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).
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