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. 2013 May 10;288(19):13669-74.
doi: 10.1074/jbc.C113.464800. Epub 2013 Apr 2.

Ascorbate induces ten-eleven translocation (Tet) methylcytosine dioxygenase-mediated generation of 5-hydroxymethylcytosine

Emily A Minor  1 , Brenda L Court, Juan I Young, Gaofeng Wang
Affiliations

Ascorbate induces ten-eleven translocation (Tet) methylcytosine dioxygenase-mediated generation of 5-hydroxymethylcytosine

Emily A Minor et al. J Biol Chem. .

Abstract

Background: Tet methylcytosine dioxygenase converts 5-mC to 5-hmC in DNA.

Results: Ascorbate significantly and specifically enhances Tet-mediated generation of 5-hmC.

Conclusion: Our findings suggest that ascorbate enhances 5-hmC generation, most likely by acting as a co-factor for Tet methylcytosine dioxygenase to generate 5-hmC.

Significance: The availability of ascorbate could have significant consequences for health and diseases by modulating the epigenetic control of genome activity. Ascorbate (vitamin C) is best known for its role in scurvy, in which the hydroxylation of collagen catalyzed by dioxygenases is incomplete due to ascorbate deficiency. Here, we report a novel function of ascorbate in the hydroxylation of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) in DNA catalyzed by Tet (ten-eleven translocation) methylcytosine dioxygenase. The content of 5-hmC is extremely low in mouse embryonic fibroblasts cultured in ascorbate-free medium. Additions of ascorbate dose- and time-dependently enhance the generation of 5-hmC, without any effects on the expression of Tet genes. Treatment with another reducer glutathione (GSH) does not change the level of 5-hmC. Further, blocking ascorbate entry into cells by phloretin and knocking down Tet (Tet1, Tet2, and Tet3) expression by short interference RNAs (siRNA) significantly inhibit the effect of ascorbate on 5-hmC. These results suggest that ascorbate enhances 5-hmC generation, most likely by acting as a co-factor for Tet methylcytosine dioxygenase to hydroxylate 5-mC. Thus, we have uncovered a novel role for ascorbate in modulating the epigenetic control of genome activity.

Keywords: 5-Hydroxymethylcytosine; 5-Methylcytosine; DNA Methylation; Enzymes; Epigenetics; Mouse Embryonic Fibroblast; Phloretin; Tet Methylcytosine Dioxygenases; Transport; Vitamin C.

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Figures

FIGURE 1.
FIGURE 1.
Ascorbate dose-dependently enhances the generation of 5-hmC in MEFs. A, immunostaining shows that ascorbate (0–1,000 μm) dose-dependently increases 5-hmC signal. Without treatment, 5-hmC signal (shown by Cy3 labeling) is absent or negligible, whereas the presence of 5-hmC is obvious after treatment with 1 μm of ascorbate for 24 h. Treatment with ascorbate (10–1,000 μm) further increases 5-hmC signal. In contrast, there is no obvious 5-hmC signal in MEFs after incubation with 100 μm GSH for 24 h. B, a representative dot-blot shows that ascorbate (0–1,000 μm) dose-dependently increases the content of 5-hmC, whereas GSH (100 μm) does not have any effects on 5-hmC. Std: 5-hmC standard (Active Motif) at 2.5, 0.5, 0.1, and 0.02 ng (from top to bottom). C, semiquantitative analysis of the dot-blot indicates that ascorbate (1–1,000 μm) increases 5-hmC up to 4-fold of the basal level (*, p < 0.05 assessed by Student's t test; data are represented as mean ± S.E.).
FIGURE 2.
FIGURE 2.
Ascorbate time-dependently enhances the generation of 5-hmC in MEFs. A, immunostaining shows that 10 μm ascorbate (0–48 h) time-dependently enhances the generation of 5-hmC in MEFs. There is no obvious 5-hmC signal in controls (0 min) and cells incubated with ascorbate for 5 or 20 min. Treatment with ascorbate for 1–48 h causes a gradual and dramatic increase of 5-hmC signal in MEFs. B, a representative dot-blot shows that ascorbate (10 μm) time-dependently (0–48 h) increases the content of 5-hmC. C, semiquantitative analysis of the dot-blot indicates that the incubation of ascorbate for 1–48 h increases 5-hmC levels (*, p < 0.05 assessed by Student's t test; data are represented as mean ± S.E.).
FIGURE 3.
FIGURE 3.
The effect of ascorbate on 5-hmC is blocked by phloretin. A, immunostaining shows that phloretin (100 μm) does not change 5-hmC signal in ascorbate-free MEFs but does block the induction of 5-hmC by ascorbate (10 μm) treatment for 24 h. B, a representative dot-blot shows that phloretin (100 μm) inhibits the effects of ascorbate (10 μm) on 5-hmC content. C, semiquantitative analysis of the dot-blot indicates that phloretin inhibits the induction of 5-hmC by ascorbate (*, p < 0.05 assessed by Student's t test; data are represented as mean ± S.E.).
FIGURE 4.
FIGURE 4.
The effect of ascorbate on 5-hmC generation is mediated by Tet methylcytosine dioxygenase. A, siRNAs targeting Tet (Tet1, Tet2, and Tet3) decrease the level of Tet mRNAs to, ∼60% for Tet1, ∼40% for Tet2, and ∼35% for Tet3 as compared with control siRNA shown by quantitative RT-PCR. (*, p < 0.05 assessed by Student's t test; data are represented as mean ± S.E.) B, immunostaining shows that knocking down the expression of Tet genes decreases the basal level of 5-hmC signal and attenuates the induction of 5-hmC by ascorbate (10 μm) treatment for 24 h. C, a representative dot-blot shows that cells with less Tet1–3 reduce the generation of 5-hmC by ascorbate (10 μm) treatment. D, semiquantitative analysis of the dot-blot indicates that Tet1–3 partially depleted cells exhibit a decrease in the basal level of 5-hmC (∼40% as compared with the controls). Ascorbate still induces an increase (∼3-fold) of 5-hmC content in Tet1–3 partially depleted cells, but this effect is much smaller as compared with its effect in control cells (*, p < 0.05 assessed by Student's t test; data are represented as mean ± S.E.). E, in addition to Fe2+ as a co-factor and 2-oxoglutarate as co-substrate, ascorbate is suggested as an additional co-factor for Tet to hydroxylate 5-mC to 5-hmC.
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