Induced Active DNA Demethylation in Mammalian Cells Using Arabidopsis Demeter 5-Methylcytosine DNA Glycosylase

Although plants and animals have significantly conserved DNA methylation machineries, their DNA demethylation systems appear to have independently evolved. In plants, the DEMETER (DME) 5-methylcytosine (5mC) DNA glycosylase family proteins directly excise 5mC from DNA, replacing it with unmethylated cytosine via the base excision repair pathway. By contrast, animals have no such equivalent enzymes, and instead, 5mC is subjected to further modifications such as hydroxylation and deamination by different classes of enzymes prior to base excision. Here we adopt the active DNA demethylation system from plants by expressing Arabidopsis DME in HEK-293T cells to induce direct 5mC excision. DME expression slowed down cell proliferation and many of cells were found arrested at the S phase. In addition, severe DNA damage was observed in DME expressing cells, probably due to the formation of single strand breaks by DME lyase activity during 5mC excision. Transcription profiles of DME expressing cells revealed that a significant number of genes were upregulated by induced DNA demethylation, and many of them were involved in stress responses. We conclude that ectopic expression of plant DNA demethylases in mammalian cells allows active DNA demethylation, which induces DNA damage and corresponding stress responses.