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).
Polycomb-group (PcG) proteins mediate repression of developmental regulators in a reversible manner, contributing to their spatiotemporally regulated expression. However, it is poorly understood how PcG-repressed genes are activated by developmental cues. This study used the mouse Meis2 gene as a model to identify a role of a tissue-specific enhancer in removing PcG from the promoter. Meis2 repression in early development depends on binding of RING1B, an essential E3 component of PcG, to its promoter, coupled with its association with another RING1B-binding site (RBS) at the 3' end of the Meis2 gene. During early midbrain development, a midbrain-specific enhancer (MBE) transiently associates with the promoter-RBS, forming a promoter-MBE-RBS tripartite interaction in a RING1-dependent manner. Subsequently, RING1B-bound RBS dissociates from the tripartite complex, leaving promoter-MBE engagement to activate Meis2 expression. This study therefore demonstrates that PcG and/or related factors play a role in Meis2 activation by regulating the topological transition of cis-regulatory elements (Kondo, 2014).
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).
Polycomb complexes establish chromatin modifications for maintaining gene repression and are essential for embryonic development in mice. This study used pluripotent embryonic stem (ES) cells to demonstrate an unexpected redundancy between Polycomb-repressive complex 1 (PRC1) and PRC2 during the formation of differentiated cells. ES cells lacking the function of either PRC1 or PRC2 can differentiate into cells of the three germ layers, whereas simultaneous loss of PRC1 and PRC2 abrogates differentiation. On the molecular level, the differentiation defect is caused by the derepression of a set of genes that is redundantly repressed by PRC1 and PRC2 in ES cells. Furthermore, it was found that genomic repeats are Polycomb targets, and it was shown that, in the absence of Polycomb complexes, endogenous murine leukemia virus elements can mobilize. This indicates a contribution of the Polycomb group system to the defense against parasitic DNA, and a potential role of genomic repeats in Polycomb-mediated gene regulation (Leeb, 2010).
The combined disruption of Ring1B and Eed results in an aberration of cell differentiation. Given the molecular differences of the activities of PRC1 and PRC2, the severity of this synthetic phenotype is unexpected. PRC1-catalyzed ubH2A has been reported to inhibit transcription elongation, suggesting a direct function in gene repression. Yet, the removal of ubH2A during mitosis is required for cell cycle progression, which precludes this modification as a heritable epigenetic mark. In contrast, H3K27me3 is transmitted through the cell cycle, but a direct function of H3K27me3 in gene repression has not been shown, and its only reported function is to recruit other Polycomb proteins such as Cbx7 or PRC2. The very different cell-biological properties of ubH2A and H3K27me3 indicate that the regulation of Polycomb-mediated silencing is a complex process. It is likely that considerable cross-talk exists between different Polycomb complexes, such as the reported binding of the PcG complex protein Rybp to ubH2A and Ring1B, and other chromatin-associated complexes. PRC1 has additional functions in replication, and it has been shown that PRC1 proteins remain bound to chromatin when replicated in an in vitro system (Leeb, 2010).
Ring1B recruitment to PcG target genes depends quantitatively on PRC2; most of the Ring1B signal on gene promoters is lost in Eed-deficient ES cells. Nevertheless, in the absence of PRC2, Ring1B remains detectable clearly over background level and is functionally relevant. In Ring1B-deficient ES cells, Suz12 and H3K27me3 signals are reduced on most nonredundantly silenced genes, but remain at wild-type levels on redundant gene promoters. These data could indicate that, to some extent, redundant gene silencing by PcG complexes reflects the recruitment of PRC1 and PRC2. However, it is believed that additional factors might also contribute to and determine gene repression of PcG target genes (Leeb, 2010).
The data show that the deletion of Ring1B and Eed in ES cells disrupts the catalytic function of PRC1 or PRC2, respectively. Although no H3K27me3 was detected, it is noted that Ezh2 protein is still present at a reduced amount and could be functional to an extent below detection. Since the same level of Ezh2 is also present in dKO cells, the differentiation defect does not arise due to further loss of Ezh2. Ring1B-/- ES cells are deficient for PRC1 function, since Ring1A, a functional homolog of Ring1B, is not expressed in ES cells and does not restore genomic ubH2A levels. In addition, Ring1B deletion leads to a loss of several PRC1 proteins, including Rybp, Cbx4, Mel18, and Bmi1. At present, it is not resolved to what extent the catalytic activity toward ubH2A and the structural role of PRC1 proteins contribute to the phenotype. From these observations, it is suggested that the function of PRC1 and PRC2 is largely eliminated by disruption of Ring1B and Eed, respectively (Leeb, 2010).
Ring1B-deficient or Eed-deficient ES cells can differentiate into cell types of all three germ layers, suggesting that the dynamic modulation of gene expression required for differentiation can be performed by a single PcG complex. However, the complete loss of PcG function abolishes the tumor formation potential of dKO ES cells. The ability of an EedGFP transgene to restore tumor formation indicates that dKO ES cells can regain pluripotency. Upon differentiation of dKO ES cells, pluripotency markers are down-regulated and differentiation markers are up-regulated. Thus, the differentiation defect of PcG-deficient ES cells does not result from a block to enter differentiation, but is due to a failure to maintain differentiated cells. This suggests that the Polycomb system is critical for fine-tuning gene expression, and that epigenetic patterns required to progress through differentiation cannot be set up in the absence of PcG regulation (Leeb, 2010).
ES cells lacking Ring1B and Eed can self-renew and maintain pluripotency marker expression. They can also contribute to the inner cell mass when injected into blastocysts. However, these ES cells are unstable and tend to spontaneously differentiate in culture. Furthermore, they fail to execute differentiation programs appropriately. Thus, Polycomb complexes stabilize ES cell identity. Only a slight decrease in the proliferation rate of dKO ES cells was measured, as compared with controls, indicating that ES cell self-renewal is largely independent of epigenetic gene regulation. This is also consistent with the finding that ES cells lacking DNA cytosine methyltransferases are viable. Recently, it has been suggested that ES cells represent the ground state of pluripotency, which would not require epigenetic regulation. These data support this notion by showing that the network of transcription factors in ES cells is stable enough to maintain self renewal in the absence of Polycomb regulation. The ability to maintain ES cells in the absence of PRC1 and PRC2 catalytic activity now provides an opportunity for studying the function of the PcG system in gene repression, chromatin organization, and genome stability. In the future, this will facilitate characterizing the role of the Polycomb system for the nuclear architecture of mammals (Leeb, 2010).
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).
Pluripotent cells develop within the inner cell mass of blastocysts, a mosaic of cells surrounded by an extra-embryonic layer, the trophectoderm. This study shows that a set of somatic lineage regulators (including Hox, Gata and Sox factors) that carry bivalent chromatin enriched in H3K27me3 and H3K4me2 are selectively targeted by Suv39h1-mediated H3K9me3 and de novo DNA methylation in extra-embryonic versus embryonic (pluripotent) lineages, as assessed both in blastocyst-derived stem cells and in vivo. This stably repressed state is linked with a loss of gene priming for transcription through the exclusion of PRC1 (Ring1B) and RNA polymerase II complexes at bivalent, lineage-inappropriate genes upon trophoblast lineage commitment. Collectively, these results suggest a mutually exclusive role for Ring1B and Suv39h1 in regulating distinct chromatin states at key developmental genes and propose a novel mechanism by which lineage specification can be reinforced during early development (Alder, 2010).
Gene expression programmes must be tightly controlled to govern cell identity and lineage choice. An inherent challenge for developing organisms is to maintain a critical balance between stable and flexible gene regulation. This is most obvious in pluripotent embryonic stem (ES) cells, which are functionally characterised by their ability to self-renew and to generate all somatic lineages when induced. Accordingly, ES cells express genes that encode self-renewal factors, while repressing many lineage-specific regulators that are ultimately required during development. A series of recent reports have indicated how Polycomb-mediated gene repression might provide short-term, and therefore flexible, epigenetic silencing of such developmental genes in pluripotent cell lines. This contrasts with long-term repression mechanisms as conferred, for example, by DNA methylation at transposons, imprinted and pluripotency-associated genes in somatic cells (Alder, 2010).
Two distinct Polycomb Repressive Complexes (PRC), PRC1 and PRC2, are known to be important for the function of ES cells. PRC2 contains Ezh2, which catalyses histone H3 lysine 27 trimethylation (H3K27me3), as well as Eed and Suz12. PRC1 contains the E3 ubiquitin ligases Ring1A (also known as Ring1) and Ring1B (Rnf2 -- Mouse Genome Informatics) that mono-ubiquitylate histone H2A lysine 119. Other PRC1 components include Bmi1, Mel18 (Pcgf6 -- Mouse Genome Informatics) and proteins of the Cbx family with H3K27 methylation affinity. Candidate-based and genome-wide studies of histone methylation in ES cells led to the remarkable finding that many PRC2-target genes not only carry the repressive H3K27me3 mark, but are also enriched for conventional indicators of active chromatin, including methylated H3K4. These so-called bivalent chromatin domains are thought to silence key developmental regulators while keeping them primed for future activation (or repression), and thus generally resolve to monovalent configurations upon differentiation, in accordance with gene expression changes. Further work showed that multipotent stem cells and some differentiated cells also possess bivalent domains, albeit perhaps fewer than in ES cells, indicating that plasticity might be maintained at loci that are required for the function and differentiation of lineage-committed cells. Consistent with gene priming, bivalent genes assemble RNA polymerase II complexes preferentially phosphorylated on Ser5 residues (poised RNAP) and are transcribed at low levels. Productive expression is, however, prevented by the action of PRC1 with conditional deletion of Ring1A/B, resulting in an upregulation of target gene expression in ES cells (from primed to overt transcription) (Alder, 2010 and references therein).
Clearly, Polycomb repressors are functionally required to prevent premature expression of primed genes and thus to stably maintain a pluripotent ES cell identity in culture. Whether ES cell epigenetic signatures can also be seen in the developing embryo and when (and how) bivalent domains are established and subsequently resolved upon lineage commitment in vivo remains to be elucidated. This study focused on the earliest stages of mouse development to address whether poised chromatin structures are unique hallmarks of the founder (pluripotent) cells of the early embryo, and to investigate the kinetics of appearance and resolution of bivalent domains during the first lineage decision event (trophectoderm formation). In vivo evidence is provided that bivalent histone marking operates in the early mouse embryo from the eight-cell up to the blastocyst stage. Unexpectedly, it was shown that bivalent domains are retained at a subset of genes encoding key somatic lineage regulators in extra-embryonic restricted cells, as assessed both in vitro and in vivo. However, and in contrast to pluripotent cells, PRC1 (Ring1B) and poised RNAP are not engaged at these PRC2 (Suz12)-bound genes, consistent with a loss of gene priming. Instead, bivalent genes become selectively targeted by Suv39h1-mediated repression upon trophoblast lineage commitment. Collectively, these results suggest a mutually exclusive role for Ring1B and Suv39h1 in specifying the fate of bivalent genes in a lineage-specific manner upon blastocyst formation (Alder, 2010).
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).
During neocortical development, neural precursor cells (NPCs, or neural stem cells) produce neurons first and astrocytes later. Although the timing of the fate switch from neurogenic to astrogenic is critical for determining the number of neurons, the mechanisms are not fully understood. This study shows that the polycomb group complex (PcG) restricts neurogenic competence of NPCs and promotes the transition of NPC fate from neurogenic to astrogenic. Inactivation of PcG by knockout of the Ring1B or Ezh2 gene or Eed knockdown prolonged the neurogenic phase of NPCs and delayed the onset of the astrogenic phase. Moreover, PcG was found to repress the promoter of the proneural gene neurogenin1 in a developmental-stage-dependent manner. These results demonstrate a role of PcG: the temporal regulation of NPC fate (Hirabayashi, 2009).
During neocortical development, the neurogenic phase normally persists for a limited time period (about 11 cell cycles on average in the mouse neocortex, and this restricted period may be a major parameter in determining the final number of neurons produced during development. PcG proteins contribute to the termination of the neurogenic phase, which normally takes place between E18.5 and E19.0 in the neocortex. Indeed, birthdating analysis showed that cells labeled by BrdU at E19.0 still contributed to upper-layer neurons at P6.5 in Ring1B- or Ezh2-deficient mice but not in control mice. Interestingly, the excess neurons produced at around the end of neurogenic phase appear to be eliminated (probably by cell death) later during postnatal development in both wild-type and Ring1B-deficient mice, suggesting that these late-born excess neurons fail to integrate into the appropriate neuronal networks and therefore cannot be supported by activity/target-dependent survival signals. In other words, the correct timing of the end of neurogenesis might help avoid production of excess (unnecessary, undesirable) neurons (Hirabayashi, 2009).
The roles of PcG in ES cells strikingly differ from those in NPCs. Components of the PcG are known to localize and repress a variety of target genes and play an essential role in the maintenance of pluripotency of ES cells by suppressing differentiation into multiple lineages. A previous report has shown that different arrays of genes are labeled with H3K27me3 in ES cells and ES-derived neuronal progenitors, suggesting that PcG targets are different between these cell types. Indeed, Ring1B deletion in late-stage neocortical NPCs preferentially increases the expression of genes associated with neuronal differentiation/development over those associated with other lineages based on microarray analyses, whereas developmental genes in multiple lineages are derepressed by Ring1B deletion in ES cells (Hirabayashi, 2009).
The fate restriction of ES cells during differentiation is accompanied by diminished occupancy of H3K27me3 at specific 'bivalent' gene promoters involved in the corresponding differentiation process, in contrast to the increased H3K27me3 at ngn loci during fate restriction of NPCs. Moreover, deletion of Ring1B or Suz12 in ES cells results in the loss of neurogenic capacity, whereas deletion of Ring1B in the late NPCs extended neurogenic capacity. These observations further support the difference of PcG functions between these cell types. Thus, this study has unveiled an in vivo role of PcG, namely, temporal (stage-dependent) fate conversion of multipotent progenitors during development (Hirabayashi, 2009).
This study found that PcG is responsible for ngn1suppression in late-stage NPCs. Since misexpression of ngn1 extends neurogenesis in late-stage NPCs, it is clear that suppression of ngn1 is a prerequisite for the neuronal-to-glial transition of NPC fate. Therefore, the suppression of ngn1 by PcG may partly account for the PcG restriction of neurogenic potential and transition to gliogenesis in the neocortex. However, it is unclear whether PcG also regulates other genes with similar functions. Ngn2 might be such a target, given that the level of H3K27me3 increases at the ngn2 locus in the late stage of neocortical NPCs. However, Ring1B deletion by itself did not cause much increase in ngn2 expression, suggesting that additional mechanisms might account for suppression of ngn2 at late stages of neocortical development (Hirabayashi, 2009).
Besides ngn1, no other proneural genes were found that were greatly upregulated by Ring1B deletion in neocortical NPCs. For instance, there was no elevation in neurogenic genes expressed in the neocortex such as Pax6, Math1, and Mash1. Among the basic helix-loop-helix or homeodomain-containing transcription factors expressed in brain, Dlx2 was significantly derepressed by Ring1B deletion. Although Dlx2 can contribute to neurogenesis in the ventral telencephalon in some contexts, it is not thought that this gene is responsible for the PcG suppression of neurogenesis in the neocortex, since Dlx2 is associated with differentiation of GABAergic interneurons rather than the glutamatergic neurons observed in the Ring1B-deficient mice. Nonetheless, a recent report has shown that the chromatin remodeling factor Mll1 suppresses the accumulation of H3K27me3 at the dlx2 locus and thus confers neurogenic potential in the adult neural stem cells. This implies a very interesting possibility that PcG participates in a common mechanism that suppresses neurogenic potential in both dorsal and ventral telencephalon in the late stages of development, and a small NPC population that escapes from this mechanism by Mll1 is selected to become adult neural stem cells that continue to produce neurons for lifetime (Hirabayashi, 2009).
Although knockdown of Bmi1, a component of PRC1, resulted in NPC loss and brain size reduction in a previous study, these phenotypes were not observed in the Ring1B-deficient mouse, implicating that Bmi1 and Ring1B may form distinct complexes that exert different functions. These functions of Bmi1 may not be related to PRC2, since it was found that brain size reduction was not seen in mice deficient for Ezh2 in the central nervous system, although H3K27me3 modification was barely seen in NPCs from these mice. Functional differences between Bmi1 and PRC2 have also been suggested in the hematopoietic system and tumors. For example, Bmi1 deletion reduced the numbers of myeloid and preB cells, whereas Eed deletion increased these cell types (Hirabayashi, 2009).
The levels of H3K27me3 gradually increase over time at the ngn1 promoter, and it is plausible that, at a certain threshold, their chromatin state becomes inactivated by PRC1, resulting in the suppression of ngn expression and the transition of NPC fate. It is proposed that the developmental-stage-dependent accumulation of H3K27me3 at specific gene loci functions as a timer to drive cell fate switching. Exactly how this accumulation occurs is not clear at present but might involve either a global increase in PcG activity or local recruitment of PcG to the ngn1 locus in late stages of neocortical NPCs. In either case, further analysis of this accumulation may shed light on the mechanism that underlies the developmental regulation of differentiation potential (Hirabayashi, 2009).
Polycomb group (PcG) genes are chromatin modifiers that mediate epigenetic silencing of target genes. PcG-mediated epigenetic silencing is implicated in embryonic development, stem cell plasticity, cell fate maintenance, cellular differentiation and cancer. However, analysis of the roles of PcG proteins in maintaining differentiation programs during vertebrate embryogenesis has been hampered due to the early embryonic lethality of several PcG knock-outs in the mouse. Zebrafish Ring1b/Rnf2, the single E3 ubiquitin ligase in the Polycomb Repressive Complex 1, critically regulates the developmental program of craniofacial cell lineages. Zebrafish ring1b mutants display a severe craniofacial phenotype, which includes an almost complete absence of all cranial cartilage, bone and musculature. This study shows that Cranial Neural Crest (CNC)-derived cartilage precursors migrate correctly into the pharyngeal arches, but fail to differentiate into chondrocytes. The results reveal a critical and specific role for Ring1b in promoting the differentiation of cranial neural crest cells into chondrocytes. The zebrafish ring1b mutant provides a molecular model for investigating mechanisms underlying craniofacial abnormalities (Van der Velden, 2013).
In the developing neocortex, neural precursor cells (NPCs) sequentially generate various neuronal subtypes in a defined order. Although the precise timing of the NPC fate switches is essential for determining the number of neurons of each subtype and for precisely generating the cortical layer structure, the molecular mechanisms underlying these switches are largely unknown. This study shows that epigenetic regulation through Ring1B, an essential component of polycomb group (PcG) complex proteins, plays a key role in terminating NPC-mediated production of subcerebral projection neurons (SCPNs). The level of histone H3 residue K27 trimethylation at and Ring1B binding to the promoter of Fezf2, a fate determinant of SCPNs, increased in NPCs as Fezf2 expression decreased. Moreover, deletion of Ring1B in NPCs, but not in postmitotic neurons, prolonged the expression of Fezf2 and the generation of SCPNs that were positive for CTIP2. These results indicate that Ring1B mediates the timed termination of Fezf2 expression and thereby regulates the number of SCPNs (Morimoto-Suzki, 2014).
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