在哺乳動(dòng)物線粒體中DNA甲基化轉(zhuǎn)移酶1,胞嘧啶甲基化,和胞嘧啶羥甲基化

哺乳動(dòng)物線粒體中DNA甲基化轉(zhuǎn)移酶1,胞嘧啶甲基化和胞嘧啶羥甲基化MitochondrialDNA(mtDNA)hasbeenreportedtocontain5-methylcytosine(5mC)atCpGdinucleotides,asinthenucleargenome,butneitherthemechanismgeneratingmtDNAmethylationnoritsfunctionalsignificanceisknown.Wenowreportthepresenceof5-hydroxymethylcytosine(5hmC)aswellas5mCinmammalianmtDNA,suggestingthatpreviousstudiesunderestimatedthelevelofcytosinemodificationinthisgenome.DNAmethyltransferase1(DNMT1)translocatestothemitochondria,drivenbyamitochondrialtargetingsequencelocatedimmediatelyupstreamofthecommonlyacceptedtranslationalstartsite.線粒體DNA(mtDNA)已經(jīng)報(bào)道在核基因組中CpG二核苷酸上包含5甲基化胞嘧啶(5mC),但是mtDNA甲基化產(chǎn)生的機(jī)制和它功能的重要性都還不知道。我們現(xiàn)在報(bào)道5hmC5羥甲基化胞嘧啶和5mC一樣存在于哺乳動(dòng)物mtDNA中，表明之前的研究低估了胞嘧啶修飾在基因組中的水平。DNA甲基化轉(zhuǎn)移酶1(DNMT1)轉(zhuǎn)移到線粒體，被一個(gè)線粒體靶序列？驅(qū)動(dòng)立刻定位到大家公認(rèn)的轉(zhuǎn)錄起始位點(diǎn)？的上游。Thistargetingsequenceisconservedacrossmammals,andtheencodedpeptidedirectsaheterologousproteintothemitochondria.DNMT1istheonlymemberofthethreeknowncatalyticallyactiveDNAmethyltransferasestargetedtothemitochondrion.MitochondrialDNMT1(mtDNMT1)bindstomtDNA,provingthepresenceofmtDNMT1inthemitochondrialmatrix.mtDNMT1expressionisup-regulatedbyNRF1andPGCla,transcriptionfactorsthatactivateexpressionofnuclear-encodedmitochondrialgenesinresponseto(響應(yīng)，反應(yīng))hypoxia,andbylosso(失去)p53,atumorsuppressorknowntoregulatemitochondrialmetabolism.AlteredmtDNMT1expressionasymmetricallyaffectsexpressionoftranscriptsfromtheheavyandlightstrandsofmtDNA.Hence,mtDNMT1appearstobe(好像是，仿佛)responsiblefor(是的原因，為負(fù)責(zé))mtDNAcytosinemethylation,fromwhich5hmCispresumedtobederived,anditsexpressioniscontrolledbyfactorsthatregulatemitochondrialfunction.這個(gè)靶序列在哺乳動(dòng)物中是保守的，編碼的多肽引導(dǎo)一個(gè)外源蛋白到線粒體上。DNMT1是三個(gè)已知催化活性的DNA甲基化轉(zhuǎn)移酶中唯個(gè)針對(duì)線粒體的。線粒體DNMT1(mtDNMT1)結(jié)合mtDNA，證明線粒體基質(zhì)中存在mtDNMT1。NRF1和PGC1a上調(diào)mtDNMT1的表達(dá)，轉(zhuǎn)錄因子激活核編碼線粒體基因的表達(dá)是對(duì)低氧的反應(yīng)，失去P53,一個(gè)腫瘤抑制因子已知可調(diào)節(jié)線粒體新陳代謝。改變mtDNMT1表達(dá)不對(duì)稱(chēng)地影響mtDNA重鏈和輕鏈的轉(zhuǎn)錄表達(dá)。因此，mtDNMT1好像是mtDNA胞嘧啶甲基化的原因，推測(cè)它衍生出5hmC，調(diào)節(jié)線粒體功能的因子控制mtDNMT1的表達(dá)。正文Inthenucleus,cytosinemethylationcooperateswith(合作，協(xié)作)N-terminalhistonemodificationstoestablishasilencedchromatinstructure(1),thus(從而)regulatingnucleargeneexpression.MethylationpatternsareestablishedinthedevelopingembryobytwodenovoDNAmethyltransferases,DNMT3aand-3b(2).Maintenanceofthispatterninsomaticcellsisbelievedtobe(被認(rèn)為是)thepredominantfunctionofDNMT1,withfunctionalcooperationevidentbetweenthetwogroupsofenzymes(3).Cytosinemethylationisessentialfor(是。。必需的)normaldevelopment,anddeletionof(缺失，刪除)DNMT1resultsinembryoniclethalityinmiceandmitoticcatastropheinculturedcells(4).在細(xì)胞核中，胞嘧啶甲基化與N末端組蛋白修飾共同協(xié)作去建立一個(gè)沉默的染色體結(jié)構(gòu)，從而調(diào)節(jié)和基因的表達(dá)。在發(fā)育的胚胎中通過(guò)兩種從頭DNA甲基化轉(zhuǎn)移酶（DNMT3a，3b）建立甲基化模式。人們認(rèn)為在體細(xì)胞中維持這種模式是DNMT1的主要功能，這兩組酶間具有明顯的功能合作。胞嘧啶甲基化是正常發(fā)育必需的，缺乏DNMT1導(dǎo)致小鼠胚胎致死和培養(yǎng)細(xì)胞有絲分裂障礙。Recently,thepresenceofsignificantlevelsof5-hydroxymethyl-cytosine(5hmC)wasdemonstratedinDNAfromneurons,brain(5),andembryonicstemcells(6).5hmCisderivedfrom5-methylcytosine(5mC)oxidationcatalyzedbytheTETfamilyofmethylcytosineoxygenases,anditsfunctionalsignificanceisunderintenseinvestigation.Thismodificationislikelyto(有可能)haveanimpacto(對(duì)于有影響)localchromatinstructure,andithasbeenproposedthat(人們已經(jīng)提議)5hmCactsa(充當(dāng)，擔(dān)任)anintermediateinactiveorpassivedemethylation(7).最近，已經(jīng)證明在神經(jīng)、腦和胚胎干細(xì)胞DNA中存在顯著水平的5hmC。5hmC來(lái)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積極調(diào)查的。這種修飾很可能對(duì)局部的染色體結(jié)構(gòu)有影響，人們已經(jīng)提議5hmC充當(dāng)了在主動(dòng)去甲基化和被動(dòng)去甲基化的中間物。CytosinemethylationofmitochondrialDNA(mtDNA)hasbeencontroversialand,remarkably,infrequentlystudied.Theearlieststudy,conductedoverthreedecadesago,reportedthattherewasnomethylationofmtDNA(8).Subsequently,lowlevelsofmethylationrestrictedtoCpGdinucleotideswerereportedinmitochondriaofseveralspecies,usingmethylation-sensitiverestrictionendonucleasecleavageandnearest-neighboranalysis(911).引人注目的是，線粒體胞嘧啶甲基化(mtDNA)是引起爭(zhēng)論和很少被研究的。大約30年前，最早的研究中報(bào)道m(xù)tDNA沒(méi)有甲基化(8)。隨后，使用甲基化敏感限制性核酸內(nèi)切酶分裂和進(jìn)行最近鄰分析，在幾個(gè)物種的線粒體中報(bào)道低水平甲基化限制在CpG二核苷酸(9-10)。MammalianmtDNAshowsasimilarlevelofCpGsuppressiontothatofnuclearDNA(12),suggestingthat5mCissusceptibleto(對(duì)敏感，易受影響)mutationinmtDNA.Todate(迄今為止),5mCistheonlymodifiedbase(修飾堿基)describedinmtDNA,butthemechanismsestablishingandmaintainingmtDNAmethylation,andthefunctionalsignificanceofthismodificationinmtDNA,arenotknown.哺乳動(dòng)物mtDNA與核DNA的CpG抑制顯示了一個(gè)相似的水平(12)，表明mtDNA中5mC容易突變。迄今為止，5mC在mtDNA中描述的唯一修飾的堿基，但是這個(gè)建立和維持mtDNA甲基化的機(jī)制，和這個(gè)修飾在mtDNA中功能性意義還是未知的。MammalianmtDNAisa16.5-kbdouble-stranded,circularmolecule,presentinmultiplecopiespermitochondrion(13).Themitochondrialgenomeencodes13oftheproteinspresentintherespiratorychaincomplexesofmammalianmitochondria,aswellastworibosomalRNAsand22transferRNAsspecifictothisorganelle.Allothermitochondrialproteins,includingthoserequiredformtDNAreplicationandtranscription,areencodedinthenucleusandtranslocatedtothemitochondriausingspecializedimportsystemswhichofteninvolveN-terminalmitochondrialtargetingsequences(MTSs)(14).Incontrasttothenucleargenome,mtDNAisnotcomplexedwithhistones.However,mtDNAispresentinprotein-containingcomplexescallednucleoids,eachcontainingmultiplecopiesofmtDNAboundtoacomplexmixtureofproteins(15).哺乳動(dòng)物mtDNA是一個(gè)16.5kb雙鏈環(huán)狀分子，每個(gè)線粒體中存在多個(gè)拷貝(13)。線粒體基因組編碼哺乳動(dòng)物線粒體呼吸鏈上13個(gè)蛋白復(fù)合體，兩個(gè)核糖體RNA和22轉(zhuǎn)運(yùn)RNA專(zhuān)門(mén)針對(duì)這個(gè)細(xì)胞器的。其他所有線粒體蛋白，包括mtDNA復(fù)制和轉(zhuǎn)錄需要的，在核中編碼并使用專(zhuān)門(mén)的導(dǎo)入系統(tǒng)轉(zhuǎn)運(yùn)到線粒體，專(zhuān)門(mén)的導(dǎo)入系統(tǒng)經(jīng)常包括N末端線粒體靶序列(MTSs)(14)。對(duì)比核基因組，mtDNA沒(méi)有與組蛋白形成復(fù)合體。然而，mtDNA存在于包含蛋白質(zhì)的復(fù)合物中，稱(chēng)之為擬核，每個(gè)包含mtDNA多個(gè)拷貝結(jié)合到一個(gè)復(fù)雜的蛋白質(zhì)混合物上(15)。Transcriptionofthemitochondrialgenomeisthoughttobe(被認(rèn)為是)coregulatedwithnuclearcomponentsoftherespiratorychaincomplexes（16）.Inmammals,oxidativestressresultsinstabilizationofperoxisomeproliferator-activatedreceptory-coactivator1a（PGC1a）,whichactivatesthetranscriptionofseveralnuclear-encodedtranscriptionfactors,includingnuclearrespiratoryfactor1（NRF1）.PGClaandNRF1formacomplexthatinturn（依次，輪流）up-regulatestranscriptionoftranscriptionfactorofactivatedmitochondria（TFAM）andmultiplemembersofmitochondrialrespiratorychaincomplexes（17）.線粒體基因組的轉(zhuǎn)錄被認(rèn)為是與呼吸鏈復(fù)合物核組成部分共同調(diào)節(jié)的（16）。哺乳動(dòng)物中，氧壓導(dǎo)致了氧化物酶體增殖物激活受體Y輔激活因子1a（PGC1a）的穩(wěn)定性，它激活幾個(gè)核編碼轉(zhuǎn)錄因子的轉(zhuǎn)錄，包括核呼吸因子（NRF1）。PGC1a和NRF1形成一個(gè)復(fù)合體依次上調(diào)激活了的線粒體轉(zhuǎn)錄因子的轉(zhuǎn)錄（TFAM）和許多線粒體呼吸鏈復(fù)合物成員（17）。Severalnuclear-encodedgenesinvolvedinmitochondrialfunction,includingPGC1a（18）,areregulatedbyDNAmethylation.Conversely,ithasbeensuggestedtha（表達(dá)觀點(diǎn)）mitochondriaareablet（能夠）influencecytosinemethylationlevelsinthenucleusbymodulatingthefluxofone-carbonunitsforthegenerationofS-adenosylmethionine,themethyldonorinDNAmethylation（19）.Thus,epigeneticregulationofnucleargeneexpressionappearstohave（似乎有）amitochondrialcomponent.ThepresenceofcytosinemethylationinmtDNAledustoquestionwhetherthisepigeneticmodificationmightplayaroleintheco-ordinatedregulationofmitochondrialgeneexpressionfrombothnuclearandmitochondrialgenomes.通過(guò)DNA甲基化調(diào)節(jié)幾個(gè)核編碼的基因包含線粒體的功能，包括PGC1a（18）。相反地，人們認(rèn)為線粒體能夠通過(guò)調(diào)制S-腺苷甲硫氨酸產(chǎn)生的一碳單位的流出，影響細(xì)胞核中胞嘧啶甲基化水平，S-腺苷甲硫氨酸為DNA甲基化的甲基供體（19）。因此，核基因表達(dá)的表觀調(diào)節(jié)似乎有線粒體的成分。mtDNA中胞嘧啶甲基化的存在使我們?nèi)ニ伎急碛^修飾可能在從核和線粒體兩個(gè)基因組對(duì)線粒體的基因表達(dá)協(xié)同調(diào)節(jié)中扮演著重要的角色。ResultsHumanandMouseDNMT1EncodeMitochondrialTargetingSequences.EarlyreportsofDNAmethylationinthemitochondrialgenome（9-11）ledustoaskwhetheroneormoreofthecatalyticallyactivemammalianDNAmethyltransferasesmightbetargetedtomitochondria.Examinationofthe5'UTRand5'flankinggenomicDNAupstreamofthepublishedtranscriptionstartsites（20）forbothhumanandmouseDNMT1revealedthatsequenceequivalentto101codonsinhumanand63codonsinmouseDNMT1wasin-framewiththehighlyconservedaminoacidsequenceofDNMT1,startingwiththeATGreported（20）tobetheprimarytranslationalstartcodon（Fig.1AandB）.結(jié)果人類(lèi)和小鼠DNMT1編碼線粒體靶序列早期報(bào)道的線粒體基因組DNA甲基化（9-11）引導(dǎo)我們?nèi)?wèn)是否有一個(gè)或者更多催化活性哺乳動(dòng)物DNA甲基化轉(zhuǎn)移酶可能是針對(duì)線粒體的。檢查人類(lèi)和小鼠DNMT1公布的轉(zhuǎn)錄起始位點(diǎn)（20）基因組DNA上游5’UTR和5’側(cè)翼區(qū)，顯示人類(lèi)101個(gè)密碼子和小鼠DNMT163個(gè)密碼子序列等效是具有高度保守氨基酸序列的DNMT1編碼框，報(bào)道的ATG起始（20）是主要的轉(zhuǎn)錄起始密碼（Fig.1A和B）。Thisupstreamsequenceincludestwoadditionalin-framecodonsformethionine,eachinamoderatecontextforribosomebinding（21）;theupstreamATGcodonsaredenotedATG1andATG2,whereasthepublishedtranslationstartisshownasATG3.RT-PCRusingsenseprimerslocatedoverATG1orATG2andanti-senseprimerscrossingtheexon1-2boundaryby4nucleotidesdetectedtranscriptscapableofencodingtheseN-terminalextensionsinhumanandmousecells.TranscriptsinitiatingupstreamofATG1inmouseandATG2(butnotATG1)inhumanmRNAwereeasilydetected(Fig.1C),suggestingtheutilizationofanup-streamtranscriptionst;siteencodinganN-terminalextension.這個(gè)上游序列包括兩個(gè)額外的甲硫氨酸編碼框密碼子，每個(gè)都在序列中間于核糖體結(jié)合(21)；上游ATG密碼子表示ATG1和ATG2，而已發(fā)布的轉(zhuǎn)錄起始是指ATG3。人類(lèi)和小鼠細(xì)胞中，RT-PCR使用上游引物位于ATG1或ATG2,下游引物橫跨外顯子1-2邊界線通過(guò)4個(gè)核苷酸檢測(cè)編碼這些N末端的延長(zhǎng)的轉(zhuǎn)錄能力。mRNA轉(zhuǎn)錄起始上游小鼠中ATG1和人類(lèi)中ATG2(不是ATG1)是容易測(cè)定的。MouseorhumanDNMT1isoformscontainingthisadditionalN-terminalsequencewerepredictedbyMitoProtII(http://ihg.gsf.de/ihg/mitoprotbettargetedt(針對(duì))themitochondriawithveryhighprobability,comparedwithproteinsbeginningatthepublishedstartcodon,ATG3(TableS1).Thegenomedatabasesalsocontainupstreamsequencesforchimpanzee,rat,andcowDNMT1;ineacfeciespneormorein-framepotentialstartcodonsencodeapeptidewithastrongprobability(>90%)ofmitochondriallocalization(TableS1).」arepredictedtoformamphiphidhelices,althoughquenceconservationbetweenthem;low,asisoftenthecase(情況常常如此，這是常有的事)formiochondrialleaderpeptidesacrossspecies.通過(guò)MitoProtH預(yù)測(cè)小鼠和人類(lèi)DNMT1亞型包括這個(gè)額外的N末端的序列(22)針對(duì)線粒體具有很高的可能性，與從公布的起始密碼子ATG3(TableS1開(kāi)始蛋白相比較?；蚪M數(shù)據(jù)庫(kù)也包括黑猩猩，大鼠和牛DNMT1上游序列；在每個(gè)物種中，一個(gè)或者更多編碼框內(nèi)潛在的起始密碼子編碼一個(gè)具有定位線粒體很大可能性(>90%)的多肽(TableS1)。預(yù)測(cè)所有形成兩性分子叫』螺旋，盡管他們之間的序列保守性很低，線粒體引導(dǎo)肽穿越物種這是常有的事。Immunoblotsofpurifieditochondriafrommouseembryonicfibroblas(MEFs)andHCT116humancoloncarcinomacellsshowedthepresenceofDNMT1(Fig.2A)butnotDNMT3aorDNMT3binthisorganelle(Fig.2B).Full-lengthDNMT1andasmallerpeptidearedetectedbyanN-terminalDNMT1antibody,suggestingthatproteolytipcrocessingoccursuponentryintothemitochondria.AbsencofthenucleaimarkerH3K4me3inthanitochondrialfractionindicatedpurityfromcontaminationbynuclearmaterial,theprimarysiteoflocalizatioofDNAmethyltransferases.來(lái)自小鼠胚胎成纖維細(xì)胞(MEFs)和人結(jié)腸癌細(xì)胞HCT116純化線粒體的免疫印跡分析顯示在這個(gè)細(xì)胞器中存在DNMT1(Fig.2A)，不存在DNMT3a或DNMT3b(Fig.2B。用一個(gè)N末端DNMT1抗體檢測(cè)全長(zhǎng)DNMT1和較小的多肽，表明蛋白酶加工發(fā)生在進(jìn)入線粒體后。線粒體斷片中缺少細(xì)胞核標(biāo)志H3K4me3顯示純化被核物質(zhì)污染，定位DNA甲基化轉(zhuǎn)移酶的主要位點(diǎn)。Weclonedthemouseanchumanleadersequences,fromATG1toostreamofATG3,in-framewiththeC-terminalGFPtagofpcDNA6.2/GFP,andtransfectectheplasmidsintoNIH/3T3fibroblasts.ConfocalmicroscopyshowedthatbothhumanandmouseleadersequencestargetedGFPtothemitochondria,indicatedbycolocalizationofMitoTrackerRedwithgreenfluorescence(Fig2C).Mitochondriainuntransfectedellwithinthesamevisuafieldemainedredinthemergedphotomicrographserving(作為，充當(dāng)，擔(dān)任)negativecontrolsforcolocalization,whereasachloramphenicolacetyltransferase(CAT)-GFPcontrolplasmidremainedcytosolic.WealsotransfectedheseconstructsLntoHCT116humancoloncarcinomacellsforimmunoblotanalysisofpurifiedmitochondriausinganti-GFPantibody(Fig.S1).我們克隆小鼠和人類(lèi)的前導(dǎo)序列，從ATG1到ATG3的上游，編碼框內(nèi)具有C末端具有GFP標(biāo)簽質(zhì)粒pcDNA6.2/GFP，轉(zhuǎn)染到NIH/3T3成纖維細(xì)胞。共聚焦顯微鏡表明人和小鼠兩者的前導(dǎo)序列將GFP定位到線粒體，通過(guò)MitoTrackerRed和綠色熒光共定位體現(xiàn)(Fig.2C)。未轉(zhuǎn)染的細(xì)胞內(nèi)的線粒體在合并的顯微照片相同視野內(nèi)保持紅色，作為共定位的陰性對(duì)照，然而氯霉素乙酰轉(zhuǎn)移酶(CAT)-GFP控制質(zhì)粒保持細(xì)胞質(zhì)基質(zhì)。我們也轉(zhuǎn)染這些結(jié)構(gòu)到人結(jié)腸癌細(xì)胞HCT116用抗GFP抗體免疫印跡分析純化的線粒體(Fig.S1)。HCT116mitochondriaclearlyaccumulatedGFP.Thus,humanandmouseleaderpeptidesrepresentbonafideMTSscapableoftrackingheterologousproteinstothisorganelle.EachMTSisabletooperateacrossspecies,indicatingfunctionalconservation.HCT116線粒體清晰地積累GFPo因此，人類(lèi)和小鼠引導(dǎo)肽體現(xiàn)這些細(xì)胞器MTSs法真實(shí)的跟蹤外源性蛋白檢測(cè)能力。每一個(gè)MTS能夠穿越物種操作，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).對(duì)氧化壓力反應(yīng)的因子調(diào)節(jié)mtDNMT1表達(dá)MatInspector在人類(lèi)和小鼠的DNMT1中預(yù)測(cè)了NRF1一個(gè)結(jié)合位點(diǎn)。這個(gè)共有序列位于越過(guò)上游編碼框起始密碼子之一，在研究的所有其他哺乳類(lèi)物種中是保守的(TableS1；Fig.3A)。在氧化壓力的條件下，共激活劑PGC1a激活并且與NRF1相互作用去上調(diào)多個(gè)核編碼的線粒體基因(17)o因此，我們瞬時(shí)轉(zhuǎn)染NRF1，PGC1a或者兩者一起轉(zhuǎn)染到HCT116細(xì)胞，通過(guò)免疫印跡分析線粒體DNMT1的水平(Fig.3B)。AsmallincreaseinmtDNMT1wasseenincellstransfectedwithNRF1orPGClaalone,whereascotransfectionwithbothPGC1aandNRF1resultedinanapproximatelyfivefoldincreaseinmtDNMT1relativetocontrol.Thus,thislocusissensitivetoregulationbyactivatorsthatrespondtooxidativestress.在單獨(dú)轉(zhuǎn)染NRF1或者PGC1a的細(xì)胞中看到mtDNMT1有一個(gè)很小的增加，然而兩者共轉(zhuǎn)染導(dǎo)致相對(duì)對(duì)照組mtDNMT1有一個(gè)大約5倍的增加。因此，這個(gè)位點(diǎn)對(duì)于氧化壓力反應(yīng)的調(diào)節(jié)子的調(diào)節(jié)是敏感的。TheNRF1bindingsiteiscoincidentwithap53consensusbindingsite(Fig.1AandB),whichwepreviouslydemonstratedto(向說(shuō)明，向證明)repressDNMT1transcription(23).Ourearlierstudyshowedathree-tosixfoldincreaseinDNMT1transcriptionfollowingeitheractivationorgeneticdeletionofp53inHCT116cellsandMEFs.Becausep53isknowntoregulatemitochondrialrespiration(24),weaskedwhetherthistumorsuppressorproteinalsoaffectedmtDNMT1mRNAexpression.NRF1結(jié)合位點(diǎn)和P53共識(shí)結(jié)合位點(diǎn)是一致的(Fig.1A和B)，這個(gè)我們之前已經(jīng)證明抑制DNMT1轉(zhuǎn)錄(23)。我們之前的研究隨著在HCT116細(xì)胞和MEFs細(xì)胞中P53的激活或者基因缺失DNMT1轉(zhuǎn)錄顯示了一個(gè)3-6倍的增加。因?yàn)镻53是人們所周知調(diào)節(jié)線粒體呼吸作用(24)，我們思考這個(gè)腫瘤抑制蛋白是否也影響mtDNMT1mRNA表達(dá)。WeusedRT-quantitative(q)PCRwithprimersthatdistinguishthemitochondrialtranscriptfromthetotalDNMT1transcript(Fig.1C);themitochondrialtranscriptcomprised1-2%ofthetotalDNMT1synthesizedinlog-phaseMEFsorHCT116cells.TherelativeabundanceofmtDNMTltranscriptincreasedsixfoldinp53-/-MEFscomparedwithWTMEFs,whereastotalDNMT1mRNAincreasedthreefold(Fig.3C),suggestingapreferentialup-regulationofthemitochondrialtranscriptincellslackingp53.ImmunoblotanalysisoftheseisogeniccellsshowedastrikingincreaseinmtDNMT1proteinwithlossofp53(Fig.3D).我們用從總的DNMT1轉(zhuǎn)錄物中區(qū)分線粒體的轉(zhuǎn)錄物的引物，進(jìn)行RT-qPCR(Fig.1C)；線粒體轉(zhuǎn)錄占對(duì)數(shù)階段MEFs或HCT116細(xì)胞總合成DNMT1的1-2%。P53-/-MEFs與野生型MEFs細(xì)胞相比mtDNMT1轉(zhuǎn)錄物的相對(duì)豐都增加了6倍，然而總的DNMT1mRNA增加了3倍(Fig.3C)，表明在缺少P53的細(xì)胞中線粒體轉(zhuǎn)錄物的一個(gè)優(yōu)先上調(diào)。這些等基因細(xì)胞的免疫印跡分析在缺乏P53細(xì)胞中mtDNMT1蛋白顯示了一個(gè)顯著的增加(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.線粒體轉(zhuǎn)錄中獨(dú)特基因的改變我們?cè)儐?wèn)mtDNMT1過(guò)表達(dá)是否反應(yīng)線粒體基因組轉(zhuǎn)錄的改變(Fig.3E)。NADH脫氫酶亞基6(ND6)，輕鏈上唯一編碼蛋白的基因，在mtDNMT1增加上顯著地低表達(dá)，表明mtDNA甲基化在抑制輕鏈轉(zhuǎn)錄中的一個(gè)作用。在重鏈上，ATPase亞基6(ATP6)和細(xì)胞色素酶C氧化酶亞基1(COX1)在他們表達(dá)水平上是不變的。然而，NADH脫氫酶亞基1(ND1)，第一條輕鏈編碼蛋白區(qū)域跟隨核糖體rRNA基因，在反應(yīng)提高的mtDNMT1中是顯著增加的。這些數(shù)據(jù)支持了一個(gè)獨(dú)特基因影響線粒體基因轉(zhuǎn)錄，正如下面討論的。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.線粒體DNMT1結(jié)合到mtDNA我們建立了一個(gè)HCT116細(xì)胞系(25)，在這個(gè)細(xì)胞系中一個(gè)內(nèi)源性DNMT1等位基因攜帶一個(gè)C末端串聯(lián)親和純化(TAP)標(biāo)簽(26)°TAP標(biāo)簽的DNMT1高效地轉(zhuǎn)移進(jìn)入線粒體(Fig.4B)。因此我們用這些細(xì)胞去詢問(wèn)mtDNMT1是否與mtDNA直接相互作用。甲醛交聯(lián)線粒體溶菌產(chǎn)物用IgG小珠子免疫沉淀(26)，用mtDNA專(zhuān)門(mén)的引物進(jìn)行qPCR(TableS2)去定量mtDNMT1和mtDNA間的相互作用。TAP標(biāo)簽細(xì)胞進(jìn)行免疫共沉淀相比沒(méi)有標(biāo)簽的細(xì)胞的免疫共沉淀與mtDNA大量的富集，除了不包含CpG二核苷酸的擴(kuò)增子，兩個(gè)細(xì)胞系都給了相等的低信號(hào)(Fig.4C)。這些數(shù)據(jù)顯示mtDNMTl和線粒體基因組相互作用依賴CpG，證實(shí)了這個(gè)蛋白定位到線粒體基質(zhì)中。InteractionwasevidentintheD-loopcontrolregion,whichcarriesthemitochondrialoriginofreplicationandpromoters,aswellasinrRNAandprotein-codingregions.Thelevelofenrichmentwasdependentonthetargetamplicon;fiveofthesixregionsprobedshowedathree-tofivefoldenrichmentofmtDNAsequences.However,qPCRoftheregioncoveringthejunctionbetween12Sand16SrRNAgenes(primer2)showedonlytwofoldenrichmentinbindingofmtDNMT1-TAP.ThedensityofCpGdinucleotidesinthisampliconis<50%thatinallotherampliconsanalyzed,suggestingthatinteractionofmtDNMT1withmtDNAisproportionaltoCpGdensity,andsupportingafunctionalroleformtDNMT1inestablishmentandmaintenanceofmtDNAmethylation.在D環(huán)控制區(qū)域相互作用是明顯的，這個(gè)區(qū)域帶有線粒體復(fù)制原點(diǎn)和啟動(dòng)子，和rRNA中編碼蛋白質(zhì)區(qū)域一樣。富集水平依賴靶擴(kuò)增子；6個(gè)區(qū)域中探測(cè)了5個(gè)，顯示mtDNA序列3-5倍的富集。然而，這個(gè)區(qū)域的qPCR引物2覆蓋12S和16SrRNA基因之間的連接，在結(jié)合mtDNMT1-TAP中顯示僅2倍富集。這個(gè)擴(kuò)增子中CpG二核苷酸密度V50%，這個(gè)也在所有其他擴(kuò)增子進(jìn)行了分析，表明mtDNMT1和mtDNA間的相互作用與CpG密度成比例的，支持了mtDNMT1在建立和維持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羥甲基化。我們用5mC或者5hmC的抗體免疫共沉淀隨機(jī)地修剪mtDNA，通過(guò)qPCR探測(cè)沉淀的DNA去確定mtDNA中這兩個(gè)修飾堿基的存在和相對(duì)豐度(Fig.5A和5B)。免疫共沉淀的樣品富集5mC相對(duì)所有區(qū)域檢測(cè)的IgG對(duì)照10-20倍。mtDNA免疫共沉淀使用抗5hmC相對(duì)IgG對(duì)照是高度富集(85-580倍)的，除了穿過(guò)D環(huán)(引物27)，他富集了38倍。針對(duì)他們各自的修飾每個(gè)抗體的特異性用對(duì)照DNA確定，對(duì)照DNA中每個(gè)胞嘧啶轉(zhuǎn)換為5mC或5hmC(Fig.S2)。mtDNA中兩個(gè)胞嘧啶修飾的存在表明早期的研究低估了線粒體基因組表觀修飾的程度。WeusedphageT45hmC-6-glucosyltransferase(6-gt)(27)todeterminethepresenceof5hmCatGla1restrictionendonucleasecleavagesites(28).ControlexperimentsusingdefinedDNAsequencescontainingcytosine,5mC,or5hmCconfirmedthatGla1cleavedonlysitesmodifiedbymethylationorhydroxymethylation,butnotsitescontainingglucosylated5hmC(Fig.S3A).我們用T4噬菌體5hmC-p-葡糖基轉(zhuǎn)移酶(8-gt)(27)去確定在Gla1限制性內(nèi)切酶切割位點(diǎn)5hmC的存在(28)。對(duì)照試驗(yàn)用確定了的DNA序列包括胞嘧啶，5mC或5hmC證明甲基化或羥甲基化修飾的Gla1唯一的切割位點(diǎn)，但是不包括葡糖基修飾的5hmC位點(diǎn)(Fig.S3A)。ProtectionofmtDNAfromcleavageby5hmCglucosylationwasassessedbyendpoint(Fig.S3BandC)andqPCR(Fig.5C).5hmCwaspresentinthreedifferentampliconsfromhumanmtDNAandtwoampliconsfrommousemtDNAorgenomicDNA.AmpliconscontainingtwoGla1restrictionsiteseach(ampliconsATP6,12S,and16S-3)showed50%protectionincomparisonwithampliconswithasingleGla1site(amplicons2and16S-2),suggestingasimilarlevelof5hmCatallrestrictionsitestested.Amouseamplicondevoidof(沒(méi)有，缺乏)Gla1sites(ATP6/COX3)wasprotectedfromcleavageirrespectiveof5hmCglucosylation(Fig.S3C).保護(hù)5hmC葡糖基化的mtDNA不被切割，通過(guò)末端(Fig.S3B和C)和qPCR(Fig.5C)進(jìn)行評(píng)估。5hmC存在與三個(gè)不同擴(kuò)增子中，人類(lèi)mtDNA和兩個(gè)小鼠mtDNA或基因組DNA擴(kuò)增子。擴(kuò)增子包括兩個(gè)Gla1限制性位點(diǎn)與帶有一個(gè)Gla1位點(diǎn)的擴(kuò)增子(擴(kuò)增子2和16S-2)相比較每個(gè)(擴(kuò)增子ATP6，12S和16S-3)顯示50%保護(hù)，表明在檢測(cè)的所有限制性位點(diǎn)上5hmC具有一個(gè)相似的水平。一個(gè)小鼠擴(kuò)增子缺乏Gla1位點(diǎn)(ATP6/COX3)不論5hmC是否葡糖基化都保護(hù)不被切割(Fig.S3C)。DiscussionCytosinemethylationofthemitochondrialgenomehasremainedlargelyoverlooked,inpartbecauseearlyreportsusingnearest-neighboranalysisindicatedthatthismodificationwaspresentatonly25%ofCpGdinucleotides(11),wellbelowthelevelofmethylationseeninthenucleus.Thedatapresentedhereshowa10-to20-foldenrichmentofmtDNAsequencesinimmunoprecipitatesusing5mCantibody,somewhatlowerthanthatusuallyobtainedfromgenomicDNA(100-foldforCpGislands).討論線粒體基因組的胞嘧啶甲基化仍然保持一個(gè)很大的空缺，部分是因?yàn)樵缙诘膱?bào)道使用最近鄰順序分析顯示這種修飾在CpG二核苷酸僅有2-5%(11)，細(xì)胞核中也甲基化水平也很低。本文這些數(shù)據(jù)呈現(xiàn)了用5mC抗體的免疫共沉淀中mtDNA序列一個(gè)10-20倍的富集，通常比基因組DNA(是CpG島的~100倍)獲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.這個(gè)很可能反應(yīng)自然線粒體基因組中CpG稀少，它不包含CpG島。這里我們用兩個(gè)獨(dú)立的實(shí)驗(yàn)證明5hmC在mtDNA中存在。因此，線粒體基因組中胞嘧啶的表觀修飾很可能比我們之前認(rèn)為的更加頻繁。在細(xì)胞核中，5hmC是由5mC通過(guò)TET家族的甲基胞嘧啶加氧酶作用產(chǎn)生的(6)。這里還沒(méi)有證明這些酶在線粒體中的存在或缺失，TET家族蛋白或基因座不包含可認(rèn)識(shí)的線粒體的目標(biāo)序列(14)。因此我們不能排除5hmC產(chǎn)生的一個(gè)不同機(jī)制的可能性，包括mtDNMT1用線粒體多功能氧化酶產(chǎn)生的甲醛將5羥甲基集團(tuán)直接共價(jià)增加到DNA胞嘧啶殘基上(29)。Theapparentlylowerenrichmentfor5hmCintheD-loopcontrolregionmostlikelyreflectsthelessefficientamplificationofalongerfragment(833bpcomparedwith112-238bp)frommtDNAshearedtoanaveragesizeof300-400bp.Howevei,theDloopexistsasastabletriple-helicalstructurecontaininganRNAprimerrequiredforinitiationofmtDNAreplication(13),andwehavefoundthisregiontoberesistanttoinvitromethylationbyM.Ssslcytosinemethyltransferase.Itisthereforepossiblethatthekineticsofepigeneticmodificationinthisregionofthemitochondrialgenomemightbedifferentfromthoseincodingregions.在D環(huán)對(duì)照區(qū)域有一個(gè)明顯較低5hmC富集最可能反應(yīng)mtDNA較長(zhǎng)片段(833bp相比較112-238bp)修剪為平均長(zhǎng)度300-400bp擴(kuò)增效率較低。然而，D環(huán)的存在作為一個(gè)穩(wěn)定的三螺旋結(jié)構(gòu)包括mtDNA復(fù)制起始所需要的一個(gè)RNA引物(13)，我們發(fā)現(xiàn)這個(gè)區(qū)域?qū)τ谕ㄟ^(guò)M.Sss1胞嘧啶甲基轉(zhuǎn)移酶體外甲基化是有抵抗的。因此，線粒體基因組的這個(gè)區(qū)域的表觀修飾動(dòng)力學(xué)可能不同于編碼區(qū)的動(dòng)力學(xué)。Thefunctionof5hmCinthenucleargenomeisnotyetclear.Ithasbeenproposedthat5hmCisanintermediatemetaboliteinactivedemethylationofthegenomebyrepairenzymes(30),inpassivedemethylationasaresultoflackofrecognitionbyenzymesinvolvedinmaintenancemethylation(31)句型,orthatitalterslocalchromatinstructurebecause5hmCisnotrecognizedby5-methylcytosine-bindingproteins(7).Theroleof5hmCinthemitochondrialgenomelikelyinvolvesoneormoreoftheseprocesses.Althoughquantitativemeasurementsoftherelativeabundanceof5hmCand5mCcanbeachievedusingmethylatedDNAimmunoprecipitation(Me-DIP),5hMe-DIF,HPLC,orenzymaticmethods,mappingthelocationanddistributionof5hmCineitherthenuclearormitochondrialgenomeisnotyettechnicallyfeasible,becausethismodifiedbaseisindistinguishablefrom5mCbybisulfitemodification(7).核基因組中的5hmC的功能還沒(méi)有很清楚。提出5hmC是通過(guò)修復(fù)酶對(duì)基因組的主動(dòng)去甲基化一個(gè)中間代謝物(30)，在被動(dòng)去甲基化中作為多維持甲基化酶缺乏認(rèn)識(shí)的一個(gè)結(jié)果(31)，或者5hmC改變當(dāng)?shù)厝旧w結(jié)構(gòu)因?yàn)?hmC是還沒(méi)有通過(guò)5甲基胞嘧啶結(jié)合蛋白認(rèn)識(shí)(7)。5hmC在線粒體基因組中的作用很可能涉及這些過(guò)程的一個(gè)或者多個(gè)。盡管5hmC和5mC的相對(duì)豐都的定量測(cè)量能夠通過(guò)使用甲基化的DNA免疫共沉淀(Me-DIP)，5hMe-DIP，HPLC，或酶催化的方法獲得，繪制5hmC在細(xì)胞核或者線粒體基因組中的位置和分布還沒(méi)有技術(shù)上的可行性，因?yàn)檫@些修飾堿基通過(guò)亞硫酸氫鹽與5mC不能區(qū)分的(7)。ThisstudyreportsamitochondrialisoformofDNAmethyltransferase1,whichistheonlymemberofthecatalyticallyactivemammalianDNAmethyltransferasefamilyfoundinthisorganelle.TheconservationofanORFencodingamitochondrialtargetingsequenceupstreamofthecommonlyacceptedtranslationalstartcodonacrossmultiplemammalianspeciessuggestsanimportantroleforthisenzymeinmitochondrialfunction.這個(gè)研究報(bào)道一個(gè)線粒體DNA甲基轉(zhuǎn)移酶1的亞型，它是在這個(gè)細(xì)胞器中發(fā)現(xiàn)的哺乳動(dòng)物DNA甲基轉(zhuǎn)移酶家族唯一具有催化活性的。保守的一個(gè)開(kāi)放閱讀框編碼的一個(gè)線粒體靶序列普遍公認(rèn)的轉(zhuǎn)錄起始密碼子上游穿過(guò)多種哺乳動(dòng)物物種顯示這個(gè)酶在線粒體功能中的一個(gè)重要作用。AlthoughDNMT1isgenerallyconsideredtobe(認(rèn)為是)themaintenanceDNAmethyltransferase,itisabletomethylatecompletelyunmethylatedDNAinvitrowithanefficiencythatexceedsthatofthedenovomethyltransferasesDNMT3aand-3b(32).Thus,DNMT1appearstobecapableofbothinitiatingandmaintainingcytosinemethylationinthenucleus,andthelackofdenovomethyltransferasesinmitochondriaimplicatesmtDNMT1inbothprocessesinthisorganelle.盡管DNMT1普遍被認(rèn)為是維持DNA的甲基轉(zhuǎn)移酶，他能在體外甲基化完全未甲基化的DNA效率超過(guò)了從頭甲基轉(zhuǎn)移酶DNMT3a和-3b的效率（32）。因此，DNMT1似乎在細(xì)胞核中具有從頭和維持胞嘧啶甲基化兩種能力。線粒體中缺乏從頭甲基轉(zhuǎn)移酶暗示DNMT1在這個(gè)細(xì)胞器中具有這兩個(gè)過(guò)程。WeshowthatmtDNMTlbindstothemitochondrialgenomeinamanner（在某種意義上，在某種程度上）proportionaltothedensityofCpGdinucleotides.OfparticularrelevanceisthebindingofmtDNMT1totheD-loopcontrolregion,whichcarriesthepromotersdrivingtranscriptioninitiationofbothheavyandlightstrands,supportingaroleformtDNMT1inregulationofmitochondrialgeneexpression.Theasymmetric,gene-specificalterationinmitochondrialtranscriptionpatternsshownheresuggestsdiverserolesformtDNMT1andcytosinemodificationinthisorganelle.我們顯示mtDNMT1結(jié)合線粒體基因組在某種程度上與CpG二核苷酸密度成比例。特別相關(guān)的是mtDNMT1與D環(huán)對(duì)照區(qū)結(jié)合，D環(huán)對(duì)照區(qū)攜帶驅(qū)動(dòng)重鏈和輕鏈兩者轉(zhuǎn)錄起始的啟動(dòng)子，支持mtDNMT1在調(diào)節(jié)線粒體基因表達(dá)中的功能。不對(duì)稱(chēng)地，本文表明在線粒體轉(zhuǎn)錄模式中獨(dú)特基因的改變，暗示在這個(gè)細(xì)胞器中mtDNMT和胞嘧啶修飾具有很多作用。DecreasedexpressionofND6ontheLstrandimpliesthatcytosinemethylationinmtDNArepressesgeneexpressionfromthelight-strandpromoter,asitdoesinthenucleus.However,increasedtranscriptionofND1withnochangeintranscriptionofATP6orCOX1raisesthepossibilityofadifferentmodeofactionontheHstrand.Abindingsiteformitochondrialterminatorfactor1（MTERF1）islocatedbetweentheendofthe16SrRNAgeneandthetranslationstartofND1（33）.MTERF1bindstobothH-strandpromoter1（HSP1）andtheterminatorbindingsite（Fig.4A）,formingatranscriptionloopthatmaintainshigh-levelproductionofrRNA.TranscriptsinitiatingatHSP2producepolycistronicmessagesencodingtheentireHstrand（13）.L鏈ND6表達(dá)減少表明mtDNA胞嘧啶甲基化抑制從輕鏈啟動(dòng)子開(kāi)始的基因表達(dá)，和在細(xì)胞核中的一樣。然而，ND1轉(zhuǎn)錄的增加沒(méi)有改變ATP6和COX1的轉(zhuǎn)錄增加重鏈上不同作用模式的可能性。線粒體終止子因子（MTERF1）結(jié)合位點(diǎn)位于16SrRNA基因末尾和ND1轉(zhuǎn)錄起始之間（33）。MTERF1結(jié)合到H鏈啟動(dòng)子1（HSP1）和終止子結(jié)合位點(diǎn)上（Fig.4A），形成一個(gè)轉(zhuǎn)錄環(huán)，它維持rRNA高水平產(chǎn)物。在HSP2轉(zhuǎn)錄起始產(chǎn)生多順?lè)醋拥男畔⒕幋a整個(gè)H鏈（13）。OurdataraisethepossibilitythatmtDNMT1,eitherthroughmodificationofCpGdinucleotidesorbydirectprotein-proteininteraction,interfereswithMTERF-dependenttranscriptiontermination,allowingread-throughfromHSP1tothenexttranscriptionalunit（ND1）withoutimpactingpolycistronicmRNAsynthesisfromHSP2.我們的數(shù)據(jù)提高了mtDNMT1的可能性，不管是通過(guò)CpG二核苷酸的修飾還是通過(guò)蛋白和蛋白質(zhì)間的直接相互作用，阻礙了依賴MTERF轉(zhuǎn)錄的終止，允許從HSP1到下一個(gè)轉(zhuǎn)錄單元（ND1）的通讀不影響從HSP2多順?lè)醋拥膍RNA合成。WeshowherethatDNMT1ispresentinthemitochondrialmatrix,boundtomtDNA,andmodifiestranscriptionofthemitochondrialgenomeinwhatappearstobeagene-specificfashion.Wereportthepresenceofboth5hmCand5mCinmtDNA,suggestingthatearlierstudiesmayhaveunderestimatedtheproportionofmodifiedcytosinesinthisgenome.Hence,mtDNMT1appearstoberesponsiblefor（是。。的原因）theestablishmentandmaintenanceofcytosinemethylationinmtDNA,fromwhich5hmCispresumablyderived.Ourdatasupportaroleforepigeneticmodificationofthemitochondrialgenomeinregulationofmitochondrialtranscription.這里我們表明DNMT1存在于線粒體基質(zhì)中，結(jié)合mtDNA,修飾線粒體基因組的轉(zhuǎn)錄，這個(gè)似乎獨(dú)特的基因方式是什么。我們報(bào)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飾在這基因組中的比例。因此，mtDNMTl似乎是在mtDNA中建立和維持胞嘧啶甲基化的原因，這很可能是由5hmC衍生來(lái)的。我們的數(shù)據(jù)支持了線粒體基因組的表觀修飾在調(diào)節(jié)線粒體轉(zhuǎn)錄中的作用。MaterialsandMethods材料和方法CellLines.HCT116p53+/+andHCT116p53-/-wereobtainedfromBertVogelstein,JohnsHopkinsUniversity(Baltimore,MD).PrimaryMEFswerepreparedfromE12.5-E13.5embryos.細(xì)胞系。HCT116p53+/+和P53-/-從JohnsHopkinsUniversity大學(xué)的BertVogelstein博士獲得(Baltimore,MD)。主要的MEFs細(xì)胞來(lái)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.質(zhì)粒和轉(zhuǎn)染。使用表S2中列舉的引物。使用隨機(jī)引物擴(kuò)增小鼠和人類(lèi)cDNAs線粒體目標(biāo)序列。小鼠NRF1cDNA獲得于美國(guó)標(biāo)準(zhǔn)菌庫(kù)然后重新克隆到pDEST26/C-FLAG0PGCla質(zhì)粒由VirginiaCommonwealthUniversity大學(xué)的GregorioGil饋贈(zèng)。使用Polyjet脂質(zhì)體(ProSci)(HCT116)或核轉(zhuǎn)染(Amaxa)(MEFandNIH/3T3)按照制造商說(shuō)明書(shū)轉(zhuǎn)染細(xì)胞，轉(zhuǎn)染48h后收獲。MitochondrialPurificationandImmunoblotAnalysis.Mitochondriawerepurifiedbydouncehomogenizationanddifferentialcentrifugationinthepresenceofcompleteproteas