1、辅抑制子 SMRT 特异地抑制孤儿核受体hB1FhLRH-1 的转录活性生物化学与生物物理NACTABIOCHIMICAetBIOPHY.S.1.C.A.S.I.N.I.C.A.2.003,35(10):897.-.9.033113ooCorepressorSMRTSpecificRepressestheTranscriptionalActivityofOrphanNuclearReceptorhBlF/blRH-1xuPingLong,KONGYuYing,XIEYouHua,WANGYuanrStateKeyLaboratoryofMoleclarBiology,InstteofBioch
2、emistryandCellBiology,ShanghaiIntittesforBiologicalScienc,theChineseAcademyofSciences,Shanghai200031,China)AbslractTheorphannuclearreceptorhBlF(alsoknownasNR5A2,LRH 一 1,FTForCPF)playsimportantrolesinregulatingtheexpressionofseveralcellularandviralgenesactivelyinvolvedinawiderangeofbiologicalprocesse
3、ssuchasthebileacidbiosynthesis,liverspecificgeneregulatorynetworkandhepatitisBvirusreplication.Theactivityofnuclearrec 印torsISregulatedbymultiplemechanisms,includingcoactivationandcorepression.Inthisstudy,itwasfoundthatthesilencingmediatortorretinoicacidreceptorandthyroidhormonerec 印 tor(SMRT)specif
4、icallyrepressesthetranscriptionalactivitvofhBlF.oneitherGAL4dependentreportersystemorthehBlFresponsive 鹏VenhancerII/c0reDr0moter.TherepressionimposedbySMRTisobservedindiff.erentcelllines.Interestingly,hBlFc0uldntinteractwithSMRTdirectly.asdemonsgatedbymammaliantwohybridanalysisorGSTpulldownassay.Tak
5、entogether,itcanbeconcludedforthefirsttimethatthetranscriptionalactivityofhBlFisregulatedspecificallybythecoreDressorSMRTviaanindirectmechanism.KeywordspromoterhB1F/hLRH 一 1;orphannuclearreceptor;repression;SMRT;HBVenhancerII/corenlllnanhepatitisBvirusenhancerIIBlbindingfactorfhB1F1hasbeenformanydes
6、ignatedasNlA2andisalsoknownasliverreceDtorhomologuel(LRH-I),CY1P7Apromoterbindingfactor(CPF)and仅一 fetoproteintranscriptionfactor(FTF).hBlFbelongstothefushitarctzufactorI(FTZFl,subfamilyofthenuclearreceDtorsuperfamily.MembersoftheFTZ.FIsubfamilypossessahighlyconservedFTZ.-FIboxlocateddownstreamfromth
7、ezincfingersintheDNA-bindingdomainandbindtotheircorrespondingsitesasmonomer.hElFmainlvexpressesinthepancreasandliver.andhasalsobeenfoundinovary.intestineandcoIon.WereportedpreyiouslythathBlFspecificallybindstheBlelementofENIIandactivatestheenhancertoregulatetheexpressionOfviralgenes1,9.Received:June
8、l7,2003Accepted:Ju9.2oo3ThisworkwassupportedbythegrantsfromtheNationalNatura1ScienceFoundationofChinafNo.3O100088),theNationalrraghTechnologyRDProgram(2001AA221261),BasicResearchProgramfromMinistryofScienceandTechnology(G1999054105),andtheQiMingXhagProjectfromShanghaiSconceandTechnologyCommittee(01Q
9、A14046)Correspondingauthors:WANGYuan:Tel,86-21-54921103;Fax,86?2154921011:e?rflall,wangyss.ac.CllXIEYouI-ha:Tel,86-21-54921105;Fax,86-21-54921011;erfl 礼Recentlv.accumulatingdataonthebiologicalfuctionsofhBlFinrecentyearshaveshownthatitwasanimportanttranscriptionalactivatorinthebileacidandcholesterolh
10、omeostasisbyregulatingtheexpressionofkeyenzymesandtransportersincludingcholesterol7 仅一hydroxylaseI,川.steroll2hydroxylase】andcholesterylestertransferprotein.AAditionaUy.hBlFalsoregulatestheexpressionofl1Bhydroxylase,aromatase14A,scavengerreceptorclassBtypeI,andseveralliverenrichedtranSCriptionalfacto
11、rssuchasF3B,F4 仅 andHNFl 仅.Sofar,littlehasbeenknownaboutthemolecularmechanismunderlyinghBlFdependentpromoteractivation.ThetranSCri)tionmediatedbynuclearreceDtorsfrequentlyrequirestherecruitmentofspecificcoactivatorsandcorepressorsthroughinteractingdomainsonboththereceDtorandcofactor.Recruitmentofcoa
12、ctivatorssuchasSRC.10rCBP/p300occursmainlythroughdirectorindirectinteractionwithactivationfunctionmodulesofnuclearreceptors,theHganddependentAF-2andNterminalAF1.Whi1erepressionisachievedbyrecruitmentofcorepresrsor.storegulatoryregionsofnuclearreceptor.Thesencingmediatorforretinoicacidreceptorandthyr
13、oidhormonereceptorfSMRT1andnuclearreceptorcorepressor(NCoR)aretwowel1.knowncorepressors,sharingahighhomologyinthe898ACTABIOCHIMICAetBIOPHYSICASINICAVbL35.No.10Nterminusl19,20.Dif_ferentnuclearreceptorsexhibitpreferenceinassociationwithNCoRorS.SF.1.aFTZF1relatedreceptor,hasbeenshowntointeractwiththeo
14、rphannuclearreceptorDAX 一 1whichintumrecruitsthecorepressorNCoR.Inthepresentstudy,thecor 印 ressorSMI 盯 wasfoundtospecificallyinhbitthetranscriptionalactivityOfhBlFinadosedependentmariner.However,mammaliantwo.hybridanalysisandGSTpull-?downassaydemons 仃 atedthatSdidnotdirectlyinteractwithhB1F,suggesti
15、ngthatthecor 印 ressorSMRTexertstherepressiononthetranscriptionalactivityoftlBlFthroughanindirectmechanism.1MaterialsandMethods1.1Pl 舔 midsconslruclionThehB1FdependentreporterpENII/CpLucwiththeenhancerIIandthecorepromoter(ENII/Cp1of耶 Vl,gJWasmadefromthepGL2basic(Promega).ThepENIIm/CpLucreportercontai
16、nsPCRin 仃 oducedpo 订 Itmutatbns(5 一 GATCAACtACaGAtCTcGAG 一 3,mutationsinlowercaseletters1thatdisruptthehB1FbindingsiteintheB1elementofENII.TheGAL4dependentreporterpG5LucwasmadebyreplacingtheCATgeneinPG5CAT(Clontech)withtheluciferasegene.TheexpressionplasmidofGAL4 一 hB1Fl4l 一495wasmadebyinsertingtheP
17、CRfragmentofhB1F(aal414951digestedwithEcoRIandaIintotheCterminusoftheGAL4DNA_bindingdomainfDBD)inthepM(Ciontech).ThepVP16 一 STcwasmadebyinsertingreceptorinteractingdomainfRID119,21ofmouseSMRTor.(residuesl97524131.digestedfrompCMV侬一凡(kindlyprovidedbyProf.RonaldM.Evans)withBglIIandPstI,intotheCterminu
18、softheactivationdomainfAD)ofVP16inthepVP16(Ciontech).TheprokaryoticexpressionplasmidofpGSThB1Fl86495wasmadebyinsertingthePCRamplifiedfragmentofhBIF(aal864951intotheSmaIsiteofthepGEX 一 3X(Phamacia).inframetotheGST.PCRamplificationswereperformedwiththehighfidelityP),robestpolymerase(TaKaRa)withprimers
19、contammgappropmterestrictionenzymesRestofacilitatecloning.AllplasmidsconstructedwithPCRfragmentswereverifiedbysequencing.1.2TnmsfeclionandluciferaseassaysCOS,HeLa,HepG2andHuh7cellsweremaintainedinDulbeccosmodifiedEaglesmedium(DMEM)supplementedwith10%fetalcalfserum(FCS).Y1cellsweregrowninF12mediumsup
20、plementedwich10%FCS.Transienttransfectionwascarriedoutata35nlnldishwiththestandardcalciumphosphateprecipitationmethodaspreviouslydescribedusing4IxgoftotalDNwith0.5gofpCMV 一 Ztonormalizethetransfectionefficiency.48haftertransfection.cellswereharvestedandlysedin1xreporterlysisbuffer(Promega).Thehcifer
21、aseactivitywasdeterminedwithluciferaseassaysystem(Promega)andthepgalactosidaseactivltywas.measuredbyastandardcole?rimetricmethod.LuciferaseactivitiesfromdifferenttransfectionswerenormalizedbytheBgalactosidaseactivities.Eachtransfectionwasperformedinduplicatedishesandrepeatedatleastthreetimes.13Wlest
22、embtangHuh7cellswereseededin35milldishesat2.510cellsperdish,andtransfectedwith0.2gexpressionplasmidofGAL4 一 hB1Fl4I-495and0.5IxgpCMV-/acZ.ForthecoexpressionofS.1IxgexpressionplasmidofSToremptypcDNA3vectorwasincludedinthetransfection.After48hpost.transfection,cellswereharvestedandasmallproportionofth
23、ecellswaskeptforthemeasurementOftheBgalactosidaseactivitytonormalizethetransfectionefficiency.Adiustedamountofthewholecellextractsweresubjectedto10%TrisglycineSDSPAGEand 仃 ansferredtonitrocelhlosemembrane(Protran.SS1.ImmunoblottiugwascarriedoutwithanantiGAL4 一 DBDmonoclonalantodySC 一 510(SantaCruZ1u
24、singtherabbitantimouseIg/RPfDAKO1asasecondaryantbody.PeroxidaseactivitywasdetectedbytheECLreactionwithWestemblotlumiuo1reagentfSantaomz).1.4MamnmlimltwohybridassayTwohvbridassayofhB1FandSMRTwasperfo?rmedinHuh7cellsusingmammalianMatchMarekrtwohybridassaykitfCiontech1accordingtothemanufacturersinstruc
25、tion.1gOfvectorforGAL4orGAL4 一 hB1Fl4I-495and1IxgofvectorforVP16orVP16.Sc(correspondingtoresiduesl97524131werecotransfectedintoHuh7cells.alongwith0.5IxgpG5Lucreporterand0.5IxgpCMV-/acZcontrO1.ThehciferaseactiviWandtheBgalactosidaseactivityweremeasuredasdescrNedinpart1-2.1.5GsTpug-downassayGSTandGSTh
26、B1Fl86495werepurifiedwiththegiutathioneSepharose4Bbeads(AmershamPharmacia)fromlysatesofBL21(DE3)(Invitrogen)culturescontaining 印 proprmteexpressionplasmidsafterinducingby0.2mmoFibIPTGfor2h.PurifiedGSTfusionproteinswerequantifiedbycomparingwiththeBSAstandardonCoomassicstainedSDSPAGE.Pulldownassaywasp
27、erformedwithpurifiedGSTfusionproteinsandthefuUlengthSMRTorSRC 一 1synthesized 加 vitrowithTNTquickcoupledtranscription/translationsystemsfPromega)inthepresenceofSJmethionine(AmershamPharmacia1ACTABIOCHIMICAetBIOPHYSICASINICAVoL35,No.10possibilities.thepENIIm/CpLucrepotterwasconstructed.inwhichthefunct
28、ionallycriticalhBlFbindingsiteintheB1elementofE11wasmutatedJ.Asexpected.hBlFCOUldbarelyact1.vatethePDIm/CpLucrepotter.CoexpressionOfStTobviouslydidnotinhlbittherel:otter(Fig-4),indicatingthatStTinhibitedtheactivityofENII/CpprimarviahBlF.ThesedatastronglysuggestedthatthereisafunctionalcorrelationbetweenhBlFandStT.pENII/CpLucpENIIrrICpLuc(A1
