Quantification of global DNA methylation level using 5-methylcytosine dioxygenase.

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. [ABSTRACT FROM AUTHOR]