Sex combs extra
DEVELOPMENTAL BIOLOGY

To determine if Sce is developmentally regulated the spatial distribution of Sce transcript and protein was examined during development. In situ hybridization using a Sce cDNA probe showed a general maternal expression in syncytial blastoderm embryos. This ubiquitous expression is maintained until stage 11. However, by stage 13 of development Sce mRNA was restricted to the neuroectoderm with no expression in the epidermis. Later on, at stage 15 of development, Sce transcripts were detected only in the central nervous system (Gorfinkiel, 2004).

Expression pattern of the Sce protein was also analyzed using an anti-Sce antibody. In Western blots, this antibody recognizes predominantly a unique band, corresponding to the mobility of a 58 kDa polypeptide. In embryos, the same expression pattern of the Sce protein was observed as the one detected by in situ hybridization. However, some differences can be observed. For example, at stage 14 of development, although no transcripts are detected in the epidermis, some Sce protein is still present in the epidermis of anterior segments. This observation suggests that Sce translation or stability might be spatially regulated (Gorfinkiel, 2004).

Ubiquitous expression of Sce in all the imaginal discs was detected by either in situ hybridization or antibody staining. This expression in the imaginal discs is in agreement with the requirement of Sce function during all stages of larval development (Beuchle, 2001; Gorfinkiel, 2004).

PcG proteins are chromosomal proteins, which show binding to discrete euchromatic sites in polytene chromosomes. Many of these binding sites overlap among different PcG proteins. The distribution of Sce protein on salivary gland polytene chromosomes was examined. About 110 euchromatic sites of antibody staining were observed in the polytene chromosomes. Fifty-one of the 110 sites overlap with Pc/Ph/Pcl/Psc binding sites; 25 overlap with Pc/Ph/Pcl sites and 6 of them are common to the subset of unique Asx sites. Among the Sce sites are those of known targets of PcG genes, such as the Ant-C and Bx-C clusters. Thus, the extensive co-localization of Sce and other PcG proteins at many chromosomal sites is in agreement with Sce being a functional partner of other PcG proteins in Drosophila (Gorfinkiel, 2004).

Effects of Mutation or Deletion

In a genetic screen for new PcG mutants, a novel Sce allele was identified, Sce33M2, which does not complement the Sce1 allele previously identified by Breen (1986). The embryonic phenotype of Sce33M2 and Sce1 mutants were analyzed and compared by staining homozygous embryos with antibodies against the protein products of the homeotic gene Ultrabithorax (Ubx). In both mutants, Ubx is misexpressed, but misexpression is more widespread in Sce1 homozygotes than in Sce33M2 homozygotes (McKeon, 1991). In the case of most PcG genes, maternally deposited wild-type products rescue homozygous mutant embryos to a certain extent, and this is also true for Sce (Breen, 1986). To analyze embryos that lack maternally deposited wild-type Sce protein, Sce mutant embryos were generated from Sce germ line clones. Sce1 homozygous embryos obtained from Sce1 mutant germ cells show the extreme PcG phenotype reported previously (Breen, 1986). In contrast, Sce33M2 homozygotes derived from Sce33M2 mutant germ cells show less severe homeotic transformations and paternally rescued Sce33M2/+ animals develop into viable adult flies. Taken together, these results suggest that Sce33M2 is a weaker allele than Sce1 (Fritsch, 2003).

Sce mutant clones in imaginal discs were analyzed for misexpression of homeotic genes. Clones of Sce1 or Sce33M2 mutant cells were induced during the first larval instar and analyzed 96 h after clone induction. In these experiments, Sce mutant cells were identified by the absence of a GFP-expressing marker gene. Both Sce1 and Sce33M2 mutant clones show misexpression of Ubx and Abdominal-B (Abd-B) protein, although misexpression is slightly weaker in Sce33M2 mutant clones compared to Sce1 mutant clones. These results show that silencing of homeotic genes requires Sce function throughout development. The clonal analysis also supports the allele classification; Sce33M2 appears to be a weaker allele than Sce1(Fritsch, 2003).

By recombination mapping, Breen (1986) located Sce1 to region 3-92 on the genetic map. This map position approximately corresponds to the chromosomal interval harboring the Ring locus in 98A. A candidate gene approach was used to test whether Sce encodes Ring, and the Ring coding sequence was examined for molecular lesions. The Ring coding region was PCR-amplified from genomic DNA prepared from Sce1 and Sce33M2 mutants and sequenced. This analysis revealed that both alleles show a distinct molecular lesion. Sce1 carries a 410 bp deletion that removes the codons for the C-terminal 113 amino acids as well as the intron and 12 nucleotides of the 3′ untranslated region downstream of the termination codon. The Ring ORF in the Sce1 allele therefore encodes a truncated Ring protein (Ring1-322) that is fused in frame to 23 novel amino acid codons that are encoded by the 3' UTR. The predicted polyadenylation signal is still present in the Sce1 allele. In Sce33M2, a single base substitution was found that changes the codon for Arg65 in the middle of the RING finger into a codon for Cys. This arginine, immediately adjacent to the histidine in the C3HC4 Ring-finger, is conserved in the mammalian RING1A and RING1B proteins and in database searches no Ring finger protein was found containing a cysteine at this position. Substitution of Arg by Cys at this position might perturb the structure of the RING finger and/or interaction with other proteins. The slightly weaker phenotype of Sce33M2 mutants compared to Sce1 mutants suggests that this protein is probably expressed and provides at least some Sce+ function. Taken together, the molecular characterization of these two Sce alleles reveals that Sce encodes the Ring protein. This locus is therefore called Sce/Ring (Fritsch, 2003).

Reconstitution of stable Drosophila PRC1 in vitro has been shown to require the Ring protein (Francis, 2001). This study shows that Sce encodes Ring and, together with earlier studies, that mutations in Sce/Ring cause misexpression of homeotic genes that is as severe as misexpression caused by mutations in Psc, ph and Pc, the other three PRC1 core components. The extreme PcG phenotype of Sce mutants provides strong support for the idea that Sce/Ring protein is strictly required for formation and function of PRC1 in Drosophila. At present, it is not known how Sce/Ring integrates into PRC1 in Drosophila but 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. The molecular lesions in Sce1 and Sce33M2 and the strong PcG phenotypes observed in both mutants suggest that both domains are needed for PcG silencing, although, at present, it is not known whether the mutant proteins encoded by either of these two alleles are expressed as stable polypeptides in vivo. Formally, the possibility that Sce1 is not a null allele cannot be excluded, although the extreme PcG phenotype suggests that it may be a null mutation with respect to homoetic gene silencing (Fritsch, 2003).

The strong PcG phenotype of Sce/Ring mutants in Drosophila provides an interesting comparison with the phenotypes of Ring1 A and Ring1 B mutant mice. Ring1 A null mutant mice show anteriorly directed transformations, whereas most other mouse PcG mutants show posteriorly directed homeotic transformations and misexpression of HOX genes (del Mar Lorente, 2000). Thus, it is not clear whether Ring1 A is needed for HOX gene silencing. Mice that are homozygous for a null mutation in Ring1 B die during gastrulation, making it difficult to assess the requirement for Ring1B in HOX gene silencing (Voncken, 2003). However, recent studies show that mice carrying a hypomorphic Ring1 B mutation show very weak PcG phenotypes and subtle misexpression of HOX genes (Suzuki, 2002). This suggests that at least RING1B is indeed required for HOX gene silencing in vertebrates. In summary, the results of this study show that Sce encodes Ring and that, in Drosophila, the Sce/Ring protein is critically required for PcG silencing during embryonic and larval development (Fritsch, 2003).

Sce1/Sce1 embryos from Sce1/+ mothers (m+, z embryos) die as first instar larvae and show very weak posteriorly directed segmental transformation. In such larvae the ventral denticle belts of A7 develop with some A8 character (Breen, 1986). Sce1/Sce1 embryos derived from Sce1/Sce1 germ-line mutant females crossed to Sce1 males (m, z embryos) show extreme posteriorly directed segmental transformation. All the thoracic and abdominal segments are transformed to A8 and head involution is blocked (Breen, 1986). Moreover, these mutants showed an anterior de-repression of the homeotic gene products such as AbdB (Fritsch, 2003). To test whether Sce1 allele is a lack of function mutation, a deficiency that uncovered Sce locus was sought. All available deficiencies were examined at 98A, a region where Drosophila Ring was located. Df(3R)IR16, whose breakpoints include 97F1-2; 98A on the cytological map, is lethal over Sce1. In addition, homozygous Sce1/Sce1 germ-line mutant females crossed to Sce1 males produce embryos that have identical phenotypes than when crossed to Df(3R)IR16 males. This result indicates that Df(3R)IR16 is a genuine deficiency for the Sce locus and suggest that Sce1 is a null allele (Gorfinkiel, 2004).

To verify that Drosophila Ring is Sce, the phenotype of Sce1 (m, z) embryos was examined when Drosophila Ring/Sce was over-expressed using arm-GAL4 driver. A complete rescue of the embryonic phenotype was observed in such embryos, which are undistinguishable from wild type embryos. In the resulting embryonic population of the same experiment, there were also zygotic rescued embryos (m, z+) that have almost wild type phenotype. To unequivocally distinguish the Sce1 embryos rescued by ectopic Drosophila Ring/Sce from the rest of the embryonic derived population, UAS-Drosophila Ring/Sce (UAS-Sce) was ectopically expressed using the paired-Gal4 line, which induces ectopic expression in alternate segments. The areas of rescued cuticle in the Sce1 (m, z embryos); prd-Gal4/UAS-Sce embryos corresponded to those of prd expression domains. This rescue was visualized by the normalized T1 and T3 denticle belts and was also observed in anterior A2 and posterior A3 denticle belts. It was then asked whether the murine Ring1/Ring1A protein would substitute for the fly Sce protein. As before, Ring/Ring1A was expressed in Sce1 (m, z) embryos using the lines arm-Gal4 and prd-GAL4 as drivers, and a rescue of the Sce phenotype was observed similar to that seen with Drosophila Ring/Sce. These results further demonstrate that Sce locus encodes for the Drosophila ortholog of vertebrate Ring1/Rnf2 genes and that the function of the Ring proteins is conserved in mice and flies (Gorfinkiel, 2004).

Polycomb group genes are required for neural stem cell survival in postembryonic neurogenesis of Drosophila

Genes of the Polycomb group (PcG) are part of a cellular memory system that maintains appropriate inactive states of Hox gene expression in Drosophila. This study investigates the role of PcG genes in postembryonic development of the Drosophila CNS. Mosaic-based MARCM techniques were used to analyze the role of these genes in the persistent larval neuroblasts and progeny of the central brain and thoracic ganglia. Proliferation in postembryonic neuroblast clones is dramatically reduced in the absence of Polycomb, Sex combs extra, Sex combs on midleg, Enhancer of zeste or Suppressor of zeste 12. The proliferation defects in these PcG mutants are due to the loss of neuroblasts by apoptosis in the mutant clones. Mutation of PcG genes in postembryonic lineages results in the ectopic expression of posterior Hox genes, and experimentally induced misexpression of posterior Hox genes, which in the wild type causes neuroblast death, mimics the PcG loss-of-function phenotype. Significantly, full restoration of wild-type-like properties in the PcG mutant lineages is achieved by blocking apoptosis in the neuroblast clones. These findings indicate that loss of PcG genes leads to aberrant derepression of posterior Hox gene expression in postembryonic neuroblasts, which causes neuroblast death and termination of proliferation in the mutant clones. These findings demonstrate that PcG genes are essential for normal neuroblast survival in the postembryonic CNS of Drosophila. Moreover, together with data on mammalian PcG genes, they imply that repression of aberrant reactivation of Hox genes may be a general and evolutionarily conserved role for PcG genes in CNS development (Bello, 2007).

Genetic analysis indicates that the PcG genes Sce, Scm, Pc, E(z) and Su(z)12 are required for postembryonic neurogenesis in the central brain and thoracic ganglia of Drosophila. In the absence of any one of these genes, several mutant phenotypes are observed in the third-instar CNS: (1) neural proliferation is dramatically reduced and only small numbers of cells are found in neuroblast clones; (2) proliferating postembryonic neuroblasts are absent in most of the mutant clones due to apoptosis; (3) posterior Hox genes are ectopically expressed in the postembryonic neuroblast lineages. It is hypothesized that these phenotypes are causally related, in that loss of PcG genes leads to ectopic Hox gene expression in postembryonic neuroblasts resulting in their premature cell death and, thereby, in drastically reduced neuroblast lineage size. Strong support for this hypothesis is provided by the fact that the mutant lineages proliferate normally if apoptosis is blocked. Corollary support for this notion is provided by the fact that Psc-Su(z)2 mutant clones, which do not show ectopic Hox gene expression, are consistently wild-type-like in size and presence of neuroblast (Bello, 2007).

Numerous genes are required for the continued mitotic activity of neuroblasts during postembryonic life. These findings provide the first demonstration that PcG genes are essential for neuroblast survival and proliferation in the postembryonic CNS. Previous work on PcG gene action during embryonic neurogenesis has demonstrated that the derepression of posterior Hox genes in PcG mutants leads to a change in the segmental determination of neuroblasts and their lineage, but not to their mitotic arrest and death. Thus, the effects of PcG gene loss on neurogenesis are context-dependent and differ during embryonic development as compared with postembryonic development. This is underscored in recent work which indicates that the PcG gene ph is essential for maintaining neuronal identity and diversity during metamorphosis (Bello, 2007).

In postembryonic development of the Drosophila CNS, a remarkable link exists between neuroblast survival and Hox gene expression. In the ventral ganglia, a neuroblast-specific pulse of abd-A during the third instar provides the cue for cell death, which limits the number of progeny produced per neuroblast. These data indicate that this mechanism, which in the wild type relates Hox gene expression to the clone size of neural stem cells, also operates in PcG mutants and is responsible for the PcG mutant phenotypes. Indeed, a general function of PcG genes in postembryonic neurogenesis may be to prevent the premature and widespread operation of this mechanism for temporal regulation of neurogenesis through termination of neuroblast life. It is noteworthy that the Hox gene-dependent activation of apoptosis within the CNS is selective for the neuroblast and does not occur when Hox genes are derepressed in neurons, either during normal development or in misexpression experiments. This explains why the neurons in PcG mutant clones, which were generated before the induction of neuroblast cell death, continue to survive despite the presence of ectopic Hox gene derepression (Bello, 2007).

These data indicate that loss of specific PcG genes in larval neuroblasts leads to ectopic Hox gene expression that is sufficient to cause neuroblast cell death. However, the PcG proteins may also contribute to neuroblast survival by repressing other unidentified target genes which, when derepressed, might result in premature death of postembryonic neuroblasts. Indeed, although deregulation of Hox gene expression is one of the hallmarks of PcG phenotypes in Drosophila, a diverse set of other target genes, including genes involved in cell cycle regulation, are controlled by PcG genes (Bello, 2007).

Interesting parallels to these findings on the role of PcG genes in neural proliferation come from studies of mammalian PcG genes, specifically of the Bmi1 gene. Bmi1 mutant mice develop ataxia, seizures and tremors in early postnatal life, and display a significant reduction in overall brain size, which is particularly severe in the granular and molecular layers of the cerebellum. Strikingly, Bmi1-deficent mice become depleted of cerebellar neural stem cells postnataly, indicating an in vivo requirement for Bmi1 in neural stem cell renewal. Bmi1 deficiency leads to increased expression of the cell cycle regulators p16Ink-4a and p19Arf (both now known as Cdkn2a - Mouse Genome Informatics), and the neurogenesis defect in the mutant mice can be partially rescued by further deleting p16Ink4a. This suggests that one way in which Bmi1 promotes the maintenance of adult stem cells is by repressing the p16Ink4a pathway. However, it is also likely that Hox gene repression through Bmi1 is involved in this process, given that loss of Bmi1 has been shown to cause a deregulation of posterior Hox gene expression in neural stem cells in vitro. Moreover, a direct molecular link between Bmi1 and Hox gene regulation has recently been discovered in mammalian development, in that the promyelocytic leukemia zinc finger (Plzf; Zbtb16 -- Mouse Genome Informatics) protein directly binds Bmi1 and recruits PcG proteins in the HoxD cluster (Bello, 2007 and references therein).

In Drosophila, the homologs of the mammalian Bmi1 gene are the PcG genes Psc and Su(z)2. Psc and Su(z)2 encode very similar proteins and are partially redundant in function, but both genes are eliminated in a deletion in the Psc-Su(z)2 line. Rather surprisingly, mutational loss of Psc-Su(z)2 does not lead to ectopic Hox gene derepression and, in consequence, does not appear to affect neuronal proliferation in the postembryonic CNS of Drosophila. This is in stark contrast to the other five PcG genes investigated, which do play important roles in proliferation control by preventing ectopic Hox gene expression and cell death in postembryonic neuroblasts. The discrepancy between murine Bmi1 and Drosophila Psc-Su(z)2 function in neuronal proliferation suggests that although a general role of PcG genes in neuronal proliferation control may be conserved between mammals and flies, conservation of gene action may not always be retained at the level of individual PcG homologs (Bello, 2007).

In terms of overall development, it is clear that one and the same PcG gene can have very different functions depending on the developmental context in which it acts. For example, as mentioned above, during embryonic neurogenesis the Drosophila Pc gene acts in tagmata-specific differentiation of neuroblasts, in contrast to its role in postembryonic neurogenesis. Moreover, in postembryonic development of imaginal discs, deletions in the Drosophila Psc-Su(z)2 genes have been shown to result in cellular hyperproliferation, which contrasts with the lack-of-proliferation phenotype of Psc-Su(z)2 mutants in postembryonic development of the CNS. Similarly, in the mouse, the Bmi1 gene has been implicated in tumor progression in mantle cell lymphoma, colorectal cancer, liver carcinomas and non-small cell lung cancer, in addition to its role in nervous system development. Nevertheless, in all of the Drosophila and mammalian phenotypes mentioned, deregulation of Hox gene expression appears to be one of the conserved and thus unifying features of PcG gene functional loss (Bello, 2007).

The role of the histone H2A ubiquitinase Sce in Polycomb repression

Polycomb group (PcG) proteins exist in multiprotein complexes that modify chromatin to repress transcription. Drosophila PcG proteins Sex combs extra (Sce; dRing) and Posterior sex combs (Psc) are core subunits of PRC1-type complexes. The Sce:Psc module acts as an E3 ligase for monoubiquitylation of histone H2A, an activity thought to be crucial for repression by PRC1-type complexes. This study created an Sce knockout allele and showed that depletion of Sce results in loss of H2A monoubiquitylation in developing Drosophila. Genome-wide profiling identified a set of target genes co-bound by Sce and all other PRC1 subunits. Analyses in mutants lacking individual PRC1 subunits reveals that these target genes comprise two distinct classes. Class I genes are misexpressed in mutants lacking any of the PRC1 subunits. Class II genes are only misexpressed in animals lacking the Psc-Su(z)2 and Polyhomeotic (Ph) subunits but remain stably repressed in the absence of the Sce and Polycomb (Pc) subunits. Repression of class II target genes therefore does not require Sce and H2A monoubiquitylation but might rely on the ability of Psc-Su(z)2 and Ph to inhibit nucleosome remodeling or to compact chromatin. Similarly, Sce does not provide tumor suppressor activity in larval tissues under conditions in which Psc-Su(z)2, Ph and Pc show such activity. Sce and H2A monoubiquitylation are therefore only crucial for repression of a subset of genes and processes regulated by PRC1-type complexes. Sce synergizes with the Polycomb repressive deubiquitinase (PR-DUB) complex to repress transcription at class I genes, suggesting that H2A monoubiquitylation must be appropriately balanced for their transcriptional repression (Gutiérrez, 2012).

This study analyzed how PRC1 regulates target genes in Drosophila to investigate how the distinct chromatin-modifying activities of this complex repress transcription in vivo. Because H2A monoubiquitylation is thought to be central to the repression mechanism of PRC1-type complexes, focus was placed on the role of Sce. The following main conclusions can be drawn from the work reported in this study. First, in the absence of Sce, bulk levels of H2A-K118ub1 are drastically reduced but the levels of the PRC1 subunits Psc and Ph are undiminished. Sce is therefore the major E3 ligase for H2A monoubiquitylation in developing Drosophila but is not required for the stability of other PRC1 subunits. Second, PRC1-bound genes fall into two classes. Class I target genes are misexpressed if any of the PRC1 subunits is removed. Class II target genes are misexpressed in the absence of Ph or Psc-Su(z)2 but remain stably repressed in the absence of Sce or Pc. At class II target genes, Ph and the Psc-Su(z)2 proteins work together to repress transcription by a mechanism that does not require Sce and Pc and is therefore independent of H2A monoubiquitylation. Third, removal of the Ph, Psc-Su(z)2 or Pc proteins results in imaginal disc tumors that are characterized by unrestricted cell proliferation. However, removal of Sce does not cause this phenotype, suggesting that this tumor suppressor activity by the PcG system does not require H2A monoubiquitylation. Finally, these analyses reveal that PRC1 subunits are essential for repressing the elB, noc, dac and pros genes outside of their normal expression domains in developing Drosophila. This expands the inventory of developmental regulator genes in Drosophila for which PcG repression has been demonstrated in a functional assay (Gutiérrez, 2012).

In the Sce33M2 allele Arg65 is mutated to Cys, but this mutant Sce protein is undetectable and therefore does not appear to be stable in vivo. The crystal structure of the Ring1B-Bmi1 complex provides a molecular explanation for this observation: the Arg70 residue in Ring1B that corresponds to Arg65 in Sce is thought to be critical for interaction with Bmi1. A likely scenario therefore is that the SceArg65Cys protein in Drosophila is unstable and is degraded because it is unable to associate with Psc or its paralog Su(z)2. Interestingly, removal of Sce protein has no detectable effect on the levels of the Psc and Ph proteins. Psc is therefore stable in the absence of its binding partner Sce. This is in contrast to the situation in mice in which Ring1B mutant ES cells show a drastic reduction in the levels of the Ring1B partner protein Bmi1 and its paralog Mel18 (Pcgf62) and also a reduction in the levels of Mph2 (Phc2) and Mpc2 (Cbx4) (Leeb, 2007). The interdependence between PRC1 subunits for protein stability is therefore different in mammals and Drosophila (Gutiérrez, 2012).

Reconstitution of the Drosophila PRC1 core complex in a baculovirus expression system suggests that Sce is important for complex stability. At present, it is not know whether the Psc, Ph and Pc proteins still form a complex in vivo in the absence of Sce. It is currently unknown whether Psc, Ph and Pc are still bound to all PRC1 target genes in the absence of Sce. However, the finding that class II genes remain repressed in the absence of Sce, even though their repression depends on Psc-Su(z)2 and Ph, argues against a crucial role of Sce in the targeting of these other PRC1 subunits to these genes. Interestingly, the repression of all class II target genes analyzed in this study always requires both the Ph and the Psc-Su(z)2 proteins. A possible explanation for this observation is that Ph and Psc-Su(2) still form a PRC1 subcomplex in the absence of Sce and that this complex is fully functional to repress class II target genes. Alternatively, it is possible that Ph and Psc-Su(z)2 repress class II target genes as components of as yet uncharacterized complexes that are distinct from PRC1 and Drosophila dRing-associated factors (dRAF) complex (Gutiérrez, 2012).

In vitro, Psc and Su(z)2 proteins compact nucleosome templates, inhibit nucleosome remodeling by SWI/SNF complexes and repress transcription on chromatin templates. The observation that repression of class II target genes requires Psc-Su(z)2 and Ph but not Pc and Sce supports the idea that the chromatin-modifying activities of Psc-Su(z)2 identified in vitro are the main mechanism by which PRC1 represses these genes. Previous structure/function analyses in Drosophila showed that the same domains of the Psc protein responsible for chromatin compaction and remodeling inhibition in vitro are crucial for HOX gene repression in vivo. Chromatin modification by Psc and Su(z)2 is therefore also crucial for repression of class I target genes. Regulation of the class I target gene en further illustrates this point. In some tissues (e.g. in the dorsal hinge region of the wing imaginal disc) repression of en requires all PRC1 core subunits, but in other tissues (e.g. in the pouch of the wing imaginal disc) en remains repressed in the absence of Sce and Pc, and only Psc-Su(z)2 and Ph seem to be crucial to keep the gene inactive. At present, the molecular mechanism of Ph is not well understood. In vitro, Ph protein has the capacity to inhibit chromatin remodeling and transcription but it does so less effectively than Psc. At the target genes analyzed in this study, Ph is required for transcriptional repression wherever Psc-Su(z)2 is required, suggesting that Ph and Psc-Su(z)2 act in concert in this repression. Nevertheless, it is possible that repression of other PRC1 target genes requires a different subset of PRC1 subunits, or that, as in the case of en, the subunit requirement for repression changes depending on the cell type (Gutiérrez, 2012).

In mammals, Ring1B and Ring1A are responsible for the bulk of H2A-K119 monoubiquitylation. Similarly, Sce generates the bulk of H2A-K118 monoubiquitylation in Drosophila, both in tissue culture cells (Lagarou, 2008) and in the developing organism (this study). The requirement for Sce at class I target genes is consistent with the idea that H2A monoubiquitylation of their chromatin is part of the repression mechanism. Repression of a subset of class I genes, namely the HOX genes, also requires the H2A deubiquitinase PR-DUB (Gayt├ín de Ayala Alonso, 2007; Scheuermann, 2010). Moreover, PR-DUB and Sce strongly synergize to repress HOX genes. Specifically, the phenotype of Sce PR-DUB double mutants suggests that H2A monoubiquitylation becomes ineffective for HOX gene repression if PR-DUB is absent. However, embryos that lack PR-DUB alone show a 10-fold increase in the bulk levels of H2A-K118ub1 and it is estimated that ~10% of all H2A molecules become monoubiquitylated in these animals. How could this conundrum be explained? One possibility is that H2A monoubiquitylation and deubiquitylation at HOX gene chromatin need to be regulated in a precisely balanced manner. However, an alternative explanation considers H2A-K118ub1 levels in the context of ubiquitin homeostasis. In particular, the high H2A-K118ub1 levels in PR-DUB mutants suggest that Sce generates widespread H2A monoubiquitylation at most Sce-bound genes and possibly also elsewhere in the genome, but that in wild-type animals PR-DUB continuously deubiquitylates H2A-K118ub1 at these locations and thereby recycles ubiquitin. The observation that PR-DUB is widely co-bound with Sce, not only at HOX but also at many other class I and class II target genes, is consistent with this idea. It is tempting to speculate that the widespread H2A monoubiquitylation in PR-DUB mutants sequesters a substantial fraction of the pool of free ubiquitin. It is therefore possible that removal of PR-DUB effectively depletes the pool of free ubiquitin in the nucleus to an extent that H2A monoubiquitylation at HOX target genes becomes inefficient and, consequently, their repression can no longer be maintained. According to this model, the crucial function of PR-DUB would not be the deubiquitylation of H2A-K118ub1 at HOX genes but rather at class II target genes and elsewhere in the genome where Sce 'wastefully' monoubiquitylates H2A (Gutiérrez, 2012).

Polycomb group genes are required to maintain a binary fate choice in the Drosophila eye

Identifying the mechanisms by which cells remain irreversibly committed to their fates is a critical step toward understanding and being able to manipulate development and homeostasis. Polycomb group (PcG) proteins are chromatin modifiers that maintain transcriptional silencing, and loss of PcG genes causes widespread derepression of many developmentally important genes. However, because of their broad effects, the degree to which PcG proteins are used at specific fate choice points has not been tested. To understand how fate choices are maintained, R7 photoreceptor neuron development has been examined in the fly eye. R1, R6, and R7 neurons are recruited from a pool of equivalent precursors. In order to adopt the R7 fate, these precursors make three binary choices. They: (1) adopt a neuronal fate, as a consequence of high receptor tyrosine kinase (RTK) activity (they would otherwise become non-neuronal support cells); (2) fail to express Seven-up (Svp), as a consequence of Notch (N) activation (they would otherwise express Svp and become R1/R6 neurons); and (3) fail to express Senseless (Sens), as a parallel consequence of N activation (they would otherwise express Sens and become R8 neurons in the absence of Svp). PcG genes were removed specifically from post-mitotic R1/R6/R7 precursors, allowing these genes' roles to be probed in the three binary fate choices that R1/R6/R7 precursors face when differentiating as R7s. This study shows that loss of the PcG genes Sce, Scm, or Pc specifically affects one of the three binary fate choices that R7 precursors must make: mutant R7s derepress Sens and adopt R8 fate characteristics. This fate transformation occurs independently of the PcG genes' canonical role in repressing Hox genes. While N initially establishes Sens repression in R7s, this study shows that N is not required to keep Sens off, nor do these PcG genes act downstream of N. Instead, the PcG genes act independently of N to maintain Sens repression in R1/R6/R7 precursors that adopt the R7 fate. It is concluded that cells can use PcG genes specifically to maintain a subset of their binary fate choices (Finley, 2015).

Identifying the mechanisms by which cells remain irreversibly committed to their fates is a critical step toward understanding and being able to manipulate development and homeostasis. Polycomb group (PcG) proteins are chromatin modifiers that maintain transcriptional silencing, and loss of PcG genes causes widespread derepression of many developmentally important genes. However, because of their broad effects, the degree to which PcG proteins are used at specific fate choice points has not been tested. To understand how fate choices are maintained, R7 photoreceptor neuron development has been examined in the fly eye . R1, R6, and R7 neurons are recruited from a pool of equivalent precursors. In order to adopt the R7 fate, these precursors make three binary choices. They: (1) adopt a neuronal fate, as a consequence of high receptor tyrosine kinase (RTK) activity (they would otherwise become non-neuronal support cells); (2) fail to express Seven-up (Svp) , as a consequence of Notch (N) activation (they would otherwise express Svp and become R1/R6 neurons); and (3) fail to express Senseless (Sens) , as a parallel consequence of N activation (they would otherwise express Sens and become R8 neurons in the absence of Svp). PcG genes were removed specifically from post-mitotic R1/R6/R7 precursors, allowing these genes' roles to be probed in the three binary fate choices that R1/R6/R7 precursors face when differentiating as R7s. This study shows that loss of the PcG genes Sce , Scm , or Pc specifically affects one of the three binary fate choices that R7 precursors must make: mutant R7s derepress Sens and adopt R8 fate characteristics. This fate transformation occurs independently of the PcG genes' canonical role in repressing Hox genes. While N initially establishes Sens repression in R7s, this study shows that N is not required to keep Sens off, nor do these PcG genes act downstream of N. Instead, the PcG genes act independently of N to maintain Sens repression in R1/R6/R7 precursors that adopt the R7 fate. It is concluded that cells can use PcG genes specifically to maintain a subset of their binary fate choices (Finley, 2015).

The GMR-FLP/MARCM system allowed allowed the removal of Sce and Scm function specifically from post-mitotic R1/R6/R7 precursors, allowing these genes' roles to be probed in the limited number of binary fate choices that R1/R6/R7 precursors face. In order to adopt the R7 fate, these precursors must choose to: (1) become neurons in response to high RTK activity-they would otherwise become non-neuronal cells; (2) fail to express Svp in response to N activity-they would otherwise become R1/R6s; and (3) fail to express Sens in response to N activity-they would otherwise become R8s. Loss of Sce or Scm from R7s specifically was found to compromises maintenance of the last of these choices. By contrast, no evidence was found that PcG genes maintain either of the other two choices. Sce mutant R7s were examined throughout larval and pupal development and none were found none misexpressed Svp, nor were Sce or Scm mutant R7s that displayed other R1/R6 characteristics found, such as large rhabdomeres positioned at the periphery of the ommatidium or expression of the R1-R6-specific rhodopsin Rh1. While loss of the Abelson kinase was recently shown to cause R neurons to lose expression of the neuronal marker Elav and switch to a non-neuronal pigment cell fate, this study found that Sce and Scm mutant R1/R6s and R7s maintain expression of Elav and the photoreceptor-specific protein Chaoptin, indicating that their commitment to a neuronal fate is also independent of PcG gene function. It is concluded that R7s use Sce and Scm to maintain repression of one but not all alternative binary fate choices (Finley, 2015).

The Sens-encoding region is bound by Pc in Drosophila embryos and by Sce in Drosophila larvae , suggesting that Sens is directly regulated by these proteins in at least some cell types. However, because of the technical difficulty in isolating sufficient quantities of chromatin specifically from R7 cells, it was not possible to determine whether PcG proteins bind the Sens locus in R7s. It remains possible, therefore, that Sce, Scm, and Pc maintain Sens repression indirectly in R7s-however, the evidence suggests that they do so independently of their canonical role in repressing Hox genes (Finley, 2015).

Considerable differences were observed in the strengths of the R7 defects caused by loss of Sce, Scm, Pc, or Psc. One possibility is that these proteins do not contribute equally to PRC1's gene-silencing ability. Indeed, the fly genome contains a second Psc-related gene that plays a redundant role with Psc in some cells, possibly accounting for the lack of defect in Psc mutant R7s. Alternatively, the different wild-type PcG proteins may perdure to different degrees within the mutant R7 clones (the cells that divide to generate the mutant R1/R6/R7 precursors contain a wild-type copy of the mutant gene). Attempts were made, but it was not possible to measure the time course of Sce and Scm protein levels in Sce and Scm mutant R7s, respectively, to test their perdurance directly. However, this thought that perdurance is likely, as this study found that Gal80 perdures until early pupal development within GMR-FLP/MARCM-induced R7 clones (Finley, 2015).

Sce and Scm were found to be required to maintain Sens repression in R7s generated either in the presence or absence of N activity. What might be regulating the deployment of Sce and Scm in these cells? One possibility is that Sce and Scm repress Sens in R1/R6/R7 precursors by default, since these cells never normally express Sens. However, it was found that neither Sce nor Scm is required to maintain the repression of Sens that is established by Svp. Alternatively, Sce and Scm may be deployed to repress Sens as part of a cell's initial commitment to the R7 fate. As mentioned above, wild-type Sce or Scm protein is likely to perdure in newly created homozygous Sce or Scm mutant R7s, respectively, leaving open the possibility that these genes are required not only for the maintenance but also for the establishment of the R7 fate. Previous work showed that the NF-YC subunit of the heterotrimeric transcription factor nuclear factor Y (NF-Y) is also required to maintain Sens repression in R7s. Like the PcG proteins, NF-YC is broadly expressed in all photoreceptor neurons and is not sufficient to cause R7s to adopt R8 fates, indicating that NF-YC is not responsible for the specific role of PcG proteins in R7s. However, the resemblance between the R7 defects caused by loss of Sce, Scm, and NF-YC suggests that NF-Y may participate in PRC1 function. In support of this possibility, loss of the NF-YA subunit from Caenorhabditis elegans also causes defects similar to those caused by loss of the PcG gene sop-2, including derepression of the Hox gene egl-5 (Finley, 2015).

PcG proteins have been shown to silence many regulators of development in addition to their canonical Hox targets, suggesting that PcG proteins are likely to play broad roles in maintaining cell fate commitments. However, whether PcG proteins are used to maintain specific binary fate choices as cells differentiate is unclear. In fact, the opposite is true during stem cell differentiation, when the repression of terminal differentiation genes by PcG proteins must instead be relieved. This paper has have identified a role for PRC1-associated PcG proteins in maintaining a specific binary fate choice made during adoption of the R7 fate-a choice that does not involve Hox gene regulation or misregulation. The same PRC1-associated proteins are not required to maintain two other binary fate choices that R7s must make. It is concluded that PcG genes are indeed used to maintain some though not all binary fate choices (Finley, 2015).


REFERENCES

Aggarwal, B. D. and Calvi, B. R. (2004). Chromatin regulates origin activity in Drosophila follicle cells. Nature 430: 372-376. PubMed Citation: 15254542

Alder, O., et al. (2010). Ring1B and Suv39h1 delineate distinct chromatin states at bivalent genes during early mouse lineage commitment. Development 137(15): 2483-92. PubMed Citation: 20573702

Balicky, E. M., Young, L., Orr-Weaver, T. L. and Bickel, S. E. (2004). A proposed role for the Polycomb group protein dRING in meiotic sister-chromatid cohesion. Chromosoma 112(5): 231-9. 14669021

Bardos, J. I., Saurin, A. J., Tissot, C., Duprez, E. and Freemont, P. S. (2000). HPC3 is a new human polycomb orthologue that interacts and associates with RING1 and Bmi1 and has transcriptional repression properties. J. Biol. Chem. 275(37): 28785-92. 1082516

Bello, B., Holbro, N. and Reichert, H. (2007). Polycomb group genes are required for neural stem cell survival in postembryonic neurogenesis of Drosophila. Development 134(6): 1091-9. Medline abstract: 17287254

Beuchle, D., Struhl, G. and Muller, J.(2001). Polycomb group proteins and heritable silencing of Drosophila Hox genes. Development 128 (2001), pp. 993-1004. Medline abstract: 14669021

Breen, T. R. and Duncan, I. M. (1986). Maternal expression of genes that regulate the bithorax complex of Drosophila melanogaster. Dev. Biol. 118: 442-456. 3098596

del Mar Lorente, M., et al. (2000). Loss- and gain-of-function mutations show a Polycomb group function for Ring1A in mice. Development 127: 5093-5100. 11060235

de Napoles, M., et al. (2004). Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation. Dev. Cell 7: 663-676. Medline abstract: 15525528

Fereres, S., Simon, R., Mohd-Sarip, A., Verrijzer, C. P. and Busturia, A. (2014). dRYBP counteracts chromatin-dependent activation and repression of transcription. PLoS One 9: e113255. PubMed ID: 25415640

Finley, J. K., Miller, A. C. and Herman, T. G. (2015). Polycomb group genes are required to maintain a binary fate choice in the Drosophila eye. Neural Dev 10: 2. PubMed ID: 25636358

Francis, N. J., Saurin, A. J., Shao, Z. and Kingston, R. E. (2001). Reconstitution of a functional core polycomb repressive complex. Mol. Cell 8: 545-556. 11583617

Francis, N. J., et al. (2009). Polycomb proteins remain bound to chromatin and DNA during DNA replication in vitro. Cell 137(1): 110-22. PubMed Citation: 19303136

Fritsch, C., Beuchle, D. and Muller, J. (2003). Molecular and genetic analysis of the Polycomb group gene Sex combs extra/Ring in Drosophila. Mech Dev. 120(8): 949-54. 12963114

Fujimura, Y., et al. (2006). Distinct roles of Polycomb group gene products in transcriptionally repressed and active domains of Hoxb8. Development 133: 2371-2381. Medline abstract: 16687444

Gaytán de Ayala Alonso, A., et al. (2007). A genetic screen identifies novel polycomb group genes in Drosophila. Genetics 176: 2099-2108. PubMed Citation: 17717194

Gutiérrez, L., et al. (2012). The role of the histone H2A ubiquitinase Sce in Polycomb repression. Development 139(1): 117-27. PubMed Citation: 22096074

Gil, J., Bernard, D., Martinez, D. and Beach, D. (2004). Polycomb CBX7 has a unifying role in cellular lifespan. Nat. Cell Biol. 6(1): 67-7. 14647293

Gorfinkiel, N., Fanti, L., Melgar, T., Garcia, E., Pimpinelli, S., Guerrero, I. and Vidal, M. (2004). The Drosophila Polycomb group gene Sex combs extra encodes the ortholog of mammalian Ring1 proteins. Mech. Dev. 121(5): 449-62. 15147763

Hemenway, C.S., Halligan, B.W. and Levy, L.S. (1998). The Bmi-1 oncoprotein interacts with dinG and MPh2: the role of RING finger domains. Oncogene 16: 2541-2547. 9627119

Hirabayashi, Y., et al. (2009). Polycomb limits the neurogenic competence of neural precursor cells to promote astrogenic fate transition. Neuron 63(5):600-13. PubMed Citation: 19755104

Kondo, T., Isono, K., Kondo, K., Endo, T. A., Itohara, S., Vidal, M. and Koseki, H. (2014). Polycomb potentiates meis2 activation in midbrain by mediating interaction of the promoter with a tissue-specific enhancer. Dev Cell 28: 94-101. PubMed ID: 24374176

Lagarou, A., et al. (2008). dKDM2 couples histone H2A ubiquitylation to histone H3 demethylation during Polycomb group silencing. Genes Dev. 22: 2799-2810. PubMed Citation: 18923078

Langlais, K. K., Brown, J. L. and Kassis, J. A. (2012). Polycomb group proteins bind an engrailed PRE in both the 'ON' and 'OFF' transcriptional states of engrailed. PLoS One 7: e48765. PubMed ID: 23139817

Lee, M. G., Norman, J., Shilatifard, A. and Shiekhattar, R. (2007). Physical and functional association of a trimethyl H3K4 demethylase and Ring6a/MBLR, a polycomb-like protein. Cell 128(5): 877-87. PubMed citation: 17320162

Leeb, M. and Wutz, A. (2007). Ring1B is crucial for the regulation of developmental control genes and PRC1 proteins but not X inactivation in embryonic cells. J. Cell Biol. 178: 219-229. PubMed Citation: 17620408

Leeb, M., et al. (2010). Polycomb complexes act redundantly to repress genomic repeats and genes. Genes Dev. 24(3): 265-76. PubMed Citation: 20123906

Levine, S.S., Weiss, A., Erdjument-Bromage, H., Shao, Z., Tempst, P. and Kingston, R.E. (2002). The core of the Polycomb repressive complex is compositionally and functionally conserved in flies and humans. Mol. Cell. Biol. 22: 6070-6078. 12167701

Marchetti, M., et al. (2003). Differential expression of the Drosophila BX-C in polytene chromosomes in cells of larval fat bodies: a cytological approach to identifying in vivo targets of the homeotic Ubx, Abd-A and Abd-B proteins. Development 130: 3683-3689. PubMed Citation: 12835385

McKeon, J. and Brock, H. W. (1991). Interactions of the Polycomb group of genes with homeotic loci of Drosophila. Roux's Arch. Dev. Biol. 199: 387-396

Morimoto-Suzki, N., Hirabayashi, Y., Tyssowski, K., Shinga, J., Vidal, M., Koseki, H. and Gotoh, Y. (2014). The polycomb component Ring1B regulates the timed termination of subcerebral projection neuron production during mouse neocortical development. Development 141: 4343-4353. PubMed ID: 25344075

Moshkin, Y. M., et al. (2001). The Bithorax complex of Drosophile melanogaster: underreplication and morphology in polytene chromosomes. Proc. Natl. Acad. Sci. 98: 570-574. PubMed Citation: 11136231

Qin, H., Wang, J., Liang, Y., Taniguchi, Y., Tanigaki, K. and Han, H. (2004). RING1 inhibits transactivation of RBP-J by Notch through interaction with LIM protein KyoT2. Nucleic Acids Res. 32(4): 1492-501. 14999091

Satijn, D. P., Gunster, M. J., van der Vlag, J., Hamer, K. M., Schul, W., Alkema, M. J. et al. (1997). RING1 is associated with the Polycomb group protein complex and acts as a transcriptional repressor. Mol. Cell. Biol. 17: 4105-4113. 9315667

Satijn, D. P. and Otte, A. P. (1999). RING1 interacts with multiple Polycomb-group proteins and displays tumorigenic activity. Mol. Cell. Biol. 19: 57-68. 9858531

Saurin, A. J., Shao, Z., Erdjument-Bromage, H., Tempst, P. and Kingston, R. E. (2001). A Drosophila Polycomb group complex includes Zeste and dTAFII proteins. Nature 412: 655-660. 11493925

Schoorlemmer, J., Marcos-Gutierrez, C., Were, F., Martinez, R., Garcia, E., Satijn, D.P. et al. (1997). Ring1A is a transcriptional repressor that interacts with the Polycomb-M33 protein and is expressed at rhombomere boundaries in the mouse hindbrain. Eur. Mol. Biol. Org. J. 16: 5930-5942. 9312051

Scheuermann, J. C., et al. (2010). Histone H2A deubiquitinase activity of the Polycomb repressive complex PR-DUB. Nature 465: 243-247. PubMed Citation: 20436459

Simoes da Silva, C. J., Fereres, S., Simon, R. and Busturia, A. (2017). Drosophila SCE/dRING E3-ligase inhibits apoptosis in a Dp53 dependent manner. Dev Biol 29(1): 81-91. PubMed ID: 28712876

Shao, Z., Raible, F., Mollaaghababa, R., Guyon, J. R., Wu, C. T., Bender, W. and Kingston, R. E. (1999). Stabilization of chromatin structure by PRC1, a Polycomb complex. Cell 98: 37-46. 10412979

Smith, M., Mallin, D. R., Simon, J. A. and Courey, A. J. (2011). Small ubiquitin-like modifier (SUMO) conjugation impedes transcriptional silencing by the polycomb group repressor Sex Comb on Midleg. J. Biol. Chem. 286(13): 11391-400. PubMed Citation: 21278366

Suzuki, M., et al. (2002). Involvement of the polycomb-group gene Ring1B in the specification of the anterior-posterior axis in mice. Development 129. 4171-4183. 12183370

Tuckfield, A., et al. (2002). Binding of the RING polycomb proteins to specific target genes in complex with the grainyhead-like family of developmental transcription factors. Mol. Cell. Biol. 22: 1936-1946. 11865070

van der Velden, Y. U., Wang, L., Querol Cano, L. and Haramis, A. P. (2013). The polycomb group protein ring1b/rnf2 is specifically required for craniofacial development. PLoS One 8: e73997. PubMed ID: 24040141

Voncken, J. W., et al. (2003). Rnf2 (Ring1b) deficiency causes gastrulation arrest and cell cycle inhibition. Proc. Natl. Acad. Sci. 100: 2468-2473. 12589020

Wang, H., Wang, L., Erdjument-Bromage, H., Vidal, M., Tempst, P., Jones, R. S. and Zhang, Y. (2004). Role of histone H2A ubiquitination in Polycomb silencing. Nature 431: 873-878. Medline abstract: 15386022


Sex combs extra: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 22 December 2017

Home page: The Interactive Fly © 2003 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.