The murine Polycomb homolog M33 is implicated in mesoderm patterning in the mouse; it acts as a transcriptional repressor in transiently transfected cells. Two murine proteins, Ring1A and Ring1B, have been identified that interact directly with the repressor domain of M33. Ring1A and Ring1B display blocks of similarity throughout their sequences, including an N-terminal RING finger domain. However, the interaction with M33 occurs through a region at the C-terminus. Ring1A represses transcription through sequences not involved in M33 binding. Ring1A protein co-localizes in nuclear domains with M33 and other Pc-G homologs, such as Bmi1. The expression of Ring1A at early stages of development is restricted to the neural tube, whereas M33 is expressed ubiquitously. Within the neural tube, Ring1A RNA is located at the rhombomere boundaries of the hindbrain. Taken together, these data suggest that Ring1A may contribute to a tissue-specific function of Pc-G-protein complexes during mammalian development (Schoorlemmer, 1997).
In a two-hybrid screen with a vertebrate Polycomb homolog as a target (Xenopus Pc), the human RING1 protein was identified as interacting with Pc. RING1 is a protein that contains the RING finger motif, a specific zinc-binding domain, which is found in many regulatory proteins. So far, the function of the RING1 protein has remained enigmatic. RING1 coimmunoprecipitates with a human Pc homolog, the vertebrate PcG protein BMI1, and HPH1, a human homolog of the PcG protein Polyhomeotic (Ph). The human polycomb homolog, (hPc2) shows an overall identity of 70% with XPc but a mere 24% identity with M33, a murine Pc homolog closely related to hPc1. Interaction between RING1 and XPc does not involve the RING finger motif. RING1 colocalizes with the vertebrate PcG proteins in nuclear domains of SW480 human colorectal adenocarcinoma and Saos-2 human osteosarcoma cells. RING1, like Pc, is able to repress gene activity when targeted to a reporter gene. These findings indicate that RING1 is associated with the human PcG protein complex and that RING1, like PcG proteins, can act as a transcriptional repressor. It is possible that RING1 is a vertebrate homolog of the product of a Drosophila PcG gene that has not yet been characterized. No Drosophila RING1 homolog has yet been described (Satijn, 1997).
Polycomb-group (PcG) proteins form large multimeric protein complexes that are involved in maintaining the transcriptionally repressive state of genes. RING1 interacts with vertebrate Polycomb (Pc) homologs and is associated with or is part of a human PcG complex. However, very little is known about the role of RING1 as a component of the PcG complex. A detailed characterization of RING1 protein-protein interactions has been undertaken. By using directed two-hybrid and in vitro protein-protein analyses, it has been demonstrated that RING1, in addition to interacting with the human Pc homolog HPC2, can also interact with itself and with the vertebrate PcG protein BMI1. Distinct domains in the RING1 protein are involved in the self-association and in the interaction with BMI1. Further, the BMI1 protein can also interact with itself. To better understand the role of RING1 in regulating gene expression, the protein was overexpressed in mammalian cells and differences in gene expression levels were analyzed. This analysis shows that overexpression of RING1 strongly represses En-2, a mammalian homolog of the well-characterized Drosophila PcG target gene engrailed. Furthermore, RING1 overexpression results in enhanced expression of the proto-oncogenes c-jun and c-fos. The changes in expression levels of these proto-oncogenes are accompanied by cellular transformation, as judged by anchorage-independent growth and the induction of tumors in athymic mice. These data demonstrate that RING1 interacts with multiple human PcG proteins, indicating an important role for RING1 in the PcG complex. Further, deregulation of RING1 expression leads to oncogenic transformation by deregulation of the expression levels of certain oncogenes (Satijn, 1999).
Experimentally-induced mutations in the C3HC4 RING finger domain of the Bmi-1 oncoprotein block its ability to induce lymphomas in mice. In this report, the role of the Bmi-1 RING finger in mediating protein-protein interactions is examined using the yeast two-hybrid system. Bmi-1 interacts directly with the RING finger protein dinG/RING1B. Heterodimerization of the two proteins requires the intact RING finger structures of both Bmi-1 and dinG. Although the RING finger domains are necessary for dimerization, they are not sufficient for this process since residues outside the C3HC4 motif are also required. Thus, binding specificity may be partly conferred by residues outside the RING motif. Both Bmi-1 and dinG interact with the Polyhomeotic protein MPh2 through binding domains apart from the RING finger. The data suggest a model whereby Bmi-1, dinG, and MPh2 form a stable heterotrimeric complex in which each protein contributes to the binding of the others (Hemerway, 1998).
Polycomb group (PcG) proteins were first described in Drosophila as factors responsible for maintaining the transcriptionally repressed state of Hox/homeotic genes in a stable and heritable manner throughout development. A growing number of vertebrate genes related to the Drosophila PcG proteins have been identified, including two Polycomb orthologues, Pc2 and M33. PcG proteins form multiprotein complexes, termed PcG bodies, that are thought to repress transcription by altering chromatin structure. HPC3 (human Polycomb 3), a novel PcG protein, was isolated in a yeast two-hybrid screen using human RING1 as bait. HPC3 shows strong sequence similarity to Drosophila Pc and also to vertebrate Pc2 and M33, particularly within the chromodomain and C-box. M33 and human Pc2 (HPC2) can interact with RING1, and HPC3 also binds to RING1. This interaction is dependent upon the HPC3 C-box but, only partially on the RING finger of RING1. In contrast to HPC2, HPC3 interactions with RING1 are only observed in vivo with covalently modified forms of RING1. HPC3 also colocalizes with other PcG proteins in human PcG bodies. Consistent with its role as a PcG member, HPC3 is able to act as a long range transcriptional silencer when targeted to a reporter gene by a heterologous DNA-binding domain. Taken together, these data suggest that HPC3 is part of a large multiprotein complex that also contains other PcG proteins and is involved in repression of transcriptional activity (Bardos, 2000).
In contrast to cancer cells and embryonic stem cells, the lifespan of primary human cells is finite. After a defined number of population doublings, cells enter in an irreversible growth-arrested state termed replicative senescence. Mutations of genes involved in immortalization can contribute to cancer. In a genetic screen for cDNAs bypassing replicative senescence of normal human prostate epithelial cells (HPrEC), CBX7 (Chromobox homolog 7) was identifed. CBX7 encodes a Polycomb protein, as shown by sequence homology, its interaction with Ring1 and its localization to nuclear Polycomb bodies. CBX7 extends the lifespan of a wide range of normal human cells and immortalizes mouse fibroblasts by downregulating expression of the Ink4a/Arf locus. CBX7 does not inter-function or colocalize with Bmi1, and both can exert their actions independently of each other as shown by reverse genetics. CBX7 expression is downregulated during replicative senescence and its ablation by short-hairpin RNA (shRNA) treatment inhibits growth of normal cells though induction of the Ink4a/Arf locus. Taken together, these data show that CBX7 controls cellular lifespan through regulation of both the p16(Ink4a)/Rb and the Arf/p53 pathways (Gil, 2004).
The Polycomb group (PcG) of proteins represses homeotic gene expression through the assembly of multiprotein complexes on key regulatory elements. The mechanisms mediating complex assembly have remained enigmatic since most PcG proteins fail to bind DNA. The human PcG protein dinG interacts with CP2, a mammalian member of the grainyhead-like family of transcription factors, in vitro and in vivo. The functional consequence of this interaction is repression of CP2-dependent transcription. The CP2-dinG interaction is conserved in evolution with the Drosophila factor Grainyhead binding to dring, the fly homolog of dinG. Electrophoretic mobility shift assays demonstrate that the Grh-dring complex forms on regulatory elements of genes whose expression is repressed by Grh but not on elements where Grh plays an activator role. These observations reveal a novel mechanism by which PcG proteins may be anchored to specific regulatory elements in developmental genes (Tuckfield, 2002).
The DNA-binding protein recombination signal binding protein-Jkappa (RBP-J) mediates transcriptional activation of the Notch intracellular domain (NIC). In the absence of transcriptional activators, RBP-J suppresses transcription by recruiting co-suppressors. KyoT2 is a LIM domain protein that inhibits the RBP-J-mediated transcriptional activation. Evidence is provided that the polycomb group protein RING1 interacts with the LIM domains of KyoT2 in yeast and mammalian cells. The interaction between KyoT2 and RING1 was detected both in vitro and in vivo. By using a co-immunoprecipitation assay, it was also shown that, though RING1 and RBP-J do not associate directly, the two molecules can be co-precipitated simultaneously by KyoT2, probably through the LIM domains and the RBP-J-binding motif of KyoT2, respectively. These results suggested the formation of a three-molecule complex consisting of RBP-J, KyoT2 and RING1 in cells. Moreover, overexpression of RING1 together with KyoT2 in cells inhibits transactivation of RBP-J by NIC. Suppression of the NIC- mediated transactivation of RBP-J by RING1 is abrogated by overexpression of KBP1, a molecule that competes with RING1 for binding to LIM domains of KyoT2, suggesting that suppression of RBP-J by RING1 is dependent on its associating with KyoT2. Taken together, these data suggested that there might be at least two ways of the KyoT2-mediated suppression of RBP-J: competition for binding sites with transactivators, and recruitment of suppressors such as RING1 (Qin, 2004).
Histone methylation is a posttranslational modification regulating chromatin structure and gene regulation. BHC110/LSD1 has been described as a histone demethylase that reverses dimethyl histone H3 lysine 4 (H3K4). This study shows that JARID1d, a JmjC-domain-containing protein, specifically demethylates trimethyl H3K4. Detailed mapping analysis revealed that besides the JmjC domain, the BRIGHT and zinc-finger-like C5HC2 domains are required for maximum catalytic activity. Importantly, isolation of native JARID1d complexes from human cells revealed the association of the demethylase with a polycomb-like protein Ring6a/MBLR. Ring6a/MBLR not only directly interacts with JARID1d but also regulates its enzymatic activity. JARID1d and Ring6a occupy human Engrailed 2 gene and regulate its expression and H3K4 methylation levels. Depletion of JARID1d enhances recruitment of the chromatin remodeling complex, NURF, and the basal transcription machinery near the transcriptional start site, revealing a role for JARID1d in regulation of transcriptional initiation through H3K4 demethylation (Lee, 2007).
Biochemical and molecular evidence suggests that the mouse Ring1A gene is a member of the PcG of genes. However, genetic evidence is needed to establish PcG function for Ring1A. To study Ring1A function a mouse line lacking Ring1A and mouse lines overexpressing Ring1A were generated. Both Ring1A(-/-)and Ring1A(+/-) mice show anterior transformations and other abnormalities of the axial skeleton, which indicates an unusual sensitivity of axial skeleton patterning to Ring1A gene dosage. Ectopic expression of Ring1A also results in dose-dependent anterior transformations of vertebral identity, many of which, interestingly, are shared by Ring1A(-/-) mice. In contrast, the alterations of Hox gene expression observed in both type of mutant mice are subtle and involve a reduced number of Hox genes. Taken together, these results provide genetic evidence for a PcG function of the mouse Ring1A gene (del Mar Lorente, 2000).
The products of the Polycomb group of genes form complexes that maintain the state of transcriptional repression of several genes with relevance to development and in cell proliferation. Ring1B, the product of the Ring1B gene (Rnf2 - Mouse Genome Informatics), has been identified by means of its interaction with the Polycomb group protein Mel18. Biochemical and genetic studies are described that were directed to understanding the biological role of Ring1B. Immunoprecipitation studies indicate that Ring1B forms part of protein complexes containing the products of other Polycomb group genes, such as Rae28/Mph1 and M33, and that this complex associates with chromosomal DNA. A mouse line bearing a hypomorphic Ring1B allele was generated that showed posterior homeotic transformations of the axial skeleton and a mild derepression of some Hox genes (Hoxb4, Hoxb6 and Hoxb8) in cells anterior to their normal boundaries of expression in the mesodermal compartment. By contrast, the overexpression of Ring1B in chick embryos results in the repression of Hoxb9 expression in the neural tube. These results, together with the genetic interactions observed in compound Ring1B/Mel18 mutant mice, are consistent with a role for Ring1B in the regulation of Hox gene expression by Polycomb group complexes (Suzuki, 2002).
The highly homologous Rnf2 (Ring1b) and Ring1 (Ring1a) proteins were identified as in vivo interactors of the Polycomb Group (PcG) protein Bmi1. Functional ablation of Rnf2 results in gastrulation arrest, in contrast to relatively mild phenotypes in most other PcG gene null mutants belonging to the same functional group, among which is Ring1. Developmental defects occur in both embryonic and extraembryonic tissues during gastrulation. The early lethal phenotype is reminiscent of that of the PcG-gene knockouts Eed and Ezh2, which belong to a separate functional PcG group and PcG protein complex. This finding indicates that these biochemically distinct PcG complexes are both required during early mouse development. In contrast to the strong skeletal transformation in Ring1 hemizygous mice, hemizygocity for Rnf2 does not affect vertebral identity. However, it does aggravate the cerebellar phenotype in a Bmi1 null-mutant background. Together, these results suggest that Rnf2 or Ring1-containing PcG complexes have minimal functional redundancy in specific tissues, despite overlap in expression patterns. The early developmental arrest in Rnf2-null embryos is partially bypassed by genetic inactivation of the Cdkn2a (Ink4aARF) locus. Importantly, this finding implicates Polycomb-mediated repression of the Cdkn2a locus in early murine development (Voncken, 2003).
In many higher organisms, 5%-15% of histone H2A is ubiquitylated at lysine 119 (uH2A). The function of this modification and the factors involved in its establishment, however, are unknown. This study demonstrates that uH2A occurs on the inactive X chromosome in female mammals and that this correlates with recruitment of Polycomb group (PcG) proteins belonging to Polycomb repressor complex 1 (PRC1). Based on these observations, the role of the PRC1 protein Ring1B and its closely related homolog Ring1A in H2A ubiquitylation was tested. Analysis of Ring1B null embryonic stem (ES) cells revealed extensive depletion of global uH2A levels. On the inactive X chromosome, uH2A was maintained in Ring1A or Ring1B null cells, but not in double knockout cells, demonstrating an overlapping function for these proteins in development. These observations link H2A ubiquitylation, X inactivation, and PRC1 PcG function, suggesting an unanticipated and novel mechanism for chromatin-mediated heritable gene silencing (de Napoles, 2004).
To address the molecular mechanisms underlying Polycomb group (PcG)-mediated repression of Hox gene expression, this study focused on the binding patterns of PcG gene products to the flanking regions of the Hoxb8 gene in expressing and non-expressing tissues. In parallel, the distribution of histone marks of transcriptionally active H3 acetylated on lysine 9 (H3-K9) and methylated on lysine 4 (H3-K4) was followed, and of transcriptionally inactive chromatin trimethylated on lysine 27 (H3-K27). Chromatin immunoprecipitation revealed that the association of PcG proteins, and H3-K9 acetylation and H3-K27 trimethylation around Hoxb8 were distinct in tissues expressing and not expressing the gene. Developmental changes of these epigenetic marks temporally coincide with the misexpression of Hox genes in PcG mutants. Functional analyses, using mutant alleles impairing the PcG class 2 component Rnf2 (Homolog of Drosophila Ring) or the Suz12 mutation decreasing H3-K27 trimethylation, revealed that interactions between class 1 and class 2 PcG complexes, mediated by trimethylated H3-K27, play decisive roles in the maintenance of Hox gene repression outside their expression domain. Within the expression domains, class 2 PcG complexes appeared to maintain the transcriptionally active status via profound regulation of H3-K9 acetylation. The present study indicates distinct roles for class 2 PcG complexes in transcriptionally repressed and active domains of Hoxb8 gene (Fujimura, 2006; full text of article).
The main outcome of this study was to show that binding of a specific, Rnf2-containing form of the class 2 PcG complex, as well as H3-K27 trimethylation marking inactive chromatin, correlates with the maintenance of transcriptional silencing of a Hox gene in developing embryos. Moreover, the results demonstrated that genetic impairment of both PcG binding, and H3-K27 trimethylation leads to Hox gene derepression, and that H3-K27 trimethylation is required for PcG binding. In addition, the establishment of differential PcG binding and histone marks in expressing and non-expressing embryonic tissues occur in the same developmental time window as when Hox genes are deregulated in PcG mutants (Fujimura, 2006).
Rnf2 association to known regulatory elements of the Hoxb8 gene is seen predominantly in transcriptionally silent anterior embryonic tissues, whereas the binding of other PcG class 2 members, Phc1 and Cbx2, is observed at all AP levels, irrespective of transcriptional status. This implies that different forms of class 2 PcG complexes bind to the Hoxb genomic region in embryonic domains where the gene is transcriptionally active and repressed. This is reminiscent of previous findings in the Engrailed/Inv/GeneVI complex in Drosophila SL-2 cells, where the Pc protein is exclusively associated with transcriptionally silent genes, while Ph and Psc are present irrespective of the transcriptional status. Therefore the complete, 'perfect' form of the class 2 PcG core complex may mediate transcriptional repression more efficiently than form(s) lacking the Rnf2 component. If this is the case, incorporation of the Rnf2 component into the complex might be a limiting process to mediate transcriptional repression and regulate its stability. It is also possible that the role of Rnf2 is mediated through its E3 ubiquitin ligase activity directed to histone H2A (Fujimura, 2006).
Transcriptional repression of Hox genes in the developing embryo has been shown to correlate with the association of Rnf2-containing class 2 PcG complexes and H3-K27 trimethylation. De-repression of Hox genes in Rnf2 and Suz12 mutant cells reveal the requirement of both Rnf2 association and H3-K27 trimethylation in the mediation of this transcriptional repression. Since Rnf2 association to Hox genes is reduced in Suz12 mutant ES cells and Rnf2 mutation alters Hox expression without changing local levels of H3-K27 trimethylation, H3-K27 trimethylation mediated by class 1 PcG complexes at Hox genes may facilitate subsequent binding of Rnf2-containing PcG complexes. Recruitment of Rnf2-containing PcG complexes may in turn prevent the access of nucleosome remodeling factors, such as SWI/SNF complex, leading to the formation of a repressed chromatin status. Therefore, molecular circuitry underlying PcG silencing of Hox genes seems to have been evolutionarily conserved between Drosophila and mammals. It is also notable that Cbx2, a homologue of Drosophila Pc, binds to Hoxb8 in transcriptionally active embryonic tissues, despite the lack of histone H3 trimethylated at K27. This is consistent with biochemical data that have shown the association of purified or reconstituted PcG complexes with the nucleosomal templates lacking histone tails. The implication of these findings is that there are at least two different means by which class 2 PcG complexes bind to the chromatin, and that the association, which involves trimethylated H3-K27, mediates the repression at the Hox genes in vivo (Fujimura, 2006).
The maintenance of regionally restricted expression of Hox genes is likely to involve H3-K9 acetylation and H3-K4 methylation. This study has shown that these modifications of the histone tail increases craniocaudally along the axis. Although the transcriptionally active posterior tissues of 9.5 dpc and older embryos are more heavily acetylated at H3-K9 than the anterior, non-Hox expressing tissues, some acetylation of H3-K9 at Hoxb8 is seen in anterior regions where Hoxb8 expression is repressed at early and later developmental stages. De-repression of Hoxb8 expression upon depletion of Rnf2 in MEFs derived from the cranial part of 9.5 dpc embryos suggests the involvement of Rnf2-containing class 2 PcG complexes to mediate this transcriptional repression. Therefore, these data suggest that the associations of Rnf2-containing PcG complexes and acetylated H3-K9 may counteract each other and cooperate to maintain the anterior boundaries of Hoxb8 expression at mid-gestational stages and later. This is consistent with the antagonistic properties of Mll and Bmi1 mutations. Moreover, the establishment of the differential binding of the Rnf2 and H3-K9 acetylation at Hoxb8 during embryogenesis temporally coincides with de-repression of that Hox gene in Bmi1/Rnf110 and Phc1/Phc2 double homozygotes, and loss of its transcription in Mll homozygotes. Intriguingly, class 2 PcG complexes, which lack the Rnf2 component, are also involved in the maintenance of H3-K9 acetylation in embryonic tissues where Hox genes are expressed. This is consistent with predominant subnuclear localization of several PcG proteins in the perichromatin compartment where most pre-mRNA synthesis takes place. The molecular mechanisms underlying this positive action remain unaddressed (Fujimura, 2006).
In conclusion, class 2 PcG gene products play distinct roles in embryonic territories, which are silent or active for Hoxb8 transcription, by forming complexes of different composition. Interaction between class 1 and class 2 PcG complexes mediated by trimethylated H3-K27 play decisive roles in Hox gene repression outside their expression domains, as seen in Drosophila. In addition, within the Hox expression domain, class 2 PcG complexes are involved in maintaining a transcriptionally active status, independent of H3-K27 trimethylation (Fujimura, 2006).
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