Genomic context differentially impacts stepwise oxidation by TET enzymes to shape DNA hydroxymethylation landscapes

Ten-Eleven Translocation (TET) enzymes reverse gene silencing imposed by 5-methylcytosine (5mC) in mammalian genomes by catalyzing the stepwise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC). 5hmC both acts as an independent epigenetic mark and promotes replication-dependent DNA demethylation by antagonizing maintenance DNA methylation. Oxidation of 5hmC to 5fC/5caC instead marks the committed step in replication-independent DNA demethylation, whereby 5fC/5caC are actively excised and reverted to unmodified cytosine.

Although prior work indicates that TET-mediated oxidation is influenced by bases neighboring 5mC, these studies have been unable to resolve more than two of the relevant species – 5mC, 5hmC, and 5fC/5caC – at the same time. As 5hmC is the key fulcrum in DNA demethylation, we established a high-throughput enzymology approach to probe these three cytosine states at base resolution simultaneously across hundreds of sites.