Ascorbic Acid Enhances Tet-Mediated 5‑Methylcytosine Oxidation and Promotes DNA Demethylation in Mammals

DNA hydroxymethylation
and its mediated DNA demethylation are critical
for multiple cellular processes, for example, nuclear reprogramming,
embryonic development, and many diseases. Here, we demonstrate that
a vital nutrient ascorbic acid (AA), or vitamin C (Vc), can directly
enhance the catalytic activity of Tet dioxygenases for the oxidation
of 5-methylcytosine (5mC). As evidenced by changes in intrinsic fluorescence
and catalytic activity of Tet2 protein caused by AA and its oxidation-resistant
derivatives, we further show that AA can uniquely interact with the
C-terminal catalytic domain of Tet enzymes, which probably promotes
their folding and/or recycling of the cofactor Fe2+. Other
strong reducing chemicals do not have a similar effect. These results
suggest that AA also acts as a cofactor of Tet enzymes. In mouse embryonic
stem cells, AA significantly increases the levels of all 5mC oxidation
products, particularly 5-formylcytosine and 5-carboxylcytosine (by
more than an order of magnitude), leading to a global loss of 5mC
(∼40%). In cells deleted of the Tet1 and Tet2 genes, AA alters neither 5mC oxidation nor the overall
level of 5mC. The AA effects are however restored when Tet2 is re-expressed
in the Tet-deficient cells. The enhancing effects of AA on 5mC oxidation
and DNA demethylation are also observed in a mouse model deficient
in AA synthesis. Our data establish a direct link among AA, Tet, and
DNA methylation, thus revealing a role of AA in the regulation of
DNA modifications.