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

Abstract

DNA methylation is a heritable epigenetic modification involved in gene silencing, imprinting, and the suppression of retrotransposons1. Global DNA demethylation occurs in the early embryo and the germ line2,3, and may be mediated by Tet (ten eleven translocation) enzymes4,5,6, which convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC)7. Tet enzymes have been studied extensively in mouse embryonic stem (ES) cells8,9,10,11,12, 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 embryo2,13, 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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Figure 1: Vitamin C induces loss of 5mC at gene promoters through a transient increase in 5hmC.
Figure 2: Vitamin-C-induced DNA demethylation leads to expression of germline genes.
Figure 3: The effects of vitamin C are Tet-dependent.
Figure 4: Vitamin C reduces DNA methylation in ES cells that is normally gained post-implantation.

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Accessions

Gene Expression Omnibus

Data deposits

Microarray and DIP-seq data have been deposited in the Gene Expression Omnibus under the accession number GSE46403.

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Acknowledgements

The authors wish to acknowledge the epigenomics and sequencing groups at the Michael Smith Genome Sciences Centre, Canada, for performing 5mC DIP and 5hmC DIP Illumina sequencing, as well as for technical support. We thank C. -J. Lin for technical advice on isolation of blastocysts. We particularly thank M. M. Dawlaty and R. Jaenisch for the Tet1−/− and Tet1−/− Tet2−/− ES cells. We also wish to thank M. Okano for the Dnmt1−/− Dnmt3a−/− Dnmt3b−/− ES cells. We thank J. S. Song for statistical advice, as well as M. Conti, S. Lomvardas, J. Costello and members of the Ramalho-Santos laboratory for critical reading of the manuscript. K.B. is a recipient of an NSF pre-doctoral fellowship and K.T.E. is supported by a California Institute of Regenerative Medicine postdoctoral training grant (TG2-01153). M.M.K. is supported by a postdoctoral fellowship from the Michael Smith Foundation for Health Research. M.C.L. is supported by a CIHR New Investigator Award. This work was funded by a CIHR grant (92093) to M.C.L. and M.H., NIH grants HD065812 and CA151535, a grant from the California Institute of Regenerative Medicine to A.R., and an NIH New Innovator Award (DP2OD004698) and R01 (OD012204) to M.R.-S.

Author information

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Authors

Contributions

M.R.-S. directed the study. K.B. and K.T.E. designed and performed experiments. K.B., K.T.E., M.M.K., M.C.L. and M.R.-S. analysed and interpreted the data. M.M.K. and M.C.L. performed bioinformatics analyses. P.G. performed bisulphite sequencing. S.M. generated the human recombinant Tet1 catalytic domain, and J.A.Z.-M. performed the in vitro Tet activity assay under the direction of A.R., who provided expertise on Tet activity. A.T. and M.H. developed and performed DIP-seq and sequencing data processing. D.J.L. provided financial support, advice, and laboratory space to K.T.E. K.B., K.T.E., M.C.L. and M.R.-S. wrote the manuscript.

Corresponding authors

Correspondence to Matthew C. Lorincz or Miguel Ramalho-Santos.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-15 and Supplementary Tables 5-6 (see separate files for Supplementary Tables 1-4). (PDF 3227 kb)

Supplementary Table 1

This table shows 5hmC and 5mC DIP-seq data at promoters in untreated and vitamin C-treated ES cells. (XLSX 5852 kb)

Supplementary Table 2

This table shows 5hmC and 5mC DIP-seq data at repetitive elements in untreated and vitamin C-treated ES cells. (XLSX 115 kb)

Supplementary Table 3

This table shows gene expression changes in untreated and vitamin C-treated ES cells by microarray. (XLSX 2512 kb)

Supplementary Table 4

This table shows 5hmC and 5mC DIP-seq data for untreated and vitamin C-treated ES cells and bis-seq data for ES cells and blastocysts at CGIs. (XLSX 2356 kb)

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Blaschke, K., Ebata, K., Karimi, M. et al. Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells. Nature 500, 222–226 (2013). https://doi.org/10.1038/nature12362

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