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

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-hydroxymethylcytosine (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 commonly accepted translational start site. 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。

This targeting sequence is conserved across mammals, and the encoded 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 expression is up-regulated by NRF1 and PGC1α, transcription factors that activate expression of nuclear-encoded mitochondrial genes in response to（响应，反应） hypoxia, and by loss of（失去） p53, a tumor suppressor known to regulate mitochondrial metabolism. Altered mtDNMT1 expression asymmetrically affects expression 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，为负责） mtDNA cytosine methylation, from which 5hmC is presumed to be derived, and its expression is controlled by factors that regulate mitochondrial function.

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甲基化转移酶中唯一一个针对线粒体的。线粒体DNMT1（mtDNMT1）结合mtDNA，证明线粒体基质中存在mtDNMT1。NRF1和PGC1α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

In the nucleus, cytosine methylation cooperates with（合作，协作） N-terminal histone modifications to establish a silenced chromatin structure(1), thus（从而） 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 in somatic cells is believed to be（被认为是） 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，删除）DNMT1 results in embryonic lethality in mice and mitotic catastrophe in cultured cells (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, the presence of significant levels of 5-hydroxymethyl-cytosine (5hmC) was demonstrated 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（对于有影响） local chromatin structure, and it has been proposed that （人们已经提议）5hmC acts as（充当，担任） an intermediate in active or passive demethylation (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。

Cytosine methylation of 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 several species, using methylation-sensitive restriction endonuclease cleavage and nearest-neighbor analysis(9 –11).

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）。 Mammalian mtDNA shows a similar level of CpG suppression to that of nuclear DNA (12), suggesting that 5mC is susceptible to（对敏感，易受影响）mutation in mtDNA. To date（迄今为止）, 5mC is the only modified base（修饰碱基） described in mtDNA, but the mechanisms establishing and maintaining mtDNA methylation, and the functional significance of this modification in mtDNA, are not known.

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。

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 present 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 mitochondria 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 copies of mtDNA bound to a complex mixture of proteins (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）。 Transcription of the mitochondrial genome is thought to be（被认为是） coregulated with

nuclear components of the respiratory chain complexes (16). In mammals, oxidative stress results in stabilization of peroxisome proliferator-activated receptor γ-coactivator 1α (PGC1 α ), which activates the transcription of several nuclear-encoded transcription factors, including nuclear respiratory factor 1 (NRF1). PGC1α and NRF1 form a complex that in turn（依次，轮流） up-regulates transcription of transcription factor of activated mitochondria (TFAM) and multiple members of mitochondrial respiratory chain complexes (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γ辅激活因子1α（PGC1α）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）。PGC1α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）。 Several nuclear-encoded genes involved in mitochondrial function, including PGC1 α (18), are regulated by DNA methylation. Conversely, it has been suggested that（表达观点） mitochondria are able to（能够） 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 gene 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PGC1α（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。 Results

Human and Mouse DNMT1 Encode Mitochondrial Targeting Sequences.

Early reports of DNA methylation in the mitochondrial genome(9 –11) led us to ask whether one or more of the catalytically active mammalian DNA methyltransferases might be targeted to mitochondria. Examination of the 5′UTR and 5′flanking genomic DNA upstream of the published transcription start sites(20) for both human and mouse DNMT1 revealed 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 be the primary translational start codon (Fig. 1 A and B). 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DNMT1 63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）。 This upstream sequence includes two additional in-frame codons for methionine, each in a moderate context for ribosome binding(21); the upstream 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 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.

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。

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（针对） 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 strong probability ( > 90 %) of mitochondrial localization (Table S1 ). All are predicted to form amphiphilic α -helices, although sequence conservation between them is low, as is often the case（情况常常如此，这是常有的事） for miochondrial leader peptides across species.

通过MitoProtⅡ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（Table S1）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（Table S1）。预测所有形成两性分子α-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。

Immunoblots of purified mitochondria from mouse embryonic fibroblasts (MEFs) and HCT116 human colon carcinoma cells showed the presence of DNMT1 (Fig. 2A) but not DNMT3a or DNMT3b 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 mitochondrial fraction indicated purity from contamination by nuclear material, the primary site of localization of DNA methyltransferases.

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。

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 the 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（作为，充当，担任） negative controls for colocalization, whereas a chloramphenicol acetyl 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).