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.

DNA羟甲基化（DNA hydroxymethylation）及其介导的DNA去甲基化（DNA demethylation）对多种细胞进程至关重要，例如细胞核重编程（nuclear reprogramming）、胚胎发育（embryonic development）以及多种疾病的发生发展。本研究证实，一种关键营养物质抗坏血酸（ascorbic acid，AA），又称维生素C（vitamin C，Vc），可直接增强Tet双加氧酶（Tet dioxygenases）对5-甲基胞嘧啶（5-methylcytosine，5mC）的氧化催化活性。通过抗坏血酸及其抗氧化衍生物对Tet2蛋白固有荧光与催化活性的影响实验证据，我们进一步发现，AA可特异性结合Tet酶的C端催化结构域（C-terminal catalytic domain），这一过程或可促进酶的折叠以及辅助因子Fe²+的循环利用。其他强还原性化学物质则无此类作用。上述结果表明，AA同时可作为Tet酶的辅助因子发挥功能。在小鼠胚胎干细胞（mouse embryonic stem cells）中，AA可显著提升所有5mC氧化产物的水平，其中5-甲酰基胞嘧啶（5-formylcytosine）与5-羧基胞嘧啶（5-carboxylcytosine）的水平提升幅度更是超过一个数量级，最终导致全基因组范围内5mC水平下降约40%。在Tet1与Tet2基因敲除的细胞中，AA既无法改变5mC的氧化过程，也不会影响5mC的总水平。但若在Tet基因缺陷的细胞中重新表达Tet2蛋白，则AA的上述调控效应可得到恢复。在AA合成缺陷的小鼠模型中，同样可观察到AA对5mC氧化与DNA去甲基化的增强效应。本研究数据明确了AA、Tet酶与DNA甲基化之间的直接关联，从而揭示了AA在DNA修饰调控中的重要作用。