InteractiveFly: GeneBrief

ebi: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

ebi (the Japanese term for 'shrimp') regulates the Epidermal growth factor receptor signaling pathway at multiple steps in Drosophila development. Mutations in ebi and Egfr lead to similar phenotypes and show genetic interactions. However, ebi does not show genetic interactions with other RTKs (e.g., torso) or with components of the canonical Ras/MAP kinase pathway. ebi encodes an evolutionarily conserved protein with a unique amino terminus, distantly related to F-box sequences, and six tandemly arranged carboxy-terminal WD40 repeats. The existence of closely related proteins in yeast, plants, and humans suggests that ebi functions in a highly conserved biochemical pathway. Proteins with related structures regulate protein degradation. Similarly, in the developing eye, ebi promotes Egfr-dependent down-regulation of Tramtrack88, an antagonist of neuronal development (Dong, 1999).

The similarity of Ebi to F-box/WD40 repeat-containing proteins and its nuclear localization suggests that Ebi may regulate Egfr signaling through degradation of nuclear proteins. Recent studies have revealed an important role for both Egfr and degradation of a specific transcription factor Tramtrack88, for R7 development. The structural similarity between Ebi and F-box/WD40-repeat proteins involved in protein degradation prompted an exploration of the relationship between ebi and Ttk88 protein levels in the developing eye. Ttk88 is expressed at very low levels in undifferentiated cells in the developing eye disc and at high levels in developing cone cell nuclei; it is not expressed in developing photoreceptor cells. The protein Phyllopod, promotes the degradation of Tramtrack. Transformation of cone cells into R7 by misexpression of phyllopod under the sevenless promoter leads to Ttk88 degradation. Ectopic R7 induction by constitutively active Egfr (Tor^DEgfr) driven by the sev promoter also leads to marked degradation of Ttk88. sev-Tor^DEgfr-induced Ttk88 degradation is dominantly suppressed by ebi. Similarly, ebi dominantly suppresses the pGMR-phyl-induced decrease in Ttk88, as well as the pGMR-phyl-induced eye phenotype (Dong, 1999).

The role of ebi in regulating Ttk88 levels in an otherwise wild-type eye disc was examined. Analysis of Ttk88 levels in the small mutant clones generated with ey-Flp reveals no obvious differences. To explore this issue further, reduction in ebi was achieved by expressing the dominant-negative form of ebi in all cells posterior to the morphogenetic furrow in an ebi heterozygous background. Dominant-negative ebi contains the amino-terminal half of the protein from amino acids 1-334 expressed under the control of the pGMR promoter. In wild-type eye discs, Ttk88 staining is not observed in a focal plane in which photoreceptor cell nuclei are located. In contrast, in mutant discs, an average of 36 ± 6 Ttk88-positive nuclei are observed in this region. Most Ttk88-positive nuclei are found 8-10 rows posterior to the morphogenetic furrow. This increase in Ttk88-positive cells also parallels a concomitant decrease in the number of cells stained with the pan-neuronal stain, anti-Elav. In wild-type eye discs, all ommatidia 8-10 rows posterior to the morphogenetic furrow have at least seven Elav-positive cells (R1-R6 and R8). However, in mutant discs, many ommatidia in this region contain less than seven stained cells. Interestingly, a considerably smaller fraction of ommatidia in rows 11-13 contain less than eight Elav-positive R cells; in wild-type discs, all clusters contain eight Elav-positive cells in this region. Hence, a reduction in ebi activity delays neuronal development and this is correlated with persistent nuclear expression of the Ttk88 protein. In summary, both ebi and Egfr promote Ttk88 down-regulation, thereby promoting neuronal development. Further work in other developmental contexts is required to assess the relationship between ebi and ttk in Egfr signaling (Dong, 1999).

Ebi appears to have at least two distinct functions; Ebi promotes EGFR-induced down-regulation of Ttk88, and independently it also promotes G1 arrest in certain cell types. It is suggested that, in some cell types, Ebi serves to coordinate cell exit with the onset of differentiation. Mutations in ebi were isolated as enhancers of an over-proliferation phenotype generated by elevated E2F/DP activity in the Drosophila eye. ebi alleles also strongly suppress a phenotype caused by the cyclin-dependent kinase inhibitor p21, restoring S phases in the second mitotic wave of the developing eye disk. Ebi physically interacts with Sina and Phyllopod, and Ebi promotes Ttk88 degradation in vitro and in S2 cells. Ectopic expression of Ttk88 inhibits differentiation in embryos and eye discs; however, this block to differentiation is insufficient to promote S phase entry in either of the situations where ebi mutations give this effect. Thus it is concluded that Ebi function limits S phase entry (Boulton, 2000).

An E2F/DP overexpression phenotype in the Drosophila eye has been used to screen randomly generated EMS and X-ray-induced mutations for alleles that are modifiers of E2F activity. The same GMR-dE2F-dDP-p35 chromosome was also used to screen a P-element collection for additional alleles that are important for this E2F-dependent phenotype. From this screen, the P-element P[w+; LacZ]K16213 was isolated as a dominant enhancer of the GMR-dE2F-dDP-p35 phenotype. K16213 enhances the general roughness of the eye, increasing the irregular arrangement of the ommatidial facets relative to the GMR-dE2F-dDP-p35 phenotype alone, and generating a large number of bristle duplications. K16213 is a lethal insertion that maps to position 21C on the left arm of chromosome II. K16213 was tested against mutant alleles that had been mapped to the 21C region and a complementation group of three alleles was found that failed to complement the P-element (CC1, CC3 and CC4). No escapers were observed from any of the possible trans-heterozygous combinations of K16213, CC1, CC3 and CC4, indicating that the mutated gene is essential for Drosophila development. Each of the three EMS alleles enhances the GMR-dE2F-dDP-p35 to a degree similar to that observed for the P-element. Excision of the P-element from K16213 reverted the lethality associated with this chromosome and eliminates its ability to modify the GMR-dE2F-dDP-p35 phenotype. Database searches using K16213 flanking sequence identified Ebi (Boulton, 2000).

Since ebi alleles has been isolated as enhancers of an E2F-dependent phenotype characterized by increased cell proliferation, an examination was carried out as to whether Ebi gene dosage can suppress eye phenotypes caused by a reduction in cell proliferation. Expression of the human cyclin-dependent kinase inhibitor p21 has been shown to strongly inhibit S phase entry in the developing eye disk, giving a rough eye phenotype characterized by reduced numbers of pigment cells and fused ommatidia. The GMR-p21 phenotype is shown here to be dominantly suppressed by mutations in Ebi. The number of ommatidia in the pGMR-p21/ebi adult eye is similar to wild type and, other than minor bristle defects, the pGMR-p21/ebi adult eye is morphologically normal (Boulton, 2000).

Wild-type eye disks exhibit a band of S phases, posterior to the morphogenetic furrow that constitutes the second mitotic wave. This wave of S phase is required to generate the full complement of cells that will be needed for subsequent differentiation events. GMR driven expression of p21 abolishes the second mitotic wave of S phases, causing the dramatic reduction in the number of cells observed in the adult eye. To examine the effect on this process of reducing the dosage of Ebi, 3rd instar larval eye disks were isolated from the various genotypes and S phase cells were labeled by BrdU incorporation. Consistent with the adult phenotype, the second mitotic wave of S phase in pGMR-p21/ebi disks is partially restored and in some cases returns to levels similar to that observed in the wild type. Thus, the normal function of Ebi is necessary in these G₁ phase cells for p21 to block S phase entry efficiently (Boulton, 2000).

The ability of ebi alleles to enhance an eye phenotype resulting from increased cell proliferation, and to suppress a phenotype resulting from reduced cell proliferation raises the possibility that Ebi might act to limit proliferation during normal development. To test this, an examination was performed to see whether loss of Ebi function would lead to inappropriate S phase entry. Stocks carrying the ebi alleles were intercrossed to generate trans-heterozygous mutants, and BrdU incorporation was used to determine the patterns of embryonic S phases. Following germ band retraction in wild-type embryos, cells within the peripheral nervous system (PNS) have completed S phase of cell cycle 16 and arrest in G₁ of cell cycle 17. In contrast, cells within the fore and hindgut enter a period of endo-reduplication. In ebi mutants, the endocycles in the gut (consistent with stage 13 embryos) occur as normal. However, cells within the PNS fail to exit the cell cycle and continue into a 17th S phase. This PNS phenotype is very similar to that observed in dacapo (dap) mutant embryos, although ebi mutants do not display the ectopic S phases seen in the epidermis of dap mutant embryos. In addition to ectopic S phases in the PNS, ebi mutants display aberrant S phase entry within the central nervous system (CNS). The ectopic S phases in the CNS are clearly visible as an expansion of the BrdU incorporation in the CNS of the mutant embryos relative to the incorporation observed in the wild type. The cell cycle defects observed in the ebi mutant could arise through a defect in cell cycle withdrawal or might reflect a delay in the normal cell cycle program. It is not possible from the above observations to distinguish definitively between these two possible models. However, if the extra S phases seen in the ebi mutants occur due to a delay in the S phase program one would expect to see a reduction in the number of S phases in the mutant at earlier stages in development. To address this possibility BrdU pulse labeling experiments were performed on wild-type and ebi mutant embryos to analyze S phases at developmental times prior to stage 13/14. Consistent with a defect in cell cycle exit at later stages, no evidence of a reduction in the number of S phases is found in the ebi mutant at developmental times prior to stage 13/14, when compared with the wild type. Thus, it seems most likely that the additional S phases observed in the ebi mutant arise from a defect in cell cycle exit and not from a delay in the S phase program (Boulton, 2000).

The neuronal-specific antibody Mab22C10 was used to analyze the integrity of neuronal lineages in ebi mutants. Consistent with the cell cycle defects described, a number of defects in neuronal differentiation were found including a reduction in peripheral neuron staining and an absence of a large number of Mab22C10-positive cells within the CNS. This phenotype has also been observed in phyllopod mutants and in embryos that over-express Ttk69 and Ttk88 from heat shock transgenes (Boulton, 2000).

One possible interpretation of the experiments described above is that Ebi has a function that limits S phase entry, and that the defects in neuronal differentiation are an indirect effect of a failure to arrest the cell cycle completely. The observations of Dong (1999), showing that Ebi regulates Ttk88 degradation, suggest an alternative interpretation. If the failure to degrade Ttk88 interferes with neuronal differentiation, then the additional S phases described above might reflect the continued proliferation of cells that have failed to differentiate, rather than a direct role in cell cycle control. A third possibility is that Ebi function is important for both cell cycle exit and for the onset of differentiation. Biochemical evidence was sought to support the idea that Ebi promotes Ttk88 degradation and then, in order to distinguish between these models, an examination was performed to see whether simply blocking neuronal differentiation by elevating Ttk88 is sufficient to cause the increase in S phase cells that occurs when Ebi levels are reduced (Boulton, 2000).

Three lines of evidence were found that link Ebi to Sina and Phyllopod-dependent degradation of Ttk88. The first line of evidence comes from transient transfection experiments. Sina and Phyl are able to target Ttk88 for ubiquitin-dependent degradation when expressed in transient transfection experiments in S2 cells. S2 cells contain high levels of endogenous Ebi. To interfere with the activity of this protein, an N-terminal fragment (EbiN) was expressed that has been used (Dong, 1999) as a dominant-negative mutant to interfere with Ttk88 degradation in the eye disk. Cells were co-transfected with pIZT vectors constitutively expressing either Cat or EbiN from the OpIE2 promotor, in the presence of metallothionine-inducible vectors containing Phyl, Sina and Ttk88-myc. Following transfection and copper induction, cells were metabolically labeled with [³⁵S]methionine and then chased with cold methionine for various time points to monitor Ttk88 degradation. In the presence of Sina and Phyl, Ttk88 is degraded with a half life of ~25 min, a process that can be blocked by the proteosome inhibitor MG132. Ttk88 degradation is blocked in cells expressing the EbiN dominant-negative form of pIZT, whereas a control pIZT vector that constitutively expressed Cat gave no effect (Boulton, 2000).

The second line of evidence stems from an in vitro degradation assay. To investigate whether Ebi is linked to Ttk88 degradation, attempts were made to reconstitute Ttk88 degradation in vitro. These studies show that Sina and Phyl are needed for Ttk degradation, mirroring their requirement in vivo. The results of the in vitro degradation assay suggested that Ebi might physically associate with Ttk88, Sina and Phyl. Ebi was found to interact strongly with Sina and Phyl, and weakly with Ttk88, when these proteins were expressed and assayed individually. When Sina, Phyl and Ttk88 are co-expressed in the same lysate, Ebi is able to pull down Ttk88 with a much higher affinity, compared with Ttk88 when expressed and bound alone. This suggests that the Ebi-Ttk interaction is indirect and may require Sina and Phyl to facilitate association. Since the ß-propeller structure formed by WD-repeat domains provides a surface for many protein-protein interactions, it is likely that this domain in Ebi provides a scaffold for Sina, Phyl and Ttk88 association (Boulton, 2000).

If stabilization of Ttk88 is responsible for the cell cycle phenotypes associated with Ebi, a consistent pattern of genetic interactions between GMR-p21 or GMR-E2F-DP-p35 and mutations in the Egfr pathway would be expected that function to regulate Ttk88 levels. However, unlike mutations in Ebi, loss-of-function alleles of egfr, gap1, raf1, ras1, mapk, yan, sina, phyl and ttk have no strong effect on either the GMR-p21 or the GMR-E2F-DP-p35 phenotype. Mutations in the Ets-domain transcription factor Pointed (pnt) enhance the GMR-E2F-DP-p35 phenotype but fail to modify the GMR-p21 phenotype. While it is possible that Ebi is the only dosage-sensitive component of the Egfr pathway whose levels affect cell proliferation, these results suggest that Ebi might have an activity that is independent of Ttk88 degradation (Boulton, 2000).

To test directly whether stabilization of Ttk88 is likely to be responsible for the changes in cell cycle control caused by ebi mutant alleles, the effects of elevating the levels of Ttk88 were tested. Ectopic expression of Ttk88 was induced by heat shock for 30 min at 39°C in embryos carrying hs-Ttk88. Following recovery, embryos were either pulsed with BrdU or aged for immunohistochemistry. The ectopic expression of Ttk88 from a heat shock-regulated transgene is sufficient to disrupt neuronal differentiation in stage 13-14 embryos. Unlike the ebi mutant embryos, BrdU incorporation demonstrates that the block to differentiation in hs-Ttk88 embryos does not result in a failure to exit the cell cycle in the PNS and CNS. Furthermore, expression of Ttk88 from a GMR transgene inhibits differentiation in the eye disc. However, the GMR-Ttk88 transgene fails to suppress the GMR-p21 eye phenotype and is unable to restore S phases in the GMR-p21 eye disc. In contrast, halving the dosage of Ebi or expressing cyclin E from the GMR promoter is sufficient to restore the second mitotic wave of S phases. It is concluded that increasing Ttk88 protein to a level where differentiation is perturbed in either the embryo or the eye disc is insufficient to promote S phase entry in either of the situations where ebi mutations give this effect. It is inferred that Ebi must have a second function, independent of Ttk88 degradation, that is important for regulating cell cycle exit (Boulton, 2000).

Thus this study shows that ebi alleles are dominant suppressors of the GMR-p21 phenotype, and that this suppression occurs because halving the gene dosage of Ebi reduces the ability of the p21 CDK inhibitor to block the second mitotic wave. The second mitotic wave represents the synchronous division of cells that have not yet become committed to a cell fate; therefore, neither the p21-induced arrest, nor its suppression by ebi alleles appears to be tied to neuronal differentiation. These results suggest that the function provided by Ebi is important for some aspects of cell cycle control (Boulton, 2000).

Although cell cycle exit and the induction of differentiation are tightly linked processes, it is evident that the role of Ebi in cell cycle control is distinct from its previously reported role in the regulation of Ttk88. Ectopic expression of Ttk88 is able to disrupt neuronal differentiation in both the embryo and the eye disk, yet Ttk88 is insufficient to promote S phase entry or to suppress the p21-induced cell cycle arrest. Thus Ebi appears to have at least two distinct functions: Ebi promotes Egfr-induced down-regulation of Ttk88, and independently, it also promotes G₁ arrest in certain cell types. In the light of these results, it is suggested that, in some cell types, Ebi could serve to coordinate cell exit with the onset of differentiation (Boulton, 2000).

What is the molecular basis of Ebi function? Closely related homologs of Ebi are found in diverse species. Two regions of these proteins that are most highly conserved, and that define this group of proteins, include an N-terminal domain that resembles an F-box sequence and the C-terminal domain that contains WD40-repeats. The conservation of a divergent F-box suggests that Ebi homologs may target associated proteins for degradation. If so, Ttk88 appears to be the most likely substrate. The expression of an N-terminal fragment of Ebi blocks the Sina-Phyllopod-induced degradation of Ttk88 in tissue culture cells and in eye imaginal disks, and the C-terminus of Ebi can physically interact with Sina-Phyl-Ttk88. Moreover, in vitro assays show that Ebi-associated proteins can promote the degradation of Ttk88. Since F-box proteins can target multiple substrates for degradation (for example, see Slimb), this would provide a simple model to explain how Ebi could independently regulate multiple processes. The results described here could be explained simply if Ebi targets Ttk88 and a positive cell cycle regulator such as E2F or cyclin E. However, no physical association between Ebi and either cyclin E or E2F/DP could be detected using the Drosophila proteins, or their human homologs, in either in vitro assays or by transient transfection. In addition, GST-Ebi beads fail to stimulate the degradation of cyclin E, E2F or dDP in the in vitro degradation assays described above. While the genetic interactions suggest that cyclin E and E2F might be the most obvious candidates, it is clear that a more systematic approach is needed to identify a suitable cell cycle regulator (Boulton, 2000).

Several observations suggest that Ebi may not act simply as a component of the conventional SCF complex. The N-terminal sequences of Ebi proteins contain many, but not all of the residues that are conserved in F-box proteins that interact with Skp1. Drosophila Ebi binds very weakly to GST-Skp1, but Ebi-Skp1 complexes could not be recovered from transfected cells. The Drosophila genome contains at least nine genes that are related to Skp1 and it is possible that Ebi interacts with a Skp-like protein. It is intriguing that, in addition to the connections between Ebi and Ttk88, Ebi homologs have also been physically linked with transcriptional repressors in two other species. Sif2, the yeast ortholog of Ebi, has been found to interact with Sir4, a protein implicated in transcriptional silencing (Cockell, 1998). The recent purification of the SMRT repressor complex reveals that it contains TBL1, a human homolog of Ebi (Guenther, 2000). While the role of the Ebi-related proteins in these complexes is uncertain, these studies raise the possibility that Ebi's role in G₁ arrest may be attributable to its association with repressor complexes. One attractive model, which combines all of these studies, is the idea that the divergent F-box in the N-terminus of Ebi recruits a specialized Skp-Cul complex to chromatin in order to selectively ubiquitylate repressor proteins. Clearly, further work is needed to test this and other models of Ebi function (Boulton, 2000).

REGULATION

Targets of Activity

An NRSF/REST-like repressor downstream of Ebi/SMRTER/Su(H) regulates eye development in Drosophila

The corepressor complex that includes Ebi and SMRTER is a target of epidermal growth factor (EGF) and Notch signaling pathways and regulates Delta (Dl)-mediated induction of support cells adjacent to photoreceptor neurons of the Drosophila eye. A mechanism is described by which the Ebi/SMRTER corepressor complex maintains Dl expression. charlatan (chn) is repressed by Ebi/SMRTER corepressor complex by competing with the activation complex that includes the Notch intracellular domain (NICD). Chn represses Dl expression and is critical for the initiation of eye development. Thus, under EGF signaling, double negative regulation mediated by the Ebi/SMRTER corepressor complex and an NRSF/REST-like factor, Chn, maintains inductive activity in developing photoreceptor cells by promoting Dl expression (Tsuda, 2006).

The corepressor complex that includes Ebi, SMRTER and Su(H) is required for expression of Dl in Drosophila photoreceptor cells. To identify genetic loci that are transcriptionally repressed by the Ebi corepressor, a screen was set up using an ectopic gene expression system (Gene Search System). Insertion of a Gene Search (GS) vector, a modified P-element carrying the Gal4 upstream activating sequence (UAS_G) near its 3' end, causes overexpression of a nearby gene under the control of the Gal4-UAS_G system. GS insertions into the chn locus were identified, whose overexpression phenotype in the eye using an eye-specific Gal4 driver (GMR-Gal4) was modified by reducing ebi activity. Thus the regulation of chn by Ebi-dependent transcriptional repression was studied (Tsuda, 2006).

In third instar larval-stage eye discs, the chn transcript is highly expressed in the morphogenetic furrow (MF), where photoreceptor differentiation initiates, but is downregulated in cells in the later stage photoreceptor development. In ebi mutant eye discs, however, chn expression becomes detectable in differentiating photoreceptor cells, and its expression in the MF is increased, suggesting that Su(H) in association with Ebi and SMRTER represses chn transcription in the eye disc (Tsuda, 2006).

To reveal the role of Su(H) as an activator, chn expression was examined when the level of Su(H) expression was reduced. Removing one copy of Su(H) suppresses the loss-of-Dl expression phenotype in ebi mutants. It was found that reducing one copy of Su(H) suppresses ectopic chn expression in ebi mutants, suggesting that ectopic expression of chn in ebi mutants is Su(H)-dependent. RT-PCR analysis of chn expression in ebi^- eye discs differing in the dosage of Su(H) gene also supported these results. Strong reduction of Su(H) expression alone reduced expression of chn in the MF; this expression became weaker and was slightly broader. The phenotype of ebi, Su(H) double mutants is almost the same as Su(H) single mutants , suggesting that Su(H) acts as an activator in the absence of Ebi. This might be due to dual functions of Su(H) as an activator or repressor. Hence, reducing the amount of Ebi in the corepressor complex involving Su(H) might convert Su(H) to an activator by permitting the replacement of the corepressor complex with NICD (Tsuda, 2006).

To reveal the molecular nature of transcriptional regulation of chn by Su(H), Su(H) target sites were sought in the genomic region of chn. Since Su(H) binds slightly degenerate sequences, it was not easy to identify the functional Su(H) binding region from a simple genomic search. An alternative approach was taken to map the chn genomic region, which is regulated by Su(H) in the normal chromosomal context. Ebi-mediated repression involves SMRTER, a corepressor that recruits histone deacetylases and induces the formation of inactive chromatin, which spreads from the site where Su(H) recruits the corepressor complex. Promoters near the Su(H)-binding site are thus expected to be downregulated in an Ebi-dependent manner. Four insertion lines of the GS vector were identified in the chn promoter region. All these GS lines caused ectopic expression of chn with consequent abnormal eye morphology when they were crossed with GMR-Gal4. If the effect of the Ebi/SMRTER corepressor complex reaches the UAS_G in those insertions, reduction of Ebi activity will derepress UAS_G and further enhance activation by GMR-Gal4. One copy of a dominant-negative construct of ebi (GMR-ebi^DN) caused only a mild defect in eye morphology and weak, if any, ectopic expression of chn. GMR-ebi^DN strongly enhanced the overexpression phenotype of chn^GS17605 and chn^GS11450, which contained GS vector insertions (-474 and -734, respectively) upstream of the transcriptional start site. However, GMR-ebi^DN failed to enhance the overexpression phenotype of other GS lines (chn^GS2112 and chn^GS17892) that were inserted downstream (+773 and +1040, respectively) of the first exon. From these results, it is concluded that Ebi-dependent transcriptional repression is targeted to the proximity of the transcriptional initiation site of the chn promoter (Tsuda, 2006).

Chn is a 1108-amino-acid protein with multiple C2H2-type zinc-finger motifs. Although no highly homologous gene within the mammalian genome could not be detected using BLAST, a small sequence of similarity between the N-terminal zinc-finger motif of Chn and the fifth zinc-finger of human NRSF/REST was found. Chn has several structural and functional similarities to human NRSF/REST, as follows. First, Chn and NRSF/REST each contain an N-terminal region with multiple zinc-finger motifs (five motifs in 264 residues in Chn and eight motifs in 251 residues in NRSF/REST), followed by a cluster of S/T-P motifs (serine or threonine followed by a proline) and a single zinc-finger motif at the C terminus. Second, the C-terminal region of NRSF/REST binds a corepressor, CoREST, which serves as an adaptor molecule to recruit a complex that imposes silencing activities. The Drosophila homolog of CoREST (dCoREST) (Andres, 1999; Dallman, 2004) can associate with the C-terminal half of Chn in cultured S2 cells. Finally, NRSF/REST binds to NRSE/RE1, a 21-bp sequence located in the promoter region of many types of neuron-restricted genes, via the N-terminal zinc-finger motifs. It was found that a recombinant protein containing the N-terminal zinc-fingers of Chn bound specifically to the NRSE/RE1 sequence in vitro. Thus, the structural similarity to NRSF/REST, binding to dCoREST and the DNA-binding specificity of Chn suggest that it is a candidate for a functional Drosophila homolog of NRSF/REST (Tsuda, 2006).

If Chn acts as a regulator of neural-related functions, as suggested for NRSF/REST, then Chn would be expected to bind to a regulatory region common to many types of neural-related genes in Drosophila. Numerous sequences similar to NRSE/RE1 were identified in the Drosophila genome, and their binding to Chn was assessed by EMSA. Using these sequences, a consensus binding sequence for Chn (Chn-binding element (CBE), 5'-BBHASMVMMVCNGACVKNNCC-3') was derived. 26 CBEs were identified within 10 kb of annotated genes from the Drosophila genome. Binding to Chn was confirmed for 18 CBEs using EMSA competition assay. Genes containing the CBE include dopamine receptor 2 (DopR2) and the potassium channel, ether-a-go-go, for which the mammalian homologs are target genes of NRSF/REST. These results suggest that the CBE is a good indicator of Chn binding sites and that Chn regulates many types of neural-related genes, as is implicated for NRSF/REST. However, it was found that divergent forms of CBE adjacent to hairy and extramacrochaetae were bound specifically by Chn. Likewise, some of the CBE sites failed to bind to Chn. Thus, a further refinement will be necessary to predict a definitive set of Chn binding sites in the Drosophila genome (Tsuda, 2006).

Although it has been established that mammalian NRSF/REST is a key regulator of neuron-specific genes, attempts to isolate invertebrate homolog of NRSF/REST have so far failed to identify a true homologous factor in invertebrates. The properties of Chn, including the similarity in DNA-binding specificity, association with CoREST and transcriptional repressor activity, suggest that Chn is a strong candidate for a functional Drosophila homolog of NRSF/REST. chn was originally identified by its requirement in the development of the PNS. This study identified a number of candidate target genes of Chn, a large fraction of which is implicated in neural function and/or gene expression. It is expected that further analysis of these candidates will provide valuable information about chn function in vivo, which may be extended to the understanding of NRSF/REST (Tsuda, 2006).

The Chn mutation blocks eye development by preventing the initiation of MF, a process requiring Notch signaling. This phenotype is likely owing to a loss of Notch function, because elevated Dl expression is known to block Notch signaling. The function of Chn during the early stage of eye development might be to regulate Notch signaling at an appropriate level by downregulating Dl. It is possible that Chn-mediated modulation places a variety of Notch functions in eye under the influence of EGFR signaling and provides flexibility in its regulation (Tsuda, 2006).

Although chn is expressed in the MF, genetic analyses show that small clones of chn mutant cells permit progression of the MF and photoreceptor differentiation. It is speculated that the repressive effect of Chn is overcome by other signals in the MF, such as hedgehog signaling, which strongly induces Dl (Tsuda, 2006).

Developing photoreceptor cells are exposed to the EGFR ligand, Spitz, and the Notch ligand, Dl, and each cell must assess the level of the two signals and respond appropriately to perform each task of photoreceptor cell specification and induction of non-neural cone cells. This question was investigated by studying the expression of Dl in photoreceptor cells. chn was identified as a direct target of Ebi/SMRTER-dependent transcriptional repression and as a repressor of Dl expression. The abrogated expression of Dl in ebi mutants was recovered by reducing one copy of chn, suggesting that the negative regulation of chn by ebi is indeed prerequisite for photoreceptor cell development (Tsuda, 2006).

Genetic data suggest that Su(H) may activate or repress chn expression. This idea is supported by data showing that Ebi/SMRTER and NICD are recruited to the promoter region of chn. The Ebi/SMRTER complex formed in this region did not contain any detectable level of the intracellular domain of Notch (NICD), suggesting that the binding of Ebi/SMRTER and NICD to this region may be mutually exclusive, and therefore it is expected that a regulatory system controls the balance between the active and repressive states of Su(H). Taken together, these results suggest that chn is a key factor in the crosstalk between two major signal transduction pathways: the EGFR-dependent pathway and the Notch/Delta-dependent pathway (Tsuda, 2006).

In the mammalian system, competition between SMRT and NICD for interaction with RBPJkappa determines the state of RBPJkappa-dependent transcriptional activity. Extracellular signaling may modulate this competition; diverse signaling pathways modulate the functions of N-CoR/SMRT. The current findings would prompt investigations of potential interaction of two repression systems of NRSF/REST and N-CoR/SMRT, and their regulation by Notch and EGF signaling in mammalian neuronal differentiation (Tsuda, 2006).

Protein Interactions

To investigate whether Ebi is linked to Ttk88, attempts were made to reconstitute Ttk88 degradation in vitro. Initial experiments showed that Ttk88 is not degraded when co-expressed with Sina and Phyl in rabbit reticulocyte lysate. In an attempt to stimulate the degradation activity, GST (glutathione S-transferase) or GST-Ebi beads were incubated together with ubiquitin, an ATP regeneration system and Sina-Phyl-Ttk co-expressed in a reticulocyte lysate. Neither GST nor GST-Ebi is able to promote Ttk88 degradation in this setting. It was speculated that Ebi may function as a bridge between the Sina-Phyl-Ttk88 complex and an activity required for ubiquitylation and subsequent degradation of Ttk88. In order to provide this missing activity, GST or GST-Ebi beads were preincubated with S2 extracts (now referred to as loaded beads) prior to performing the degradation reaction. Loaded GST-Ebi beads are unable to degrade Ttk88 when expressed alone. However, when Sina and Phyl are co-translated with Ttk88, loaded GST-Ebi beads are able to promote degradation of TtK88 by a mechanism that is blocked by the proteosome inhibitor, LLnL, whereas loaded GST beads cannot. The need for Sina and Phyl for Ttk degradation mirrors their requirement in vivo. In vitro-translated E2F, dDP or cyclin E is not degraded in any of the experiments described. In order to map the region of Ebi that associates with the activity required for Ttk88 degradation, loaded GST-EbiN and GST-EbiC beads were pre-incubated and then the degradation assay was performed. Interestingly, neither half of Ebi alone is capable of targeting Ttk88 for degradation. These data suggest that the full-length Ebi protein may act to bring the Sina-Phyl-Ttk88 complex and a ubiquitylation activity into close proximity (Boulton, 2000).

The results of the in vitro degradation assay suggest that Ebi might physically associate with Ttk88, Sina and Phyl. This was tested using the in vitro translated Sina, Phyl and Ttk88, and the GST-Ebi fusion proteins described above. GST-Ebi was found to interact strongly with Sina and Phyl, and weakly with Ttk88, when these proteins were expressed and assayed individually. The GST control shows no association. Interestingly, when Sina, Phyl and Ttk88 are co-expressed in the same lysate, GST-Ebi is able to pull down Ttk88 with a much higher affinity compared with when Ttk88 is expressed and bound alone. This suggests that the Ebi-Ttk interaction is indirect and may require Sina and Phyl to facilitate association. To define the region of Ebi required for Sina, Phyl and Ttk association, GST-EbiN (N-terminal domain) and GST-EbiC (C-terminal WD-repeat domain) fusion proteins were tested for binding. GST-EbiN does not associate with any of the proteins, whereas GST-EbiC is able to bind to all three proteins, although with a slightly reduced affinity when compared with the full-length protein. Since the ß-propeller structure formed by WD-repeat domains provides a surface for many protein-protein interactions, it is possible that this domain in Ebi provides a scaffold for Sina, Phyl and Ttk88 association. To provide further evidence for these interactions, pIZT-Ebi with pIZT-V5His-Sina or pIZT-V5His-Phyl, or all three constructs were transiently co-transfected into S2 cells. Sina and Phyl (His₆- and V5-tagged) were purified from lysates from the various transfected populations by Ni-NTA agarose chromatography. The beads were then subjected to Western blotting using a monoclonal antibody to Ebi. Ebi was found to co-precipitate with Sina and Phyl but was not observed in the untransfected control (Boulton, 2000).

The Notch and Epidermal growth factor receptor (Egfr) pathways both regulate proliferation and differentiation, and the cellular response to each is often influenced by the other. A mechanism is described that links them in a sequential fashion, in the developing compound eye of Drosophila. Egfr activation induces photoreceptor (R cell) differentiation and promotes R cell expression of Delta. This Notch ligand then induces neighboring cells to become nonneuronal cone cells. ebi and strawberry notch (sno) regulate Egfr-dependent Delta transcription by antagonizing a repressor function of Suppressor of Hairless [Su(H)]. Sno binds to Su(H), and Ebi, an F-box/WD40 protein, forms a complex with Su(H) and the corepressor Smrter. Egfr-activated transcriptional derepression requires ebi and sno, is proteasome-dependent, and correlates with the translocation of Smrter to the cytoplasm (Tsuda, 2002).

The Notch signaling pathway plays multiple roles in eye development. At the morphogenetic furrow, the proneural protein Atonal facilitates the expression of Dl in the R8 cell. The first step of ommatidial assembly involves lateral inhibition between equivalent cells, but successive steps are inductive, arising from an already differentiated cell to its uncommitted neighbors. The Notch pathway is involved in the regulation of both of these processes. Similarly, the Egfr ligand, Spi, expressed in R8, activates the receptor in neighbors allowing them to assume their respective R1–R7 cell fates. Subsequently, these R cells express Spi, and as described in this study, they also express Dl in response to Egfr activation. The cone cells receive an Egfr signal and a Notch signal from the R cells and this combination is critical for the assumption of their fate. Later, after their fate is determined, these cone cells, too, will express Delta, which is important for pigment cell induction. Presumably, the level of the Egfr signal rises in the cone cells with time, and as a threshold of Egfr activation is surpassed, the proteasome mediated arm of the pathway becomes effective causing derepression of Su(H) and expression of functional levels of Dl sufficient for pigment cell development. Thus, a temporally and spatially positioned combination of parallel and sequential Egfr/Notch signals is important for the successive induction of cell types in the eye (Tsuda, 2002).

An interesting interplay between Egfr and Notch pathways is also seen during vulval induction in C. elegans. Cells that are close to the anchor cell assume the primary developmental fate, while those farther away become secondary cells. The development of the secondary cell fate shows many similarities with cone cell development. Both secondary and cone cells primarily require high levels of Notch signal and a low-level activation of the Egfr signaling pathway. Genetic studies support one of two alternative models for the development of the secondary cell fate. In the first model, the graded activation of Egfr (Let23) mediated by the expression of its ligand Lin3 in the anchor cell and lateral Notch (Lin12) signaling imparts a secondary cell fate. Alternatively, the signal mediated by Lin3 is required for the specification of the primary cell, which in turn induces secondary cells through the Notch pathway. The latter model is similar to the sequential activation mechanism describe in this study for cone cell development. It will be interesting to determine if in C. elegans, the Egfr (Let23) pathway activates an as yet unidentified Notch (Lin12) ligand in primary cells that is then used to induce secondary cell fate (Tsuda, 2002 and references therein).

Evidence from mammalian systems has suggested that CBF1, the mammalian homolog of Su(H), is a component of a large repressor complex. The activation function of CBF1 results from a displacement of repressive components (such as HDAC) by the intracellular domain of Notch which converts Su(H) into a transcriptional activator. Genetic analysis of the embryonic midline and the pupal bristle complexes in Drosophila have also supported a switch from Su(H)-mediated repression to activation. A second mechanism for relieving Su(H) mediated repression is through Sno, Ebi, and the Egfr pathway. In response to the Egfr signal, Ebi, an F-box protein, presumably causes a proteasome-mediated degradation of an unknown component of the Su(H) inhibitory complex. Mammalian TBL1 (Ebi) can function downstream of the tumor suppressor gene, p53, in the degradation of the ß-catenin protein in a novel ubiquitin-dependent degradation pathway involving Siah, the mammalian homolog of the Drosophila Sina protein. Similarly, Drosophila Ebi can also act in combination with Sina to degrade protein targets. More generally, phosphorylation by MAPK downstream of RTK pathways is known to trigger proteasome-mediated degradation of target proteins. In addition to Ebi, a core component of the proteasome, encoded by l(3)73Ai gene, is also important for expression of Dl. The simplest model is that in response to Egfr signaling, one or more of the many components in the large Su(H)/SMRTER repression complex becomes a target of a proteasome-mediated degradation process (Tsuda, 2002).

The studies presented here also show that the corepressor SMRTER is redistributed from the nucleus to the cytoplasm in an Egfr/Sno/Ebi dependent manner. These results are in complete agreement with the role of the corresponding mammalian protein SMRT in its function as a repressor. Like Su(H), nuclear hormone receptors such as retinoic acid receptor and thyroid hormone receptor can function as both repressors and activators. SMRT has been shown to be phosphorylated in response to an RTK signal. This leads to translocation of SMRT out of the nucleus. Thus, steroid hormone receptors lose their ability to repress but not activate transcription. In an in vivo example, the Egfr/Sno/Ebi pathway promotes the dissociation of the Su(H)/SMRTER repressor complex and causes the nuclear export of SMRTER. As a result, target genes such as Dl are derepressed (Tsuda, 2002).

Notch signaling can take place between cells that are equivalent at the time the signal initiates, or it can occur between a signaling cell that is different from the cell receiving the signal. Traditionally, the first kind of process has been referred to as lateral inhibitory Notch signaling and the second as an inductive Notch pathway. These studies suggest that the fundamental difference between these two processes is not due to differences in molecular components of the pathway downstream from activated Notch, but rather due to the mechanism that controls the expression of the ligand, Dl. For lateral inhibitory Notch pathways, a mechanism involving a feedback loop and proneural genes is at the core of Dl/Notch regulation. Starting with an equipotent group, an asymmetric signaling system is created, in which the signaling cell expressing high levels of Dl, assumes a differentiated fate and prevents its neighbors from adopting an identical fate. All available evidence suggests that the Egfr pathway, Sno, and Ebi do not control Dl expression in such lateral inhibitory processes mediated by Notch. In contrast, this study shows that in inductive processes controlled by Notch signaling, Dl expression is controlled by Egfr, Ebi, and Sno and apparently not by proneural genes. For example, no known proneural gene (Ac/sc, amos, or atonal) is expressed in R cells that contact the cone cells (i.e., R1-R7) and express Dl. This is also true for cells at the dorsoventral boundary of the wing disc where Notch signaling directly activates vestigial expression through Su(H) binding to the enhancer and in the mesectodermal cells of stage 6 embryos where the Notch pathway has been implicated in controlling the expression of single minded at the midline. Instead, all of these cells in the eye, wing, and embryo receive an Egfr signal that likely controls Dl expression. Indeed, the late expression of Dl in R cells does not involve feedback from the cone cells but instead involves the derepression of Dl expression in a Notch-independent manner. This is different from the early expression of Dl that is required for the selection of R8 cells at the furrow through a lateral inhibitory signal (Tsuda, 2002).

This study highlights the function of two unusual proteins, Sno and Ebi, in controlling the expression of Dl. Mammalian Ebi (TBL1) interacts with a SMRT/HDAC complex as also supported by this study in Drosophila. There are two human and three mouse genes similar to Sno identified by genome projects. The function of the mammalian Sno proteins is unknown. Whether the mammalian proteins also function upstream of the Notch pathway, as they do in Drosophila, remains to be established. Given the conservation of developmental pathways between Drosophila and mammals, this may not be an unreasonable expectation (Tsuda, 2002).

A Drosophila Smyd4 homologue is a muscle-specific transcriptional modulator involved in development

SET and MYND domain (Smyd) proteins are involved in the transcriptional regulation of cellular proliferation and development in vertebrates. However, the in vivo functions and mechanisms by which these proteins act are poorly understood. This study used biochemical and genetic approaches to study the role of a Smyd protein in Drosophila. Eleven Drosophila genes were identified that encode Smyd proteins. CG14122 encodes a Smyd4 homologue that has been named dSmyd4. dSmyd4 repressed transcription and recruited class I histone deacetylases (HDACs). A region of dSmyd4 including the MYND domain interacted directly with approximately 150 amino acids at the N-termini of dHDAC1 (Rpd3) and dHDAC3. dSmyd4 interacts selectively with Ebi, a component of the dHDAC3/SMRTER co-repressor complex. During embryogenesis dSmyd4 was expressed throughout the mesoderm, with highest levels in the somatic musculature. Muscle-specific RNAi against dSmyd4 resulted in depletion of the protein and lead to severe lethality. Eclosion is the final moulting stage of Drosophila development when adult flies escape from the pupal case. 80% of dSmyd4 knockdown flies were not able to eclose, resulting in late pupal lethality. However, many aspects of eclosion were still able to occur normally, indicating that dSmyd4 is likely to be involved in the development or function of adult muscle. Repression of transcription by dSmyd4 and the involvement of this protein in development suggests that aspects of Smyd protein function are conserved between vertebrates and invertebrates (Thompson, 2008).

The large number of Smyd family members in Drosophila may allow these proteins to assume a repertoire of functions, or ensure redundancy between family members during development. Further analysis of vertebrate genomes may also reveal larger numbers of Smyd proteins than had previously been anticipated. Studies in vertebrates show that individual Smyd proteins control gene expression in order to fulfil varied functions during development. The tissue specific expression patterns of Drosophila Smyd family members suggest that these proteins may play equivalent roles in the development of specific tissues in this species (Thompson, 2008).

dSmyd4 represses transcription and recruits HDACs in a manner analogous to vertebrate Smyd1 and Smyd2. This study gives additional insight into the HDAC co-repressors that are involved in repression by dSmyd4. dSmyd4 was shown to interact with both dHDAC3 and Ebi, components of the SMRTER co-repressor complex. This contrasts with mammalian Smyd2 protein, which interacts with the Sin3A-HDAC complex. No interaction could be detected between dSmyd4 and HDAC1-containing NuRD, CoREST and Sin3A co-repressors by immunoprecipitation. Nevertheless, a common feature of dSmyd4 and vertebrate Smyd2 and Smyd1 is the association of a potential methyltransferase with histone deacetylase activity in a single complex. This implies that a primary role of these proteins is to co-ordinate changes in modification status at their target sites (Thompson, 2008).

This paper has described a direct interaction between dSmyd4 and the N-terminal regions of dHDAC1 and dHDAC3. There is a high level of identity between Drosophila and vertebrate class I HDACs, especially at the N-termini where this interaction occurs, therefore this interaction may be relevant to recruitment of HDACs by Smyd family members in other species. The recruitment of HDAC co-repressor complexes by MYND domains is also of clinical importance. AML/MTG8 fusions lead to the aberrant recruitment of HDAC co-repressor complexes in the development of leukaemia. The MTG8 MYND domain interacts with components of these complexes, but the interaction between the MYND domain of MTG8 and HDACs is poorly described. The novel interaction described in this study may also apply to other interactions such as these (Thompson, 2008).

The cytoplasmic over-expression pattern of dSmyd4 resembles that of vertebrate Smyd2, providing another parallel between vertebrate and invertebrate Smyd proteins. However, a more relevant indicator of biological function is the distribution of endogenous protein. Endogenous dSmyd4 is predominantly nuclear in S2 cells. The strong cytoplasmic localisation of dSmyd4 in embryos suggests that in addition to its activity as a transcriptional repressor, dSmyd4 may perform additional cytoplasmic functions, for example the methylation of non-histone substrates. This raises additional parallels with Smyd2, since a cytoplasmic role has been suggested for this protein. The cell-type dependent localisation of endogenous dSmyd4 raises interesting questions about how the localisation of dSmyd4 is regulated. The subcellular localisation of human Smyd3 is regulated in a cell cycle dependent manner and analogous developmental regulation may be required for the function of other Smyd proteins such as dSmyd4 (Thompson, 2008).

Knockdown of dSmyd4 in muscle tissue resulted in reduced rates of survival. dSmyd4 was expressed during embryogenesis, yet the majority of knockdown flies died at the late pupal stage suggesting that there is a greater requirement for dSmyd4 in processes involved in adult myogenesis. This may be due to redundancy between Smyd proteins during embryogenesis since CG8503 and CG18136 are also expressed in muscle tissue at this time. The majority of knockdown flies were not able to escape from the pupal case but performed other eclosion behaviours normally. The neural networks and signalling required for eclosion therefore appear to be intact, indicating that dSmyd4 is likely to play a role in controlling muscle development or function. Identifying the precise nature of the eclosion defect caused by dSmyd4 knockdown will be an important step in understanding the function of this and other Smyd proteins in the development of multicellular organisms. Much is known about the transcription factors involved in Drosophila muscle development but little is understood about how chromatin structure is regulated during this process. dSmyd4 is a good candidate to direct chromatin remodelling during muscle development. Smyd1 is required for cardiac development in vertebrates and a number of other Drosophila Smyd proteins appear to be specifically expressed in muscle. These results suggest that members of the Smyd family play conserved roles in muscle development in both vertebrate and invertebrate species. Drosophila provides a tractable system for the analysis of gene function, for example testing genetic interactions with other genes involved in muscle development. Analysis of mutants in dSmyd4 and other Smyd genes using this approach may also shed light on conserved aspects of Smyd function in vertebrates (Thompson, 2008).

This study presents the first analysis of both Smyd proteins in Drosophila and of a Smyd4 homologue. It appears that aspects of mechanism and function are conserved between Drosophila and vertebrate Smyd proteins. The repression of transcription by SMRTER complex recruitment and the requirement of dSmyd4 for survival highlight the importance of this protein family as transcriptional modulators of developmental processes (Thompson, 2008).

Drosophila Ebi mediates Snail-dependent transcriptional repression through HDAC3-induced histone deacetylation

The Drosophila Snail protein is a transcriptional repressor that is necessary for mesoderm formation. This study identified the Ebi protein as an essential Snail co-repressor. In ebi mutant embryos, Snail target genes are derepressed in the presumptive mesoderm. Ebi and Snail interact both genetically and physically. A Snail domain was identified that is sufficient for Ebi binding and functions independently of another Snail co-repressor, Drosophila CtBP. This Ebi interaction domain is conserved among all insect Snail-related proteins, is a potent repression domain and is required for Snail function in transgenic embryos. In mammalian cells, the Ebi homologue TBL1 is part of the NCoR/SMRT-HDAC3 (histone deacetylase 3) co-repressor complex. It was found that Ebi interacts with Drosophila HDAC3, and that HDAC3 knockdown or addition of a HDAC inhibitor impairs Snail-mediated repression in cells. In the early embryo, Ebi is recruited to a Snail target gene in a Snail-dependent manner, which coincides with histone hypoacetylation. These results demonstrate that Snail requires the combined activities of Ebi and CtBP, and indicate that histone deacetylation is a repression mechanism in early Drosophila development (Qi, 2008).

Previous studies have suggested that CtBP mediates transcriptional repression by Snail in the early embryo. However, disruption of Snail repressor activity in ebi mutant embryos cannot be due to an indirect effect on CtBP, based on several observations. Comparable CtBP protein levels were detected in ebi mutant and wt embryos using a CtBP-specific antibody. In addition, the ebi mutation did not affect the function of the Kr repressor, which also requires CtBP, on the NEE-lacZ reporter gene. Furthermore, the segment polarity gene engrailed that is indirectly regulated by CtBP-dependent repressors such as Kr and Knirps is normally expressed in ebi mutant embryos, indicating that CtBP activity is not disrupted by the ebi mutation (Qi, 2008).

Moreover, the Ebi interaction motif (Sna 1-40) that does not bind to CtBP in vitro still has repression activity in S2 cells and in transgenic embryos, suggesting that Ebi functions directly as a cofactor for Snail through a physical association. Removing this motif from the Snail protein abolishes its repression activity. This result is consistent with data that used snail transgenes to rescue snail^−/− embryos. It was found that Snail lacking amino acids 6-25 fails to repress sim expression in snail^−/− embryos. It was proposed that this part of Snail might be involved in nuclear localization of the protein. However, this study demonstrated that the mutant protein is normally localized to nuclei in S2 cells and embryos. This suggests that mutant Snail loses the ability to repress because it is unable to interact with Ebi. Taken together, it is concluded that Ebi specifically regulates Snail-mediated repression through a new, CtBP-independent pathway (Qi, 2008).

This study suggests that Snail mediates repression through two pathways, a CtBP-dependent and an Ebi-dependent pathway. Several repression activities in one protein could contribute qualitatively or quantitatively to repression. In some cases, different target genes are repressed through distinct co-repressors. By contrast, the CtBP-dependent and -independent repression activities in Knirps and Hairless exerts an effect quantitatively. The current experiments show that in the presumptive mesoderm, repression of several Snail target genes requires both CtBP and Ebi, that Snail recruits both CtBP and Ebi to the same rho enhancer and that CtBP and Ebi can interact simultaneously with Snail. Deletion of either the Ebi or CtBP interaction motifs impairs Snail function in transgenic mis-expression and rescue assays. Furthermore, derepression of Snail target genes is not complete in either ebi or CtBP mutant embryos. In ebi mutant embryos, and snail mutant embryos rescued with Snail lacking amino acids 6-25, gene repression is impaired but ventral furrow formation and mesoderm invagination normal, which is similar to the situation in the snail hypomorphic allele V2. Taken together, these results strongly suggest that Snail requires both Ebi and CtBP for full repressor activity (Qi, 2008).

By what mechanism does Ebi contribute to repression? Previous studies have shown that Ebi and its mammalian homologue TBL1 can function through two different complexes, the NCoR-SMRT-HDAC3 complex and a Sina E3 ubiquitin ligase complex. Tests were performed to see if ubiquitin-dependent protein degradation is involved in Snail-mediated repression by adding a proteasome inhibitor to Tet-Sna-expressing cells. No change in luciferase activity was observed in response to this drug. This indicates that proteasomal degradation is not necessary for Snail repressor activity, which is also supported by the lack of rho derepression in sina germline clone mutant embryos (Qi, 2008).

It is well established that histone deacetylation correlates with transcription repression. Local deacetylation of histones by HDAC3 results in repression of gene transcription. HDAC3 was purified as a core subunit of the NCoR-TBL1 (Ebi) complex in mammalian cells, suggesting that histone deacetylation is functionally linked to the activity of this complex. Although the composition of a similar complex in Drosophila has not been determined, a physical association and functional connection between Ebi and SMRTER have been reported. In this study, it was found that HDAC3 and Ebi associate and that both are required for Snail repression domain function in S2 cells, as determined by RNAi and inhibition of HDAC activity. The observation that Sna 1-245 and 1-245Δ5-25 are resistant to TSA treatment implies that these proteins can repress by an Ebi-independent mechanism. Taken together, these results suggest that Ebi-dependent repression requires histone deacetylation, whereas CtBP-dependent repression does not in this assay (Qi, 2008).

In contrast to the situation in embryos where the Ebi interaction domain and the CtBP interaction domain in Snail cooperate, in the cell culture assay these domains (1-40 and 1-245Δ5-25) can repress transcription independently of one another. It is surprising, therefore, that Ebi or HDAC3 RNAi weakly relieved repression by Sna 1-245 containing both repression domains, and produce stronger effects together with CtBP RNAi. This indicates that repression in the cell culture assay may involve further components (Qi, 2008).

The role of SMRTER in this process remains to be determined. Unfortunately, SMRTER knock down by RNAi results in cell cycle arrest and is cell lethal, precluding an investigation of its function in Snail-mediated repression. However, it has been shown that mammalian NCoR and SMRT contain a deacetylase-activating domain (DAD) that is essential for catalytic activity of HDAC3. The DAD is evolutionarily conserved and present also in SMRTER. Presumably, Drosophila HDAC3 also requires SMRTER binding for activation of its enzymatic activity. Moreover, the association between TBL1 and HDAC3 in mammalian cells is bridged by SMRT. For these reasons, it is likely that the Ebi-HDAC3 complex also includes SMRTER. Reciprocal BLAST searches also reveal a homologue of the SMRT-NCoR complex core component GPS2 in Drosophila. It appears that the composition and dependence on histone deacetylation by HDAC3 for SMRT-NCoR complex function has been evolutionarily conserved (Qi, 2008).

Ever since the discovery that Rpd3 is a HDAC over 10 years ago, a strong link between histone hypoacetylation and transcriptional repression has been established. In Drosophila, five HDACs of the class I and II type, and five Sir2-like HDACs are present. However, it is not known whether regulation of histone acetylation contributes to transcriptional control during the rapid nuclear divisions in early Drosophila embryogenesis. Although Rpd3 or the Mi-2-Rpd3 complex has been implicated in repression by the Even-skipped, Runt, Knirps, Tramtrack and Hunchback repressor proteins, and as part of Groucho and Atrophin co-repressor complexes, a direct role of histone deacetylation in repression has not been established in these instances. A recent report has invoked regulation of transcription elongation in repression by the pair-rule proteins Runt and Fushi-tarazu in early embryos. In this case, no change in histone acetylation was observed on the target gene in transcriptionally active cells compared with inactive cells. By contrast, this study demonstrates that H3 becomes hypoacetylated at a Snail-regulated enhancer in the presence of Snail, and suggests that histone deacetylation participates in Snail-mediated repression based on a cell culture assay. This is the first evidence that histone deacetylation may be involved in cell-fate specification during Drosophila embryo development (Qi, 2008).

Vertebrate Snail proteins contain a different conserved motif, the SNAG domain in their very N termini. The Snail SNAG domain is necessary to recruit a Sin3A-HDAC1/HDAC2 co-repressor complex to the E-cadherin promoter, which is sensitive to the HDAC inhibitor TSA. This indicates that both vertebrate and insect Snail proteins rely on histone deacetylation for their repressor function, but that they recruit different co-repressor complexes. Whereas vertebrate Snail depends on Sin3A-HDAC1/HDAC2, insect Snail proteins require an Ebi-HDAC3 complex for maximal activity (Qi, 2008).

DEVELOPMENTAL BIOLOGY

Embryonic

The localization of Ebi protein was determined using antibodies to three different epitopes, as well as in a transgenic line carrying an ebi genomic construct tagged with a Myc epitope. The distribution of Ebi protein determined with these different reagents was identical. Ebi is widely expressed in nuclei of the embryo and larvae. Staining is largely, if not exclusively, nuclear. Double staining of salivary gland nuclei with anti-Myc antibodies to detect Myc-tagged Ebi and the DNA stain DAPI demonstrate that Ebi is not associated with chromatin but, rather, is distributed in a reticular pattern throughout the nucleoplasm (Dong, 2000).

Combinatorial signaling in the specification of primary pigment cells in the Drosophila eye

In the developing eye of Drosophila, the EGFR and Notch pathways integrate in a sequential, followed by a combinatorial, manner in the specification of cone-cell fate. This study demonstrates that the specification of primary pigment cells requires the reiterative use of the sequential integration between the EGFR and Notch pathways to regulate the spatiotemporal expression of Delta in pupal cone cells. The Notch signal from the cone cells then functions in the direct specification of primary pigment-cell fate. EGFR requirement in this process occurs indirectly through the regulation of Delta expression. Combined with previous work, these data show that unique combinations of only two pathways -- Notch and EGFR -- can specify at least five different cell types within the Drosophila eye (Nagaraj, 2007).

Unlike photoreceptor R cells, cone cells do not express Delta at the third instar stage of development. However, these same cone cells express Delta at the pupal stage. In addition, correlated with this Delta expression, the upregulation of phosphorylated MAPK was observed in these cells. This is very similar to the earlier events seen in R cells during larval development, in which the activation of MAPK causes the expression of Delta. Also, as in the larval R cells, the pupal upregulation of Delta in cone cells is transcriptional. A Delta-lacZ reporter construct, off in the larval cone cell, is detected in the corresponding pupal cone cells. To determine whether EGFR is required for the activation of Delta in the pupal cone cells, the temperature-sensitive allele EGFR^ts1 was used. Marked clones of this allele were generated in the eye disc using ey-flp at permissive conditions and later, in the mid-pupal stages, shifted the larvae to a non-permissive temperature. Cells mutant for EGFR, but not their adjacent wild-type cells, showed a loss of Delta expression. However, both mutant and wild-type tissues showed normal cone-cell development, as judged by Cut (a cone-cell marker) expression. As supporting evidence, ectopic expression of a dominant-negative version of EGFR (EGFR^DN) in cone cells using spa-Gal4 after the cells have already undergone initial fate specification also causes a complete loss of Delta expression without compromising the expression of the cone-cell-fate-specification marker (Nagaraj, 2007).

Gain-of-function studies further support the role of EGFR signaling in the regulation of Delta expression in cone cells. Although weak EGFR activation is required for cone-cell fate, activated MAPK is not detectable in cone-cell precursors of the third instar larval eye disc. When spa-Gal4 (prepared by cloning the 7.1 kb EcoRI genomic fragment of D-Pax2) is used to express an activated version of EGFR in larval cone cells, detectable levels of MAPK activation in these cells were found and the consequent ectopic activation of Delta in the larval cone cells occurred. Taken together, these gain- and loss-of-function studies show that, during pupal stages, EGFR is required for the activation of Delta. However, this Delta expression is not essential for the maintenance of cone-cell fate (Nagaraj, 2007).

In larval R cells, the activation of Delta transcription in response to EGFR signaling is mediated by two novel nuclear proteins, Ebi and Sno. To determine the role of these genes in wild-type pupal-cone-cell Delta expression, sno and ebi function were selectively blocked in the pupal eye disc. A heteroallellic combination of the temperature-sensitive allele sno^E1 and the null allele sno⁹³ⁱ exposed to a non-permissive temperature for 12 hours caused a significant reduction in Delta expression. Similarly, a dominant-negative version of ebi also caused the loss of Delta expression. Importantly, pupal eye discs of neither spa-Gal4, UAS-ebi^DN nor sno^E1/sno⁹³ⁱ showed any perturbation in cone-cell fate, as judged by the expression of Cut. Thus, as in the case of larval R cells, the loss of ebi and sno in the pupal cone cells causes the loss of Delta expression without causing a change in cone-cell fate (Nagaraj, 2007).

To test whether the expression of Delta in pupal cone cells is required for the specification of primary pigment cells, N^ts pupae were incubated at a non-permissive temperature for 10 hours during pupal development and pigment-cell differentiation was monitored using BarH1 (also known as Bar) expression as a marker. Loss of Notch signaling during the mid-pupal stages caused a loss of Bar, further demonstrating the requirement of Notch signaling in the specification of primary pigment-cell fate. Similarly, when the 54CGal4 driver line, which is activated in pigment cells, was used to drive the expression of a dominant-negative version of Notch, pupal eye discs lost primary pigment-cell differentiation, again suggesting an autonomous role for Notch in pigment-cell precursors. In neither the N^ts nor the 54C-Gal4, UAS-N^DN genetic background was perturbation observed in cone-cell fate specification. It is concluded that Delta activation mediated by EGFR-Sno-Ebi in pupal cone cells is essential for neighboring pigment-cell fate specification (Nagaraj, 2007).

Delta-protein expression in pupal cone cells is initiated at 12 hours and is downregulated by 24 hours of pupal development. To determine the functional significance of this downregulation, the genetic combination spa-Gal4/UAS-Delta was used, in which Delta is expressed in the same cells as in wild type, but is not temporally downregulated. Whereas, in wild type, a single hexagonal array of pigment cells surrounded the ommatidium, in the pupal eye disc of spa-Gal4, UAS-Delta flies, multiple rows of pigment cells were observed surrounding each cluster. Furthermore, in wild type, only two primary pigment cells were positive for Bar expression in each cluster, whereas, in spa-Gal4, UAS-Delta pupal eye discs, ectopic expression of Bar was evident in the interommatidial cells. Therefore, the temporal regulation of Notch signaling and its activation, as well as its precise downregulation, are essential for the proper specification of primary pigment-cell fate (Nagaraj, 2007).

By contrast to the autonomous requirement for Notch signaling in primary pigment cells, the function of the EGFR signal appears to be required only indirectly in the establishment of primary pigment-cell fate through the regulation of Delta expression in the pupal cone cells. When a dominant-negative version of EGFR was expressed using hsp70-Gal4 at 10-20 hours after pupation, no perturbation was observed in the specification of primary pigment cells, as monitored by the expression of the homeodomain protein Bar. By contrast, the expression of dominant-negative Notch under the same condition resulted in the loss of Bar-expressing cells. Thus, in contrast to Notch, blocking EGFR function at the time of primary pigment-cell specification does not block the differentiation of these cells. Importantly, blocking EGFR function in earlier pupal stages caused the loss of Delta expression in cone cells and the consequent loss of pigment cells. Based on these observations, it is concluded that, in the specification of primary pigment-cell fate, the Notch signal is required directly in primary pigment cells, whereas EGFR function is required only indirectly (through the regulation of Delta) in cone cells (Nagaraj, 2007).

The Runt-domain protein Lz functions in the fate specification of all cells in the developing eye disc arising from the second wave of morphogenesis. At a permissive temperature (25°C), lz^TS114 pupal eye discs showed normal differentiation of primary pigment cells. lz^TS114 is a sensitized background in which the Lz protein is functional at a threshold level. When combined with a single-copy loss of Delta, a dosage sensitive interaction caused the loss of primary pigment cells. By contrast, under identical conditions, a single-copy loss of EGFR function had no effect on the proper specification of primary pigment-cell fate. This once again supports the notion that the specification of primary pigment cells directly requires Lz and Notch, whereas EGFR is required only indirectly to activate Delta expression in cone cells (Nagaraj, 2007).

This study highlights two temporally distinct aspects of EGFR function in cone cells. First, this pathway is required for the specification of cone-cell fate at the larval stage, and EGFR is then required later in the pupal cone cell for the transcriptional activation of Delta, converting the cone cell into a Notch-signaling cell. Delta that was expressed in the cone cell through the activation of the Notch pathway functioned in combination with Lz in a cell autonomous fashion and promoted the specification of the primary pigment-cell fate (Nagaraj, 2007).

Studies using overexpressed secreted Spitz have shown that ectopic activation of the EGFR signal in all cells of the pupal eye disc results in excess primary pigment cells. This study shows that EGFR activation in the pupal eye disc is required for the transcriptional activation of Delta in cone cells, but that the loss of EGFR function at the time when primary pigment cells are specified does not perturb their differentiation. It is concluded that the ectopic primary pigment cells seen in an activated-EGFR background result from the ectopic activation of Delta, which then signals adjacent cells and promotes their differentiation into primary pigment cells. Indeed, it has been shown that excessive Delta activity results in the over specification of primary pigment cells. The results are also consistent with the previous observation that the EGFR target gene Argos is not expressed in primary pigment cells in pupal eye discs. Additionally, loss of EGFR function in pupal eye discs does not perturb the normal patterning of interommatidial bristle development, which develop even later than the primary pigment cells (Nagaraj, 2007).

The elucidation of the Sevenless pathway for the specification of R7 led to the suggestion that different cell types within the developing eye in Drosophila will require combinations of dedicated signaling pathways for their specification. However, studies from several laboratories have suggested that the Sevenless pathway seems to be an exception, in that cell-fate-specification events usually require reiterative combinations of a very small number of non-specific signals. Cone-cell fate is determined by the sequential integration of the EGFR and Notch pathways in R cells followed by the parallel integration of the EGFR and Notch pathways in cone-cell precursors. This study shows that the most important function of EGFR in the specification of primary pigment cells is to promote the transcriptional activation of Delta in cone cells through the EGFR-Ebi-Sno-dependent pathway. The sequential integration of the EGFR and Notch pathways, first used in the larval stage for Delta activation in R cells, is then reused a second time in cone cells to regulate the spatiotemporal expression of Delta, converting the cone cells at this late developmental stage to Notch-signaling cells. Delta present in the cone cell then signals the adjacent undifferentiated cells for the specification of primary pigment cells. For this process, the Notch pathway functions directly with Lz but indirectly with EGFR. Through extensive studies of this system it now seems conclusive that different spatial and temporal combinations of Notch and EGFR applied at different levels can generate all the signaling combinations needed to specify the neuronal (R1, R6, R7) and nonneuronal (cone, pigment) cells in the second wave of morphogenesis in the developing eye disc (Nagaraj, 2007).

The EGFR and Notch pathways are sequentially integrated, in a manner similar to that described here, in multiple locations during Drosophila development. In the development of wing veins, EGFR that is activated in the pro-vein cells causes the expression of Delta, which then promotes the specification of inter-vein cells. Similarly, these two pathways are sequentially integrated in the patterning of embryonic and larval PNS, and during muscle development. Indeed, there are striking similarities between the manner in which the EGFR and Notch pathways are integrated in the developmental program in the C. elegans vulva and the Drosophila eye. During vulval fate specification in the C. elegans hermaphrodite gonad, anchor cells are a source of EGFR signal (Lin3), which induces the specification of the nearest (P6) cell to the primary cell fate from within a group of six equipotent vulval precursor cells (VPC). This high level of EGFR activation induces the transcriptional activation of Notch ligands in the primary cells in what can be considered sequential integration of the two pathways - the Notch signal from the primary cell both inhibits EGFR activity in the VPCS on either side of P6.p and also promotes the secondary cell fate. Thus, the reiterative integration of these two signals, in series and in parallel, can be used successfully to specify multiple cell fates in different animal species. Given that the RTK and Notch pathways function together in many vertebrate developmental systems, it is likely that similar networks will be used to generate diverse cell fates using only a small repertoire of signaling pathways (Nagaraj, 2007).

EFFECTS OF MUTATION

ebi mutations have been identified in a screen for enhancers of an eye mutant called roughex, which plays a key role in regulating cell cycle progression in the developing eye. As a consequence of cell cycle defects, photoreceptor differentiation and pattern formation in the eye are disrupted. Whereas cell cycle regulators enhance and suppress the primary cell cycle phenotype, mutations in other loci, such as Star and Epidermal growth factor receptor, only modify the differentiation phenotype, and not the earlier cell cycle defects. Like Star and Egfr, ebi enhances the differentiation phenotype. These observations led to a consideration of the relationship between the Egfr signaling pathway and ebi. Evidence shows that ebi participates in Egfr signaling pathways. ebi^E4, ebi^E90, and ebi^P7 are null, strong, and weak alleles, respectively (Dong, 1999).

That ebi functions in the Egfr pathway was initially suggested by phenotypes of a viable heteroallelic combination of ebi (i.e., ebi^P7/ebi^E90). These flies exhibit phenotypes similar to weak loss-of-function Egfr alleles (i.e., Egfr^top1/Egfr^f2) including partial female sterility resulting from partially ventralized eggs, wing vein defects, short bristles, and abnormal eyes (i.e., rough eyes). Further evidence that ebi participates in the Egfr pathway was provided by genetic interactions between ebi and Egfr components. For instance, flies carrying two different alleles of Egfr (Egfr^top1/Egfr^f2) have a weak rough-eye phenotype, which is enhanced in flies that are heterozygous for ebi. ebi and Egfr mutant embryos are also similar. Homozygous ebi null mutant embryos (ebi^E4) exhibit a tail-up or U-shaped embryo with head defects. Embryos lacking both the zygotic and maternal contributions of ebi were created using ovo^D and FRT/FLP-induced recombination. This results in a more severe phenotype, including the loss of ventral denticle belt structures and a tightly curled morphology indicating a marked failure in germ-band retraction. Severe head defects are also observed. In contrast to Egfr mutants, some residual ventral cuticular structures remain in embryos lacking both the zygotic and maternal contributions of ebi (Dong, 1999).

Loss of ebi also affects Egfr-dependent expression of genes in the embryo. The Egfr ligand Spitz is expressed along the ventral midline and induces expression of different target genes, including fasciclin III (fasIII) and orthodenticle (otd), in cells located in more lateral positions. In zygotic null Egfr mutants both otd and FasIII expression are lost. In wild-type stage 11/12 embryos, FasIII protein is broadly distributed in the visceral mesoderm and in a bilaterally symmetric cluster of cells flanking the midline of the ventral ectoderm. In ebi mutant embryos lacking both maternal and zygotic contribution, FasIII expression is largely abolished, although some residual patches of staining remain. Egfr-independent expression of FasIII in the anterior-most region of the embryo is unaffected in ebi mutants. In wild-type stage 10/11 embryos, otd mRNA is expressed in the preantennal head region and in the ventral-most ectoderm. In ebi mutant embryos, otd expression is markedly reduced. These data suggest that ebi may be a component in the Egfr signal transduction pathway. To assess whether ebi encodes a hitherto unidentified regulator in the Ras/MAP kinase pathway, its role in the Torso RTK pathway was assessed. Torso controls the development of the anterior and posterior termini of the embryo. Ras, Raf, MEK, and MAPK participate in both the Egfr and Torso RTK pathways. The expression of Torso target genes huckebein (hkb) and tailless (tll) in embryos entirely deficient in ebi (i.e., lacking both maternal and zygotic ebi) is indistinguishable from wild type. In summary, ebi mutant phenotypes assessed using both molecular and morphological criteria are similar to Egfr mutations. Furthermore, ebi does not function in all RTK pathways, since Torso-induced terminal development is ebi independent. These data indicate that ebi, either directly or indirectly, regulates Egfr signaling. As a step toward understanding the role of ebi in the context of a specific developmental process, the role of ebi in R7 development in the compound eye was assessed through both genetic and molecular studies (Dong, 1999).

The R7 equivalence group comprises five cells competent to become R7 neurons. They are the R7 precursor cell and the precursors to the four cone cells. Cone cell precursor cells can be induced to become R7 cells by ectopic activation of the R7 inductive pathway in these cells. Transformation of cone cells into R7 cells leads to a disorganized adult eye or a so-called rough-eye phenotype. The ability of loss-of-function ebi mutations to suppress this transformation was assessed in various genetic backgrounds. Whereas ebi dominantly suppresses R7 development induced by the activated Egfr expressed in the R7 equivalence group under the control of the sev enhancer (sev-Tor^DEgfr), it does not suppress R7 development induced by the activated Sev receptor (sev-Tor^DSev, SevS11, or activated forms of Ras, Raf, and MAPK. Hence, ebi is required for the transformation of cone cell precursors into R7 neurons by the activated Egfr (Dong, 1999).

To assess whether ebi participates in the induction of the R7 precursor cell into an R7 neuron, a genetically sensitized background in which only some 15%-20% of the R7 precursors become R7 neurons was used. The R7 inductive signal is attenuated by using a strong hypomorphic allele of sev (sev^E4) and a weak gain-of-function mutation in the Ras activator, encoded by the Son of sevenless gene, Sos^JC2. Aside from the loss of the majority of the R7 cells, development of the eye in this genetic background is otherwise indistinguishable from wild type. ebi is a dominant enhancer of this phenotype, as are Egfr loss-of-function mutations. These data are consistent with studies demonstrating a requirement for both the Egfr and Sev receptor in R7 induction. Hence, ebi is required for induction of the R7 precursor cell into an R7 neuron and for transformation of cone cell precursors into R7 in response to ectopic activation of Egfr. Ttk88 down-regulation is required for R7 induction of the R7 precursor cell. This is supported by the finding that Ttk88 mutations are dominant suppressors of the Sev^E4;Sos^JC2/+ phenotype (Dong, 1999).

To assess the role of ebi on R7 development in an otherwise wild-type background, attempts were made to generate homozygous null mutant clones. Such clones could not be generated using X-ray and heat shock Flp-induced mitotic recombination. Hence, like Egfr, ebi is required for cell proliferation and/or survival during the proliferative phase of disc development. To increase the efficiency of Flp-induced mitotic recombination, a Flp source driven by the eyeless (ey) promoter was used. The ey promoter drives expression from the earliest cell divisions in the eye primordium until the last cell division of precursor cells in the third instar. This results in the production of multiple mutant clones throughout development. Mutant clones in the eye disc have been recognized by the loss of Ebi immunoreactivity. Rather small clones have been observed: clusters within these clones contain differentiating R cells. Each cluster contains a single R8 cell (i.e., stained with antibody to the Boss protein), and early clusters appear normal. Although clusters containing eight neurons form, disorganized clusters containing fewer differentiated neurons are also observed (Dong, 1999).

Adult ommatidia containing homozygous mutant cells are frequently highly disorganized and show a marked reduction in R cells. Mutant R cells, including R7 cells, are seen in adult mosaic ommatidia; some 80% of these cells show an altered cellular morphology. Hence, although ebi is required for R7 development in a genetically sensitized background, R7 neurons can develop in an ebi mutant. Although the formal possibility that these R7 neurons develop because of perdurance of Ebi protein in the R7 precursor cell cannot be ruled out, these data strongly suggest that R7 cells can form in an ebi-independent fashion, though less efficiently than in wild type. These data are consistent with ebi subserving a redundant function in R7 development. To gain clues to the molecular pathways regulated by ebi, the gene was cloned and sequenced (Dong, 1999).

This transcription unit encodes a protein of 700 amino acids with a carboxy-terminal segment containing six WD40 repeats. The ebi^E4 and ebi^E90 alleles result in missense mutations. In ebi^E4 the methionine encoded by codon 1 is changed to an isoleucine, and in ebi^E90 a highly conserved cysteine, located at amino acid 510 between WD40 repeats 3 and 4, is changed to a tyrosine (Dong, 1999).

In vivo function of a novel Siah protein in Drosophila: Genetic interactions with Ebi

The Siah proteins, mammalian homologues of the Drosophila Sina protein, function as E3 ubiquitin ligase enzymes and target a wide range of cellular proteins for degradation. This study investigated the in vivo function of the fly protein, Sina-Homologue (SinaH), which is highly similar to Sina. Flies that completely lack SinaH are viable and in combination with a mutation in the gene, Ebi, show an extra dorsal central bristle phenotype. SinaH and Ebi can interact with each other both in vivo and in vitro suggesting that they act in the same physical complex. Flies that lack both Sina and Sina-Homologue were also created and show visible eye and bristle phenotypes, which can be explained by an inability to degrade the neuronal repressor, Tramtrack. No evidence was found for redundancy in the function of Sina and SinaH (Cooper, 2007).

The Siah E3 ubiquitin ligases have been shown to have many important functions in mammals and can target a diverse array of substrates for degradation. There are two Siah-like proteins in the Drosophila genome: Sina and the newly identified Sina-Homologue for which no mutant has existed. A defined mutation using homologous recombination to completely remove the SinaH gene was created in order to investigate the in vivo function of this potentially interesting gene. Flies that lack the SinaH gene do not show any visible phenotype in the adult. However, when homozygous, this SinaH¹ allele interacts with a heterozygous Ebi^k16213 allele to give extra dorsal central bristles. Although the formation of extra dorsal central bristles is weak, it is a specific effect of removal of SinaH and lowering the amount of Ebi (Cooper, 2007).

As well as this genetic interaction, it was also shown that SinaH and Ebi proteins physically associate both in vitro and in vivo in S2 cells, consistent with the proteins being members of the same complex. The proteins seem to interact more strongly in vivo in the co-immunoprecipitation experiment compared with in vitro assays, suggesting that other components, which might be functionally relevant, could be present within the Ebi/SinaH complex. The lack of a visible phenotype in the SinaH mutant flies could suggest that removal of SinaH alone can be compensated by other members of the complex or it may be functionally redundant with other genes. The clear extra DC bristle phenotype when there are reduced amounts of Ebi, suggests that it is only when Ebi becomes limiting within the complex, that this compensatory mechanism is not sufficient, and the bristle phenotype is visible. This suggests that a possible role of a SinaH/Ebi containing complex in vivo is to restrict the ability to form dorsal central bristles (Cooper, 2007).

Evidence indicates that a role of the SinaH/Ebi complex is to target substrates for ubiquitination and proteasome-dependent degradation. SinaH has high homology with Sina, which can act as part of an E3 ubiquitin ligase complex containing Ebi to cause ubiquitination and degradation of Tramtrack 69. If SinaH acts in a similar manner to Sina, one explanation could be an inability to degrade Tramtrack, but this would result in fewer bristles being formed rather than additional bristles, since this neuronal repressor would inhibit SOP cell formation. This suggests that SinaH/Ebi is acting on different substrates to Sina. In humans, Siah proteins, and the homologue of Ebi (TBL1) can act together to degrade a component of the Wnt (Wg) signalling pathway, β-catenin (Armadillo). Removal of SinaH and reducing levels of Ebi might therefore cause the stabilisation of Armadillo, and the increased Wg signalling may up-regulate proneural activity in the DC cluster. Siah proteins have also been implicated in cell cycle control, and Ebi has a role in repression of the cell cycle transition between G1 and S phase. Another possibility is that increased cell division within the proneuronal cluster could result in additional SOP cell formation. However, the exact mechanism and substrates of the SinaH/Ebi complex involved in DC bristle formation is yet to be determined (Cooper, 2007).

The mouse Siah1a and Siah2 are synthetically lethal which suggests a high level of redundancy between these Siah proteins. To test if there is also redundancy between Siah proteins in flies, Both Sina and SinaH genes were removed together. Interestingly, the resultant fly displayed phenotypes very similar to flies which only lacked Sina, and could be attributed to mis/over expression of Tramtrack. Sina and SinaH therefore have distinct phenotypes suggesting that they have different roles in flies, consistent with their dissimilar expression patterns during development. Sina appears to have higher expression in the embryo and larvae whereas in this study, it was shown that SinaH mRNA is mainly expressed in later developmental stages and in males. Given that the mouse knockout of Siah1a is sterile and defective in spermatogenesis, there may still be other roles of SinaH that are yet to be uncovered and in flies, such roles can be compensated for by other E3 ubiquitin ligases or members of the complexes (Cooper, 2007).

Genetic characterization of ebi reveals its critical role in Drosophila wing growth.

The ebi gene of Drosophila has been implicated in diverse signalling pathways, cellular functions and developmental processes. However, a thorough genetic analysis of this gene has been lacking and the true extent of its biological roles is unclear. This study characterize eleven ebi mutations and found that ebi has a novel role in promoting growth of the wing imaginal disc: viable combinations of mutant alleles give rise to adults with small wings. Wing discs with reduced EBI levels are correspondingly small and exhibit down-regulation of Notch target genes. Furthermore, EBI was shown to colocalize on polytene chromosomes with Smrter (SMR), a transcriptional corepressor, and Suppressor of Hairless (SU(H)), the primary transcription factor involved in Notch signalling. Interestingly, the mammalian orthologs of ebi, transducin β-like 1 (TBL1) and TBL-related 1 (TBLR1), function as corepressor/coactivator exchange factors and are required for transcriptional activation of Notch target genes. It is hypothesized that EBI acts to activate (de-repress) transcription of Notch target genes important for Drosophila wing growth by functioning as a corepressor/coactivator exchange factor for SU(H) (Marygold, 2011; full text of article).

EVOLUTIONARY HOMOLOGS

Several regions of the Saccharomyces cerevisiae genome are subject to position-dependent transcriptional repression mediated by a multi-component nucleosome-binding complex of silent information regulator proteins (Sir2p, Sir3p and Sir4p). These proteins are present in limiting amounts in the nucleus and are targeted to specific chromosomal regions by interaction with sequence-specific DNA-binding factors. Different sites of repression compete for Sir complexes, although it is not known how Sir distribution is regulated. In a screen for factors that interact with Sir4p amino terminus, SIF2 has been cloned; this protein encodes a WD40-repeat-containing factor that disrupts telomeric silencing when overexpressed. In contrast to deletion of SIR4, SIF2 deletion improves telomeric repression, suggesting that under normal conditions Sif2p antagonizes Sir4p function at telomeres. Sif2p overexpression alters the subnuclear localization of Sir4p, but not its protein expression level, suggesting that Sif2p may recruit Sir4p to nontelomeric sites or repression. The sif2 mutant strains are hypersensitive to a range of stress conditions, but do not have decreased viability and do not alter repression in the rDNA. In conclusion, Sif2p resembles the Sir4p regulatory proteins Sir1p and Uth4p in that it competes for the functional assembly of Sir4p at telomeres, yet unlike Sir1p or Uth4p, it does not target Sir4p to either mating-type or rDNA loci (Cockell, 1998).

A novel gene, transducin (beta)-like 1 (TBL1, Drosophila homolog: Ebi), has been identified in the Xp22.3 genomic region, that shows high homology with members of the WD-40-repeat protein family. The gene contains 18 exons spanning approximately 150 kb of the genomic region adjacent to the ocular albinism gene (OA1) on the telomeric side. However, unlike OA1, TBL1 is transcribed from telomere to centromere. Northern analysis indicates that TBL1 is ubiquitously expressed, with two transcripts of approximately 2.1 kb and 6.0 kb. The open reading frame encodes a 526-amino acid protein, which shows the presence of six beta-transducin repeats (WD-40 motif) in the C-terminal domain. The homology with known beta-subunits of G proteins and other WD-40-repeat containing proteins is restricted to the WD-40 motif. Genomic analysis has revealed that the gene is either partly or entirely deleted in patients carrying Xp22.3 terminal deletions. The complexity of the contiguous gene-syndrome phenotype shared by these patients depends on the number of known disease genes involved in the deletions. Interestingly, one patient carrying a microinterstitial deletion involving the 3' portion of both TBL1 and OA1 shows the OA1 phenotype associated with X-linked late-onset sensorineural deafness. An involvement of TBL1 in the pathogenesis of the ocular albinism with late-onset sensorineural deafness phenotype is postulated (Bassi, 1999).

The corepressor SMRT mediates repression by thyroid hormone receptor (TR) as well as other nuclear hormone receptors and transcription factors. A novel SMRT-containing complex has been isolated from HeLa cells. This complex contains transducin beta-like protein 1 (TBL1), whose gene is mutated in human sensorineural deafness. It also contains HDAC3, a histone deacetylase not previously thought to interact with SMRT. TBL1 displays structural and functional similarities to Tup1 and Groucho corepressors, sharing their ability to interact with histone H3. In vivo, TBL1 is bridged to HDAC3 through SMRT and can potentiate repression by TR. Intriguingly, loss-of-function TRbeta mutations cause deafness in mice and humans. These results define a new TR corepressor complex with a physical link to histone structure and a potential biological link to deafness (Guether, 2000).

Evidence is presented that both corepressors SMRT and N-CoR exist in large protein complexes with estimated sizes of 1.5-2 MDa in HeLa nuclear extracts. Using a combination of conventional and immunoaffinity chromatography, a SMRT complex has been isolated and histone deacetylase 3 (HDAC3) and transducin (beta)-like I (TBL1), a WD-40 repeat-containing protein, have been identified as the subunits of the purified SMRT complex. The HDAC3-containing SMRT and N-CoR complexes can bind to unliganded thyroid hormone receptors (TRs) in vitro. In Xenopus oocytes, both SMRT and N-CoR also associate with HDAC3 in large protein complexes and injection of antibodies against HDAC3 or SMRT/N-CoR leads to a partial relief of repression by unliganded TR/RXR. These findings thus establish both SMRT and N-CoR complexes as bona fide HDAC-containing complexes and shed new light on the molecular pathways by which N-CoR and SMRT function in transcriptional repression (Li, 2000).

Destruction of ß-catenin is regulated through phosphorylation-dependent interactions with the F box protein ß-TrCP. A novel pathway for ß-catenin degradation was discovered involving mammalian homologs of Drosophila Sina (Siah), which bind ubiquitin ß-conjugating enzymes, and Ebi, an F box protein that binds ß-catenin independent of the phosphorylation sites recognized by ß-TrCP. A series of protein interactions were identified in which Siah is physically linked to Ebi by association with a novel Sgt1 homolog SIP that binds Skp1, a central component of Skp1-Cullin-F box complexes. Expression of Siah is induced by p53, revealing a way of linking genotoxic injury to destruction of ß-catenin, thus reducing activity of Tcf/LEF transcription factors and contributing to cell cycle arrest (Matsuzawa, 2001).

A pathway linking the RING protein Siah-1 to the F box protein Ebi has been mapped and it has been shown that Ebi can bind ß-catenin. Unlike ß-TrCP, however, which requires GSK3ß-mediated phosphorylation of ß-catenin on serine 33 and serine 37, Ebi interacts with ß-catenin independently of these phosphorylation sites. Also, the Siah binding protein SIP associates with complexes containing Ebi but not ß-TrCP, suggesting differences compared to previously characterized E3 ubiquitin ligase complexes, where E2 enzymes are supplied via Cullin-mediated interactions with RING-containing proteins such as Rbx-1/Roc-1. Recent identification of interactions between Siah-1 and the ß-catenin binding protein APC suggest that this scaffold protein represents a point of common intersection of the Wnt and Siah-1 pathways for ß-catenin degradation (Matsuzawa, 2001).

Two alternative pathways for regulation of ß-catenin levels are presented, involving different F box proteins (Ebi versus ß-TrCP). One pathway is initiated by increases in the expression of Siah-family proteins, which can be induced, for example, by p53 in response to DNA damage, and involves sequential protein interactions with SIP, Skp1, and Ebi. Ebi binds ß-catenin, thus recruiting it to the Siah-1-SIP-Skp1 complex for polyubiquitination and subsequent proteosome-mediated degradation. Siah-1 binds the E2 UbcH5. The other pathway is regulated by Wnt signals (Dsh) and possibly PI3K/Akt. This pathway is phosphorylation dependent and involves GSK3ß-induced phosphorylation of Ser-33 and Ser-37 on ß-catenin, allowing ß-TrCP binding, resulting in recruitment of ß-catenin to Skp1-Cullin-1- ß-TrCP complexes (SCF). Cullin-1, in collaboration with other proteins, supplies this SCF complex with E2s, such as UbcH3. APC is required for both pathways as a scaffold protein, binding ß-catenin via one domain and also binding Siah-1 and GSK3ß (Matsuzawa, 2001).

In the fly, Sina recruits E2s to Phyllopod/Tramtrack complexes, targeting Tramtrack for ubiquitination. The ebi-gene product also binds Tramtrack and promotes its degradation in vitro and when expressed in insect cells in culture. Loss-of-function mutations of ebi cause Tramtrack accumulation and prevent R7 cell differentiation. Similar to ß-TrCP, the ebi gene of Drosophila encodes an F box/WD-40-repeat protein with sequence homology to Cdc4 (yeast), Sel-10 (C. elegans), and Slimb (Drosophila), suggesting that it provides a functional connection between a Sina-regulated pathway and SCF complexes. How this linkage between Sina and SCF complexes is achieved, however, has been unclear (Matsuzawa, 2001).

The finding that SIP functions as a molecular bridge between the human homologs of Sina and the SCF-component Skp1 provides evidence of a physical linkage between components of these two ubiquitin ligase systems, thus corroborating the genetic evidence from Drosophila that these two pathways for targeted protein degradation interact. The Drosophila ortholog of SIP is also capable of bridging the fly Skp1 and Sina proteins in three-hybrid experiments. Thus, an evolutionarily conserved network of protein interactions exists in which Siah-1 (Sina) binds to SIP, which in turn binds to Skp1, which binds Ebi (Matsuzawa, 2001).

p53 can induce expression of Siah-family genes in mammals, establishing p53 as one factor capable of invoking Siah-dependent pathways for protein degradation. Siah-family proteins are normally maintained at a relatively low level through ubiquitination-dependent protein turnover, where human Siah-1 and Siah-2 promote their own degradation through interactions of their RING domains with E2s. This therefore suggests that activation of p53 leads to a burst of Siah-1 mRNA and protein production, triggering the Siah/SIP/Skp1/Ebi pathway for ß-catenin degradation. In contrast to Siah-family proteins, it seems unlikely that SIP, Skp1, or Ebi are limiting components of this pathway, since overexpression of them has little effect on ß-catenin levels (Matsuzawa, 2001).

Though p53-mediated degradation of ß-catenin correlates with cell cycle arrest, it remains to be established whether these events are functionally linked. Activation of Tcf/LEF-family transcription factors by ß-catenin is known to induce expression of cyclin D1, c-myc, and other genes important for cell proliferation, making it plausible that ß-catenin degradation is linked to p53-mediated cell cycle arrest. However, given the role established for the cyclin-dependent kinase inhibitor p21^Waf1 in mediating G1 arrest induced by p53, it is unclear whether a parallel pathway for ß-catenin degradation would be required. Circumstances have been described where p53 fails to induce cell cycle arrest despite inducing p21^Waf1 expression, raising the question of whether p21^Waf1 is necessary but insufficient for p53-mediated G1 arrest. Recently, a genetic interaction between ebi and p21^Waf1 has been identified using an assay in Drosophila where flies are engineered to ectopically express human p21^Waf1 in the developing eye disc (Boulton, 2000). Specifically, mutant alleles of ebi abrogated inhibition of S phase entry by p21^Waf1, implying a need for Ebi in p21-mediated cell cycle arrest. Flies with mutant ebi also display ectopic S phases and overproliferation phenotypes (Boulton, 2000), further implying a role for ebi in growth suppression. Defects in cell cycle arrest in ebi mutants, however, do not necessarily implicate ß-catenin/Armadillo. For example, p53 can induce degradation of c-Myb through a proteosome-dependent mechanism partly mediated by Siah (Tanikawa, 2000). Thus, Ebi may have other targets in addition to ß-catenin that are relevant to mechanisms of p53-mediated cell cycle arrest. Future experiments should explore whether the fly homolog of p53 is linked to an ebi-dependent pathway for cell cycle arrest entailing degradation of Armadillo. In the M1 cell model, p53 induces both G1 arrest and apoptosis. Though Ebi(DeltaF)-expressing M1 cells may exhibit some delay in p53-induced apoptosis, this could result indirectly because of failed G1 arrest. Moreover, Siah-1 often fails to induce apoptosis when overexpressed in cells. However, links of Siah to apoptosis can occur under some circumstances, as demonstrated by the observation that coexpression of Siah-1 with a Siah binding protein Pw1/Peg3 causes apoptosis, whereas neither Siah-1 nor Pw1/Peg3 alone are sufficient. Mutations affecting components of the Wnt-signaling pathway are commonly observed in human cancers, resulting in aberrant accumulation of ß-catenin and activation of Tcf/LEF-target genes. Wnt-family ligands, frizzled-family receptors, and the signaling proteins downstream of these define one mechanism for regulating ß-catenin levels. However, additional inputs into pathways controlling ß-catenin turnover have recently been identified, including a mitogen-activated protein kinase pathway involving a Tak1 homolog and Nemo-like kinases in C. elegans and a cell adhesion-dependent pathway involving integrin-linked kinase. The findings reported here reveal yet another pathway for regulating ß-catenin levels that is linked at least in part to p53-dependent responses to genotoxic injury. It is speculated that loss of p53 or components of the Siah/SIP/Skp/Ebi pathway for ß-catenin destruction may contribute to aberrant ß-catenin accumulation in cancers (Matsuzawa, 2001).

REFERENCES

Search PubMed for articles about Drosophila ebi

Andres, M. E, et al. (1999). CoREST: a functional corepressor required for regulation of neural-specific gene expression. Proc. Natl. Acad. Sci. 96: 9873-9878. 10449787

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