在哺乳动物线粒体中DNA甲基化转移酶1,胞嘧啶甲基化,和胞嘧啶羟甲基化

哺乳动物线粒体中DNA甲基化转移酶1,胞嘧啶甲基化和胞嘧啶羟甲基化MitochondrialDNA(mtDNA)hasbeenreportedtocontain5-methylcytosine(5mC)atCpGdinucleotides,asinthenucleargenome,butneitherthemechanismgeneratingmtDNAmethylationnoritsfunctionalsignificanceisknown.Wenowreportthepresenceof5-hydroxymethylcytosine(5hmC)aswellas5mCinmammalianmtDNA,suggestingthatpreviousstudiesunderestimatedthelevelofcytosinemodificationinthisgenome.DNAmethyltransferase1(DNMT1)translocatestothemitochondria,drivenbyamitochondrialtargetingsequencelocatedimmediatelyupstreamofthecommonlyacceptedtranslationalstartsite.线粒体DNA(mtDNA)已经报道在核基因组中CpG二核苷酸上包含5甲基化胞嘧啶(5mC),但是mtDNA甲基化产生的机制和它功能的重要性都还不知道。我们现在报道5hmC5羟甲基化胞嘧啶和5mC一样存在于哺乳动物mtDNA中，表明之前的研究低估了胞嘧啶修饰在基因组中的水平。DNA甲基化转移酶1(DNMT1)转移到线粒体，被一个线粒体靶序列？驱动立刻定位到大家公认的转录起始位点？的上游。Thistargetingsequenceisconservedacrossmammals,andtheencodedpeptidedirectsaheterologousproteintothemitochondria.DNMT1istheonlymemberofthethreeknowncatalyticallyactiveDNAmethyltransferasestargetedtothemitochondrion.MitochondrialDNMT1(mtDNMT1)bindstomtDNA,provingthepresenceofmtDNMT1inthemitochondrialmatrix.mtDNMT1expressionisup-regulatedbyNRF1andPGCla,transcriptionfactorsthatactivateexpressionofnuclear-encodedmitochondrialgenesinresponseto(响应，反应)hypoxia,andbylosso(失去)p53,atumorsuppressorknowntoregulatemitochondrialmetabolism.AlteredmtDNMT1expressionasymmetricallyaffectsexpressionoftranscriptsfromtheheavyandlightstrandsofmtDNA.Hence,mtDNMT1appearstobe(好像是，仿佛)responsiblefor(是的原因，为负责)mtDNAcytosinemethylation,fromwhich5hmCispresumedtobederived,anditsexpressioniscontrolledbyfactorsthatregulatemitochondrialfunction.这个靶序列在哺乳动物中是保守的，编码的多肽引导一个外源蛋白到线粒体上。DNMT1是三个已知催化活性的DNA甲基化转移酶中唯个针对线粒体的。线粒体DNMT1(mtDNMT1)结合mtDNA，证明线粒体基质中存在mtDNMT1。NRF1和PGC1a上调mtDNMT1的表达，转录因子激活核编码线粒体基因的表达是对低氧的反应，失去P53,一个肿瘤抑制因子已知可调节线粒体新陈代谢。改变mtDNMT1表达不对称地影响mtDNA重链和轻链的转录表达。因此，mtDNMT1好像是mtDNA胞嘧啶甲基化的原因，推测它衍生出5hmC，调节线粒体功能的因子控制mtDNMT1的表达。正文Inthenucleus,cytosinemethylationcooperateswith(合作，协作)N-terminalhistonemodificationstoestablishasilencedchromatinstructure(1),thus(从而)regulatingnucleargeneexpression.MethylationpatternsareestablishedinthedevelopingembryobytwodenovoDNAmethyltransferases,DNMT3aand-3b(2).Maintenanceofthispatterninsomaticcellsisbelievedtobe(被认为是)thepredominantfunctionofDNMT1,withfunctionalcooperationevidentbetweenthetwogroupsofenzymes(3).Cytosinemethylationisessentialfor(是。。必需的)normaldevelopment,anddeletionof(缺失，删除)DNMT1resultsinembryoniclethalityinmiceandmitoticcatastropheinculturedcells(4).在细胞核中，胞嘧啶甲基化与N末端组蛋白修饰共同协作去建立一个沉默的染色体结构，从而调节和基因的表达。在发育的胚胎中通过两种从头DNA甲基化转移酶（DNMT3a，3b）建立甲基化模式。人们认为在体细胞中维持这种模式是DNMT1的主要功能，这两组酶间具有明显的功能合作。胞嘧啶甲基化是正常发育必需的，缺乏DNMT1导致小鼠胚胎致死和培养细胞有丝分裂障碍。Recently,thepresenceofsignificantlevelsof5-hydroxymethyl-cytosine(5hmC)wasdemonstratedinDNAfromneurons,brain(5),andembryonicstemcells(6).5hmCisderivedfrom5-methylcytosine(5mC)oxidationcatalyzedbytheTETfamilyofmethylcytosineoxygenases,anditsfunctionalsignificanceisunderintenseinvestigation.Thismodificationislikelyto(有可能)haveanimpacto(对于有影响)localchromatinstructure,andithasbeenproposedthat(人们已经提议)5hmCactsa(充当，担任)anintermediateinactiveorpassivedemethylation(7).最近，已经证明在神经、脑和胚胎干细胞DNA中存在显著水平的5hmC。5hmC来源于TET蛋白家族的甲基胞嘧啶加氧酶催化5mC的氧化，他的功能的重要性是下面积极调查的。这种修饰很可能对局部的染色体结构有影响，人们已经提议5hmC充当了在主动去甲基化和被动去甲基化的中间物。CytosinemethylationofmitochondrialDNA(mtDNA)hasbeencontroversialand,remarkably,infrequentlystudied.Theearlieststudy,conductedoverthreedecadesago,reportedthattherewasnomethylationofmtDNA(8).Subsequently,lowlevelsofmethylationrestrictedtoCpGdinucleotideswerereportedinmitochondriaofseveralspecies,usingmethylation-sensitiverestrictionendonucleasecleavageandnearest-neighboranalysis(911).引人注目的是，线粒体胞嘧啶甲基化(mtDNA)是引起争论和很少被研究的。大约30年前，最早的研究中报道mtDNA没有甲基化(8)。随后，使用甲基化敏感限制性核酸内切酶分裂和进行最近邻分析，在几个物种的线粒体中报道低水平甲基化限制在CpG二核苷酸(9-10)。MammalianmtDNAshowsasimilarlevelofCpGsuppressiontothatofnuclearDNA(12),suggestingthat5mCissusceptibleto(对敏感，易受影响)mutationinmtDNA.Todate(迄今为止),5mCistheonlymodifiedbase(修饰碱基)describedinmtDNA,butthemechanismsestablishingandmaintainingmtDNAmethylation,andthefunctionalsignificanceofthismodificationinmtDNA,arenotknown.哺乳动物mtDNA与核DNA的CpG抑制显示了一个相似的水平(12)，表明mtDNA中5mC容易突变。迄今为止，5mC在mtDNA中描述的唯一修饰的碱基，但是这个建立和维持mtDNA甲基化的机制，和这个修饰在mtDNA中功能性意义还是未知的。MammalianmtDNAisa16.5-kbdouble-stranded,circularmolecule,presentinmultiplecopiespermitochondrion(13).Themitochondrialgenomeencodes13oftheproteinspresentintherespiratorychaincomplexesofmammalianmitochondria,aswellastworibosomalRNAsand22transferRNAsspecifictothisorganelle.Allothermitochondrialproteins,includingthoserequiredformtDNAreplicationandtranscription,areencodedinthenucleusandtranslocatedtothemitochondriausingspecializedimportsystemswhichofteninvolveN-terminalmitochondrialtargetingsequences(MTSs)(14).Incontrasttothenucleargenome,mtDNAisnotcomplexedwithhistones.However,mtDNAispresentinprotein-containingcomplexescallednucleoids,eachcontainingmultiplecopiesofmtDNAboundtoacomplexmixtureofproteins(15).哺乳动物mtDNA是一个16.5kb双链环状分子，每个线粒体中存在多个拷贝(13)。线粒体基因组编码哺乳动物线粒体呼吸链上13个蛋白复合体，两个核糖体RNA和22转运RNA专门针对这个细胞器的。其他所有线粒体蛋白，包括mtDNA复制和转录需要的，在核中编码并使用专门的导入系统转运到线粒体，专门的导入系统经常包括N末端线粒体靶序列(MTSs)(14)。对比核基因组，mtDNA没有与组蛋白形成复合体。然而，mtDNA存在于包含蛋白质的复合物中，称之为拟核，每个包含mtDNA多个拷贝结合到一个复杂的蛋白质混合物上(15)。Transcriptionofthemitochondrialgenomeisthoughttobe(被认为是)coregulatedwithnuclearcomponentsoftherespiratorychaincomplexes（16）.Inmammals,oxidativestressresultsinstabilizationofperoxisomeproliferator-activatedreceptory-coactivator1a（PGC1a）,whichactivatesthetranscriptionofseveralnuclear-encodedtranscriptionfactors,includingnuclearrespiratoryfactor1（NRF1）.PGClaandNRF1formacomplexthatinturn（依次，轮流）up-regulatestranscriptionoftranscriptionfactorofactivatedmitochondria（TFAM）andmultiplemembersofmitochondrialrespiratorychaincomplexes（17）.线粒体基因组的转录被认为是与呼吸链复合物核组成部分共同调节的（16）。哺乳动物中，氧压导致了氧化物酶体增殖物激活受体Y辅激活因子1a（PGC1a）的稳定性，它激活几个核编码转录因子的转录，包括核呼吸因子（NRF1）。PGC1a和NRF1形成一个复合体依次上调激活了的线粒体转录因子的转录（TFAM）和许多线粒体呼吸链复合物成员（17）。Severalnuclear-encodedgenesinvolvedinmitochondrialfunction,includingPGC1a（18）,areregulatedbyDNAmethylation.Conversely,ithasbeensuggestedtha（表达观点）mitochondriaareablet（能够）influencecytosinemethylationlevelsinthenucleusbymodulatingthefluxofone-carbonunitsforthegenerationofS-adenosylmethionine,themethyldonorinDNAmethylation（19）.Thus,epigeneticregulationofnucleargeneexpressionappearstohave（似乎有）amitochondrialcomponent.ThepresenceofcytosinemethylationinmtDNAledustoquestionwhetherthisepigeneticmodificationmightplayaroleintheco-ordinatedregulationofmitochondrialgeneexpressionfrombothnuclearandmitochondrialgenomes.通过DNA甲基化调节几个核编码的基因包含线粒体的功能，包括PGC1a（18）。相反地，人们认为线粒体能够通过调制S-腺苷甲硫氨酸产生的一碳单位的流出，影响细胞核中胞嘧啶甲基化水平，S-腺苷甲硫氨酸为DNA甲基化的甲基供体（19）。因此，核基因表达的表观调节似乎有线粒体的成分。mtDNA中胞嘧啶甲基化的存在使我们去思考表观修饰可能在从核和线粒体两个基因组对线粒体的基因表达协同调节中扮演着重要的角色。ResultsHumanandMouseDNMT1EncodeMitochondrialTargetingSequences.EarlyreportsofDNAmethylationinthemitochondrialgenome（9-11）ledustoaskwhetheroneormoreofthecatalyticallyactivemammalianDNAmethyltransferasesmightbetargetedtomitochondria.Examinationofthe5'UTRand5'flankinggenomicDNAupstreamofthepublishedtranscriptionstartsites（20）forbothhumanandmouseDNMT1revealedthatsequenceequivalentto101codonsinhumanand63codonsinmouseDNMT1wasin-framewiththehighlyconservedaminoacidsequenceofDNMT1,startingwiththeATGreported（20）tobetheprimarytranslationalstartcodon（Fig.1AandB）.结果人类和小鼠DNMT1编码线粒体靶序列早期报道的线粒体基因组DNA甲基化（9-11）引导我们去问是否有一个或者更多催化活性哺乳动物DNA甲基化转移酶可能是针对线粒体的。检查人类和小鼠DNMT1公布的转录起始位点（20）基因组DNA上游5’UTR和5’侧翼区，显示人类101个密码子和小鼠DNMT163个密码子序列等效是具有高度保守氨基酸序列的DNMT1编码框，报道的ATG起始（20）是主要的转录起始密码（Fig.1A和B）。Thisupstreamsequenceincludestwoadditionalin-framecodonsformethionine,eachinamoderatecontextforribosomebinding（21）;theupstreamATGcodonsaredenotedATG1andATG2,whereasthepublishedtranslationstartisshownasATG3.RT-PCRusingsenseprimerslocatedoverATG1orATG2andanti-senseprimerscrossingtheexon1-2boundaryby4nucleotidesdetectedtranscriptscapableofencodingtheseN-terminalextensionsinhumanandmousecells.TranscriptsinitiatingupstreamofATG1inmouseandATG2(butnotATG1)inhumanmRNAwereeasilydetected(Fig.1C),suggestingtheutilizationofanup-streamtranscriptionst;siteencodinganN-terminalextension.这个上游序列包括两个额外的甲硫氨酸编码框密码子，每个都在序列中间于核糖体结合(21)；上游ATG密码子表示ATG1和ATG2，而已发布的转录起始是指ATG3。人类和小鼠细胞中，RT-PCR使用上游引物位于ATG1或ATG2,下游引物横跨外显子1-2边界线通过4个核苷酸检测编码这些N末端的延长的转录能力。mRNA转录起始上游小鼠中ATG1和人类中ATG2(不是ATG1)是容易测定的。MouseorhumanDNMT1isoformscontainingthisadditionalN-terminalsequencewerepredictedbyMitoProtII(http://ihg.gsf.de/ihg/mitoprotbettargetedt(针对)themitochondriawithveryhighprobability,comparedwithproteinsbeginningatthepublishedstartcodon,ATG3(TableS1).Thegenomedatabasesalsocontainupstreamsequencesforchimpanzee,rat,andcowDNMT1;ineacfeciespneormorein-framepotentialstartcodonsencodeapeptidewithastrongprobability(>90%)ofmitochondriallocalization(TableS1).」arepredictedtoformamphiphidhelices,althoughquenceconservationbetweenthem;low,asisoftenthecase(情况常常如此，这是常有的事)formiochondrialleaderpeptidesacrossspecies.通过MitoProtH预测小鼠和人类DNMT1亚型包括这个额外的N末端的序列(22)针对线粒体具有很高的可能性，与从公布的起始密码子ATG3(TableS1开始蛋白相比较。基因组数据库也包括黑猩猩，大鼠和牛DNMT1上游序列；在每个物种中，一个或者更多编码框内潜在的起始密码子编码一个具有定位线粒体很大可能性(>90%)的多肽(TableS1)。预测所有形成两性分子叫』螺旋，尽管他们之间的序列保守性很低，线粒体引导肽穿越物种这是常有的事。Immunoblotsofpurifieditochondriafrommouseembryonicfibroblas(MEFs)andHCT116humancoloncarcinomacellsshowedthepresenceofDNMT1(Fig.2A)butnotDNMT3aorDNMT3binthisorganelle(Fig.2B).Full-lengthDNMT1andasmallerpeptidearedetectedbyanN-terminalDNMT1antibody,suggestingthatproteolytipcrocessingoccursuponentryintothemitochondria.AbsencofthenucleaimarkerH3K4me3inthanitochondrialfractionindicatedpurityfromcontaminationbynuclearmaterial,theprimarysiteoflocalizatioofDNAmethyltransferases.来自小鼠胚胎成纤维细胞(MEFs)和人结肠癌细胞HCT116纯化线粒体的免疫印迹分析显示在这个细胞器中存在DNMT1(Fig.2A)，不存在DNMT3a或DNMT3b(Fig.2B。用一个N末端DNMT1抗体检测全长DNMT1和较小的多肽，表明蛋白酶加工发生在进入线粒体后。线粒体断片中缺少细胞核标志H3K4me3显示纯化被核物质污染，定位DNA甲基化转移酶的主要位点。Weclonedthemouseanchumanleadersequences,fromATG1toostreamofATG3,in-framewiththeC-terminalGFPtagofpcDNA6.2/GFP,andtransfectectheplasmidsintoNIH/3T3fibroblasts.ConfocalmicroscopyshowedthatbothhumanandmouseleadersequencestargetedGFPtothemitochondria,indicatedbycolocalizationofMitoTrackerRedwithgreenfluorescence(Fig2C).Mitochondriainuntransfectedellwithinthesamevisuafieldemainedredinthemergedphotomicrographserving(作为，充当，担任)negativecontrolsforcolocalization,whereasachloramphenicolacetyltransferase(CAT)-GFPcontrolplasmidremainedcytosolic.WealsotransfectedheseconstructsLntoHCT116humancoloncarcinomacellsforimmunoblotanalysisofpurifiedmitochondriausinganti-GFPantibody(Fig.S1).我们克隆小鼠和人类的前导序列，从ATG1到ATG3的上游，编码框内具有C末端具有GFP标签质粒pcDNA6.2/GFP，转染到NIH/3T3成纤维细胞。共聚焦显微镜表明人和小鼠两者的前导序列将GFP定位到线粒体，通过MitoTrackerRed和绿色荧光共定位体现(Fig.2C)。未转染的细胞内的线粒体在合并的显微照片相同视野内保持红色，作为共定位的阴性对照，然而氯霉素乙酰转移酶(CAT)-GFP控制质粒保持细胞质基质。我们也转染这些结构到人结肠癌细胞HCT116用抗GFP抗体免疫印迹分析纯化的线粒体(Fig.S1)。HCT116mitochondriaclearlyaccumulatedGFP.Thus,humanandmouseleaderpeptidesrepresentbonafideMTSscapableoftrackingheterologousproteinstothisorganelle.EachMTSisabletooperateacrossspecies,indicatingfunctionalconservation.HCT116线粒体清晰地积累GFPo因此，人类和小鼠引导肽体现这些细胞器MTSs法真实的跟踪外源性蛋白检测能力。每一个MTS能够穿越物种操作，表明功能保守。mtDNMTlExpressionIsRegulatedbyFactorsThatRespondtoOxidativeStress.MatInspector(http://www.genomatix.de/en/index.html)predictedabindingsiteforNRF1inbothhumanandmouseDNMT1.Thisconsensussequencewaslocated(位于)overoneoftheupstreamin-framestartcodonsandwasconservedinallothermammalianspeciesstudied(TableS1;Fig.3A).Underconditionsofoxidativestress,thecoactivatorPGClaactivatesandinteractswithNRF1toup-regulatemultiplenuclear-encodedmitochondrialgenes(17).Accordingly,wetransientlytransfectedNRF1,PGC1a,orbothtogetherintoHCT116cellsandanalyzedthelevelsofmitochondrialDNMT1(mtDNMT1)byimmunoblot(Fig.3B).对氧化压力反应的因子调节mtDNMT1表达MatInspector在人类和小鼠的DNMT1中预测了NRF1一个结合位点。这个共有序列位于越过上游编码框起始密码子之一，在研究的所有其他哺乳类物种中是保守的(TableS1；Fig.3A)。在氧化压力的条件下，共激活剂PGC1a激活并且与NRF1相互作用去上调多个核编码的线粒体基因(17)o因此，我们瞬时转染NRF1，PGC1a或者两者一起转染到HCT116细胞，通过免疫印迹分析线粒体DNMT1的水平(Fig.3B)。AsmallincreaseinmtDNMT1wasseenincellstransfectedwithNRF1orPGClaalone,whereascotransfectionwithbothPGC1aandNRF1resultedinanapproximatelyfivefoldincreaseinmtDNMT1relativetocontrol.Thus,thislocusissensitivetoregulationbyactivatorsthatrespondtooxidativestress.在单独转染NRF1或者PGC1a的细胞中看到mtDNMT1有一个很小的增加，然而两者共转染导致相对对照组mtDNMT1有一个大约5倍的增加。因此，这个位点对于氧化压力反应的调节子的调节是敏感的。TheNRF1bindingsiteiscoincidentwithap53consensusbindingsite(Fig.1AandB),whichwepreviouslydemonstratedto(向说明，向证明)repressDNMT1transcription(23).Ourearlierstudyshowedathree-tosixfoldincreaseinDNMT1transcriptionfollowingeitheractivationorgeneticdeletionofp53inHCT116cellsandMEFs.Becausep53isknowntoregulatemitochondrialrespiration(24),weaskedwhetherthistumorsuppressorproteinalsoaffectedmtDNMT1mRNAexpression.NRF1结合位点和P53共识结合位点是一致的(Fig.1A和B)，这个我们之前已经证明抑制DNMT1转录(23)。我们之前的研究随着在HCT116细胞和MEFs细胞中P53的激活或者基因缺失DNMT1转录显示了一个3-6倍的增加。因为P53是人们所周知调节线粒体呼吸作用(24)，我们思考这个肿瘤抑制蛋白是否也影响mtDNMT1mRNA表达。WeusedRT-quantitative(q)PCRwithprimersthatdistinguishthemitochondrialtranscriptfromthetotalDNMT1transcript(Fig.1C);themitochondrialtranscriptcomprised1-2%ofthetotalDNMT1synthesizedinlog-phaseMEFsorHCT116cells.TherelativeabundanceofmtDNMTltranscriptincreasedsixfoldinp53-/-MEFscomparedwithWTMEFs,whereastotalDNMT1mRNAincreasedthreefold(Fig.3C),suggestingapreferentialup-regulationofthemitochondrialtranscriptincellslackingp53.ImmunoblotanalysisoftheseisogeniccellsshowedastrikingincreaseinmtDNMT1proteinwithlossofp53(Fig.3D).我们用从总的DNMT1转录物中区分线粒体的转录物的引物，进行RT-qPCR(Fig.1C)；线粒体转录占对数阶段MEFs或HCT116细胞总合成DNMT1的1-2%。P53-/-MEFs与野生型MEFs细胞相比mtDNMT1转录物的相对丰都增加了6倍，然而总的DNMT1mRNA增加了3倍(Fig.3C)，表明在缺少P53的细胞中线粒体转录物的一个优先上调。这些等基因细胞的免疫印迹分析在缺乏P53细胞中mtDNMT1蛋白显示了一个显著的增加(Fig.3D)。Gene-SpecificChangesinMitochondrialTranscription.WeaskedwhetherthismtDNMT1overexpressionwasreflectedinanalterationintranscriptionofthemitochondrialgenome(Fig.3E).NADHdehydrogenasesubunit6(ND6),theonlyprotein-codinggeneonthelight(L)strand,wassignificantlyunderexpressedinresponsetoincreasedmtDNMT1,suggestingaroleformtDNAmethylationinrepressionofL-strandtranscription.Ontheheavy(H)strand,ATPasesubunit6(ATP6)andcytochromecoxidasesubunit1(COX1)wereunalteredintheirexpressionlevels.However,NADHdehydrogenasesubunit1(ND1),thefirstH-strandprotein-codingregionfollowingtheribosomalRNAgenes,wassignificantlyincreasedinresponsetoelevatedmtDNMT1.Thesedatasupportagene-specificeffectonmitochondrialgenetranscription,asdiscussedbelow.线粒体转录中独特基因的改变我们询问mtDNMT1过表达是否反应线粒体基因组转录的改变(Fig.3E)。NADH脱氢酶亚基6(ND6)，轻链上唯一编码蛋白的基因，在mtDNMT1增加上显著地低表达，表明mtDNA甲基化在抑制轻链转录中的一个作用。在重链上，ATPase亚基6(ATP6)和细胞色素酶C氧化酶亚基1(COX1)在他们表达水平上是不变的。然而，NADH脱氢酶亚基1(ND1)，第一条轻链编码蛋白区域跟随核糖体rRNA基因，在反应提高的mtDNMT1中是显著增加的。这些数据支持了一个独特基因影响线粒体基因转录，正如下面讨论的。MitochondrialDNMT1IsBoundtomtDNA.WecreatedanHCT116cellline(25)inwhichoneendogenousalleleofDNMT1carriesaC-terminaltandem-affinitypurification(TAP)tag(26).TAP-taggedDNMT1translocatedefficientlytomitochondria(Fig.4B).WethereforeusedthesecellstoaskwhethermtDNMT1interacteddirectlywithmtDNA.Formaldehyde-crosslinkedmitochondriallysateswereimmunoprecipitatedwithIgGbeads(26),andqPCRwithprimersspecificformtDNA(TableS2)wasusedtoquantitatetheinteractionbetweenmtDNMT1andmtDNA.ImmunoprecipitatesfromTAP-taggedcellsweresubstantiallyenrichedformtDNAincomparisonwithimmunoprecipitatesfromuntaggedcells,exceptforanampliconcontainingnoCpGdinucleotides,whichgaveequallylowsignalfrombothcelllines(Fig.4C).ThesedatasuggestCpG-dependentinteractionofmtDNMT1withthemitochondrialgenomeandconfirmthelocalizationofthisproteintothemitochondrialmatrix.线粒体DNMT1结合到mtDNA我们建立了一个HCT116细胞系(25)，在这个细胞系中一个内源性DNMT1等位基因携带一个C末端串联亲和纯化(TAP)标签(26)°TAP标签的DNMT1高效地转移进入线粒体(Fig.4B)。因此我们用这些细胞去询问mtDNMT1是否与mtDNA直接相互作用。甲醛交联线粒体溶菌产物用IgG小珠子免疫沉淀(26)，用mtDNA专门的引物进行qPCR(TableS2)去定量mtDNMT1和mtDNA间的相互作用。TAP标签细胞进行免疫共沉淀相比没有标签的细胞的免疫共沉淀与mtDNA大量的富集，除了不包含CpG二核苷酸的扩增子，两个细胞系都给了相等的低信号(Fig.4C)。这些数据显示mtDNMTl和线粒体基因组相互作用依赖CpG，证实了这个蛋白定位到线粒体基质中。InteractionwasevidentintheD-loopcontrolregion,whichcarriesthemitochondrialoriginofreplicationandpromoters,aswellasinrRNAandprotein-codingregions.Thelevelofenrichmentwasdependentonthetargetamplicon;fiveofthesixregionsprobedshowedathree-tofivefoldenrichmentofmtDNAsequences.However,qPCRoftheregioncoveringthejunctionbetween12Sand16SrRNAgenes(primer2)showedonlytwofoldenrichmentinbindingofmtDNMT1-TAP.ThedensityofCpGdinucleotidesinthisampliconis<50%thatinallotherampliconsanalyzed,suggestingthatinteractionofmtDNMT1withmtDNAisproportionaltoCpGdensity,andsupportingafunctionalroleformtDNMT1inestablishmentandmaintenanceofmtDNAmethylation.在D环控制区域相互作用是明显的，这个区域带有线粒体复制原点和启动子，和rRNA中编码蛋白质区域一样。富集水平依赖靶扩增子；6个区域中探测了5个，显示mtDNA序列3-5倍的富集。然而，这个区域的qPCR引物2覆盖12S和16SrRNA基因之间的连接，在结合mtDNMT1-TAP中显示仅2倍富集。这个扩增子中CpG二核苷酸密度V50%，这个也在所有其他扩增子进行了分析，表明mtDNMT1和mtDNA间的相互作用与CpG密度成比例的，支持了mtDNMT1在建立和维持mtDNA甲基化中的功能作用。5-HydroxymethylcytosineIsPresentinmtDNA.WeimmunoprecipitatedrandomlyshearedmtDNAwithanantibodyto5mCor5hmCandprobedtheprecipitatedDNAbyqPCRtodeterminethepresenceandrelativeabundanceofthesetwomodifiedbasesinmtDNA(Fig.5AandB).Immunoprecipitatedsampleswereenriched10-to20-foldfor5mCrelativetoIgGcontrolforallregionstested.mtDNAimmunoprecipitatedusinganti-5hmCwashighlyenriched(85-to580-fold)relativetoIgGcontrols,exceptacrosstheDloop(primer27),whichwasenriched38-fold.ThespecificityofeachantibodyforitsrespectivemodificationwasconfirmedusingcontrolDNAinwhicheverycytosinewasconvertedtoeither5mCor5hmC(Fig.S2).ThepresenceofbothcytosinemodificationsinmtDNAsuggeststhatearlierstudiesunderestimatedthedegreeofepigeneticmodificationofthemitochondrialgenome.mtDNA中存在5羟甲基化。我们用5mC或者5hmC的抗体免疫共沉淀随机地修剪mtDNA，通过qPCR探测沉淀的DNA去确定mtDNA中这两个修饰碱基的存在和相对丰度(Fig.5A和5B)。免疫共沉淀的样品富集5mC相对所有区域检测的IgG对照10-20倍。mtDNA免疫共沉淀使用抗5hmC相对IgG对照是高度富集(85-580倍)的，除了穿过D环(引物27)，他富集了38倍。针对他们各自的修饰每个抗体的特异性用对照DNA确定，对照DNA中每个胞嘧啶转换为5mC或5hmC(Fig.S2)。mtDNA中两个胞嘧啶修饰的存在表明早期的研究低估了线粒体基因组表观修饰的程度。WeusedphageT45hmC-6-glucosyltransferase(6-gt)(27)todeterminethepresenceof5hmCatGla1restrictionendonucleasecleavagesites(28).ControlexperimentsusingdefinedDNAsequencescontainingcytosine,5mC,or5hmCconfirmedthatGla1cleavedonlysitesmodifiedbymethylationorhydroxymethylation,butnotsitescontainingglucosylated5hmC(Fig.S3A).我们用T4噬菌体5hmC-p-葡糖基转移酶(8-gt)(27)去确定在Gla1限制性内切酶切割位点5hmC的存在(28)。对照试验用确定了的DNA序列包括胞嘧啶，5mC或5hmC证明甲基化或羟甲基化修饰的Gla1唯一的切割位点，但是不包括葡糖基修饰的5hmC位点(Fig.S3A)。ProtectionofmtDNAfromcleavageby5hmCglucosylationwasassessedbyendpoint(Fig.S3BandC)andqPCR(Fig.5C).5hmCwaspresentinthreedifferentampliconsfromhumanmtDNAandtwoampliconsfrommousemtDNAorgenomicDNA.AmpliconscontainingtwoGla1restrictionsiteseach(ampliconsATP6,12S,and16S-3)showed50%protectionincomparisonwithampliconswithasingleGla1site(amplicons2and16S-2),suggestingasimilarlevelof5hmCatallrestrictionsitestested.Amouseamplicondevoidof(没有，缺乏)Gla1sites(ATP6/COX3)wasprotectedfromcleavageirrespectiveof5hmCglucosylation(Fig.S3C).保护5hmC葡糖基化的mtDNA不被切割，通过末端(Fig.S3B和C)和qPCR(Fig.5C)进行评估。5hmC存在与三个不同扩增子中，人类mtDNA和两个小鼠mtDNA或基因组DNA扩增子。扩增子包括两个Gla1限制性位点与带有一个Gla1位点的扩增子(扩增子2和16S-2)相比较每个(扩增子ATP6，12S和16S-3)显示50%保护，表明在检测的所有限制性位点上5hmC具有一个相似的水平。一个小鼠扩增子缺乏Gla1位点(ATP6/COX3)不论5hmC是否葡糖基化都保护不被切割(Fig.S3C)。DiscussionCytosinemethylationofthemitochondrialgenomehasremainedlargelyoverlooked,inpartbecauseearlyreportsusingnearest-neighboranalysisindicatedthatthismodificationwaspresentatonly25%ofCpGdinucleotides(11),wellbelowthelevelofmethylationseeninthenucleus.Thedatapresentedhereshowa10-to20-foldenrichmentofmtDNAsequencesinimmunoprecipitatesusing5mCantibody,somewhatlowerthanthatusuallyobtainedfromgenomicDNA(100-foldforCpGislands).讨论线粒体基因组的胞嘧啶甲基化仍然保持一个很大的空缺，部分是因为早期的报道使用最近邻顺序分析显示这种修饰在CpG二核苷酸仅有2-5%(11)，细胞核中也甲基化水平也很低。本文这些数据呈现了用5mC抗体的免疫共沉淀中mtDNA序列一个10-20倍的富集，通常比基因组DNA(是CpG岛的~100倍)获得的富集的稍微低一些。ThislikelyreflectstheCpG-sparsenatureofthemitochondrialgenome,whichdoesnotcontainCpGislands.Wedemonstrateherethepresenceof5hmCinmtDNAusingtwoindependentassays.Thus,epigeneticmodificationofcytosinesinthemitochondrialgenomeislikelymuchmorefrequentthanpreviouslybelieved.Inthenucleus,5hmCisgeneratedfrom5mCbytheactionoftheTETfamilyofmethylcytosineoxygenases(6).Thereisnotyetevidenceregardingthepresenceorabsenceoftheseenzymesinmitochondria,andtheTETfamilyproteinsorlocidonotcontainrecognizablemitochondrialtargetingsequences(14).Wethereforecannotruleou(排除，取消)thepossibilityofadifferentmechanismforthegenerationof5hmC,includingcovalentadditionof5-hydroxymethylgroupsdirectlytoDNAcytosineresiduesbymtDNMT1(29)usingformaldehydegeneratedfrommitochondrialmixed-functionoxidases.这个很可能反应自然线粒体基因组中CpG稀少，它不包含CpG岛。这里我们用两个独立的实验证明5hmC在mtDNA中存在。因此，线粒体基因组中胞嘧啶的表观修饰很可能比我们之前认为的更加频繁。在细胞核中，5hmC是由5mC通过TET家族的甲基胞嘧啶加氧酶作用产生的(6)。这里还没有证明这些酶在线粒体中的存在或缺失，TET家族蛋白或基因座不包含可认识的线粒体的目标序列(14)。因此我们不能排除5hmC产生的一个不同机制的可能性，包括mtDNMT1用线粒体多功能氧化酶产生的甲醛将5羟甲基集团直接共价增加到DNA胞嘧啶残基上(29)。Theapparentlylowerenrichmentfor5hmCintheD-loopcontrolregionmostlikelyreflectsthelessefficientamplificationofalongerfragment(833bpcomparedwith112-238bp)frommtDNAshearedtoanaveragesizeof300-400bp.Howevei,theDloopexistsasastabletriple-helicalstructurecontaininganRNAprimerrequiredforinitiationofmtDNAreplication(13),andwehavefoundthisregiontoberesistanttoinvitromethylationbyM.Ssslcytosinemethyltransferase.Itisthereforepossiblethatthekineticsofepigeneticmodificationinthisregionofthemitochondrialgenomemightbedifferentfromthoseincodingregions.在D环对照区域有一个明显较低5hmC富集最可能反应mtDNA较长片段(833bp相比较112-238bp)修剪为平均长度300-400bp扩增效率较低。然而，D环的存在作为一个稳定的三螺旋结构包括mtDNA复制起始所需要的一个RNA引物(13)，我们发现这个区域对于通过M.Sss1胞嘧啶甲基转移酶体外甲基化是有抵抗的。因此，线粒体基因组的这个区域的表观修饰动力学可能不同于编码区的动力学。Thefunctionof5hmCinthenucleargenomeisnotyetclear.Ithasbeenproposedthat5hmCisanintermediatemetaboliteinactivedemethylationofthegenomebyrepairenzymes(30),inpassivedemethylationasaresultoflackofrecognitionbyenzymesinvolvedinmaintenancemethylation(31)句型,orthatitalterslocalchromatinstructurebecause5hmCisnotrecognizedby5-methylcytosine-bindingproteins(7).Theroleof5hmCinthemitochondrialgenomelikelyinvolvesoneormoreoftheseprocesses.Althoughquantitativemeasurementsoftherelativeabundanceof5hmCand5mCcanbeachievedusingmethylatedDNAimmunoprecipitation(Me-DIP),5hMe-DIF,HPLC,orenzymaticmethods,mappingthelocationanddistributionof5hmCineitherthenuclearormitochondrialgenomeisnotyettechnicallyfeasible,becausethismodifiedbaseisindistinguishablefrom5mCbybisulfitemodification(7).核基因组中的5hmC的功能还没有很清楚。提出5hmC是通过修复酶对基因组的主动去甲基化一个中间代谢物(30)，在被动去甲基化中作为多维持甲基化酶缺乏认识的一个结果(31)，或者5hmC改变当地染色体结构因为5hmC是还没有通过5甲基胞嘧啶结合蛋白认识(7)。5hmC在线粒体基因组中的作用很可能涉及这些过程的一个或者多个。尽管5hmC和5mC的相对丰都的定量测量能够通过使用甲基化的DNA免疫共沉淀(Me-DIP)，5hMe-DIP，HPLC，或酶催化的方法获得，绘制5hmC在细胞核或者线粒体基因组中的位置和分布还没有技术上的可行性，因为这些修饰碱基通过亚硫酸氢盐与5mC不能区分的(7)。ThisstudyreportsamitochondrialisoformofDNAmethyltransferase1,whichistheonlymemberofthecatalyticallyactivemammalianDNAmethyltransferasefamilyfoundinthisorganelle.TheconservationofanORFencodingamitochondrialtargetingsequenceupstreamofthecommonlyacceptedtranslationalstartcodonacrossmultiplemammalianspeciessuggestsanimportantroleforthisenzymeinmitochondrialfunction.这个研究报道一个线粒体DNA甲基转移酶1的亚型，它是在这个细胞器中发现的哺乳动物DNA甲基转移酶家族唯一具有催化活性的。保守的一个开放阅读框编码的一个线粒体靶序列普遍公认的转录起始密码子上游穿过多种哺乳动物物种显示这个酶在线粒体功能中的一个重要作用。AlthoughDNMT1isgenerallyconsideredtobe(认为是)themaintenanceDNAmethyltransferase,itisabletomethylatecompletelyunmethylatedDNAinvitrowithanefficiencythatexceedsthatofthedenovomethyltransferasesDNMT3aand-3b(32).Thus,DNMT1appearstobecapableofbothinitiatingandmaintainingcytosinemethylationinthenucleus,andthelackofdenovomethyltransferasesinmitochondriaimplicatesmtDNMT1inbothprocessesinthisorganelle.尽管DNMT1普遍被认为是维持DNA的甲基转移酶，他能在体外甲基化完全未甲基化的DNA效率超过了从头甲基转移酶DNMT3a和-3b的效率（32）。因此，DNMT1似乎在细胞核中具有从头和维持胞嘧啶甲基化两种能力。线粒体中缺乏从头甲基转移酶暗示DNMT1在这个细胞器中具有这两个过程。WeshowthatmtDNMTlbindstothemitochondrialgenomeinamanner（在某种意义上，在某种程度上）proportionaltothedensityofCpGdinucleotides.OfparticularrelevanceisthebindingofmtDNMT1totheD-loopcontrolregion,whichcarriesthepromotersdrivingtranscriptioninitiationofbothheavyandlightstrands,supportingaroleformtDNMT1inregulationofmitochondrialgeneexpression.Theasymmetric,gene-specificalterationinmitochondrialtranscriptionpatternsshownheresuggestsdiverserolesformtDNMT1andcytosinemodificationinthisorganelle.我们显示mtDNMT1结合线粒体基因组在某种程度上与CpG二核苷酸密度成比例。特别相关的是mtDNMT1与D环对照区结合，D环对照区携带驱动重链和轻链两者转录起始的启动子，支持mtDNMT1在调节线粒体基因表达中的功能。不对称地，本文表明在线粒体转录模式中独特基因的改变，暗示在这个细胞器中mtDNMT和胞嘧啶修饰具有很多作用。DecreasedexpressionofND6ontheLstrandimpliesthatcytosinemethylationinmtDNArepressesgeneexpressionfromthelight-strandpromoter,asitdoesinthenucleus.However,increasedtranscriptionofND1withnochangeintranscriptionofATP6orCOX1raisesthepossibilityofadifferentmodeofactionontheHstrand.Abindingsiteformitochondrialterminatorfactor1（MTERF1）islocatedbetweentheendofthe16SrRNAgeneandthetranslationstartofND1（33）.MTERF1bindstobothH-strandpromoter1（HSP1）andtheterminatorbindingsite（Fig.4A）,formingatranscriptionloopthatmaintainshigh-levelproductionofrRNA.TranscriptsinitiatingatHSP2producepolycistronicmessagesencodingtheentireHstrand（13）.L链ND6表达减少表明mtDNA胞嘧啶甲基化抑制从轻链启动子开始的基因表达，和在细胞核中的一样。然而，ND1转录的增加没有改变ATP6和COX1的转录增加重链上不同作用模式的可能性。线粒体终止子因子（MTERF1）结合位点位于16SrRNA基因末尾和ND1转录起始之间（33）。MTERF1结合到H链启动子1（HSP1）和终止子结合位点上（Fig.4A），形成一个转录环，它维持rRNA高水平产物。在HSP2转录起始产生多顺反子的信息编码整个H链（13）。OurdataraisethepossibilitythatmtDNMT1,eitherthroughmodificationofCpGdinucleotidesorbydirectprotein-proteininteraction,interfereswithMTERF-dependenttranscriptiontermination,allowingread-throughfromHSP1tothenexttranscriptionalunit（ND1）withoutimpactingpolycistronicmRNAsynthesisfromHSP2.我们的数据提高了mtDNMT1的可能性，不管是通过CpG二核苷酸的修饰还是通过蛋白和蛋白质间的直接相互作用，阻碍了依赖MTERF转录的终止，允许从HSP1到下一个转录单元（ND1）的通读不影响从HSP2多顺反子的mRNA合成。WeshowherethatDNMT1ispresentinthemitochondrialmatrix,boundtomtDNA,andmodifiestranscriptionofthemitochondrialgenomeinwhatappearstobeagene-specificfashion.Wereportthepresenceofboth5hmCand5mCinmtDNA,suggestingthatearlierstudiesmayhaveunderestimatedtheproportionofmodifiedcytosinesinthisgenome.Hence,mtDNMT1appearstoberesponsiblefor（是。。的原因）theestablishmentandmaintenanceofcytosinemethylationinmtDNA,fromwhich5hmCispresumablyderived.Ourdatasupportaroleforepigeneticmodificationofthemitochondrialgenomeinregulationofmitochondrialtranscription.这里我们表明DNMT1存在于线粒体基质中，结合mtDNA,修饰线粒体基因组的转录，这个似乎独特的基因方式是什么。我们报道了mtDNA中存在5mC和5hmC，表明之前的研究很可能低估了胞嘧啶修饰在这基因组中的比例。因此，mtDNMTl似乎是在mtDNA中建立和维持胞嘧啶甲基化的原因，这很可能是由5hmC衍生来的。我们的数据支持了线粒体基因组的表观修饰在调节线粒体转录中的作用。MaterialsandMethods材料和方法CellLines.HCT116p53+/+andHCT116p53-/-wereobtainedfromBertVogelstein,JohnsHopkinsUniversity(Baltimore,MD).PrimaryMEFswerepreparedfromE12.5-E13.5embryos.细胞系。HCT116p53+/+和P53-/-从JohnsHopkinsUniversity大学的BertVogelstein博士获得(Baltimore,MD)。主要的MEFs细胞来自E12.5-E13.5胚胎。PlasmidsandTransfections.PrimersusedarelistedinTableS2.Mitochondrialtargetingsequenceswereamplifiedfromrandom-primedhumanandmousecDNAs.MurineNRF1cDNAwasobtainedfromtheAmericanTypeCultureCollectionandreclonedintopDEST26/C-FLAG.PGClaplasmidwasagiftfromGregorioGil,VirginiaCommonwealthUniversity.CellsweretransfectedusingPolyjetliposomes(ProSci)(HCT116)ornucleofection(Amaxa)(MEFandNIH/3T3)accordingtothemanufacturers'specifications,andwereharvested48haftertransfection.质粒和转染。使用表S2中列举的引物。使用随机引物扩增小鼠和人类cDNAs线粒体目标序列。小鼠NRF1cDNA获得于美国标准菌库然后重新克隆到pDEST26/C-FLAG0PGCla质粒由VirginiaCommonwealthUniversity大学的GregorioGil馈赠。使用Polyjet脂质体(ProSci)(HCT116)或核转染(Amaxa)(MEFandNIH/3T3)按照制造商说明书转染细胞，转染48h后收获。MitochondrialPurificationandImmunoblotAnalysis.Mitochondriawerepurifiedbydouncehomogenizationanddifferentialcentrifugationinthepresenceofcompleteproteas