Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Polycomb PHF19 binds H3K36me3 and recruits PRC2 and demethylase NO66 to embryonic stem cell genes during differentiation

Abstract

Polycomb group proteins are repressive chromatin modifiers with essential roles in metazoan development, cellular differentiation and cell fate maintenance. How Polycomb proteins access active chromatin to confer transcriptional silencing during lineage transitions remains unclear. Here we show that the Polycomb repressive complex 2 (PRC2) component PHF19 binds trimethylated histone H3 Lys36 (H3K36me3), a mark of active chromatin, via its Tudor domain. PHF19 associates with the H3K36me3 demethylase NO66, and it is required to recruit the PRC2 complex and NO66 to stem cell genes during differentiation, leading to PRC2-mediated trimethylation of histone H3 Lys27 (H3K27), loss of H3K36me3 and transcriptional silencing. We propose a model whereby PHF19 functions during mouse embryonic stem cell differentiation to transiently bind the H3K36me3 mark via its Tudor domain, forming essential contact points that allow recruitment of PRC2 and H3K36me3 demethylase activity to active gene loci during their transition to a Polycomb-repressed state.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: PHF19 recruits the H3K36me3 demethylase NO66 to chromatin to repress gene transcription.
Figure 2: The Phf19 Tudor domain binds H3K36me3, but Phf19 colocalizes with PRC2 and H3K27me3, not H3K36me3, in mES cells.
Figure 3: No66 binds to Polycomb-repressed genes in mES cells.
Figure 4: Phf19 controls PRC2 and No66 target occupancy in mES cells.
Figure 5: Phf19 is required for PRC2 and No66 recruitment and embryonic stem cell differentiation.
Figure 6: A functional Phf19 Tudor domain is required for the de novo recruitment of PRC2 and No66 during embryonic stem cell differentiation.
Figure 7: Model for Polycomb Phf19 function in embryonic stem (ES) cells and during differentiation.

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. Bracken, A.P. & Helin, K. Polycomb group proteins: navigators of lineage pathways led astray in cancer. Nat. Rev. Cancer 9, 773–784 (2009).

    CAS  Article  Google Scholar 

  2. Pietersen, A.M. & van Lohuizen, M. Stem cell regulation by Polycomb repressors: postponing commitment. Curr. Opin. Cell Biol. 20, 201–207 (2008).

    CAS  Article  Google Scholar 

  3. Simon, J.A. & Kingston, R.E. Mechanisms of polycomb gene silencing: knowns and unknowns. Nat. Rev. Mol. Cell Biol. 10, 697–708 (2009).

    CAS  Article  Google Scholar 

  4. Sauvageau, M. & Sauvageau, G. Polycomb group proteins: multi-faceted regulators of somatic stem cells and cancer. Cell Stem Cell 7, 299–313 (2010).

    CAS  Article  Google Scholar 

  5. Margueron, R. & Reinberg, D. The Polycomb complex PRC2 and its mark in life. Nature 469, 343–349 (2011).

    CAS  Article  Google Scholar 

  6. Cao, R. et al. Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science 298, 1039–1043 (2002).

    CAS  Article  Google Scholar 

  7. Kuzmichev, A., Nishioka, K., Erdjument-Bromage, H., Tempst, P. & Reinberg, D. Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste protein. Genes Dev. 16, 2893–2905 (2002).

    CAS  Article  Google Scholar 

  8. Gao, Z. et al. PCGF Homologs, CBX Proteins, and RYBP define functionally distinct PRC1 family complexes. Mol. Cell 45, 344–356 (2012).

    CAS  Article  Google Scholar 

  9. Tavares, L. et al. RYBP-PRC1 complexes mediate H2A ubiquitylation at Polycomb target sites independently of PRC2 and H3K27me3. Cell 148, 664–678 (2012).

    CAS  Article  Google Scholar 

  10. Wang, H. et al. Role of histone H2A ubiquitination in Polycomb silencing. Nature 431, 873–878 (2004).

    CAS  Article  Google Scholar 

  11. Francis, N.J., Kingston, R.E. & Woodcock, C.L. Chromatin compaction by a Polycomb group protein complex. Science 306, 1574–1577 (2004).

    CAS  Article  Google Scholar 

  12. de Napoles, M. et al. Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation. Dev. Cell 7, 663–676 (2004).

    CAS  Article  Google Scholar 

  13. Shao, Z. et al. Stabilization of chromatin structure by PRC1, a Polycomb complex. Cell 98, 37–46 (1999).

    CAS  Article  Google Scholar 

  14. Nekrasov, M. et al. Pcl-PRC2 is needed to generate high levels of H3–K27 trimethylation at Polycomb target genes. EMBO J. 26, 4078–4088 (2007).

    CAS  Article  Google Scholar 

  15. O'Connell, S. et al. Polycomblike PHD fingers mediate conserved interaction with enhancer of zeste protein. J. Biol. Chem. 276, 43065–43073 (2001).

    CAS  Article  Google Scholar 

  16. Tie, F., Prasad-Sinha, J., Birve, A., Rasmuson-Lestander, A. & Harte, P.J. A 1-megadalton ESC/E(Z) complex from Drosophila that contains polycomblike and RPD3. Mol. Cell. Biol. 23, 3352–3362 (2003).

    CAS  Article  Google Scholar 

  17. Savla, U., Benes, J., Zhang, J. & Jones, R.S. Recruitment of Drosophila Polycomb-group proteins by Polycomblike, a component of a novel protein complex in larvae. Development 135, 813–817 (2008).

    CAS  Article  Google Scholar 

  18. Boulay, G., Rosnoblet, C., Guerardel, C., Angrand, P.O. & Leprince, D. Functional characterization of human Polycomb-like 3 isoforms identifies them as components of distinct EZH2 protein complexes. Biochem. J. 434, 333–342 (2011).

    CAS  Article  Google Scholar 

  19. Hunkapiller, J. et al. Polycomb-like 3 promotes polycomb repressive complex 2 binding to CpG islands and embryonic stem cell self-renewal. PLoS Genet. 8, e1002576 (2012).

    CAS  Article  Google Scholar 

  20. Walker, E., Manias, J.L., Chang, W.Y. & Stanford, W.L. PCL2 modulates gene regulatory networks controlling self-renewal and commitment in embryonic stem cells. Cell Cycle 10, 45–51 (2011).

    CAS  Article  Google Scholar 

  21. Li, X. et al. Mammalian Polycomb-like Pcl2/Mtf2 is a novel regulatory component of PRC2 that can differentially modulate polycomb activity both at the Hox gene cluster and at Cdkn2a genes. Mol. Cell. Biol. 31, 351–364 (2011).

    CAS  Article  Google Scholar 

  22. Casanova, M. et al. Polycomblike 2 facilitates the recruitment of PRC2 Polycomb group complexes to the inactive X chromosome and to target loci in embryonic stem cells. Development 138, 1471–1482 (2011).

    CAS  Article  Google Scholar 

  23. Sarma, K., Margueron, R., Ivanov, A., Pirrotta, V. & Reinberg, D. Ezh2 requires PHF1 to efficiently catalyze H3 lysine 27 trimethylation in vivo. Mol. Cell. Biol. 28, 2718–2731 (2008).

    CAS  Article  Google Scholar 

  24. Chan, D.W. et al. Unbiased proteomic screen for binding proteins to modified lysines on histone H3. Proteomics 9, 2343–2354 (2009).

    CAS  Article  Google Scholar 

  25. Wysocka, J. et al. A PHD finger of NURF couples histone H3 lysine 4 trimethylation with chromatin remodelling. Nature 442, 86–90 (2006).

    CAS  Article  Google Scholar 

  26. Kouzarides, T. Chromatin modifications and their function. Cell 128, 693–705 (2007).

    CAS  Article  Google Scholar 

  27. Schmitges, F.W. et al. Histone methylation by PRC2 is inhibited by active chromatin marks. Mol. Cell 42, 330–341 (2011).

    CAS  Article  Google Scholar 

  28. Voigt, P. et al. Asymmetrically modified nucleosomes. Cell 151, 181–193 (2012).

    CAS  Article  Google Scholar 

  29. Pasini, D. et al. Coordinated regulation of transcriptional repression by the RBP2 H3K4 demethylase and Polycomb-repressive complex 2. Genes Dev. 22, 1345–1355 (2008).

    CAS  Article  Google Scholar 

  30. Schmitz, S.U. et al. Jarid1b targets genes regulating development and is involved in neural differentiation. EMBO J. 30, 4586–4600 (2011).

    CAS  Article  Google Scholar 

  31. Lagarou, A. et al. dKDM2 couples histone H2A ubiquitylation to histone H3 demethylation during Polycomb group silencing. Genes Dev. 22, 2799–2810 (2008).

    CAS  Article  Google Scholar 

  32. Kooistra, S.M. & Helin, K. Molecular mechanisms and potential functions of histone demethylases. Nat. Rev. Mol. Cell Biol. 13, 297–311 (2012).

    CAS  Article  Google Scholar 

  33. Sinha, K.M., Yasuda, H., Coombes, M.M., Dent, S.Y. & de Crombrugghe, B. Regulation of the osteoblast-specific transcription factor Osterix by NO66, a Jumonji family histone demethylase. EMBO J. 29, 68–79 (2010).

    CAS  Article  Google Scholar 

  34. Hansen, K.H. et al. A model for transmission of the H3K27me3 epigenetic mark. Nat. Cell Biol. 10, 1291–1300 (2008).

    CAS  Article  Google Scholar 

  35. Maurer-Stroh, S. et al. The Tudor domain 'Royal Family': Tudor, plant Agenet, Chromo, PWWP and MBT domains. Trends Biochem. Sci. 28, 69–74 (2003).

    CAS  Article  Google Scholar 

  36. Bracken, A.P., Dietrich, N., Pasini, D., Hansen, K.H. & Helin, K. Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions. Genes Dev. 20, 1123–1136 (2006).

    CAS  Article  Google Scholar 

  37. Pasini, D., Bracken, A.P., Hansen, J.B., Capillo, M. & Helin, K. The Polycomb group protein Suz12 is required for embryonic stem cell differentiation. Mol. Cell. Biol. 3769–3779 (2007).

  38. Mikkelsen, T.S. et al. Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448, 553–560 (2007).

    CAS  Article  Google Scholar 

  39. Zhou, V.W., Goren, A. & Bernstein, B.E. Charting histone modifications and the functional organization of mammalian genomes. Nat. Rev. Genet. 12, 7–18 (2011).

    Article  Google Scholar 

  40. Yuan, W. et al. H3K36 methylation antagonizes PRC2-mediated H3K27 methylation. J. Biol. Chem. 286, 7983–7989 (2011).

    CAS  Article  Google Scholar 

  41. Blackledge, N.P. et al. CpG islands recruit a histone H3 lysine 36 demethylase. Mol. Cell 38, 179–190 (2010).

    CAS  Article  Google Scholar 

  42. Shen, X. et al. EZH1 mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining stem cell identity and executing pluripotency. Mol. Cell 32, 491–502 (2008).

    CAS  Article  Google Scholar 

  43. Montgomery, N.D. et al. The murine Polycomb group protein Eed is required for global histone H3 lysine-27 methylation. Curr. Biol. 15, 942–947 (2005).

    CAS  Article  Google Scholar 

  44. Peng, J.C. et al. Jarid2/Jumonji coordinates control of PRC2 enzymatic activity and target gene occupancy in pluripotent cells. Cell 139, 1290–1302 (2009).

    Article  Google Scholar 

  45. Pasini, D. et al. JARID2 regulates binding of the Polycomb repressive complex 2 to target genes in ES cells. Nature 464, 306–310 (2010).

    CAS  Article  Google Scholar 

  46. Landeira, D. et al. Jarid2 is a PRC2 component in embryonic stem cells required for multi-lineage differentiation and recruitment of PRC1 and RNA polymerase II to developmental regulators. Nat. Cell Biol. 12, 618–624 (2010).

    CAS  Article  Google Scholar 

  47. Ku, M. et al. Genomewide analysis of PRC1 and PRC2 occupancy identifies two classes of bivalent domains. PLoS Genet. 4, e1000242 (2008).

    Article  Google Scholar 

  48. Garbe, J.C. et al. Molecular distinctions between stasis and telomere attrition senescence barriers shown by long-term culture of normal human mammary epithelial cells. Cancer Res. 69, 7557–7568 (2009).

    CAS  Article  Google Scholar 

  49. Bracken, A.P. et al. The Polycomb group proteins bind throughout the INK4A-ARF locus and are disassociated in senescent cells. Genes Dev. 21, 525–530 (2007).

    CAS  Article  Google Scholar 

  50. Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).

    Article  Google Scholar 

  51. Pasini, D., Bracken, A.P., Jensen, M.R., Lazzerini Denchi, E. & Helin, K. Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity. EMBO J. 23, 4061–4071 (2004).

    CAS  Article  Google Scholar 

  52. Bracken, A.P. et al. EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer. EMBO J. 22, 5323–5335 (2003).

    CAS  Article  Google Scholar 

  53. Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858 (1996).

    CAS  Article  Google Scholar 

  54. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We are indebted to members of the Bracken laboratory for their valuable comments on the manuscript. We thank the Conway Mass Spectrometry Resource at University College Dublin. Work in the Bracken lab is supported by Science Foundation Ireland under the Principal Investigator Career Advancement Award (SFI PICA SFI/10/IN.1/B3002), the Health Research Board under the Health Research Awards 2010 (HRA_POR/2010/124) and the Irish Research Council for Science, Engineering and Technology (IRCSET).

Author information

Authors and Affiliations

Authors

Contributions

G.L.B. and A.P.B. designed the research. G.L.B. performed most of the experiments. G.G., K.W. and G.C. prepared, performed and analyzed Flag-HA–PHF19 MS samples. D.J.O. performed all SPR analyses. E.J. contributed to immunoprecipitations of Flag-HA–PHF19. L.P. and X.P. performed the initial PHF19 binding assay. S.A.T., M.C.J. and N.J. helped with protein purification and initial SPR experiments. C.M.E. performed H&E staining of embryoid bodies and quantifications thereof. A.J.L. and B.J.L. sequenced ChIP-seq material and E.J.D. analyzed ChIP-seq data. K.M.S. contributed the NO66 antibody and advised on its use. G.L.B. and A.P.B. wrote the manuscript.

Corresponding author

Correspondence to Adrian P Bracken.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 (PDF 3535 kb)

Supplementary Table 1

Mass spectrometry (MS) of FLAG–HA–PHF19 in human cells. (XLSX 170 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Brien, G., Gambero, G., O'Connell, D. et al. Polycomb PHF19 binds H3K36me3 and recruits PRC2 and demethylase NO66 to embryonic stem cell genes during differentiation. Nat Struct Mol Biol 19, 1273–1281 (2012). https://doi.org/10.1038/nsmb.2449

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2449

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing