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Molecular analysis of PRC2 recruitment to DNA in chromatin and its inhibition by RNA

Abstract

Many studies have revealed pathways of epigenetic gene silencing by Polycomb repressive complex 2 (PRC2) in vivo, but understanding the underlying molecular mechanisms requires biochemistry. Here we analyze interactions of reconstituted human PRC2 with nucleosome complexes. Histone modifications, the H3K27M cancer mutation, and inclusion of JARID2 or EZH1 in the PRC2 complex have unexpectedly minor effects on PRC2–nucleosome binding. Instead, protein-free linker DNA dominates the PRC2–nucleosome interaction. Specificity for CG-rich sequences is consistent with PRC2 occupying CG-rich DNA in vivo. PRC2 preferentially binds methylated DNA regulated by its AEBP2 subunit, suggesting how DNA and histone methylation collaborate to repress chromatin. We find that RNA, known to inhibit PRC2 activity, is not a methyltransferase inhibitor per se. Instead, RNA sequesters PRC2 from nucleosome substrates, because PRC2 binding requires linker DNA, and RNA and DNA binding are mutually exclusive. Together, we provide a model for PRC2 recruitment and an explanation for how actively transcribed genomic regions bind PRC2 but escape silencing.

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Figure 1: RNA controls H3K27 methylation status by preventing PRC2 binding to nucleosomes.
Figure 2: Histone modifications have small effects on PRC2 affinity for nucleosome substrates in vitro.
Figure 3: Nucleosome-free linker DNA dictates PRC2 binding to nucleosomes.
Figure 4: PRC2 binds long-linker dinucleosomes preferentially.
Figure 5: PRC2 preferentially binds CG-rich and CpG-methylated DNA.
Figure 6: JARID2 enhances PRC2 histone methyltransferase activity but does not prevent eviction by RNA.
Figure 7: A model for PRC2–chromatin–RNA interactions and regulation of epigenetic silencing.

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References

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Müller, J. & Bienz, M. Long range repression conferring boundaries of Ultrabithorax expression in the Drosophila embryo. EMBO J. 10, 3147–3155 (1991).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Mihaly, J., Mishra, R.K. & Karch, F. A conserved sequence motif in Polycomb-response elements. Mol. Cell 1, 1065–1066 (1998).

    Article  CAS  PubMed  Google Scholar 

  4. Zhao, J., Sun, B.K., Erwin, J.A., Song, J.-J. & Lee, J.T. Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome. Science 322, 750–756 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kaneko, S., Son, J., Bonasio, R., Shen, S.S. & Reinberg, D. Nascent RNA interaction keeps PRC2 activity poised and in check. Genes Dev. 28, 1983–1988 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Davidovich, C., Zheng, L., Goodrich, K.J. & Cech, T.R. Promiscuous RNA binding by Polycomb repressive complex 2. Nat. Struct. Mol. Biol. 20, 1250–1257 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Davidovich, C. et al. Toward a consensus on the binding specificity and promiscuity of PRC2 for RNA. Mol. Cell 57, 552–558 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Wang, X. et al. Targeting of polycomb repressive complex 2 to RNA by short repeats of consecutive guanines. Mol. Cell 65, 1056–1067.e5 (2017).

    Article  CAS  PubMed  Google Scholar 

  9. Cifuentes-Rojas, C., Hernandez, A.J., Sarma, K. & Lee, J.T. Regulatory interactions between RNA and polycomb repressive complex 2. Mol. Cell 55, 171–185 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Beltran, M. et al. The interaction of PRC2 with RNA or chromatin is mutually antagonistic. Genome Res. http://dx.doi.org/10.1101/gr.197632.115 2016).

  11. Margueron, R. et al. Role of the polycomb protein EED in the propagation of repressive histone marks. Nature 461, 762–767 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  13. Lewis, P.W. et al. Inhibition of PRC2 activity by a gain-of-function H3 mutation found in pediatric glioblastoma. Science 340, 857–861 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Xu, C. et al. Binding of different histone marks differentially regulates the activity and specificity of polycomb repressive complex 2 (PRC2). Proc. Natl. Acad. Sci. USA 107, 19266–19271 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Jiao, L. & Liu, X. Structural basis of histone H3K27 trimethylation by an active polycomb repressive complex 2. Science 350, aac4383 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Nekrasov, M., Wild, B. & Müller, J. Nucleosome binding and histone methyltransferase activity of Drosophila PRC2. EMBO Rep. 6, 348–353 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Mendenhall, E.M. et al. GC-rich sequence elements recruit PRC2 in mammalian ES cells. PLoS Genet. 6, e1001244 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Dyer, P.N. et al. Reconstitution of nucleosome core particles from recombinant histones and DNA. Methods Enzymol. 375, 23–44 (2004).

    Article  CAS  PubMed  Google Scholar 

  19. Yuan, W. et al. Dense chromatin activates Polycomb repressive complex 2 to regulate H3 lysine 27 methylation. Science 337, 971–975 (2012).

    Article  CAS  PubMed  Google Scholar 

  20. Martin, C., Cao, R. & Zhang, Y. Substrate preferences of the EZH2 histone methyltransferase complex. J. Biol. Chem. 281, 8365–8370 (2006).

    Article  CAS  PubMed  Google Scholar 

  21. Müller, J. et al. Histone methyltransferase activity of a Drosophila Polycomb group repressor complex. Cell 111, 197–208 (2002).

    Article  PubMed  Google Scholar 

  22. Sanulli, S. et al. Jarid2 methylation via the PRC2 complex regulates H3K27me3 deposition during cell differentiation. Mol. Cell 57, 769–783 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Nikitina, T., Wang, D., Gomberg, M., Grigoryev, S.A. & Zhurkin, V.B. Combined micrococcal nuclease and exonuclease III digestion reveals precise positions of the nucleosome core/linker junctions: implications for high-resolution nucleosome mapping. J. Mol. Biol. 425, 1946–1960 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Widlak, P. & Garrard, W.T. Unique features of the apoptotic endonuclease DFF40/CAD relative to micrococcal nuclease as a structural probe for chromatin. Biochem. Cell Biol. 84, 405–410 (2006).

    Article  CAS  PubMed  Google Scholar 

  25. Schones, D.E. et al. Dynamic regulation of nucleosome positioning in the human genome. Cell 132, 887–898 (2008).

    Article  CAS  PubMed  Google Scholar 

  26. Radman-Livaja, M. & Rando, O.J. Nucleosome positioning: how is it established, and why does it matter? Dev. Biol. 339, 258–266 (2010).

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Bird, A. DNA methylation patterns and epigenetic memory. Genes Dev. 16, 6–21 (2002).

    Article  CAS  PubMed  Google Scholar 

  29. Meissner, A. et al. Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature 454, 766–770 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Buck-Koehntop, B.A. et al. Molecular basis for recognition of methylated and specific DNA sequences by the zinc finger protein Kaiso. Proc. Natl. Acad. Sci. USA 109, 15229–15234 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Son, J., Shen, S.S., Margueron, R. & Reinberg, D. Nucleosome-binding activities within JARID2 and EZH1 regulate the function of PRC2 on chromatin. Genes Dev. 27, 2663–2677 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Li, G. et al. Jarid2 and PRC2, partners in regulating gene expression. Genes Dev. 24, 368–380 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Davidovich, C. & Cech, T.R. The recruitment of chromatin modifiers by long noncoding RNAs: lessons from PRC2. RNA 21, 2007–2022 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Holoch, D. & Margueron, R. Mechanisms regulating PRC2 recruitment and enzymatic activity. Trends Biochem. Sci. 42, 531–542 (2017).

    Article  CAS  PubMed  Google Scholar 

  36. Justin, N. et al. Structural basis of oncogenic histone H3K27M inhibition of human polycomb repressive complex 2. Nat. Commun. 7, 11316 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Choi, J. et al. DNA binding by PHF1 prolongs PRC2 residence time on chromatin and thereby promotes H3K27 methylation. Nat. Struct. Mol. Biol. http://dx/doi.org/10.1038/nsmb.3488 (2017).

  38. Kaneko, S., Son, J., Shen, S.S., Reinberg, D. & Bonasio, R. PRC2 binds active promoters and contacts nascent RNAs in embryonic stem cells. Nat. Struct. Mol. Biol. 20, 1258–1264 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Riising, E.M. et al. Gene silencing triggers polycomb repressive complex 2 recruitment to CpG islands genome wide. Mol. Cell 55, 347–360 (2014).

    Article  CAS  PubMed  Google Scholar 

  40. Piunti, A. et al. Therapeutic targeting of polycomb and BET bromodomain proteins in diffuse intrinsic pontine gliomas. Nat. Med. 23, 493–500 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Portoso, M. et al. PRC2 is dispensable for HOTAIR-mediated transcriptional repression. EMBO J. 36, 981–994 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Wu, H. et al. Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function in transcriptional regulation in mouse embryonic stem cells. Genes Dev. 25, 679–684 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Fenouil, R. et al. CpG islands and GC content dictate nucleosome depletion in a transcription-independent manner at mammalian promoters. Genome Res. 22, 2399–2408 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Deaton, A.M. & Bird, A. CpG islands and the regulation of transcription. Genes Dev. 25, 1010–1022 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Lynch, M.D. et al. An interspecies analysis reveals a key role for unmethylated CpG dinucleotides in vertebrate Polycomb complex recruitment. EMBO J. 31, 317–329 (2012).

    Article  CAS  PubMed  Google Scholar 

  46. Bartke, T. et al. Nucleosome-interacting proteins regulated by DNA and histone methylation. Cell 143, 470–484 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. King, A.D. et al. Reversible regulation of promoter and enhancer histone landscape by DNA methylation in mouse embryonic stem cells. Cell Rep. 17, 289–302 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Jin, B. et al. DNMT1 and DNMT3B modulate distinct polycomb-mediated histone modifications in colon cancer. Cancer Res. 69, 7412–7421 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Viré, E. et al. The Polycomb group protein EZH2 directly controls DNA methylation. Nature 439, 871–874 (2006).

    Article  PubMed  CAS  Google Scholar 

  50. Grijzenhout, A. et al. Functional analysis of AEBP2, a PRC2 Polycomb protein, reveals a Trithorax phenotype in embryonic development and in ESCs. Development 143, 2716–2723 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Sengupta, A.K., Kuhrs, A. & Müller, J. General transcriptional silencing by a Polycomb response element in Drosophila. Development 131, 1959–1965 (2004).

    Article  CAS  PubMed  Google Scholar 

  52. Klymenko, T. et al. A Polycomb group protein complex with sequence-specific DNA-binding and selective methyl-lysine-binding activities. Genes Dev. 20, 1110–1122 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Schuettengruber, B., Chourrout, D., Vervoort, M., Leblanc, B. & Cavalli, G. Genome regulation by polycomb and trithorax proteins. Cell 128, 735–745 (2007).

    Article  CAS  PubMed  Google Scholar 

  54. Mito, Y., Henikoff, J.G. & Henikoff, S. Histone replacement marks the boundaries of cis-regulatory domains. Science 315, 1408–1411 (2007).

    Article  CAS  PubMed  Google Scholar 

  55. Shen, X. et al. Jumonji modulates polycomb activity and self-renewal versus differentiation of stem cells. Cell 139, 1303–1314 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  56. 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  PubMed  PubMed Central  Google Scholar 

  57. Kaneko, S. et al. Interactions between JARID2 and noncoding RNAs regulate PRC2 recruitment to chromatin. Mol. Cell 53, 290–300 (2014).

    Article  CAS  PubMed  Google Scholar 

  58. Nguyen, U.T.T. et al. Accelerated chromatin biochemistry using DNA-barcoded nucleosome libraries. Nat. Methods 11, 834–840 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank C. J. Lim and other members of the Cech lab for useful conversations. We thank C. Davidovich (Monash University, Clayton, Australia) for initial trials of expression and purification of JARID2 and for stimulating discussion. We thank K. Luger (University of Colorado Boulder) and members of her laboratory—especially U. M. Muthurajan and P. Dyer—for providing plasmids and helpful discussion of reconstituting nucleosomes. We thank J. Müller and his group (MPI Martiensreid, Germany) and Beat Fierz (EPFL, Lausanne, Switzerland) for sharing unpublished data and discussions. T.W.M. is supported by National Institutes of Health grants R37-GM086868 and PO1-CA196539. T.R.C. is supported by the Howard Hughes Medical Institute.

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X.W., R.D.P., and T.R.C. designed the experiments. X.W. and R.D.P. carried out experiments. A.R.G. carried out protein purification. Z.Z.B., E.J.G. and T.W.M. carried out the synthesis of modified histone H3. X.W., R.D.P., and T.R.C. wrote the manuscript.

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Correspondence to Thomas R Cech.

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T.R.C. is on the board of directors of Merck, Inc., which provides no funding for his research.

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Wang, X., Paucek, R., Gooding, A. et al. Molecular analysis of PRC2 recruitment to DNA in chromatin and its inhibition by RNA. Nat Struct Mol Biol 24, 1028–1038 (2017). https://doi.org/10.1038/nsmb.3487

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