Genomic sequence 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. We reveal distinct context dependencies for 5hmC generation versus decay, with flanking bases differentially impacting 5hmC persistence. Notably, we demonstrate these predictable preferences in duplexed DNA are lost in single-stranded DNA, mechanistically linking these features to TET-mediated base-flipping. Furthermore, analysis of genomic 5hmC patterns uncovers evidence of sequence context bias in cellular 5hmC accumulation, suggesting a role for TET-intrinsic preferences in shaping cellular epigenetic landscapes.