Methylation of C EBPα by PRMT1 Inhibits Its Tumor-Suppressive Function in Breast Cancer Li-Ming Liu.pdf

该【Methylation of C EBPα by PRMT1 Inhibits Its Tumor-Suppressive Function in Breast Cancer Li-Ming Liu 】是由【袭人】上传分享，文档一共【33】页，该文档可以免费在线阅读，需要了解更多关于【Methylation of C EBPα by PRMT1 Inhibits Its Tumor-Suppressive Function in Breast Cancer Li-Ming Liu 】的内容，可以使用淘豆网的站内搜索功能，选择自己适合的文档，以下文字是截取该文章内的部分文字，如需要获得完整电子版，请下载此文档到您的设备，方便您编辑和打印。:..AuthorManuscriptPublishedOnlineFirstonApril23,2019;DOI:--18-:MethylationofαC/EBPbyPRMT1inhibitsitstumorsuppressivefunctioninbreastcancerAuthors:Li-mingLiu1,2,#,Wen-zhengSun1,2,#,Xue-zheFan1,2,Ya-liXu3,Mo-binCheng1,2,*,andYeZhang1,2,*Affiliations:1StateKeyLaboratoryofMedicalMolecularBiology,ChineseAcademyofMedicalSciences&PekingUnionMedicalCollege,Beijing100005,China2DepartmentofBiochemistryandMolecularBiology,InstituteofBasicMedicalSciences,ChineseAcademyofMedicalSciences&PekingUnionMedicalCollege,Beijing100005,China3DepartmentofBreastSurgery,PekingUnionMedicalCollegeHospital,ChineseAcademyofMedicalSciences&PekingUnionMedicalCollege,Beijing100730,China#Theseauthorscontributedequallytothiswork.*Co-correspondingauthors:Mo-binChengandYeZhang5DongdanSantiaoBeijing100005,ChinaTel:(8610)69155939;Fax:(8610)65269665Email:******@.(MC);******@.(YZ)Runningtitle:PRMT1methylatesC/EBPαtoregulateCyclinD1Competinginterests:,2019.?2019AmericanAssociationforCancerResearch.:..AuthorManuscriptPublishedOnlineFirstonApril23,2019;DOI:--18-,,littleisknownabouttheroleofargininemethylationinregulatingtheantiproliferationactivityofC/(PRMT1)-sequencinganalysisrevealedthatknockdownofPRMT1inbreastcancerpaniedbyadecreaseintheexpressionofpro-,tandemaffinitypurificationfollowedbymassspectrometryponentoftheC/(R35,R156,andR165).PRMT1-dependentmethylationofC/EBPαpromotedtheexpressionofcyclinD1byblockingtheinteractionbetweenC/EBPαanditsco-repressorHDAC3,-:,2019.?2019AmericanAssociationforCancerResearch.:..AuthorManuscriptPublishedOnlineFirstonApril23,2019;DOI:--18-(PRMT)familyisamajorposttranslationalmodificationthatregulatesmultiplecellularprocesses(1-3).PRMT1isthoughttoperform85%ofPRMTactivityinmammaliancells(4).ManynonhistonesubstratesofPRMT1areinvolvedinbiologicalprocessessuchastranscriptionalregulationandcellsignaling(1,5),andmethylationofhistoneH4atarginine3(H4R3me2a)byPRMT1activatesgeneregulation(6,7).PRMT1-mediatedargininemethylationofFOXO1enhancesitstransactivationfunctionbystabilizingtheFOXO1protein(8).PRMT1alsofunctionsasacoactivatorofRUNX1toactivateitstargetgenesbydisruptingtheinteractionbetweenRUNX1andSIN3A(9).GiventheextensivesubstratesofPRMT1incells,aberrantexpressionofPRMT1hasbeenimplicatedinthepathogenesisofseveraldiseases,includingcancer(3).PRMT1upregulation(10-12)oraberrantsplicing(13),(C/EBPs)areafamilyofbasicleucinezipper(bZIP)DNAbindingproteinsthatregulatethetranscriptionofseveraltissue-specificgenesandsomegrowth-relatedgenes(14).C/EBPαisthefoundingmemberoftheC/EBPsfamilyandcontainsaC-terminalbZIPandN-terminaltransactivationdomain(TAD1-3)(15,16).C/EBPαhastwodistinctisoforms,p42andp30,withvaryingN--p30lacksthemajortransactivationdomain(1-120aa)andcanneutralizethetranscriptionalactivityofC/EBPα-p42(17,18).C/-proteininteractionsandregulationofitsbindingproteins,suchasp21(19),CDK4/6(20),andE2F(21,22).C/EBPαalsofunctionsasatranscriptionalfactortoactivateorrepresssomegrowth-relatedgenes,suchasN-Myc(23),Sox4(24)andBMI1(25),(PTMs).C/EBPαisdecoratedwithvariousPTMs,includingphosphorylation(26-29),SUMOylation(30-32)andacetylation(33).MostofthesemodificationsrepressC/,2019.?2019AmericanAssociationforCancerResearch.:..AuthorManuscriptPublishedOnlineFirstonApril23,2019;DOI:--18-,littleisknownabouttheroleofargininemethylationinregulatingtheantiproliferationactivityofC/,wereportthatC/EBPαdirectlyinteractswithandisargininemethylatedbyPRMT1;methylatedC/,,-MB-231(Cat.#000014),MDA-MB-435(),MDA-MB-468(Cat.#000249),MCF7(Cat.#000013),SK-BR-3(Cat.#000085),HEK-293T(Cat.#000091),HeLa(Cat.#000011),BT549(Cat.#000336)andMCF10A(Cat.#000406)celllineswerepurchasedfromChineseNationalInfrastructureofCellLineResource(Beijing,China).Beforetheexperiments,%transfectefficiencywithGFP--PCRMycoplasmaTestKit(Cat.#20-700-20)aspreviouslydescribed(34).MDA-MB-231,MDA-MB-435,MDA-MB-468,MCF7,SK-BR-3,HEK-293T,HeLaandBT549cellsweregrowninDMEM(o’sModifiedEagleMedium)supplementedwith10%FBSand100U/mlpenicillin-(1:1)mediumsupplementedwith5%horseserum,,10μg/mlinsulin,20ng/mlepidermalgrowthfactor,,100U/mlpenicillin-(TNBC-1andTNBC-2)weregrowninDMEM/F12(1:1)mediumsupplemented5%FBS,,5μg/mlinsulin,10ng/mlepidermalgrowthfactor,10ng/mlcholeratoxin,25μg/mlAdenine,5μMRockinhibitorand100U/mlpenicillin-(SML1559,Sigma,,MO),aspecificinhibitorofPRMT1(35)atconcentrationof0,20,40,,2019.?2019AmericanAssociationforCancerResearch.:..AuthorManuscriptPublishedOnlineFirstonApril23,2019;DOI:--18-(07-404)andasymmetricdimethyl-arginine(ASYM25,anti-Rme2,09-814)werefromMillipore(Billerica,MA);antibodyforC/EBPα(2295)wasfromCST(Danvers,MA);antibodyforCyclinD1(ab134175)wasfromAbcam(Cambridge,UK);antibodiesforActin(sc-47778),GAPDH(sc-166574),andGST(sc-138)werefromSantaCruzBiotechnology(SantaCruz,CA);antibodyforFLAG(F3165)wasfromSigma;antibodiesforFLAG(PM020),Myc(M047-3,and562),andGFP(598)werefromMBL(Japan);antibodyforHDAC3(A2139)wasfromAbclonal(Wuhan,China).PlasmidsExpressionplasmidsofC/EBPα-pcDNA6,C/EBPβ-pcDNA6,C/EBPδ-pcDNA6,C/EBPγ-pcDNA6,HDAC1-pcDNA6,HDAC2-pcDNA6andHDAC3-pcDNA6weredescribedpreviously(36).HDAC1andHDAC3codingregionwerefurtherclonedintoMyc-taggedpCMV--3tag7andpGEX4T-(ΔTAD1,1-120,ΔbZIP,p30,bZIP)wereamplifiedbyPCRusingC/EBPα-(Biomed,Beijing,China).PRMT1cDNAwasamplifiedbyPCRfromHEK-293TcellscDNAsandclonedintopcDNA6andpCMV-(E153Q)andC/EBPαmutants(R35K,R156K,R165K,R35/156/165K,R156F,R35/156/165F,R289A)weregeneratedbyPCR-basedsite-,extendingfrom-808to+133relativetothetranscriptionstartsite,wasclonedintothepGL3-basicluciferasereportervector,andwasusedastemplatestosubcloneandgenerateaseriesofpromoterdeletionsofCyclinD1(-304/+133,-113/+133,-8/+133,-113/+15).PromoterofCyclinD1withdisruptedpotentialC/EBPαbindingsitewasconstructedusingPCR-basedsite-(37).PRMT1knockdown(shPRMT1),,2019.?2019AmericanAssociationforCancerResearch.:..AuthorManuscriptPublishedOnlineFirstonApril23,2019;DOI:--18--().Lentiviruseswerecollected48hpost--MB-231cellswereinfectedusinglentivirusexpressingshPRMT1orshGFPwith8μg/-,cellswerewashedbyPBStwice,%×103cell/wellinquintuplicatein96-?AQueousOneSolutionCellProliferationAssay(MTS).Briefly,solutionreagentwasaddedtoeachwellandincubatedat37°,(onlymedium)-×DMEM,10%FBS,%°,thetoplayerwaspoured,consistingof3×103shPRMT1orshGFPMDA-MB-231cellssuspendedin2mlof1×%×,%crystalvioletandcounted(>200μmindiameter)-healingassayCellswereplatedingrowthmediumin6-,,2019.?2019AmericanAssociationforCancerResearch.:..AuthorManuscriptPublishedOnlineFirstonApril23,2019;DOI:--18--udemicewerepurchased,×105shPRMT1orshGFPMDA-MB-231cellssuspendedin100μlPBSwereinjectedsubcutaneouslyintonudemice(8miceforeachgroup).,=L×W2/2(V,volume;L,length;W,widthoftumor).,2×106cellsweretrypsinized,washedtwiceinice-coldPBS,andfixedincold75%alcoholat4°Covernight.