Functionally distinct roles for TET-oxidized 5-methylcytosine bases in somatic reprogramming to pluripotency

Active DNA demethylation via ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming in cell state transitions. TET enzymes catalyze up to three successive oxidations of 5-methylcytosine (5mC), generating 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). Although these bases are known to contribute to distinct demethylation pathways, the lack of tools to uncouple these sequential oxidative events has constrained our mechanistic understanding of the role of TETs in chromatin reprogramming. Here, we describe the first application of biochemically engineered TET mutants that unlink 5mC oxidation steps, examining their effects on somatic cell reprogramming. We show that only TET enzymes proficient for oxidation to 5fC/5caC can rescue the reprogramming potential of Tet2-deficient mouse embryonic fibroblasts. This effect correlated with rapid DNA demethylation at reprogramming enhancers and increased chromatin accessibility later in reprogramming. These experiments demonstrate that DNA demethylation through 5fC/5caC has roles distinct from 5hmC in somatic reprogramming to pluripotency. [Display omitted] •TET mutants that stall oxidation at 5hmC uncouple active DNA demethylation pathways•TET-mediated oxidation to 5fC/5caC, but not 5hmC, promotes iPSC reprogramming efficiency•Novel base resolution-sequencing methods reveal distinctive roles for 5hmC from 5fC/5caC•5fC/5caC is the major driver of DNA demethylation during iPSC reprogramming Caldwell et al. use novel TET catalytic mutants that uncouple two distinct mechanisms of active DNA demethylation: passive loss of 5hmC and direct excision of 5fC/5caC. They identify the 5fC/5caC pathway as the major driver of DNA demethylation during iPSC reprogramming, with important roles in gene regulation and chromatin reorganization.