1、哺乳動物線粒體中DNA甲基化轉移酶1，胞嘧啶甲基化和胞嘧啶羥甲基化Mitochondrial DNA (mtDNA) has been reported to contain 5-methylcytosine (5mC) at CpG dinucleotides, as in the nuclear genome, but neither the mechanism generating mtDNA methylation nor its functional signi ficance is known. We now report the presence of 5-hydroxymeth

2、ylcytosine (5hmC) as well as 5mC in mammalian mtDNA, suggesting that previous studies underestimated the level of cytosine modification in this genome. DNA methyltransferase 1 (DNMT1) translocates to the mitochondria,driven by a mitochondrial targeting sequence located immediately upstream of the co

3、mmonly accepted translational start site.線粒體DNA（mtDNA）已經報道在核基因組中CpG二核苷酸上包含5甲基化胞嘧啶（5mC），但是mtDNA甲基化產生的機制和它功能的重要性都還不知道。我們現在報道5hmC5羥甲基化胞嘧啶和5mC一樣存在于哺乳動物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甲基化轉移酶1（DNMT1）轉移到線粒體，被一個線粒體靶序列？驅動立刻定位到大家公認的轉錄起始位點？的上游。This targeting sequence is conserved across mammals, and the enco

4、ded peptide directs a heterologous protein to the mitochondria. DNMT1 is the only member of the three known catalytically active DNA methyltransferases targeted to the mitochondrion. Mitochondrial DNMT1 (mtDNMT1) binds to mtDNA, proving the presence of mtDNMT1 in the mitochondrial matrix. mtDNMT1 ex

5、pression is up-regulated by NRF1 and PGC1, transcription factors that activate expression of nuclear-encoded mitochondrial genes in response to（響應，反應） hypoxia, and by loss of（失去） p53, a tumor suppressor known to regulate mitochondrial metabolism. Altered mtDNMT1 expression asymmetrically affects exp

6、ression of transcripts from the heavy and light strands of mtDNA. Hence, mtDNMT1 appears to be（好像是，仿佛） responsible for（是的原因，為負責） mtDNA cytosine methylation, from which 5hmC is presumed to be derived, and its expression is controlled by factors that regulate mitochondrial function.這個靶序列在哺乳動物中是保守的，編碼的

7、多肽引導一個外源蛋白到線粒體上。DNMT1是三個已知催化活性的DNA甲基化轉移酶中唯一一個針對線粒體的。線粒體DNMT1（mtDNMT1）結合mtDNA，證明線粒體基質中存在mtDNMT1。NRF1和PGC1上調mtDNMT1的表達，轉錄因子激活核編碼線粒體基因的表達是對低氧的反應，失去P53，一個腫瘤抑制因子已知可調節線粒體新陳代謝。改變mtDNMT1表達不對稱地影響mtDNA重鏈和輕鏈的轉錄表達。因此，mtDNMT1好像是mtDNA胞嘧啶甲基化的原因，推測它衍生出5hmC，調節線粒體功能的因子控制mtDNMT1的表達。正文In the nucleus, cytosine methylati

8、on cooperates with（合作，協作） N-terminal histone modifications to establish a silenced chromatin structure(1), thus（從而） regulating nuclear gene expression. Methylation patterns are established in the developing embryo by two de novo DNA methyltransferases, DNMT3a and -3b (2). Maintenance of this pattern

9、 in somatic cells is believed to be（被認為是） the predominant function of DNMT1, with functional cooperation evident between the two groups of enzymes (3). Cytosine methylation is essential for（是。必需的） normal development, and deletion of （缺失，刪除）DNMT1 results in embryonic lethality in mice and mitotic cat

10、astrophe in cultured cells (4).在細胞核中，胞嘧啶甲基化與N末端組蛋白修飾共同協作去建立一個沉默的染色體結構，從而調節和基因的表達。在發育的胚胎中通過兩種從頭DNA甲基化轉移酶（DNMT3a，3b）建立甲基化模式。人們認為在體細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育必需的，缺乏DNMT1導致小鼠胚胎致死和培養細胞有絲分裂障礙。Recently, the presence of significant levels of 5-hydroxymethyl-cytosine (5hmC) was demonstrat

11、ed in DNA from neurons, brain(5), and embryonic stem cells (6). 5hmC is derived from 5-methylcytosine (5mC) oxidation catalyzed by the TET family of methylcytosine oxygenases, and its functional significance is under intense investigation. This modification is likely to（有可能） have an impact on（對于有影響）

12、 local chromatin structure, and it has been proposed that （人們已經提議）5hmC acts as（充當，擔任） an intermediate in active or passive demethylation (7).最近，已經證明在神經、腦和胚胎干細胞DNA中存在顯著水平的5hmC。5hmC來源于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查的。這種修飾很可能對局部的染色體結構有影響，人們已經提議5hmC充當了在主動去甲基化和被動去甲基化的中間物。Cytosine methylation of

13、mitochondrial DNA (mtDNA) has been controversial and, remarkably, infrequently studied. The earliest study, conducted over three decades ago, reported that there was no methylation of mtDNA (8). Subsequently, low levels of methylation restricted to CpG dinucleotides were reported in mitochondria of

14、several species, using methylation-sensitive restriction endonuclease cleavage and nearest-neighbor analysis(9 11).引人注目的是，線粒體胞嘧啶甲基化（mtDNA）是引起爭論和很少被研究的。大約30年前，最早的研究中報道mtDNA沒有甲基化（8）。隨后，使用甲基化敏感限制性核酸內切酶分裂和進行最近鄰分析，在幾個物種的線粒體中報道低水平甲基化限制在CpG二核苷酸（9-10）。Mammalian mtDNA shows a similar level of CpG suppression

15、 to that of nuclear DNA (12), suggesting that 5mC is susceptible to（對敏感，易受影響）mutation in mtDNA. To date（迄今為止）, 5mC is the only modified base（修飾堿基） described in mtDNA, but the mechanisms establishing and maintaining mtDNA methylation, and the functional significance of this modification in mtDNA, are

16、 not known.哺乳動物mtDNA與核DNA的CpG抑制顯示了一個相似的水平（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，但是這個建立和維持mtDNA甲基化的機制，和這個修飾在mtDNA中功能性意義還是未知的。Mammalian mtDNA is a 16.5-kb double-stranded, circular molecule, present in multiple copies per mitochondrion (13).The mitochondrial genome encodes 13 of the proteins p

17、resent in the respiratory chain complexes of mammalian mitochondria, as well as two ribosomal RNAs and 22 transfer RNAs specific to this organelle. All other mitochondrial proteins, including those required for mtDNA replication and transcription, are encoded in the nucleus and translocated to the m

18、itochondria using specialized import systems which often involve N-terminal mitochondrial targeting sequences (MTSs) (14). In contrast to the nuclear genome, mtDNA is not complexed with histones. However, mtDNA is present in protein-containing complexes called nucleoids, each containing multiple cop

19、ies of mtDNA bound to a complex mixture of proteins (15).哺乳動物mtDNA是一個16.5kb雙鏈環狀分子，每個線粒體中存在多個拷貝（13）。線粒體基因組編碼哺乳動物線粒體呼吸鏈上13個蛋白復合體，兩個核糖體RNA和22轉運RNA專門針對這個細胞器的。其他所有線粒體蛋白，包括mtDNA復制和轉錄需要的，在核中編碼并使用專門的導入系統轉運到線粒體，專門的導入系統經常包括N末端線粒體靶序列（MTSs）（14）。對比核基因組，mtDNA沒有與組蛋白形成復合體。然而，mtDNA存在于包含蛋白質的復合物中，稱之為擬核，每個包含mtDNA多個拷貝結合

20、到一個復雜的蛋白質混合物上（15）。Transcription of the mitochondrial genome is thought to be（被認為是） coregulated with nuclear components of the respiratory chain complexes (16). In mammals, oxidative stress results in stabilization of peroxisome proliferator-activated receptor -coactivator 1 (PGC1 ), which activates

21、the transcription of several nuclear-encoded transcription factors, including nuclear respiratory factor 1 (NRF1). PGC1 and NRF1 form a complex that in turn（依次，輪流） up-regulates transcription of transcription factor of activated mitochondria (TFAM) and multiple members of mitochondrial respiratory ch

22、ain complexes (17).線粒體基因組的轉錄被認為是與呼吸鏈復合物核組成部分共同調節的（16）。哺乳動物中，氧壓導致了氧化物酶體增殖物激活受體輔激活因子1（PGC1）的穩定性，它激活幾個核編碼轉錄因子的轉錄，包括核呼吸因子（NRF1）。PGC1和NRF1形成一個復合體依次上調激活了的線粒體轉錄因子的轉錄（TFAM）和許多線粒體呼吸鏈復合物成員（17）。Several nuclear-encoded genes involved in mitochondrial function, including PGC1 (18), are regulated by DNA methylati

23、on. Conversely, it has been suggested that（表達觀點） mitochondria are able to（能夠） influence cytosine methylation levels in the nucleus by modulating the flux of one-carbon units for the generation of S-adenosylmethionine, the methyl donor in DNA methylation (19). Thus, epigenetic regulation of nuclear g

24、ene expression appears to have （似乎有）a mitochondrial component. The presence of cytosine methylation in mtDNA led us to question whether this epigenetic modification might play a role in the co-ordinated regulation of mitochondrial gene expression from both nuclear and mitochondrial genomes.通過DNA甲基化調

25、節幾個核編碼的基因包含線粒體的功能，包括PGC1（18）。相反地，人們認為線粒體能夠通過調制S-腺苷甲硫氨酸產生的一碳單位的流出，影響細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，核基因表達的表觀調節似乎有線粒體的成分。mtDNA中胞嘧啶甲基化的存在使我們去思考表觀修飾可能在從核和線粒體兩個基因組對線粒體的基因表達協同調節中扮演著重要的角色。ResultsHuman and Mouse DNMT1 Encode Mitochondrial Targeting Sequences.Early reports of DNA methylation in the

26、mitochondrial genome(9 11) led us to ask whether one or more of the catalytically active mammalian DNA methyltransferases might be targetedto mitochondria. Examination of the 5UTR and 5flanking genomic DNA upstream of the published transcription start sites(20) for both human and mouse DNMT1 reveale

27、d that sequence equivalent to 101 codons in human and 63 codons in mouse DNMT1 was in-frame with the highly conserved amino acid sequence of DNMT1, starting with the ATG reported (20) to bethe primary translational start codon (Fig. 1 A and B). 結果人類和小鼠DNMT1編碼線粒體靶序列早期報道的線粒體基因組DNA甲基化（9-11）引導我們去問是否有一個或

28、者更多催化活性哺乳動物DNA甲基化轉移酶可能是針對線粒體的。檢查人類和小鼠DNMT1公布的轉錄起始位點（20）基因組DNA上游5UTR和5側翼區，顯示人類101個密碼子和小鼠DNMT1 63個密碼子序列等效是具有高度保守氨基酸序列的DNMT1編碼框，報道的ATG起始（20）是主要的轉錄起始密碼（Fig.1A和B）。This upstream sequence includes two additional in-frame codons for methionine, each in a moderate context for ribosome binding(21); the upstre

29、am ATG codons are denoted ATG1 and ATG2,whereas the published translation start is shown as ATG3. RT-PCR using sense primers located over ATG1 or ATG2 and anti-sense primers crossing the exon 1-2 boundary by 4 nucleotides detected transcripts capable of encoding these N-terminal extensions in human

30、and mouse cells. Transcripts initiating upstream of ATG1 in mouse and ATG2 (but not ATG1) in human mRNA were easily detected (Fig. 1C), suggesting the utilization of an up-stream transcription start site encoding an N-terminal extension.這個上游序列包括兩個額外的甲硫氨酸編碼框密碼子，每個都在序列中間于核糖體結合（21）；上游ATG密碼子表示ATG1和ATG2，

31、而已發布的轉錄起始是指ATG3。人類和小鼠細胞中，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4個核苷酸檢測編碼這些N末端的延長的轉錄能力。mRNA轉錄起始上游小鼠中ATG1和人類中ATG2（不是ATG1）是容易測定的。Mouse or human DNMT1 isoforms containing this additional N-terminal sequence were predicted by MitoProt II (http:/ihg.gsf.de/ihg/mitoprot.html) (22) to be targeted to（針對）

32、the mitochondria with very high probability, compared with proteins beginning at the published start codon, ATG3 ( Table S1 ). The genome databases also contain upstream sequences for chimpanzee, rat,and cow DNMT1; in each species, one or more in-frame potential start codons encode a peptide with a

33、strong probability ( > 90 %) of mitochondrial localization (Table S1 ). All are predicted to form amphiphilic -helices, although sequence conservation between them is low, as is often the case（情況常常如此，這是常有的事） for miochondrial leader peptides across species.通過MitoProt預測小鼠和人類DNMT1亞型包括這個額外的N末端的序列（22）

34、針對線粒體具有很高的可能性，與從公布的起始密碼子ATG3（Table S1）開始蛋白相比較。基因組數據庫也包括黑猩猩，大鼠和牛DNMT1上游序列；在每個物種中，一個或者更多編碼框內潛在的起始密碼子編碼一個具有定位線粒體很大可能性（90%）的多肽（Table S1）。預測所有形成兩性分子-螺旋，盡管他們之間的序列保守性很低，線粒體引導肽穿越物種這是常有的事。Immunoblots of purified mitochondria from mouse embryonic fibroblasts (MEFs) and HCT116 human colon carcinoma cells showe

35、d the presence of DNMT1 (Fig. 2A) but not DNMT3a orDNMT3b in this organelle (Fig. 2 B). Full-length DNMT1 and a smaller peptide are detected by an N-terminal DNMT1 antibody, suggesting that proteolytic processing occurs upon entry into the mitochondria. Absence of the nuclear marker H3K4me3 in the m

36、itochondrial fraction indicated purity from contamination by nuclear material, the primary site of localization of DNA methyltransferases.來自小鼠胚胎成纖維細胞（MEFs）和人結腸癌細胞HCT116純化線粒體的免疫印跡分析顯示在這個細胞器中存在DNMT1(Fig.2A)，不存在 DNMT3a或DNMT3b（Fig.2B）。用一個N末端DNMT1抗體檢測全長DNMT1和較小的多肽，表明蛋白酶加工發生在進入線粒體后。線粒體斷片中缺少細胞核標志H3K4me3顯示純

37、化被核物質污染，定位DNA甲基化轉移酶的主要位點。We cloned the mouse and human leader sequences, from ATG1 to upstream of ATG3, in-frame with the C-terminal GFP tag of pcDNA6.2/GFP, and transfected the plasmids into NIH/3T3 fibroblasts. Confocal microscopy showed that both human and mouse leader sequences targeted GFP to t

38、he mitochondria,indicated by colocalization of MitoTracker Red with green fluorescence (Fig. 2 C). Mitochondria in untransfected cells within the same visual field remained red in the merged photomicrographs, serving as（作為，充當，擔任） negative controls for colocalization, whereas a chloramphenicol acetyl

39、 transferase (CAT)-GFP control plasmid remained cytosolic. We also transfected these constructs into HCT116 human colon carcinoma cells for immunoblot analysis of purified mitochondria using anti-GFP antibody ( Fig. S1).我們克隆小鼠和人類的前導序列，從ATG1到ATG3的上游，編碼框內具有C末端具有GFP標簽質粒pcDNA6.2/GFP，轉染到NIH/3T3成纖維細胞。共聚焦顯

40、微鏡表明人和小鼠兩者的前導序列將GFP定位到線粒體，通過MitoTracker Red和綠色熒光共定位體現（Fig.2C）。未轉染的細胞內的線粒體在合并的顯微照片相同視野內保持紅色，作為共定位的陰性對照，然而氯霉素乙酰轉移酶(CAT)-GFP控制質粒保持細胞質基質。我們也轉染這些結構到人結腸癌細胞HCT116用抗GFP抗體免疫印跡分析純化的線粒體（Fig.S1）。HCT116 mitochondria clearly accumulated GFP. Thus, human and mouse leader peptides represent bona fide MTSs capable o

41、f tracking heterologous proteins to this organelle. Each MTS is able to operate across species, indicating functional conservation.HCT116線粒體清晰地積累GFP。因此，人類和小鼠引導肽體現這些細胞器MTSs法真實的跟蹤外源性蛋白檢測能力。每一個MTS能夠穿越物種操作，表明功能保守。mtDNMT1 Expression Is Regulated by Factors That Respond to Oxidative Stress. MatInspector (

42、http:/ /www.genomat ix.de/en/index .html) predicted a binding site for NRF1 in both human and mouse DNMT1.This consensus sequence was located（位于）over one of the upstream in-frame start codons and was conserved in all other mammalian species studied ( Table S1 ;Fig.3 A). Under conditions of oxidative

43、 stress, the coactivator PGC1 activates and interacts with NRF1 to up-regulate multiple nuclear-encoded mitochondrial genes (17).Accordingly, we transiently transfected NRF1, PGC1 , or both together into HCT116 cells and analyzed the levels of mitochondrial DNMT1 (mtDNMT1) by immunoblot (Fig. 3B). 對

44、氧化壓力反應的因子調節mtDNMT1表達MatInspector在人類和小鼠的DNMT1中預測了NRF1一個結合位點。這個共有序列位于越過上游編碼框起始密碼子之一，在研究的所有其他哺乳類物種中是保守的（Table S1；Fig.3A）。在氧化壓力的條件下，共激活劑PGC1激活并且與NRF1相互作用去上調多個核編碼的線粒體基因（17）。因此，我們瞬時轉染NRF1，PGC1或者兩者一起轉染到HCT116細胞，通過免疫印跡分析線粒體DNMT1的水平（Fig.3B）。A small increase in mtDNMT1 was seen in cells transfected with NRF1

45、or PGC1 alone, whereas cotransfection with both PGC1 and NRF1 resulted in an approximately fivefold increase in mtDNMT1 relative to control. Thus, this locus is sensitive to regulation by activators that respond to oxidative stress.在單獨轉染NRF1或者PGC1的細胞中看到mtDNMT1有一個很小的增加，然而兩者共轉染導致相對對照組mtDNMT1有一個大約5倍的增加

46、。因此，這個位點對于氧化壓力反應的調節子的調節是敏感的。The NRF1 binding site is coincident with a p53 consensus binding site (Fig. 1 A and B), which we previously demonstrated to（向說明，向證明）repress DNMT1 transcription (23). Our earlier study showed a three- to sixfold increase in DNMT1 transcription following either activation o

47、r genetic deletion of p53 in HCT116 cells and MEFs. Because p53 is known to regulate mitochondrial respiration (24), we asked whether this tumor suppressor protein also affected mtDNMT1 mRNA expression.NRF1結合位點和P53共識結合位點是一致的（Fig.1A和B），這個我們之前已經證明抑制DNMT1轉錄（23）。我們之前的研究隨著在HCT116細胞和MEFs細胞中P53的激活或者基因缺失DNM

48、T1轉錄顯示了一個3-6倍的增加。因為P53是人們所周知調節線粒體呼吸作用（24），我們思考這個腫瘤抑制蛋白是否也影響mtDNMT1 mRNA表達。We used RT-quantitative(q)PCR with primers that distinguish the mitochondrial transcript from the total DNMT1 transcript (Fig. 1 C); the mitochondrial transcript comprised 1 2% of the total DNMT1 synthesized in log-phase MEFs

49、or HCT116 cells. The relative abundance of mtDNMT1 transcript increased sixfold in p53/ MEFs compared with WT MEFs, whereas total DNMT1 mRNA increased threefold (Fig. 3C), suggesting a preferential up-regulation of the mitochondrial transcript in cells lacking p53. Immunoblot analysis of these isoge

50、nic cells showed a striking increase in mtDNMT1 protein with loss of p53 (Fig. 3D).我們用從總的DNMT1轉錄物中區分線粒體的轉錄物的引物，進行RT-qPCR（Fig.1C）；線粒體轉錄占對數階段MEFs或HCT116細胞總合成DNMT1的1-2%。P53-/- MEFs與野生型MEFs細胞相比mtDNMT1轉錄物的相對豐都增加了6倍，然而總的DNMT1 mRNA增加了3倍（Fig.3C），表明在缺少P53的細胞中線粒體轉錄物的一個優先上調。這些等基因細胞的免疫印跡分析在缺乏P53細胞中mtDNMT1蛋白顯示了一

51、個顯著的增加（Fig.3D）。Gene-Specific Changes in Mitochondrial Transcription. We asked whether this mtDNMT1 overexpression was reflected in an alteration in transcription of the mitochondrial genome (Fig.3E).NADH dehydrogenase subunit 6 (ND6), the only protein-coding gene on the light (L) strand, was signifi

52、cantly underexpressed in response to increased mtDNMT1, suggesting a role for mtDNA methylation in repression of L-strand transcription. On the heavy(H) strand, ATPase subunit 6 (ATP6) and cytochrome c oxidase subunit 1 (COX1) were unaltered in their expression levels.However, NADH dehydrogenase sub

53、unit 1 (ND1), the first H-strand protein-coding region following the ribosomal RNA genes,was significantly increased in response to elevated mtDNMT1.These data support a gene-specific effect on mitochondrial gene transcription, as discussed below.線粒體轉錄中獨特基因的改變我們詢問mtDNMT1過表達是否反應線粒體基因組轉錄的改變（Fig.3E）。NA

54、DH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，表明mtDNA甲基化在抑制輕鏈轉錄中的一個作用。在重鏈上，ATPase亞基6（ATP6）和細胞色素酶C氧化酶亞基1（COX1）在他們表達水平上是不變的。然而，NADH脫氫酶亞基1（ND1），第一條輕鏈編碼蛋白區域跟隨核糖體rRNA基因，在反應提高的mtDNMT1中是顯著增加的。這些數據支持了一個獨特基因影響線粒體基因轉錄，正如下面討論的。Mitochondrial DNMT1 Is Bound to mtDNA. We created an HCT116 cell line (25) in which o

55、ne endogenous allele of DNMT1 carries a C-terminal tandem-affinity purification (TAP) tag (26). TAP-tagged DNMT1 translocated efficiently to mitochondria (Fig.4B). We therefore used these cells to ask whether mtDNMT1 interacted directly with mtDNA. Formaldehyde-crosslinked mitochondrial lysates were

56、 immunoprecipitated with IgG beads(26), and qPCR with primers specific for mtDNA (Table S2 ) was used to quantitate the interaction between mtDNMT1 and mtDNA. Immunoprecipitates from TAP-tagged cells were substantially enriched for mtDNA in comparison with immunoprecipitates from untagged cells, exc

57、ept for an amplicon containing no CpG dinucleotides, which gave equally low signal from both cell lines (Fig.4C). These data suggest CpG-dependent interaction of mtDNMT1 with the mitochondrial genome and confirm the localization of this protein to the mitochondrial matrix. 線粒體DNMT1結合到mtDNA我們建立了一個HCT

58、116細胞系（25），在這個細胞系中一個內源性DNMT1等位基因攜帶一個C末端串聯親和純化（TAP）標簽（26）。TAP標簽的DNMT1高效地轉移進入線粒體（Fig.4B）。因此我們用這些細胞去詢問mtDNMT1是否與mtDNA直接相互作用。甲醛交聯線粒體溶菌產物用IgG小珠子免疫沉淀（26），用mtDNA專門的引物進行qPCR（Table S2）去定量mtDNMT1和mtDNA間的相互作用。TAP標簽細胞進行免疫共沉淀相比沒有標簽的細胞的免疫共沉淀與mtDNA大量的富集，除了不包含CpG二核苷酸的擴增子，兩個細胞系都給了相等的低信號（Fig.4C）。這些數據顯示mtDNMT1和線粒體基因組相

59、互作用依賴CpG，證實了這個蛋白定位到線粒體基質中。Interaction was evident in the D-loop control region,which carries the mitochondrial origin of replication and promoters, as well as in rRNA and protein-coding regions. The level of enrichment was dependent on the target amplicon; five of the six regions probed showed a three- to fivefold enrichment of mtDNA sequences. However, qPCR of the region covering the junction between 12S and 16S rRNA genes (primer 2) showed only