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

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。