Rybp/yaf2-prc1 complexes and histone h1-dependent chromatin compaction mediate propagation of h2ak119ub1 during cell division

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. Access through your institution Buy or

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RESTORATION OF H3K27ME3 DURING DNA REPLICATION BY CHROMATIN COMPACTION Article Open access 10 July 2023 STABLE INHERITANCE OF H3.3-CONTAINING NUCLEOSOMES DURING MITOTIC CELL DIVISIONS

Article Open access 06 May 2022 HISTONE DEACETYLATION PRIMES SELF-PROPAGATION OF HETEROCHROMATIN DOMAINS TO PROMOTE EPIGENETIC INHERITANCE Article 05 September 2022 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 * 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  * Reinberg, D. & Vales, L. D. Chromatin domains rich in inheritance. _Science_ 361,

33–34 (2018). CAS  PubMed  Google Scholar  * 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  * Jiang, D. & Berger, F. DNA replication-coupled histone modification maintains Polycomb gene silencing in plants. _Science_ 357,

1146–1149 (2017). CAS  PubMed  Google Scholar  * Audergon, P. N. et al. Restricted epigenetic inheritance of H3K9 methylation. _Science_ 348, 132–135 (2015). CAS  PubMed  PubMed Central 

Google Scholar  * 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  * 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  * Ragunathan, K., Jih, G. & Moazed, D. Epigenetic inheritance uncoupled from sequence-specific

recruitment. _Science_ 348, 1258699 (2015). PubMed  Google Scholar  * 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  * 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  * 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  * Wang, C., Zhu, B. & Xiong, J. Recruitment and reinforcement: maintaining epigenetic

silencing. _Sci. China Life Sci._ 61, 515–522 (2018). PubMed  Google Scholar  * 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  * 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  * Grunstein, M. Yeast heterochromatin: regulation of its assembly and inheritance by histones. _Cell_ 93, 325–328 (1998).

CAS  PubMed  Google Scholar  * 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  * Oksuz, O.

et al. Capturing the onset of PRC2-mediated repressive domain formation. _Mol. Cell_ 70, 1149–1162 (2018). e1145. CAS  PubMed  Google Scholar  * 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  * 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  * 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  * 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  * 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  * 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  * 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  * Wang, H. et al. Role of histone H2A ubiquitination in

Polycomb silencing. _Nature_ 431, 873–878 (2004). CAS  PubMed  Google Scholar  * 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  * 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  * 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  * 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  * 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  * 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  * 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  * 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  * 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  * 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  * 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  * 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  * Arrigoni, R. et al. The Polycomb-associated protein Rybp is a ubiquitin binding protein. _FEBS Lett._ 580, 6233–6241 (2006). CAS  PubMed 

Google Scholar  * 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  * 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  * 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  * Jackson, V. & Chalkley, R. Histone segregation on replicating

chromatin. _Biochemistry_ 24, 6930–6938 (1985). CAS  PubMed  Google Scholar  * 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  * 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  * 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  * Voigt, P. et al. Asymmetrically modified nucleosomes. _Cell_ 151, 181–193 (2012). CAS 

PubMed  PubMed Central  Google Scholar  * CDi Croce, L. & Helin, K. Transcriptional regulation by Polycomb group proteins. _Nat. Struct. Mol. Biol._ 20, 1147–1155 (2013). Google Scholar

  * 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  * 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  * 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  * 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  * 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  * 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  * 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  * 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  * 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  * 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  * 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  * 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  *

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  * 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  * Yao, M. et al. PCGF5 is required for neural differentiation of embryonic stem cells. _Nat. Commun._ 9, 1463 (2018). PubMed  PubMed Central  Google Scholar  * 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  *

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  * Ying, Q. L. & Smith, A. G. Defined conditions for neural commitment and differentiation. _Methods Enzymol._ 365, 327–341 (2003). CAS  PubMed  Google Scholar  Download

references 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. AUTHOR INFORMATION Author notes

* These authors contributed equally: Jicheng Zhao, Min Wang. AUTHORS AND AFFILIATIONS * 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 * University of

the Chinese Academy of Sciences, Beijing, China Min Wang, Luyuan Chang, Aoqun Song, Cuifang Liu, Tiantian Zhang, Bing Zhu & Guohong Li * China Agricultural University, Beijing, China

Wenjun Huang * Department of Cell Biology, Tianjin Medical University, Tianjin, China Xudong Wu * Tsinghua Center for Life Sciences, School of Medicine and School of Life Sciences, Tsinghua

University, Beijing, China Xiaohua Shen Authors * Jicheng Zhao View author publications You can also search for this author inPubMed Google Scholar * Min Wang View author publications You

can also search for this author inPubMed Google Scholar * Luyuan Chang View author publications You can also search for this author inPubMed Google Scholar * Juan Yu View author publications

You can also search for this author inPubMed Google Scholar * Aoqun Song View author publications You can also search for this author inPubMed Google Scholar * Cuifang Liu View author

publications You can also search for this author inPubMed Google Scholar * Wenjun Huang View author publications You can also search for this author inPubMed Google Scholar * Tiantian Zhang

View author publications You can also search for this author inPubMed Google Scholar * Xudong Wu View author publications You can also search for this author inPubMed Google Scholar *

Xiaohua Shen View author publications You can also search for this author inPubMed Google Scholar * Bing Zhu View author publications You can also search for this author inPubMed Google

Scholar * Guohong Li View author publications You can also search for this author inPubMed Google Scholar 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. CORRESPONDING AUTHOR Correspondence to Guohong Li.

ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional

claims in published maps and institutional affiliations. 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 _Rybp__TF/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. Source data 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,

_Rybp__TF/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. Source data EXTENDED DATA FIG. 8 THE PROLIFERATION AND APOPTOSIS OF CELL LINES. A, Cell proliferating curves of wild type, _Rybp__KO_, _Rybp__TF/AA_ and H1-TKO

mESCs (n=8 biologically independent samples). Data represents mean ± s.d. B, Representative flow cytometry of apoptosis of wild type, _Rybp__KO_, RybpTF/AA and H1-TKO mESCs out of 3

independent experiments. Statistical source data are available in Source Data. Source data EXTENDED DATA FIG. 9 OVERLAPPING ROLES OF RYBP-PRC1 AND HISTONE H1. A, A scatter plot showing the

fold changes of H2AK119ub1 peaks in _Rybp__TF/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 _Rybp__TF/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, _Rybp__TF/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, _Rybp__KO_, _Rybp__TF/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 _Rybp__TF/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. Source data SUPPLEMENTARY INFORMATION REPORTING

SUMMARY SUPPLEMENTARY TABLES Supplementary Table 1 List of the neural differentiation-related genes locating at H2AK119ub1 reduction overlapping peaks in _Rybp_T31A/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. SOURCE DATA SOURCE DATA FIG. 1 Unprocessed

western blots SOURCE DATA FIG. 2 Unprocessed western blots SOURCE DATA FIG. 2 Statistical source data SOURCE DATA FIG. 3 Unprocessed western blots SOURCE DATA FIG. 3 Statistical source data

SOURCE DATA FIG. 4 Unprocessed western blots SOURCE DATA FIG. 4 Statistical source data SOURCE DATA FIG. 5 Statistical source data SOURCE DATA FIG. 6 Unprocessed western blots SOURCE DATA

FIG. 6 Statistical source data SOURCE DATA FIG. 7 Statistical source data SOURCE DATA EXTENDED DATA FIG. 1 Unprocessed western blots, Unprocessed agarose electrophoresis gel, Unprocessed

Coomassie-stained gels SOURCE DATA EXTENDED DATA FIG. 1 Statistical source data SOURCE DATA EXTENDED DATA FIG. 2 Unprocessed western blots SOURCE DATA EXTENDED DATA FIG. 2 Statistical source

data SOURCE DATA EXTENDED DATA FIG. 3 Unprocessed western blots, Unprocessed agarose electrophoresis gel SOURCE DATA EXTENDED DATA FIG. 3 Statistical source data SOURCE DATA EXTENDED DATA

FIG. 4 Unprocessed western blots SOURCE DATA EXTENDED DATA FIG. 4 Statistical source data SOURCE DATA EXTENDED DATA FIG. 5 Unprocessed western blots SOURCE DATA EXTENDED DATA FIG. 5

Statistical source data SOURCE DATA EXTENDED DATA FIG. 6 Statistical source data SOURCE DATA EXTENDED DATA FIG. 7 Statistical source data SOURCE DATA EXTENDED DATA FIG. 8 Statistical source

data SOURCE DATA EXTENDED DATA FIG. 9 Statistical source data RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received: 16 January 2019 * Accepted: 14 February 2020 * Published: 23 March 2020 * Issue Date: April 2020 * DOI:

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