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MitoTracker Red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）。
HCT116 mitochondria clearly accumulated GFP. Thus, human and mouse leader peptides represent bona fide MTSs capable of tracking heterologous proteins to this organelle. Each MTS is able to operate across species, indicating functional conservation.
HCT116线粒体清晰地积累GFP。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。 mtDNMT1 Expression Is Regulated by Factors That Respond to Oxidative Stress.
MatInspector (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 stress, the coactivator PGC1α activates and interacts with NRF1 to up-regulate multiple nuclear-encoded mitochondrial genes (17).Accordingly, we transiently transfected NRF1, PGC1 α , or both together into HCT116 cells and analyzed the levels of mitochondrial DNMT1 (mtDNMT1) by immunoblot (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（Table S1；Fig.3A）。在氧化压力的条件下，共激活剂PGC1α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）。因此，我们瞬时转染NRF1，PGC1α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）。 A small increase in mtDNMT1 was seen in cells transfected with NRF1 or PGC1α alone, whereas cotransfection with both PGC1 α 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.
在单独转染NRF1或者PGC1α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。
The NRF1 binding site is coincident with a p53 consensus binding site (Fig. 1 A and B), which we previously demonstrated to（向说明，向证明）repress DNMT1 transcription (23). Our earlier study showed a three- to sixfold increase in DNMT1 transcription following either activation or 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mtDNMT1 mRNA表达。
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 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 isogenic cells showed a striking increase in mtDNMT1 protein with loss of p53 (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DNMT1 mRNA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-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 significantly 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 subunit 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. 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。
Mitochondrial DNMT1 Is Bound to mtDNA.
We created an HCT116 cell line (25) in which one 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 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, except 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. 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（Table S2）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mtDNMT1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。
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 twofold enrichment in binding of mtDNMT1-TAP. The density of CpG dinucleotides in this amplicon is <50% that in all other amplicons analyzed, suggesting that interaction of mtDNMT1 with mtDNA is proportional to CpG density, and supporting a functional role for mtDNMT1 in establishment and maintenance of mtDNA methylation.
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16S rRNA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＜50%，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-Hydroxymethylcytosine Is Present in mtDNA.
We immunoprecipitated randomly sheared mtDNA with an antibody to 5mC or 5hmC and probed the precipitated DNA by qPCR to determine the presence and relative abundance of these two modified bases in mtDNA (Fig. 5A and B). Immunoprecipitated samples were enriched 10- to 20-fold for 5mC relative to IgG control for all regions tested. mtDNA immunoprecipitated using anti-5hmC was highly enriched (85- to 580-fold) relative to IgG controls,except across the D loop (primer 27), which was enriched 38-fold. The specificity of each antibody for its respective modification was confirmed using control DNA in which every cytosine was converted to either 5mC or 5hmC (Fig. S2). The presence of both cytosine modifications in mtDNA suggests that earlier studies underestimated the degree of epigenetic modification of the mitochondrial genome.
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。
We used phage T4 5hmC-β -glucosyltransferase (β-gt) (27) to determine the presence of 5hmC at Gla1 restriction endonuclease cleavage sites (28). Control experiments using defined DNA sequences containing cytosine, 5mC, or 5hmC confirmed that Gla1 cleaved only sites modified by methylation or hydroxymethylation, but not sites containing glucosylated 5hmC (Fig.S3A). 我们用T4噬菌体5hmC-β-葡糖基转移酶（β-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）。
Protection of mtDNA from cleavage by 5hmC glucosylation was assessed by endpoint (Fig. S3B and C) and qPCR (Fig. 5C). 5hmC was present in three different amplicons from human mtDNA and two amplicons from mouse mtDNA or genomic DNA. Amplicons containing two Gla1 restriction sites each (amplicons ATP6, 12S,and 16S-3) showed 50% protection in comparison with amplicons with a single Gla1 site (amplicons 2 and 16S-2), suggesting a similar level of 5hmC at all restriction sites tested. A mouse amplicon devoid of （没有，缺乏）Gla1 sites (ATP6/COX3) was protected from cleavage irrespective of 5hmC glucosylation (Fig. S3 C).
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）。 Discussion
Cytosine methylation of the mitochondrial genome has remained largely overlooked, in part because early reports using nearest-neighbor analysis indicated that this modification was present at only 2 –5% of CpG dinucleotides (11), well below the level of methylation seen in the nucleus. The data presented here show a 10- to 20-fold enrichment of mtDNA sequences in immunoprecipitates using 5mC antibody, somewhat lower than that usually obtained from genomic DNA (～100-fold for CpG islands).
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。
This likely reflects the CpG-sparse nature of the mitochondrial genome, which does not contain CpG islands. We demonstrate here the presence of 5hmC in mtDNA using two independent assays. Thus, epigenetic modification of cytosines in the mitochondrial genome is likely much more frequent than previously believed. In the nucleus, 5hmC is generated from 5mC by the action of the TET family of methylcytosine oxygenases (6).There is not yet evidence regarding the presence or absence of these enzymes in mitochondria, and the TET family proteins or loci do not contain recognizable mitochondrial targeting sequences (14). We therefore cannot rule out（排除，取消）the possibility of a different mechanism for the generation of 5hmC, including covalent addition of 5-hydroxymethyl groups directly to DNA cytosine residues by mtDNMT1 (29) using formaldehyde generated from mitochondrial mixed-function oxidases.
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）。
The apparently lower enrichment for 5hmC in the D-loop control region most likely reflects the
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