Covalent modification of histones is important in regulating chromatin dynamics and transcription. One example of such modification is ubiquitination, which mainly occurs on histones H2A and H2B. Although recent studies have uncovered the enzymes involved in histone H2B ubiquitination and a 'cross-talk' between H2B ubiquitination and histone methylation, the responsible enzymes and the functions of H2A ubiquitination are unknown. This study reports the purification and functional characterization of an E3 ubiquitin ligase complex that is specific for histone H2A. The complex, termed hPRC1L (human Polycomb repressive complex 1-like), is composed of several Polycomb-group proteins including Ring1, Ring2, Bmi1 and HPH2. hPRC1L monoubiquitinates nucleosomal histone H2A at lysine 119. Reducing the expression of Ring2 results in a dramatic decrease in the level of ubiquitinated H2A in HeLa cells. Chromatin immunoprecipitation analysis has demonstrated colocalization of dRing with ubiquitinated H2A at the PRE and promoter regions of the Drosophila Ubx gene in wing imaginal discs. Removal of dRing in SL2 tissue culture cells by RNA interference results in loss of H2A ubiquitination concomitant with derepression of Ubx. Thus, these studies identify the H2A ubiquitin ligase, and link H2A ubiquitination to Polycomb silencing (Wang, 2004).
The PRC1 complex contains Psc, Pc, Ph, and Sce proteins. Among these components of the PRC1 complex, it is known that Psc interacts directly with Ph and Pc and that Psc and Ph interact homotypically. Murine and human Ring1/Ring1A and Rnf2/Ring1B interact directly not only with the mammalian homologs of Pc, M33 and Pc2 (Satijn, 1997: Schoorlemmer, 1997), but also with orthologs of Psc such as Bmi1 (Satijn, 1999) and Mel18. In addition, Rnf2/Ring1B interacts with mPH2, a Ph homolog (Hemenway, 1998). To see whether the conservation of the patterns of pairwise interactions between Drosophila PcG protein and their mammalian counterparts also include Sce, its association with Pc, Psc and Ph was studied using an in vitro protein binding assay (Gorfinkiel, 2004).
The complete Sce coding sequence (amino acids 1-435, Sce), and derivatives containing the domains HD1 [Sce amino acids 1-274, Sce(N)] or HD2 and HD3 [amino acids 274-435, Sce(C)] were fused to the glutathione S-transferase (GST) gene, and the resulting hybrid proteins were expressed in Escherichia coli. GST-Sce binds specifically Pc and Psc, but not Ph. Sce(C), but not Sce(N), binds Pc. This shows that Sce binding to Pc occurs through its HD2 and HD3 domains, as previously shown for mammalian Ring1 and Pc proteins. Moreover, the Pc variant lacking the conserved carboxyl domain (PcΔC) does not bind to Sce, a result consistent with previous findings in mammals showing that such domain is responsible for the binding of Pc to Ring. However, binding to Psc occurs preferentially to Sce(N), showing that the interaction between Sce and Psc involves the same domains as the interaction between mammalian Rings and Bmi1 proteins. Sce does not interact with the conserved domain of Ph (amino acids 1297-1576), which mediates homo and heterotypic interactions. Although mouse Rinf2/Ring1B binds Ph (1297-1576), an interaction between Sce and regions in the rest of the Ph protein is still potentially possible. These results indicate that of the interactions among mammalian Ring1/Rnf2 proteins and PcG proteins, at least those between Ring and Pc and Psc are conserved in Drosophila (Gorfinkiel, 2004).
ORD protein is required for accurate chromosome segregation during male and female meiosis in Drosophila melanogaster. Null ord mutations result in random segregation of sister chromatids during both meiotic divisions because cohesion is completely abolished prior to kinetochore capture of microtubules during meiosis I. Previous analyses of mutant ord alleles have led to a proposal that the C-terminal half of the ORD protein mediates protein-protein interactions that are essential for sister-chromatid cohesion. To identify proteins that interact with ORD, a yeast two-hybrid screen was conducted using an ORD bait and isolated dRING, a core subunit of the Drosophila Polycomb repressive complex 1. A missense mutation in ORD completely ablates the two-hybrid interaction with dRING and prevents nuclear retention of the mutant ORD protein in male meiotic cells. Using affinity-purified antibodies generated against full-length recombinant dRING, it is demonstrated that dRING protein is expressed in the male and female gonads and colocalizes extensively with ORD on the chromatin of primary spermatocytes during G2 of meiosis. These results suggest a novel role for the Polycomb group protein dRING and are consistent with the model that interaction of dRING and ORD is required to promote the proper segregation of meiotic chromosomes (Balicky, 2004).
The transcriptional status of a gene can be maintained through multiple rounds of cell division during development. This epigenetic effect is believed to reflect heritable changes in chromatin folding and histone modifications or variants at target genes, but little is known about how these chromatin features are inherited through cell division. A particular challenge for maintaining transcription states is DNA replication, which disrupts or dilutes chromatin-associated proteins and histone modifications. PRC1-class Polycomb group protein complexes, consisting of four core PcG subunits, polyhomeotic (Ph), posterior sex combs (PSC), dRING, and Polycomb (Pc), are essential for development and are thought to heritably silence transcription by altering chromatin folding and histone modifications. It is not known whether these complexes and their effects are maintained during DNA replication or subsequently re-established. When PRC1-class Polycomb complex-bound chromatin or DNA is replicated in vitro, Polycomb complexes remain bound to replicated templates. Retention of Polycomb proteins through DNA replication may contribute to maintenance of transcriptional silencing through cell division (Francis, 2009).
The data suggest that PCC is not released into solution during passage of the DNA replication fork. Furthermore, nucleosomes facilitate PCC binding to and retention on templates, but are not essential for either. The finding that PCC can be maintained on either chromatin or naked DNA is interesting in light of the finding that PREs are sites of rapid histone turnover and can be depleted of nucleosomes (Francis, 2009).
One model for the transfer of PCC during DNA replication is that the complex remains in direct contact with DNA during passage of the DNA replication fork. Contacts between PcG proteins and nucleosomes or DNA could be disrupted in front of the replication fork, but replaced by contacts with nucleosomes or DNA behind the replication fork. This mechanism has been proposed for transfers of histone-DNA contacts during replication and transcription in vitro. PCC can likely contact multiple nucleosomes or a long stretch of DNA, which may allow the complex to remain on chromatin when some template contacts are disrupted. A second model is that PCC interacts with the replication machinery, either directly or through intermediary factors. These interactions could retain PCC near DNA during replication, even if direct DNA contacts are disrupted, allowing rapid rebinding of PCC to newly replicated chromatin. Consistent with this idea, several chromatin-modifying proteins can interact with components of the DNA replication machinery (Francis, 2009).
The inhibition of DNA and chromatin replication by PCC in vitro raises the question of how PcG-bound regions are replicated if PRC1-class complexes are indeed continuously bound. If PCC inhibits replication initiation but not elongation, as the results suggest, then PRC1-class complexes would limit replication only if they were bound near replication origins (Francis, 2009).
Intriguingly, targeting of Pc to a replication origin in Drosophila that mediates developmental chorion gene amplification in follicle cells decreased gene amplification (Aggarwal, 2004) and PcG-silenced regions of polytene chromosomes (such as Hox gene clusters) are underreplicated, although this involves additional genes such as Suppressor of DNA Underreplication (Marchetti, 2003; Moshkin, 2001; Francis, 2009 and references therein).
Reduction of PcG protein levels leads to reactivation of their target genes, suggesting that these genes are continuously susceptible to transcriptional activation. It may therefore be important that PRC1-class complexes, which can directly repress transcription, maintain constant association with genes marked for silencing (Francis, 2009).
It was surprising to find that H3K27me3 is not essential for maintaining PRC1-class complexes through DNA replication in vitro. It is possible that retention of parental PRC1-class complexes and recruitment of new complexes are mechanistically distinct because no evidence was found for recruitment of new PCC during replication, and in vivo data suggest that PSC is present on newly replicated chromatin but that additional PSC is recruited after replication. This may be similar to histone proteins in that it is thought that parental histones are transferred randomly to the two daughter strands, followed by deposition of new histones by replication-coupled assembly complexes. In vivo data raise the possibility that recruitment of new PRC1 is not directly coupled to DNA replication; perhaps it involves H3K27me3 (Francis, 2009).
In these experiments, PCC interacts with chromatin through mass action, but in vivo, PRC1-class complexes are specifically targeted to PREs. It is hypothesized that the stable association of PCC with chromatin observed in this study reflects how the complex could behave once it is recruited to a PRE, but it will be important to test this mechanism in a system where PCC is targeted (Francis, 2009).
In conclusion, the ability of parental PCC to be transferred to daughter chromatin may help explain how PcG-mediated repression established by transiently acting factors can be propagated through cell generations. These data also suggest that maintenance of chromatin regulatory proteins through DNA replication might be an important mechanism of epigenetic inheritance (Francis, 2009).
The Drosophila protein Sex Comb on Midleg (Scm) is a member of the Polycomb group (PcG), a set of transcriptional repressors that maintain silencing of homeotic genes during development. Recent findings have identified PcG proteins both as targets for modification by the small ubiquitin-like modifier (SUMO) protein and as catalytic components of the SUMO conjugation pathway. This study found that the SUMO-conjugating enzyme Ubc9 binds to Scm and that this interaction, which requires the Scm C-terminal sterile α motif (SAM) domain, is crucial for the efficient sumoylation of Scm. Scm is associated with the major Polycomb response element (PRE) of the homeotic gene Ultrabithorax (Ubx), and efficient PRE recruitment requires an intact Scm SAM domain. Global reduction of sumoylation augments binding of Scm to the PRE. This is likely to be a direct effect of Scm sumoylation because mutations in the SUMO acceptor sites in Scm enhance its recruitment to the PRE, whereas translational fusion of SUMO to the Scm N terminus interferes with this recruitment. In the metathorax, Ubx expression promotes haltere formation and suppresses wing development. When SUMO levels are reduced, decreased expression of Ubx and partial haltere-to-wing transformation phenotypes were observed. These observations suggest that SUMO negatively regulates Scm function by impeding its recruitment to the Ubx major PRE (Smith, 2011).
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