Tet2 Catalyzes Stepwise 5-Methylcytosine Oxidation by an Iterative and de novo Mechanism

[Image: see text] Modification of cytosine-guanine dinucleotides (CpGs) is a key part of mammalian epigenetic regulation and helps shape cellular identity. Tet enzymes catalyze stepwise oxidation of 5-methylcytosine (mC) in CpGs to 5-hydroxymethylcytosine (hmC), or onward to 5-formylcytosine (fC) or 5-carboxylcytosine (caC). The multiple mC oxidation products, while intricately linked, are postulated to play independent epigenetic roles, making it critical to understand how the products of stepwise oxidation are established and maintained. Using highly sensitive isotope-based studies, we newly show that Tet2 can yield fC and caC by iteratively acting in a single encounter with mC-containing DNA, without release of the hmC intermediate, and that the modification state of the complementary CpG has little impact on Tet2 activity. By revealing Tet2 as an iterative, de novo mC oxygenase, our study provides insight into how features intrinsic to Tet2 shape the epigenetic landscape.