A High-Density Map for Navigating the Human Polycomb Complexome
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Resource A High-Density Map for Navigating the Human Polycomb Complexome Graphical Abstract Authors Simon Hauri, Federico Comoglio, Makiko Seimiya, ..., Renato Paro, Matthias Gstaiger, Christian Beisel Correspondence [email protected] (M.G.), [email protected] (C.B.) In Brief Polycomb group (PcG) proteins mediate gene silencing and epigenetic memory in higher eukaryotes. By systematically mapping the human PcG complexome, Hauri et al. resolve Polycomb subcomplexes at high resolution and identify two human PRC2 and two PR- DUB complexes. Furthermore, genomic profiling reveals segregation of PRC1 and PR-DUB target genes. Highlights Accession Numbers d 1,400 high-confidence interactions reveal the modular GSE51673 organization of human PcG proteins d Detailed dissection of PRC1 and PRC2 subcomplexes d Two human PR-DUB complexes contain the glycosyltransferase OGT1 d PR-DUB and PRC1 bind largely distinct sets of target genes Hauri et al., 2016, Cell Reports 17, 583–595 October 4, 2016 ª 2016 The Authors. http://dx.doi.org/10.1016/j.celrep.2016.08.096 Cell Reports Resource A High-Density Map for Navigating the Human Polycomb Complexome Simon Hauri,1,2,7,8 Federico Comoglio,3,7,9 Makiko Seimiya,3 Moritz Gerstung,3,10 Timo Glatter,1,11 Klaus Hansen,4 Ruedi Aebersold,1,5 Renato Paro,3,6 Matthias Gstaiger,1,2,* and Christian Beisel3,12,* 1Department of Biology, Institute of Molecular Systems Biology, ETH Zurich,€ 8093 Zurich,€ Switzerland 2Competence Center Personalized Medicine UZH/ETH, 8044 Zurich,€ Switzerland 3Department of Biosystems Science and Engineering, ETH Zurich,€ 4058 Basel, Switzerland 4Biotech Research and Innovation Centre (BRIC) and Centre for Epigenetics, University of Copenhagen, 2200 Copenhagen, Denmark 5Faculty of Science, University of Zurich,€ 8057 Zurich,€ Switzerland 6Faculty of Sciences, University of Basel, 4056 Basel, Switzerland 7Co-first author 8Present address: Department of Clinical Sciences, Lund University, 221 00 Lund, Sweden 9Present address: Department of Haematology, Cambridge Institute for Medical Research and Wellcome Trust/MRC Cambridge Stem Cell Institute, University of Cambridge, CB2 0XY Cambridge, UK 10Present address: European Bioinformatics Institute (EMBL-EBI), Wellcome Genome Campus, CB10 1SD Hinxton, UK 11Present address: Mass Spectrometry and Proteomics, Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany 12Lead Contact *Correspondence: [email protected] (M.G.), [email protected] (C.B.) http://dx.doi.org/10.1016/j.celrep.2016.08.096 SUMMARY metazoans, including genes affiliated to fundamental signaling pathways (Ringrose, 2007). Biological processes regulated by Polycomb group (PcG) proteins are major determi- PcG proteins encompass cell differentiation, tissue regenera- nants of gene silencing and epigenetic memory in tion, and cancer cell growth (Jaenisch and Young, 2008; Maur- higher eukaryotes. Here, we systematically mapped ange et al., 2006; Sparmann and van Lohuizen, 2006). the human PcG complexome using a robust affinity The PcG system consists of multimeric repressive protein purification mass spectrometry approach. Our high- complexes containing distinct chromatin modifying activities, density protein interaction network uncovered a which impact transcriptional regulation by modulating chromatin structures. In Drosophila, five distinct PcG complexes displaying diverse range of PcG complexes. Moreover, our different biochemical functions have been reported. The Poly- analysis identified PcG interactors linking them to comb repressive complex (PRC) 2 contains enhancer of zeste, the PcG system, thus providing insight into the which trimethylates lysine 27 of histone H3 (H3K27me3) (Cao molecular function of PcG complexes and mecha- et al., 2002; Muller€ et al., 2002), while the PRC1 subunit Poly- nisms of recruitment to target genes. We identified comb provides binding specificity to H3K27me3 through its two human PRC2 complexes and two PR-DUB deu- chromo-domain (Fischle et al., 2003; Min et al., 2003). PRC1 biquitination complexes, which contain the O-linked also contains the dRing protein, which catalyzes the mono-ubiq- N-acetylglucosamine transferase OGT1 and several uitination of histone H2A on lysine 118 (H2AUb1), thereby block- transcription factors. Finally, genome-wide profiling ing RNA polymerase II activity (de Napoles et al., 2004; Stock of PR-DUB components indicated that the human et al., 2007; Wang et al., 2004). The Pleiohomeotic (Pho, PR-DUB and PRC1 complexes bind distinct sets of Drosophila homolog of mammalian YY1) repressive complex PhoRC combines DNA- and histone tail-binding specificities target genes, suggesting differential impact on (Klymenko et al., 2006), the PRC1-related dRing-associated cellular processes in mammals. factors complex (dRAF) contains the H3K36-specific histone de- methylase dKDM2 (Lagarou et al., 2008), and the Polycomb INTRODUCTION repressive deubiquitinase (PR-DUB) targets H2AUb1 (Scheuer- mann et al., 2010). Cell division requires faithful replication of the genome and resto- Although the core components of Drosophila PcG complexes ration of specific chromatin states that form the basis of epige- seem fixed, we and others have shown that they can be co-pu- netic memory (Sarkies and Sale, 2012). Polycomb group (PcG) rified with different sets of accessory proteins, thus increasing proteins were originally identified in Drosophila melanogaster the diversity of the PcG system (Furuyama et al., 2004; Klymenko as stably maintaining the repressed state of homeotic genes et al., 2006; Saurin et al., 2001; Strubbe€ et al., 2011). Epigenomic throughout development and are key players in this process. profiling has revealed that distinct PcG complexes target largely Numerous studies have since established a central role for overlapping gene sets in Drosophila, and mechanistic details of PcG proteins in the dynamic control of hundreds of targets in PcG recruitment to target genes are beginning to emerge (Beisel Cell Reports 17, 583–595, October 4, 2016 ª 2016 The Authors. 583 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). A Double Affinity LC-MS/MS Identification Contaminant Subcomplex 64 Baits Flp-In HEK293 Cells Purification LTQ Orbitrap XL X!Tandem + TPP Filtering Assignments Gene of interest 490 HCIPs N +Tet 174 AP-MS WD -Score 1400 interactions Hierarchical Strep FRT experiments Clustering HA 72 AP-MS control ensity TetO t (n > 2 biological experiments in CMV Hygro replicates) FRT recombination m/z Streptactin αHA B C CSK22 PHC2 CBX6 PRC1 PHC1 PCGF2 CSK21 CBX7 CBX8 PCGF3 PRC1.1 DCAF7 CBX2 PHC3 TCP PCGF5 PRC1.3/PRC1.5 OGT1 PR-DUB PCGF4 WDR5 CBX4 DCAF7 YAF2 ASXL2MLL RING1 RYBP RYBP RING2 MLL RING2 YAF2 RING1 ASXL1 LMBL2 PRC1.6 BAP1 E2F6 OGT1 MAX PCGF6 TFDP1 LCOR MAX LMBL4 PCGF1 E2F6 HP1 (CBX1/3/5) ZMYM4 19S INO80 proteasome -complexes LMBL3 SKP1 TFDP1 SUV92 YY1 CBX3 TRIPC LMBL1 WDR5 PR-DUB TCP 19S proteasome CBX5 CBX1 HP1 SKP1 ZN462 ZN211 EZH1 C10orf12 EHMT1 PHF19 EZH2 SUV91 LMBL1 SUZ12 CAF1B 20S proteasome AEBP2 DSN1 LCOR C17orf96 ZMYM4 YY1-INO80 PHF1 PRC2 EED Z518A MTF2 RBBP7 SENP7 LINC, NURF, NURD, SIN3 SMBT1 JARD2 PRC1.2/PRC1.4 RBBP4 LINC NURF PRC2 NURD SIN3 D E REXONMYCL1 MNTMXI1 ARGI1 SPR1B RB PRC1.6 MAD1 MYC MAD4 MAD3 RYBP RYBP CBX3 PRC1.3/5 RING1 PCGF5 RING1 MAX WDR5 CCSK22SK22 GALT8 FBRS MK67I PCGF6 MGAP CBX1 DDX54 FBSL DCAF7 CSK2BC SURF6 AUTS2 GSCR2 RING2 TFDP1 LMBL2 CCSK21SK21 TCPW FA53C DYR1B DYR1A YAF2 E2F6 RING2 PCGF3 ZN503 SWAHA DIAP1 TROAPSWAHC ZN703 E2F1E2F4 GEMI ERLN2 USMG5 TGM3 TFDP2 POF1B AMY1 COMD4 YAF2 PFD4 PSME3 PFD6 E2F5 E2F3ARF3 CAND1 AT2A2 APOD SMR3B CYTS COMD6 PFD2 PFD3 PFD5 E2F2DIC ABCD3 MCIN DHE3 GSTP1 CYTT COMD1 DNA binding proteins NTPCR AKAP8 S10AB COMD8 Figure 1. Systematic Profiling of Human PcG Protein Complexes (A) Workflow for native protein complex purifications from Flp-In HEK293 T-REx cells. Open reading frames of 64 bait proteins were cloned into an expression vector containing a tetracycline inducible CMV promoter, Strep-HA fusion tag, and FRT sites. The proteins were affinity purified from whole cell extracts of isogenic cell lines, trypsinized, and identified by tandem mass spectrometry on an LTQ Orbitrap XL. HCIPs were hierarchically clustered to infer protein complex compositions. (B) Hierarchical clustering of HCIPs. Clusters of PcG and non-PcG complexes are labeled in red and blue, respectively. The inset shows the location of PRC1.1, PRC1.3/PRC1.5, and the four core proteins RYBP/YAF2 and RING1/2. The clusters defined by single baits are indicated in green. Spearman’s rank correlation coefficient based dissimilarities are color coded as indicated (top right). (legend continued on next page) 584 Cell Reports 17, 583–595, October 4, 2016 and Paro, 2011; Enderle et al., 2011; Oktaba et al., 2008; teractions and 490 proteins considerably refines the topology of Scheuermann et al., 2010; Schuettengruber et al., 2009). the human PRC1 and PRC2 network, including their relation with In contrast, the mammalian PcG system is less well defined the heterochromatin silencing system, and uncovers additional and appears to be significantly more complex. Each Drosophila interaction partners. Furthermore, we determined the composi- PcG subunit has up to six human homologs, which combinatori- tion of the human PR-DUB. We found that this highly diverse ally assemble in different complexes (Gao et al., 2012; Mar- complex contains MBD proteins, FOXK transcription factors, gueron et al., 2008; Shen et al., 2008). The six homologs of the and OGT1 (OGT), an O-linked N-acetylglucosamine (O-GlcNAC) Drosophila PRC1 core protein Psc, PCGF1–6, purify together transferase implicated in PcG silencing in Drosophila (Gambetta with RING2 (RNF2), the homolog of dRing, in different complexes et al., 2009). Finally, chromatin profiling of PR-DUB components named PRC1.1–PRC1.6, and each of them associates with spe- and comparison with chromatin maps of PcG proteins indicate cific additional components (Gao et al., 2012; Sa´ nchez et al., that in contrast to Drosophila, PRC1 and PR-DUB regulate 2007). These PRC1 complexes are further distinguished by the distinct sets of genes in human cells.