Tet-Mediated Formation of 5-Carboxylcytosine and Its Excision by TDG in Mammalian DNA

TLDR

DNA methylation to 5‑methylcytosine and its conversion to 5‑hydroxymethylcytosine by Tet enzymes is widespread, yet the mechanisms for reversing these modifications remain unclear. Tet enzymes oxidize 5‑methylcytosine and 5‑hydroxymethylcytosine to 5‑carboxylcytosine, which is then specifically recognized and excised by thymine‑DNA glycosylase (TDG). The study demonstrates that Tet‑mediated oxidation of 5‑methylcytosine and 5‑hydroxymethylcytosine to 5‑carboxylcytosine, followed by TDG excision, constitutes an active DNA demethylation pathway, as TDG depletion in embryonic stem cells leads to 5‑caC accumulation.

Abstract

The prevalent DNA modification in higher organisms is the methylation of cytosine to 5-methylcytosine (5mC), which is partially converted to 5-hydroxymethylcytosine (5hmC) by the Tet (ten eleven translocation) family of dioxygenases. Despite their importance in epigenetic regulation, it is unclear how these cytosine modifications are reversed. Here, we demonstrate that 5mC and 5hmC in DNA are oxidized to 5-carboxylcytosine (5caC) by Tet dioxygenases in vitro and in cultured cells. 5caC is specifically recognized and excised by thymine-DNA glycosylase (TDG). Depletion of TDG in mouse embyronic stem cells leads to accumulation of 5caC to a readily detectable level. These data suggest that oxidation of 5mC by Tet proteins followed by TDG-mediated base excision of 5caC constitutes a pathway for active DNA demethylation.