BIOLOGICAL OVERVIEW

In Drosophila, the Polycomb group (PcG) of genes is required for the maintenance of homeotic gene repression during development. The Drosophila ortholog of the products of the mammalian Ring1/Ring1A and Rnf2/Ring1B genes has been characterized. Drosophila Ring corresponds to Sex combs extra (Fritsch, 2003: Gorfinkiel, 2004), a previously described PcG gene. Ring/Sce is expressed and required throughout development and the extreme Pc embryonic phenotype due to the lack of maternal and zygotic Sce can be rescued by ectopic expression of Ring/Sce. This phenotypic rescue is also obtained by ectopic expression of the murine Ring1/Ring1A, suggesting a functional conservation of the proteins during evolution. In addition, Ring/Sce binds to about 100 sites on polytene chromosomes, 70% of which overlap those of other PcG products such as Polycomb, Posterior sex combs and Polyhomeotic, and 30% of which are unique. Ring/Sce interacts directly with PcG proteins, because it occurs in mammals (Gorfinkiel, 2004).

Drosophila is part of a protein complex that monoubiquitinates nucleosomal histone H2A. Reducing the expression of mammalian Ring2 results in a dramatic decrease in the level of ubiquitinated H2A in HeLa cells. Chromatin immunoprecipitation analysis has demonstrated colocalization of Drosophila Ring with ubiquitinated H2A at the polycomb response elements and promoter regions of the Drosophila Ubx gene in wing imaginal discs. Removal of Drosophila Ring in SL2 tissue culture cells by RNA interference results in loss of H2A ubiquitination concomitant with derepression of Ubx. These studies identify the H2A ubiquitin ligase, and link H2A ubiquitination to Polycomb silencing (Wang, 2004).

Genetic analysis in Drosophila has unveiled a repression function required for proper regulation of the homeotic genes that determine segmental identities. A large number of genes, collectively known as the Polycomb group of genes (PcG), participate in such a repressive activity. Thus, mutations in the PcG genes lead to homeotic phenotype associated with the indiscriminate expression of genes from the Bithorax complex (BX-C) and/or Anntenapedia complex (ANT-C). PcG related genes have been identified in plants and in vertebrates, and mutations in these genes are, among others, associated with homeotic phenotypes. The PcG are thought to be required for the maintenance of transcriptionally repressed states of the Hox genes, but not for the initiation of their repression. Other transcriptional repressors of the gap and pair rule groups, transiently expressed during development, are responsible for this initiation of repression (Gorfinkiel, 2004).

The molecular mechanism(s) of PcG function is (are) unknown. Several lines of evidence, however, indicate that PcG products work together in multimeric protein complexes in which individual PcG proteins interact with other PcG proteins through conserved domains. Biochemical fractionation of Drosophila nuclear extracts shows two major multimeric complexes. One, termed Polycomb Repressive Complex 1 (PRC1) has a size of about 2 MDa, contains the PcG products Polycomb (Pc), Polyhomeotic (Ph), Posterior sex combs (Psc), Sex combs on midleg (Scm) and Drosophila Ring, some components of the basal transcriptional machinery (TAFs) and other polypeptides (Shao, 1999 and Saurin, 2001). Another complex, of about 600 kDa in size, does not contain any of the above proteins, but instead comprises the products of the extra sex combs (esc), Enhancer of zeste [E(z)] and Suppressor of zeste 12 [Su(Z)12] genes. In contrast to the lack of enzymatic activities associated with the PRC1 complex, the so-called Esc-E(z) complex has histone deacetylase and histone methyltransferase activities. A complex-based function is consistent with the synergistic genetic interactions between any two PcG genes. Additionally, the PcG products are chromosomal proteins that bind specific sites, visualized on salivary gland polytene chromosomes. Many of these binding sites are common for several PcG proteins. The large number of chromosomal sites that bind PcG proteins suggests that the homeotic complexes, BX-C and ANT-C, are only some of many target loci regulated by PcG (Gorfinkiel, 2004).

Repression by PcG proteins occurs through Polycomb response elements (PRE), which are regulatory DNA sequences harbouring functional binding sites for PcG proteins. Until recently, PREs were identified in a few loci, including the homeotic genes of the BX-C and ANT-C complexes. Recently, computational methods have been used in Drosophila to predict PREs on a genome wide scale identifying about 170 candidate PREs, which map to a variety of loci involved in development and cell proliferation. PREs have a modular structure and bind PcG complexes of different composition. How these complexes are targeted to DNA is not known. PREs have DNA binding sites for proteins such as GAGA factor, Zeste and Pleiohomeotic (Pho), which is the only PcG product able to bind DNA. However, Pho is found only in PcG complexes at the earliest stages of Drosophila development. The molecular mechanism(s) by which the PcG repression function uses multimeric complexes is not known (Gorfinkiel, 2004).

In a search for new mammalian PcG genes, Ring1/Ring1A and Rnf2/Ring1B, two mouse genes have been found whose products interact both in vitro and in two hybrid assays with Pc, Psc and Ph homologs (Hemenway, 1998; Satijn, 1999; Satijn, 1997; Schoorlemmer, 1997). Ring1/Ring1A and Rnf2/Ring1B proteins are part of a PRC1 complex isolated from mammalian cells (Levine, 2002). The Drosophila PRC1 complex also contains the ortholog of vertebrate Ring1 proteins, which seems to play an essential role in the in vitro reconstitution of a PRC1 core complex together with Pc, Psc and Ph (Francis, 2001). In mice, null or hypomorphic mutations in the Ring1/Ring1A or Rnf2/Ring1B genes, respectively, show axial skeleton alterations consistent with a PcG function (del Mar Lorente, 2000; Suzuki, 2002; Gorfinkiel, 2004 and references therein).

The product of the Drosophila melanogaster Ring gene (Ring) has been identified as Sex combs extra (Fritsch, 2003; Gorfinkiel, 2004), one of the molecularly uncharacterized PcG mutants in Drosophila. Over-expression of Ring/Sce and also of the murine Ring1/Ring1A can rescue the extreme Pc embryonic phenotype derived from the lack of maternal and zygotic Sce¹, suggesting a functional conservation of the Drosophila and vertebrate proteins during evolution. In addition, Ring/Sce encodes a chromosomal protein that binds to more than 100 specific sites. Direct interactions between Ring/Sce and PcG proteins take place through the same domains as the interactions between their mammalian counterparts (Gorfinkiel, 2004).

The EST databases of the BDGP were searched with either murine Ring/Ring1A or Rnf2/Ring1B cDNAs and two overlapping cDNAs (LD3177 and LD6636) were identified. The complete sequence of cDNA LD3177 was almost identical to a cDNA sequence termed Ring deposited in the databanks (CG5595) (Gorfinkiel, 2004).

By in situ hybridization to polytene chromosomes, Drosophila Ring was located at the end of the long arm of chromosome 3 in section 98A. Interestingly, Sce, a non-molecularly characterized PcG gene, which was defined by a single mutant allele Sce¹, had been mapped by recombination to the 3-92 interval (Breen, 1986). The proximity of such an interval to the cytological localization of the Drosophila Ring gene encouraged the exploration of a possible identity between the Sce and the Drosophila Ring gene (Gorfinkiel, 2004).

The genomic DNA was sequenced from Sce¹ heterozygous embryos corresponding to the Drosophila Ring coding region. Comparing these sequences with the wild type, a deletion of 410 bp was found that removed the codons for the C-terminal 113 amino acids, a small intron and 12 nucleotides of the 3' untranslated region after the termination codon. Therefore, the Sce¹ allele conceptually encodes a truncated Ring protein that is fused in frame to 23 novel amino acids at the C-terminal part of the protein. Fritsch (2003) has made an identical observation. Drosophila Ring will therefore be referred to as Sce (Gorfinkiel, 2004).

In agreement with the presence of Ring in embryonic PcG complexes the data support a PcG function for the Ring protein. Mice bearing null (Ring1/Ring1A) or hypomorphic (Rnf2/Ring1B) mutations show an involvement of the Ring genes in the patterning of the antero-posterior axis (del Mar Lorente, 2000; Suzuki, 2002). However, in contrast to mutations in other vertebrate PcG genes, the alterations of the axial skeleton seen in the Ring mutant mice could not be associated clearly to a deregulation of Hox genes (del Mar Lorente, 2000). Therefore, the role of vertebrate Ring proteins as genuine PcG proteins is strengthened by the data showing a genetic evidence for a PcG function for Sce (Gorfinkiel, 2004).

Cross-species complementation experiments with PcG genes show contrasting results. Thus, M33, the mouse ortholog of Drosophila Pc, was shown to rescue the Pc mutant phenotype in early embryos. However, eed, the mouse ortholog of Drosophila extra sex combs (esc) is not only unable to rescue the embryonic lethality of esc embryos but shows a dominant negative effect on the leg transformation phenotype of esc mutants. It has been suggested that the activity of eed in Drosophila cells is related to its inability to interact with E(z). With respect to Ring, mouse Ring1/Ring1A rescues the cuticle phenotype of Sce embryos, indicating that in early development, at least, the function of Ring is conserved between mice and flies. This might be due to the structural conservation of Ring proteins. The three domains conserved in Ring1/Ring1A and Rnf2/Ring1B are also present in Sce and constitute about 57% of this protein. Whereas the size and degree of conservation of the domains HD2 and HD3 are similar to other protein motifs identified in fly and vertebrate PcG proteins, domain HD1 is somewhat exceptional. This is a 147 amino acids domain in which 78% are identical in fly and vertebrate proteins, particularly in the RING finger motif. An indication of the relevance of the functionality of this region of Ring proteins is the Sce^33M2 allele which shows a phenotype much milder than that of Sce¹ but that is due to a Ring protein with a single amino acid alteration in that region (Fritsch, 2003). The overall structural conservation between Ring proteins seems to dictate a conservation of interaction with other PcG proteins. In addition, Sce interacts with Pc and Psc. In fact, the core of a PRC1 complex isolated from human cells is compositionally similar to that of flies and the biochemical activity of both complexes is similar (Gorfinkiel, 2004).

Despite this conservation, it is possible that Sce serve diverse functions in late development. For example, expression of the mouse M33 protein in flies does not rescue the Pc adult phenotype. Experiments have not addressed the activity of vertebrate Ring proteins at these later developmental stages and, therefore, whether vertebrate Ring proteins can fully substitute for Sce needs to be approached experimentally (Gorfinkiel, 2004).

Previous genetic and biochemical evidence has shown that PcG proteins act as protein complex(es). Sce interacts directly with Pc and Psc, but not with a Ph-fragment, which binds mouse Rnf2/Ring1B. In addition, immunolocalization studies show that Sce binds to approximately 100 sites, which are in part shared by Pc, Psc, Ph, Pcl and Asx binding site, including the ANT-C and BX-C complexes. These results are consistent with the presence of Sce in the PRC1 complex. However, almost a third of the sites that bind Sce do not bind any of the other PcG proteins. This contrasts with the observation that most Sce molecules in cell extracts are found complexed with PcG proteins in the PRC1 complex (Saurin, 2001). The discrepancy, however, may be related to the fact that the characterized PRC1 has been isolated from Drosophila embryos, whereas the Sce chromosomal sites correspond to binding sites in salivary glands from larvae. Psc, another component of the PRC1 complex, is also found in sites, which do not have Pc/Ph/Pcl. It is worth noting that, despite the ability of Sce to interact with Psc, no Sce is found at these unique Psc sites. Nevertheless, some of these sites correspond with predicted PREs. Therefore, the partial overlapping patterns of Sce and other PcG binding sites suggest the existence of different Polycomb complexes in a tissue specific and developmentally controlled manner. An indication of complexes containing subsets of PcG proteins comes from studies in vertebrates where Drosophila Ring proteins are found together with other polypeptides but not Pc or Ph homologs (Gorfinkiel, 2004).

An intriguing result of the studies on the chromosomal binding sites of Sce is that, in contrast to all PcG genes so far studied, the cytological localization of the Sce gene is free of any PcG protein. The absence of PcG proteins at 98A, therefore, suggests that Sce is regulated somehow differently from other PcG loci (Gorfinkiel, 2004).

In summary, the PcG gene Sce encodes the Drosophila ortholog of mammalian Ring proteins. Sce gene binds to Pc and Psc and is a chromosomal protein associated with many sites in polytene chromosomes, which also bind PcG proteins. Finally, Sce is expressed and required throughout development. The extreme Pc phenotype of Sce embryos is rescued by ectopic expression of Drosophila Ring/Sce and Ring1/Ring1A suggesting that the function of these proteins in conserved between flies and mammals, at least in the early stages of fly development (Gorfinkiel, 2004).

Drosophila SCE/dRING E3-ligase inhibits apoptosis in a Dp53 dependent manner

The Polycomb group (PcG) of proteins control developmental gene silencing and are highly conserved between flies and mammals. PcG proteins function by controlling post-translational modification of histones, such as ubiquitylation, which impacts chromatin compaction and thus gene transcription. Changes in PcG cellular levels have drastic effects on organismal development and are involved in the generation of human pathologies such as cancer. However, the mechanisms controlling their levels of expression and their physiological effects are only partially understood. This work describes the effects of modulating levels of SCE/dRING (Sex combs extra), a conserved E3 ubiquitin ligase and member of the PcG known to mono-ubiquitylate histone H2A. Inactivation of Sce induces apoptosis, an effect that is decreased in the absence of Dp53 function. However, over-expression of SCE produce no developmental effects but inhibits DP53-induced apoptosis. Thus, Sce functions as a Dp53-dependent apoptosis inhibitor. The SCE inhibition of DP53-induced apoptosis requires Ring and YY1 Binding Protein (dRYBP), an ubiquitin binding protein and member of the PcG. Moreover, this inhibition of apoptosis involves the reduction of DP53 protein levels. Finally, high levels of SCE inhibit X-ray induced apoptosis as well as the apoptosis associated with tumor growth. It is proposed that SCE, together with dRYBP, inhibits apoptosis either by epigenetically regulating Dp53 transcription or by controlling the stabilization of DP53 protein levels thus promoting its ubiquitylation for proteaosomal degradation. This function may generate a homeostatic balance between apoptosis and proliferation during development that provides cell survival during the initiation and progression of disease processes (Simoes da Silva, 2017).

These results identify a novel apoptotic role for the SCE protein. The results show that Sce functions as a repressor of apoptosis, as inactivation of Sce promotes apoptosis. This novel function of Sce has gone unidentified most likely because the strong effect of Sce inactivation on the de-repression of UBX expression in the wing has masked its apoptotic function. This activity is revealed when Ubx and Sce are simultaneously inactivated (Simoes da Silva, 2017).

Components of the PRC1 complex, such as PH and PSC/SU(Z)2, have been classified as tumor suppressors because, when inactivated, they induce tumor growth in the wing disc. Inactivation of Sce in the wing disc does not induce tumor growth thus adding complexity to the analysis of PcG targets. Interestingly, SCE-mediated repression of apoptosis requires Dp53 indicated by the decrease in apoptosis when both Sce and Dp53 were inactivated and the response in the activation of the P53R-GFP reporter expression construct when Sce function was inactivated (Simoes da Silva, 2017).

The results on the impact of high SCE levels also support its proposed anti-apoptotic function. This analysis showed SCE and dRYBP, an ubiquitin binding protein previously found to interact genetically and physically with SCE (Fereres, 2014), participates in the inhibition of apoptosis. Curiously, over-expression of SCE produces no developmental phenotype in the wings and the SCE-mediated inhibition of apoptosis is revealed only under stress conditions such as high levels of DP53, X-ray treatment or tumorogenesis. Moreover, human RYBP has been shown to induce apoptosis in transformed cells but not in normal cells. It will be very interesting to investigate if human SCE (Ring1b/RNF2) is involved in human RYBP's apoptotic activity, which could make the combination of RNF2 and RYBP a potential cancer therapeutic candidate (Simoes da Silva, 2017).

The results indicate that SCE-mediated inhibition of apoptosis both requires Dp53 and also modulates DP53 levels, as measured by the activation of P53R-GFP, P53-GFP-FLAG and by DP53 immuno-staining. Thus, the results show that SCE inhibits apoptosis through the modulation of DP53 levels by a mechanism that is, as yet, not clear (Simoes da Silva, 2017).

The well described functions of the PcG proteins and E3-ubiquitin ligases suggest two mechanisms through which SCE could modulate DP53 levels: 1) SCE, as part of the PRC1 complex, could epigenetically act to regulate Dp53 transcription; and 2) SCE, as an E3 ubiquitin ligase, could control the stabilization of DP53 protein levels by promoting its ubiquitylation for proteaosomal degradation. Several observations combined with results from the experiments described in this study support a mechanism by which SCE inhibits apoptosis by promoting DP53 protein degradation. There is no evidence for the presence of Polycomb Response Elements (PREs) in the Dp53 gene, as SCE protein was not seen to bind to Dp53 genomic sequences in wing imaginal discs by ChiP experiments. Further, RNF2, the vertebrate SCE counterpart, has been shown to function as a p53 E3-ubiquitin ligase. The current results show that SCE requires Dp53 to inhibit apoptosis and that inactivation of Sce function increases DP53 protein levels as measured by the activation of a P53R-GFP reporter construct. Further, high levels of SCE produce no phenotype in wing cells, implying that it has no effect on transcription in wing disc cells. Importantly, the results indicate that high levels of SCE modulate apoptosis and DP53 protein levels. It would be interesting to know whether, and to which degree, each of the possible mechanisms contribute to the modulation of DP53 cellular levels mediated by high levels of SCE (Simoes da Silva, 2017).

In Drosophila, SCE function has been associated with protein mono-ubiquitylation rather than poly-ubiquitylation. Further studies are needed to determine the SCE function in the mono- or poly-ubiquitylation of DP53 in the fly. In vertebrates, RNF2 appears to have poly-ubiquitylation activity and, also in vertebrates, MDM2, the major E3-ligase for p53 poly-ubiquitylation, is able to mono- and poly-ubiquitylate p53. In the fly, these functions could be provided by either SCE itself or other E3-ligases acting on DP53, such as Bonus, Synoviolyn or CORP, or via through the cooperation of E3-ligases such as PSC, a member of the PRC1 complex known to have transcriptional independent E3-ligase activity on Cyclin B (Simoes da Silva, 2017).

This study has also shown that SCE is capable of controlling both hyperplasic growth induced by high levels of ABRUPT and neoplasic growth induced by inactivation of polyhomeotic function. Each of these is achieved by reducing apoptosis (Simoes da Silva, 2017).

Curiously neither high levels of DIAP1 nor inactivation of Dp53 function were capable of inhibiting apoptosis induced by either over-expression of ABRUPT or by inactivation of polyhomeotic. Thus, in these tumor conditions high levels of SCE efficiently inhibit apoptosis without inducing proliferation. Further studies will be needed to characterize the mechanisms underlying the SCE-dependent inhibition of apoptosis. In vertebrates, as elevated RNF2 levels have been found in many tumors, RNF2 has been proposed to function as an oncogene via its ability to decrease levels of p53. In Drosophila, high levels of SCE do not produce overgrowth during normal development or during tumoral growth. The results show that SCE acts to inhibit apoptosis in the fly, a function that may include oncogenic activity. This oncogenic activity may be based on the regulation of DP53 levels, as proposed for vertebrates. It appears that in Drosophila the mechanisms of DP53 regulation may also involve SCE, highlighting the evolutionarily conservation of these mechanisms and the utility of the fly for their study (Simoes da Silva, 2017).

GENE STRUCTURE

cDNA clone length - 1457 bp

Exons - 2

Bases in 3' UTR - 137

PROTEIN STRUCTURE

Amino Acids - 435

Structural Domains

Studies on the mammalian RING1 proteins suggest that the C-terminal domain of RING1B interacts with the C-terminal repressor domain of M33, the mammalian homologue of Drosophila Pc (Schoorlemmer, 1997). Similarly, in yeast two-hybrid assays, the RING finger of RING1B has been found to interact with the RING finger of Bmi-1, the orthologue of Psc in the mammalian PRC1 complex (Hemenway, 1998; Satijn, 1999; Levine, 2002). Hence, it appears that RING1 proteins contain two functional domains, an N-terminal RING finger domain that interacts with RING finger proteins such as Bmi-1 and a C-terminal domain that interacts with M33/Pc (Fritsch, 2003).

The comparison between the fly and murine Ring proteins revealed a high degree of conservation. Thus, the three domains (HD1, HD2 and HD3) identified in the murine (and human) proteins are also identified in the fly protein. These domains are separated, as in the mammalian proteins, by non-conserved sequences. Therefore, 78% of the 147 amino acids N-terminal domain (HD1), which contains a Ring finger, are identical between the fly and either of the murine Ring1 proteins. Conservation at the other two domains is lower: 53% and 60% identity with HD2 of Ring1/Ring1A and Rnf2/Ring1B, respectively, and 46% identity between Drosophila Ring HD3 and either HD3 of the murine Ring1 proteins. Curiously, the HD2 of Drosophila Ring is interrupted by a stretch of 11 amino acids (Gorfinkiel, 2004).

date revised: 30 June 2005

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