Abstract

DNA methylation is a key epigenetic modification involved in regulating gene expression and maintaining genomic integrity. Here we inactivated all three catalytically active DNA methyltransferases (DNMTs) in human embryonic stem cells (ESCs) using CRISPR/Cas9 genome editing to further investigate the roles and genomic targets of these enzymes. Disruption of DNMT3A or DNMT3B individually as well as of both enzymes in tandem results in viable, pluripotent cell lines with distinct effects on the DNA methylation landscape, as assessed by whole-genome bisulfite sequencing. Surprisingly, in contrast to findings in mouse, deletion of DNMT1 resulted in rapid cell death in human ESCs. To overcome this immediate lethality, we generated a doxycycline-responsive tTA-DNMT1* rescue line and readily obtained homozygous DNMT1-mutant lines. However, doxycycline-mediated repression of exogenous DNMT1* initiates rapid, global loss of DNA methylation, followed by extensive cell death. Our data provide a comprehensive characterization of DNMT-mutant ESCs, including single-base genome-wide maps of the targets of these enzymes.

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Acknowledgements

We thank all members of the Meissner laboratory, in particular C. Sindhu for helpful discussion and Z.D. Smith for critical feedback on the manuscript. We thank K. Musunuru (Harvard University) for providing the CRIPSR/Cas9 plasmids and thank Q. Ding from the Musunuru laboratory for technical support. J.L. was supported by a postdoctoral fellowship from the Human Frontiers Science Program. J.K.J. is supported by US NIH Director's Pioneer Award DP1 GM105378. A.M. is a New York Stem Cell Foundation Robertson Investigator. The work was funded by a US NIH (National Institute of General Medical Sciences) grant (P01GM099117) and the New York Stem Cell Foundation.

Author information

Author notes

    • Jing Liao
    •  & Rahul Karnik

    These authors contributed equally to this work.

Affiliations

  1. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

    • Jing Liao
    • , Rahul Karnik
    • , Hongcang Gu
    • , Michael J Ziller
    • , Kendell Clement
    • , Alexander M Tsankov
    • , Veronika Akopian
    • , Casey A Gifford
    • , Julie Donaghey
    • , Christina Galonska
    • , Ramona Pop
    • , William Mallard
    • , John L Rinn
    • , Andreas Gnirke
    •  & Alexander Meissner
  2. Harvard Stem Cell Institute, Cambridge, Massachusetts, USA.

    • Jing Liao
    • , Rahul Karnik
    • , Michael J Ziller
    • , Kendell Clement
    • , Alexander M Tsankov
    • , Veronika Akopian
    • , Casey A Gifford
    • , Julie Donaghey
    • , Christina Galonska
    • , Ramona Pop
    • , William Mallard
    • , John L Rinn
    •  & Alexander Meissner
  3. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.

    • Jing Liao
    • , Rahul Karnik
    • , Michael J Ziller
    • , Kendell Clement
    • , Alexander M Tsankov
    • , Veronika Akopian
    • , Casey A Gifford
    • , Julie Donaghey
    • , Christina Galonska
    • , Ramona Pop
    • , William Mallard
    • , John L Rinn
    •  & Alexander Meissner
  4. Department of Pathology, Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

    • Deepak Reyon
    • , Shengdar Q Tsai
    •  & J Keith Joung

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Contributions

J.L. and A.M. designed and conceived the study. J.L. generated all the cell lines and performed the experiments. R.K. performed the analysis. H.G. generated the WGBS libraries. A.G. and A.M. supervised the DNA methylation experiments. M.J.Z. and K.C. performed some analysis and assisted in the general data processing. A.M.T. and V.A. performed the experiments with the Scorecard assay. C.A.G. and J.D. assisted in endoderm and hepatocyte differentiation. C.G. performed the dot blot assay. R.P. supported the RNA profiling and FACS analysis. D.R., S.Q.T. and J.K.J. designed and generated the TALENs. W.M. and J.L.R. performed expression analysis. J.L., R.K. and A.M. interpreted the data and wrote the manuscript.

Competing interests

J.K.J. is a consultant for Horizon Discovery. J.K.J. has financial interests in Editas Medicine and Transposagen Biopharmaceuticals. The interests of J.K.J. were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies.

Corresponding author

Correspondence to Alexander Meissner.

Integrated supplementary information

Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–7 and Supplementary Tables 4 and 5

Excel files

  1. 1.

    Supplementary Table 1

    List of DMRs in knockout cell lines (1-kb tiles and CpG islands).

  2. 2.

    Supplementary Table 2

    GO analysis for enrichment of regions hypomethylated in DNMT3A–/–; DNMT3B–/– cells.

  3. 3.

    Supplementary Table 3

    Cell cycle analysis after DNMT1* withdrawal.

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DOI

https://doi.org/10.1038/ng.3258

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