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Chromatin-modifying enzymes as modulators of reprogramming

Nature volume 483, pages 598–602 (2012)Cite this article

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Abstract

Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodelling1. Whereas several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming2,3, the role of specific chromatin-modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used short hairpin RNAs (shRNAs) to target genes in DNA and histone methylation pathways, and identified positive and negative modulators of iPSC generation. Whereas inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase EZH2, reduced reprogramming efficiency, suppression of SUV39H1, YY1 and DOT1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase DOT1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for KLF4 and c-Myc (also known as MYC). Inhibition of DOT1L early in the reprogramming process is associated with a marked increase in two alternative factors, NANOG and LIN28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. DOT1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors.

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Figure 1: Screening for inhibitors and enhancers of reprogramming.
Figure 2: DOT1L inhibition enhances reprogramming efficiency and substitutes for KLF4 and Myc.
Figure 3: NANOG and LIN28 are required for enhancement of reprogramming by DOT1L inhibition.
Figure 4: Genome-wide analysis of H3K79me2 marks during reprogramming.

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Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

The microarray and ChIP-seq data have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO) and are accessible through GEO Series accession numbers GSE29253 and GSE35791.

Change history

  • 29 March 2012

    Author name for B.O.M. was corrected.

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Acknowledgements

We thank G. Hu and S. J. Elledge for providing the MSCV-PM vector, K. Ng and M. W. Lensch for teratoma injections and assessment and S. Loewer for discussions. We also thank E. Olhava and Epizyme Inc. for synthesizing and providing the DOT1L inhibitor, EPZ004777. G.Q.D. is an investigator of the Howard Hughes Medical Institute. Research was funded by grants from the US National Institutes of Health (NIH) to S.A.A. (CA140575) and G.Q.D., and the CHB Stem Cell Program.

Author information

Authors and Affiliations

  1. Division of Pediatric Hematology and Oncology, Stem Cell Transplantation Program, Manton Center for Orphan Disease Research, Children’s Hospital Boston and Dana Farber Cancer Institute, Boston, 02115, Massachusetts, USA

    Tamer T. Onder, Anne Cherry, Patrick Cahan, Juli Unternaehrer & George Q. Daley

  2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Tamer T. Onder, Anne Cherry, Patrick Cahan, Juli Unternaehrer & George Q. Daley

  3. Harvard Stem Cell Institute, Cambridge, 02138, Massachusetts, USA

    Tamer T. Onder, Anne Cherry, Nan Zhu, Kathrin M. Bernt, Patrick Cahan, Juli Unternaehrer, Scott A. Armstrong & George Q. Daley

  4. Stem Cell Program, Children’s Hospital Boston, Boston, 02115, Massachusetts, USA

    Tamer T. Onder, Anne Cherry, Patrick Cahan, Juli Unternaehrer & George Q. Daley

  5. German Cancer Research Center, Heidelberg, 69120, Germany,

    Nergis Kara

  6. Division of Hematology/Oncology, Children’s Hospital, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Amit U. Sinha, Nan Zhu, Kathrin M. Bernt, Scott A. Armstrong & George Q. Daley

  7. Department of Pediatric Oncology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Amit U. Sinha, Nan Zhu, Kathrin M. Bernt & Scott A. Armstrong

  8. Department of Molecular Biology and Genetics, Bilkent University, Ankara, 06800, Turkey

    B. Ogan Mancarci

  9. Department of Biology, Massachusetts Institute of Technology, Cambridge, 02142, Massachusetts, USA

    Piyush B. Gupta & Eric S. Lander

  10. Whitehead Institute for Biomedical Research, Cambridge, 02142, Massachusetts, USA

    Piyush B. Gupta

  11. The Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, 02142, Massachusetts, USA

    Eric S. Lander

  12. Department of Systems Biology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Eric S. Lander

  13. Division of Hematology, Brigham and Women's Hospital, Boston, 02115, Massachusetts, USA

    George Q. Daley

  14. Howard Hughes Medical Institute, Chevy Chase, 20815, Maryland, USA

    George Q. Daley

Authors
  1. Tamer T. Onder
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Contributions

T.T.O. performed project planning, experimental work, data interpretation and preparation of the manuscript. N.K., A.C, N.Z., J.U. and B.O.M. performed experimental work. P.C. and A.U.S. participated in data analysis. K.M.B. and S.A.A. provided critical materials and participated in the preparation of the manuscript. P.B.G. and E.S.L., participated in data acquisition, data interpretation and preparation of the manuscript. G.Q.D. supervised research and participated in project planning, data interpretation and preparation of the manuscript.

Corresponding author

Correspondence to George Q. Daley.

Ethics declarations

Competing interests

S.A.A. is a consultant for Epizyme Inc. G.Q.D. is a member of the scientific advisory boards and holds stock in or receives consulting fees from the following companies: Johnson & Johnson, Verastem, Epizyme, iPierian, Solasia KK and MPM Capital, LLP.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-22. (PDF 1698 kb)

Supplementary Table 1

This table contains a list of all the shRNA sequences used. (XLS 21 kb)

Supplementary Table 2

This table contains a list of qRT-PCR primers used. (XLS 11 kb)

Supplementary Table 3

This table contains genes upregulated and downregulated upon Dot1L inhibition during reprogramming based on gene expression profiling. (XLS 56 kb)

Supplementary Table 4

This table contains the enrichment scores for H3K79me2 and H3K27me3 Chip-seq and lists of genes significantly enriched in the indicated cell populations. (XLS 6867 kb)

Supplementary Table 5

This table shows gene sets significantly enriched in the gene set overlap analysis. (XLS 635 kb)

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Onder, T., Kara, N., Cherry, A. et al. Chromatin-modifying enzymes as modulators of reprogramming. Nature 483, 598–602 (2012). https://doi.org/10.1038/nature10953

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