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• W2783403873 abstract "•BCOR depletion induced endodermal and mesodermal differentiation of hESCs•BCOR, PRC1.1, and PRC2 components co-occupied promoters of differentiation genes•Deletions in the C terminus of BCOR abolished BCOR-PRC1.1 assembly at targets•The N terminus of BCOR repressed transcription independently of the C terminus Polycomb group proteins regulate self-renewal and differentiation in many stem cell systems. When assembled into two canonical complexes, PRC1 and PRC2, they sequentially deposit H3K27me3 and H2AK119ub histone marks and establish repressive chromatin, referred to as Polycomb domains. Non-canonical PRC1 complexes retain RING1/RNF2 E3-ubiquitin ligases but have unique sets of accessory subunits. How these non-canonical complexes recognize and regulate their gene targets remains poorly understood. Here, we show that the BCL6 co-repressor (BCOR), a member of the PRC1.1 complex, is critical for maintaining primed pluripotency in human embryonic stem cells (ESCs). BCOR depletion leads to the erosion of Polycomb domains at key developmental loci and the initiation of differentiation along endoderm and mesoderm lineages. The C terminus of BCOR regulates the assembly and targeting of the PRC1.1 complex, while the N terminus contributes to BCOR-PRC1.1 repressor function. Our findings advance understanding of Polycomb targeting and repression in ESCs and could apply broadly across developmental systems. Polycomb group proteins regulate self-renewal and differentiation in many stem cell systems. When assembled into two canonical complexes, PRC1 and PRC2, they sequentially deposit H3K27me3 and H2AK119ub histone marks and establish repressive chromatin, referred to as Polycomb domains. Non-canonical PRC1 complexes retain RING1/RNF2 E3-ubiquitin ligases but have unique sets of accessory subunits. How these non-canonical complexes recognize and regulate their gene targets remains poorly understood. Here, we show that the BCL6 co-repressor (BCOR), a member of the PRC1.1 complex, is critical for maintaining primed pluripotency in human embryonic stem cells (ESCs). BCOR depletion leads to the erosion of Polycomb domains at key developmental loci and the initiation of differentiation along endoderm and mesoderm lineages. The C terminus of BCOR regulates the assembly and targeting of the PRC1.1 complex, while the N terminus contributes to BCOR-PRC1.1 repressor function. Our findings advance understanding of Polycomb targeting and repression in ESCs and could apply broadly across developmental systems. Embryonic stem cells (ESCs) can self-renew in culture while retaining the potential to form the full repertoire of cell types found in the body. In mouse ESCs (mESCs), maintenance of the naive pluripotent state requires activation of the LIF and BMP4 pathways, whose effector proteins STAT3 and SMAD1/5/8 cooperate with the core pluripotency factors NANOG, OCT4, and SOX2 and accessory factors such as ESRRB, TBX3, and SALL4 to maintain the expression of pluripotency-associated genes and repress pro-differentiation genes (Martello and Smith, 2014Martello G. Smith A. The nature of embryonic stem cells.Annu. Rev. Cell Dev. Biol. 2014; 30: 647-675Crossref PubMed Scopus (272) Google Scholar). Additionally, Polycomb repressive complex 1 (PRC1) and PRC2 occupy the promoters of developmental genes and are regarded as key components of the repressor network in ESCs (Boyer et al., 2006Boyer L.A. Plath K. Zeitlinger J. Brambrink T. Medeiros L.A. Lee T.I. Levine S.S. Wernig M. Tajonar A. Ray M.K. et al.Polycomb complexes repress developmental regulators in murine embryonic stem cells.Nature. 2006; 441: 349-353Crossref PubMed Scopus (2036) Google Scholar, Lee et al., 2006Lee T.I. Jenner R.G. Boyer L.A. Guenther M.G. Levine S.S. Kumar R.M. Chevalier B. Johnstone S.E. Cole M.F. Isono K. et al.Control of developmental regulators by Polycomb in human embryonic stem cells.Cell. 2006; 125: 301-313Abstract Full Text Full Text PDF PubMed Scopus (1867) Google Scholar). EZH1/2, the catalytic subunits of the PRC2 complex, methylate histone H3 at Lys27 (H3K27me3), whereas the RING1/RNF2 (RING1A/RING1B) subunits of the PRC1 complex ubiquitinate histone H2A at Lys119 (H2AK119ub) (Simon and Kingston, 2013Simon J.A. Kingston R.E. Occupying chromatin: Polycomb mechanisms for getting to genomic targets, stopping transcriptional traffic, and staying put.Mol. Cell. 2013; 49: 808-824Abstract Full Text Full Text PDF PubMed Scopus (534) Google Scholar). The current canonical model proposes that PRC2 is recruited to target sites via DNA-binding proteins, such as JARID2, resulting in the deposition and propagation of H3K27me3 around the target site. This mark is recognized by the CBX subunit of the PRC1 complex (Margueron and Reinberg, 2011Margueron R. Reinberg D. The Polycomb complex PRC2 and its mark in life.Nature. 2011; 469: 343-349Crossref PubMed Scopus (2229) Google Scholar). Once recruited to a target, PRC1 deposits H2AK119ub and contributes to chromatin compaction, clustering of PRC-bound regions and gene silencing (Eskeland et al., 2010Eskeland R. Leeb M. Grimes G.R. Kress C. Boyle S. Sproul D. Gilbert N. Fan Y. Skoultchi A.I. Wutz A. Bickmore W.A. Ring1B compacts chromatin structure and represses gene expression independent of histone ubiquitination.Mol. Cell. 2010; 38: 452-464Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, Fischle et al., 2003Fischle W. Wang Y. Jacobs S.A. Kim Y. Allis C.D. Khorasanizadeh S. Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb and HP1 chromodomains.Genes Dev. 2003; 17: 1870-1881Crossref PubMed Scopus (790) Google Scholar, Kundu et al., 2017Kundu S. Ji F. Sunwoo H. Jain G. Lee J.T. Sadreyev R.I. Dekker J. Kingston R.E. Polycomb repressive complex 1 generates discrete compacted domains that change during differentiation.Mol. Cell. 2017; 65: 432-446Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, Wang et al., 2004Wang H. Wang L. Erdjument-Bromage H. Vidal M. Tempst P. Jones R.S. Zhang Y. Role of histone H2A ubiquitination in Polycomb silencing.Nature. 2004; 431: 873-878Crossref PubMed Scopus (1288) Google Scholar, Wani et al., 2016Wani A.H. Boettiger A.N. Schorderet P. Ergun A. Münger C. Sadreyev R.I. Zhuang X. Kingston R.E. Francis N.J. Chromatin topology is coupled to Polycomb group protein subnuclear organization.Nat. Commun. 2016; 7: 10291Crossref PubMed Scopus (121) Google Scholar). In addition to canonical PRC1 complexes (cPRC1), non-canonical PRC1 complexes (ncPRC1) may also play a role in pluripotency maintenance. They contain the RING1/RNF2 proteins but differ in their accessory subunit composition and do not depend on H3K27me3 for targeting (Gao et al., 2012Gao Z. Zhang J. Bonasio R. Strino F. Sawai A. Parisi F. Kluger Y. Reinberg D. PCGF homologs, CBX proteins, and RYBP define functionally distinct PRC1 family complexes.Mol. Cell. 2012; 45: 344-356Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar, Tavares et al., 2012Tavares L. Dimitrova E. Oxley D. Webster J. Poot R. Demmers J. Bezstarosti K. Taylor S. Ura H. Koide H. et al.RYBP-PRC1 complexes mediate H2A ubiquitylation at polycomb target sites independently of PRC2 and H3K27me3.Cell. 2012; 148: 664-678Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar). Recent reports suggest that KDM2B, a component of the ncPRC1.1 complex with H3K36me2 demethylase activity and a non-methylated CpG-binding domain, may contribute to complex targeting and repression of differentiation genes in mESCs (Blackledge et al., 2014Blackledge N.P. Farcas A.M. Kondo T. King H.W. McGouran J.F. Hanssen L.L. Ito S. Cooper S. Kondo K. Koseki Y. et al.Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation.Cell. 2014; 157: 1445-1459Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, Farcas et al., 2012Farcas A.M. Blackledge N.P. Sudbery I. Long H.K. McGouran J.F. Rose N.R. Lee S. Sims D. Cerase A. Sheahan T.W. et al.KDM2B links the Polycomb repressive complex 1 (PRC1) to recognition of CpG islands.eLife. 2012; 1: e00205Crossref PubMed Scopus (326) Google Scholar, He et al., 2013He J. Shen L. Wan M. Taranova O. Wu H. Zhang Y. Kdm2b maintains murine embryonic stem cell status by recruiting PRC1 complex to CpG islands of developmental genes.Nat. Cell Biol. 2013; 15: 373-384Crossref PubMed Scopus (229) Google Scholar, Tavares et al., 2012Tavares L. Dimitrova E. Oxley D. Webster J. Poot R. Demmers J. Bezstarosti K. Taylor S. Ura H. Koide H. et al.RYBP-PRC1 complexes mediate H2A ubiquitylation at polycomb target sites independently of PRC2 and H3K27me3.Cell. 2012; 148: 664-678Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, Wu et al., 2013Wu X. Johansen J.V. Helin K. Fbxl10/Kdm2b recruits polycomb repressive complex 1 to CpG islands and regulates H2A ubiquitylation.Mol. Cell. 2013; 49: 1134-1146Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). Unlike murine models, regulatory mechanisms in human ESCs (hESCs) are poorly understood. These cells are thought to resemble a primed pluripotent state found in the murine epiblast (Brons et al., 2007Brons I.G. Smithers L.E. Trotter M.W. Rugg-Gunn P. Sun B. Chuva de Sousa Lopes S.M. Howlett S.K. Clarkson A. Ahrlund-Richter L. Pedersen R.A. Vallier L. Derivation of pluripotent epiblast stem cells from mammalian embryos.Nature. 2007; 448: 191-195Crossref PubMed Scopus (1549) Google Scholar, Tesar et al., 2007Tesar P.J. Chenoweth J.G. Brook F.A. Davies T.J. Evans E.P. Mack D.L. Gardner R.L. McKay R.D. New cell lines from mouse epiblast share defining features with human embryonic stem cells.Nature. 2007; 448: 196-199Crossref PubMed Scopus (1677) Google Scholar). Self-renewal in hESCs requires the basic fibroblast growth factor (bFGF) and transforming growth factor β (TGF-β) pathways, whose effector proteins, SMAD2/3 and ERK1/2, interface with the transcriptional network in the nucleus (Göke et al., 2013Göke J. Chan Y.S. Yan J. Vingron M. Ng H.H. Genome-wide kinase-chromatin interactions reveal the regulatory network of ERK signaling in human embryonic stem cells.Mol. Cell. 2013; 50: 844-855Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, Vallier et al., 2009Vallier L. Mendjan S. Brown S. Chng Z. Teo A. Smithers L.E. Trotter M.W. Cho C.H. Martinez A. Rugg-Gunn P. et al.Activin/Nodal signalling maintains pluripotency by controlling Nanog expression.Development. 2009; 136: 1339-1349Crossref PubMed Scopus (320) Google Scholar, Xu et al., 2005Xu R.H. Peck R.M. Li D.S. Feng X. Ludwig T. Thomson J.A. Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells.Nat. Methods. 2005; 2: 185-190Crossref PubMed Scopus (829) Google Scholar). The core factors NANOG, OCT4, and SOX2/3 are reorganized into different modules to suppress epiblast cell fates (Wang et al., 2012Wang Z. Oron E. Nelson B. Razis S. Ivanova N. Distinct lineage specification roles for NANOG, OCT4, and SOX2 in human embryonic stem cells.Cell Stem Cell. 2012; 10: 440-454Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). However, homologs of transcriptional regulators of mESCs, such as ESRRB and TBX3, are not expressed in hESCs. Instead, PRDM14, FOXO1, and LSD1, all of which appear to be dispensable for mESC self-renewal, have been identified as regulators of hESC pluripotency (Adamo et al., 2011Adamo A. Sesé B. Boue S. Castaño J. Paramonov I. Barrero M.J. Izpisua Belmonte J.C. LSD1 regulates the balance between self-renewal and differentiation in human embryonic stem cells.Nat. Cell Biol. 2011; 13: 652-659Crossref PubMed Scopus (242) Google Scholar, Chia et al., 2010Chia N.Y. Chan Y.S. Feng B. Lu X. Orlov Y.L. Moreau D. Kumar P. Yang L. Jiang J. Lau M.S. et al.A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity.Nature. 2010; 468: 316-320Crossref PubMed Scopus (364) Google Scholar, Zhang et al., 2011Zhang X. Yalcin S. Lee D.F. Yeh T.Y. Lee S.M. Su J. Mungamuri S.K. Rimmelé P. Kennedy M. Sellers R. et al.FOXO1 is an essential regulator of pluripotency in human embryonic stem cells.Nat. Cell Biol. 2011; 13: 1092-1099Crossref PubMed Scopus (197) Google Scholar). Here, we combine functional, biochemical, and genomics approaches to screen for additional regulators of primed pluripotency in humans. We identify the BCL6 corepressor (BCOR) as a critical factor for hESC maintenance and show that BCOR defines a subtype of the PRC1 complexes with distinct recruitment and repression mechanisms that are essential for silencing differentiation programs in hESCs. To identify additional regulators of hESCs, we performed microarray profiling of H1 hESC line differentiating in the presence of retinoic acid. We selected 59 genes with functions related to transcription and chromatin that were expressed in hESCs but downregulated upon differentiation (Figure S1A). Interrogating this gene set with small hairpin RNAs (shRNAs) identified five genes whose depletion led to a loss of proliferative capacity, morphological changes, and the induction of differentiation markers (Figures S1B–S1E). Of these positive hits, PRDM14 and FOXO1 have been reported as hESC regulators (Chia et al., 2010Chia N.Y. Chan Y.S. Feng B. Lu X. Orlov Y.L. Moreau D. Kumar P. Yang L. Jiang J. Lau M.S. et al.A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity.Nature. 2010; 468: 316-320Crossref PubMed Scopus (364) Google Scholar, Zhang et al., 2011Zhang X. Yalcin S. Lee D.F. Yeh T.Y. Lee S.M. Su J. Mungamuri S.K. Rimmelé P. Kennedy M. Sellers R. et al.FOXO1 is an essential regulator of pluripotency in human embryonic stem cells.Nat. Cell Biol. 2011; 13: 1092-1099Crossref PubMed Scopus (197) Google Scholar), while the three remaining genes (ZFP42, ZNF649, and BCOR) have not been implicated in hESC maintenance. While knockdown of FOXO1, ZFP42, and ZNF649 in H7 and H9 cells failed to induce differentiation (data not shown), depletion of BCOR induced a robust, consistent loss-of-proliferation phenotype in all three cell lines tested (Figures 1A–1C). Thus far, only four factors (NANOG, OCT4, SOX2/3, and PRDM14) have been shown to be required in multiple hESC lines. We examined the consequences of BCOR knockdown (shBCOR) in hESCs. There was no increase in apoptotic or dead cells in shBCOR-hESC cultures compared to cells transduced with a non-targeting control vector (control ESCs) (Figure 1D). Cell-cycle analysis revealed an increase in the duration of the G1 phase, while the S phase was shortened (Figure 1E). shBCOR hESCs were morphologically distinct from control hESCs (Figure 1F). shBCOR hESCs also exhibited an abnormal balance of pluripotency factors. RNA and protein levels of POU5F1/OCT4 were unaltered, while NANOG was upregulated and SOX2 was reduced (Figure 1G). Immunofluorescence analyses confirmed the induction of mesoderm and endoderm markers in shBCOR hESCs (Figure 1H). For validation, we derived a BCOR rescue line (BCORR hESCs) in which endogenous BCOR was inactivated via a frameshift mutation while doxycycline (Dox)-inducible FLAG-BCOR and rtTA transgenes introduced into the AAVS1 locus allowed for self-renewal in the presence of Dox (Figure 1I). Upon Dox removal, BCOR was lost, and the cells had a phenotype similar to shBCOR ESCs (Figures 1J–1L). These analyses identify BCOR as a core component of the hESC regulatory network governing the pluripotent state. BCOR was initially identified as a corepressor that interacts with the B cell transcription factor BCL6 (Huynh et al., 2000Huynh K.D. Fischle W. Verdin E. Bardwell V.J. BCoR, a novel corepressor involved in BCL-6 repression.Genes Dev. 2000; 14: 1810-1823Crossref PubMed Google Scholar). In several cell types, BCOR co-purifies with a non-canonical PRC1.1 complex (Béguelin et al., 2016Béguelin W. Teater M. Gearhart M.D. Calvo Fernández M.T. Goldstein R.L. Cárdenas M.G. Hatzi K. Rosen M. Shen H. Corcoran C.M. et al.EZH2 and BCL6 cooperate to assemble CBX8-BCOR complex to repress bivalent promoters, mediate germinal center formation and lymphomagenesis.Cancer Cell. 2016; 30: 197-213Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, Gao et al., 2012Gao Z. Zhang J. Bonasio R. Strino F. Sawai A. Parisi F. Kluger Y. Reinberg D. PCGF homologs, CBX proteins, and RYBP define functionally distinct PRC1 family complexes.Mol. Cell. 2012; 45: 344-356Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar, Gearhart et al., 2006Gearhart M.D. Corcoran C.M. Wamstad J.A. Bardwell V.J. Polycomb group and SCF ubiquitin ligases are found in a novel BCOR complex that is recruited to BCL6 targets.Mol. Cell. Biol. 2006; 26: 6880-6889Crossref PubMed Scopus (269) Google Scholar, Oliviero et al., 2015Oliviero G. Munawar N. Watson A. Streubel G. Manning G. Bardwell V. Bracken A.P. Cagney G. The variant Polycomb Repressor Complex 1 component PCGF1 interacts with a pluripotency sub-network that includes DPPA4, a regulator of embryogenesis.Sci. Rep. 2015; 5: 18388Crossref PubMed Scopus (34) Google Scholar). To identify BCOR-interacting proteins in hESCs, we performed FLAG immunoprecipitation (IP) from BCORR hESCs followed by mass spectrometry. While BCL6 was not identified in these IPs, the PRC1.1 components PCGF1, RNF2, RING1, RYBP, KDM2B, and USP7 and the heat shock protein HSPD1 were among the top hits. PRC2 and canonical PRC1-specific proteins were not detected (Figures 2A and 2B ; Table S1). Next, we carried out sucrose-gradient centrifugation of nuclear extracts from BCORR hESCs. In the presence of Dox, BCOR, RNF2, PCGF1, and KDM2B co-fractionated in the megadalton-size range, suggesting the existence of the common complex (Figure 2C). Upon removal of Dox, the distributions of KDM2B and PCGF1 shifted toward lower-molecular-weight fractions, indicating that BCOR interacts with both proteins and may link KDM2B to the PRC1.1 core, which consists of RNF2, PCGF1, and RYBP. While the majority of HSPD1 fractionated outside the range of the BCOR complexes, a small amount was detected in high-molecular-weight fractions also containing BCOR, RNF2, PCGF1, and KDM2B. To study the assembly of the BCOR-PRC1.1 complexes, we overexpressed individual components in HEK293FT cells, followed by FLAG-affinity capture. The PRC1.1 core was recovered in both FLAG-BCOR and FLAG-KDM2B IPs. Both BCOR and the PRC1.1 core were recovered in FLAG-KDM2B IP, but the interaction between KDM2B and the core was lost when BCOR was removed (Figure S2A). HSPD1 was recovered in all reconstitution experiments that contained BCOR but did not associate with either the PRC1.1 core or the KDM2B/SKP1 subcomplex, indicating a specific interaction with BCOR (Figure S2B). To validate these results, we performed co-IP in BCORR hESCs maintained with or without Dox. Both KDM2B and the PRC1.1 core were recovered when either KDM2B or RNF2 was used as bait in the presence of Dox. Similar interactions were also observed in the absence of Dox (Figure S2C). Because the expression of the BCOR homolog BCORL1 in hESCs is very low (Figure S3A), we concluded that the minimal KDM2B-PRC1.1 complexes, which have been reported in mESCs and Drosophila embryos (Lagarou et al., 2008Lagarou A. Mohd-Sarip A. Moshkin Y.M. Chalkley G.E. Bezstarosti K. Demmers J.A. Verrijzer C.P. dKDM2 couples histone H2A ubiquitylation to histone H3 demethylation during Polycomb group silencing.Genes Dev. 2008; 22: 2799-2810Crossref PubMed Scopus (208) Google Scholar, Wu et al., 2013Wu X. Johansen J.V. Helin K. Fbxl10/Kdm2b recruits polycomb repressive complex 1 to CpG islands and regulates H2A ubiquitylation.Mol. Cell. 2013; 49: 1134-1146Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar), could also form in hESCs in the absence of BCOR. No HSPD1 was detected in KDM2B and RNF2 IPs, in either the presence or absence of Dox/BCOR, indicating that the HSPD1-BCOR-PRC1.1 complexes are not abundant (Figure S2C). To determine how the genomic distribution of BCOR correlates with the distributions of the components of the PRC1/2 complexes, DNA accessibility, and transcriptional state, we mapped the binding of BCOR, KDM2B, PCGF1, RNF2, RYBP, and the PRC1.1-associated chromatin marks H2AK119ub and H3K36me2. Published chromatin immunoprecipitation sequencing (ChIP-seq) data for the PRC2 component SUZ12 and H3K27me3, the canonical PRC1 subunit CBX2, which is abundant in hESCs (Figure S3A), and marks of chromatin accessibility and transcription such as DNase I, H3K4me3, H3K79me3, and POL2-Ser5p/7p phosphorylation states were added to this dataset. Because BCOR binding exhibited a strong bias toward the promoter regions (Figure S2B), our analyses were focused on the 20-kb intervals centered on the transcription start sites (TSSs). K-means clustering with these 16 parameters separated all 57,728 annotated promoter regions into three distinct groups (Figure 2D). The strong-binding group contained 2,610 promoters with the highest PRC1.1 levels. Developmental regulators, particularly transcription factors of different families, were overrepresented in this group (Figure S2C). Promoters in this group were also highly enriched for SUZ12, H3K27me3, and CBX2, in agreement with reports demonstrating co-occupancy of cPRC1, ncPRC1, and PRC2 at many genomic loci (Blackledge et al., 2014Blackledge N.P. Farcas A.M. Kondo T. King H.W. McGouran J.F. Hanssen L.L. Ito S. Cooper S. Kondo K. Koseki Y. et al.Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation.Cell. 2014; 157: 1445-1459Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, Tavares et al., 2012Tavares L. Dimitrova E. Oxley D. Webster J. Poot R. Demmers J. Bezstarosti K. Taylor S. Ura H. Koide H. et al.RYBP-PRC1 complexes mediate H2A ubiquitylation at polycomb target sites independently of PRC2 and H3K27me3.Cell. 2012; 148: 664-678Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar). The weak-binding group contained 14,277 promoters with lower levels of PRC1.1 components. This group had a more diverse ontology with enrichment for cell division, mRNA splicing, protein transport, and other housekeeping cellular processes (Figure S2C). Promoters in both the strong- and the weak-binding groups had elevated levels of marks associated with transcriptional activation such as DNase I hypersensitivity, H3K4me3, H3K79me2, and POL2-Ser5p/7p, in contrast to promoters in the no-binding group (40,841 regions), the majority of which were transcriptionally inert regions. Further analyses revealed strong correlation between PRC1.1 and PRC2 components in the strong-binding group (Figure 2E). PRC1.1 and PRC2 binding in the strong group correlated positively with DNase I hypersensitivity and POL2-Ser5p signals, but not with POL2-Ser7p and H3K79me2, indicating that the majority of genes in this group may be primed, but not transcribed. In contrast, there was a positive correlation between PRC1.1 and POL2-Ser5p/7p in the weak-binding group (Figure 2E). It has been shown that PRC targets showing co-occupancy of PRC1 and POL2-Ser2p in bulk analyses are not co-bound but are fluctuating between actively transcribed and repressed states (Brookes et al., 2012Brookes E. de Santiago I. Hebenstreit D. Morris K.J. Carroll T. Xie S.Q. Stock J.K. Heidemann M. Eick D. Nozaki N. et al.Polycomb associates genome-wide with a specific RNA polymerase II variant, and regulates metabolic genes in ESCs.Cell Stem Cell. 2012; 10: 157-170Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). A significant fraction of BCOR-PRC1.1 weak-binding group targets in hESCs very likely correspond to such fluctuating targets. RNA-sequencing (RNA-seq) analyses in shBCOR and control hESCs showed that expression levels of 1,571 genes, of which 1,082 (68.9%) were BCOR-PRC1.1 direct targets, were significantly altered. The transcriptional response was biased toward the upregulated genes, 30% of which were BCOR-PRC1.1 targets from the strong-binding group (Figure S2D). The upregulated gene group was enriched for gene ontologies associated with primitive streak formation, endoderm differentiation, epithelial-to-mesenchymal transition, BMP4, TGF-β, and WNT signaling, suggesting that derepression of these genes following BCOR depletion is the major cause of differentiation (Figure S2E). Not all BCOR-PRC1.1 targets in the strong-binding group are derepressed in shBCOR hESCs. To identify parameters correlating with the promoter response to BCOR depletion, we compared the levels of 16 parameters shown in Figure 2D at upregulated, downregulated, and non-responsive targets within each binding group. For the strong-binding group, the levels of BCOR and other PRC1.1 components were not significantly different. However, the levels of RNF2, H2AK119ub, SUZ12, and H3K27me3 were on average higher at non-responsive targets (Figures 2G and S2H). These data suggest that BCOR-independent recruitment of the PRC2/cPRC1 complexes at non-responsive targets could counteract gene activation resulting from BCOR loss. Together, our mass spectrometry, chromatin localization, and gene expression data indicate that the BCOR-PRC1.1 complex represses key developmental regulators in hESCs. To probe the role of BCOR in PRC1.1 recruitment and function, we performed ChIP-qPCR analyses in shBCOR hESCs. We selected a set of BCOR targets from the strong-binding group that were associated with particularly large Polycomb domains. This set included the BCOR-responsive targets EOMES, T, FOXA2, SOX17, and GATA6 and the non-responsive targets GSX2, EN2, NEUROD1, SOX1, and VAX2 (Figure S4A). Depletion of BCOR resulted in decreased RNF2 and KDM2B binding and was accompanied by the loss of H2AK119ub and H3K27me3 at both responsive and non-responsive targets, while a gain of H3K4me3 was observed only at BCOR-responsive genes (Figure 3A). Time-course analyses in BCORR hESCs differentiating in the absence of Dox revealed that BCOR was sharply depleted at all loci tested after Dox removal. A decrease in RNF2, H3K27me3, and H2AK119ub followed, while KDM2B enrichment was lost more gradually (Figure 3B). Non-responsive targets exhibited similar trends, but of a lesser magnitude. Furthermore, after their initial decline, the RNF2 and H3K27me3 levels at these targets were partially restored (Figure 3B). The coordinated recovery of H3K27me3 and RNF2 levels suggest that the PRC2/cPRC1 complexes at non-responsive targets could be recruited independently of BCOR and the PRC1.1 complex. Indeed, ChIP-qPCR analyses of CBX2 alongside with RYBP in BCORR hESCs maintained without Dox demonstrated a significant decrease in both CBX2 and RYBP levels at BCOR-responsive targets. At non-responsive targets, CBX2 was largely retained, and RYBP was lost at some (GSX2 and NEUROD1) but retained at other (SOX1 and VAX2) targets that appear to recruit RYBP independently of BCOR (Figure 3C). Because the loss of RNF2, H2AK119ub, and H3K27me3 at BCOR-responsive targets after Dox removal occurs slower than the loss of BCOR itself, we tested whether the effects of BCOR depletion on PRC1 and PRC2 levels are secondary to transcriptional activation of BCOR targets. We used shRNA to deplete BCOR in hESCs that had a Dox-inducible dCas9-KRAB repressor integrated into the AAVS1 locus (Figure S4B). Using lentiviral single guide RNAs (sgRNAs), we tethered this repressor to the TSS region of the EOMES gene. In the presence of Dox, dCas9-KRAB inhibited EOMES transcription. The EOMES-regulated endodermal genes SOX17 and FOXA2, which are BCOR-PRC1.1 targets, were also downregulated. ChIP-qPCR analyses in these cells demonstrated that reduced transcription did not prevent decay of H3K27me3, CBX2, RNF2, or RYBP at any of these three loci (Figures S4B–S4D). Thus, it is unlikely that the loss of PRC1.1 and PRC2 components at BCOR-responsive genes occurs solely due to activation of transcription. These data indicate that the presence of BCOR at BCOR-responsive targets is crucial for the recruitment of the PRC1.1 complex, the maintenance of H3K27me3, and the recruitment of the cPRC1 complexes. Studies in mESCs have indicated that the CXXC domain of KDM2B is involved in recruiting the PRC1.1 complex to unmethylated CpG islands (Blackledge et al., 2014Blackledge N.P. Farcas A.M. Kondo T. King H.W. McGouran J.F. Hanssen L.L. Ito S. Cooper S. Kondo K. Koseki Y. et al.Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation.Cell. 2014; 157: 1445-1459Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, Farcas et al., 2012Farcas A.M. Blackledge N.P. Sudbery I. Long H.K. McGouran J.F. Rose N.R. Lee S. Sims D. Cerase A. Sheahan T.W. et al.KDM2B links the Polycomb repressive complex 1 (PRC1) to recognition of CpG islands.eLife. 2012; 1: e00205Crossref PubMed Scopus (326) Google Scholar, He et al., 2013He J. Shen L. Wan M. Taranova O. Wu H. Zhang Y. Kdm2b maintains murine embryonic stem cell status by recruiting PRC1 complex to CpG islands of developmental genes.Nat. Cell Biol. 2013; 15: 373-384Crossref PubMed Scopus (229) Google Scholar, Wu et al., 2013Wu X. Johansen J.V. Helin K. Fbxl10/Kdm2b recruits polycomb repressive complex 1 to CpG islands and regulates H2A ubiquitylation.Mol. Cell. 2013; 49: 1134-1146Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). If the same holds true in hESCs, depletion of KDM2B should phenocopy the BCOR depletion phenotype by triggering the loss of Polycomb domains, induction of BCOR targets, and initiation of differentiation. To test this prediction, we knocked down KDM2B in hESCs with shRNA. Surprisingly, differentiation did not occur (Figures S3A–S3D). Similar results were obtained with CRISPR/Cas9-generated KDM2B-KO hESCs, which" @default.
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• W2783403873 title "A Non-canonical BCOR-PRC1.1 Complex Represses Differentiation Programs in Human ESCs" @default.
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