Vitamin C induces Tet-dependent DNA demethylation in ESCs to promote a blastocyst-like state

DNA methylation is a heritable epigenetic modification involved in gene silencing, imprinting, and the suppression of retrotransposons(1). Global DNA demethylation occurs in the early embryo and the germline(2,3) and may be mediated by Tet (ten-eleven-translocation) enzymes(4–6), which convert 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC)(7). Tet enzymes have been extensively studied in mouse embryonic stem cells (ESCs)(8–12), which are generally cultured in the absence of Vitamin C (VitC), a potential co-factor for Fe(II) 2-oxoglutarate dioxygenase enzymes like Tets. Here we report that addition of VitC to ESCs promotes Tet activity leading to a rapid and global increase in hmC. This is followed by DNA demethylation of numerous gene promoters and up-regulation of demethylated germline genes. Tet1 binding is enriched near the transcription start site (TSS) of genes affected by VitC treatment. Importantly, VitC, but not other antioxidants, enhances the activity of recombinant Tet1 in a biochemical assay and the VitC-induced changes in hmC and mC are entirely suppressed in Tet1/2 double knockout (Tet DKO) ESCs. VitC has the strongest effects on regions that gain methylation in cultured ESCs compared to blastocysts and in vivo are methylated only after implantation. In contrast, imprinted regions and intracisternal A-particle (IAP) retroelements, which are resistant to demethylation in the early embryo(2,13), are resistant to VitC-induced DNA demethylation. Collectively, this study establishes VitC as a direct regulator of Tet activity and DNA methylation fidelity in ESCs.