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 (UASG) near its 3' end, causes overexpression of a nearby gene under the control of the Gal4-UASG 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 UASG in those insertions, reduction of Ebi activity will derepress UASG and further enhance activation by GMR-Gal4. One copy of a dominant-negative construct of ebi (GMR-ebiDN) caused only a mild defect in eye morphology and weak, if any, ectopic expression of chn. GMR-ebiDN strongly enhanced the overexpression phenotype of chnGS17605 and chnGS11450, which contained GS vector insertions (-474 and -734, respectively) upstream of the transcriptional start site. However, GMR-ebiDN failed to enhance the overexpression phenotype of other GS lines (chnGS2112 and chnGS17892) 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).
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).
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).
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).
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