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

Gene name - ebi

Synonyms -

Cytological map position - 21C2

Function - Scaffolding protein

Keywords - dHDAC3/SMRTER co-repressor complex, Egfr pathway, protein degradation, cell cycle

Symbol - ebi

FlyBase ID: FBgn0263933

Genetic map position -

Classification - Trp-Asp (WD) repeats protein

Cellular location - nuclear

NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Lim, Y.M., Yagi, Y. and Tsuda, L. (2015). Cellular defense and sensory cell survival require distinct functions of ebi in Drosophila. PLoS One 10: e0141457. PubMed: 26524764
The innate immune response and stress-induced apoptosis are well-established signaling pathways related to cellular defense. NF-κB and AP-1 are redox-sensitive transcription factors that play important roles in those pathways. This study shows that Ebi, a Drosophila homolog of the mammalian co-repressor molecule transducin β-like 1 (TBL1), variously regulates the expression of specific genes that are targets of redox-sensitive transcription factors. In response to different stimuli, Ebi activates gene expression to support the acute immune response in fat bodies, whereas Ebi represses genes that are involved in apoptosis in photoreceptor cells. Thus, Ebi seems to act as a regulatory switch for genes that are activated or repressed in response to different external stimuli. These results offer clear in vivo evidence that the Ebi-containing co-repressor complex acts in a distinct manner to regulate transcription that is required for modulating the output of various processes during Drosophila development.
Nguyen, M.B., Vuong, L.T. and Choi, K.W. (2016). Ebi modulates wing growth by ubiquitin-dependent downregulation of Crumbs in Drosophila. Development 143: 3506-3513. PubMed ID: 27702784
Notch signaling at the dorsoventral (DV) boundary is essential for patterning and growth of wings in Drosophila. The WD40 domain protein Ebi has been implicated in the regulation of Notch signaling at the DV boundary. This study shows that Ebi regulates wing growth by antagonizing the function of the transmembrane protein Crumbs (Crb). Ebi physically binds to the extracellular domain of Crb (Crbext), and this interaction is specifically mediated by WD40 repeats 7-8 of Ebi and a laminin G domain of Crbext. Wing notching resulting from reduced levels of Ebi is suppressed by decreasing the Crb function. Consistent with this antagonistic genetic relationship, Ebi knockdown in the DV boundary elevates the Crb protein level. Furthermore, Ebi is required for downregulation of Crb by ubiquitylation. Taken together, the study proposes that the interplay of Crb expression in the DV boundary and ubiquitin-dependent Crb downregulation by Ebi provides a mechanism for the maintenance of Notch signaling during wing development.


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 (TorDEgfr) driven by the sev promoter also leads to marked degradation of Ttk88. sev-TorDEgfr-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).


cDNA clone length - 3307

Bases in 5' UTR - 318

Exons - 3

Bases in 3' UTR - 883


Amino Acids - 700

Structural Domains

ebi-related human cDNA sequences and genomic sequences from S. cerevisiae and Arabidopsis thaliana, were identified in the database. Because the initial human expressed sequence tag was not complete, additional cDNAs were isolated from adult human spleen cDNA library and sequenced. Both an amino-terminal 89-amino-acid segment and the carboxy-terminal WD40 repeats of fly Ebi correspond remarkably well to these regions in the mammalian, plant, and yeast genes; the amino-terminal 89 amino acids and the WD40 repeat region share 81%, 34%, and 51% identity with the human, yeast, and plant sequences, respectively. In addition to these conserved regions the fly protein is predicted to contain an insertion of 160 amino acids between the amino terminus and the WD40 repeats (Dong, 2000).

The bipartite structure of Ebi is reminiscent of three proteins involved in protein degradation: Cdc4 from S. cerevisiae, Slimb from Drosophila, and Sel-10 from C. elegans. All three proteins contain an amino-terminal F-box and carboxy-terminal WD40 repeats; these proteins have been shown (Cdc4) or proposed (Slimb and Sel-10) to target proteins for degradation by linking them to a ubiquitin-conjugase complex. Although the amino-terminal domain of Ebi is divergent from the Cdc4 F box (as is Slimb), it shares weak sequence and structural homology. The amino-terminal half of the F box is more highly related to ebi than the carboxy-terminal region. The periodic spacing of hydrophobic residues in both Ebi and F-box sequences is consistent with these regions assuming an alpha-helical amphipathic conformation. Three residues in the amino-terminal region of the Cdc4 F box are known to be required for binding to Skp1 (a component in the E3 complex). These amino acids are conserved in Eb, and correspond to residues P45, I52, and L57 in the Ebi sequence (Dong, 2000).

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

date revised: 1 February 2001

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