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

Nature volume 500, pages 222–226 (2013)Cite this article

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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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Gene Expression Omnibus

Data deposits

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

References

  1. Bird, A. DNA methylation patterns and epigenetic memory. Genes Dev. 16, 6–21 (2002)

    Article  CAS  PubMed  Google Scholar 

  2. Smith, Z. D. et al. A unique regulatory phase of DNA methylation in the early mammalian embryo. Nature 484, 339–344 (2012)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  3. Seisenberger, S. et al. The dynamics of genome-wide DNA methylation reprogramming in mouse primordial germ cells. Mol. Cell 48, 849–862 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Gu, T.-P. et al. The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. Nature 477, 606–610 (2011)

    Article  ADS  CAS  PubMed  Google Scholar 

  5. Yamaguchi, S. et al. Tet1 controls meiosis by regulating meiotic gene expression. Nature 492, 443–447 (2012)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  6. Hackett, J. A. et al. Germline DNA demethylation dynamics and imprint erasure through 5-hydroxymethylcytosine. Science 339, 448–452 (2013)

    Article  ADS  CAS  PubMed  Google Scholar 

  7. Tahiliani, M. et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324, 930–935 (2009)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  8. Pastor, W. A. et al. Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells. Nature 473, 394–397 (2011)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  9. Wu, H. et al. Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells. Nature 473, 389–393 (2011)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  10. Williams, K. et al. TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity. Nature 473, 343–348 (2011)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ficz, G. et al. Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation. Nature 473, 398–402 (2011)

    Article  ADS  CAS  PubMed  Google Scholar 

  12. Koh, K. P. et al. Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells. Cell Stem Cell 8, 200–213 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Kobayashi, H. et al. Contribution of intragenic DNA methylation in mouse gametic DNA methylomes to establish oocyte-specific heritable marks. PLoS Genet. 8, e1002440 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Marks, H. et al. The transcriptional and epigenomic foundations of ground state pluripotency. Cell 149, 590–604 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Loenarz, C. & Schofield, C. J. Expanding chemical biology of 2-oxoglutarate oxygenases. Nature Chem. Biol. 4, 152–156 (2008)

    Article  CAS  Google Scholar 

  16. Wang, P. J., McCarrey, J. R., Yang, F. & Page, D. C. An abundance of X-linked genes expressed in spermatogonia. Nature Genet. 27, 422–426 (2001)

    Article  PubMed  CAS  Google Scholar 

  17. Fouse, S. D. et al. Promoter CpG methylation contributes to ES cell gene regulation in parallel with Oct4/Nanog, PcG complex, and histone H3 K4/K27 trimethylation. Cell Stem Cell 2, 160–169 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Karimi, M. M. et al. DNA methylation and SETDB1/H3K9me3 regulate predominantly distinct sets of genes, retroelements, and chimeric transcripts in mES cells. Cell Stem Cell 8, 676–687 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wang, T. et al. The histone demethylases Jhdm1a/1b enhance somatic cell reprogramming in a vitamin-C-dependent manner. Cell Stem Cell 9, 575–587 (2011)

    Article  CAS  PubMed  Google Scholar 

  20. Dawlaty, M. M. et al. Tet1 is dispensable for maintaining pluripotency and its loss is compatible with embryonic and postnatal development. Cell Stem Cell 9, 166–175 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Dawlaty, M. M. et al. Combined deficiency of Tet1 and Tet2 causes epigenetic abnormalities but is compatible with postnatal development. Dev. Cell 24, 310–323 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Borgel, J. et al. Targets and dynamics of promoter DNA methylation during early mouse development. Nature Genet. 42, 1093–1100 (2010)

    Article  CAS  PubMed  Google Scholar 

  23. Tang, F. et al. Tracing the derivation of embryonic stem cells from the inner cell mass by single-cell RNA-Seq analysis. Cell Stem Cell 6, 468–478 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Leitch, H. G. et al. Naive pluripotency is associated with global DNA hypomethylation. Nature Struct. Mol. Biol. 20, 311–316 (2013)

    Article  CAS  Google Scholar 

  25. Chung, T.-L. et al. Vitamin C promotes widespread yet specific DNA demethylation of the epigenome in human embryonic stem cells. Stem Cells 28, 1848–1855 (2010)

    Article  CAS  PubMed  Google Scholar 

  26. Stadtfeld, M. et al. Ascorbic acid prevents loss of Dlk1-Dio3 imprinting and facilitates generation of all-iPS cell mice from terminally differentiated B cells. Nature Genet. 44, 398–405 (2012)

    Article  CAS  PubMed  Google Scholar 

  27. Doege, C. A. et al. Early-stage epigenetic modification during somatic cell reprogramming by Parp1 and Tet2. Nature 488, 652–655 (2012)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  28. Gao, Y. et al. Replacement of Oct4 by Tet1 during iPSC induction reveals an important role of DNA methylation and hydroxymethylation in reprogramming. Cell Stem Cell 12, 453–469 (2013)

    Article  CAS  PubMed  Google Scholar 

  29. Minor, E. A., Court, B. L., Young, J. I. & Wang, G. Ascorbate induces Ten-eleven translocation (Tet) methylcytosine dioxygenase-mediated generation of 5-hydroxymethylcytosine. J. Biol. Chem. 288, 13669–13674 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Tan, L. & Shi, Y. G. Tet family proteins and 5-hydroxymethylcytosine in development and disease. Development 139, 1895–1902 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Ying, Q.-L. et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  32. Kumaki, Y., Oda, M. & Okano, M. QUMA: quantification tool for methylation analysis. Nucleic Acids Res. 36, W170–W175 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Morin, R. D. et al. Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma. Nature 476, 298–303 (2011)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  34. Wiegand, K. C. et al. ARID1A mutations in endometriosis-associated ovarian carcinomas. N. Engl. J. Med. 363, 1532–1543 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Li, H., Ruan, J. & Durbin, R. Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res. 18, 1851–1858 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Fejes, A. P. et al. FindPeaks 3.1: a tool for identifying areas of enrichment from massively parallel short-read sequencing technology. Bioinformatics 24, 1729–1730 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Kent, W. J., Zweig, A. S., Barber, G., Hinrichs, A. S. & Karolchik, D. BigWig and BigBed: enabling browsing of large distributed datasets. Bioinformatics 26, 2204–2207 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L. & Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nature Methods 5, 621–628 (2008)

    Article  CAS  PubMed  Google Scholar 

  40. Pepke, S., Wold, B. & Mortazavi, A. Computation for ChIP-seq and RNA-seq studies. Nature Methods 6, S22–S32 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Pruitt, K. D. & Maglott, D. R. RefSeq and LocusLink: NCBI gene-centered resources. Nucleic Acids Res. 29, 137–140 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Younesy, H. et al. An interactive analysis and exploration tool for epigenomic data. Computer Graphics Forum (Eurographics Conference on Visualization (EuroVis) 2013) 32 (number 3), 92–93 (2013)

    Google Scholar 

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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

Author notes

  1. Kathryn Blaschke and Kevin T. Ebata: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Obstetrics and Gynecology and Center for Reproductive Sciences, Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, 35 Medical Center Way, San Francisco, 94143, California, USA

    Kathryn Blaschke, Kevin T. Ebata, Diana J. Laird & Miguel Ramalho-Santos

  2. Department of Medical Genetics, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada,

    Mohammad M. Karimi, Preeti Goyal & Matthew C. Lorincz

  3. BC Cancer Agency, Canada’s Michael Smith Genome Sciences Centre, 675 West 10th Avenue, Vancouver, British Columbia V5Z 1L3, Canada,

    Mohammad M. Karimi, Angela Tam & Martin Hirst

  4. La Jolla Institute for Allergy and Immunology and Sanford Consortium for Regenerative Medicine, La Jolla, 92037, California, USA

    Jorge A. Zepeda-Martínez, Sahasransu Mahapatra & Anjana Rao

  5. Department of Microbiology and Immunology, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada,

    Martin Hirst

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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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