Excision of 5-Hydroxymethylcytosine By DEMETER DNA Glycosylase from Arabidopsis

In plants and animals, 5-methylcytosine (5mC) serves as an epigenetic mark to repress gene expression and plays important roles in gene imprinting and transposon silencing. Mammals also have 5-hydroxymethylcytosine (5hmC), resulting from hydroxylation of 5mC by TET family-enzymes. 5hmC is abundant in mouse Purkinje neurons and embryonic stem cells, and regarded as an important intermediate during active DNA demethylation in mammals. However, it has not been clearly demonstrated that 5hmC is also present in plants. In Arabidopsis, DEMETER (DME) DNA glycosylase efficiently removes 5mC, which results in DNA demethylation and transcriptional activation of target genes. Here we showed that 5hmC was also excised by DME in vitro. We also performed a two dimensional thin layer chromatography analysis to verify whether 5hmC is present in Arabidopsis. However, we detected no or very little, if any, 5hmC from the Arabidopsis genome, suggesting that it is very unlikely for plants to have 5hmC as another substrate for active DNA demethylation. Our results support that both plants and animals have 5mC in common but DNA demethylation systems have evolved independently.