Ascorbate-induced generation of 5-hydroxymethylcytosine is unaffected by varying levels of iron and 2-oxoglutarate - PubMed (original) (raw)

Ascorbate-induced generation of 5-hydroxymethylcytosine is unaffected by varying levels of iron and 2-oxoglutarate

Kevin M Dickson et al. Biochem Biophys Res Commun. 2013.

Abstract

Tet (ten-eleven translocation) methylcytosine dioxygenases, which belong to the iron and 2-oxoglutarate (2OG)-dependent dioxygenase superfamily, convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. We recently reported that ascorbate (vitamin C) induces Tet-mediated generation of 5hmC. To initially delineate the role of ascorbate on 5hmC generation, we analyzed whether the effect of ascorbate is dependent upon the conditions of other components involved in the hydroxylation of 5mC catalyzed by Tet. We found that removing iron from the culture medium did not affect the induction of 5hmC by ascorbate (10 μM) in mouse embryonic fibroblasts (MEFs). The effect of ascorbate did not involve an increased expression of Tet1-3 or isocitrate dehydrogenases (IDH1-2), the enzymes responsible for producing 2OG. Interestingly, MEFs cultured with different concentrations of glucose, a major precursor of 2OG, exhibited nearly identical responses to ascorbate treatment. Further, blocking the uptake of the reduced form of vitamin C, ascorbic acid, through the sodium-dependent vitamin C transporters (SVCTs) inhibited the effect of ascorbate on 5hmC. However, inhibition of the facilitative glucose transporters (GLUTs), which mediate the incorporation of the oxidized form of vitamin C, dehydroascorbic acid (DHA), did not modify the ability of ascorbate to induce 5hmC generation. These results indicate that the effect of ascorbate on 5hmC is not dependent upon iron uptake, the expression of Tet and IDH, or the production of 2OG, suggesting that ascorbate may directly participate in the generation of 5hmC, most likely as a cofactor of Tet.

Keywords: 2-Oxoglutarate; 5-Hydroxymethylcytosine; Ascorbate; Glucose; Iron; Isocitrate dehydrogenase; Sodium-dependent vitamin C transporter; Tet methylcytosine dioxygenase.

Copyright © 2013 Elsevier Inc. All rights reserved.

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Figures

Fig. 1

Fig. 1

The generation of 5hmC induced by ascorbate is independent on the iron uptake by MEFs. (A) Immunostaining shows that the 5hmC signal induced by ascorbate (10 µM) treatments for 24 hr is at a similar level in MEFs cultured with different concentrations (0, 0.25, 2.5µM) of iron. (B) A representative dot-blot shows that ascorbate (10 µM) increases the content of 5hmC in MEFs cultured with different concentrations (0, 0.25, 2.5µM) of iron. (C) Semiquantitative analysis of the dot-blot indicates that ascorbate (10 µM) increases 5hmC at a similar level (~4 fold of the basal level) in MEFs cultured with different concentrations (0, 0.25, 2.5µM) of iron. (P > 0.05 assessed by t test. Date are represented as mean ± SEM).

Fig. 2

Fig. 2

Glucose in the medium does not change the generation of 5hmC induced by ascorbate treatment. (A) Immunostaining shows that glucose (0, 5.56, 25 mM) does not change 5hmC signal induced by ascorbate (10 µM) treatment for 24 hr. (B) A representative dot-blot shows that ascorbate (10 µM) increases the content of 5hmC in MEFs cultured with different concentrations (0, 5.56, 25 mM) of glucose. (C) Semiquantitative analysis of the dot-blot indicates that ascorbate (10 µM) increases 5hmC at a similar level (~4 fold of the basal level) in MEFs cultured with different concentrations (0, 5.56, 25 mM) of glucose. (P > 0.05 assessed by t test. Date are represented as mean ± SEM).

Fig. 3

Fig. 3

Ascorbate treatment does not change the expression of Tet and IDH genes. (A) Quantitative PCR shows that ascorbate (0 – 1,000 µM) does not significantly change the expression of Tet genes in MEFs. (P > 0.05 assessed by t test. Date are represented as mean ± SEM). (B) Quantitative PCR shows that ascorbate (0 – 1,000 µM) does not significantly change the expression of IDH genes in MEFs. (P > 0.05 assessed by t test. Date are represented as mean ± SEM).

Fig. 4

Fig. 4

The effect of ascorbate on 5hmC is partially reduced by sulfinpyrazone. (A) Immunostaining shows that both sulfinpyrazone (2 mM) and cytochalasin B (20 µM) do not change 5hmC signal in ascorbate-free MEFs. Only sulfinpyrazone blocks the induction of 5hmC by ascorbate (10 µM) treatment for 24 hr. (B) A representative dot-blot shows that sulfinpyrazone (2 mM), but not cytochalasin B (20 µM), inhibits the effects of ascorbate (10 µM) on 5hmC content. (C) Semiquantitative analysis of the dot-blot indicates that sulfinpyrazone (2 mM), but not cytochalasin B (20 µM), inhibits the induction of 5hmC by ascorbate (* P < 0.05 assessed by t test. Date are represented as mean ± SEM).

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