DNA methylation is one of the best studied epigenetic modifications. Alteration of the global DNA methylation level occurs in abnormal cells, such as those associated with cancers and Alzheimer’s disease. Several assays are used to determine the global DNA methylation level, including the bisulfite-based assay, high-performance liquid chromatography (HPLC)–based assay, enzyme-linked immunosorbent assay (ELISA), and methyl acceptance assay. However, these assays require several cumbersome steps to detect methylation levels. We developed a simpler enzymatic assay for the quantification of the global DNA methylation level using the Ten-eleven translocation (TET) protein. TET proteins mediate DNA demethylation through the oxidation of 5-methylcytosine (5mC) in CpG in mammalian cells. Succinate is produced during this oxidation reaction, and the amount of succinate produced correlates to the global DNA methylation level. The catalytic domain of the TET2 was expressed in Escherichia coli (E. coli), and the purified TET2 catalytic domain was reacted with human genomic DNA. The reaction solution was used for enzymatic succinate quantification with no purification step. The results showed that the succinate produced through TET-mediated oxidation increased with increasing global DNA methylation levels in human genomic DNA, which was determined using the bisulfite method. These results show that the global DNA methylation level is quantifiable by measuring the amount of succinate produced by the TET2-mediated 5mC oxidation reaction.

Graphical abstract

DNA 甲基化是研究得最好的表观遗传修饰之一。整体 DNA 甲基化水平的改变发生在异常细胞中，例如与癌症和阿尔茨海默病相关的细胞。几种测定方法用于确定整体 DNA 甲基化水平，包括基于亚硫酸氢盐的测定、基于高效液相色谱 (HPLC) 的测定、酶联免疫吸附测定 (ELISA) 和甲基接受测定。然而，这些检测需要几个繁琐的步骤来检测甲基化水平。我们开发了一种更简单的酶促检测方法，用于使用 10-11 易位 (TET) 蛋白对整体 DNA 甲基化水平进行量化。TET 蛋白通过氧化哺乳动物细胞 CpG 中的 5-甲基胞嘧啶 (5mC) 来介导 DNA 去甲基化。在这个氧化反应中产生琥珀酸，产生的琥珀酸数量与全球 DNA 甲基化水平相关。TET2 的催化域表达于大肠杆菌（E.coli），纯化的 TET2 催化结构域与人类基因组 DNA 反应。反应溶液用于无纯化步骤的酶促琥珀酸定量。结果表明，通过 TET 介导的氧化产生的琥珀酸随着人类基因组 DNA 中整体 DNA 甲基化水平的增加而增加，这是使用亚硫酸氢盐方法测定的。这些结果表明，整体 DNA 甲基化水平可以通过测量由 TET2 介导的 5mC 氧化反应产生的琥珀酸量来量化。

图形概要