepeptideswereincubatedwith0.
25mCi[3H]acetylcoenzymeA(Amersham)and0.
1mgofpuriedGCN5-GSTacetyltransferase(HAT)domainina30mlacetylationbuffercontaining50mMTris-HCl(pH8.
0),50mMNaCl,10%glycerol,0.
5mMEDTA,1mMdithiothreitol,10mMsodiumbuytrateand1mMphenylmethylsulfonyluoride.
Reactionmixtureswereincubatedat30°Cfor45min.
Subsequently,thepeptideswerespottedontoP81lterpaper(Whatman).
Thelterpaperwasair-driedandmeasuredforacetylationactivityusingliquidscintillationcounting.
MassspectrometricanalysisperformedontheinvitroacetylatedC/EBPapeptidesthatcoverK298,K302andK326lysineresiduesconrmedacetylationofthesethreeresiduesanduncoveredanadditionalacetylationsiteatK304(SupplementaryFig.
1e–g).
ButasacetylationatK304wasnotseenintheinvitroacetyltransferaseassaywithK304inoverlappingpeptides(SupplementaryTable1forP-7andP-8;Fig.
1f,SupplementaryFig.
1e–g),nofurtherstudiesonthislysineresiduewerepursued.
Liquidchromatographymassspectrometryanalysis.
Peptidesampleswereanalysedusingnano-HPLCcoupledtoanLTQVelos(ThermoFisherScientic).
PeptidesweretrappedontoaC18pre-columnandseparatedonananalyticalcolumnusinga1or2-hgradientrangingfrom2%to8%acetonitrile/0.
1%formicacid,followedbya5mingradientrangingfrom8%to80%acetonitrile/0.
1%formicacidandstayedat80%acetonitrile/0.
1%formicacidfor20min.
MSscansrangedfrom310to1,400m/z,AGCtarget3E4,andmaximuminjectiontimeof50ms.
The10mostintenseionswithanionintensityabove1,000andachargestateexcludingoneweresequentiallyisolatedtoamaximumAGCtargetvalueof4E4foramaximal100msandfragmentedbyElectronTransferDissociation(ETD).
ThefollowingparameterswereemployedforETD:thereagentionsourcetemperatureof160°C,reagentionsourceemissioncurrentof50mA,reagentionsourceelectronenergyof70.
00V,reagentionsourcechemicalionisationof20p.
s.
i.
,reagentvial1iontimeof50,reagentvial1AGCtargetof1E5,reagentvial2iontimeof50,reagentvial2AGCtargetof1E5,supplementalactivationenergyof15.
Adynamicexclusionlistwasappliedusinganexclusionlistsizeof500,onerepeatcount,repeatdurationof45s,exclusiondurationof30saswellasamasswidthof1.
0lowand1.
5high.
Expirationcountwasdisabled.
Dataprocessinganddatabasesearch.
RawleprocessingforthepeptidesampleswascarriedoutusingMascotDaemon(version2.
3.
2,MatrixScience).
Dataimportlterforprecursormasseswasfrom700to4,000Da,withaminimumscanpergroupof1andaminimumpeakcountof10.
Mascotsearchwasperformedusingthecustommadedatabaseconsistingofthreepeptidesequencesonly:RLRKRVEQLSRC,IAVRKSRDKAKQRCandRKSRDKAKQRNVEC.
Noenzymecleavagewasspecied.
Acetyl(C),Acetyl(K),Acetyl(N-term),Acetyl(S),Oxidation(M)weresetasvariablemodications.
Tolerancefortheprecursormasseswas2Daandforfragments0.
8Da.
ThehighestscoreofMS/MSspectrumforeachintactuniqueacetylatedpeptide(withaminimummascotscoreof25)wasmanuallyvalidated.
Generationofsite-specicacetylationantibodies.
Acetylation-specicC/EBPaantiserawasraisedinrabbitsagainstkeyholelimpethaemocyanin(KLH)-conjugatedpeptides,whichcomprisedofthefollowingresiduesofhumanC/EBPa:IAVRKAcSRDKAKQR(Ac-K298antiserum),IAVRKSRDKAcAKQR(Ac-K302antiserum)andRLRKAcRVEQLSR(Ac-K326antiserum),withaC-terminalcysteineresidueaddedtothepeptidesequence.
AdditionofcysteinewasfortheconjugationofthesyntheticpeptidetothecarrierproteinKLH.
Antiserawerepuriedbybindingtoacetylatedpeptide-conjugatedSulfolinkgel(Pierce)andsubsequentlypassedthroughanadditionalnon-acetylatedpeptide-conjugatedSulfolinkgeltoeliminatenon-acetylation-specicantibodies.
Puriedacetylation-specicantibodieswereusedatadilutionof1:1,000forwesternblotting.
ImmunoblottingandImmunoprecipitation.
Forwhole-celllysis,1107cellswerelysedwithradioimmunoprecipitationassaybuffer.
Forsubcellularfractionation,nuclearandcytosolicfractionswerepreparedusingNuclearExtractKitfromActiveMotif.
ProteinconcentrationswerequantitatedwithBioradProteinAssay.
Proteinswereseparatedon10%SDS-PAGEgels.
Immunoblotswereincubatedwithprimaryantibody(SupplementaryTable5)overnightat4°C,followedbyasecondaryhorseradishperoxidase-conjugatedantibodyatroomtemperaturefor1h.
ForimmunoprecipitationofC/EBPa,800mgofwholecelllysatewasused.
radioimmunoprecipitationassaybufferwasdilutedfourtimesandusedasimmunoprecipitationbufferwithantibody.
Lysatewaspre-clearedwithrabbitIgGantibody(CellSignalingTechnology)andDynabeadsProteinA(Invitrogen),whileantibodyforimmunoprecipitationwasboundtoDynabeadsProteinAfor1hat4°C.
Subsequently,thepre-clearedlysatewasincubatedwiththebeads-boundantibodyfor3hwithconstantrotationat4°C.
Beads-boundimmunoprecipitateswerewashedthricewithimmunoprecipitationbuffer.
Co-immunoprecipiationofFLAG-taggedproteinswithM2agarosebeads(Sigma)wasperformedaccordingtomanufacturer'sinstructions.
Briey,1mgofwholecelllysatewaslysedinlysisbuffer(50mMTris-HCl,150mMNaCl,1mMEDTA,1%TritonX-100)andincubatedwithanti-FLAGM2agarosebeadsovernightat4°C.
Beads-boundimmunoprecipitateswerewashedthricewithTBS.
Proteinswereelutedfromthebeadsunderacidicconditionswith0.
1Mglycine,pH2.
3atroomtemperaturefor5minwithconstantshaking.
Elutantwasneutralizedwith1/10volumeof0.
5MTris,pH7.
4with1.
5MNaClandanalysedwithimmunoblotting.
Computationalmethods.
ThecrystalstructureoftheC/EBPa-DNAcomplex(knownasC/EBPaWT-DNA)wasdownloadedfromtheProteinDataBank(PDBcode:1NWQ)18.
TogeneratethecoordinatesfortheacetylationmimeticC/EBPa-K2QcomplexedwithDNA(K2Q-DNA),K298andK302residueswerereplacedwithglutamineusingtheUCSFChimerapackage45.
TheGLNrotamersfromtheDunbracklibrarywereused46.
Theacetylatedlysineresidues(K2Ac)werebuiltmanuallywithUCSFChimera.
TheH–N–COdihedralangleofallfouracetylatedlysineresiduesweresetto0°intherstacetylatedmodel(hereaftertermedasK2Ac_a-DNA)basedonthemostpopulatedstructuresderivedfromcomputations47.
However,theacetylatedlysineresiduesinrecentlypublishedARTICLENATURECOMMUNICATIONS|DOI:10.
1038/ncomms1096810NATURECOMMUNICATIONS|7:10968|DOI:10.
1038/ncomms10968|www.
nature.
com/naturecommunicationscrystalstructureswerefoundtohavetheH–N–COdihedralangleswithboth0°and180°(Fig.
3b)48–50.
Thus,theH–N–COdihedralangleofallfouracetylatedlysineresiduesweresetto180°inthesecondacetylatedmodel(K2Ac_b-DNA).
Thebackboneatomsofthefourmodelsshowedsimilaructuationsduringthe3.
2nslongMDsimulations(SupplementaryFig.
3).
However,longerMDtrajectoriesareneededtomeaningfullydiscussthestabilityofthefourmodels.
Thescopeofthecurrentcomputationalstudyistogaininsightintohowdifferentmutationsaffectprotein–DNA-bindingenergyandprotein-DNAhydrogenbonding.
Forthispurpose,therelativelyshortMDtrajectoriespresentedhereshouldbesufcient51.
Theff03molecularmechanics(MM)forceeldwasassignedforallresidues52.
Theacetylationofpartialchargesderivedforusewiththeff03forceeldweretakenfromliteraturewithcorrespondingatomnamesandatomtypes47.
TheforceeldparametersweresetupusingtheleapprogrammeoftheAmberTools13package53.
Nacounterionswereaddedtoneutralizetheprotein–DNAmodelsystems.
Thecrystallographicwatermoleculeswereretainedinallfourmodelsystems.
Eachprotein–DNAmodelcomplexwasplacedintoapre-equilibratedTIP3Ptruncatedoctahedralwaterbox54.
ThecrystallographicwatermoleculeswereincludedintheTIP3Psolventboxeswhichextendedatleast12fromtheprotein-DNAcomplex.
MMminimizationsandMDsimulationswerecarriedoutwiththesanderprogrammeoftheAMBER12package53.
TheMDsimulationswereprecededbythreeminimizationsteps:(1)soluteatomswereheldxedwhilesolventmoleculeswererelaxed(1000stepsofsteepestdescentand1,000stepsofconjugategradientminimization);(2)solventatomswereheldxedwhilesolutemoleculeswererelaxed(1,000stepsofsteepestdescentand1,000stepsofconjugategradientminimization);(3)bothsolventandsolutewereoptimizedwithoutanyrestraints(1,000stepsofsteepestdescentand4000stepsofconjugategradientminimization).
Afterminimization,atwo-stepMDsimulationwascarriedoutonallsystemswitha2kcalmol12restraintonthesoluteatoms:(1)eachsystemwasheatedfrom0to300Koveraperiodof50psinthecanonical(NVT)ensemble;(2)eachsystemwasrelaxedfor50psintheisothermic-isobaric(NPT)ensemble(T300KandP1atm).
Finally,productionMDsimulationswererunfor3.
2nsintheNPTensemble(T300KandP1atm).
Forcecalculationswereperformedwithperiodicboundaryconditionsanda9cutoffwasusedfornon-bondedinteractions.
CovalentbondsconnectinghydrogenatomswereconstrainedwithSHAKE55.
Acollisionfrequencyof3ps–1wasusedintheLangevinthermostattomaintainthesystemtemperature56.
Atimestepof2fswasusedinallMDsimulations.
Atomcoordinatesweresavedevery10psduringtheMDsimulations(100framesns1).
TherandomnumbergeneratorseedwasalteredateveryrestartoftheMDsimulations.
ThepolardesolvationfreeenergywascalculatedwiththeMMPBSA.
pyprogram57.
Thelast3nsoftheMDtrajectorieswereusedforMolecularMechanics/PoissonBoltzmannSurfaceArea(MM/PBSA)calculationsandhydrogenbondanalysis.
TheradiioptimizedbyTanetal.
58wereusedwitha0.
5gridspacing,a1.
4solventproberadius,aproteindielectricconstantof1.
0,andasolventdielectricconstantof80.
Duetotheexpensivecomputationaldemand,entropieswerenotconsidered.
MM/PBSAcalculationscanyieldsatisfactoryresultswithouttheinclusionofentropy59.
GenerationofstableK562celllines.
TogenerateWTormutantC/EBPalines,K562cellswerestablytransfectedbyelectroporationwiththerespectivepBabe-C/EBPa-ERfusionconstructs.
Briey,2106cellswereelectroporatedinanAmaxaapparatuswith5mgofSca-1linearizedplasmid.
At48hpost-transfection,cellsweresubjectedtoselectionwith0.
5mgml1puromycinand50cellswereseededper96-wellplate.
C/EBPa-ERnucleartranslocationwasinducedbyadditionof5mMofb-estradiol(Sigma,E2758).
Cellswereassessed4dayspostinductionfordifferentiationbyCD11bsurfacemarkerexpression,morphologyandneutrophilicenzymaticactivity4.
Multipleindividualclonesforeachtypeofmutantwerepickedandanalysedbywesternblottoconrmexpressionofthefusionprotein.
Theresultsfromtworepresentativeclonesarepresentedinthisstudy.
Assessmentofgranulocyticdifferentiation.
Nitrobluetetrazoliumanalysiswith2.
5105cellswasdoneaspreviouslydescribed4.
Thepercentageofnitrobluetetrazolium-positivecellswasquantiedbycountingatleast100cellsforeachmutantunderbrighteldmicroscopy(Nikon).
CellmorphologywasassessedbyWright-Giemsastaining.
Cytospinpreparationof2.
5105cellsweredoneaspreviouslydescribed60.
ForstainingofCD11bsurfacemarker,1106cellswerestainedwithCD11bantibody(SupplementaryTable6)andanalysedusingLSRIIowcytometry(BDBiosciences).
TheresultingdatawereprocessedwithFlowJoVersion7.
6.
4software(TreeStar).
shRNAlentiviruspackagingandtransduction.
Lentiviralplasmids(pLKO.
1-puro)encodingGCN5-specicshRNA(TRCN0000038879(shGCN5#1),TRCN0000294386(shGCN5#2),TRCN0000307319(shGCN5#3))andnon-targetingcontrolshRNA(SHC0002(NTC#1),SHC007(NTC#2))werepurchasedfromSigmaMISSION.
Lentiviruspackagingwasperformedin293Tcellsbyco-transfectingshRNAlentiviralplasmidswithpCMV-dR8.
91andpCMV-VSVGusingLipofectamine2000.
ForGCN5knockdown,cellswereexposedtoviralparticleswithmultiplicitiesofinfection(MOI)rangingfrom1to2,inthepresenceof8mgml1polybrene(SantaCruz)for24h.
Cellswereselectedinmediacontaining1mgml1puromycin(Sigma)at48hposttransduction,andcheckedforknockdownefciencybyimmunoblottingat2weeksposttransduction.
RetroviraltransductionofCebpaD/DLSKcells.
AnimalworkwasdoneatNUSwithapprovalfromIACUC.
C/EBPaconditionalknockout(Cebpa/)andMx1-Cremicehavebeendescribedpreviously2.
LiquidcultureofGCN5LSKcells.
AnimalworkwasdoneatNUSwithapprovalfromIACUC.
Gcn5D/DVav-iCreknockoutmiceweregeneratedbycrossingGcn5/andVav-iCremice25,26.
Cells(5,000–10,000)wereaddedtogrowinStemlineIIhematopoieticstemcellexpansionmedium(Sigma-Aldrich)supplementedwith1Penicillin-Streptomycin,1anti-mycoticcocktail,6ngml1IL-3,10ngml1IL-6,20ngml1stemcellfactor(SCF),20ngml1G-CSFand10%FBS.
Cellsweregrownfor7daysbeforeproceedingwithFACSandmorphologicalanalysis.
Electrophoreticmobilityshiftassay.
EMSAwasdoneaspreviouslydescribed4.
Briey,EMSAwasperformedwithnuclearextractsfromK562C/EBPalines.
TheoligonucleotidesusedarederivedfromtheG-CSFreceptorpromoter(57to38bp),thesequencesareasfollows(C/EBPbindingsitesareunderlined):upperstrand,50-CTAGGGCTTGCGCAATCTATATTCG-30,lowerstand,50-CGAATATAGATTGCGCAAGCCCTA-30.
EMSAswereperformedbyincubating10mgofnuclearextractswith50,000countsperminutedouble-strandedoligonucleotidesina25mlreactionmixturecontaining10mMHEPES-KOHbuffer(pH7.
9),50mMKCl,2.
5mMMgCl2,1mMDTT,10%glycerol,1mgbovineserumalbumin(BSA)and0.
5mgpoly(dI-dC)onicefor20min.
Forsupershiftassay,2mgofanti-C/EBPaantibody(14AA)oranti-ER(HC-20)wasaddedtothebindingreaction.
Bindingreactionswereresolvedona6%non-denaturingpolyacrylamidegelwith0.
5TBE(0.
089MTrisborateand0.
002MEDTA)andelectrophoresedat140Vat4°C.
Chromatinimmunoprecipitation.
ChIPwasperformedwith2mganti-ERantibodiesonK562C/EBPa-ERcells(2107cells).
Briey,cellswereinducedwith5mMb-estradiolfor45min,xedin1%formaldehydefor5minatroomtemperatureandsubsequentlyquenchedwithadditionofglycinetoanalconcentrationof200mM.
CellswerelysedwithBufferL1(50mMTrispH8.
0,2mMEDTA,0.
1%NP-40,10%glycerol)for5minonice,centrifugedfor10minat850g.
ThenuclearpelletwasresuspendedinBufferL2(50mMTrispH8.
0,5mMEDTA,1%SDS).
CrosslinkedchromatinwassonicatedusingBioruptor(Diagenode)at'high'amplitude,12son/30sofffor16cycles,andcentrifugedfor10minat17,000g.
Sonicatedmaterialwasdilutedwith9volumesofBufferDB(50mMTris,200mMNaCl,5mMEDTA,0.
5%NP-40)andpre-clearedwithsalmonspermDNA(Sigma),rabbitIgG(SantaCruz)andDynabeadsProteinA(Invitrogen).
Pre-clearedchromatinwasimmunoprecipitatedovernightwithERa(HC-20)antibody,followedbyadditionofDynabeadsProteinAfor30minat4°C.
Beadswerewashedthricewithwashingbuffer(WB)(20mMTrispH8.
0,500mMNaCl,2mMEDTA,0.
1%SDSand1%NP-40),oncewithWBplus500mMLiCl,thricewithbufferTE.
Boundchromatinwaselutedfromthebeadswith100mlofelutionbuffer(1xTE,2%SDS).
Afterreverse-crosslinkingwithRNaseAandproteinaseKdigestion,DNAwaspuriedbyQiaquickPCRpuricationkitandelutedwith50mlofTEbuffer.
PrimersusedareindicatedinSupplementaryTable7.
qPCRwascarriedoutwithGoTaqqPCRmastermix(Promega)andanalysedwithaCorbettRotorgenesystem.
Relativeenrichmentiscalculatedovertheinactiveinvolucrinlocus.
Dimerizationassay.
293Tcellswereco-transfectedwith10mgofV5-taggedWTormutantversionsofC/EBPaand10mgofFLAG-taggedWTC/EBPa,celllysateswerecollected24haftertransfection.
Anti-FLAGM2Afnitygel(Sigma)wasusedtoco-immunoprecipitateFLAG-taggedproteins.
Sampleswerekeptunderconstantrotationfor2hat4°C.
Beads-boundimmunoprecipitateswerewashedthricewithTBSbufferandelutedinSDS-PAGEsamplebufferforimmunoblottinganalysis.
Geneexpressionanalysis.
Rawmicroarraygeneexpressiondata(CELles)forAMLandnormalBMCD34samplesweretakenfromGSE13159(ref.
61)andGSE19429(ref.
62),respectively.
WeusedtheRMAmodeltoextractthegeneintensityfrommultipleprobeintensities,whilethecross-arraydatanormalizationwasperformedusingtheCross-Correlationmethod63.
Statisticalanalysis.
Thetwo-tailed,Student'st-testwasusedtocomparebetweentwogroups.
Asterisk(*)indicatesPvalueo0.
05,**indicatesPvalueo0.
01,***indicatesPvalueo0.
001inthegures.
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AcknowledgementsWethankPerePuigserver(DanaFarberCancerInstitute,Boston,USA)forGCN5andPCAFconstructs,GeorgW.
Bornkamm(HelmholtzCenter,Munich,Germany)forp300construct.
WeeJooChng(CancerScienceInstitute,Singapore)forAMLsamplesandcelllines,Yoon-PinLim(NationalUniversityofSingapore,Singapore)andShirlyChongPohKuan(NationalUniversityofSingapore,Singapore)forsyntheticpeptidesanalysisusedforrabbitimmunization,AlanFriedman(JohnHopkinsUniversity,Maryland,USA)forthepBabe-ERvector,AxelImhof(Ludwig-Maximilians-University,Munich,Germany)forGST-taggedGCN5HATdomainexpressionconstruct,SharonY.
R.
Dent(MDAndersonCancerCenter,Texas,USA)forGcn5/mice,MotomiOsato(CancerScienceInstitute,Singapore)forVav-iCremice,CelestinaChinAiQi(CancerScienceInstitute,Singapore)forproofreadingofthemanuscript,andmembersoftheTenenlaboratoryformanyhelpfuldiscussionsandtechnicalsupport.
ThisresearchissupportedbytheNationalResearchFoundationSingaporeandtheSingaporeMinistryofEducationunderitsResearchCentresofExcellenceinitiative,SingaporeTranslationalResearchAwardfromtheSingaporeNationalMedicalResearchCouncil(NMRC/STaR/0001/2008toD.
G.
T)andgrantsfromtheSingaporeMinistryofEducationandNationalResearchFoundationandtheNationalInstitutesofHealth(CA66996andHL112719toD.
G.
T).
ThisresearchwassupportedbytheMax-EderProgramoftheDeutscheKrebshilfe(#110659toO.
W.
).
ThemolecularmodellingstudieswerefundedbytheEuropeanSocialFund(ESF)oftheEuropeanUnion(EU)andtheFreeStateofSaxony(M.
B.
S.
).
AuthorcontributionsD.
G.
T.
supervisedtheproject;H.
S.
K.
,R.
S.
W.
,A.
N.
,A.
K.
E.
,D.
B.
andD.
G.
T.
conceivedanddesignedthestudy;H.
S.
K.
,R.
S.
W.
,A.
N.
,D.
R.
,S.
T.
andD.
B.
performedexperiments;M.
B.
S.
performedmolecularmodellingstudies;H.
Y.
performedmicroarraydataanalysis;S.
W.
andJ.
G.
performedmassspectrometryexperimentsandassistedinanalysis;resourcesprovidedbyO.
W.
andD.
G.
T.
;H.
S.
K.
,R.
S.
W.
,A.
N.
,E.
L.
,D.
B.
andD.
G.
T.
wrotethepaper.
Allauthorsdiscussedtheresultsandcommentedonthemanuscript.
AdditionalinformationSupplementaryInformationaccompaniesthispaperathttp://www.
nature.
com/naturecommunicationsCompetingnancialinterests:Theauthorsdeclarenocompetingnancialinterests.
Reprintsandpermissioninformationisavailableonlineathttp://npg.
nature.
com/reprintsandpermissions/Howtocitethisarticle:Bararia,D.
etal.
AcetylationofC/EBPainhibitsitsgranulopoieticfunction.
Nat.
Commun.
7:10968doi:10.
1038/ncomms10968(2016).
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