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

Nature Structural & Molecular Biology volume 19, pages 1273–1281 (2012)Cite this article

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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.

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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.

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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).

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Authors and Affiliations

  1. The Smurfit Institute of Genetics, Trinity College Dublin, Ireland

    Gerard L Brien, Emilia Jerman, Siobhán A Turner, Chris M Egan, Eiseart J Dunne & Adrian P Bracken

  2. Conway Institute, University College Dublin, Ireland

    Guillermo Gambero, David J O'Connell, Maike C Jurgens, Kieran Wynne, Amanda J Lohan, Neil Ferguson, Brendan J Loftus & Gerard Cagney

  3. Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA

    Lianhua Piao & Xiaobing Shi

  4. Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA

    Krishna M Sinha

  5. The Adelaide & Meath Hospital, including the National Children's Hospital, Dublin, Ireland

    Adrian P Bracken

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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.

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Correspondence to Adrian P Bracken.

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Supplementary Table 1

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

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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

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