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• W2000672112 abstract "RING1B, a Polycomb Group (PcG) protein, binds methylated chromatin through its association with another PcG protein called Polycomb (Pc). However, RING1B can associate with nonmethylated chromatin suggesting an alternate mechanism for RING1B interaction with chromatin. Here, we demonstrate that two proteins with little sequence identity between them, the Pc cbox domain and RYBP, bind the same surface on the C-terminal domain of RING1B (C-RING1B). Pc cbox and RYBP each fold into a nearly identical, intermolecular beta sheet with C-RING1B and a loop structure which are completely different in the two proteins. Both the beta sheet and loop are required for stable binding and transcription repression. Further, a mutation engineered to disrupt binding on the Drosophila dRING1 protein prevents chromatin association and PcG function in vivo. These results suggest that PcG targeting to different chromatin locations relies, in part, on binding partners of C-RING1B that are diverse in sequence and structure. RING1B, a Polycomb Group (PcG) protein, binds methylated chromatin through its association with another PcG protein called Polycomb (Pc). However, RING1B can associate with nonmethylated chromatin suggesting an alternate mechanism for RING1B interaction with chromatin. Here, we demonstrate that two proteins with little sequence identity between them, the Pc cbox domain and RYBP, bind the same surface on the C-terminal domain of RING1B (C-RING1B). Pc cbox and RYBP each fold into a nearly identical, intermolecular beta sheet with C-RING1B and a loop structure which are completely different in the two proteins. Both the beta sheet and loop are required for stable binding and transcription repression. Further, a mutation engineered to disrupt binding on the Drosophila dRING1 protein prevents chromatin association and PcG function in vivo. These results suggest that PcG targeting to different chromatin locations relies, in part, on binding partners of C-RING1B that are diverse in sequence and structure. Crystal structure of C-RING1B/cbx7 cbox complex cbx7 cbox beta sheet and loop are required for binding C-RING1B and gene repression RYBP binds to the same surface on C-RING1B as cbx7 cbox Drosophila RING1 protein-protein interaction mutations hinders its chromatin binding The Polycomb Group (PcG) of gene silencers are chromatin-associated multiprotein complexes that maintain the genomic program of cells (Schwartz and Pirrotta, 2008Schwartz Y.B. Pirrotta V. Polycomb complexes and epigenetic states.Curr. Opin. Cell Biol. 2008; 20: 266-273Crossref PubMed Scopus (229) Google Scholar). In stem cells, PcG complexes bind to hundreds of genomic loci, where they repress genes that promote differentiation (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 (1927) 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 (1789) Google Scholar). A fundamental question regarding PcG function is how these complexes bind specifically to so many sites. Such targeting would appear to require highly specific and elaborate regulatory mechanisms since it is ultimately responsible for maintaining the intricate balance between pluripotency and differentiation. Several of the multiprotein PcG complexes have been isolated and shown to have distinct repressive functions. Polycomb repression complex 1 (PRC1) is composed of four core components: Polycomb (Pc), RING1A or RING1B (dRING1 or Sex combs extra in Drosophila), Bmi-1 or Mel-18 (Posterior sex combs, Psc, in Drosophila), and Polyhomeotic (Ph) (Francis et al., 2001Francis N.J. Saurin A.J. Shao Z. Kingston R.E. Reconstitution of a functional core polycomb repressive complex.Mol. Cell. 2001; 8: 545-556Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, Shao et al., 1999Shao Z. Raible F. Mollaaghababa R. Guyon J.R. Wu C.T. Bender W. Kingston R.E. Stabilization of chromatin structure by PRC1, a Polycomb complex.Cell. 1999; 98: 37-46Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar). In vitro experiments suggest that PRC1 may mediate repression through chromatin compaction and the inhibition of chromatin remodeling enzymes (Francis et al., 2001Francis N.J. Saurin A.J. Shao Z. Kingston R.E. Reconstitution of a functional core polycomb repressive complex.Mol. Cell. 2001; 8: 545-556Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, Francis et al., 2004Francis N.J. Kingston R.E. Woodcock C.L. Chromatin compaction by a polycomb group protein complex.Science. 2004; 306: 1574-1577Crossref PubMed Scopus (576) Google Scholar, Shao et al., 1999Shao Z. Raible F. Mollaaghababa R. Guyon J.R. Wu C.T. Bender W. Kingston R.E. Stabilization of chromatin structure by PRC1, a Polycomb complex.Cell. 1999; 98: 37-46Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar). Alternatively, the ligation of ubiquitin to lysine 119 of histone H2A (Wang et al., 2004aWang 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 (1157) Google Scholar), which is catalyzed by a heterodimer formed by the RING finger domains of RING1B and Bmi-1 (Buchwald et al., 2006Buchwald G. van der Stoop P. Weichenrieder O. Perrakis A. van Lohuizen M. Sixma T.K. Structure and E3-ligase activity of the Ring-Ring complex of Polycomb proteins Bmi1 and Ring1b.EMBO J. 2006; 25: 2465-2474Crossref PubMed Scopus (315) Google Scholar, Li et al., 2006Li Z. Cao R. Wang M. Myers M.P. Zhang Y. Xu R.M. Structure of a BMI-1-ring1B polycomb group ubiquitin ligase complex.J. Biol. Chem. 2006; 281: 20643-20649Crossref PubMed Scopus (169) Google Scholar), may maintain RNA polymerase II in an inactive, but poised position at repressed genes (Stock et al., 2007Stock J.K. Giadrossi S. Casanova M. Brookes E. Vidal M. Koseki H. Brockdorff N. Fisher A.G. Pombo A. Ring1-mediated ubiquitination of H2A restrains poised RNA polymerase II at bivalent genes in mouse ES cells.Nat. Cell Biol. 2007; 9: 1428-1435Crossref PubMed Scopus (493) Google Scholar). A potential way for these two distinct repressive mechanisms of PRC1 could exist was recently suggested by the identification of a PRC1-like complex in Drosophila called dRAF (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 (188) Google Scholar). The dRAF complex is distinct from PRC1 but still contains two of the PRC1 core components (Psc and dRING1) and is a competent Ub ligase. Thus, it may be possible for the PRC1-like dRAF complex to function as a ubiquitin E3 ligase while PRC1 may function to create higher order repressive chromatin structures. What is largely unresolved with regards to PcG function are the precise mechanisms of targeting PcG complexes to hundreds of locations within genomes. PcG complexes bind to cis-regulatory DNA elements called Polycomb response elements (PREs). In Drosophila, a PcG protein called Pleiohomeotic (Pho), the only PcG protein capable of binding a specific DNA sequence, plays a key role in directing PRC1 to the PREs (Wang et al., 2004bWang L. Brown J.L. Cao R. Zhang Y. Kassis J.A. Jones R.S. Hierarchical recruitment of polycomb group silencing complexes.Mol. Cell. 2004; 14: 637-646Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). Pho can cooperate with PRC1 to bind PREs that are depleted of histones forming a repressive structure whereby the DNA is wrapped around the Pho/PRC1 complex (Mohd-Sarip et al., 2005Mohd-Sarip A. Cleard F. Mishra R.K. Karch F. Verrijzer C.P. Synergistic recognition of an epigenetic DNA element by Pleiohomeotic and a Polycomb core complex.Genes Dev. 2005; 19: 1755-1760Crossref PubMed Scopus (75) Google Scholar, Mohd-Sarip et al., 2006Mohd-Sarip A. van der Knaap J.A. Wyman C. Kanaar R. Schedl P. Verrijzer C.P. Architecture of a polycomb nucleoprotein complex.Mol. Cell. 2006; 24: 91-100Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Pho is also a component of a distinct PcG complex called PhoRC which includes the PcG protein dSfmbt (Klymenko et al., 2006Klymenko T. Papp B. Fischle W. Kocher T. Schelder M. Fritsch C. Wild B. Wilm M. Muller J. A Polycomb group protein complex with sequence-specific DNA-binding and selective methyl-lysine-binding activities.Genes Dev. 2006; 20: 1110-1122Crossref PubMed Scopus (286) Google Scholar). The methylated histone binding ability of the dSfmbt MBT domain (Grimm et al., 2009Grimm C. Matos R. Ly-Hartig N. Steuerwald U. Lindner D. Rybin V. Muller J. Muller C.W. Molecular recognition of histone lysine methylation by the Polycomb group repressor dSfmbt.EMBO J. 2009; 28: 1965-1977Crossref PubMed Scopus (61) Google Scholar, Klymenko et al., 2006Klymenko T. Papp B. Fischle W. Kocher T. Schelder M. Fritsch C. Wild B. Wilm M. Muller J. A Polycomb group protein complex with sequence-specific DNA-binding and selective methyl-lysine-binding activities.Genes Dev. 2006; 20: 1110-1122Crossref PubMed Scopus (286) Google Scholar) may allow PhoRC to utilize the combination of the DNA binding ability of Pho and methylated histone binding of dSfmbt to bind specifically to hundreds of sites within the Drosophila genome (Oktaba et al., 2008Oktaba K. Gutierrez L. Gagneur J. Girardot C. Sengupta A.K. Furlong E.E. Muller J. Dynamic regulation by polycomb group protein complexes controls pattern formation and the cell cycle in Drosophila.Dev. Cell. 2008; 15: 877-889Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). While Pho is clearly important for PRE PcG targeting, a PRE prediction algorithm using the DNA binding sequence of Pho and other PcG-associated proteins (Ringrose et al., 2003Ringrose L. Rehmsmeier M. Dura J.M. Paro R. Genome-wide prediction of Polycomb/Trithorax response elements in Drosophila melanogaster.Dev. Cell. 2003; 5: 759-771Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar) failed to detect most PcG binding sites identified from several Drosophila genome-wide studies (Negre et al., 2006Negre N. Hennetin J. Sun L.V. Lavrov S. Bellis M. White K.P. Cavalli G. Chromosomal distribution of PcG proteins during Drosophila development.PLoS Biol. 2006; 4: e170Crossref PubMed Scopus (196) Google Scholar, Schwartz et al., 2006Schwartz Y.B. Kahn T.G. Nix D.A. Li X.Y. Bourgon R. Biggin M. Pirrotta V. Genome-wide analysis of Polycomb targets in Drosophila melanogaster.Nat. Genet. 2006; 38: 700-705Crossref PubMed Scopus (454) Google Scholar, Tolhuis et al., 2006Tolhuis B. Muijrers I. de Wit E. Teunissen H. Talhout W. van Steensel B. van Lohuizen M. Genome-wide profiling of PRC1 and PRC2 Polycomb chromatin binding in Drosophila melanogaster.Nat. Genet. 2006; 38: 694-699Crossref PubMed Scopus (286) Google Scholar). Predicting PREs in vertebrate genomes have proved to be a challenging task and the identification of two mammalian PREs have only been recently reported (Sing et al., 2009Sing A. Pannell D. Karaiskakis A. Sturgeon K. Djabali M. Ellis J. Lipshitz H.D. Cordes S.P. A vertebrate Polycomb response element governs segmentation of the posterior hindbrain.Cell. 2009; 138: 885-897Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, Woo et al., 2010Woo C.J. Kharchenko P.V. Daheron L. Park P.J. Kingston R.E. A region of the human HOXD cluster that confers polycomb-group responsiveness.Cell. 2010; 140: 99-110Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). Like the PREs in Drosophila, the mammalian PREs contain DNA sequences that are recognized by the homolog of Pho, Yin Yang 1 (YY1). The vertebrate PRC1 component that likely plays a key role in directing PRC1 to the YY1 binding sites is RING1A or RING1B. The C-terminal domain of RING1A or RING1B (C-RING1A or C-RING1B) binds to the C-terminal region of an adaptor protein called RYBP (RING1 YY1 binding protein) while a nonoverlapping N-terminal portion of RYBP associates with YY1 (Garcia et al., 1999Garcia E. Marcos-Gutierrez C. del Mar Lorente M. Moreno J.C. Vidal M. RYBP, a new repressor protein that interacts with components of the mammalian Polycomb complex, and with the transcription factor YY1.EMBO J. 1999; 18: 3404-3418Crossref PubMed Scopus (180) Google Scholar). This type of protein-protein interaction would allow targeting of PRC1 to specific DNA sequences in the mammalian genome. The important role played by RYBP in PcG function is reflected in its requirement for repression mediated through the mammalian PRE (Woo et al., 2010Woo C.J. Kharchenko P.V. Daheron L. Park P.J. Kingston R.E. A region of the human HOXD cluster that confers polycomb-group responsiveness.Cell. 2010; 140: 99-110Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). RING1B also plays a role in an alternative mechanism of targeting PRC1 to chromatin. The posttranslational trimethylation of histone H3 at lysine 27 (H3K27Me3) is important in PRC1 targeting. The N-terminal chromo domain of Pc binds H3K27Me3 (Cao et al., 2002Cao R. Wang L. Wang H. Xia L. Erdjument-Bromage H. Tempst P. Jones R.S. Zhang Y. Role of histone H3 lysine 27 methylation in Polycomb-group silencing.Science. 2002; 298: 1039-1043Crossref PubMed Scopus (2572) 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 (752) Google Scholar, Min et al., 2003Min J. Zhang Y. Xu R.M. Structural basis for specific binding of Polycomb chromodomain to histone H3 methylated at Lys 27.Genes Dev. 2003; 17: 1823-1828Crossref PubMed Scopus (474) Google Scholar), providing one avenue by which PRC1 can be targeted to chromatin. The C-terminal cbox domain of Pc directly associates with C-RING1B (Schoorlemmer et al., 1997Schoorlemmer J. Marcos-Gutierrez C. Were F. Martinez R. Garcia E. Satijn D.P. Otte A.P. Vidal M. Ring1A is a transcriptional repressor that interacts with the Polycomb-M33 protein and is expressed at rhombomere boundaries in the mouse hindbrain.EMBO J. 1997; 16: 5930-5942Crossref PubMed Scopus (129) Google Scholar), facilitating assembly of PRC1 at the histone posttranslational modification. It should be noted that the chromo domain from other vertebrate Pc orthologs are capable of binding different histone methylations besides H3K27Me3 (Bernstein et al., 2006Bernstein E. Duncan E.M. Masui O. Gil J. Heard E. Allis C.D. Mouse polycomb proteins bind differentially to methylated histone H3 and RNA and are enriched in facultative heterochromatin.Mol. Cell. Biol. 2006; 26: 2560-2569Crossref PubMed Scopus (370) Google Scholar). For example, cbx7 can bind both H3K9Me3 as well as H3K27Me3. Because of this and perhaps of alternative targeting mechanisms for PRC1, there are many instances where RING1B is bound to chromatin at locations other than H3K27Me3. There are several examples of this phenomenon. (1) In preimplantation embryos, paternal heterochromatin that lacks H3K27Me3 can still bind maternal RING1B and other PRC1 components (Puschendorf et al., 2008Puschendorf M. Terranova R. Boutsma E. Mao X. Isono K. Brykczynska U. Kolb C. Otte A.P. Koseki H. Orkin S.H. et al.PRC1 and Suv39h specify parental asymmetry at constitutive heterochromatin in early mouse embryos.Nat. Genet. 2008; 40: 411-420Crossref PubMed Scopus (238) Google Scholar). (2) In embryonic stem cells that are deficient in the enzyme that methylates H3K27 and thus lack H3K27Me3, RING1B can still bind to the X chromosome (Schoeftner et al., 2006Schoeftner S. Sengupta A.K. Kubicek S. Mechtler K. Spahn L. Koseki H. Jenuwein T. Wutz A. Recruitment of PRC1 function at the initiation of X inactivation independent of PRC2 and silencing.EMBO J. 2006; 25: 3110-3122Crossref PubMed Scopus (295) Google Scholar). (3) In mouse embryonic stem cells, RING1B was found to bind to the promoter region of 244 genes (of the 1219 total genes that exhibit RING1B binding) where the H3K27Me3 was not detected (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 (1927) Google Scholar). While it is possible that RING1B is present at these sites but not detected, alternative recruitment mechanisms may also be in play. Moreover, the Drosophila ortholog dRING1 has been shown to localize to sites on polytene chromosomes that are independent of other PRC1 components (Gorfinkiel et al., 2004Gorfinkiel N. Fanti L. Melgar T. Garcia E. Pimpinelli S. Guerrero I. Vidal M. The Drosophila Polycomb group gene Sex combs extra encodes the ortholog of mammalian Ring1 proteins.Mech. Dev. 2004; 121: 449-462Crossref PubMed Scopus (32) Google Scholar). C-RING1B was predicted to have (Sanchez-Pulido et al., 2008Sanchez-Pulido L. Devos D. Sung Z.R. Calonje M. RAWUL: a new ubiquitin-like domain in PRC1 ring finger proteins that unveils putative plant and worm PRC1 orthologs.BMC Genomics. 2008; 9: 308Crossref PubMed Scopus (90) Google Scholar) and confirmed to possess a ubiquitin fold (Bezsonova et al., 2009Bezsonova I. Walker J.R. Bacik J.P. Duan S. Dhe-Paganon S. Arrowsmith C.H. Ring1B contains a ubiquitin-like docking module for interaction with Cbx proteins.Biochemistry. 2009; 48: 10542-10548Crossref PubMed Scopus (32) Google Scholar) but how it is capable of having multiple binding partners remains unknown. Here, we provide evidence suggesting how RING1B, and by extension, PRC1, can be specifically targeted to different sites in the genome. We show that two different binding partners to C-RING1B, the Pc cbox domain and RYBP, can be very different in their sequence and bind with different conformations to RING1B. Yet, despite these differences, these proteins bind to the same binding site on RING1B. This finding has important implications for the types of multiprotein PRC1-like complexes that can form in vivo and how they might be targeted to chromatin. In order to better understand the protein-protein interactions of C-RING1B, we first determined the structure of C-RING1B bound to a Pc cbox domain. Of the five vertebrate Pc orthologs, we chose the cbox domain from cbx7 for crystallization because of its strong affinity for C-RING1B (Kd = 9.2 nM) (Wang et al., 2008Wang R. Ilangovan U. Robinson A.K. Schirf V. Schwarz P.M. Lafer E.M. Demeler B. Hinck A.P. Kim C.A. Structural transitions of the RING1B C-terminal region upon binding the polycomb cbox domain.Biochemistry. 2008; 47: 8007-8015Crossref PubMed Scopus (16) Google Scholar). To facilitate crystallization, we used C-RING1B and cbx7 cbox residues (223–333 and 219–248, respectively) which showed the least amount of conformational flexibility when in complex with each other based on NMR relaxation measurements (Figures 1A and 1B ; see Figure S1 available online). We determined the crystal structure of the C-RING1B/cbx7 219–248 complex and refined it to 1.7 Å resolution (Figures 1C–1E; Table S1). In the crystal, there are two C-RING1B/cbx7 cbox complexes in the asymmetric unit associated via the interaction between each of their long central helices. Our mutagenesis data (Demeler et al., 2010Demeler B. Brookes E. Wang R. Schirf V. Kim C.A. Characterization of reversible associations by sedimentation velocity with ultrascan.Macromolec. Biosci. 2010; 10: 755-782Google Scholar) as well as the structure of C-RING1B alone (Bezsonova et al., 2009Bezsonova I. Walker J.R. Bacik J.P. Duan S. Dhe-Paganon S. Arrowsmith C.H. Ring1B contains a ubiquitin-like docking module for interaction with Cbx proteins.Biochemistry. 2009; 48: 10542-10548Crossref PubMed Scopus (32) Google Scholar) indicate that this interaction is the homodimerization interface of C-RING1B when C-RING1B is devoid of either the Pc cbox or RYBP. Because this interaction occurs only in the absence of a binding partner (Czypionka et al., 2007Czypionka A. de los Panos O.R. Mateu M.G. Barrera F.N. Hurtado-Gomez E. Gomez J. Vidal M. Neira J.L. The isolated C-terminal domain of Ring1B is a dimer made of stable, well-structured monomers.Biochemistry. 2007; 46: 12764-12776Crossref PubMed Scopus (44) Google Scholar, Wang et al., 2008Wang R. Ilangovan U. Robinson A.K. Schirf V. Schwarz P.M. Lafer E.M. Demeler B. Hinck A.P. Kim C.A. Structural transitions of the RING1B C-terminal region upon binding the polycomb cbox domain.Biochemistry. 2008; 47: 8007-8015Crossref PubMed Scopus (16) Google Scholar), the two C-RING1B molecules in the asymmetric unit are likely the result of the high concentration of protein required for crystallization and not reflective of what occurs inside cells. Our analysis of the structure is thus focused on a single heterodimer unit. The C-RING1B structure is composed of an extensive beta sheet region and a long central helix that extends from residue Val 256 to Glu 277. Residues 219–238 of the cbx7 cbox domain form an antiparallel beta sheet which makes an extended, intermolecular beta sheet with C-RING1B. The combined beta sheet structure packs against the C-RING1B central helix. In the cbox beta sheet (Figure 1D), the side-chain aromatic ring of cbx7 Phe 234 is most notable as it stacks against the side-chain aromatic ring of RING1B Tyr 262. Additionally, cbx7 beta sheet residues Thr 223, Ile 225, Ala 227, and Val 232 are clustered around Phe 234, packing against a hydrophobic pocket formed by RING1B residues Ile 248, Thr 250, Ala 254, His 258, Leu 259, Tyr 262, and Val 265. In addition to the backbone hydrogen bonds between C-RING1B and cbx7 that comprise the intermolecular beta sheet structure, there is a side-chain mediated polar interaction between cbx7 Glu 236 and RING1B residues Arg 246 and Tyr 262. Furthermore, cbx7 residues 239–248 form a loop structure that contacts C-RING1B (Figure 1E). In the cbox loop region, two consecutive cbx7 Phe residues, 243 and 244, pack into a hydrophobic pocket formed by RING1B residues Val 229, Tyr 247, and Pro 324. The side chains of RING1B residues Lys 249 and Glu 227 are in an extended conformation and are hydrogen bonded to the backbone atoms of the cbox loop residues. These extended conformations allow the methylene groups on these residues to help form the binding pocket for Phe 243 and 244. The guanidinium group of cbx7 Arg 247 forms a salt bridge interaction with the RING1B Glu 227 side chain while also hydrogen bonding to the carboxamide group of RING1B Gln 322. We next tested whether both the beta sheet and the loop structure of the cbox domain are necessary for binding. We introduced mutations into C-RING1B that alter either the beta sheet binding site (Tyr262Ala and His258Ala) or the cbox loop binding site (Val227Ala and Tyr247Ala) and then tested each of these mutant proteins for binding to the cbx7 cbox domain using a native gel binding assay (Figure 2A ). These mutations are of surface residues and do not disrupt the tertiary fold (Figure S2). The mutant C-RING1B proteins showed diminished ability to bind cbx7 cbox, as indicated by the significant amounts of residual unbound cbx7 cbox that was observed for all the mutants except for Val229Ala (compare the bands for faster migrating unbound cbx7 cbox in Figure 2A lane 7 for wild-type to the corresponding bands in lanes 8–11). We also performed the complementary experiment of testing the binding of cbx7 cbox proteins mutated at the beta sheet and loop regions. When cbx7 cbox was mutated in the beta sheet, Phe234Asp, there was no binding to C-RING1B (Figure 2B, lane 7). The cbx7 loop mutation Phe244Asp was found to be capable of associating with C-RING1B, but with reduced affinity, as indicated by the presence of a substantial quantity of unbound cbx7 protein (Figure 2B, compare the bands for cbx7 in lanes 6 and 8). We further tested the role of the cbox loop interaction with C-RING1B by deleting cbx7 loop residues altogether. Deleting the loop of cbx7 cbox completely disrupted binding (Figure 2B, lane 9). The results of these mutagenesis studies are consistent with our NMR data which showed that Pc cbox domains were unstructured in the absence of C-RING1B (Wang et al., 2008Wang R. Ilangovan U. Robinson A.K. Schirf V. Schwarz P.M. Lafer E.M. Demeler B. Hinck A.P. Kim C.A. Structural transitions of the RING1B C-terminal region upon binding the polycomb cbox domain.Biochemistry. 2008; 47: 8007-8015Crossref PubMed Scopus (16) Google Scholar). NMR conformational dynamics measurements revealed that upon binding C-RING1B, both the beta sheet and the loop structure of the cbx7 cbox domain become ordered (Figure S1A). Previously published transcription assays using recombinant Pc proteins targeted to reporter genes had shown that the cbox domain is required for repression (Bardos et al., 2000Bardos J.I. Saurin A.J. Tissot C. Duprez E. Freemont P.S. HPC3 is a new human polycomb orthologue that interacts and associates with RING1 and Bmi1 and has transcriptional repression properties.J. Biol. Chem. 2000; 275: 28785-28792Crossref PubMed Scopus (60) Google Scholar, Bunker and Kingston, 1994Bunker C.A. Kingston R.E. Transcriptional repression by Drosophila and mammalian Polycomb group proteins in transfected mammalian cells.Mol. Cell. Biol. 1994; 14: 1721-1732Crossref PubMed Scopus (111) Google Scholar, Muller, 1995Muller J. Transcriptional silencing by the Polycomb protein in Drosophila embryos.EMBO J. 1995; 14: 1209-1220Crossref PubMed Scopus (118) Google Scholar). We wondered whether specific, structure-guided point mutations within the cbox domain designed to disrupt binding to C-RING1B would hinder the ability of Pc to repress transcription. To this end, we used a transcription assay carried out in Drosophila S2 cells using the full-length Drosophila Pc (dPc) protein (Figure 2C). Using this assay, we found that the dPc that was targeted to an exogenous metallothionine promoter (MTp) was able to repress transcription of the luciferase reporter gene. In contrast, there was elevated luciferase activity with a dPc that was similarly expressed but not targeted to the MTp (Figure 2D). We also introduced mutations into dPc cbox that are equivalent to the cbx7 cbox mutations that hinder cbx7 cbox binding to RING1B (discussed above). The cbox beta sheet mutation Ile367Ala (equivalent to cbx7 Phe 234) and the loop residue mutant Phe377Ala (Phe 244 in cbx7) were both found to be incapable of repressing the expression of the reporter gene as compared to wild-type. Taking all of the in vitro and in vivo results together, we conclude that both the cbox beta sheet and loop structures are required to form a stable complex with C-RING1B, which in turn is required for full transcription repression. Evidence indicates a strong likelihood that RING1B targeting is dependent on the identity of the binding partner(s) of C-RING1B. For example, multiprotein complexes that include RING1B and RYBP (or its homolog YAF2) also contain proteins like E2F6, E2F2, E2F3, hGABPβ, and YY1, all of which have DNA binding domains (Garcia et al., 1999Garcia E. Marcos-Gutierrez C. del Mar Lorente M. Moreno J.C. Vidal M. RYBP, a new repressor protein that interacts with components of the mammalian Polycomb complex, and with the transcription factor YY1.EMBO J. 1999; 18: 3404-3418Crossref PubMed Scopus (180) Google Scholar, Ogawa et al., 2002Ogawa H. Ishiguro K. Gaubatz S. Livingston D.M. Nakatani Y. A complex with chromatin modifiers that occupies E2F- and Myc-responsive genes in G0 cells.Science. 2002; 296: 1132-1136Crossref PubMed Scopus (596) Google Scholar, Sawa et al., 2002Sawa C. Yoshikawa T. Matsuda-Suzuki F. Delehouzee S. Goto M. Watanabe H. Sawada J. Kataoka K. Handa H. YEAF1/RYBP and YAF-2 are functionally distinct members of a cofactor family for the YY1 and E4TF1/hGABP transcription factors.J. Biol. Chem. 2002; 277: 22484-22490Crossref PubMed Scopus (40) Google Scholar, Schlisio et al., 2002Schlisio S. Halperin T. Vidal M. Nevins J.R. Interaction of YY1 with E2Fs, mediated by RYBP, provides a mechanism for specificity of E2F function.EMBO J. 2002; 21: 5775-5786Crossref PubMed Scopus (170) Google Scholar, Trimarchi et al., 2001Trimarchi J.M. Fairchild B. Wen J. Lees J.A. The E2F6 transcription factor is a component of the mammalian Bmi1-containing polycomb complex.Proc. Natl. Acad. Sci. USA. 2001; 98: 1519-1524Crossref PubMed Scopus (211) Google Scholar, Zheng et al., 2001Zheng L. Schickling O. Peter M.E. Lenardo M.J. The death effector domain-associated factor plays distinct regulatory roles in the nucleus and cytoplasm.J. Biol. Chem. 2001; 276: 31945-31952Crossref PubMed Scopus (71) Google Scholar). If the binding target of RING1B is a specific DNA sequence and not methylated chromatin, then C-RING1B would have to bind RYBP with 1:1 stoichiometry while not allowing C-RING1B to bind the Pc cbox domains. If the stoichiometry of the interactions is greater than 1:1 utilizing multiple binding surfaces on C-RING1B, then a single RING1B protein could simultaneously bind to two different sites. Since there is little sequence similarity between RYBP and the Pc cbox domains (Figure 1A), it is difficult to predict how RYBP binds to C-RING1B. We performed a series of" @default.
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• W2000672112 date "2010-08-01" @default.
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• W2000672112 title "Polycomb Group Targeting through Different Binding Partners of RING1B C-Terminal Domain" @default.
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