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RYBP/YAF2-PRC1 complexes and histone H1-dependent chromatin compaction mediate propagation of H2AK119ub1 during cell division

Nature Cell Biology volume 22, pages 439–452 (2020)Cite this article

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Abstract

Stable propagation of epigenetic information is important for maintaining cell identity in multicellular organisms. However, it remains largely unknown how mono-ubiquitinated histone H2A on lysine 119 (H2AK119ub1) is established and stably propagated during cell division. In this study, we found that the proteins RYBP and YAF2 each specifically bind H2AK119ub1 to recruit the RYBP–PRC1 or YAF2–PRC1 complex to catalyse the ubiquitination of H2A on neighbouring nucleosomes through a positive-feedback model. Additionally, we demonstrated that histone H1-compacted chromatin enhances the distal propagation of H2AK119ub1, thereby reinforcing the inheritance of H2AK119ub1 during cell division. Moreover, we showed that either disruption of RYBP/YAF2–PRC1 activity or impairment of histone H1-dependent chromatin compaction resulted in a significant defect of the maintenance of H2AK119ub1. Therefore, our results suggest that histone H1-dependent chromatin compaction plays a critical role in the stable propagation of H2AK119ub1 by RYBP/YAF2–PRC1 during cell division.

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Fig. 1: RYBP–PRC1 is recruited to chromatin by the direct interaction between H2AK119ub1 and RYBP.
Fig. 2: RYBP–PRC1 propagates H2AK119ub1 via a positive-feedback loop.
Fig. 3: H1-compacted chromatin facilitates H2AK119ub1 by RYBP–PRC1.
Fig. 4: Reduction of H2AK119ub1 is largely independent of H3K27me3 in Rybp mutant and H1 TKO mESCs.
Fig. 5: Inheritance of H2AK119ub1 after washing out target signal with Dox.
Fig. 6: Role of H2AK119ub1 in gene expression.
Fig. 7: Overlapping functions of RYBP–PRC1 and histone H1.

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Data availability

The ChIP-Seq and RNA-Seq data that support the findings of this study have been deposited in the Sequence Read Archive BioProject under accession code PRJNA604675. These data have also been deposited in the Genome Sequence Archive of the BIG Data Center, Beijing Institute of Genomics (BIG), Chinese Academy of Sciences (http://bigd.big.ac.cn/gsa), under accession code CRA001837. Mass spectrometry data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD017105. Previously published ChIP-Seq data that were re-analysed here are available in the Gene Expression Omnibus under accession codes GSM2051877, GSM1180182, GSM1180179, GSM1180181, GSM1124783 and GSM1300602. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

References

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  3. Dodd, I. B., Micheelsen, M. A., Sneppen, K. & Thon, G. Theoretical analysis of epigenetic cell memory by nucleosome modification. Cell 129, 813–822 (2007).

    CAS  PubMed  Google Scholar 

  4. Jiang, D. & Berger, F. DNA replication-coupled histone modification maintains Polycomb gene silencing in plants. Science 357, 1146–1149 (2017).

    CAS  PubMed  Google Scholar 

  5. Audergon, P. N. et al. Restricted epigenetic inheritance of H3K9 methylation. Science 348, 132–135 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Coleman, R. T. & Struhl, G. Causal role for inheritance of H3K27me3 in maintaining the OFF state of a Drosophila HOX gene. Science 356, eaai8236 (2017).

    PubMed  PubMed Central  Google Scholar 

  7. Liu, N. et al. Recognition of H3K9 methylation by GLP is required for efficient establishment of H3K9 methylation, rapid target gene repression, and mouse viability. Genes Dev. 29, 379–393 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    PubMed  Google Scholar 

  9. Wang, X. & Moazed, D. DNA sequence-dependent epigenetic inheritance of gene silencing and histone H3K9 methylation. Science 356, 88–91 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Yu, R., Wang, X. & Moazed, D. Epigenetic inheritance mediated by coupling of RNAi and histone H3K9 methylation. Nature 558, 615–619 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Laprell, F., Finkl, K. & Muller, J. Propagation of Polycomb-repressed chromatin requires sequence-specific recruitment to DNA. Science 356, 85–88 (2017).

    CAS  PubMed  Google Scholar 

  12. Wang, C., Zhu, B. & Xiong, J. Recruitment and reinforcement: maintaining epigenetic silencing. Sci. China Life Sci. 61, 515–522 (2018).

    PubMed  Google Scholar 

  13. Margueron, R. & Reinberg, D. Chromatin structure and the inheritance of epigenetic information. Nat. Rev. Genet. 11, 285–296 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  17. Oksuz, O. et al. Capturing the onset of PRC2-mediated repressive domain formation. Mol. Cell 70, 1149–1162 (2018). e1145.

    CAS  PubMed  Google Scholar 

  18. Fan, Y. et al. Histone H1 depletion in mammals alters global chromatin structure but causes specific changes in gene regulation. Cell 123, 1199–1212 (2005).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  21. Boettiger, A. N. et al. Super-resolution imaging reveals distinct chromatin folding for different epigenetic states. Nature 529, 418–422 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Song, F. et al. Cryo-EM study of the chromatin fiber reveals a double helix twisted by tetranucleosomal units. Science 344, 376–380 (2014).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  26. Kundu, S. et al. Polycomb repressive complex 1 generates discrete compacted domains that change during differentiation. Mol. Cell 65, 432–446.e5 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 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  PubMed  PubMed Central  Google Scholar 

  28. 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).

    CAS  PubMed  Google Scholar 

  29. Tardat, M. et al. Cbx2 targets PRC1 to constitutive heterochromatin in mouse zygotes in a parent-of-origin-dependent manner. Mol. Cell 58, 157–171 (2015).

    CAS  PubMed  Google Scholar 

  30. Kalb, R. et al. Histone H2A monoubiquitination promotes histone H3 methylation in Polycomb repression. Nat. Struct. Mol. Biol. 21, 569–571 (2014).

    CAS  PubMed  Google Scholar 

  31. Bernstein, E. et al. Mouse Polycomb proteins bind differentially to methylated histone H3 and RNA and are enriched in facultative heterochromatin. Mol. Cell Biol. 26, 2560–2569 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Eskeland, R. et al. Ring1B compacts chromatin structure and represses gene expression independent of histone ubiquitination. Mol. Cell 38, 452–464 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    PubMed  PubMed Central  Google Scholar 

  35. Wu, X., Johansen, J. V. & Helin, K. Fbxl10/Kdm2b recruits Polycomb repressive complex 1 to CpG islands and regulates H2A ubiquitylation. Mol. Cell 49, 1134–1146 (2013).

    CAS  PubMed  Google Scholar 

  36. He, J. et al. Kdm2b maintains murine embryonic stem cell status by recruiting PRC1 complex to CpG islands of developmental genes. Nat. Cell Biol. 15, 373–384 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Almeida, M. et al. PCGF3/5-PRC1 initiates Polycomb recruitment in X chromosome inactivation. Science 356, 1081–1084 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  39. Muller, M. M., Fierz, B., Bittova, L., Liszczak, G. & Muir, T. W. A two-state activation mechanism controls the histone methyltransferase Suv39h1. Nat. Chem. Biol. 12, 188–193 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Schalch, T., Duda, S., Sargent, D. F. & Richmond, T. J. X-ray structure of a tetranucleosome and its implications for the chromatin fibre. Nature 436, 138–141 (2005).

    CAS  PubMed  Google Scholar 

  41. Hisada, K. et al. RYBP represses endogenous retroviruses and preimplantation- and germ line-specific genes in mouse embryonic stem cells. Mol. Cell Biol. 32, 1139–1149 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Jackson, V. & Chalkley, R. Histone segregation on replicating chromatin. Biochemistry 24, 6930–6938 (1985).

    CAS  PubMed  Google Scholar 

  43. McKnight, S. L. & Miller, O. L. Jr Electron microscopic analysis of chromatin replication in the cellular blastoderm Drosophila melanogaster embryo. Cell 12, 795–804 (1977).

    CAS  PubMed  Google Scholar 

  44. Jackson, V. & Chalkley, R. A new method for the isolation of replicative chromatin: selective deposition of histone on both new and old DNA. Cell 23, 121–134 (1981).

    CAS  PubMed  Google Scholar 

  45. Xu, M. et al. Partitioning of histone H3–H4 tetramers during DNA replication-dependent chromatin assembly. Science 328, 94–98 (2010).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  47. CDi Croce, L. & Helin, K. Transcriptional regulation by Polycomb group proteins. Nat. Struct. Mol. Biol. 20, 1147–1155 (2013).

    Google Scholar 

  48. Farcas, A. M. et al. KDM2B links the Polycomb repressive complex 1 (PRC1) to recognition of CpG islands. eLife 1, e00205 (2012).

    PubMed  PubMed Central  Google Scholar 

  49. Stielow, B., Finkernagel, F., Stiewe, T., Nist, A. & Suske, G. MGA, L3MBTL2 and E2F6 determine genomic binding of the non-canonical Polycomb repressive complex PRC1.6. PLoS Genet. 14, e1007193 (2018).

    PubMed  PubMed Central  Google Scholar 

  50. Hsieh, T. H. et al. Mapping nucleosome resolution chromosome folding in yeast by micro-C. Cell 162, 108–119 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Risca, V. I., Denny, S. K., Straight, A. F. & Greenleaf, W. J. Variable chromatin structure revealed by in situ spatially correlated DNA cleavage mapping. Nature 541, 237–241 (2017).

    CAS  PubMed  Google Scholar 

  52. Seale, R. L. Rapid turnover of the histone–ubiquitin conjugate, protein A24. Nucleic Acids Res. 9, 3151–3158 (1981).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Moussa, H. F. et al. Canonical PRC1 controls sequence-independent propagation of Polycomb-mediated gene silencing. Nat. Commun. 10, 1931 (2019).

    PubMed  PubMed Central  Google Scholar 

  54. Endoh, M. et al. Histone H2A mono-ubiquitination is a crucial step to mediate PRC1-dependent repression of developmental genes to maintain ES cell identity. PLoS Genet. 8, e1002774 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Illingworth, R. S. et al. The E3 ubiquitin ligase activity of RING1B is not essential for early mouse development. Genes Dev. 29, 1897–1902 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Pengelly, A. R., Kalb, R., Finkl, K. & Muller, J. Transcriptional repression by PRC1 in the absence of H2A monoubiquitylation. Genes Dev. 29, 1487–1492 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Kallin, E. M. et al. Genome-wide uH2A localization analysis highlights Bmi1-dependent deposition of the mark at repressed genes. PLoS Genet. 5, e1000506 (2009).

    PubMed  PubMed Central  Google Scholar 

  58. Qin, W. et al. DNA methylation requires a DNMT1 ubiquitin interacting motif (UIM) and histone ubiquitination. Cell Res. 25, 911–929 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Tsuboi, M. et al. Ubiquitination-independent repression of PRC1 targets during neuronal fate restriction in the developing mouse neocortex. Dev. Cell 47, 758–772.e5 (2018).

    CAS  PubMed  Google Scholar 

  60. Cohen, I. et al. PRC1 fine-tunes gene repression and activation to safeguard skin development and stem cell specification. Cell Stem Cell 22, 726–739.e7 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Van den Boom, V. et al. Non-canonical PRC1.1 targets active genes independent of H3K27me3 and is essential for leukemogenesis. Cell Rep. 14, 332–346 (2016).

    CAS  PubMed  Google Scholar 

  62. Yao, M. et al. PCGF5 is required for neural differentiation of embryonic stem cells. Nat. Commun. 9, 1463 (2018).

    PubMed  PubMed Central  Google Scholar 

  63. Endoh, M. et al. PCGF6-PRC1 suppresses premature differentiation of mouse embryonic stem cells by regulating germ cell-related genes. eLife 6, e21064 (2017).

    PubMed  PubMed Central  Google Scholar 

  64. Chen, P. et al. H3.3 actively marks enhancers and primes gene transcription via opening higher-ordered chromatin. Genes Dev. 27, 2109–2124 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Ying, Q. L. & Smith, A. G. Defined conditions for neural commitment and differentiation. Methods Enzymol. 365, 327–341 (2003).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We are grateful to E. Campos and C. Wu for critical reading and discussion of our manuscript, T. Yao (National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences) for preparing materials and ordering reagents, and J. Wang and M. Zhang (Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences) for SILAC mass spectrometry. This work was supported by grants to G.L. from the National Science Fund for Distinguished Young Scholars (31525013), Ministry of Science and Technology of China (2017YFA0504202), National Natural Science Foundation of China (31630041 and 31521002), Chinese Academy of Sciences Strategic Priority Program (XDB19040202) and Key Research Program on Frontier Science (QYZDY-SSW-SMC020). The work was also supported by a HHMI International Research Scholar grant (55008737) to G.L. and a grant from the National Natural Science Foundation of China (31400657 to J.Z.). All electron microscopy and fluorescent images were collected and processed at the Center for Bio-imaging, Core Facility for Protein Sciences, Institute of Biophysics and Chinese Academy of Sciences. We are also indebted to the colleagues whose work could not be cited due to the limitation of space.

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  1. These authors contributed equally: Jicheng Zhao, Min Wang.

Authors and Affiliations

  1. National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Jicheng Zhao, Min Wang, Luyuan Chang, Juan Yu, Aoqun Song, Cuifang Liu, Tiantian Zhang, Bing Zhu & Guohong Li

  2. University of the Chinese Academy of Sciences, Beijing, China

    Min Wang, Luyuan Chang, Aoqun Song, Cuifang Liu, Tiantian Zhang, Bing Zhu & Guohong Li

  3. China Agricultural University, Beijing, China

    Wenjun Huang

  4. Department of Cell Biology, Tianjin Medical University, Tianjin, China

    Xudong Wu

  5. Tsinghua Center for Life Sciences, School of Medicine and School of Life Sciences, Tsinghua University, Beijing, China

    Xiaohua Shen

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Contributions

J.Z. and M.W. carried out the experiments and assembled the figures. L.C. and J.Y. performed the bioinformatics analysis. W.H. and T.Z. performed the purification and prepared ubiquitinated histone H2A. A.S. and C.L. assisted with the expression of recombinant proteins, construction of plasmids and generation of stable cell lines. B.Z., X.S. and X.W. contributed the reagents and helped to discuss the project. G.L. conceived of and supervised the project. J.Z., M.W. and G.L. analysed the data and wrote the manuscript. All authors read and commented on the manuscript.

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Correspondence to Guohong Li.

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Extended data

Extended Data Fig. 1 RYBP-PRC1 is recruited to chromatin by the direct interaction between H2AK119ub1 and RYBP.

a, Representative 15% SDS-PAGE of H2A and H2AK119ub1 octamers out of 3 independent experiments. b, Representative 1.2% agarose gel electrophoresis of H2A- and H2AK119ub1- mononucleosomes out of 3 independent experiments. 187 bp 601 DNA is labelled by biotin for capturing by streptavidin-beads. c, ChIP-qPCR analysis of RING1B, H2AK119ub1 and RYBP levels at selected gene loci in Ring1b wild type (-4-OHT) and knockout (+4-OHT) cells (n=3 biologically independent samples). Data represents mean ± s.d. ChIP enrichments are normalized to input. d, Representative immunoblot of bindings of RYBP and RYBPTF/AA to RING1B/GST-BMI1 complex by GST pull-down assay out of 3 independent experiments. e, Representative coomassie-stained gels of purified recombinant proteins and protein complexes out of 3 independent experiments. Unprocessed blots and statistical source data are available in Source Data.

Source data

Extended Data Fig. 2 Changes of RING1B occupancy in Rybp-/-, Eed-/- and Eed-/-/Rybp-/- mESCs.

a, A Venn diagram showing the overlap of H2AK119ub1 (orange) and RYBP (purple) peaks in mESCs. Numbers represent the total peaks of H2AK119ub1 (orange) and RYBP (purple), respectively. b, Representative immunoblot of RING1B, CBX7, EZH2 and H2AK119ub1 in wild type and Rybp knockout cells out of 3 independent experiments. c, A scatter plot shows changes of RING1B reads density upon RYBP deletion in mESCs. d, ChIP-qPCR analysis for RING1B at selected gene loci in wild type, Rybp knockout and RybpTF/AA mESCs, IgG as negative control (n=3 biologically independent samples). Data represents mean ± s.d. ChIP enrichments are normalized to input. e, Heat map analysis of RING1B across RING1B enriched peaks (±3 kb) in wild type, Rybp-/-, Eed-/- and Eed-/-/Rybp-/- mESCs, ranked from highest to lowest ChIP-seq signal of RING1B in wild type mESCs. f, ChIP-seq cumulative enrichment deposition centered at peak summit of RING1B in wild type, Rybp-/-, Eed-/- and Eed-/-/Rybp-/- mESCs. The ChIP-seq assays in a, c, e, f have been performed in duplicates using two biologically independent samples (once per sample). g, ChIP-qPCR analysis of RING1B at selected gene loci in wild type, Rybp-/-, Eed-/- and Eed-/-/Rybp-/- mESCs, IgG served as ChIP negative control (n=3 biologically independent samples). Data represents mean ± s.d. ChIP enrichments are normalized to input. Unprocessed blots and statistical source data are available in Source Data.

Source data

Extended Data Fig. 3 RYBP-PRC1 propagates H2AK119ub1 by a positive-feedback loop.

a, Representative EM images of 12×-polynucleosome arrays (by metal-shadowing method). b, A schematic illustrating the octamers in chromatin assembly. c, Representative immunoblot of in vitro ubiquitination assay for measuring the activity of RYBP-PRC1 on mononucleosome and polynucleosome substrates out of 3 independent experiments. Triangles represent RYBP-PRC1 from low to high level, also in (e). d, Representative immunoblot of in vitro ubiquitination assay for measuring the effect of H2AK119ub1 on RYBP-PRC1 activity in trans out of 3 independent experiments. 12×-H2AKK/RR- or 12×-H2AK119ub1-polynucleosomes were mixed with 12×-H2A-polynucleosome substrates immediately prior ubiquitination reactions in substrate I or in substrate II, respectively. e, Representative immunoblot of in vitro ubiquitination assay for measuring the ubiquitin E3 ligase activity of RYBP-PRC1 on 12×-H2AKK/RR- and 12×-H2A-polynucleosomes out of 3 independent experiments. f, A schematic illustrating the strategy for generating 12×-polynucleosome substrates by sequential ligation of tetranucleosomes. g, Representative EM images of the tetranucleosomes for ligation assays. h, Representative EM images of 12×-H2A-polynucleosomes generated by sequential ligation of tetranucleosomes. Scale bar in a, g and h is 50 nm. The EM in a, g and h was performed in triplicates with three biologically independent samples (once per sample). i, Representative 1.2% agarose gel electrophoresis of 12×-H2A-polynucleosomes generated by sequential ligation of tetranucleosomes out of 3 independent experiments. j, A schematic illustrating the TetR-tetO targeting system. RING1B-TetR-HA is recruited by specific binding of TetR to 8xtetO arrays. RYBP-FLAG or RYBPTF/AA-FLAG was overexpressed to promote the spreading of H2AK119ub1 around 8xtetO sites. k, ChIP-qPCR analysis of HA (TetR-HA), FLAG and H2AK119ub1 across the 8xtetO sites in TetR-tetO targeting mESCs with TetR-HA transient transfection (n=3 biologically independent samples). Data represents mean ± s.d. ChIP enrichments are normalized to input. Unprocessed blots and statistical source data are available in Source Data.

Source data

Extended Data Fig. 4 The effect of Yaf2 on propagation of H2AK119ub1.

a, Heat map analysis of H2AK119ub1 across H2AK119ub1 enriched peaks (±3 kb) in wild type, Rybp-/-, Yaf2-/- and Rybp-/-/Yaf2-/- mESCs, ranked from highest to lowest ChIP-seq signal of H2AK119ub1 in wild type mESCs. b, ChIP-seq cumulative enrichment deposition centered at peak summit of H2AK119ub1 in wild type, Rybp-/-, Yaf2-/- and Rybp-/-/Yaf2-/- mESCs. The ChIP-seq assays in a, b have been performed in duplicatesusing two biologically independent samples (once per sample). c, ChIP-qPCR analysis of H2AK119ub1 at selected gene loci in wild type, Rybp-/-, Yaf2-/- and Rybp-/-/Yaf2-/- mESCs (n=3 biologically independent samples). Data represents mean ± s.d. IgG served as ChIP negative control. ChIP enrichments are normalized to input. d, Representative immunoblot of YAF2, RING1B, H2AK119ub1, RYBP and H3 in wild type, Rybp-/-, Yaf2-/- and Rybp-/-/Yaf2-/- mESCs out of 3 independent experiments. e, Representative quantitative-immunoblot of H2AK119ub1 in Rybp-/-/Yaf2-/-, Rybp-/- and Yaf2-/- mESCs out of 3 independent experiments. f, Quantitative signals of H2AK119ub1 immunoblot by Image J, normalized to H3 signal (n=4 biologically independent experiments). Data represents mean ± s.d. Unprocessed blots and statistical source data are available in Source Data.

Source data

Extended Data Fig. 5 H1-compacted chromatin facilitates H2AK119ub1 by RYBP-PRC1.

a, A schematic illustrating the octamers and proteins in chromatin assembly. b, Representative EM images of 12×-ploynucleosomes (by metal-shadowing method, top) and H1-compacted chromatin (by negative staining method, bottom) out of 3 independent experiments. A schematic of substrates is shown at the top of the panel. Scale bar is 50 nm. c, Representative immunoblot of in vitro ubiquitination assay for measuring the ubiquitin E3 ligase activity of RYBP-PRC1 on 12×-polynucleosome and H1-compacted chromatin substrates out of 3 independent experiments. Modified and un-modified octamers were mixed immediately prior chromatin assembly in a ratio of 1:2, also in (d~f). H1e was added into substrates II to compact chromatin fibers. d, Representative immunoblot of in vitro ubiquitination assay for measuring the activity of RYBP-PRC1 on mononucleosome substrates with or without H1 out of 3 independent experiments. e, Representative immunoblot of in vitro ubiquitination assay for measuring the effect of nucleosome stacks in H1-compacted chromatin on long-distance propagation of H2AK119ub1 out of 3 independent experiments. Substrate octamers were replaced by un-modified H2Bmut octamers in substrate II or by un-modified H4no-N octamers in substrate III, respectively, also in (f). H1e was added to compact chromatin. f, Representative immunoblot of in vitro ubiquitination assay for measuring the activity of RYBP-PRC1 on H2A-, H2Bmut- and H4no-N-12×-polynucleosome arrays out of 3 independent experiments. g, Representative immunoblot of H1 in wild type and H1-TKO mESCs out of 3 independent experiments, H3 as internal reference. h, ChIP-qPCR analysis of H2AK119ub1 levels at selected gene loci in wild type and H1-TKO mESCs (n=3 biologically independent experiments). Data represents mean ± s.d. IgG served as ChIP negative control. ChIP enrichments are normalized to input. Unprocessed blots and statistical source data are available in Source Data.

Source data

Extended Data Fig. 6 Inheritance of H2AK119ub1 after washing out target signal by Dox.

a, ChIP-qPCR analysis of HA (left) and H2AK119ub1 (right) across the 8xtetO integrating locus in TetR-tetO targeting mESCs, either treated by Dox 36 hrs or untreated. Dox was added 24 hrs after transient transfection of RING1B-TetR-HA (n=3 biologically independent samples). Data represents mean ± s.d. ChIP enrichments are normalized to input. b, ChIP-qPCR analysis of H2AK119ub1 across the 8xtetO integrating locus in TetR-tetO targeting mESCs with RYBP-TetR-HA stable expression, either treated by dox 24 hrs, 48 hrs and 72 hrs or untreated (n=3 biologically independent samples). Data represents mean ± s.d. ChIP enrichments are normalized to input. c, ChIP-qPCR analysis of H2AK119ub1 across the 8xtetO integrating locus in TetR-tetO targeting mESCs, either treated by Dox 36 hrs or untreated (n=3 biologically independent samples). Data represents mean ± s.d. ChIP enrichments are normalized to input. d, ChIP-qPCR analysis of H2AK119ub1 across the 8xtetO integrating locus in TetR-tetO targeting mESCs with RYBP-TetR-HA or RYBPTF/AA-TetR-HA stable expression, either treated by Dox 36 hrs or untreated, IgG served as ChIP negative controls (n=3 biologically independent samples). Data represents mean ± s.d. ChIP enrichments are normalized to input. Statistical source data are available in Source Data.

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Extended Data Fig. 7 The role of RYBP-PRC1-propagated H2AK119ub1 in regulating gene expression.

a, A schematic illustrating the reporter gene system in TetR-tetO targeting cells. De-targeting is achieved by adding Dox. The switch of CYP26A1 promoter between “OFF” and “ON” is achieved by adding atRA. Single-cell EGFP signal is detected by flow cytometry. b, Relative EGFP signals of TetR-tetO targeting mESCs with or without RING1B-TetR-HA stable expression, evaluated by flow cytometry (n=3 biologically independent samples). Data represents mean ± s.d. mESCs were treated by 1uM atRA after a time course of 1ug/ml Dox treatment, also in (b–e). c, % of EGFP positive cells of TetR-tetO targeting mESCs with or without RING1B-TetR-HA stable expression, counted by flow cytometry (n=3 biologically independent samples). Data represents mean ± s.d. d, Relative EGFP signals of wild type and H1-TKO TetR-tetO targeting mESCs with RYBP-TetR-HA stable expression (n=3 biologically independent samples). Data represents mean ± s.d. e, % of EGFP positive cells of wild type and H1-TKO cell lines with RYBP-TetR-HA stable expression (n=3 biologically independent samples). Data represents mean ± s.d. f, Histogram of EGFP signals of different treated cells. g, ChIP-qPCR analysis of HA-AID-RYBP at selected target gene after a time course of IAA treatment (n=3 biologically independent samples). Data represents mean ± s.d. h, ChIP-qPCR analysis of RING1B at selected target gene after a time course of IAA treatment (n=3 biologically independent samples). Data represents mean ± s.d. IgG in g, h served as ChIP negative control. i, Volcano Plots depicting the gene expression changes at whole-genome mRNA level, RybpTF/AA vs wild type mESCs (n=3 biologically independent samples). P-value determined by two-side student’s t- test. No adjustments for multiple comparisons. Statistical source data are available in Source Data.

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Extended Data Fig. 8 The proliferation and apoptosis of cell lines.

a, Cell proliferating curves of wild type, RybpKO, RybpTF/AA and H1-TKO mESCs (n=8 biologically independent samples). Data represents mean ± s.d. b, Representative flow cytometry of apoptosis of wild type, RybpKO, RybpTF/AA and H1-TKO mESCs out of 3 independent experiments. Statistical source data are available in Source Data.

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Extended Data Fig. 9 Overlapping roles of RYBP-PRC1 and histone H1.

a, A scatter plot showing the fold changes of H2AK119ub1 peaks in RybpTF/AA and H1-TKO mESCs, both relative to wild type mESCs. Red dots represent peaks with H2AK119ub1 reduction > 1.5-fold. b, Venn diagrams depicting the overlap genes identified as showing a greater than 2-fold expression change both in RybpTF/AA and H1-TKO mESCs, relative to wild type mESCs (n=3 biologically independent samples). Gene numbers with expression changes were shown, respectively. c, Representative immunofluorescence images of NANOG and TUBB3 during NPC differentiation in wild type, RybpTF/AA and H1-TKO mESCs out of 3 independent experiments. DNA was stained by DAPI. Scale bars are 50μm. d, RT-qPCR analysis of expression of selected genes during mNPCs differentiation in wild type, RybpKO, RybpTF/AA and H1-TKO mESCs, normalized with reference control of GAPDH and to wild-type mESCs (n=3 biologically independent samples). Data represents mean ± s.d. e, Venn diagrams depicting the overlap genes identified as showing a greater than 2-fold expression change both in RybpTF/AA and H1-TKO mNPCs, relative to wild type mNPCs (n=3 biologically independent samples). Gene numbers with expression changes were shown, respectively. Statistical source data are available in Source Data.

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Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Table 1 List of the neural differentiation-related genes locating at H2AK119ub1 reduction overlapping peaks in RybpT31A/F32A and H1 TKO mESCs, based on Gene Ontology analysis. Supplementary Table 2 Antibodies, primers, cell lines and reagents. a, Antibodies used in western blots, immunofluorescence and ChIP-Seq. b, The sequence of primer pairs used in RT-qPCR and ChIP-qPCR. c, List of cell lines generated in our laboratory or from other labs. d, List of commercial reagents.

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Zhao, J., Wang, M., Chang, L. 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). https://doi.org/10.1038/s41556-020-0484-1

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