Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells - PubMed (original) (raw)

. 2013 Aug 8;500(7461):222-6.

doi: 10.1038/nature12362. Epub 2013 Jun 30.

Kevin T Ebata, Mohammad M Karimi, Jorge A Zepeda-Martínez, Preeti Goyal, Sahasransu Mahapatra, Angela Tam, Diana J Laird, Martin Hirst, Anjana Rao, Matthew C Lorincz, Miguel Ramalho-Santos

Affiliations

Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells

Kathryn Blaschke et al. Nature. 2013.

Abstract

DNA methylation is a heritable epigenetic modification involved in gene silencing, imprinting, and the suppression of retrotransposons. Global DNA demethylation occurs in the early embryo and the germ line, and may be mediated by Tet (ten eleven translocation) enzymes, which convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Tet enzymes have been studied extensively in mouse embryonic stem (ES) cells, which are generally cultured in the absence of vitamin C, a potential cofactor for Fe(II) 2-oxoglutarate dioxygenase enzymes such as Tet enzymes. Here we report that addition of vitamin C to mouse ES cells promotes Tet activity, leading to a rapid and global increase in 5hmC. This is followed by DNA demethylation of many gene promoters and upregulation of demethylated germline genes. Tet1 binding is enriched near the transcription start site of genes affected by vitamin C treatment. Importantly, vitamin C, but not other antioxidants, enhances the activity of recombinant Tet1 in a biochemical assay, and the vitamin-C-induced changes in 5hmC and 5mC are entirely suppressed in Tet1 and Tet2 double knockout ES cells. Vitamin C has a stronger effect on regions that gain methylation in cultured ES cells compared to blastocysts, and in vivo are methylated only after implantation. In contrast, imprinted regions and intracisternal A particle retroelements, which are resistant to demethylation in the early embryo, are resistant to vitamin-C-induced DNA demethylation. Collectively, the results of this study establish vitamin C as a direct regulator of Tet activity and DNA methylation fidelity in ES cells.

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Figures

Figure 1

Figure 1. Vitamin C induces loss of mC at gene promoters via a transient increase in hmC

a, Immunofluorescence for hmC. Scale bar is 200 µm. b, Global hmC and mC levels assayed by dot blot. c, Graph shows fold change in DIP-seq reads (RPKM) at methylated promoters (n = 1045) for VitC-treated cells relative to untreated cells. Values are mean ± s.e.m. d, Overlap of methylated promoters that gain hmC and those that lose mC. P value calculated using Fisher’s exact test. e, Genome browser view of Dazl.f, Bisulfite sequencing of promoters. Open circles = unmethylated, closed circles = methylated. g, Scatter plot of methylated promoters comparing change in methylation (Z score) versus CpG content. Red circles = imprinted genes. h, Graphs show fold change in RPKM at retrotransposons for VitC-treated cells relative to untreated cells. Values are mean ± s.e.m.

Figure 2

Figure 2. Vitamin C-induced DNA demethylation leads to expression of germline genes

a, Number of genes differentially expressed following VitC treatment (2-fold and P < 0.05 by t-test). b, Chromosomal distribution of up-regulated genes. c, Gene ontology analysis of up-regulated genes. d, Box plot showing basal promoter methylation levels (RPKM) in untreated ESCs for all genes on the microarray (n = 18,023), up-regulated genes (n = 102), down-regulated genes (n = 48), all germline genes (n = 865), up-regulated germline genes (n = 8), and down-regulated germline genes (n = 3). e, Box plot showing the extent of VitC-induced demethylation (Z score) at gene promoters categorized as in panel d. The box plots have Tukey whiskers, a line for the median, and edges for the 25th/75th percentile. ****P < 0.0001 by ANOVA throughout the figure.

Figure 3

Figure 3. The effects of Vitamin C are Tet-dependent

a, Dose-dependent effect of VitC on in vitro Tet activity. n = 3 technical replicates, values are mean ± s.d. b, Dot blot for hmC following a 12 hr VitC treatment. c, hmC (top) and mC (bottom) DIP-qPCR following a 12 or 72 hr VitC treatment, respectively. d, Gene expression at 72 hrs of VitC treatment. qRT-PCR data expressed relative to untreated wild-type cells. For c, d, n = 2 biological replicates, values are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 by t-test throughout the figure.

Figure 4

Figure 4. Vitamin C reduces DNA methylation in ESCs that is normally gained post-implantation

a, Overlap of methylated CGIs in ESCs from this study (mC DIP-seq, RPKM >0.5) and ref. (whole genome bisulfite (bis)-seq, >25% methylated CpGs). Only CGIs found to be methylated in both data sets were used for subsequent analysis in b–d. b, The box plot shows CGI methylation levels in ESCs and blastocysts (values from ref. , ****P < 0.0001 by t-test). **c**, CGIs were first categorized as having mC loss with VitC (>75% loss of mC at 72 hrs, n = 23) or mC maintenance with VitC (<25% loss of mC at 72 hrs, n = 14) in ESCs, then plotted for difference in methylation between untreated ESCs and blastocysts. CGIs demethylated upon VitC treatment show significantly greater ESC hypermethylation compared to CGIs resistant to VitC (**P < 0.01 by t-test). d, Genome browser views of a gene from each category described in panel c. e, Methylation levels during development of all genes categorized in panel c for which data exist (ref. 2). f, Gene expression in ESCs cultured with or without VitC compared to E3.5 blastocysts. Data expressed relative to untreated ESCs. n = 2 biological replicates, values are mean ± s.e.m.

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