Recognition of UbcH5c and the nucleosome by the Bmi1/Ring1b ubiquitin ligase complex

The Polycomb repressive complex 1 (PRC1) mediates gene silencing, in part by monoubiquitination of histone H2A on lysine 119 (uH2A). Bmi1 and Ring1b are critical components of PRC1 that heterodimerize via their N-terminal RING domains to form an active E3 ubiquitin ligase. We have determined the crystal structure of a complex between the Bmi1/Ring1b RING-RING heterodimer and the E2 enzyme UbcH5c and find that UbcH5c interacts with Ring1b only, in a manner fairly typical of E2-E3 interactions. However, we further show that the Bmi1/Ring1b RING domains bind directly to duplex DNA through a basic surface patch unique to the Bmi1/Ring1b RING-RING dimer. Mutation of residues on this interaction surface leads to a loss of H2A ubiquitination activity. Computational modelling of the interface between Bmi1/Ring1b-UbcH5c and the nucleosome suggests that Bmi1/Ring1b interacts with both nucleosomal DNA and an acidic patch on histone H4 to achieve specific monoubiquitination of H2A. Our results point to a novel mechanism of substrate recognition, and control of product formation, by Bmi1/Ring1b.     

Structure of a Bmi-1-Ring1B polycomb group ubiquitin ligase complex

Polycomb group proteins Bmi-1 and Ring1B are core subunits of the PRC1 complex, which plays important roles in the regulation of Hox gene expression, X-chromosome inactivation, tumorigenesis, and stem cell self-renewal. The RING finger protein Ring1B is an E3 ligase that participates in the ubiquitination of lysine 119 of histone H2A, and the binding of Bmi-1 stimulates the E3 ligase activity. We have mapped the regions of Bmi-1 and Ring1B required for efficient ubiquitin transfer and determined a 2.5-A structure of the Bmi-1-Ring1B core domain complex. The structure reveals that Ring1B "hugs" Bmi-1 through extensive RING domain contacts and its N-terminal tail wraps around Bmi-1. The two regions of interaction have a synergistic effect on the E3 ligase activity. Our analyses suggest a model where the Bmi-1-Ring1B complex stabilizes the interaction between the E2 enzyme and the nucleosomal substrate to allow efficient ubiquitin transfer.     

Structure and E3-ligase activity of the Ring-Ring complex of polycomb proteins Bmi1 and Ring1b

Polycomb group proteins Ring1b and Bmi1 (B-cell-specific Moloney murine leukaemia virus integration site 1) are critical components of the chromatin modulating PRC1 complex. Histone H2A ubiquitination by the PRC1 complex strongly depends on the Ring1b protein. Here we show that the E3-ligase activity of Ring1b on histone H2A is enhanced by Bmi1 in vitro. The N-terminal Ring-domains are sufficient for this activity and Ring1a can replace Ring1b. E2 enzymes UbcH5a, b, c or UbcH6 support this activity with varying processivity and selectivity. All four E2s promote autoubiquitination of Ring1b without affecting E3-ligase activity. We solved the crystal structure of the Ring-Ring heterodimeric complex of Ring1b and Bmi1. In the structure the arrangement of the Ring-domains is similar to another H2A E3 ligase, the BRCA1/BARD1 complex, but complex formation depends on an N-terminal arm of Ring1b that embraces the Bmi1 Ring-domain. Mutation of a critical residue in the E2/E3 interface shows that catalytic activity resides in Ring1b and not in Bmi1. These data provide a foundation for understanding the critical enzymatic activity at the core of the PRC1 polycomb complex, which is implicated in stem cell maintenance and cancer.     

The Polycomb protein and E3 ubiquitin ligase Ring1B harbors an IRES in its highly conserved 5' UTR

Ring1B is an essential member of the highly conserved Polycomb group proteins, which orchestrate developmental processes, cell growth and stem cell fate by modifying local chromatin structure. Ring1B was found to be the E3 ligase that monoubiquitinates histone H2A, which adds a new level of chromatin modification to Polycomb group proteins. Here we report that Ring1B belongs to the exclusive group of proteins that for their translation depend on a stable 5' UTR sequence in their mRNA known as an Internal Ribosome Entry Site (IRES). In cell transfection assays the Ring1B IRES confers significantly higher expression levels of Ring1B than a Ring1B cDNA without the IRES. Also, dual luciferase assays show strong activity of the Ring1B IRES. Although our findings indicate Ring1B can be translated under conditions where cap-dependent translation is impaired, we found the Ring1B IRES to be cap-dependent. This raises the possibility that translational control of Ring1B is a multi-layered process and that translation of Ring1B needs to be maintained under varying conditions, which is in line with its essential role as an E3 ligase for monoubiquitination of histone H2A in the PRC1 Polycomb protein complex.     

A phosphorylated form of Mel-18 targets the Ring1B histone H2A ubiquitin ligase to chromatin

Recent studies have shown that PRC1-like Polycomb repressor complexes monoubiquity-late chromatin on histone H2A at lysine residue 119. Here we have analyzed the function of the polycomb protein Mel-18. Using affinity-tagged human MEL-18, we identify a polycomb-like complex, melPRC1, containing the core PRC1 proteins, RING1/2, HPH2, and CBX8. We show that, in ES cells, melPRC1 can functionally substitute for other PRC1-like complexes in Hox gene repression. A reconstituted subcomplex containing only Ring1B and Mel-18 functions as an efficient ubiquitin E3 ligase. This complex ubiquitylates free histone substrates nonspecifically but is highly specific for histone H2A lysine 119 in the context of nucleosomes. Mutational analysis demonstrates that while Ring1B is required for E3 function, Mel-18 directs this activity to H2A lysine 119 in chromatin. Moreover, this substrate-targeting function of Mel-18 is dependent on its prior phosphorylation at multiple residues, providing a direct link between chromatin modification and cell signaling pathways.     

Enhancement of BRCA1 E3 ubiquitin ligase activity through direct interaction with the BARD1 protein

The breast and ovarian cancer-specific tumor suppressor RING finger protein BRCA1 has been identified as an E3 ubiquitin (Ub) ligase through in vitro studies, which demonstrated that its RING finger domain can autoubiquitylate and monoubiquitylate histone H2A when supplied with Ub, E1, and UBC4 (E2). Here we report that the E3 ligase activity of the N-terminal 110 amino acid residues of BRCA1, which encodes a stable domain containing the RING finger, as well as that of the full-length BRCA1, was significantly enhanced by the BARD1 protein (residues 8-142), whose RING finger domain itself lacked Ub ligase activity in vitro. The results of mutagenesis studies indicate that the enhancement of BRCA1 E3 ligase activity by BARD1 depends on direct interaction between the two proteins. Using K48A and K63A Ub mutants, we found that BARD1 stimulated the formation of both Lys(48)- and Lys(63)-linked poly-Ub chains. However, the enhancement of BRCA1 autoubiquitylation by BARD1 mostly resulted in poly-Ub chains linked through Lys(63), which could potentially activate biological pathways other than BRCA1 degradation. We also found that co-expression of BRCA1 and BARD1 in living cells increased the abundance and stability of both proteins and that this depended on their ability to heterodimerize.     

Regulation of the Polycomb protein RING1B ubiquitination by USP7

The E3 ubiquitin ligase RING1B plays an important role in Polycomb-mediated gene silencing by monoubiquitinating histone H2A. Both the activity and stability of RING1B are controlled by ubiquitination in two distinct manners. Self ubiquitination of RING1B generates K6, K27 and K48-based mixed polyubiquitin chain, and is required for its activity as a ligase. On the other hand, its proteasomal degradation is mediated by another ligase; E6-AP catalyzes the formation of K48-based chains. Since these two modes of ubiquitination target the same lysine residues and are therefore mutually exclusive, an important mode of regulation of RING1B should be at the level of deubiquitination. Here we identify USP7 as a deubiquitinating enzyme that regulates the ubiquitination state of RING1B. RING1B interacts with USP7, which is mediated in part by its RING domain. In addition, USP7 was found in a complex with other Polycomb proteins, suggesting a broad role in regulating these complexes. Although, USP7 directly and specifically deubiquitinates RING1B in vitro and in vivo, it does not discriminate between the activating and proteolysis-targeting modes of ubiquitination, and therefore has a stabilizing effect on RING1B.     

Switches, excitable responses and oscillations in the Ring1B/Bmi1 ubiquitination system

In an active, self-ubiquitinated state, the Ring1B ligase monoubiquitinates histone H2A playing a critical role in Polycomb-mediated gene silencing. Following ubiquitination by external ligases, Ring1B is targeted for proteosomal degradation. Using biochemical data and computational modeling, we show that the Ring1B ligase can exhibit abrupt switches, overshoot transitions and self-perpetuating oscillations between its distinct ubiquitination and activity states. These different Ring1B states display canonical or multiply branched, atypical polyubiquitin chains and involve association with the Polycomb-group protein Bmi1. Bistable switches and oscillations may lead to all-or-none histone H2A monoubiquitination rates and result in discrete periods of gene (in)activity. Switches, overshoots and oscillations in Ring1B catalytic activity and proteosomal degradation are controlled by the abundances of Bmi1 and Ring1B, and the activities and abundances of external ligases and deubiquitinases, such as E6-AP and USP7.     

Kaposi's sarcoma-associated herpesvirus LANA-1 interacts with the short variant of BRD4 and releases cells from a BRD4- and BRD2/RING3-induced G1 cell cycle arrest

The Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen 1 (LANA-1) is required for the replication of episomal viral genomes. Regions in its N-terminal and C-terminal domains mediate the interaction with host cell chromatin. Several cellular nuclear proteins, e.g., BRD2/RING3, histones H2A and H2B, MeCP2, DEK, and HP1alpha, have been suggested to mediate this interaction. In this work, we identify the double-bromodomain proteins BRD4 and BRD3/ORFX as additional LANA-1 interaction partners. The carboxy-terminal region of the short variant of BRD4 (BRD4S) containing the highly conserved extraterminal domain directly interacts with an element in the LANA-1 carboxy-terminal domain. We show that ectopically expressed BRD4S and BRD2/RING3 delay progression into the S phase of the cell cycle in epithelial and B-cell lines and increase cyclin E promoter activity. LANA-1 partly releases epithelial and B cells from a BRD4S- and BRD2/RING3-induced G1 cell cycle arrest and also promotes S-phase entry in the presence of BRD4S and BRD2/RING3. This is accompanied by a reduction in BRD4S-mediated cyclin E promoter activity. Our data are in keeping with the notion that the direct interaction of KSHV LANA-1 with BRD4 and other BRD proteins could play a role in the G1/S phase-promoting functions of KSHV LANA-1. Further, our data support a model in which the LANA-1 C terminus contributes to a functional attachment to acetylated histones H3 and H4 via BRD4 and BRD2, in addition to the recently demonstrated direct interaction (A. J. Barbera, J. V. Chodaparambil, B. Kelley-Clarke, V. Joukov, J. C. Walter, K. Luger, and K. M. Kaye, Science 311:856-861, 2006) of the LANA-1 N terminus with histones H2A and H2B.     

RYBP represses endogenous retroviruses and preimplantation- and germ line-specific genes in mouse embryonic stem cells

Polycomb repressive complexes (PRCs) are important chromatin regulators of embryonic stem (ES) cell function. RYBP binds Polycomb H2A monoubiquitin ligases Ring1A and Ring1B and has been suggested to assist PRC localization to their targets. Moreover, constitutive inactivation of RYBP precludes ES cell formation. Using ES cells conditionally deficient in RYBP, we found that RYBP is not required for maintenance of the ES cell state, although mutant cells differentiate abnormally. Genome-wide chromatin association studies showed RYBP binding to promoters of Polycomb targets, although its presence is dispensable for gene repression. We discovered, using Eed-knockout (KO) ES cells, that RYBP binding to promoters was independent of H3K27me3. However, recruiting of PRC1 subunits Ring1B and Mel18 to their targets was not altered in the absence of RYBP. In contrast, we have found that RYBP efficiently represses endogenous retroviruses (murine endogenous retrovirus [MuERV] class) and preimplantation (including zygotic genome activation stage)- and germ line-specific genes. These observations support a selective repressor activity for RYBP that is dispensable for Polycomb function in the ES cell state. Also, they suggest a role for RYBP in epigenetic resetting during preimplantation development through repression of germ line genes and PcG targets before formation of pluripotent epiblast cells.     

The RING finger protein RNF8 recruits UBC13 for lysine 63-based self polyubiquitylation

The heterodimeric ubiquitin conjugating enzyme (E2) UBC13-UEV mediates polyubiquitylation through lysine 63 of ubiquitin (K63), rather than lysine 48 (K48). This modification does not target proteins for proteasome-dependent degradation. Searching for potential regulators of this variant polyubiquitylation we have identified four proteins, namely RNF8, KIA00675, KF1, and ZNRF2, that interact with UBC13 through their RING finger domains. These domains can recruit, in addition to UBC13, other E2s that mediate canonical (K48) polyubiquitylation. None of these RING finger proteins were known previously to recruit UBC13. For one of these proteins, RNF8, we show its activity as a ubiquitin ligase that elongates chains through either K48 or K63 of ubiquitin, and its nuclear co-localization with UBC13. Thus, our screening reveals new potential regulators of non-canonical polyubiquitylation.     

The ubiquitin-conjugating enzymes UbcH7 and UbcH8 interact with RING finger/IBR motif-containing domains of HHARI and H7-AP1.

Ubiquitinylation of proteins appears to be mediated by the specific interplay between ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s). However, cognate E3s and/or substrate proteins have been identified for only a few E2s. To identify proteins that can interact with the human E2 UbcH7, a yeast two-hybrid screen was performed. Two proteins were identified and termed human homologue of Drosophila ariadne (HHARI) and UbcH7-associated protein (H7-AP1). Both proteins, which are widely expressed, are characterized by the presence of RING finger and in between RING fingers (IBR) domains. No other overt structural similarity was observed between the two proteins. In vitro binding studies revealed that an N-terminal RING finger motif (HHARI) and the IBR domain (HHARI and H7-AP1) are involved in the interaction of these proteins with UbcH7. Furthermore, binding of these two proteins to UbcH7 is specific insofar that both HHARI and H7-AP1 can bind to the closely related E2, UbcH8, but not to the unrelated E2s UbcH5 and UbcH1. Although it is not clear at present whether HHARI and H7-AP1 serve, for instance, as substrates for UbcH7 or represent proteins with E3 activity, our data suggests that a subset of RING finger/IBR proteins are functionally linked to the ubiquitin/proteasome pathway.     

Bmi1 cooperates with Dnmt1-associated protein 1 in gene silencing

Polycomb group (PcG) proteins are involved in gene silencing through chromatin modifications. Among polycomb repressive complexes (PRCs), PRC1 exhibits H2A-K119 ubiquitin E3 ligase activity. However, the molecular mechanisms underlying PRC1-mediated gene silencing remain largely obscure. In this study, we found that Bmi1 directly interacts with Dnmt-associated protein 1 (Dmap1), which has been characterized to associate with the maintenance DNA methyltransferase, Dnmt1. Bmi1 was demonstrated to form a ternary complex with Dmap1 and Dnmt1 with Dmap1 in the central position. Chromatin immunoprecipitations confirmed the ternary complex formation within the context of the PRC1 at the Bmi1 target loci. Loss of Dmap1 binding to the Bmi1 target loci was tightly associated with derepressed gene expression in Bmi1-/- cells. Dmap1 knockdown exhibited the same impact as Bmi1 knockout did on the expression of Bmi1 targets, including Hox genes. Collectively, our findings suggest that Bmi1 incorporates Dmap1 in polycomb gene silencing.     

The PP4R1 subunit of protein phosphatase PP4 targets TRAF2 and TRAF6 to mediate inhibition of NF-κB activation

TRAFs constitute a family of proteins that have been implicated in signal transduction by immunomodulatory cellular receptors and viral proteins. TRAF2 and TRAF6 have an E3-ubiquitin ligase activity, which is dependent on the integrity of their RING finger domain and it has been associated with their ability to activate the NF-κB and AP1 signaling pathways. A yeast two-hybrid screen with TRAF2 as bait, identified the regulatory subunit PP4R1 of protein phosphatase PP4 as a TRAF2-interacting protein. The interaction of TRAF2 with PP4R1 depended on the integrity of the RING finger domain of TRAF2. PP4R1 could interact also with the TRAF2-related factor TRAF6 in a RING domain-dependent manner. Exogenous expression of PP4R1 inhibited NF-κB activation by TRAF2, TRAF6, TNF and the Epstein–Barr virus oncoprotein LMP1. In addition, expression of PP4R1 downregulated IL8 induction by LMP1, whereas downregulation of PP4R1 by RNA interference enhanced the induction of IL8 by LMP1 and TNF. PP4R1 could mediate the dephosphorylation of TRAF2 Ser11, which has been previously implicated in TRAF2-mediated activation of NF-κB. Finally, PP4R1 could inhibit TRAF6 polyubiquitination, suggesting an interference with the E3 ubiquitin ligase activity of TRAF6. Taken together, our data identify a novel mechanism of NF-κB pathway inhibition which is mediated by PP4R1-dependent targeting of specific TRAF molecules.