Functionally distinct roles for TET-oxidized 5-methylcytosine bases in somatic reprogramming to pluripotency [ATAC-seq]

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 TET's role in reprogramming. Here, we employ biochemically engineered TET mutants to examine their effects on the reprogramming efficiency of mouse embryonic fibroblasts (MEFs). We show that only TET mutants proficient for oxidation to 5fC/5caC are able to rescue the reprogramming potential of Tet2 -deficient MEFs. This effect correlated with an increased capacity to drive DNA demethylation at reprogramming enhancers, ultimately accelerating chromatin opening in these regions. Together, these experiments demonstrate that DNA demethylation through 5fC/5caC intermediates is critical for iPSC reprogramming. Overall design: ATAC-seq of Dox-inducible Tet2-/- ; OKSM-rtTA mouse embryonic fibroblasts (MEFs) transduced with empty vector (pMXs), Tet2-WT, or Tet2-E (stalling mutant) at three timepoints of iPSC reprogramming (Day 0, Day 5, and Day 10), each with four replicates.