5Hydroxymethylcytosin

5-Hydroxymethylcytosine (5hmC) is a modified form of the DNA base cytosine produced by oxidation of 5-methylcytosine (5mC). The oxidation is carried out by the TET family of dioxygenases (TET1, TET2, TET3), which require Fe2+ and α-ketoglutarate as cofactors. In mammalian genomes, 5hmC is enriched in neural tissues and embryonic stem cells and tends to be found at gene bodies and enhancers. It is considered both an intermediate in active DNA demethylation and, in its own right, a potential epigenetic mark with regulatory functions.

In the demethylation pathway, 5hmC can be further oxidized to 5-formylcytosine (5fC) and then to 5-carboxylcytosine (5caC). These derivatives can be excised by thymine DNA glycosylase (TDG) and replaced with unmodified cytosine, completing demethylation.

Because standard bisulfite sequencing cannot distinguish 5mC from 5hmC, specialized methods have been developed, including oxidative bisulfite sequencing (oxBS-Seq) and TAB-seq, along with chemical labeling approaches to map 5hmC at base resolution.

Biological significance of 5hmC includes its association with transcriptional activity and developmental regulation. Altered 5hmC levels have been reported in cancer and certain neurological disorders, and its distribution is thought to influence gene expression programs in development and differentiation.

History: 5hmC was identified in mammalian DNA in 2009 as a product of TET-mediated oxidation of 5mC, marking a shift in understanding of DNA methylation dynamics and epigenetic regulation.