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

less efficient amplification of a longer fragment(833 bp compared with 112 –238 bp) from mtDNA sheared to an average size of 300–400 bp. However, the D loop exists as a stable triple-helical structure containing an RNA primer required for initiation of mtDNA replication (13), and we have found this region to be resistant to in vitro methylation by M.Sss1 cytosine methyltransferase. It is therefore possible that the kinetics of epigenetic modification in this region of the mitochondrial genome might be different from those in coding regions.

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。

The function of 5hmC in the nuclear genome is not yet clear. It has been proposed that 5hmC is an intermediate metabolite in active demethylation of the genome by repair enzymes (30), in passive demethylation as a result of lack of recognition by enzymes involved in maintenance methylation (31)句型, or that it alters local chromatin structure because 5hmC is not recognized by 5-methylcytosine-binding proteins (7). The role of 5hmC in the mitochondrial genome likely involves one or more of these processes. Although quantitative measurements of the relative abundance of 5hmC and 5mC can be achieved using methylated DNA immunoprecipitation (Me-DIP), 5hMe-DIP, HPLC, or enzymatic methods, mapping the location and distribution of 5hmC in either the nuclear or mitochondrial genome is not yet technically feasible, because this modified base is indistinguishable from 5mC by bisulfite modification (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）。

This study reports a mitochondrial isoform of DNA methyltransferase 1, which is the only member of the catalytically active mammalian DNA methyltransferase family found in this organelle. The conservation of an ORF encoding a mitochondrial targeting sequence upstream of the commonly accepted translational start codon across multiple mammalian species suggests an important role for this enzyme in mitochondrial function.

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。

Although DNMT1 is generally considered to be （认为是）the maintenance DNA methyltransferase, it is able to methylate completely unmethylated DNA in vitro with an efficiency that exceeds that of the de novo methyltransferases DNMT3a and -3b (32). Thus,DNMT1 appears to be capable of both initiating and maintaining cytosine methylation in the nucleus, and the lack of de novo methyltransferases in mitochondria implicates mtDNMT1 in both processes in this organelle.

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。

We show that mtDNMT1 binds to the mitochondrial genome in a manner（在某种意义上，在某种程度上） proportional to the density of CpG dinucleotides. Of particular relevance is the binding of mtDNMT1 to the D-loop control region, which carries the promoters driving transcription initiation of both heavy and light strands, supporting a role for mtDNMT1 in regulation of mitochondrial gene expression. The asymmetric, gene-specific alteration in mitochondrial transcription patterns shown here suggests diverse roles for mtDNMT1 and cytosine modification in this organelle.

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。

Decreased expression of ND6 on the L strand implies that cytosine methylation in mtDNA represses gene expression from the light-strand promoter, as it does in the nucleus. However, increased transcription of ND1 with no change in transcription of ATP6 or COX1 raises the possibility of a different mode of action on the H strand. A binding site for mitochondrial terminator factor 1 (MTERF1) is located between the end of the 16S rRNA gene and the translation start of ND1 (33). MTERF1 binds to both H-strand promoter 1(HSP1) and the terminator binding site (Fig.4A), forming a transcription loop that maintains high-level production of rRNA. Transcripts initiating at HSP2 produce polycistronic messages encoding the entire H strand (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16S rRNA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）。

Our data raise the possibility that mtDNMT1, either through modification of CpG dinucleotides or by direct protein–protein interaction, interferes with MTERF-dependent transcription termination, allowing read-through from HSP1 to the next transcriptional unit (ND1) without impacting polycistronic mRNA synthesis from HSP2. 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。

We show here that DNMT1 is present in the mitochondrial matrix, bound to mtDNA, and modifies transcription of the mitochondrial genome in what appears to be a gene-specific fashion. We report the presence of both 5hmC and 5mC in mtDNA , suggesting that earlier studies may have underestimated the proportion of modified cytosines in this genome. Hence,mtDNMT1 appears to be responsible for（是。。的原因） the establishment and maintenance of cytosine methylation in mtDNA, from which 5hmC is presumably derived. Our data support a role for epigenetic modification of the mitochondrial genome in regulation of mitochondrial transcription.

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，mtDNMT1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。 Materials and Methods 材料和方法

Cell Lines. HCT116 p53+/+ and HCT116 p53?/?were obtained from Bert Vogelstein, Johns Hopkins University (Balti more, MD). Primary MEFs were prepared from E12.5 –E13.5 embryos. 细胞系。HCT116 p53+/+和P53?/? 从Johns Hopkins University大学的Bert Vogelstein博士获得(Balti more, MD)。主要的MEFs细胞来自E12.5-E13.5胚胎。

Plasmids and Transfections. Primers used are listed in Table S2. Mitochondrial targeting sequences were amplified from random-primed human and mouse cDNAs. Murine NRF1 cDNA was obtained from the American Type Culture Collection and recloned into pDEST26/C-FLAG. PGC1α plasmid was a gift from Gregorio Gil, Virginia Commonwealth University. Cells were transfected using Polyjet liposomes (ProSci) (HCT116) or nucleofection (Amaxa) (MEF and NIH/3T3) according to the manufacturers’ specifications, and were harvested 48h after transfection.

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NRF1 cDNA获得于美国标准菌库然后重新克隆到pDEST26/C-FLAG。PGC1α质粒由 Virginia Commonwealth University大学的Gregorio Gil馈赠。使用Polyjet 脂质体(ProSci) (HCT116) 或核转染(Amaxa) (MEF and NIH/3T3)按照制造商说明书转染细胞，转染48h后收获。 Mitochondrial Purification and Immunoblot Analysis . Mitochondria were purified by dounce homogenization and differential centrifugation in the presence of complete protease inhibitors (Roche) (34). Proteins were resolved on 4 – 15% gradient SDS/PAGE gels. Antibodies used were anti-DNM T1 amino acids 1–10 (Abcam), anti-tubulin and anti –voltage-dependent anion carrier (VDAC) (Pierce), anti-H3K4me3 (Upstate Biotechnology), anti-DNMT3a and anti-DNMT3b (Imgenex), anti-GFP (Invitrogen), and anti-TAP (Open Biosystems). Protein was loaded onto SDS/PAGE gels to approximate equal cell equivalents, so that an equal signal for each compartment-specific antibody was obtained (whole-cell lysate, 75μg; cytosol, 25μg; mitochondria, 18μg).

线粒体纯化和免疫印迹分析。通过dounce均质化和差速离心在完整的蛋白酶抑制剂（Roche）存在时纯化线粒体（34）。蛋白在4-15%梯度SDS/PAGE凝胶分离。抗体用的是抗DNMT1氨基酸1-10（Abcam），抗微管蛋白和抗电压依赖性阴离子载体（VDAC）（Pierce），抗H3K4me3（Upstate Biotechnology），抗DNMT3a和抗DNMT3b（Imgenex），抗GFP（Invitrogen），和抗TAP（Open Biosystems）。向SDS/PAGE凝胶上样大致相等的细胞等量蛋白，以便获得特定分隔抗体的一个相等的信号（whole-cell lysate, 75μg; cytosol, 25μg; mitochondria, 18μg）。 Confocal Microscopy. NIH/3T3 cells were plated onto poly-L-lysine-coated coverslips and fixed with 4% paraformaldehyde 48h after transfection . Cells were stained with 1nM MitoTracker Red (Molecular Probes) for 15 min washed three times with PBS, and mounted onto glass slides with ProLong Gold antifade reagent with DAPI (Invitrogen). Microscopy was carried out using a Leica TCS-SP2 AOBS confocal scanning microscope.

共聚焦显微镜。转染48h后的NIH/3T3细胞接种到涂有多聚L赖氨酸的玻片上，用4%多聚甲醛固定。细胞用1nM MitoTracker Red (Molecular Probes)染色15min后用PBS洗3遍，然

后用DAIP ProLong Gold antifade reagent安装在载玻片上（Invitrogen）。显微镜检查使用Leica TCS-SP2 AOBS 共聚焦显微镜。

Gene Expression. Total RNA was isolated using TRIzol (Invitrogen), DNase I-treated, and reverse-transcribed with SuperScript III (Invitrogen ) and random hexamers. Gene expression was determined using qPCR with Quantitect SYBR Green PCR Mastermix (Qiagen). Values were normalized to 18S rRNA for mitochondrial transcription or to β-actin for mtDNMT1 quantitation. 基因表达。用TRLzoL（Invitrogen）提取总RNA，DNaseⅠ处理，用SuperScript III （Invitrogen）反转录和随机引物。Quantitect SYBR Green PCR Mastermix （Qiagen）进行qPCR检测基因的表达。线粒体转录的值被18SrRNA标准化或mtDNMT1定量被β-actin标准化。

Statistics. Statistical analyses of differential mitochondrial gene expression profiles were performed using a random-effects ANOVA随机方差分析 using the statistical package JMP version 7.0 (SAS). The least-squares mean for each gene for WT and p53?/ ?MEFs was obtained along with standard errors, and each dataset was normalized to 18S rRNA expression. Three independent sets of biological samples were analyzed for each gene, with triplicate technical replicates in each sample. The ANOVA model included technical replicates as nested effects and biological replicates as random effects. The corresponding 95% confidence intervals (C.I.) were obtained using statistical methods for transformations(Delta method). All other qPCR experiments include multiple replicates from two or more independent experiments, normalized to relevant controls or to input DNA. SDs were computed using the formulation.

统计分析。不同线粒体基因表达图谱的数据分析用JMP version 7.0 (SAS)统计软件包进行随机方差分析。WT和 p53?/ ?MEFs细胞每个基因的最小二乘法伴随着标准误差获得，每个数据按照18SrRNA表达进行标准化。三个独立生物样本集进行每个基因的分析，每个样本进行三个技术重复。方差分析模式包括技术重复作为嵌套阈和随机生物学重复。用转换(Delta method)统计学方法获得相当于95%可信区间（C.I.）。所有其他的qPCR实验包括2到更多个独立实验的多个重复，按照相关对照或输入的DNA进行标准化。SDs是用下面公式计算的。 Mitochondrial Immunoprecipitation. Purified mitochondria from DNMT1-TAP and nontagged HCT116 cells were formaldehyde-cross-linked, lysed, and immunoprecipitated as described (35). Mitochondrial DNA was sheared using a Diagenode Bioruptor water bath sonicator to an average length of 400bp.We used 750μg mitochondrial extract in each mitochondrial immunoprecipitation (mtIP) . IgG beads were equilibrated in lysis buffer and added to mitochondrial lysates for overnight incubation at 4°C to isolate DNMT1-TAP/DNA complexes. 线粒体免疫共沉淀。DNMT1-TAP和没有标签HCT116细胞中纯化的线粒体被甲醛交联，细胞溶解和按照文献中免疫共沉淀（35）。线粒体DNA用 Diagenode Bioruptor水浴超音波样品震碎仪修剪为平均长度为400bp的片段。每个线粒体免疫共沉淀中（mtIP）我们使用750ug的线粒体提取物。IgG小珠子平衡到细胞溶解buffer中，加入线粒体溶解产物4℃孵育过夜分离DNMT1-TAP/DNA复合物。

Immunoprecipitated samples were processed as described (35) and purified DNA was analyzed by qPCR with 1μL mtIP DNA and Quantitect SYBR Green Mastermix. The a bundance of mtDNA was determined from a standard curve of purified mtDNA and is expressed as ng mtDNA immunoprecipitated. Values obtained for non-TAP-tagged HCT116 cells represent nonspecific

background. Primers used were from Lu et al. (35)(fragments of 800 – 900 bp, primers 1, 2, 3, and 27) or as listed in Table S2(fragments < 200bp). 免疫共沉淀样本按照文献中一样处理（35），用1μL mtIP DNA 和Quantitect SYBR Green Mastermix对纯化的DNA进行qPCR分析。mt DNA的丰度通过纯化mtDNA的一个标准曲线确定，mtDNA免疫共沉淀用ng表示。non-TAP-tagged HCT116 cells代表非特异性背景获得值。引物使用Lu et al. (35)(fragments of 800 – 900 bp, primers 1, 2, 3, and 27) or as listed in Table S2(fragments < 200bp)。

Mitochondrial 5mC and 5hmC Immunoprecipitation. Purified mtDNA (4 μg) was sheared to an average length of 400bp and rotated overnight at 4°C with 2ug IgG, anti-5mC, or anti-5hmC (Active Motif). The specificity of both antibodies for their respective cytosine modifications in DNA was verified using defined DNA substrates synthesized in the presence of dCTP, 5m-dCTP, or 5hm-dCTP (Active Motif). Precleared protein-G beads (Amersham) were used as previously described (23) to immunoprecipitate the antibody/DNA complexes, and DNA was purified from immunoprecipitates using proteinase K,organic extraction, and precipitation(23). The abundance of mtDNA was determined from a standard curve of purified mtDNA and is expressed as ng mtDNA immunoprecipitated relative to input values.

线粒体5mC和5hmC免疫共沉淀。纯化的mtDNA（4ug）被修剪为平均长度400bp与2ug IgG，抗5mC或抗5hmC（Active Motif）4℃旋转过夜。使用已经确定的DNA物质dCTP, 5m-dCTP, 或5hm-dCTP (Active Motif)合成，进行证明两个抗体对DNA中他们各自的胞嘧啶修饰是特异性的。已经证明蛋白G小珠子(Amersham)使用按照之前抗体/DNA复合物免疫共沉淀的描述（23）进行，使用蛋白酶K，有机萃取和沉淀的方法从免疫共沉淀中纯化DNA（23）。从一个纯化的mtDNA标准曲线中确定mtDNA丰度，用ng表示mtDNA免疫共沉淀相对于输入的值。 Sequence-Specific Detection of 5hmC.The presence of 5hmC at Gla1 restriction sites was determined using a Quest 5hmC Detection Kit (Zymo Research) as described by the manufacturer using 80ng mtDNA or total cellular DNA. Gla1 cleaves DNA only when restriction-site cytosines are methylated or hydroxymethylated; glucosylation of 5hmC residues results in protection from Gla1 cleavage. Control DNAs used to validate the assay were from Active Motif.

5hm特意序列的检测。5hmC在Gla1限制性位点上的存在是Quest 5hmC Detection Kit (Zymo Research)确定的，用80ng mtDNA或总细胞DNA按照说明书操作。Gla1分割DNA仅当限制性位点胞嘧啶是甲基化或者羟甲基化时；葡糖基化5hmC残基导致Gla1不被分割。使用对照DNAs去证实试剂盒的检测。