[go: up one dir, main page]
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.

  • Article
  • Published:

Read–write mechanisms of H2A ubiquitination by Polycomb repressive complex 1

Nature (2024)Cite this article

Subjects

Abstract

Epigenetic inheritance of silent chromatin domains is fundamental to cellular memory during embryogenesis, but it must overcome the dilution of repressive histone modifications during DNA replication1. One such modification, histone H2A lysine 119 monoubiquitination (H2AK119Ub), needs to be re-established by the Polycomb repressive complex 1 (PRC1) E3 ligase to restore the silent Polycomb domain2,3. However, the exact mechanism behind this restoration remains unknown. Here, combining cryo-electron microscopy (cryo-EM) and functional approaches, we characterize the read–write mechanism of the non-canonical PRC1-containing RYBP (ncPRC1RYBP). This mechanism, which functions as a positive-feedback loop in epigenetic regulation4,5, emphasizes the pivotal role of ncPRC1RYBP in restoring H2AK119Ub. We observe an asymmetrical binding of ncPRC1RYBP to H2AK119Ub nucleosomes, guided in part by the N-terminal zinc-finger domain of RYBP binding to residual H2AK119Ub on nascent chromatin. This recognition positions the RING domains of RING1B and BMI1 on the unmodified nucleosome side, enabling recruitment of the E2 enzyme to ubiquitinate H2AK119 within the same nucleosome (intra-nucleosome read–write) or across nucleosomes (inter-nucleosome read–write). Collectively, our findings provide key structural and mechanistic insights into the dynamic interplay of epigenetic regulation, highlighting the significance of ncPRC1RYBP in H2AK119Ub restoration to sustain repressive chromatin domains.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: The structure of ncPRC1RYBP on singly modified H2AK119Ub nucleosome reveals an asymmetric mode of binding.
Fig. 2: Two distinct acidic patch interactions and ubiquitin binding stabilize the ncPRC1RYBP complex on the nucleosome, suggesting an intra-nucleosome read–write mechanism.
Fig. 3: Structural and biochemical characterization of the intra-nucleosome read–write mechanism by ncPRC1RYBP.
Fig. 4: The structure of ncPRC1RYBP on H2AK119Ub dinucleosome offers insights into inter-nucleosome read–write mechanism.

Similar content being viewed by others

Data availability

The cryo-EM maps have been deposited in the Electron Microscopy Data Bank (EMDB) with the following accession codes: EMDB-46728 for the overall map of ncPRC1RYBP bound to singly modified H2AK119Ub nucleosome (map 1), EMDB-46729 for map with the best-resolved NZF of RYBP of ncPRC1RYBP bound to singly modified H2AK119Ub nucleosome (map 2), EMDB-46730 for map with the best-resolved RING1B–BMI1 of ncPRC1RYBP bound to singly modified H2AK119Ub nucleosome (map 3) and EMDB-46731 the composite map of maps 1, 2 and 3 (map 14). EMDB-46732 for ncPRC1RYBP bound to doubly modified H2AK119Ub nucleosome (map 4). EMDB-46822 for ncPRC1RYBP Δlinker mutant bound to singly modified H2AK119Ub nucleosome (map 5) and EMDB-46823 for ncPRC1RYBP bound to unmodified nucleosome (map 6). EMDB-46733 for the overall map of ncPRC1RYBP bound to symmetric H2AK119Ub dinucleosome (map 7), EMDB-46734 for map focused on RYBP–ubiquitin of ncPRC1RYBP bound to symmetric H2AK119Ub dinucleosome (map 8), and EMDB-46735 for map focused on RING1B–BMI1 of ncPRC1RYBP bound to symmetric H2AK119Ub dinucleosome (map 9). EMDB-46771 for the overall map of ncPRC1RYBP bound to H2AK119Ub–H1.4 chromatosome (map 10). EMDB-46736 for the overall map of ncPRC1RYBP bound to asymmetric-H2AK119Ub dinucleosome (map 11), EMDB-46737 for map focused on RYBP–ubiquitin of ncPRC1RYBP bound to asymmetric-H2AK119Ub dinucleosome (map 12), and EMDB-46738 for map focused on RING1B–BMI1 of ncPRC1RYBP bound to asymmetric-H2AK119Ub dinucleosome (map 13). The atomic coordinates for the structures have been deposited in the PDB with the accession code 9DBY for ncPRC1RYBP bound to the singly modified H2AK119Ub nucleosome, 9DDE for the ncPRC1RYBP bound to chromatosome, 9DG3 for the RYBP ∆linker mutant bound to singly modified H2AK119Ub nucleosome and 9DGG for the ncPRC1RYBP bound to the unmodified nucleosome. Plasmid reagents are available upon request. Source data are provided with this paper.

References

  1. Stewart-Morgan, K. R., Petryk, N. & Groth, A. Chromatin replication and epigenetic cell memory. Nat. Cell Biol. 22, 361–371 (2020).

    Article  CAS  PubMed  Google Scholar 

  2. Flury, V. et al. Recycling of modified H2A-H2B provides short-term memory of chromatin states. Cell 186, 1050–1065.e19 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  ADS  CAS  PubMed  Google Scholar 

  4. Reinberg, D. & Vales, L. D. Chromatin domains rich in inheritance. Science 361, 33–34 (2018).

    Article  ADS  CAS  PubMed  Google Scholar 

  5. Zhao, J. et al. RYBP/YAF2–PRC1 complexes and histone H1-dependent chromatin compaction mediate propagation of H2AK119Ub1 during cell division. Nat. Cell Biol. 22, 439–452 (2020).

    Article  CAS  PubMed  Google Scholar 

  6. Wenger, A. et al. Symmetric inheritance of parental histones governs epigenome maintenance and embryonic stem cell identity. Nat. Genet. 55, 1567–1578 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Cheedipudi, S., Genolet, O. & Dobreva, G. Epigenetic inheritance of cell fates during embryonic development. Front. Genet. 5, 19 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Blackledge, N. P. et al. Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation. Cell 157, 1445–1459 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Taherbhoy, A. M., Huang, O. W. & Cochran, A. G. BMI1–RING1B is an autoinhibited RING E3 ubiquitin ligase. Nat. Commun. 6, 7621 (2015).

    Article  ADS  PubMed  Google Scholar 

  10. Schoorlemmer, J. et al. Ring1A is a transcriptional repressor that interacts with the Polycomb-M33 protein and is expressed at rhombomere boundaries in the mouse hindbrain. EMBO J. 16, 5930–5942 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Buchwald, G. et al. Structure and E3-ligase activity of the Ring–Ring complex of Polycomb proteins Bmi1 and Ring1b. EMBO J. 25, 2465–2474 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 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).

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  15. Fursova, N. A. et al. Synergy between variant PRC1 complexes defines Polycomb-mediated gene repression. Mol. Cell 74, 1020–1036.e8 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Rose, N. R. et al. RYBP stimulates PRC1 to shape chromatin-based communication between Polycomb repressive complexes. eLife 5, e18591 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Morey, L., Aloia, L., Cozzuto, L., Benitah, S. A. & Di Croce, L. RYBP and Cbx7 define specific biological functions of polycomb complexes in mouse embryonic stem cells. Cell Rep. 3, 60–69 (2013).

    Article  CAS  PubMed  Google Scholar 

  19. Garcia, E., Marcos-Gutierrez, C., del Mar Lorente, M., Moreno, J. C. & Vidal, M. RYBP, a new repressor protein that interacts with components of the mammalian Polycomb complex, and with the transcription factor YY1. EMBO J. 18, 3404–3418 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Eid, A. & Torres-Padilla, M. E. Characterization of non-canonical Polycomb repressive complex 1 subunits during early mouse embryogenesis. Epigenetics 11, 389–397 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Wang, R. et al. Polycomb group targeting through different binding partners of RING1B C-terminal domain. Structure 18, 966–975 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Arrigoni, R. et al. The Polycomb-associated protein Rybp is a ubiquitin binding protein. FEBS Lett. 580, 6233–6241 (2006).

    Article  CAS  PubMed  Google Scholar 

  23. Wang, B. et al. Structure and ubiquitin interactions of the conserved zinc finger domain of Npl4. J. Biol. Chem. 278, 20225–20234 (2003).

    Article  CAS  PubMed  Google Scholar 

  24. Alam, S. L. et al. Ubiquitin interactions of NZF zinc fingers. EMBO J. 23, 1411–1421 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hansen, K. H. et al. Erratum: A model for transmission of the H3K27me3 epigenetic mark. Nat. Cell Biol. 10, 1484–1484 (2008).

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  27. Iglesias, N. et al. Automethylation-induced conformational switch in Clr4 (Suv39h) maintains epigenetic stability. Nature 560, 504–508 (2018).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  28. Poepsel, S., Kasinath, V. & Nogales, E. Cryo-EM structures of PRC2 simultaneously engaged with two functionally distinct nucleosomes. Nat. Struct. Mol. Biol. 25, 154–162 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Cutter DiPiazza, A. R. et al. Spreading and epigenetic inheritance of heterochromatin require a critical density of histone H3 lysine 9 tri-methylation. Proc. Natl Acad. Sci. USA 118, e2100699118 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Yu, J. R. et al. The H3K36me2 writer–reader dependency in H3K27M-DIPG. Sci. Adv. 7, eabg7444 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  31. Grunstein, M. Yeast heterochromatin: regulation of its assembly and inheritance by histones. Cell 93, 325–328 (1998).

    Article  CAS  PubMed  Google Scholar 

  32. Ragunathan, K., Jih, G. & Moazed, D. Epigenetic inheritance uncoupled from sequence-specific recruitment. Science 348, 1258699 (2015).

    Article  PubMed  Google Scholar 

  33. Blackledge, N. P. & Klose, R. J. The molecular principles of gene regulation by Polycomb repressive complexes. Nat. Rev. Mol. Cell Biol. 22, 815–833 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Ciapponi, M., Karlukova, E., Schkolziger, S., Benda, C. & Muller, J. Structural basis of the histone ubiquitination read–write mechanism of RYBP–PRC1. Nat. Struct. Mol. Biol. 31, 1023–1027 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. McGinty, R. K., Henrici, R. C. & Tan, S. Crystal structure of the PRC1 ubiquitylation module bound to the nucleosome. Nature 514, 591–596 (2014).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  36. Ai, H. S. et al. Synthetic E2–Ub–nucleosome conjugates for studying nucleosome ubiquitination. Chem 9, 1221–1240 (2023).

    Article  CAS  Google Scholar 

  37. Li, Z. et al. Structure of a Bmi-1–Ring1B Polycomb group ubiquitin ligase complex. J. Biol. Chem. 281, 20643–20649 (2006).

    Article  CAS  PubMed  Google Scholar 

  38. Hauri, S. et al. A high-density map for navigating the human Polycomb complexome. Cell Rep. 17, 583–595 (2016).

    Article  CAS  PubMed  Google Scholar 

  39. Kaufman, P. D. & Rando, O. J. Chromatin as a potential carrier of heritable information. Curr. Opin. Cell Biol. 22, 284–290 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Cao, R., Tsukada, Y. & Zhang, Y. Role of Bmi-1 and Ring1A in H2A ubiquitylation and Hox gene silencing. Mol. Cell 20, 845–854 (2005).

    Article  CAS  PubMed  Google Scholar 

  41. Bentley, M. L. et al. Recognition of UbcH5c and the nucleosome by the Bmi1/Ring1b ubiquitin ligase complex. EMBO J. 30, 3285–3297 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  43. Kulathu, Y., Akutsu, M., Bremm, A., Hofmann, K. & Komander, D. Two-sided ubiquitin binding explains specificity of the TAB2 NZF domain. Nat. Struct. Mol. Biol. 16, 1328–1330 (2009).

    Article  CAS  PubMed  Google Scholar 

  44. Escobar, T. M., Loyola, A. & Reinberg, D. Parental nucleosome segregation and the inheritance of cellular identity. Nat. Rev. Genet. 22, 379–392 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Flury, V. & Groth, A. Safeguarding the epigenome through the cell cycle: a multitasking game. Curr. Opin. Genet. Dev. 85, 102161 (2024).

    Article  CAS  PubMed  Google Scholar 

  46. Tamburri, S. et al. Histone H2AK119 mono-ubiquitination is essential for Polycomb-mediated transcriptional repression. Mol. Cell 77, 840–856.e5 (2020).

  47. Witus, S. R. et al. BRCA1/BARD1 intrinsically disordered regions facilitate chromatin recruitment and ubiquitylation. EMBO J. 42, e113565 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Valencia-Sanchez, M. I. et al. Structural basis of Dot1L stimulation by histone H2B lysine 120 ubiquitination. Mol. Cell 74, 1010–1019.e6 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. 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 

  50. Zhou, B. R. et al. Distinct structures and dynamics of chromatosomes with different human linker histone isoforms. Mol. Cell 81, 166–182.e6 (2021).

    Article  CAS  PubMed  Google Scholar 

  51. Long, L., Furgason, M. & Yao, T. Generation of nonhydrolyzable ubiquitin-histone mimics. Methods 70, 134–138 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Thomas, J. F. et al. Structural basis of histone H2A lysine 119 deubiquitination by Polycomb repressive deubiquitinase BAP1/ASXL1. Sci. Adv. 9, eadg9832 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Ge, W. et al. Basis of the H2AK119 specificity of the Polycomb repressive deubiquitinase. Nature 616, 176–182 (2023).

    Article  ADS  CAS  PubMed  Google Scholar 

  54. Armache, K. J., Garlick, J. D., Canzio, D., Narlikar, G. J. & Kingston, R. E. Structural basis of silencing: Sir3 BAH domain in complex with a nucleosome at 3.0 A resolution. Science 334, 977–982 (2011).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  55. Dao, H. T., Liu, H., Mashtalir, N., Kadoch, C. & Muir, T. W. Synthesis of oriented hexasomes and asymmetric nucleosomes using a template editing process. J. Am. Chem. Soc. 144, 2284–2291 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Machida, S. et al. Structural basis of heterochromatin formation by human HP1. Mol. Cell 69, 385–397.e8 (2018).

    Article  CAS  PubMed  Google Scholar 

  57. Lee, C. H. et al. Distinct stimulatory mechanisms regulate the catalytic activity of Polycomb repressive complex 2. Mol. Cell 70, 435–448.e5 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Hall, M. D. et al. Fluorescence polarization assays in high-throughput screening and drug discovery: a review. Methods. Appl. Fluoresc. 4, 022001 (2016).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  59. Stark, H. GraFix: stabilization of fragile macromolecular complexes for single particle cryo-EM. Methods Enzymol. 481, 109–126 (2010).

    Article  CAS  PubMed  Google Scholar 

  60. Grau, D. et al. Structures of monomeric and dimeric PRC2:EZH1 reveal flexible modules involved in chromatin compaction. Nat. Commun. 12, 714 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  61. Valencia-Sanchez, M. I. et al. The structure of a virus-encoded nucleosome. Nat. Struct. Mol. Biol. 28, 413–417 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Cheng, A. et al. Leginon: new features and applications. Protein Sci. 30, 136–150 (2021).

    Article  CAS  PubMed  Google Scholar 

  64. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  66. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  67. Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).

    Article  CAS  PubMed  Google Scholar 

  68. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  69. Pettersen, E. F. et al. UCSF Chimera-a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  70. Vijay-Kumar, S., Bugg, C. E. & Cook, W. J. Structure of ubiquitin refined at 1.8 A resolution. J. Mol. Biol. 194, 531–544 (1987).

    Article  CAS  PubMed  Google Scholar 

  71. Evans, R. et al. Protein complex prediction with AlphaFold-Multimer. Preprint at bioRxiv https://doi.org/10.1101/2021.10.04.463034 (2021).

  72. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    Article  ADS  PubMed  Google Scholar 

  73. Dombrowski, M., Engeholm, M., Dienemann, C., Dodonova, S. & Cramer, P. Histone H1 binding to nucleosome arrays depends on linker DNA length and trajectory. Nat. Struct. Mol. Biol. 29, 493–501 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. DeLano, W. The PyMOL Molecular Graphics System, version 1.3r1 (Schrödinger, 2020).

  75. Meng, E. C. et al. UCSF ChimeraX: tools for structure building and analysis. Protein Sci. 32, e4792 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors thank G. Narlikar, B. Al-Sady and members of the Armache laboratory for helpful suggestions and discussions; W. Rice, B. Wang and K. Huihui for helping with data collection at NYU Langone Health’s Cryo-Electron Microscopy Laboratory, as well as the NYU Microscopy Laboratory; colleagues and staff at Simons Electron Microscopy Center at the New York Structural Biology Center for their support in the data collection; and the HPC Core at NYU Langone Health for computer access and support. Work in the Armache laboratory is supported by grants from the Mark Foundation for Cancer Research and the National Institutes of Health (NIH) (R01GM115882 and R01CA266978).

Author information

Author notes

  1. Brian A. Sosa

    Present address: MOMA Therapeutics, Cambridge, MA, USA

Authors and Affiliations

  1. Department of Biochemistry and Molecular Pharmacology, New York University Grossman School of Medicine, New York, NY, USA

    Victoria Godínez López, Marco Igor Valencia-Sánchez, Stephen Abini-Agbomson, Jonathan F. Thomas, Rachel Lee, Pablo De Ioannes, Brian A. Sosa & Karim-Jean Armache

  2. Department of Biochemistry and Molecular Biology and The Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, USA

    Jean-Paul Armache

Authors
  1. Victoria Godínez López
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marco Igor Valencia-Sánchez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Stephen Abini-Agbomson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jonathan F. Thomas
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Rachel Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Pablo De Ioannes
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Brian A. Sosa
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jean-Paul Armache
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Karim-Jean Armache
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

V.G.L., M.I.V.-S. and K.-J.A. conceptualized and designed the study. V.G.L. conducted the biochemical and structural experiments. J.F.T., S.-A.A., R.L., B.A.S. and P.D.I. generated various reagents. J.-P.A., M.I.V.-S. and V.G. contributed to cryo-EM data analysis. V.G., M.I.V.-S. and K.-J.A. interpreted the data and wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Karim-Jean Armache.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature thanks Rob Klose and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 The NZF domain and R-fingers of RYBP and YAF2 are highly conserved.

(a) Schematic representation of the RYBP domains (top) and sequence of human RYBP (bottom) highlighting the position of key residues. Positively charged lysines are represented in the linker as boxes. The linker region, present in RYBP but absent in YAF2 is denoted as “RYBP-specific region”. (b) Bar diagram of RYBP, the mutants used in this work and the paralog YAF2. (c) Multiple sequence alignment of RYBP and its paralog YAF2. The NZF domain is shaded in blue, highlighting in dark blue the cysteines that coordinate the Zinc atom. The dark blue rectangle marks the residues T31/F32 that interact with Ub, and the residues of R-finger that interact with the acidic patch are shown in yellow. The linker connecting the NZF domain with the C-terminal domain is shown in green. The C-terminal domain of RYBP/YAF2 (not visible in structures presented here), which interacts with the RING1B RAWL domain is shown in cyan, with residues that interact with RING1B highlighted. The structure visible in our maps is shown as solid lines and disordered regions in dashed lines above the sequence.

Extended Data Fig. 2 Cryo-EM analysis of the ncPRC1RYBP bound to doubly modified H2AK119Ub nucleosome.F.

(a) Raw cryo-EM images of ncPRC1RYBP complex bound to doubly modified H2AK119Ub nucleosome with selected particles of the complex in blue circles. (b) Representative 2D class averages were calculated from the final subset of particles. (c) FSC plot of the 3.41 Å ncPRC1RYBP bound to doubly modified H2AK119Ub nucleosome complex, between two independently refined half maps (measured at FSC = 0.143). (d) 3D-FSC plot of the 3.41 Å ncPRC1RYBP bound to doubly modified H2AK119Ub nucleosome complex, between two independently refined half maps (measured at FSC = 0.143). (e) Two representative views of the 3D reconstruction of ncPRC1RYBP bound to doubly modified H2AK119Ub nucleosome complex (Map 4). (f) Euler angle distribution of assignment of particles used to generate the final 3D reconstruction at 3.41 Å. The length of every cylinder is proportional to the number of particles assigned to the specific orientation. (g) Local resolution heat map for the reconstruction of ncPRC1RYBP complex bound to the doubly modified H2AK119Ub nucleosome.

Extended Data Fig. 3 Cryo-EM analysis of the ncPRC1RYBP bound to singly modified H2AK119Ub nucleosome complex.

(a) Raw cryo-EM images of ncPRC1RYBP complex bound to the singly modified H2AK119Ub nucleosome. with selected particles of the complex in blue circles. (b) Representative 2D class averages were calculated from the final subset of particles (Map 1). (c) FSC plot between two independently refined half maps (measured at FSC = 0.143) of the 2.80 Å ncPRC1RYBP bound to singly modified H2AK119Ub nucleosome complex. (d) 3D-FSC plot of the 2.80 Å ncPRC1RYBP bound to singly modified H2AK119Ub nucleosome complex between two independently refined half maps. (e) Two representative views of the 3D reconstruction of ncPRC1RYBP complex bound to singly modified H2AK119Ub nucleosome (Map 1). (f) Euler angle distribution of assignment of particles used to generate the final 3D reconstruction of the 2.80 Å complex. The length of every cylinder is proportional to the number of particles assigned to the specific orientation. (g) Local resolution heat map for the reconstruction of ncPRC1RYBP complex bound to the singly modified H2AK119Ub nucleosome.

Extended Data Fig. 4 Close-up of the cryo-EM map and interfaces in ncPRC1RYBP complex bound to the singly modified H2AK119Ub nucleosome.

Selected views of the model fit to the cryo-EM maps for the structure are shown. (a) RING1B:acidic patch interactions (left) and cryo-EM map in the region (right). (b) NZF of RYBP:acidic patch interactions (left) and cryo-EM map in the region (right). (c) NZF of RYBP:Ub interface (left) and cryo-EM map in the region (right). (d) BMI1:histones H3,H4 and H2B interface (left) and cryo-EM map in the region (right).

Extended Data Fig. 5 Cryo-EM analysis of the ncPRC1RYBP bound to doubly modified H2AK119Ub chromatosome.

(a) Raw cryo-EM images of ncPRC1RYBP bound to doubly modified H2AK119Ub chromatosome with selected particles of the complex in blue circles. (b) Representative 2D class averages were calculated from the final subset of particles. (c) FSC plot of the 3.20 Å ncPRC1RYBP bound to doubly modified H2AK119Ub chromatosome complex between two independently refined half maps (measured at FSC = 0.143). (d) 3D-FSC plot of the 3.20 Å ncPRC1RYBP bound to doubly modified H2AK119Ub chromatosome complex between two independently refined half maps (measured at FSC = 0.143). (e) Two representative views of the 3D reconstruction of ncPRC1RYBP bound to doubly modified H2AK119Ub chromatosome (Map 10). (f) Euler angle distribution of assignment of particles used to generate the final 3D reconstruction of the 3.20 Å complex. The length of every cylinder is proportional to the number of particles assigned to the specific orientation. (g) Local resolution heat map of the reconstruction of ncPRC1RYBP bound to doubly modified H2AK119Ub chromatosome.

Extended Data Fig. 6 Cryo-EM analysis of the ncPRC1RYBP ∆linker mutant bound to singly modified H2AK119Ub nucleosome.

(a) Raw cryo-EM images of ncPRC1RYBP ∆linker mutant bound to singly modified H2AK119Ub nucleosome with selected particles of the complex in blue circles. (b) Representative 2D class averages were calculated from the final subset of particles. (c) FSC plot of the 3.46 Å ncPRC1RYBP ∆linker mutant bound to singly modified H2AK119Ub nucleosome complex between two independently refined half maps (measured at FSC = 0.143). (d) 3D-FSC plot of the 3.46 Å ncPRC1RYBP ∆linker mutant bound to singly modified H2AK119Ub nucleosome complex between two independently refined half maps (measured at FSC = 0.143). (e) Two representative views of the 3D reconstruction of ncPRC1RYBP ∆linker mutant bound to singly modified H2AK119Ub nucleosome complex (Map 5). (f) Euler angle distribution of assignment of particles used to generate the final 3D reconstruction of the 3.46 Å complex. The length of every cylinder is proportional to the number of particles assigned to the specific orientation. (g) Local resolution heat map of the reconstruction of ncPRC1RYBP complex ∆linker mutant bound to singly modified H2AK119Ub nucleosome.

Extended Data Fig. 7 Quantitative fluorescent in vitro E3-ligase activity assays.

(a) Fluorescently-labeled ubiquitin was made by covalent fluorescein labeling of Cyso-Ub (ubiquitin with cysteine added at the N-terminus). The activity of ncPRC1 was tested with the wt Ubiquitin, Cyso-Ub, and CysoUb-fluorescein. The assay was monitored with the fluorescein channel (top) in the ChemiDoc, as well as with Coomassie staining (bottom). (b) Controls for ubiquitination: reactions in absence of ATP (lane 1), ubiquitin (lane 2) or RING1B/BMI1 (lane 3) do not show ubiquitination compared with lanes 4 and 5 where RING1B/BMI1 and ncPRC1RYBP are included. (c) Normalization curve for H2AK119Ub quantification. Multiple concentrations of Cyso-Ub were loaded in the gel representing the total amount of nucleosome in molarity. Consecutive dilutions were done to generate the curve. Each point was done in triplicate (n = 3) and monitored in the fluorescein channel in the ChemiDoc with 0.1 s of exposure. (d-f) Time course of E3-ligase activity assay with the catalytic core (RING1B/BMI1) and ncPRC1RYBP on (d) unmodified nucleosome (e) asymmetric H2AK119Ub dinucleosome and (f) hybrid unmodified/H2AK119Ub 12 N nucleosome array show the increase of H2AK119Ub over time. Data are mean ± s.d. from n = 3 independent experiments.

Source Data

Extended Data Fig. 8 Fluorescent quantitative in vitro E3-ligase activity assays and fluorescent polarization binding assays.

(a-c) Time course E3-ligase activity assays of ncPRC1 complexes on different substrates. Each point was monitored in the fluorescein channel in the ChemiDoc with 0.1 s of exposure. The Fluorescein signal was adjusted and normalized to the normalization plot (Extended Data Fig. 7). (a) Time course of E3-ligase activity assay for the acidic patch mutant (R47A/R53A) on unmodified (left) and singly modified H2AK119Ub nucleosome (right). (b) Time course of E3-ligase activity assay for the RYBP linker mutants and YAF2 on unmodified (left) and singly modified H2AK119Ub nucleosome (right). (c) Time course E3-ligase activity assays for the ncPRC1 enzymes on asymmetric dinucleosome (left) and hybrid unmodified/H2AK119Ub 12 N nucleosome array which has 3:1 unmodified and H2AK119Ub octamer (right). (d) Fluorescence polarization binding assays of ncPRC1RYBP, RYBP alone, and RYBP K/A linker (K75-134A) mutant alone with the unmodified, fluorescently-labeled nucleosome. Data are mean ± s.d. from n = 3 independent experiments except for the ncPRC1RYBP R47A/R53A on dinucleosome substrate n = 1 (c).

Source Data

Extended Data Fig. 9 Cryo-EM analysis of the ncPRC1RYBP bound to symmetric H2AK119Ub dinucleosome complex.

(a) Raw cryo-EM images of ncPRC1RYBP bound to symmetric H2AK119Ub dinucleosome complex with selected particles of the complex in blue circles. (b) Representative 2D class averages calculated from the final subset of particles. (c-e) Two representative views of the 3D reconstruction of ncPRC1RYBP bound to symmetric H2AK119Ub dinucleosome complex. (c) Overall, 11.81 Å cryo-EM map showing the architecture of the dinucleosome (Map 7); (d) Cryo-EM map at 4.69 Å focused on RING1B/BMI1 nucleosome (Map 9) (Figs. S12 and S14); (e) Cryo-EM map at 4.24 Å focused on the RYBP/Ub nucleosome (Map 8) (Figs. S12 and S14). (f-h) Euler angle distribution of assignment of particles used to generate the final 3D reconstruction of the complex corresponding to the reconstructions shown in c-e. The length of every cylinder is proportional to the number of particles assigned to the specific orientation. (i-k) FSC plots of the ncPRC1RYBP bound to H2AK119Ub symmetric dinucleosome complex for the cryo-EM maps shown in c-e, the FSC plots are calculated between the two independently refined half maps (measured at FSC = 0.143).

Extended Data Fig. 10 Cryo-EM analysis of the ncPRC1RYBP bound to asymmetric H2AK119Ub dinucleosome complex.

(a) Raw cryo-EM images of ncPRC1RYBP bound to asymmetric H2AK119Ub dinucleosome complex with selected particles of the complex in blue circles. (b) Representative 2D class averages calculated from the final subset of particles. (c-e) Two representative views of the 3D reconstruction of ncPRC1RYBP bound to asymmetric H2AK119Ub dinucleosome complex. (c) Overall 7.91 Å cryo-EM map showing the architecture of the dinucleosome (Map 11); (d) cryo-EM map at 3.61 Å focused on RING1B/BMI1 nucleosome (Map 13) (Figs. S13 and S15); (e) cryo-EM map at 6.19 Å focused on the RYBP/Ub nucleosome (Map 12) (Figs. S13 and S15). (f-h) Euler angle distribution of assignment of particles used to generate the final 3D reconstruction of the complex corresponding to the reconstructions shown in c-e. The length of every cylinder is proportional to the number of particles assigned to the specific orientation. (i-k) FSC plots of the ncPRC1RYBP bound to asymmetric H2AK119Ub dinucleosome complex for the cryo-EM maps shown in c-e, the FSC plots are calculated between the two independently refined half maps (measured at FSC = 0.143).

Extended Data Table 1 Cryo-EM data collection, refinement, and validation statistics of PRC1RYBP-bound to different substrates
Full size table

Supplementary information

Supplementary Information

Supplementary Figs. 1–16 and Supplementary Tables 1–3.

Reporting Summary

Source data

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

López, V.G., Valencia-Sánchez, M.I., Abini-Agbomson, S. et al. Read–write mechanisms of H2A ubiquitination by Polycomb repressive complex 1. Nature (2024). https://doi.org/10.1038/s41586-024-08183-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41586-024-08183-5

Search

Advanced 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