Article

TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells

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Abstract

TET enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which can lead to DNA demethylation. However, direct connections between TET-mediated DNA demethylation and transcriptional output are difficult to establish owing to challenges in distinguishing global versus locus-specific effects. Here we show that TET1, TET2 and TET3 triple-knockout (TKO) human embryonic stem cells (hESCs) exhibit prominent bivalent promoter hypermethylation without an overall corresponding decrease in gene expression in the undifferentiated state. Focusing on the bivalent PAX6 locus, we find that increased DNMT3B binding is associated with promoter hypermethylation, which precipitates a neural differentiation defect and failure of PAX6 induction during differentiation. dCas9-mediated locus-specific demethylation and global inactivation of DNMT3B in TKO hESCs partially reverses the hypermethylation at the PAX6 promoter and improves differentiation to neuroectoderm. Taking these findings together with further genome-wide methylation and TET1 and DNMT3B ChIP–seq analyses, we conclude that TET proteins safeguard bivalent promoters from de novo methylation to ensure robust lineage-specific transcription upon differentiation.

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Acknowledgements

We thank the WCMC Epigenomics core for ERRBS and 5mC MassARRAY analysis and the MSKCC Integrated Genomics core for performing RNA-seq and WGBS. We also thank L. Studer and J. Tchieu for advice on neuroectoderm differentiation, E. Apostolou and M. Donohoe for comments on the manuscript, and M. Goll and members of the Huangfu laboratory for insightful discussions and critical reading of the manuscript. This study was funded in part by the Tri-Institutional Stem Cell Initiative (2016-032), New York State Stem Cell Science (NYSTEM C029156) and an MSKCC Cancer Center Support grant (P30 CA008748). N.V. is supported by the Weill Graduate School of Medical Sciences at the Cornell University/The Rockefeller University/Sloan Kettering Institute Tri-Institutional MD–PhD Program.

Author information

Author notes

    • Federico González

    Present address: Institute for Bioengineering of Catalonia (IBEC), Barcelona, Spain

  1. Nipun Verma and Heng Pan contributed equally to this work.

Affiliations

  1. Developmental Biology Program, Sloan Kettering Institute, New York, NY, USA

    • Nipun Verma
    • , Abhijit Shukla
    • , Qing V. Li
    • , Virginia Teijeiro
    • , Federico González
    •  & Danwei Huangfu
  2. Weill Graduate School of Medical Sciences at Cornell University/The Rockefeller University/Sloan Kettering Institute Tri-Institutional MD–PhD Program, New York, NY, USA

    • Nipun Verma
    •  & Bobbie Pelham-Webb
  3. Department of Physiology and Biophysics, Englander Institute for Precision Medicine, Institute for Computational Biomedicine, Weill Cornell Medical College, New York, NY, USA

    • Heng Pan
    •  & Olivier Elemento
  4. Weill Cornell Graduate School of Medical Sciences, Weill Cornell Medical College, New York, NY, USA

    • Heng Pan
    •  & Virginia Teijeiro
  5. Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, Howard Hughes Medical Institute, University of Chicago, Chicago, IL, USA

    • Louis C. Doré
    •  & Chuan He
  6. Louis V. Gerstner Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    • Qing V. Li
  7. Cancer Biology and Genetics Program, Sloan Kettering Institute, New York, NY, USA

    • Andrei Krivtsov
  8. Department of Oncological Sciences and Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    • Chan-Jung Chang
    •  & Eirini P. Papapetrou

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Contributions

N.V. and D.H. devised experiments and interpreted results. N.V. performed most experiments and collected data. H.P. and O.E. performed computational analysis on WGBS, ERRBS, RNA-seq, ChIP–seq and 5hmC profiling. L.C.D. and C.H. performed 5hmC profiling, sequencing and analysis, and mass spectrometry. A.K. generated libraries for WGBS. A.S., Q.V.L., B.P.-W., V.T., F.G., E.P.P. and C.-J.C. assisted with additional experiments. N.V. and D.H. wrote the manuscript; all other authors provided editorial advice.

Competing interests

5hmC-Seal has been licensed to Active Motif and Epican by the University of Chicago. This statement is relevant to C.H.

Corresponding authors

Correspondence to Olivier Elemento or Danwei Huangfu.

Integrated Supplementary Information

Supplementary information

  1. Supplementary Text and Figures 

    Supplementary Figures 1–8, Supplementary Tables 1–6 and Supplementary Note

  2. Life Sciences Reporting Summary

  3. Supplementary Data 1

    Hyper-DMRs for TKO as compared to WT HUES8 hESCs by WGBS. The number of differentially methylated CpGs per hyper-DMR is provided along with the methylation difference between WT and TKO HUES8 hESCs. Data from one run of WGBS are presented

  4. Supplementary Data 2

    DNA methylation levels at bivalent promoters in WT and TKO HUES8 hESCs by WGBS. The average methylation at each bivalent promoter as well as the methylation difference (TKO – WT) and the fold change (TKO/WT) is provided. The definitions used for promoters are provided in the Methods. Data from one run of WGBS are presented

  5. Supplementary Data 3

    Hyper-DMRs for TKO as compared to WT HUES8 hESCs by ERRBS. The number of differentially methylated CpGs per hyper-DMR is provided along with the methylation difference between WT and TKO HUES8 hESCs. The combined analysis of two replicates of ERRBS is presented

  6. Supplementary Data 4

    Hyper-DMRs for TKO as compared to WT MEL-1 hESCs by ERRBS. The number of differentially methylated CpGs per hyper-DMR is provided along with the methylation difference between WT and TKO MEL-1 hESCs. The combined analysis of two replicates of ERRBS is presented

  7. Supplementary Data 5

    DNA methylation levels at bivalent promoters in WT and TKO HUES8 hESCs by ERRBS. The average methylation at each bivalent promoter as well as the methylation difference (TKO – WT) and the fold change (TKO/WT) is provided. The definitions used for promoters are provided in the Methods. The combined analysis of two replicates of ERRBS is presented

  8. Supplementary Data 6

    DNA methylation levels at bivalent promoters in WT and TKO MEL-1 hESCs by ERRBS. The average methylation at each bivalent promoter as well as the methylation difference (TKO – WT) and the fold change (TKO/WT) is provided. The definitions used for promoters are provided in the Methods. The combined analysis of two replicates of ERRBS is presented

  9. Supplementary Data 7

    Differentially expressed genes between TKO and WT HUES8 hESCs. The log2-transformed fold change (TKO/WT), P value and adjusted P value (Benjamini–Hochberg correction) are provided. The combined analysis of three replicates of RNA-seq is presented

  10. Supplementary Data 8

    DNA methylation levels at bivalent promoters in WT, TKO and QKO HUES8 hESCs and in WT and TKO MEL-1 hESCs by ERRBS. The average methylation at each bivalent promoter is provided. The individual replicates of the ERRBS are provided