Protein Interactions

Groucho protein binds specifically to Hairy and also to Hairy-related bHLH proteins encoded by deadpan and the Enhancer of split complex. The C-terminal WRPW motif present in all these bHLH proteins is essential for this interaction (Paroush, 1994).

Hairy, Deadpan and E(SPL) proteins have three evolutionarily conserved domains required for their function: the bHLH, Orange, and WRPW domains. However, the suppression of Scute activity by Hairy does not require the WRPW domain. The Orange domain is an important functional domain that confers specificity among members of the Hairy/E(SPL) family. A Xenopus Hairy homology conserves not only Hairy's structure but also its biological activity. Transcriptional repression by the Hairy/E(SPL) family of bHLH proteins involves two separable mechanisms: repression of specific transcriptional activators (such as Scute, through the bHLH and Orange domains) and repression of other activators (such as the Groucho protein) via interaction of the C-terminal WRPW motif with corepressors (Dawson, 1995).

The repression by Groucho and Hairy-like proteins requires the S/P (Ser-Pro-rich) domain of Groucho and the conserved carboxl-terminal Trp-Arg-Pro-Trp (WRPW) sequence of the Hairy-like transcription factor for direct interactions between Groucho and Hairy-like proteins. In addition to the S/P domain, a WD40 motif is found in the C-terminal half of Groucho. The WD40 motif is a loosely conserved repeat of 40 amino acids, separated by a Trp-Asp dipeptide sequence. The other sequence recognized in the Drosophila protein is a CcN motif, containing Cdc3 and casein kinase sites and a possible nuclear localization signal. A glutamine-rich region, the Q domain, is conserved as well in mammalian Groucho homologs. Both the Q domain and the WD40 domain are highly conserved, suggesting that they perform a highly conserved function. The S/P domain is less well conserved, implying that each Groucho family member binds preferentially to a particular Hairy-like transcription factor. Groucho-related genes in mammals also include one gene that encodes a truncated homolog of Drosophila, containing only the Q domain and part of the S/P sequence. The short protein may act to regulate the activity of the longer protein. The Q domain is used for dimerization between Groucho family members. Groucho proteins are able to dimerize through the Q domain; that dimerization requires a core of 50 amino acids. Surprising, the dimerization does not require the leucine zipper located within the Q domain. It is suggested that the ability of Groucho proteins to dimerize is conserved, and that the presence of truncated Groucho proteins in mammals provides yet an additional level of transcriptional regulation in mammalian systems (Pinto, 1996).

Runt domain family members are defined based on the presence of the 128-amino-acid Runt domain, which is necessary and sufficient for sequence-specific DNA binding. There exists an evolutionarily conserved protein-protein interaction between Runt domain proteins and the corepressor Groucho. However, the interaction is independent of the Runt domain and can be mapped to a 5-amino-acid sequence, VWRPY, present at the C terminus of all Runt domain proteins. Drosophila runt and groucho interact genetically; the in vivo repression of a subset of Runt-regulated genes is dependent on the interaction with Groucho and is sensitive to Groucho dosage. Runt's repression of one gene, engrailed, is independent of VWRPY and Groucho, thus demonstrating alternative mechanisms for repression by Runt domain proteins (Aronson, 1997).

Unlike other transcriptional regulatory proteins that interact with Groucho, Runt domain proteins are known to activate transcription. The distinction between the Runt domain consensus (VWRPY) and the Hairy-related/HES consensus (WRPW) raises a question: are the C-termini of these families interchangeable? The ability of Runt domain proteins to activate transcription suggests that the interaction with Groucho is regulated: when Runt domain proteins assemble on a promoter that is to be activated, Groucho must either be absent or in a context where it cannot exert its repressive effects. The difference between the Groucho-recruiting C-termini of the Hairy-related/HES family and the Runt domain family may be the difference between a constitutive Groucho interaction and one that is regulated (Aronson, 1997).

An investigation was carried out of the molecular mechanisms underlying the functions of Groucho and its mammalian homologs, the transducin-like Enhancer of split (TLE) proteins. A minority fraction of nuclear Groucho/TLEs are associated with chromatin in live cells; they co-purify with isolated histones. Affinity chromatography and far Western blotting studies show further that native Groucho/TLE proteins interact specifically with histone H3 and not with other core histones. This interaction is mediated by the H3 amino-terminal domain previously shown by genetic analysis in yeast to be essential for the role of H3 in transcriptional silencing. Groucho/TLEs form oligomeric structures in vivo. These combined findings suggest that transcription complexes containing Groucho/TLEs may associate with chromatin through interactions with the amino terminus of histone H3. These interactions may be propagated along the chromosome due to the ability of Groucho/TLEs to participate in higher order structures (Palaparti, 1997).

In vivo functional assays were used to test whether different repressor activities are mediated by the Groucho (Gro) corepressor in the Drosophila embryo. Previously, Gro was proposed to mediate repression by the Hairy-related family of basic helix-loop-helix proteins. Repression by Hairy requires Gro. Specifically, Groucho is required for Hairy repression of Sex lethal transcription. Although Hairy is not normally involved in sex determination, Groucho's misexpression at early blastoderm stages mimics Deadpan's activity and inhibits Sxl transcription. Hairy is unable to repress Sxl transcription in groucho mutant embryos. Replacement of the carboxyl terminus of Hairy by the Eve repressor domain generates a Groucho-independent function for Hairy. This result also provides direct evidence that the failure of Hairy to repress Sxl in gro embryos results from a loss of activity of the C-terminal WRPW domain, strongly suggesting that the latter acts through Gro in vivo (Jimenez, 1997)

A repressor domain from the Engrailed (En) homeodomain protein is also Gro dependent. HairyEn was created, a chimeric protein including Hairy1-286 fused to a repressor domain from the En segmentation protein (amino acids 168-298). HairyEn behaves as a strong repressor of Sex lethal. Previous results suggest that ftz is a direct target of repression by Hairy/Gro. Repression of fushi tarazu by HairyEn is Groucho dependent. The latter result correlates with an ability of this En domain to bind to Gro in vitro. In contrast, repressor regions from the Even-skipped, Snail, Kruppel, and Knirps transcription factors are effective in the absence of Gro. A conserved element (called eh1), present in other homeodomain proteins (such as Goosecoid) plays an important role in the ability of En to bind to Gro. Mutants in Groucho lacking the WD repeat region suggest that the WD repeats plays a role in the interaction with Hairy, although it is clear that both halves of Gro are required for full interaction with Hairy and Engrailed. These results show that Gro is not generally required for repression, but acts as a specific corepressor for a fraction of negative regulators, including Hairy and En (Jimenez, 1997).

Active transcriptional repression has been characterized as a function of many regulatory factors. Repression facilitates combinatorial regulation of gene expression by allowing repressors to be dominant over activators under certain conditions. The Engrailed protein uses two distinct mechanisms to repress transcription. One is predominant under normal transient transfection assay conditions in cultured cells. The second is predominant in an in vivo active repression assay. Two repression domains (region 4 immediately flanking the EN HD and region6) are more potent in transient transfections of cultured cells than in vivo. The domain mediating the in vivo activity (region 3 or eh1, not closely associated with the HD in the primary sequence) is highly conserved throughout several classes of homeoproteins and interacts specifically with the Groucho corepressor. When both regions 4 and 6 are deleted, very little activity remains in culture, while repression in vivo is still strong. Thus, in vivo, region 3 contributes the predominant repression activity, while in transient transfection assays in culture, regions 4 and 6 contribute the predominant activity. While eh1 shows only weak activity in transient transfections, much stronger activity is seen in culture when an integrated target gene is used. In this assay, the relative activities of different repression domains closely parallel those seen in vivo, with eh1 showing the predominant activity. Reducing the amounts of repressor and target gene in a transient transfection assay also increases the sensitivity of the assay to the Groucho interaction domain, albeit to a lesser extent. This suggests that the Groucho interaction domain utilizes rate-limiting components that are relatively low in abundance. Since Groucho itself is abundant in these cells, the results suggest that a limiting component is recruited effectively by the repressor-corepressor complex only on the integrated target gene (Tolkunova, 1998).

The fact that multiple domains contribute to repression activity in the two assays and the likelihood that they utilize distinct mechanisms suggests that the evolution of En has involved strong selection for repression function. This possibility is reinforced by the observation that none of the deletion derivatives show significant activation function on appropriate reporter genes in culture, either alone or in combination with other activators, even when all identified repression domains are removed. Indeed, preliminary data suggest that even the En homeodomain contributes to repression activity in the normal En molecule, since single domain deletions that significantly affect repression activity in the context of the Ftz homeodomain do not affect the repression activity of En itself to the same degree. The idea that En might be primarily a repressor in vivo conflicts, on the surface, with results from ectopic expression assays in embryos, in which En has been shown to induce expression of its own gene, as well as with the positive regulatory action of En on hedgehog. These interactions might be indirect, through repression of a repressor; this is suggested by the results presented here. However, it remains possible that protein-protein interactions allow En to have a net positive regulatory effect on some direct target genes. It is worthy of note in this context that a similar positive autoregulatory effect of Even-skipped, a strong repressor in both cell culture assays and in vitro, has been attributed to indirect effects in vivo, involving repression of other repressors (Tolkunova, 1998).

The Drosophila Groucho (Gro) protein is a corepressor required by a number of DNA-binding transcriptional repressors. Comparison of Gro with its homologues in other eukaryotic organisms reveals that Gro contains, in addition to a conserved C-terminal WD repeat domain, a conserved N-terminal domain, which has previously been implicated in transcriptional repression. The N-terminal region of Gro is highly conserved among the members of the Gro family of corepressor proteins. Sequence analysis of the conserved N-terminal region predicts two alpha-helices (residues 24 to 52 and 73 to 100) in this region of Gro that have a high propensity to form coiled coils. Sequence alignment with Gro homologs from Caenorhabditis elegans, Xenopus, zebra fish, rats, mice, and humans shows that the two putative amphipathic helices and the region between them are highly conserved in all Gro family members. The first of these amphipathic helices is potential leucine zipper domain in other Gro family proteins. By using the consensus sequence produced, this alignment was used to search protein databases, it was found that the first conserved motif does indeed have high sequence similarity with leucine zipper motifs present in the proto-oncogene products Maf-1, c-Fos, and N-Myc. In addition, the two motifs as well as the intervening sequence exhibit high sequence homology to multiple regions of two coiled-coil proteins: the murine homolog of the leukemia-associated PML isoform 1 and human centromeric protein E. Because of their similarity to leucine zippers, these motifs are referred to as leucine zipper-like (LZL) motifs. Helical-wheel projections of the two LZL motifs reveal that both segments contain 4-3 hydrophobic heptad repeats, since almost all residues at the a and d positions are hydrophobic in nature. In addition, potential interhelical salt bridges in the first LZL motif may further contribute to the stability and specificity of a potential parallel coiled-coil structure. It has been determined, via a variety of hydrodynamic measurements as well as protein cross-linking, that native Gro is a tetramer in solution and that tetramerization is mediated by the two LXL motifs found in the N-terminal region. Genetic analyses have demonstrated that Gro is maternally required for conversion of Dorsal from an activator to a repressor. In addition, Dorsal and Twist proteins together can synergistically activate transcription in SL2 cells. To test the possibility that Gal4-Gro can override this synergistic activation, a luciferase reporter vector bearing five copies of a regulatory module containing both the Dorsal and Twist binding sites was prepared, and was inserted just upstream of the herpes simplex virus thymidine kinase core promoter. Two versions of this vector, with and without Gal4 binding sites upstream of the Dorsal and Twist binding sites, were prepared. Cotransfection of expression vectors encoding Dorsal and Twist with these reporters results in 30- to 50-fold activation. The addition of wild-type Gal4-Gro expression vectors results in strong Gal4 binding site-dependent repression (>15-fold) of the Dorsal-Twist-activated transcription. Therefore, when directly tethered to the promoter, Gro acts in a dominant fashion to repress the synergistic activation promoted by Dorsal and Twist. The effects of the Gro point mutations on repression were analyzed next. Gal4-GroL38,87P is unable to repress Dorsal-Twist-activated transcription, while both Gal4-GroL38P and Gal4-GroL87P retain partial binding site-dependent repression activity. Point mutations in the leucine zipper-like motifs that block tetramerization, also block repression by Gro, as assayed in cultured Drosophila cells with Gal4-Gro fusion proteins. The heterologous tetramerization domain from p53 fully substitutes for the Gro tetramerization domain in transcriptional repression. These findings suggest that oligomerization is essential for Gro-mediated repression and that the primary function of the conserved N-terminal domain is to mediate this oligomerization (Chen, 1998).

Surprisingly small peptide motifs can confer critical biological functions. One example is the WRPW tetrapeptide present in the Hairy family of transcriptional repressors, that mediates recruitment of the Groucho (Gro) corepressor to target promoters. Engrailed (En) is another repressor that requires association with Gro for its function. En lacks a WRPW motif; instead, it contains another short conserved sequence, the En homology region 1 (eh1)/GEH motif, that is likely to play a role in tethering Gro to the promoter. A repressor domain from the Goosecoid (Gsc) developmental regulator is characterized that includes an eh1/GEH-like motif. This motif is found within the N-terminal half of the protein. The motif is 17 amino acids long and includes a 7-amino-acid core and ten flanking residues that are partly conserved among Gsc proteins. The flanking residues do not show significant similarity to the equivalent region of other eh1/GEH-containing proteins. Interestingly, a Phe residue in the core motif distinguishes the eh1/GEH motif from a related sequence known as the octapeptide, which is present in several paired-domain and homeodomain proteins. This domain (GscR) mediates efficient repression in Drosophila blastoderm embryos and repression by GscR requires Gro function. GscR and Gro interact in vitro, and the eh1/GEH motif is necessary and sufficient for the interaction and for in vivo repression. Because WRPW- and eh1/GEH-like motifs are present in different proteins and in many organisms, the results suggest that interactions between short peptides and Gro represent a widespread mechanism of repression. Finally, whether Gro is part of a stable multiprotein complex in the nucleus was investigated. These results indicate that Gro does not form stable associations with other proteins but that it may be able to assemble into homomultimeric complexes (Jimenez, 1999).

The precellular Drosophila embryo contains approximately 10 well characterized transcriptional repressors. At least half are short-range repressors that must bind within 100 bp of either upstream activators or the core transcription complex to inhibit (or quench) gene expression. The two long-range repressors can function over distances of 1 kilobase or more to silence transcription. Previous studies have shown that three of the five short-range repressors interact with a common corepressor protein, dCtBP. In contrast, the two long-range repressors, Hairy and Dorsal, recruit a different corepressor protein, Groucho. Hairy also was shown to interact with dCtBP, thereby raising the possibility that Groucho and dCtBP are components of a common corepressor complex. To investigate this issue, wild-type and mutant forms of Hairy were misexpressed in transgenic embryos. Evidence is presented that Hairy-mediated repression depends on the Groucho interaction sequence (WRPW) but not the weak dCtBP motif (PLSLV) present in the native protein. Conversion of the PLSLV motif into an optimal dCtBP interaction sequence (PLDLS) disrupts the activity of an otherwise normal Hairy protein. These results suggest that dCtBP and Groucho mediate separate pathways of transcriptional repression and that the two proteins can inhibit one another when both bind the same repressor (Zhang, 1999).

The removal of the weak dCtBP interaction motif (PLSLV) does not impair Hairy-mediated repression of Sxl, fkh, hkb, and tll. If anything, removal of this motif augments Hairy function. This observation suggests that the binding of dCtBP somehow interferes with Groucho-mediated repression. Additional support for this view stems from the observation that the PLDLS/WRPW protein, which contains an optimal dCtBP motif, is inactive and fails to repress any of the target genes that were examined. The simplest interpretation of these results is that the dCtBP and Groucho corepressors interfere with one another when both are bound to Hairy. Such antagonistic interactions are supported by previous genetic studies, which suggest that lowering the dose of maternal dCtBP products can partially suppress the embryonic phenotypes of hairy mutants. The P-SLV-K and WRPW motifs are separated by just nine amino acid residues within the C terminus of the Hairy protein. When dCtBP and Groucho both bind, they might be unable to interact with additional corepressors or with their target proteins in the core transcription complex (Zhang, 1999).

Drosophila C-terminal binding protein (dCtBP) and Groucho have been identified as Hairy-interacting proteins required for embryonic segmentation and Hairy-mediated transcriptional repression. While both dCtBP and Groucho are required for proper Hairy function, their properties are very different. As would be expected for a co-repressor, reduced Groucho activity enhances the hairy mutant phenotype. In contrast, reduced dCtBP activity suppresses it. dCtBP can function as either a co-activator or co-repressor of transcription in a context-dependent manner. The regions of dCtBP required for activation and repression are separable. mSin3A-histone deacetylase complexes (see Drosophila Sin3A) are altered in the presence of dCtBP and dCtBP interferes with both Groucho and Mad transcriptional repression. Similar to CtBP's role in attenuating E1A's oncogenicity, it is proposed that dCtBP can interfere with corepressor-histone deacetylase complexes, thereby attenuating transcriptional repression. Hairy defines a new class of proteins that requires both CtBP and Groucho co-factors for proper function (Phippen, 2000).

The homeodomain transcription factor NK-4 acts as either a transcriptional activator or repressor and interacts with the p300 coactivator and the groucho corepressor

tinman encodes an NK-2 class homeodomain transcription factor that is required for development of the Drosophila dorsal mesoderm, including heart. Genetic evidence suggests its important role in mesoderm subdivision, yet the properties of Tinman as a transcriptional regulator and the mechanism of gene transcription by Tinman are not completely understood. Tinman can activate or repress target genes in cultured cells, based on evaluation of functional domains that are conserved between the tinman genes of Drosophila melanogaster and Drosophila virilis. Using GAL4-tinman fusion constructs, a transcriptional activation domain (amino acids 1-110) and repression domains (amino acids 111-188 and the homeodomain) have been mapped and an inhibitory function for the homeodomain has been found upon transactivation by Tinman (Choi, 1999).

Tinman is regulated by Twist and autoregulates its own promoter. The properties of Tin as a transcription factor were assessed using tinman P1 and P1E2m promoters and truncated forms of the Tin expression vectors (d8 and d6). The P1 reporter contains Tin-responsive elements (the E2 cluster) and is activated by Tin. The P1E2m reporter contains mutated Tin binding sites but otherwise is exactly the same as the wild-type P1 reporter, which also contains several weak Tin binding sites. Tin can activate the P1 reporter (6-fold activation). In contrast, Tin down-regulates the P1E2m reporter gene (3-fold repression). In this case, Tin binds to weak binding sites and represses the P1E2m reporter gene. These results indicate that Tin can act either as a transcriptional activator or repressor, depending on the context of the reporters (P1 or P1E2m). These phenomena are dependent on the functional domains of Tin. For example, deletion of the amino-terminal region of Tin abrogates activation of the P1 reporter, indicating that the amino terminus (aa 1-110) of Tin is required for transcriptional activation. Indeed, this Tin mutant (d8) represses gene expression of both the P1 and the P1E2m reporter. Further deletion of Tin (construct d6) relieves this repression, irrespective of the reporter gene used. These results suggest that the region following the amino terminus of Tin (aa 111-188) is required for the repressor activity of Tin. Taken together, these results indicate that, depending on the context of the target genes (for example P1 or P1E2m), Tin can act as either a transcriptional activator or repressor and that these different transcriptional activities are dependent on functional domains of Tin (Choi, 1999).

Tinman-dependent transactivation is augmented by the p300 coactivator; Tinman physically interacts with p300 via the activation domain. In addition, cotransfection experiments indicate that the repressor activity of Tinman is strongly enhanced by the Groucho corepressor. Using immunoprecipitation and in vitro pull-down assays, Tinman is shown to directly interact with the Groucho corepressor, for which the homeodomain is required. Together, these results indicate that Tinman can act as either a transcriptional activator or repressor. The first evidence of Tinman interactions with the p300 coactivator and the Groucho corepressor is provided (Choi, 1999).

Elbow and Noc define a family of zinc finger proteins controlling morphogenesis of specific tracheal branches: ElB associates with the Groucho protein

The elbow B (elB) gene encodes a conserved nuclear protein with a single zinc finger. Expression of ElB is restricted to a specific subset of tracheal cells, namely the dorsal branch and the lateral trunks. Stalled or aberrant migration of these branches is observed in elB mutant embryos. Conversely, ElB misexpression in the trachea gives rise to absence of the visceral branch and an increase in the number of cells forming the dorsal branch. These results imply that the restricted expression of ElB contributes to the specification of distinct branch fates, as reflected in their stereotypic pattern of migration. Since elB loss-of-function tracheal phenotypes are reminiscent of defects in Dpp signaling, the relationship between ElB and the Dpp pathway was examined. By using pMad antibodies that detect the activation pattern of the Dpp pathway, it has been shown that Dpp signaling in the trachea is not impaired in elB mutants. In addition, expression of the Dpp target gene kni is unaltered. The opposite is true as well, because expression of elB is independent of Dpp signaling. ElB thus defines a parallel input, which determines the identity of the lateral trunk and dorsal branch cells. No ocelli (Noc) is the Drosophila protein most similar to ElB. Mutations in noc give rise to a similar tracheal phenotype. Noc is capable of associating with ElB, suggesting that they can function as a heterodimer. ElB also associates with the Groucho protein, indicating that the complex has the capacity to repress transcription of target genes. Indeed, in elB or noc mutants, expanded expression of tracheal branch-specific genes is observed (Dorfman, 2002).

The Groucho protein is known to mediate long-range transcriptional repression, and to associate with DNA-binding proteins bearing a number of motifs, including FKPY. This sequence is conserved in ElB, Noc and the two human homologs. Thus the capacity of ElB to associate with Groucho was tested. Indeed, specific association was detected. CtBP promotes short-range repression and is known to associate with DNA-binding proteins containing the PxDLSxR/K/H motif. Such a motif is not found in ElB or Noc, and the GST-ElB fusion protein shows only negligible precipitation of labeled CtBP. This result strongly suggests that the ElB/Noc complex represses transcription of target genes directly, by recruiting Groucho to these sites (Dorfman, 2002).

Two modes of recruitment of E(spl) repressors onto target genes

The decision of ectodermal cells to adopt the sensory organ precursor fate in Drosophila is controlled by two classes of basic-helix-loop-helix transcription factors: the proneural Achaete (Ac) and Scute (Sc) activators promote neural fate, whereas the E(spl) repressors suppress it. E(spl) proteins m7 and mgamma are potent inhibitors of neural fate, even in the presence of excess Sc activity and even when their DNA-binding basic domain has been inactivated. Furthermore, these E(spl) proteins can efficiently repress target genes that lack cognate DNA binding sites, as long as these genes are bound by Ac/Sc activators. This activity of E(spl)m7 and mgamma correlates with their ability to interact with proneural activators, through which they are probably tethered on target enhancers. Analysis of reporter genes and sensory organ (bristle) patterns reveals that, in addition to this indirect recruitment of E(spl) onto enhancers via protein-protein interaction with bound Ac/Sc factors, direct DNA binding of target genes by E(spl) also takes place. Irrespective of whether E(spl) are recruited via direct DNA binding or interaction with proneural proteins, the co-repressor Groucho is always needed for target gene repression (Giagtzoglou, 2003).

E(spl) proteins are known to recruit the co-repressor Groucho in order to silence target genes. It is conceivable that when E(spl) proteins exert their repressive effect by interacting with proneural proteins, a different mechanism might be at play, such as occlusion of the transcriptional activation domain of proneural activators. It was therefore of interest to address whether Gro is needed to mediate repression when E(spl) proteins are indirectly bound to DNA. To this end, expression of UAS-sc was driven together with UAS-E(spl)m7 in a mosaic background containing patches homozygous for the severe groE48 allele and the response of the EE4-lacZ reporter was assayed. This reporter is repressed by E(spl)m7 exclusively via protein-mediated recruitment. Indeed in gro+ territory little or no expression was observed, as expected; however, within mutant clones EE4-lacZ was strongly expressed. Therefore, E(spl) proteins employ a Gro-dependent repression mechanism regardless of mode of recruitment on target genes (Giagtzoglou, 2003).

The requirement for Gro was corroborated by cuticle phenotype: groE48 clones produce tufts of bristles on the notum, a result of the breakdown of lateral inhibition during SOP commitment. Although ubiquitous expression of E(spl)m7 abolishes bristles, when it was induced in groE48 clones in an ap-Gal4; UAS-E(spl)m7 background (which abolishes bristles throughout the notum), patches of high bristle density were recovered in a bald notum. This suggests that ectopic (as well as normally expressed) E(spl)m7 cannot repress endogenous target genes in the absence of Gro, just as it cannot repress the artificial EE4-lacZ target. Finally, a UAS-E(spl)m7deltaW transgene, which lacks the C-terminal tryptophane of the Gro-binding WRPW motif, was completely inactive in both bristle suppression and reporter gene repression. A corollary from these experiments is that E(spl)m7 does not function by sequestering proneural activators off DNA. The latter activity should have no requirement for a co-repressor like Gro, since physical removal of activators should suffice to turn target genes off (Giagtzoglou, 2003).

Local action of long-range repressors in the Drosophila embryo

Two corepressors have been identified in the early Drosophila embryo: Groucho and dCtBP. Both proteins are recruited to the DNA template by interacting with short peptide motifs conserved in a variety of sequence-specific transcriptional repressors. Once bound to DNA, Groucho appears to mediate long-range repression, while dCtBP directs short-range repression. The short-range Krüppel repressor is converted into a long-range repressor by replacing the dCtBP interaction motif (PxDLSxH) with a Groucho motif (WRPW). The resulting chimeric repressor causes a different mutant phenotype from that of the native Krüppel protein when misexpressed in transgenic embryos. The different patterning activities can be explained on the basis of long-range silencing within the hairy 5' regulatory region. The analysis of a variety of synthetic transgenes provides evidence that Groucho-dependent long-range repressors do not always cause the dominant silencing of linked enhancers within a complex cis-regulatory region. A 'hot chromatin' model is suggested, whereby repressors require activators to bind DNA (Nibu, 2001).

Complex enhancers direct stripes and bands of gene expression in the early Drosophila embryo. These enhancers are typically 300 bp-1 kb in length and contain clustered binding sites for transcriptional activators and repressors. Different enhancers can work independently of one another within a common cis-regulatory region to direct composite patterns of gene expression. For example, the seven-stripe even-skipped (eve) expression pattern is activated by five separate enhancers located 5' and 3' of the transcription unit. The ability of these enhancers to function in an autonomous fashion depends on short-range transcriptional repressors that work over distances of <100 bp to inhibit, or quench, upstream activators. The binding of the Krüppel repressor to the stripe 2 enhancer does not interfere with the activity of the stripe 3 enhancer since Krüppel mediates repression only when positioned near upstream activators. Consquently, Krüppel quenches Bicoid activators within the stripe 2 enhancer without interfering with the D-Stat activators bound to the stripe 3 enhancer (Nibu, 2001).

There are several short-range repressors in the early embryo, including Krüppel, Snail, Knirps and Giant. Most or all of these repressors interact with a common corepressor protein, dCtBP, which is the Drosophila homolog of a human protein that was found to attenuate the oncogenic activities of the adenovirus E1A protein. dCtBP is maternally expressed and ubiquitously distributed throughout early embryos. A variety of studies suggest that the dCtBP corepressor protein is recruited to the DNA template by interacting with a conserved sequence motif contained in most or all sequence-specific short-range repressors: PxDLSxK/R/H. There is emerging evidence that mammalian CtBP proteins also function as corepressors, although it is not known currently whether the mammalian repressors (e.g. bKLF, Ikaros and ZEB-1) only function over short distances (Nibu, 2001).

A number of repressors can work when positioned far from upstream activators and the core promoter. For example, the binding of the Hairy repressor to a modified rhomboid lateral stripe enhancer (NEE) can cause the dominant silencing of a linked mesoderm-specific enhancer, even when the two enhancers are separated by >1 kb in the 5' cis -regulatory region. Hairy interacts with a second ubiquitous corepressor protein, Groucho. Hairy-Groucho interactions depend on a conserved sequence motif at the Hairy C-terminus: WRPW. These studies suggest that the dCtBP corepressor protein mediates short-range repression, while Groucho mediates long-range repression. The present study provides additional support for this possibility (Nibu, 2001).

The long-range action of the Groucho corepressor poses a potential problem with regard to enhancer autonomy in complex promoter regions. In principle, the binding of a Groucho-dependent repressor could result in the dominant silencing of all enhancers located in the 5' and 3' regulatory regions of a target gene. This imposes a potentially severe constraint on the evolution of complex patterns of gene activity. To investigate this issue, the activities have been examined of chimeric repressor proteins that contain the DNA-binding domains of the short-range Krüppel or Snail repressors and the Groucho interaction sequences in the long-range Hairy repressor. These chimeric repressors were expressed in specific regions of transgenic embryos using defined, heterologous enhancers. The Krüppel-Hairy fusion protein causes altered patterns of segmentation gene expression that are consistent with the notion that Hairy-Groucho interactions convert Krüppel into a long-range repressor. However, the abnormal rhomboid expression pattern obtained with a similar Snail-Hairy fusion protein suggests that it does not function as a dominant silencer, but instead causes the local repression of a single enhancer. The subsequent analysis of a number of synthetic transgenes provides direct evidence that the long-range Hairy repressor does not always cause the dominant silencing of linked enhancers (Nibu, 2001).

A comparison of the altered patterns of hairy expression obtained with the twi-Krüppel and twi-Krüppel-hairy transgenes provides evidence that dCtBP and Groucho mediate short- and long-range repression, respectively. The twi-Krüppel transgene causes the repression of hairy stripe 6, but not stripe 5. Previous studies have shown that the stripe 6 enhancer contains optimal, high-affinity Krüppel operator sites that can be occupied by the low levels of Krüppel produced in ventral regions by the twi-Krüppel transgene. These low levels appear to be insufficient to bind the low-affinity sites within the hairy stripe 5 enhancer and, consequently, the native Krüppel protein works as a short-range repressor to inhibit stripe 6 expression without affecting stripe 5 expression. In contrast, the twi-Krüppel-hairy transgene leads to the repression of both stripes 5 and 6. The binding of the Krüppel-Hairy fusion repressor to the stripe 6 enhancer appears to cause the dominant silencing of the neighboring stripe 5 enhancer over a distance of ~2.5 kb in the hairy 5' regulatory region. An implication of these observations is that different repression domains exert distinct influences on embryonic patterning. Replacing the PxDLSxH motif (native Krüppel) with WRPW (Krüppel-Hairy) changes the regulatory activity of the Krüppel repressor (Nibu, 2001).

The Snail-Hairy fusion protein represses the rhomboid lateral stripes, but fails to repress the amnioserosa pattern. In contrast, the same Hairy repression domain permits Krüppel to function as a dominant silencer within the hairy 5' regulatory region. There are several possible explanations for the failure of the Snail-Hairy repressor to silence rhomboid expression in the amnioserosa. Perhaps there is competition between dCtBP bound to the Snail moiety and Groucho bound to the Hairy moiety within the fusion protein. The Krüppel-Hairy fusion protein was mutagenized to eliminate the dCtBP motif (PEDLSMH), whereas the Snail-Hairy fusion protein retains both dCtBP sequences. Previous studies suggest that the conversion of the weak dCtBP interaction motif near the Hairy C-terminus, PLSLVIK, into an optimal motif, PLDLSIK, disrupts the repressor function of an otherwise normal Hairy protein. This result was taken as evidence that the dCtBP and Groucho corepressors interfere with one another when bound to closely linked motifs within the Hairy C-terminus. An argument against this explanation for the behavior of the Snail-Hairy fusion protein stems from the observation that the binding of Hairy to a modified NEE is sufficient to repress a linked mesoderm enhancer (twist PE), but not a similarly spaced race enhancer. Similarly, the binding of Hairy to a modified race enhancer fails to silence the mesoderm enhancer (Nibu, 2001).

It is proposed that Hairy can only bind active or 'open' enhancers. The NEE is activated by the maternal Dorsal nuclear gradient and, consequently, it might contain activator proteins in both ventral and lateral regions of early embryos. As a result, the binding of Hairy to the modified h-NEE-h enhancer can lead to the dominant silencing of a linked mesoderm enhancer (twist PE). In contrast, there is no Dorsal activator in dorsal regions of the early embryo, thereby rendering the h-NEE-h enhancer in a closed or condensed state. This absence of activator might preclude the binding of Hairy so that the race enhancer is not silenced. Similarly, the race enhancer is probably activated by transcription factors that are restricted to dorsal regions, such as Zen and Smads. These activators are absent in ventral regions and, consequently, Hairy may be unable to bind the h-race-h enhancer and silence linked enhancers such as the twist PE (Nibu, 2001).

The altered pattern of hairy expression caused by the Krüppel-Hairy fusion protein can be interpreted in the context of this 'hot chromatin' model. There is evidence that hairy stripes 5, 6 and 7 are activated by a posterior gradient of the Caudal activator. The binding of the Krüppel-Hairy fusion protein to the optimal Krüppel operator sites in the stripe 6 enhancer would be expected to silence the neighboring stripe 5 enhancer due to the open conformation of the stripe 6 enhancer in those regions of the embryo where stripe 5 is expressed. Thus, the Caudal activator might bind to both enhancers in the position of stripe 5, thereby rendering the stripe 6 enhancer accessible to the Krüppel-Hairy fusion protein (Nibu, 2001).

The dependence of repressors on activators might restrain long-range repressors and permit enhancer autonomy. This dependence might reflect the inherent properties of activators and repressors. Some activators recruit enzymes that decondense chromatin, and this may be essential for the binding of repressors in vivo. Short-range repression has been put forward as an important mechanism for enhancer autonomy. It is suggested that a second mechanism involves the reliance of repressors on activators for binding to target enhancers (Nibu, 2001).

Hairy transcriptional repression targets and cofactor recruitment in Drosophila

Members of the widely conserved Hairy/Enhancer of split family of basic Helix-Loop-Helix repressors are essential for proper Drosophila and vertebrate development and are misregulated in many cancers. While a major step forward in understanding the molecular mechanism(s) surrounding Hairy-mediated repression was made with the identification of Groucho, Drosophila C-terminal binding protein (dCtBP), and Drosophila silent information regulator 2 (dSir2) as Hairy transcriptional cofactors, the identity of Hairy target genes and the rules governing cofactor recruitment are relatively unknown. The chromatin profiling method DamID was used to perform a global and systematic search for direct transcriptional targets for Drosophila Hairy and the genomic recruitment sites for three of its cofactors: Groucho, dCtBP, and dSir2. Each of the proteins was tethered to Escherichia coli DNA adenine methyltransferase, permitting methylation proximal to in vivo binding sites in both Drosophila Kc cells and early embryos. This approach identified 40 novel genomic targets for Hairy in Kc cells, as well as 155 loci recruiting Groucho, 107 loci recruiting dSir2, and wide genomic binding of dCtBP to 496 loci. DamID profiling was adapted such that tightly gated collections of embryos (2-6 h) could be used, and 20 Hairy targets related to early embryogenesis were found. As expected of direct targets, all of the putative Hairy target genes tested show Hairy-dependent expression and have conserved consensus C-box-containing sequences that are directly bound by Hairy in vitro. The distribution of Hairy targets in both the Kc cell and embryo DamID experiments corresponds to Hairy binding sites in vivo on polytene chromosomes. Similarly, the distributions of loci recruiting each of Hairy's cofactors are detected as cofactor binding sites in vivo on polytene chromosomes. Fifty-nine putative transcriptional targets of Hairy were identified. In addition to finding putative targets for Hairy in segmentation, groups of targets were found suggesting roles for Hairy in cell cycle, cell growth, and morphogenesis, processes that must be coordinately regulated with pattern formation. Examining the recruitment of Hairy's three characterized cofactors to their putative target genes revealed that cofactor recruitment is context-dependent. While Groucho is frequently considered to be the primary Hairy cofactor, it is associated with only a minority of Hairy targets. The majority of Hairy targets are associated with the presence of a combination of dCtBP and dSir2. Thus, the DamID chromatin profiling technique provides a systematic means of identifying transcriptional target genes and of obtaining a global view of cofactor recruitment requirements during development (Bianchi-Frias, 2004).

The 59 putative Hairy targets identified correspond to bands of Hairy immunostaining on polytene chromosomes, suggesting that the polytene chromosome staining faithfully represents Hairy binding. Polytene chromosomes are functionally similar in transcriptional activity and display factor/cofactor binding properties similar to chromatin of diploid interphase cells, despite their DNA endoreplication (Bianchi-Frias, 2004).

Since the microarray chips used contained roughly half of Drosophila cDNAs, the actual number of Hairy targets was estimaed to be approximately twice that number (i.e., 118 targets). This predicted number of Hairy targets is close to the approximately 120 strongly staining sites observed on polytene chromosomes. Of the 59 putative Hairy targets identified in both the Kc cell and embryo DamID experiments, 58 correspond to bands of Hairy staining on the polytene chromosomes, suggesting that polytene chromosome staining is representing Hairy binding sites without regard to tissue specificity. It is not yet clear what is limiting Hairy accessibility in different tissues or why Hairy's access does not appear to be limited in salivary glands. It may be that polytene chromosome organization necessitates a looser chromatin structure or that the large number of factors that seem to be endogenously expressed in salivary glands affects accessibility. Ultimately, additional confirmation of the DamID and polytene staining correspondence will require microarray tiling chips containing overlapping genomic DNA fragments; however, such genomic DNA tiling chips are currently unavailable (Bianchi-Frias, 2004).

DNA methylation by tethered Dam has been shown to spread up to a few kilobases from the point where it is brought to the DNA. It was of concern in the beginning that Hairy targets might be missed if the DNA fragments of 2.5 kb or less that were recovered for probes were far away from the start of the transcribed region, especially since the Drosophila microarray chip used was generated using full-length cDNAs. Indeed, Hairy has been described as a long-range repressor; it is likely to bind at a distance from the transcription start site. However, the targets identified by DamID in both Kc cells and in embryos correspond closely to the Hairy staining pattern on polytene chromosomes. As is the case for Hairy, the distribution of DamID-identified loci that recruit the long-range repression-mediating Groucho corepressor corresponds well with the distribution of Groucho binding sites on polytene chromosomes. These results suggest that there is a higher-order structure to the promoter that is allowing factors that bind far upstream of the transcription start site to have physical access to the transcribed region (i.e., DNA looping) or that Hairy does not bind as far away from the transcription start site as it had been proposed to do (Bianchi-Frias, 2004).

Hairy is needed at multiple times during development, where it has primarily been associated with the regulation of cell fate decisions. During embryonic segmentation, ftz has long been thought to be a direct Hairy target. However, the order of appearance of ftz stripes is not inversely correlated with those of Hairy, as would be expected if ftz stripes are generated by Hairy repression. While it was not possible to assess ftz as a direct Hairy target using DamID, no evidence was found for ftz being a direct Hairy target based on the association of Hairy with polytene chromosomes. Indeed, the evidence suggesting that ftz is a direct target of Hairy is based on timing, i.e., that there is not enough time for another factor to be involved. Since the half-life of the pair-rule gene products is very short (less than 5 min), it is possible that additional factors could be acting and that the interaction between Hairy and ftz is indirect (Bianchi-Frias, 2004).

Interestingly, one of the Hairy targets identified in embryos is the homeobox-containing transcriptional regulator, prd. Pair-rule genes have been split into two groups: primary pair-rule genes mediate the transition from nonperiodic to reiterated patterns via positional cues received directly from the gap genes, whereas secondary pair-rule genes take their patterning cues from the primary pair-rule genes and in turn regulate the segment polarity and homeotic gene expression. The transcriptional regulator prd was originally categorized as a secondary pair-rule gene since its expression is affected by mutations in all other known pair-rule genes. However, prd stripes were subsequently shown to require gap gene products for their establishment, and the prd locus has the modular promoter structure associated with primary pair-rule genes. Thus prd has properties of both primary and secondary pair-rule genes and is a good candidate to directly mediate Hairy's effects on segmentation. Hairy can specifically bind to C-box sequences in the prd promoter and interacts genetically with prd. Further experiments will be required to determine if Paired in turn binds to the ftz promoter, such that the order of regulation would be Hairy > prd > ftz (Bianchi-Frias, 2004).

In addition to identifying potential targets for Hairy in segmentation, targets were identified that implicate Hairy in other processes including cell cycle, cell growth, and morphogenesis. The group of targets implicating Hairy in the regulation of morphogenesis includes: concertina, a G-alpha protein involved in regulating cell shape changes during gastrulation; kayak, the Drosophila Fos homolog involved in morphogenetic processes such as follicle cell migration, dorsal closure, and wound healing; pointed and mae, both of which function in the ras signaling pathway to control aspects of epithelial morphogenesis; egh, a novel, putative secreted or transmembrane protein proposed to play a role in epithelial morphogenesis, and Mipp1, a phosphatase required for proper tracheal development (Bianchi-Frias, 2004).

Hairy has been thought to be involved mostly in the regulation of cell fate decisions. However, mosaic experiments in the eye imaginal disc have suggested that Hairy may also play a role in the regulation of cell cycle or cell growth. Consistent with this, another group of Hairy targets implicates Hairy in the regulation of cell cycle or cell growth; this group includes stg, the Drosophila Cdc25 homolog; dacapo, a cyclin-dependent kinase inhibitor related to mammalian p27kip1/p21waf1; IDGF2, a member of a newly identified family of growth-promoting glycoproteins, and ImpL2, a steroid-responsive gene of the secreted immunoglobulin superfamily that functions as a negative regulator of insulin signaling. Consistent with a role for Hairy in growth signaling, mammalian HES family proteins have been linked to insulin signaling (Bianchi-Frias, 2004).

Since cells that are dividing or proliferating cannot simultaneously undergo the cell shape changes and cell migrations required for morphogenetic movements, Hairy may be required to transiently pause the cell cycle in a spatially and temporally defined manner, thereby allowing the cell fate decisions regulated by the transcription cascade to be completed. Since Hairy is itself spatially and temporally expressed, Hairy must be only one of several genes necessary to orchestrate these processes. While much progress has been made in understanding the regulatory networks governing pattern formation, cell proliferation, and morphogenesis, and while it is clear that they must be integrated, the details surrounding their coordination have not yet been elucidated. Thus, the putative Hairy targets identified are consistent with known processes involving Hairy and suggest that in addition to regulating pattern formation, Hairy plays a role in transiently repressing other events, perhaps in order to coordinate cell cycle events with the segmentation cascade. Further experiments will be needed to determine how these different roles for Hairy fit together (Bianchi-Frias, 2004).

The numbers of loci that recruit Groucho, dCtBP, and dSir2 cofactors are consistent with the breadth of interaction they have been shown to exhibit. One hundred and fifth-five loci were identified that recruit Groucho and, as expected, roughly twice as many sites were found on polytene chromosomes. Although Groucho was the first Hairy cofactor identified and its interaction site is often described as Hairy's 'major' repression motif, Groucho is associated with only a minority of Hairy targets in Kc cells. Groucho's dominance as a cofactor during segmentation may reflect a preference for Groucho in the reporter assays used previously to assess corepressor activity, or it may be more heavily recruited to Hairy's targets during segmentation. In the future it will be interesting to determine the loci that recruit Groucho in early embryos and, because Groucho binds a number of other repressors, which, if any, of these factors recruits Groucho as its major cofactor (Bianchi-Frias, 2004).

CtBP was identified as a repressive co-factor, first on the basis of its binding to the C-terminal region of E1A, and in Drosophila by its association with the developmental repressors Hairy and Knirps. CtBP is an integral component in a variety of multiprotein transcriptional complexes. It has been shown to function as a context-dependent cofactor, having both positive and negative effects on transcriptional repression depending upon the repressor to which it is recruited. More than 40 different repressors have been shown to recruit CtBP. Consistent with this wide recruitment of CtBP, 496 loci that recruit dCtBP were found by DamID profiling and roughly twice that many sites on polytene chromosomes. A global protein-protein interaction study has shown that the binding partners for Groucho and dCtBP are largely nonoverlapping. This, along with the near exclusivity of Groucho and dCtBP binding as assayed by DamID and polytene chromosome staining, makes it unlikely that both cofactors work together as a general rule and strengthens the possibility that the binding of each of these factors assembles different protein complexes that are, for the most part, mutually exclusive (Bianchi-Frias, 2004).

dSir2 was only very recently identified as a corepressor for Hairy and other HES family members. 107 loci were identified by DamID profiling that recruit dSir2 and roughly twice that many sites on polytene chromosomes. Surprisingly, the distribution of loci recruiting dSir2 identified by DamID profiling, as well as dSir2's staining on polytene chromosomes, shows regional binding specificity. This binding specificity may be a reflection of the different nuclear compartments in which these regions of the chromosomes are found. Sir2 has been described mostly as a protein involved in heterochromatic silencing rather than in euchromatic repression. The number of dSir2 euchromatic sites observed is similar to that of Groucho, suggesting that euchromatic repressors (in addition to HES family members) are likely to recruit Sir2. Consistent with this, a recent report has described a role for mammalian Sir2 in repressing the muscle cell differentiation program. The region-specific binding of dSir2 might reflect a difference in the types of factors it can associate with, or the association of dSir2 with particular chromosomal regions or nuclear domains (Bianchi-Frias, 2004).

Interestingly, dCtBP and dSir2 recruitment are largely overlapping, and this association continues outside of those loci where Hairy binds: 90% of dSir2-recruiting loci also recruit dCtBP. dCtBP and dSir2 are unique among transcriptional coregulators in that they both encode NAD+-dependent enzymatic activities. As NAD and NADH levels within the cell exist in closely regulated equilibrium, it is possible that dCtBP and dSir2 function as NAD/NADH redox sensors. In this way, the cell could use coenzyme metabolites to coordinate the transcriptional activity of differentiation-specific genes with the cellular redox state (Bianchi-Frias, 2004).

Protein Interactions: Groucho and Huckebein

The Groucho corepressor mediates negative transcriptional regulation in association with various DNA-binding proteins in diverse developmental contexts. Groucho has been implicated in Drosophila embryonic terminal patterning: it is required to confine tailless and huckebein terminal gap gene expression to the pole regions of the embryo. An additional requirement for Groucho in this developmental process has been revealed by establishing that Groucho mediates repressor activity of the Huckebein protein. Putative Huckebein target genes are derepressed in embryos lacking maternal groucho activity and biochemical experiments demonstrate that Huckebein physically interacts with Groucho. Using an in vivo repression assay, a functional repressor domain in Huckebein that has been identified contains an FRPW tetrapeptide, similar to the WRPW Groucho-recruitment domain found in Hairy-related repressor proteins. Mutations in Huckebein's FRPW motif abolish Groucho binding and in vivo repression activity, indicating that binding of Groucho through the FRPW motif is required for the repressor function of Huckebein. Thus Groucho-repression regulates sequential aspects of terminal patterning in Drosophila (Goldstein, 1999).

One proposed Hkb target is the snail (sna) gene, which is transcribed in the ventral-most portion of the embryo. sna expression is thought to be excluded from the posterior pole by hkb activity. Accordingly, sna and hkb expression domains abut in cellularizing wild-type embryos, whereas sna expression extends to, and includes, the posterior pole of hkb mutant embryos. In torD embryos, hkb expression expands towards the center of the embryo and the sna domain correspondingly retracts. By contrast, in gromat- embryos, the expression of sna does not respect the sna posterior border and spreads to the pole, overlapping extensively with the hkb expression domain. The expression of the T-related gene brachyenteron (byn; also called Trg) also seems to be repressed by Hkb. byn is not expressed at the most posterior region of wild-type (or torD) embryos, whereas it extends throughout the posterior cap of hkb mutant embryos, consistent with hkb setting the posterior limit of byn expression. However, it is found that byn is ectopically expressed at the posterior tip of gromat- embryos. Together, these results suggest that gro is, directly or indirectly, necessary for hkb repressor functions (Goldstein, 1999).

To establish whether Hkb can function as a repressor, a HairyHkb chimera was constructed by replacing the C terminus of Hairy with Hkb's N-terminal 195 amino acids (lacking the Hkb Sp1-like zinc-finger DNA-binding domain). When expressed under the regulation of the hb promoter, the HairyHkb chimera causes effective repression of Sxl (normally a target of Hairy) in the anterior region of female embryos. Furthermore, this repression also causes female-specific lethality, probably due to the role of Sxl in dosage compensation. These results indicate that Hkb contains a potent repression domain within its N-terminal 195 aminoacids (Goldstein, 1999).

Protein Interactions: Groucho and Capicua

capicua (cic) is involved in gene repression in Drosophila terminal and dorsoventral patterning. Cic functions in two Groucho-dependent repressor processes inactivated by Torso signaling. Therefore, it was asked whether Cic interacts with Gro in vitro. Three different fragments of Cic (amino-terminal, central, and carboxy-terminal) were expressed in bacteria as GST fusions and their ability to bind radiolabeled Gro protein was assayed. The carboxy-terminal portion of Cic interacts with Gro, whereas the amino-terminal and central regions of the protein show little or no binding. The binding of Cic to Gro is weaker than that of Hairy, but stronger than the Dorsal/Gro interaction in the same assay. The interaction of Cic with Gro does not depend on the conserved carboxy-terminal domain of Cic, indicating that this domain mediates another aspect of Cic function. Taken together, the results support the idea that Cic and Gro form a repressor complex inactivated by Torso signaling during terminal and dorsoventral patterning (Jimenez, 2000)

Protein Interactions: Groucho and Rpd3

The Drosophila gene groucho encodes a transcriptional corepressor that plays critical roles in many development processes. In an effort to illuminate the mechanism of Gro-mediated repression, Gro was employed as an affinity reagent to purify Gro-binding proteins from embryonic nuclear extracts. One of these proteins was found to be the histone deacetylase Rpd3. Protein-protein interaction assays suggest that Gro and Rpd3 form a complex in vivo and that they interact directly via the glycine/proline rich (GP) domain in Gro. Cell culture assays demonstrate that Rpd3 potentiates repression by the GP domain. Furthermore, experiments employing a histone deacetylase inhibitor, as well as a catalytically inactive form of Rpd3, imply that histone deacetylase activity is required for efficient Gro-mediated repression. Finally, mutations in gro and rpd3 have synergistic effects on embryonic lethality and pattern formation. These findings support the view that Gro mediates repression, at least in part, by the direct recruitment of the histone deacetylase Rpd3 to the template, where it can modulate local chromatin structure. They also provide evidence for a specific role of Rpd3 in early development (Chen, 1999).

Immunoprecipitation assays suggest that endogenous Gro and Rpd3 are associated in Drosophila nuclei, as antibodies against Gro coprecipitate Gro and Rpd3 from both Drosophila embryonic and S2 cell nuclear extracts. In each case, ~10%-20% of Rpd3 in the nuclear extracts has been found to precipitate with Gro. In vitro histone deacetylase assays using 3H-acetyl-labeled histones as the substrate indicates that ~20% of the histone deacetylase activity in the embryonic extracts coprecipitates with Gro. The histone deacetylase activity in the crude extracts and in the Gro immunoprecipitate is largely inhibited by the histone deacetylase inhibitor trichostatin A (TSA), as is the activity of purified recombinant Rpd3 (Chen, 1999).

To map the domain(s) of Gro responsible for the interaction with Rpd3, a series of truncated forms of Gro was constructed. When epitope tagged Groucho (M2-Gro) lacking the carboxy-terminal WD repeat domain (M2GroN420) is coexpressed with epitope tagged Rpd3 (H6Rpd3) in insect cells, M2GroN420 and H6Rpd3 copurify on both Ni2+-NTA-agarose and anti-Flag affinity beads. Therefore, the amino-terminal region of Gro is necessary, whereas the WD repeat domain is dispensable for the Rpd3 interaction (Chen, 1999).

The domain(s) in the amino-terminal region of Gro required for the Rpd3 interaction were further mapped by incubating in vitro-translated 35S-labeled Gro deletions with anti-Flag affinity beads containing purified M2Rpd3. Those 35S-labeled Gro variants that contain the glycine/proline-rich (GP) domain bind to beads containing purified M2Rpd3 but not to beads alone. Conversely, those 35S-labeled Gro variants lacking the GP domain fail to associate with M2Rpd3. In addition, a GST pull-down assay using purified GST-Gro fusion proteins confirms that the GP domain of Gro is required for the interaction with Rpd3. Furthermore, the deletion of the amino-terminal glutamine-rich (Q) domain of Gro severely reduces the affinity of the interaction. In conclusion, these findings suggest that the GP domain is required for the interaction, whereas the Q domain, which is required for Gro tetramerization, significantly stimulates the interaction (Chen, 1999).

To address whether the interaction between Gro and Rpd3 is functional in Gro-mediated repression, it was first determined if histone deacetylase activity is important for transcriptional repression by Gro in cultured cells. Gro strongly represses activated transcription in S2 cells when directly targeted to a promoter by fusion to the DNA binding domain (DBD) of the yeast transcription factor Gal4. The Gal4-Gro fusion strongly represses the transcriptional activation promoted by the combination of Dorsal and Twist, when expression vectors encoding these factors were cotransfected with a luciferase reporter (G5DE5tkLuc) driven by a herpes simplex virus thymidine core promoter, an artificial enhancer element containing multimerized Dorsal and Twist binding sites (Dl-Ebox), and multimerized Gal4-binding sites (USAG) (Chen, 1999).

Transfected S2 cells were treated with the histone deacetylase inhibitor TSA to determine whether deacetylase activity is important for Gal4-Gro-mediated repression. TSA treatment dramatically reduces Gal4-Gro-mediated repression, suggesting that histone deacetylation does contribute to this repression. Introduction of TSA results in a small (less than 2-fold) increase in the level of reporter activity in the absence of Gal4-Gro, but a much larger (up to 20-fold) increase in the level of reporter activity in the presence of Gal4-Gro. As a result, the calculated repression by Gal4-Gro decreases from 25-fold in the absence of TSA to ~3-fold in the presence of 300 nM TSA. The residual repression observed at high concentrations of TSA suggests that although full repression of transcription by Gro requires histone deacetylase activity, Gro may also utilize histone deacetylase-independent mechanisms for transcriptional repression (Chen, 1999).

In agreement with the finding that the GP domain of Gro is required for the interaction with Rpd3, this GP domain functions as a repression domain, when fused to the Gal4 DBD and the tetramerization domain (TD) of p53 (construct G4TDGP). The p53 TD, which itself does not repress transcription when fused to the Gal4 DBD, was utilized in place of the Gro TD to avoid the repression activity that is believed to result from the association of Gal4-Gro TD fusion with endogenous full-length Gro. In the absence of the p53 TD, the Gal4-GP domain fusion failed to repress transcription. These findings suggest that efficient repression by the GP domain of Gro requires tetramerization. This agrees with the finding that the Q domain is required for efficient binding between Gro and Rpd3 and with results showing that efficient repression by Gro requires a functional tetramerization domain (Chen, 1999 and references therein).

To further determine whether histone deacetylase activity is critical for the function of the Gro GP domain, a single point mutation in Rpd3 was generated in which a highly conserved histidine residue (H196) is replaced with a phenylalanine (H196F). Consistent with studies on mammalian histone deacetylases, the mutation decreases the specific activity of the enzyme by about sevenfold. Additionally, using a GST pull-down assay, both Rpd3WT and Rpd3H196F were found to bind to Gro with comparable affinity. Unlike Rpd3WT, which represses transcription three- to fourfold when fused to the Gal4 DBD, the Gal4-Rpd3H196F fusion fails to repress transcription in a similar assay. Therefore, Rpd3H196F represents an enzymatically inactive form of Drosophila histone deacetylase that binds to Gro with the same affinity as wild-type Rpd3 (Chen, 1999).

Although the Gal4-GP domain fusion protein does not repress activate transcription on its own due to the lack of a tetramerization domain, the Gal4-GP fusion is able to synergize with cotransfected wild-type Rpd3 to repress transcription but does not synergize with the mutant catalytically inactive form of Rpd3. These results strongly suggest that the GP domain contributes to Gro-mediated repression by interacting directly with the histone deacetylase Rpd3 and that the histone deacetylase activity of Rpd3 is essential for its ability to contribute to Gro-mediated repression (Chen, 1999).

gro and rpd3 interact genetically. gro is a maternally required gene with critical roles in multiple developmental processes, including anterior/posterior and dorsal/ventral pattern formation, neurogenesis, and sex determination. To determine whether or not Rpd3 could possibly interact with Gro to help mediate these processes, the distribution of this protein was examined in ovaries and embryos. Immunostaining of wild-type ovaries indicates that Rpd3 protein is ubiquitously present in the nuclei of both nurse and follicle cells throughout oogenesis. In addition, Rpd3 protein is detected in all the nuclei of the syncytial blastoderm embryo. Rpd3 protein levels drop significantly and remain low during gastrulation and at later stages of embryogenesis. However, a high level of spatially restricted expression is observed in the head region of stage 9-10 embryos (Chen, 1999).

Embryos heterozygous for a P element insertion in rpd3 show reduced levels of Rpd3 staining in the head, whereas homozygous embryos display no detectable expression in the head. In addition, this P1633 insertion fails to complement a deletion that removes the rpd3 gene entirely, Df(3L)10H. Therefore, P1633 likely represents a null or strong hypomorphic allele of rpd3 (Chen, 1999).

To look for evidence for an interaction between maternally expressed gro and rpd3, the frequency of unhatched embryos produced by females carrying various combinations of gro and rpd3 alleles was scored (in this experiment two gro alleles were used: groE48, a strong hypomorphic allele and groBX22, a null allele). Approximately 3%-4% of embryos produced by mothers singly heterozygous for either groBX22, groE48, P1633, or Df(3L)10H fail to hatch. This frequency is not significantly greater than that observed for embryos derived from wild-type mothers. However, embryos laid by females doubly heterozygous for gro and rpd3 alleles [groBX22/P1633, groE48/P1633, groBX22/Df(3L)10H, and groE48/Df(3L)10H] show a dramatic increase in embryonic lethality---16%-30% of the embryos fail to hatch. These synergistic effects on embryonic lethality suggest that gro and rpd3 function together during Drosophila oogenesis and/or embryogenesis (Chen, 1999).

Cuticles prepared from the unhatched progeny derived from females singly heterozygous for either gro or rpd3 are indistinguishable from wild type. However, those unhatched embryos produced by mothers doubly heterozygous for gro and rpd3 often display cuticles with striking abnormalities in anterior/posterior pattern formation. The majority of the unhatched embryos (>70%) generated by the groE48/P1633 and groBX22/P1633 trans-heterozygous mothers display replacement of anterior embryonic segments by a mirror-image duplication of the three to five posterior-most segments. Mirror-image duplicated structures include denticle belts and posterior spiracles. The remaining unhatched embryos were normal or only had minor defects in head structures. The unhatched embryos collected from the groBX22/Df(3L)10H, and groE48/Df(3L)10H trans-heterozygous mothers display more severe cuticle phenotypes. Most (50%-60%) have no cuticle, whereas 5%-10% show a mirror-image duplication of the posterior spiracle and disordered denticle belts. The remaining unhatched embryos display cuticles that are nearly wild type or have minor defects in head structures (Chen, 1999).

The cuticle phenotype of embryos derived from mosaic females containing P1633 homozygous germ-line clones was examined. The majority of the embryos (>65%) lacking maternally expressed rpd3 fail to hatch, and most of those unhatched embryos (>80%) exhibit variable pair-rule segmentation defects. Observed in particular was the partial or complete fusion of adjacent denticle belts resulting in embryos with five to eight thoracic and abdominal segments. In addition, variable, often severe, defects were observed in the head skeleton. Gro has been shown to interact physically with the pair-rule gene products Hairy and Runt and to be required for their function as transcriptional repressors. Therefore, the pair-rule phenotype observed in embryos lacking maternal Rpd3 suggests that like Gro, Rpd3 may also be involved in the repression mediated by certain pair-rule gene products (Chen, 1999).

In addition to Rpd3, Drosophila histone H1 has also been found to associate with Gro. H1 is a linker histone that stabilizes higher-order chromatin structure. Several previous findings suggest that the interaction between Gro and H1 may be functionally relevant to Gro-mediated repression. Drosophila H1 was purified and identified as a general inhibitor of transcription by RNA polymerase II in vitro . Genetic analysis has indicated that H1 does not appear to affect global transcription; instead, H1 functions as a gene-specific transcriptional repressor. In addition, the expression of genes encoding various H1 subtypes is developmentally regulated. Thus, it is possible that the interaction between Gro and H1 functions in a developmentally regulated manner to facilitate chromatin condensation and/or establish a repressive chromatin enviroment for transcription (Chen, 1999 and references therein).

The findings reported here, as well as those from previous studies showing that Gro family proteins make specific contacts with the amino-terminal tails of core histones (Palaparti, 1997), suggest that Gro represses transcription by inducing a silenced chromatin conformation. Thus it is proposed that after a direct interaction with DNA-binding transcription factors brings Gro to the template, the known ability of Gro to oligomerize, together with the known favorable interactions between Gro and core histones and/or histone H1, results in the nucleation of a Gro polymer that spreads along the template. Template-bound Gro may then provide an interface for recruitment to the template of key chromatin-modifying enzymes including histone deacetylases. These enzymes may then serve to modulate local higher order chromatin structure to establish a transcriptionally silenced domain. It is known that the interaction between Tup1 (a possible yeast homolog of Gro) and core histones is actually enhanced by histone deacetylation (Edmondson, 1996), so it is possible that histone deacetylation also serves to facilitate the further recruitment of the corepressor to the template, thereby reenforcing the transcriptionally repressed state (Chen, 1999).

Although histone deacetylation contributes to Gro-mediated repression, there are likely to be additional mechanisms by which Gro represses transcription. For example, cotransfection experiments show that Gro possesses some repression activity that is resistant to the deacetylase inhibitor TSA. In addition, the WD repeat domain of Gro functions as a weak repression domain (Fisher, 1996), and this repression activity appears to be independent of Rpd3. Consistent with the idea that corepressors can modulate transcription by multiple mechanisms, histone deacetylase-independent repression has also been observed for the Sin3 corepressor. It will thus be interesting to determine the identities of additional Gro-interacting proteins to see if they provide any clues to these possible alternative mechanisms of repression (Chen, 1999).

Protein Interactions: Groucho and Pangolin

The HMG-box protein Pangolin (Drosophila Tcf) can function as either an activator or a repressor of Wingless-responsive genes depending on the state of the Wingless signaling pathway and the availability of Armadillo, Pangolin's coactivator. Mutations of Tcf-binding sites in the promoters of Drosophila Ultrabithorax or Xenopus siamois reduce the level of gene expression in the normal expression domain of the animal, showing that Pangolin and its vertebrate homolog act as gene activators. In Drosophila, signal transduction from Wingless stabilizes cytosolic Armadillo, which then forms a bipartite transcription factor with Pangolin and activates expression of Wingless-responsive genes. In the absence of Armadillo, Pangolin acts as a transcriptional repressor of Wingless-responsive genes, and Groucho acts as a corepressor in this process. Reduction of Pangolin activity partially suppresses wingless and armadillo mutant phenotypes, leading to derepression of Wingless-responsive genes. wingless null mutants completely lose epidermal engrailed expression before stage 10, but in homozygous wg embryos that are heterozygous for pangolin, some cells maintain en expression. This corroborates a repressive role for Pangolin in cells in which the Wg signalling pathway is not active. Reduction of Armadillo levels causes Pangolin to act as a repressor. Dominant negative Pangolin, lacking the Armadillo-binding regions, acts as a constitutive repressor. Furthermore, overexpression of wild-type Pangolin enhances the phenotype of a weak wingless allele. Finally, mutations in the Drosophila groucho gene also suppress wingless and armadillo mutant phenotypes since Groucho physically interacts with Pangolin and is required for its full repressor activity. When the N-terminal region of Groucho is expressed in cultured cells, it localizes to the cytoplasm. Coexpression of either human Tcf-1 or Pangolin results in the localization of this truncated Groucho to the nucleus, consistent with a physical association between the proteins. Full-length Gro is constitutively nuclear, and as such, is not informative in this assay. The recruitment of truncated Groucho by Pangolin is very similar to the recruitment of beta-catenin, a known Tcf-binding partner. groucho mutations show dose-senstive interactions with both wg and arm. Reducing the dose of maternal Gro suppresses the wg null phenotype, whereas reduction of paternal Gro has no effect. Pangolin repression is shown to requires Groucho. Deletion analysis defines a minimal region in hTCF-1 (amino acids 176-359) that is capable of binding to Grg-5; this domain is separable from the Armadillo (Arm)-interaction domain (amino acids 4-63). XGrg-5, which lacks the C-terminal WD40 repeats of the longer Grg proteins, enhances the transcriptional activity of suboptimal amounts of Arm-XTcf-3 complexes. mGrg-5 has no intrinsic transactivation properties when fused to a Gal4 DNA-binding domain. The enhancement of transcription by XGrg-5 could probably be attributed to its interference with the repressive effects of endogenous Gro proteins. A deletion mutant of XTcf-3 that lacks the Grg-interaction domain is a tenfold more potent transcriptional activator than its wild-type counterpart, confirming the activity of endogenous corepressors of Tcf factors. Therefore, it is proposed that the balance between the activity of Gro and Arm controls cell-fate choice by the Wnt pathway in both vertebrates and invertebrates (Cavallo, 1998).

Osa-containing Brahma chromatin remodeling complexes are required for the repression of Wingless target genes

The Wingless signaling pathway directs many developmental processes in Drosophila by regulating the expression of specific downstream target genes. The product of the trithorax group gene osa is required to repress such genes in the absence of the Wingless signal. The Wingless-regulated genes nubbin, Distal-less, and decapentaplegic and a minimal enhancer from the Ultrabithorax gene are misexpressed in osa mutants and repressed by ectopic Osa. Osa-mediated repression occurs downstream of the up-regulation of Armadillo but is sensitive both to the relative levels of activating Armadillo/Pangolin and repressing Groucho/Pangolin complexes that are present, and to the responsiveness of the promoter to Wingless. Osa functions as a component of the Brahma chromatin-remodeling complex; other components of this complex are likewise required to repress Wingless target genes. These results suggest that altering the conformation of chromatin is an important mechanism by which Wingless signaling activates gene expression (Collins, 2000).

In addition to transducing the wg signal in a complex with Arm, Pan is also required for the active repression of Wg target genes in the absence of the Wg signal. This repression requires the association of Pan with the corepressor Groucho (Gro). Gro functionally interacts with the histone deacetylase Rpd3, and this interaction is important for at least some of the repressive activity of Gro. Thus, both Osa-containing Brm complexes and Pan/Gro/Rpd3 complexes repress the expression of Wg target genes and probably mediate this repression by altering the local chromatin architecture at the promoters of these genes. Consistent with this, reduction of gro or rpd3 dosage reduces the ability of Osa to repress nub. The loss of nub expression caused by expression of UAS-Osa with ap-Gal4 is significantly rescued in wing discs homozygous for a hypomorphic allele of rpd3. Also, larvae transheterozygous for osaeld308 and groE48 often ectopically express nub in the wing disc, and 40% of transheterozygous adults have notum-to-wing transformations. These phenotypes are not seen when osa or gro single mutants are crossed to wild-type flies (Collins, 2000).

Interestingly, Gro has been shown to interact with the N-terminal tail of histone H3 and with the histone deacetylase Rpd3, and it has therefore been proposed that Gro mediates repression by altering chromatin structure. Consistent with this, a strong genetic interaction exists between osa and gro that suggests that their activities in repressing Wg target genes are closely related. Although it has not previously been reported that Rpd3 functions in the repression of wg target genes, reducing the function of rpd3 can partly rescue the loss of nub expression caused by the overexpression of Osa. Rpd3 is therefore important for the repression of Wg target genes; testing whether it is essential awaits the isolation of null alleles (Collins, 2000).

The loss of either osa or gro leads to ectopic expression of Wg target genes; thus, the activity of one is not sufficient to repress the expression of these genes without the activity of the other. Osa and Gro may, therefore, be mediating the same repressive event rather than acting in parallel. Interestingly, human SWI/SNF forms a repressor complex with Rb and the histone deacetylase HDAC. This complex interacts with the cyclin E promoter through the binding of Rb to E2F-1 and represses E2F-1 activation of cyclin E expression. This suggests the intriguing possibility that Osa and the Brm complex function in a larger repressor complex containing Gro and Rpd3 and that this complex is recruited to Wg target genes though the binding of Gro to Pan. However, Gro acts as a corepressor for a large number of transcription factors, and Osa cannot be required for all repression mediated by Gro because loss of osa does not result in neurogenic phenotypes like those caused by the loss of gro. Further research is needed to determine if Gro and/or Rpd3 can directly interact with components of the Brm complex and, if so, what determines the specificity of this interaction (Collins, 2000 and references therein).

A model for the regulation of gene expression by components of the Wg pathway is presented. The chromatin remodeling activity of the OsaBrm complex is required to maintain the chromatin at the promoters of wg-responsive genes in a repressive conformation. This would prohibit the association of other transcription factors with their binding sites and prevent the recruitment of components of the basal transcription machinery. Osa/Brm complexes may be recruited to Wg-responsive genes through an association with Pan/Gro/Rpd3 complexes. In response to the Wg signal, Arm is stabilized and accumulates in the cytosol. This accumulation of cytosolic Arm permits Arm to translocate to the nucleus and displace Gro from Pan and, in so doing, relieve the repression mediated by Gro, Rpd3, and Osa/Brm complexes. Arm may also promote a more open chromatin conformation by recruiting the HAT activity of dCBP, thus permitting the association of other transcription factors with their binding sites. Also, the stimulation of the DNA-bending activity of Pan by Arm may bring distantly spaced transcription factors into juxtaposition to promote the activation of gene expression. In the absence of osa, the chromatin is maintained in a more open and less repressive conformation. This would permit other transcription factors to interact with their binding sites at lower concentrations than would otherwise be possible. Under these conditions, the low levels of Arm that are always present in the cell may be sufficient to promote the activation of gene expression without the Wg signal (Collins, 2000).

Protein Interactions: Groucho and Even skipped and Extra-extra

Groucho acts as a co-repressor for several Drosophila DNA binding transcriptional repressors. Several of these proteins have been found to contain both Groucho-dependent and -independent repression domains, but the extent to which this distinction has functional consequences for the regulation of different target genes is not known. The product of the pair-rule gene even skipped contains a Groucho-independent repression activity. Outside the Groucho-independent repression domain of Eve, a conserved C-terminal motif (LFKPY), similar to motifs that mediate Groucho interaction in Hairy, Runt and Hückebein, has been identified. Eve interacts with Groucho in yeast and in vitro, and groucho and even skipped genetically interact in vivo. Eve with a mutated Groucho interaction motif, which abolishes binding to Groucho, shows a significantly reduced ability to rescue the eve null phenotype when driven by the complete eve regulatory region. Replacing this motif with a heterologous Groucho interaction motif restores the rescuing function of Eve in segmentation. Further functional assays demonstrate that the Eve C terminus acts as a Groucho-dependent repression domain in early Drosophila embryos. This novel repression domain is active on two target genes that are normally repressed by Eve at different concentrations: paired and sloppy paired. When the Groucho interaction motif is mutated, repression of each target gene is reduced to a similar extent, with some activity remaining. Thus, the ability of Eve to repress different target genes at different concentrations does not appear to involve differential recruitment or function of Groucho. The accumulation of multiple domains of similar function within a single protein may be a common evolutionary mechanism that fine-tunes the level of activity for different regulatory functions (Kobayashi, 2001a).

When comparing the activities of altered proteins in their normal context in vivo, it is desirable to have a reliable dose-sensitive assay for function. eve is weakly haplo-insufficient, showing a reduced viability when only one copy is present. Although most single copy embryos have no apparent defects at the end of embryogenesis, most of them do have abnormally narrow odd-numbered parasegments at early stages of development. Conversely, introducing extra copies of eve using the rescuing transgene results in abnormally wide odd-numbered parasegments. The hypomorphic (pair-rule) eve phenotype arises from odd-numbered parasegments that are severely reduced in width, and are unstable, so that at later stages they are eliminated by processes that repair patterning defects. Thus, one copy of the endogenous gene is near a threshold of sufficiency for generating stable parasegments, and the spacing of parasegments is a dose-dependent assay for early eve function (Kobayashi, 2001a).

Two copies of the wild-type eve transgene, inserted at various chromosomal locations, do not completely rescue the parasegment spacing defects of eve null mutants. Rather, a homozygous transgene phenocopies heterozygous eve, while a single (heterozygous) eve transgene produces a hypomorphic phenotype. This provides a means with which to compare the activities of altered Eve proteins expressed in their normal pattern, in either one or two copies. When the early parasegment spacing was examined in eve mutant embryos rescued by a homozygous transgene with a point mutation in the Groucho interaction domain (GID), it was found that odd-numbered parasegments were reduced, closely resembling an eve hypomorph. This was consistently observed with each of the GID mutants that abolished the Gro interaction. In contrast, in transgenic flies in which the wild-type Eve motif was replaced with the Hairy GID, odd-numbered parasegments were normal, or sometimes even slightly increased relative to even-numbered parasegments. Thus, the parasegment spacing parallels the relative strength of the in vitro interaction with Gro. The similarity in phenotype produced by one copy of the wild-type transgene and two copies of the GID-mutant transgene suggests that the activity of Eve is reduced about twofold by mutation of the GID. The more complete rescue by the Hairy GID-containing protein suggests that it has a stronger activity than does wild-type Eve, consistent with in vitro interaction data and the embryonic pattern rescue. However, the apparently more complete rescue of embryonic pattern was not fully reflected in the ability to rescue to adulthood (Kobayashi, 2001a).

What is the significance of the two distinct Eve repression activities, only one of which is dependent on Gro? Gro could be required to repress a subset of Eve targets, whereas repression of other target genes might be Gro independent. Alternatively, the two repression activities might function cooperatively, in which case both activities might be required for repression of each target gene. Extensive molecular and genetic studies have identified several target genes that are likely to be directly repressed by Eve. The best characterized of these genes are sloppy paired (slp), paired (prd) and odd skipped (odd). The posterior boundaries of expression of slp and prd correspond to the anterior and posterior borders, respectively, of the odd-numbered en stripes. As these en stripes shift posteriorly, both when the dose of gro is reduced and when the GID is mutated, the boundaries of slp and prd may be coordinately shifted. slp and prd expression were examined in embryos rescued with a GID-mutated transgene. Both slp and prd expression were expanded in the eve domains, relative to wild-type embryos. The degree of expansion of each gene correlates with the shift of en stripes. Furthermore, both the width of individual en stripes and their spacing are very similar to those in eve hypomorphs. Thus, the removal of the GID has an effect that is similar to that of a general reduction of eve activity on both targets, slp and prd. This expansion of slp and prd expression is reversed, in each case, when the Eve GID motif is replaced by that of Hairy. These results suggest that Gro is required by Eve to a similar degree for its repression activity on each of these genes (Kobayashi, 2001a).

Repression of another Eve target gene (odd) is required for the establishment of the even-numbered (ftz-dependent) en stripes. Intriguingly, these are established more or less normally in embryos rescued by the GID-mutated transgene, and examination of odd expression in those embryos has showen it to be normal in the even-numbered parasegments. However, repression of odd and the establishment of even-numbered en stripes are also normal when eve function is reduced in other ways (e.g. in the hypomorph), suggesting that a lower threshold of Eve activity is required for this eve function than for proper repression of slp and prd. Therefore, this assay did not allow for a full assessment of the contribution of Gro to odd repression by Eve (Kobayashi, 2001a).

dHb9 (FlyBase designation: Extra-extra [Exex]), the Drosophila homolog of vertebrate Hb9, encodes a factor central to motorneuron (MN) development. Exex regulates neuronal fate by restricting expression of Lim3 and Even-skipped, two homeodomain (HD) proteins required for development of distinct neuronal classes. Exex and Lim3 are activated independently of one another in a virtually identical population of ventrally and laterally projecting MNs. Surprisingly, Exex represses Lim3 cell nonautonomously in a subset of dorsally projecting MNs, revealing a novel role for intercellular signaling in the establishment of neuronal fate in Drosophila. Evidence is provided that Exex and Eve regulate one another's expression through Groucho-dependent crossrepression. This mutually antagonistic relationship bears similarity to the crossrepressive relationships between pairs of HD proteins that pattern the vertebrate neural tube (Broihier, 2002).

The mutually exclusive expression patterns of Eve and Exex and the ability of Exex to repress Eve led to an investigation of whether Eve exhibits a reciprocal ability to repress Exex. Whether eve represses exex was tested by following Exex in eve1D mutant embryos. This temperature-sensitive allele allowed the circumvention of the early requirement for eve during embryonic segmentation. On average, two ectopic Exex-positive neurons were observed in each hemisegment of eve mutant embryos. The position of these neurons identifies one as RP2 and the other as likely aCC or pCC. Therefore, eve exhibits a reciprocal ability to repress exex in a subset of dorsally projecting MNs (Broihier, 2002).

During segmentation, Eve has been shown to act as a transcriptional repressor and contains two domains with repressive capability -- one dependent on the corepressor Groucho (Gro) and one Gro independent. To determine whether Eve requires Gro to repress Exex in the CNS, Exex expression was assayed in eve null embryos that contain an eve transgene deleted for the Gro-interaction domain. In these embryos, Exex is derepressed in RP2 and one of the corner cells. Since this phenotype is essentially identical to that of eve1D mutants, it is concluded that Eve represses Exex in a Gro-dependent manner. These results demonstrate that Eve/Evx proteins act through Gro to regulate cell fate in the CNS (Broihier, 2002).

Protein Interactions: Groucho and Dorsal

The Dorsal morphogen acts as both an activator and a repressor of transcription in the Drosophila embryo in order to regulate the expression of dorsal/ventral patterning genes. Circumstantial evidence has suggested that Dorsal is an intrinsic activator and that additional factors (corepressors) convert it into a repressor. These corepressors, however, have previously eluded definitive identification. Via the analysis of embryos lacking the maternally encoded Groucho corepressor and via protein-binding assays it has been shown that recruitment of Groucho to the template by protein:protein interactions is required for the conversion of Dorsal from an activator to a repressor. Specifically, Groucho is required for the Dorsal, mediated repression of Zerknullt and Decapentaplegic. Groucho is not required for the spatially regulated expression of genes that are activated by Dorsal such as twist and snail. Groucho is therefore a critical component of the dorsal/ventral patterning system (Dubnicoff, 1997).

Activation and repression by the C-terminal domain of Dorsal

In the Drosophila embryo, Dorsal, a maternally expressed Rel family transcription factor, regulates dorsoventral pattern formation by activating and repressing zygotically active fate-determining genes. Dorsal is distributed in a ventral-to-dorsal nuclear concentration gradient in the embryo, the formation of which depends upon the spatially regulated inhibition of Dorsal nuclear uptake by Cactus. Using maternally expressed Gal4/Dorsal fusion proteins, the mechanism of activation and repression by Dorsal has been explored. A fusion protein containing the Gal4 DNA-binding domain fused to full-length Dorsal is distributed in a nuclear concentration gradient that is similar to that of endogenous Dorsal, despite the presence of a constitutively active nuclear localization signal in the Gal4 domain. Whether this fusion protein activates or represses reporter genes depends upon the context of the Gal4-binding sites in the reporter. A Gal4/Dorsal fusion protein lacking the conserved Rel homology domain of Dorsal, but containing the non-conserved C-terminal domain also mediates both activation and repression, depending upon Gal4-binding site context. A region close to the C-terminal end of the C-terminal domain has homology to a repression motif in Engrailed -- the eh1 motif. Deletion analysis indicates that this region mediates transcriptional repression and binding to Groucho, a co-repressor known to be required for Dorsal-mediated repression. As has previously been shown for repression by Dorsal, activation by Dorsal, in particular by the C-terminal domain, is modulated by the maternal terminal pattern-forming system (Flores-Saaib, 2001).

The results presented here show that just as Dorsal sites function in a context-dependent manner in the presence of endogenous Dorsal, so too do Gal4 sites function in a context-dependent manner in the presence of a Gal4/Dorsal fusion protein. When Gal4/Dorsal*/nt1 binds to multiple tandemly repeated Gal4 sites upstream of a core promoter, the result is activation. In contrast, when Gal4/Dorsal*/nt1 binds a modified dpp VRR in which two critical Dorsal-binding sites have been replaced by Gal4-binding sites, the result is repression. Thus, bringing Dorsal to its target sites is sufficient for both activation and repression -- the rel homology domain (RHD) itself need not be directly engaged with the DNA. Perhaps Dorsal, other DNA-bound repressors (the assistant repressors) and co-repressors such as Gro cooperatively assemble at the ventral silencer to form a 'silencesome'. As might be expected if silencer function required the assembly of such a complex, silencing by the zen VRR is crucially dependent upon the spacing between the sites for the DNA-binding proteins. Changing the spacing (by a non-integral multiple of the DNA helical repeat distance) severely abrogates silencing, presumably by rotating DNA-bound proteins onto opposite faces of the helix. Very similar spacing effects have been observed for enhancesomes (Flores-Saaib, 2001).

The co-repressor Gro, which is required for Dorsal-mediated repression, interacts with the Dorsal RHD. This finding is consistent with the observation that truncated forms of Dorsal consisting of little more than the RHD are able to mediate partial repression of target genes such as zen and dpp. However, the repression directed by the RHD alone is weak relative to that directed by full-length Dorsal and it is therefore not surprising to discover an additional Gro-interaction domain in Dorsal, this one in the CTD. Although the CTD is not conserved between Rel family proteins, the Dorsal-related immunity factor (Dif) can partially substitute for Dorsal during embryogenesis. In addition, patterning of the chick limb may involve the regulation by NF-kappaB of the vertebrate orthologs of Dorsal-target genes. Given these similarities in function, how is it possible to explain the apparent absence of the eh1-like repression domain from Dorsal-homologs such as Dif and NF-kappaB? One possibility is that Rel family protein-mediated transcriptional repression is of relatively minor importance to pattern formation. This is possible because other redundant mechanisms involving Short gastrulation (Sog)-family inhibitors exist to ensure that Dpp-orthologs will not be active at inappropriate positions along the dorsal/ventral axis of the metazoan embryo. The additional Gro-interacting repression domain in the Dorsal CTD may have arisen relatively recently, perhaps as an evolutionary adaptation to allow more complete or more reliable repression of dpp and other genes that interact with dpp to pattern the dorsal ectoderm (Flores-Saaib, 2001).

The studies presented here suggest that potent repression by Dorsal does require a region with homology to the eh1 motif. Thus, Engrailed and Dorsal may use a similar interface to recruit Gro. In this respect, it is interesting to note that Engrailed and Dorsal actually have a ~150 amino acid region of similarity, with the eh1 motif at the C-terminal end of this region. The similar region contains polyalanine stretches, which is a characteristic associated with other repression domains. Perhaps this extended region of similarity plays some role in repression beyond that played by the eh1 motif (e.g. the recruitment of another co-repressor). While Dorsal can function as either an activator or repressor, Engrailed and all other previously characterized repressors containing eh1 motifs appear to be dedicated repressors. The conserved phenylalanine in the eh1 domain is required for efficient Gro recruitment and transcriptional repression. The absence of this phenylalanine in the Dorsal motif could explain the ability of Dorsal to act as either an activator or a repressor depending upon binding site context. Perhaps this 'defect' in the Dorsal eh1 motif prevents Dorsal from recruiting Gro without help from other nearby DNA-bound repressor proteins (assistant repressors). In this respect, it is very interesting to note that Hairy family proteins, which are dedicated repressors, use a C-terminal WRPW motif to recruit Gro, while Runt family proteins, which can function as both activators and repressors, recruit Gro, at least in part, via a C-terminal WRPY motif. Perhaps the conversion of the C-terminal tryptophan to a tyrosine weakens Gro recruitment thereby allowing Runt family proteins to function as either activators or repressors depending upon binding site context (Flores-Saaib, 2001).

Consistent with previous experiments showing that the CTD contributes to transcriptional activation in Drosophila S2 cells and in vitro, this domain mediates activation in embryos. Transcriptional activation by the CTD may be mediated by the previously described interactions of this domain with TAFII110 and TAFII60. Interestingly, the deletion that removes the eh1-like motif and prevents repression by the CTD also results in reduced transcriptional activation. There are multiple possible explanations for this observation. Perhaps Gro has some role in activation in addition to repression. This is reminiscent of studies suggesting that Tup1, a possible yeast ortholog of Gro, functions in both activation and repression. Alternatively, it is possible that the activation and repression domains overlap in the CTD, but that they function via completely different co-regulators. If this is true, then one might expect the binding of the co-repressor and the co-activator to be mutually exclusive, thus ensuring that Dorsal cannot function at cross-purposes by simultaneously recruiting a co-activator and a co-repressor (Flores-Saaib, 2001).

When Gal4/CTD is targeted to the anterior end of the embryo, the resulting zone of repression does not include the anterior pole of the embryo. A key finding in the study of this phenomenon came with the discovery and analysis of capicua (cic), a gene that encodes an HMG-box family transcription factor. In addition to being required for terminal pattern formation, Cic is also required for efficient Dorsal-mediated repression. The finding that Cic appears to be degraded in response to Tor activation suggests that Cic may be a direct target of the terminal pattern forming system. Previous evidence also hinted at a role for the terminal system in modulating Dorsal-mediated activation. When an artificial anterior-to-posterior gradient of Dorsal is established in the embryo, activation of a reporter gene under the control of the proximal twi VAR does not extend to the anterior pole of the embryo. This effect has attributed to the possible presence of Tor response elements in the twi VAR. However, as reported here, even when activation is mediated by nothing but tandem Dorsal sites, this activation is still inhibited at the termini of the embryo by Tor. Likewise, Tor also blocks activation by Gal4/CTD through multiple Gal4 sites. Since these artificial reporters are unlikely to contain Tor response elements distinct from the Dorsal or Gal4 sites, it is likely that the Tor pathway interferes directly with Dorsal-mediated activation, either by modifying Dorsal itself or by modifying a co-activator required for Dorsal activity. Consistent with the possibility that Dorsal itself is the direct target of the terminal system, it has been found that elimination of Tor activity results in an increase in the lower SDS-PAGE mobility form of Dorsal. Because phosphorylation usually decreases SDS-PAGE mobility, this finding suggests that Tor activation might result in the dephosphorylation of Dorsal, either by inactivating a Dorsal kinase or by activating a Dorsal phosphatase. In addition to blocking the activation of Dorsal target genes directly, the terminal system also blocks their activation indirectly, since huckebein, a zygotic target of the terminal system, clearly directs sna repression at the poles. Thus, there appear to be multiple perhaps partially redundant mechanisms to ensure that mesodermal determinants such as twi and sna will not be inappropriately expressed at the poles (Flores-Saaib, 2001 and references therein).

The effect of a tor gain-of-function mutation on activation by Dorsal and the Gal4/Dorsal fusion is not what would be predicted based upon the simple idea that Tor inhibits Dorsal-mediated activation. Instead of resulting in a further retraction of expression from the pole of the embryo, the gain-of-function mutation causes no obvious change in the size of the anterior gap. In addition, this mutation results in an expansion towards the posterior of Gal4/CTD-driven activation and a broadening in the D4/lacZ expression domain. These findings appear to be consistent with a model in which Tor has two completely different effects on Dorsal-mediated activation, inhibiting it at the poles and strengthening it away from the poles. This is precisely what has been observed for the interaction between Bcd and the terminal system. Thus, the effects of Tor on activation may be very general. How Tor is able to function in these two opposite ways depending upon position in the embryo is not clear (Flores-Saaib, 2001 and references therein).

Protein Interactions: Groucho and Brinker

A Dpp activity gradient specifies multiple thresholds of gene expression in the dorsal ectoderm of the early embryo. Some of these thresholds depend on a putative repressor, Brinker, which is expressed in the neurogenic ectoderm in response to the maternal Dorsal gradient and Dpp signaling. In this study it is shown that Brinker is a sequence-specific transcriptional repressor. It binds the consensus sequence, TGGCGc/tc/t, and interacts with the Groucho corepressor through a conserved sequence motif, FKPY. An optimal Brinker binding site is contained within an 800-bp enhancer from the tolloid gene, which has been identified as a genetic target of the Brinker repressor. A tolloid-lacZ transgene containing point mutations in this site exhibits an expanded pattern of expression, suggesting that Brinker directly represses tolloid transcription (Zhang, 2001).

Brinker is the fourth sequence-specific repressor that has been shown to interact with Groucho through the tetrapeptide motif, aromatic-basic-pro-aromatic. The first version of this motif that was identified is WRPW, located at the carboxyl terminus of the Hairy repressor. The related WRPY motif was subsequently shown to mediate Runt-Groucho interactions, and FRPW permits Huckebein to bind Groucho. The Brinker repression domain identified in this study, FKPY, conforms to the other three Groucho motifs except for the lysine residue at position 2 (Zhang, 2001).

Genetic studies are consistent with the occurrence of Brinker-Groucho interactions in the early embryo. The tail-up and pannier expression patterns appear to expand into lateral regions of embryos derived from groucho germ-line clones. It is conceivable that Brinker mediates both Groucho-dependent and Groucho-independent transcriptional repression because the removal of the FKPY motif does not abolish the ability of an otherwise normal stripe2-Brinker transgene to repress dpp expression. The residual activity of the mutagenized transgene might be mediated by cryptic Groucho interaction motifs in Brinker. Alternatively, Brinker might repress certain target enhancers via competition between Smad activators and the Brinker repressor to overlapping DNA-binding sites. A similar situation has been described for the Kruppel and Knirps repressors. They require the dCtBP corepressor to regulate some, but perhaps not all, target genes. The Groucho and dCtBP corepressors might be required only when activators and repressors bind to distinct, nonoverlapping sites within a target enhancer (Zhang, 2001).

Dpp-Brinker interactions represent a particularly vivid example of how sequence-specific transcriptional repressors can limit inductive interactions by extracellular signaling molecules. Brinker helps promote neurogenesis by blocking Dpp signaling in the neurogenic ectoderm. It might also work as a gradient repressor to subdivide the dorsal ectoderm into dorsal epidermis and amnioserosa. There are other examples of repressors limiting signaling pathways. High levels of the Spaetzle ligand lead to optimal activation of the Toll-Dorsal signaling pathway and the induction of the Snail repressor in the presumptive mesoderm of early embryos. Snail prevents high levels of Spaetzle from activating neurogenic genes (e.g., Brinker, achaete-scute, and rhomboid) in the mesoderm (Zhang, 2001 and references therein).

The interplay between extracellular signaling molecules and localized transcriptional repressors is reminiscent of the segmentation pathway in the early Drosophila embryo. Pair-rule stripes of gene expression are established by broadly distributed transcriptional activators, such as Bicoid and Stat. The stripe borders are formed by localized gap repressors, including Hunchback, Kruppel, and Knirps. Similarly, the activation of tolloid and pannier might depend on broadly distributed Smad proteins, whereas the lateral limits of the expression patterns are established by the localized Brinker repressor. It is likely that vertebrates also employ one or more transcriptional repressors to restrict TGF-beta signaling interactions (Zhang, 2001).

Brinker requires two corepressors for maximal and versatile repression in Dpp signalling

Responses to graded Dpp activity requires an input from a complementary and opposing gradient of Brinker (Brk), a transcriptional repressor protein encoded by a Dpp target gene. Brk harbours a functional and transferable repression domain, through which it recruits the corepressors Groucho and CtBP. By analysing transcriptional outcomes arising from the genetic removal of these corepressors, and by ectopically expressing Brk variants in the embryo, it has been demonstrated that these corepressors are alternatively used by Brk for repressing some Dpp-responsive genes, whereas for repressing other distinct target genes they are not required. These results show that Brk utilizes multiple means to repress its endogenous target genes, allowing repression of a multitude of complex Dpp target promoters (Hasson, 2001).

Gro is ubiquitously expressed in the adult wing and mutations in gro have been identified in genetic screens for modifiers of various wing and eye phenotypes, implicating Gro in advanced developmental stages. Indeed, Gro has been ascribed at least one specific role in the establishment of wing configuration, as a corepressor for the Enhancer of split basic-helix-loop-helix proteins acting downstream of Notch signaling in D/V wing patterning. To assess whether Gro also contributes in hitherto unrecognized ways to wing A/P axis formation, the expression of wing-patterning genes was examined in marked clones of cells that either ectopically overexpress, or are mutant for, gro. Overexpression of gro should enhance the silencing of genes normally repressed by Gro-dependent transcriptional regulators while, reciprocally, the loss of gro should result in derepression, and therefore in the ectopic induction of these genes (Hasson, 2001).

In the wing imaginal disc, cells in the posterior compartment are programmed by the engrailed selector gene product to secrete Hedgehog (Hh), which induces dpp in a stripe of anterior cells along the A/P boundary. Dpp then acts as a long-range morphogen that governs patterning across the entire imaginal disc field. To determine whether Gro participates in the implementation of Hh signaling, clones overexpressing gro, or clones that are homozygous for the strong groE48 allele, were stained for dpp-lacZ expression. In all clones, even those overlapping with the Hh activity domain, there are no noticeable alterations in the dpp expression pattern, indicating that Gro is not required downstream of Hh for dpp transcriptional regulation. In striking contrast, however, three distinct targets of the Dpp pathway, expressed either in the wing pouch [optomotor-blind (omb) and vestigial (vg) or in the periphery of the wing disc (brk)], are repressed in clones overexpressing gro. Expression of omb-lacZ, as well as that of a lacZ reporter driven by vg's Dpp-responsive enhancer (vgQ-lacZ), is completely abrogated in these clones, whereas expression of brk-lacZ is only reduced. All three Dpp targets are repressed in a cell autonomous manner, i.e. only in the clones but never in adjacent cells. These results, together with an extensive gro loss-of-function clonal analysis detailed below, implicate Gro specifically as a downstream effector of Dpp signaling (Hasson, 2001).

Recent genetic and molecular studies have shown that brk encodes a repressor acting downstream of the Dpp pathway, which helps define the low end of the Dpp gradient. In particular, the Dpp targets omb and vgQ are both derepressed in brk- mutant clones and in brk- wing imaginal discs, suggesting that they are normally subjected to Brk repression. More directly, Brk binds to specific sequences within defined omb and vgQ enhancer elements, bringing about their silencing by outcompeting the Mad-Medea complex, or some other activator, from binding to overlapping DNA sites (Hasson, 2001).

That putative Brk target genes are repressed in clones of cells with increased gro dosage strongly suggests that Brk is a Gro-dependent repressor. Accordingly, Brk's proposed repression domain (RD) harbours a potential Gro recruitment motif (FKPY), similar to the Gro-binding domains defined in the repressors Hairy (WRPW), Runt (WRPY) and Huckebein (FRPW), and identical to that in Even-skipped (Eve). Brk also contains a CtBP-binding domain (PMDLSLG. Brk is shown, in fact, to be able to interact physically with both Gro and CtBP, and the functional relevance of these associations to Brk's in vivo repressor capacity are addressed (Hasson, 2001).

To demonstrate Brk's ability to associate with the two corepressors in vitro, the protein's putative RD (amino acids 369-541) was fused to glutathione S-transferase (GST), and it was incubated with radioactively labelled Gro or CtBP. In GST pull-down assays, Brk's RD (BrkRD) readily retains [35S]methionine-labelled Gro. To test further the specificity of this interaction, three mutant derivatives of the BrkRD, fused to the GST moiety, were generated in which the Gro recruitment domain (BrkRDmutG; FKPY to FEAY), the core of the CtBP-binding motif (BrkRDmutC; DLS to AAA) or both (BrkRDmutC/G) were altered. Brk's binding to Gro is impaired by the modifications in the FKPY motif. Significantly, however, Gro associates with the GST- BrkRDmutC construct as strongly as it does with the native GST-BrkRD fusion. GST-BrkRD also binds labelled CtBP in vitro and, although the binding of Brk to CtBP is weak in this assay, the specificity of the interaction is clearly evident: the association between the two proteins is abolished by mutations in the CtBP recruitment domain but is unaffected by alterations in the Gro recruitment motif (Hasson, 2001).

Brk has been reported to negate transcription by competing with activators, such as Mad/Medea, for overlapping DNA target sites, thereby preventing activators' access to target promoters. The direct interactions of Brk with Gro and CtBP, however, suggest that Brk acts in a more instructive manner. While in the former 'passive' mechanism Brk is expected to rely solely on its competitive DNA-binding activity, the latter 'active' mechanism predicts that it accommodates an innate RD that depends on the recruitment of corepressors (Hasson, 2001).

To establish whether Brk contains a functional RD that can silence gene expression, separable from its DNA-binding domain, an in vivo assay was employed that relies on repression of the sex-determining Sex-lethal (Sxl) gene by ectopic expression of the pair-rule gene hairy. Sxl is normally expressed only in female embryos whereas, in males, it is repressed by Deadpan (Dpn), an autosomally encoded Hairy-related repressor protein. When Hairy is expressed prematurely, under the hunchback (hb) promoter, it mimics Dpn's repressor function and eradicates Sxl transcription in the anterior of syncytial blastoderm female embryos. Because Sxl is essential for dosage compensation in females, this repression subsequently leads to female-specific lethality. A form of Hairy, lacking its own RD, is inert in this assay. However, fusion of heterologous RDs to the truncated Hairy protein restores its ability to repress Sxl. Indeed, the equivalent expression of a hb-Hairy-BrkRD transgene results in an effective repression of Sxl in the anterior halves of female embryos and female-specific lethality ensues. Thus, the region in Brk spanning the Gro- and CtBP-binding domains promotes potent repression in embryos (Hasson, 2001).

The ability to selectively disrupt Brk binding to each individual corepressor allowed the exploration of the dependence of its repressor potential on Gro and/or CtBP in vivo. Since both Gro- and CtBP-mediated repression can be detected in the Sxl-repression assay, truncated Hairy was fused to the three derivatives of the Brk RD, mutated in the Gro, CtBP or both recruitment motifs and placed under hb promoter regulation. In female embryos expressing Hairy-BrkRDmutC, Sxl is substantially repressed, although not as effectively as by Hairy-BrkRD. Furthermore, this repression still leads to statistically significant female-specific lethality. Thus, blocking CtBP binding does not completely abolish activity of the Brk RD. In comparison, mutating the Gro recruitment domain causes only residual Sxl repression and no apparent female-specific lethality. Finally, Sxl expression is seen throughout female embryos expressing hb-Hairy-BrkRDmutC/G, and no female-specific lethality is observed. Thus, Brk relies mainly on Gro for repressing Sxl. Nevertheless, since mutating the CtBP recruitment motif in Brk's RD attenuates Sxl repression, it is concluded that, for full potency as a negative transcriptional regulator, Brk requires both corepressors (Hasson, 2001).

These data indicate that the interactions between Brk and the corepressors Gro and CtBP are indispensable for maximal repression of Sxl in vivo. Whether Brk requires both cofactors for repression of its endogenous target genes was examined. For repression of distinct target genes, Brk requires Gro and/or CtBP differentially, presumably as a function of specific promoter topology and architecture (Hasson, 2001).

Brk competes with an activator for binding to an omb wing enhancer, suggesting that, for this promoter, Brk should act independently of corepressors. Consistent with this, omb-lacZ is not ectopically expressed in cells homozygous for groE48 (hereafter referred to as gro- clones), nor is it affected by CtBP loss-of-function clones, generated using the l(3)87De-10 allele (CtBP-), or by CtBP-, gro- double mutant clones). Thus, single and double mutant clones for gro and CtBP do not phenocopy the omb derepression seen in brk- clones, implying that Brk can repress omb even in the absence of these corepressors. Repression of the Dpp target gene spalt (sal) is also independent of Gro and CtBP. Nonetheless, in gro overexpression clones, omb is repressed, suggesting that, even for the omb promoter, Gro reinforces Brk repressor function (Hasson, 2001).

To establish whether Brk represses vgQ via Gro, CtBP or both, vgQ-lacZ expression was monitored in gro- and CtBP- single, and CtBP-; gro- double mutant clones. In this instance, a mandatory requirement for Gro, but not for CtBP is found; in gro- clones, vgQ is upregulated. Importantly, as is the case for brk- clones, the cell-autonomous upregulation of vgQ is seen only in gro- clones close to the periphery of the disc, suggesting that the observed effects are Brk dependent. In contrast, in CtBP- mutant clones vgQ expression is downregulated, in the Brk territory but also outside it, at the centre of the disc, indicating that these effects are Brk independent and that CtBP is positively required for vg expression. CtBP-;gro- double mutant clones show a composite effect: ectopic expression and upregulation of vgQ in clones in the brk expression domain, and a phenotype resembling that of CtBP- clones at the middle of the disc, where brk is not expressed. Thus, Brk repression of vgQ is Gro- but not CtBP-dependent (Hasson, 2001).

omb and vgQ expression is completely shut off in clones of cells overexpressing gro, whereas that of brk is only reduced, suggesting that Brk might be repressing its own transcription via a negative autoregulatory loop. To establish whether, in negating its own expression, Brk is assisted by Gro and/or CtBP, gro- and CtBP- single, or CtBP-, gro- double mutant clones were stained for brk-lacZ expression. brk is never ectopically expressed in any of the single mutant clones, whereas ectopic brk expression is clearly observable in double mutant clones. Thus, in the absence of one corepressor, repression is adequately mediated by the other, suggesting that negative autoregulation by Brk is robust, relying on either Gro or CtBP (Hasson, 2001).

Strikingly, the effects on brk expression are seen only in double mutant clones found at the periphery of the disc, but not at the center where Shn is active, supporting the notion that the effects are, indeed, Brk- but not Shn-dependent. Furthermore, the fact that double mutant clones at the middle of the disc do not ectopically express brk suggests that Shn-mediated repression of brk transcription must be taking place even in the absence of both corepressors (Hasson, 2001).

To be able to compare Brk's dependence on Gro and CtBP in the embryo, full-length Brk was expressed in its native form or with its corepressor-binding domains mutated, using UAS-brk transgenes driven by maternal GAL4 . This experimental design is inapplicable for studying Brk's targets in the wing, since ectopic expression of brk prevents proliferation and survival of imaginal disc cells, but is nevertheless effective in the embryo. Ectopic Brk represses zen and dpp in mid- to late-cellularizing embryos but not earlier, so endogenous Brk targets were analyzed in transgenic embryos at comparable stages of development. Ectopic expression of all three mutant forms of Brk in embryos brings about repression of zen to the same extent as does native Brk. This result suggests that Brk represses zen independently of corepressors. In contrast, Brk requires corepressors for negating transcription of both tolloid (tld) and dpp. Thus, abolishing Brk's interactions with Gro (BrkmutG), but not with CtBP (BrkmutC), completely relieves tld repression, indicating that Brk repression of tld is strictly Gro dependent, as is repression of pannier (pnr). Similarly, dpp is repressed in embryos expressing BrkmutC, but is still transcribed in embryos expressing Brk with its Gro recruitment motif mutated. In the case of dpp, however, CtBP must also be contributing to Brk repression, since the level of dpp expression is significantly lower in BrkmutG-expressing embryos, in comparison with wild-type embryos or embryos expressing BrkmutC/G. Thus it is concluded that, for repression of dpp, Brk rests mainly on Gro, yet for maximal repressor activity it also requires CtBP. The data indicate that Brk utilizes different means of repression for silencing its downstream targets in the embryo, as in the adult (Hasson, 2001).

Gro and CtBP mediate gene silencing in qualitatively different ways. Gro potentiates long-range repressors that function at a distance and that are able to block, in a dominant fashion, complex modular promoters consisting of multiple enhancer elements. In contrast, CtBP-dependent short-range repressors inhibit activators only locally, thereby permitting enhancer autonomy in a compound promoter. By virtue of its ability to recruit both Gro and CtBP, together with its capacity to outcompete pMad and other activators from binding DNA, Brk is competent to repress a multitude of complex Dpp target promoters, which receive positive inputs from manifold signaling pathways. It is proposed that, for promoters with low-affinity Mad-binding sites, the driving repressor force is direct competition between Brk and pMad for DNA binding, whereas for Dpp target promoters that contain high-affinity Mad-binding sites, corepressors are essential for mediating Brk repression. For this latter class of promoters, Brk relies on one or both of its cognate corepressors, depending on the particular promoter topology (Hasson, 2001).

Brk utilizes a self-reliant mechanism, which need not depend on tethered corepressors, by competing with activators over coinciding DNA-binding sites. In the absence of both Gro and CtBP, Brk represses not only omb and zen, but also sal, suggesting that the Brk-binding site(s) in the sal promoter overlap with those employed by activators. Transcription of both sal and vgQ requires activation by Mad, yet, although both promoters are exposed to identical levels of pMad, the sal expression domain is spatially more restricted than that of vgQ, presumably because activation of sal requires higher levels of pMad than that of vgQ. Hence, 'passive' competition-based repression should efficiently block activation of sal but may not be sufficient for promoters like vgQ, which are activated even by low amounts of Mad. For silencing such promoters, alternative mechanisms such as recruitment of corepressors have evolved and are employed (Hasson, 2001).

Brk represses its distinct endogenous target genes by recruiting Gro and/or CtBP differentially. For the silencing of many target promoters, Gro alone is sufficient (vg, tld and pnr) but, for fully repressing others, Brk depends on both corepressors. Thus, in the case of dpp and Sxl, when CtBP is lacking, a decrease in Brk's overall repressor capacity is apparent and, in the absence of Gro, repression is almost completely impaired. Importantly, for negating its own transcription, Brk can utilize either corepressor (Hasson, 2001).

The majority of activator and repressor binding sites in most Dpp-responsive enhancers have yet to be precisely mapped. It is nevertheless proposed that lengthy and complex promoters, which respond to several signaling inputs, will be found to be strictly silenced in a Gro-dependent manner. Thus, in repressing the vgQ enhancer, a composite cis-acting regulatory sequence with multiple elements that integrate information relayed by the dpp, wingless and EGF receptor signaling pathways, Brk is fully reliant on Gro, but not on CtBP. For other more simple promoters, short-range repression should be adequate and will be mediated by either corepressor, as exemplified by the robust Brk autoregulation, for which either Gro or CtBP is sufficient; CtBP and Gro are presumably interchangeable in this context, compensating for each other's absence (Hasson, 2001).

Significantly, the overexpression of gro results in ectopic omb repression, suggesting that, even for promoters that are switched off in a 'passive', competitive manner, excess Gro can over-potentiate Brk-mediated negative transcriptional regulation. Thus, Gro and/or CtBP might reinforce Brk repression of those promoters on which it initially acts by competing with activators for binding to DNA, via recruitment of histone deacetylases and alterations to chromatin structure, or by some other mechanism (Hasson, 2001).

In summary, these data suggest that Brk uses multiple means to negate target gene expression, such as competition and the varied recruitment of long- and short-range corepressors. It is proposed that this versatility is, biologically, most significant given Brk's role in Dpp signaling, since it facilitates the negative regulation of diverse, complex Dpp target promoters (Hasson, 2001).

Groucho and Hairless

The DNA-binding transcription factor Suppressor of Hairless [Su(H)] functions as an activator during Notch (N) pathway signaling, but can act as a repressor in the absence of signaling. Hairless (H), a novel Drosophila protein, binds to Su(H) and has been proposed to antagonize N signaling by inhibiting DNA binding by Su(H). In vitro, H directly binds two corepressor proteins, Groucho (Gro) and dCtBP. Reduction of gro or dCtBP function enhances H mutant phenotypes and suppresses N phenotypes in the adult mechanosensory bristle. This activity of gro is surprising, because it is directed oppositely to its traditionally defined role as a neurogenic gene. Su(H)-H complexes can bind to DNA with high efficiency in vitro. Furthermore, a H-VP16 fusion protein causes dominant-negative phenotypes in vivo, a result consistent with the proposal that H functions in transcriptional repression. Taken together, these findings indicate that 'default repression' of N pathway target genes by an unusual adaptor/corepressor complex is essential for proper cell fate specification during Drosophila peripheral nervous system development (Barolo, 2002b).

H is a novel protein, with no known vertebrate homologs. However, the H gene has been identified in three members of the order Diptera: Drosophila melanogaster, D. hydei, and the mosquito Anopheles gambiae. H is surprisingly poorly conserved among these three species: It shares 63% identity between D. melanogaster and D. hydei (diverged ~65 Mya), and 33% identity between Drosophila and Anopheles (diverged ~260 Mya). The rapid divergence of the H protein sequence readily allows the identification of short conserved motifs, which are presumably important for H function. Two such regions occur in a part of H that is required for its interaction with Su(H) in vitro (Barolo, 2002b).

Another conserved motif in the H protein is YSIxxLLG, which is perfectly conserved from Drosophila to Anopheles. This sequence resembles certain examples of the 'eh1' type of Gro-binding domain found in many transcriptional repressor proteins. Among eh1 domains, the 'octapeptide' motifs in the Pax 2/5/8 proteins, which have been shown to directly mediate repression by recruiting Gro-family corepressors, show the greatest similarity to this region of H. In addition, the extreme C-terminal sequence of H, PLNLSKH, includes a match to the consensus binding site for the CtBP corepressor, Px(D/N)LS. The PLNLS motif, fully conserved from Drosophila to Anopheles, exactly matches motifs found in four vertebrate CtBP-binding transcription factors. H also contains three lengthy alanine-repeat domains: AAAVAAAAAAAAA, AAAAAAAAAA, and AAVAAA AAAAAA. Alanine repeats and alanine-rich regions are common in transcriptional repression domains, and are found in many repressor proteins. However, these repeats are reduced or absent in the D. hydei and A. gambiae H proteins: this suggests that they may not make an essential contribution to H function (Barolo, 2002b).

A gel retardation experiment reported by Brou (1994), indicating that H can inhibit the binding of Su(H) to DNA in vitro, has strongly influenced interpretations of genetic studies of H, Su(H), and N. A DNA-binding-inhibition model of H function is indeed consistent with both loss- and gain-of-function genetic data demonstrating that H affects cell fate in a manner antagonistic to N signaling, including the N-stimulated transcriptional activation function of Su(H). However, the recent discovery of Su(H)-mediated transcriptional repression has forced a reconsideration of this simple model, since it makes incorrect predictions about the effect of H on a cell fate that is dependent on the repression function of Su(H). It is proposed that the genetic data on cell fate are instead consistent with a different role for H: facilitating transcriptional repression by Su(H) (Barolo, 2002b).

During the socket/shaft cell fate decision in adult mechanosensory bristle development, the cell that responds to N signaling takes the socket fate, while its sister cell, in which N signal transduction is blocked by the Numb protein, takes the shaft fate. Overexpression of Su(H), or loss of H function, during the socket/shaft decision causes both cells to adopt the socket fate; conversely, overexpression of H, or loss of Su(H) function, results in two shaft cells. Autorepression by Su(H) in shaft cells is important for maintaining the shaft cell fate. The corepressors Gro and dCtBP are important for specification of the shaft cell, a fate that is inhibited by N signaling and depends on both H activity and Su(H)-mediated repression. Reduction of gro or dCtBP function strongly enhances the effects of both reduction of H activity and loss of Su(H) repression, and suppresses the effects of reduced N signaling in the bristle lineage. It is therefore concluded that Gro and dCtBP, along with H and transcriptional repression mediated by Su(H), act in the opposite direction from the N signaling pathway during the socket/shaft cell fate decision, in that they promote the fate (shaft) that is inhibited by N signaling. The observation that both gro and dCtBP heterozygotes show a weak dominant (haploinsufficient) shaft-to-socket cell fate conversion phenotype is further confirmation of an important role for both corepressors in promoting the shaft cell fate. These results represent the first in vivo functional evidence for the involvement of Gro and dCtBP in transcriptional repression mediated by Su(H) (Barolo, 2002b).

Genetic analyses show that gro loss-of-function mutations enhance the effects of reduced H activity on two N-mediated cell fate decisions, the socket/shaft decision and the epidermal/SOP decision, while reduction of gro activity suppresses the effects of N loss of function on the socket/shaft and pIIA/pIIB cell fate decisions. In addition, gro has a weak haploinsufficient bristle loss phenotype, resembling an excess of N signaling. A role for gro in promoting the SOP cell fate is surprising, because gro was originally identified as a 'neurogenic' gene that acts to inhibit the SOP fate downstream of N signaling, in its capacity as a corepressor for bHLH transcriptional repressor proteins encoded by N target genes in the Enhancer of split gene complex [E(spl)-C]. In fact, gro was named after the phenotype of flies homozygous for gro1, a weak hypomorphic allele: bushy tufts of bristles over the eyes caused by a failure of N-mediated lateral inhibition of the SOP fate. At least one E(spl)-C bHLH repressor gene appears to be directly repressed by Su(H) in SOPs; the proposal that Gro promotes the SOP fate by cooperating with H to repress N target genes in this cell is currently being tested. If proved, this would represent a novel and complex form of regulation, in which Gro inhibits the SOP fate in all but one cell of the proneural cluster by partnering with the E(spl)-C bHLH repressors, and simultaneously promotes the SOP fate in one neighboring cell by preventing the expression of its own partners (Barolo, 2002b).

The current results support the hypothesis that H antagonizes N signaling by acting as an adaptor molecule between the transcription factor Su(H) and the corepressor proteins Gro and dCtBP. This model entails an unusual mechanism of repression: DNA-binding transcriptional repressors that recruit CtBP or the Gro family of corepressors generally do so via direct protein-protein interactions, although evidence for CtBP recruitment by non-DNA-binding proteins has been reported. In mammalian cells, the corepressors SMRT and CIR bind directly to the Su(H) homolog CBF1 (Barolo, 2002b).

In contrast to a DNA-binding inhibition model for H function, an adaptor/corepressor model explains why H counters NIC/Su(H)-mediated activation, but not Su(H)-mediated repression. Like previous views of H function, this model presumes competition between Su(H)-binding partners, in this case between NIC-containing activation complexes and H/Gro/dCtBP repression complexes. NIC activation complexes are likely to include the Mastermind (Mam) protein, and may also include the p300 coactivator. In the presence of N signaling, Su(H)/NIC/Mam complexes presumably replace Su(H)/H/Gro/dCtBP complexes on target genes, and convert Su(H) from a repressor to an activator. Whether this occurs by simple affinity-based competition for binding to Su(H), or by a mechanism involving active impairment of the H/Su(H) interaction, is unknown. Under an adaptor/corepressor model, the H mutant phenotype results from derepression of Su(H)/N target genes in cells lacking N pathway activity, thus mimicking an increase in N signaling. The H overexpression phenotype may be explained by the displacement of NIC-containing activation complexes by an excess of H-containing repression complexes, thus repressing NIC/Su(H) target genes in cells that respond to the N signal (Barolo, 2002b).

It has recently become apparent that the transcriptional target genes of at least six major developmental signaling pathways are in many cases subject to 'default repression'; that is, binding sites for signal-regulated transcription factors, which mediate activation during signaling events, mediate repression in the absence of signaling (for review, see Barolo, 2002a). Each of these pathways uses a different mechanism to switch from repression to activation upon stimulation of the pathway, but in each case, the effect seems to be the same: restricting the expression of pathway target genes to cells that receive active signaling. The results of this study strongly suggest that H contributes to default repression in the N pathway by directly recruiting the corepressors Gro and dCtBP to Su(H), and that formation of H/Su(H) repression complexes is crucial for the establishment of two N-inhibited cell fates, the SOP and shaft cell fates. Default repression, therefore, appears to be as important as signal-dependent activation for proper cell fate specification in this developmental context (Barolo, 2002b).

Hairless-mediated repression of Notch target genes requires the combined activity of Groucho and CtBP corepressors

Notch signal transduction centers on a conserved DNA-binding protein called Suppressor of Hairless [Su(H)] in Drosophila species. In the absence of Notch activation, target genes are repressed by Su(H) acting in conjunction with a partner, Hairless, which contains binding motifs for two global corepressors, CtBP and Groucho (Gro). Usually these corepressors are thought to act via different mechanisms; complexed with other transcriptional regulators, they function independently and/or redundantly. This study investigated the requirement for Gro and CtBP in Hairless-mediated repression. Unexpectedly, it was found that mutations inactivating one or the other binding motif can have detrimental effects on Hairless similar to those of mutations that inactivate both motifs. These results argue that recruitment of one or the other corepressor is not sufficient to confer repression in the context of the Hairless-Su(H) complex; Gro and CtBP need to function in combination. In addition, this study demonstrates that Hairless has a second mode of repression that antagonizes Notch intracellular domain and is independent of Gro or CtBP binding (Nagel, 2005).

To test the repressive effects of Hairless in the absence of NICD, Hairless ability to inhibit transcription in the presence of Grainyhead (Grh) was tested. The Notch response (NRE) reporter contains binding sites for the transcriptional activator Grh that stimulate transcription fourfold in the absence of NICD and increase the stimulation seen in the presence of NICD. Addition of full-length Hairless inhibits these effects, reducing transcription in the presence of Grh alone by 50%. Furthermore, this inhibitory effect is dependent on Su(H), as indicated by a lack of repression of HDeltaS, and requires both CtBP and Gro, since Hairless proteins with either interaction domain mutated (HDeltaC, H*C, HDeltaG, H*G) lose most of their repressive activity. Again, the levels of activity with the single mutants are similar to the levels seen with the double-mutant forms of the protein (HDeltaGC, H*GC) and all resulted in >90% of the expression seen with Grh. These experiments suggest that Hairless has two modes of repression, one that operates by repressing the transcriptional machinery through its recruitment of global corepressors and a second that operates by directly antagonizing NICD (Nagel, 2005).

These data confirm therefore that both Gro and CtBP can function as corepressors with Hairless, and indeed both factors are necessary for full repression by Hairless on the NRE; preventing the interaction with one or the other factor severely compromises Hairless activity. This is in apparent contrast to the effects on vgBE-LacZ, for which only Gro appears essential. Furthermore, the two cofactors appear to act together, since Hairless proteins lacking both interaction motifs retains a level of repression that is comparable to the results seen upon removing either alone (Nagel, 2005).

Previous studies of CtBP and Gro have argued that they mediate repression in qualitatively different ways, although both are thought to recruit histone deacetylases. Gro has predominantly been associated with so-called long-range repression, as it operates to dominantly silence modular enhancers. In contrast, CtBP appears to act in a local way to inhibit activators that are bound nearby. However, these models do not appear compatible with a combined requirement for Gro and CtBP in Hairless-mediated repression. Furthermore, direct fusion of a Gro interaction domain to the Su(H) protein is sufficient to convert it into a potent repressor, as described for other transcriptional regulators. Why should Gro and CtBP therefore be interdependent in the context of Hairless recruitment? One simple explanation would be that one or the other corepressor is needed to specifically counteract NICD activation. For example, CtBP interferes with recruitment of p300, a histone acetyltransferase that is reported to interact with mammalian NICD. However, the data suggest that CtBP and Gro are both needed to repress Grh even in the absence of NICD, arguing that each corepressor can only perform a subset of its functions in the context of Hairless. Maybe the two corepressors recruit different enzymatic activities that are needed together to promote repression. If the Hairless complex were incompatible with oligomerization of Gro, which is reported to be important for stable repression, Gro might be able to recruit histone deacetylases but not to promote spreading of the repression complex. And if CtBP, which in mammals has been found complexed with methyl transferases as well as deacetylases, could recruit only histone methyl transferases, the corepressors would each confer a critical component on the Hairless complex. A more complete understanding of the molecular functions of Gro and CtBP in the context of chromatin dynamics and transcription complexes will be needed to determine why Hairless requires their coordinate activities in many developmental scenarios, as has been shown in this study (Nagel, 2005).

Lateral inhibition in proneural clusters: cis-regulatory logic and default repression by Suppressor of Hairless

Lateral inhibition, wherein a single cell signals to its neighbors to prevent them from adopting its own fate, is the best-known setting for cell-cell communication via the Notch (N) pathway. During peripheral neurogenesis in Drosophila, sensory organ precursor (SOP) cells arise within proneural clusters (PNCs), small groups of cells endowed with SOP fate potential by their expression of proneural transcriptional activators. SOPs use N signaling to activate in neighboring PNC cells the expression of multiple genes that inhibit the SOP fate. These genes respond transcriptionally to direct regulation by both the proneural proteins and the N pathway transcription factor Suppressor of Hairless [Su(H)], and their activation is generally highly asymmetric; i.e., only in the inhibited (non-SOP) cells of the PNC, and not in SOPs. The substantially higher proneural protein levels in the SOP put this cell at risk of inappropriately activating the SOP-inhibitory genes, even without input from N-activated Su(H). This is prevented by direct 'default' repression of these genes by Su(H), acting through the same binding sites Su(H) uses for activation in non-SOPs. Derepression of even a single N pathway target gene in the SOP can extinguish the SOP cell fate. Finally, crucial roles are defined for the adaptor protein Hairless and the co-repressors Groucho and CtBP in conferring repressive activity on Su(H) in the SOP. This work elucidates the regulatory logic by which N signaling and the proneural proteins cooperate to create the neural precursor/epidermal cell fate distinction during lateral inhibition (Castro, 2005).

Su(H) is known to act as a transcriptional repressor in another context during sensory organ development; namely, the socket/shaft sister cell fate decision in the bristle lineage. Auto-repression of Su(H) is necessary to prevent inappropriate high-level activation of the gene in the shaft cell, which in turn can cause this cell (which does not respond to N signaling) to adopt the N-responsive socket cell fate. The biochemical basis of transcriptional repression by Su(H) has been studied in some detail in this setting. Specifically, the Hairless (H) protein has been shown to act as an adaptor that recruits the transcriptional corepressor proteins Gro and CtBP to Su(H), thus conferring repressive activity (Castro, 2005).

Earlier work can be interpreted to suggest that a similar protein complex might mediate repression by Su(H) in the SOP. At several macrochaete and many microchaete positions on the adult fly, simultaneous reduction of the doses of Hairless and gro in an otherwise wild-type background leads to significant bristle loss; this is due to a failure of commitment to the SOP cell fate. A plausible interpretation of these findings is that H and Gro are normally part of a repressive Su(H)-containing complex in the SOP, and that reduction of their doses sufficiently compromises the repressive activity as to partially de-repress N pathway target genes like E(spl)m8, leading to failure of SOP specification. As a test of this model, it was thought that it might be possible to detect such de-repression of a suitable reporter gene. This expectation was borne out. Late third-instar wing discs from wild-type larvae or larvae heterozygous for null alleles of either Hairless or gro only rarely exhibit detectable activity of an E(spl)malpha-GFP reporter transgene in SOPs. By contrast, wing discs from larvae doubly heterozygous for null alleles of both Hairless and gro show substantial frequencies of ectopic GFP expression in SOPs. Moreover, the SOP expression observed in the double heterozygotes is considerably stronger than that detected rarely in a wild-type background. These results demonstrate that normal levels of Hairless and gro activity are required for the Su(H)-dependent repression of N pathway target genes in SOPs, and are consistent with the participation of a Su(H)-H-Gro-containing protein complex in this repression (Castro, 2005).

Broad overexpression of Hairless (including in proneural clusters) during lateral inhibition causes a 'neurogenic' phenotype; that is, the appearance of supernumerary bristles surrounding normal bristles. This phenotype is readily understood in light of the model described above; namely, that Hairless normally serves to recruit Gro and CtBP to Su(H) for its repressive activity in the SOP. Overexpression of Hairless in the N-responsive non-SOP cells of the PNC would be expected to elevate their levels of the repressive form of Su(H), causing repression of N pathway target genes that would normally be activated by the Su(H)-NIC-Mam complex. This in turn would result in a partial failure of lateral inhibition and the commitment of additional cells in the PNC to the SOP fate, giving rise to ectopic bristles in the adult (Castro, 2005).

A key prediction of the model is that the ability of Hairless to bind Gro (via the motif YSIHSLLG) and CtBP (via the motif PLNLSKH) should be required for the SOP fate-promoting activity of H. This prediction was tested by using an E(spl)malpha GAL4 driver to express different forms of H specifically in the non-SOP cells of the PNCs. The orbital region of the adult fly head is a particularly favorable territory in which to assay the production of supernumerary bristles by H overexpression. Expression of a wild-type UAS-Hairless transgene results in the appearance of an average of approximately four ectopic bristles in the orbital region. This activity is significantly impaired by mutating either the Gro recruitment motif (UAS-H[Gm]) or the CtBP-binding motif (UAS-H deltaC), suggesting that both co-repressors make a functional contribution. Loss of both motifs (UAS-H[Gm] deltaC) essentially abolishes the capacity of Hairless to promote ectopic bristle development in this assay. These results are strongly consistent with the interpretation that the SOP cell's requirement for Hairless activity is based on the recruitment by Hairless of Gro and CtBP to confer repressive activity on Su(H), thus preventing inappropriate expression of inhibitory N pathway target genes (Castro, 2005).

It is concluded that discrete transcriptional cis-regulatory modules, bearing binding sites for both Su(H) and the proneural proteins, direct the non-SOP-only expression pattern of E(spl)-C genes in PNCs. Mutation of the Su(H) sites in these modules results in an inversion of this pattern of activity, including both the loss of most non-SOP expression and the appearance of strong ectopic expression in SOPs. These observations reveal a dual role for Su(H) in the PNC: as a direct, N-activated transcriptional activator of E(spl)-C genes in non-SOP cells, and as a direct transcriptional repressor of the same genes in the SOP. The issue was addressed as to whether Su(H)-mediated repression of E(spl)-C genes in the SOP is important developmentally. The experiments with wild-type and Sm versions of an E(spl)m8 genomic DNA transgene demonstrate that it is. Failure to repress this single bHLH repressor gene is sufficient to extinguish the SOP fate (marked by Sens) at a frequency significantly greater than that observed with a repressible (wild-type) transgene. Evidence is provided that the Hairless protein is responsible for conferring repressive activity on Su(H) in the SOP, by recruiting the co-repressors Gro and CtBP. It is suggested that the Hairless null phenotype widespread, irreversible loss of the SOP fate in an E(spl)-C-dependent manner, offers the best indication of the developmental consequences of relieving Su(H)-mediated repression of all E(spl)-C genes in the SOP (Castro, 2005).

A specific configuration of Su(H)-binding sites known as the Suppressor of Hairless Paired Site (SPS) has been shown to be essential for transcriptional synergy between proneural proteins and Su(H) in driving specific expression in PNCs. The results reported in this study on transcriptional regulation of E(spl)malpha contradict this conclusion with regard to the function of the SPS. The strong expression of E(spl)malpha in the non-SOP cells of the PNC depends crucially on cooperation between proneural activators and Su(H), yet none of the Su(H) sites of this gene are in the SPS configuration. Thus, until the mechanistic basis for proneural/Su(H) synergy is more fully elucidated, it is thought that the term 'Su(H) plus proneural' remains the most accurate and most general description of the PNC cis-regulatory code (Castro, 2005).

Direct repression of E(spl)-C genes in the SOP during lateral inhibition is a conspicuous example of what has been termed 'default repression', a property of developmental signaling pathways whereby pathway target genes are repressed by a signal-regulated transcription factor in the absence of signaling. It is proposed that default repression has evolved in order to prevent inappropriate (signal-independent) activation of pathway target genes in cells that express local activators but do not respond to the signal. Indeed, the SOP is in particular need of default repression because it is characterized (perhaps unusually) by elevated accumulation of the local activators for the PNC, the proneural proteins. That Su(H) can keep N pathway target genes off in SOPs even in the face of exceptionally high local activator levels is testament to the efficacy of default repression as a regulatory strategy (Castro, 2005).

It is now clear that default repression by Su(H) is a crucial feature of the operation of the N pathway in all three of the developmental situations in which it is known to function: lateral inhibition (this study), binary cell fate decisions in lineages, and formation of tissue boundaries. This conclusion is based on an analysis, in all three cases, of the consequences of mutating Su(H)- binding sites in one or more N pathway-activated genes; it is emphasized that attribution of a default repression activity to a signal-regulated transcription factor can be made only after such cis-regulatory experiments have been performed. It is likely that default repression by Su(H) is an integral part of N pathway function during Drosophila development (Castro, 2005).

The studies presented here, when combined with earlier reports, illuminate a prominent feature of the transcriptional regulation of gene expression and cell fate during lateral inhibition in Drosophila. It is now clear that three key regulatory factors [the proneural proteins (Ac and Sc), Su(H) and Gro] each have dual, and oppositely directed, functions in the SOP versus the non-SOP cells of the PNC during lateral inhibition. The proneural proteins are strictly required for the SOP cell fate, at least in part because they directly activate genes that promote or execute this fate, such as sens, phyllopod and ac itself. But, proneural proteins also have a vital role in non-SOPs as direct activators of genes, including those of the E(spl)-C, that are involved in inhibiting the SOP fate. Su(H) also has crucial, but opposing, functions in the SOP [as a direct default repressor of SOP-inhibitory E(spl)-C genes] and in the non-SOPs (as an essential direct activator of these same genes in response to N signaling). Finally, evidence is presented strongly supporting the hypothesis that Gro is likewise a 'double agent' during lateral inhibition: in the non-SOPs, where it serves as the co-repressor for the E(spl)-C bHLH repressor proteins to inhibit the SOP fate (its traditional function in the process), whereas in the SOP it partners with Su(H) via H to effect default repression and thus protect the SOP fate. The regulatory machinery underlying lateral inhibition is all the more elegant for its versatility and economy (Castro, 2005).

Groucho and Vnd

A nuclear concentration gradient of the maternal transcription factor Dorsal establishes three tissues across the dorsal-ventral axis of precellular Drosophila embryos: mesoderm, neuroectoderm, and dorsal ectoderm. Subsequent interactions among Dorsal target genes subdivide the mesoderm and dorsal ectoderm. The subdivision of the neuroectoderm by three conserved homeobox genes, ventral nervous system defective (vnd), intermediate neuroblasts defective (ind), and muscle segment homeobox (msh) has been investigated. These genes divide the ventral nerve cord into three columns along the dorsal-ventral axis. Sequential patterns of vnd, ind, and msh expression are established prior to gastrulation and evidence is presented that these genes respond to distinct thresholds of the Dorsal gradient. Maintenance of these patterns depends on cross-regulatory interactions, whereby genes expressed in ventral regions repress those expressed in more dorsal regions. This 'ventral dominance' includes regulatory genes that are expressed in the mesectoderm and mesoderm. At least some of these regulatory interactions are direct. For example, the misexpression of vnd in transgenic embryos represses ind and msh, and the addition of Vnd binding sites to a heterologous enhancer is sufficient to mediate repression. The N-terminal domain of Vnd contains a putative eh1 repression domain that binds Groucho in vitro. Mutations in this domain diminish Groucho binding and also attenuate repression in vivo. The significance of ventral dominance is discussed with respect to the patterning of the vertebrate neural tube, and ventral dominance is compared with the previously observed phenomenon of posterior prevalence, which governs sequential patterns of Hox gene expression across the anterior-posterior axis of metazoan embryos (Cowden, 2003).

Further evidence that Vnd is a repressor was obtained using an in vivo repression assay in transgenic embryos. The N-terminal region of Vnd contains a putative eh1 Groucho-interaction motif, FxIxxIL. This eh1 motif is present in two known transcriptional repressors, Engrailed and Goosecoid. It is also found in the Ind and Msh proteins. GST pull-down assays suggest that this motif mediates interaction between Vnd and Groucho. A GST-VEH1 fusion protein containing amino acid residues 183 to 226 from Vnd binds S35-labeled Groucho protein produced via in vitro translation. This binding is lost when the GST-Vnd fusion protein is mutagenized to replace the phenylalanine in the FxIxxIL motif with an alanine. Various positive and negative controls were included in these experiments. For example, Groucho does not bind a GST-Ind fusion protein containing the Ind homeodomain. Weak binding is observed with a GST-Eve fusion protein containing the FKPY Groucho-interaction motif (Cowden, 2003 and references therein).

At the molecular level, members of the NKx2.2 family of transcription factors establish neural compartment boundaries by repressing the expression of homeobox genes specific for adjacent domains. The Drosophila homologue, vnd, interacts genetically with the high-mobility group protein, Dichaete, in a manner suggesting co-operative activation. However, evidence for direct interactions and transcriptional activation is lacking. This study presents molecular evidence for the interaction of Vnd and Dichaete that leads to the activation of target gene expression. Two-hybrid interaction assays indicate that Dichaete binds the Vnd homeodomain, and additional Vnd sequences stabilize this interaction. In addition, Vnd has two activation domains that are typically masked in the intact protein. Whether vnd can activate or repress transcription is context-dependent. Full-length Vnd, when expressed as a Gal4 fusion protein, acts as a repressor containing multiple repression domains. A divergent domain in the N-terminus, not found in vertebrate Vnd-like proteins, causes the strongest repression. The co-repressor, Groucho, enhances Vnd repression, and these two proteins physically interact. The data presented indicate that the activation and repression domains of Vnd are complex, and whether Vnd functions as a transcriptional repressor or activator depends on both intra- and inter-molecular interactions (Yu, 2005; full text of article).

Conserved interaction of Ind with Groucho

Ind-Gsh-type homeodomain proteins are critical to patterning of intermediate domains in the developing CNS; yet, the molecular basis for the activities of these homeodomain proteins is not well understood. This study identifies domains within the Ind protein that are responsible for transcriptional repression, as well as those required for its interaction with the co-repressor, Groucho. To do this, a combination of chimeric transient transfection assays, co-immunoprecipitation and in vivo expression assays are utilized. Ind’s candidate Eh1 domain is shown to be essential to the embryonic repression activity of this protein, and that Groucho interacts with Ind via this domain. However, when activity is assayed in transient transfection assays using Ind-Gal4 DNA binding domain chimeras to determine domain activity, the repression activity of the Eh1 domain is minimal. This result is similar to previous results on the transcription factors, Vnd and Engrailed. Furthermore, the Eh1 domain is necessary, but not sufficient, for binding to Groucho; the C terminus of Ind, including the homeodomain also affects the interaction with this co-repressor in co-immunoprecipitations. Finally, this study shows that aspects of the cross-repressive activities of Ind/Gsh2-Ey/Pax6 are evolutionarily conserved. Taken together, these results point to conserved mechanisms used by Gsh/Ind-type homeodomain protein in regulating the expression of target genes (Van Ohlen, 2007).

The data presented in this study indicate that the capacity of ind to repress target gene expression is conferred not only by its ability to interact with Groucho through its Eh1 domain, but also by secondary domains, which include the C terminus of the protein, wherein resides the homeodomain. Indeed, deletion of Ind's C terminus affects the repressor activity of Ind in Gal4-Ind chimeric assays in tissue culture. Ind's physical interaction with Groucho suggests that this transcription factor uses redundant protein-protein interactions to exert maximal repressor activity (Van Ohlen, 2007).

Ind's candidate Eh1 domain is required for Ind-mediated repression in embryos and in vitro. Apart from the homeodomain, this is the only Ind region that is highly conserved between flies and vertebrates. Moreover, the co-immunoprecipitation data indicate that Ind's secondary structure is important for efficient Groucho binding. The fact that the full requirement for Ind's Eh1 domain is masked in the transient transfection assay can be explained by this observation, which coincidentally parallels previous findings for the Eh1 domain of Engrailed, Nkx6, and Vnd using chimeric transfection assays. Previous studies have shown that the sequestering of Groucho to its DNA-bound transcription factor target, Dorsal, requires secondary DNA binding proteins, including Dead Ringer and Cut. Potentially, the binding of Ind to DNA via the Gal4 DBD, rather than the homeodomain, results in an altered Ind conformation relative to when the native protein contacts its DNA target via its homeodomain. This could in turn result in less efficient Groucho binding to the chimeric Gal4-Ind proteins in the transfection assay. Indeed, the transcription factor, Pax 2, must be bound to its bone fide Pax 2 target for Groucho recruitment (Van Ohlen, 2007).

Dichaete and Sox neuro interact genetically with ind. An ac enhancer represses expression of that gene, when tested in a reporter assay in transgenic embryos. It contains 3 Ind binding sites adjacent to a single Dichaete binding site. When all four sites are mutated, the reporter is partially de-repressed relative to the wild-type reporter in transgenic embryos. In addition, Ind physically interacts with Dichaete in a yeast two-hybrid expression assay. These results, and the demonstration that Ind interacts with the co-repressor, Groucho, possibly explain the relatively weak Ind over-expression phenotype, despite strong expression of the transgene. Perhaps the limited (wild-type) availability of Groucho and Dichaete in cells that over-express Ind leads to the titration, and depletion, of these essential co-repressors, such that some ectopic Ind molecules cannot exert their regulatory effects maximally (Van Ohlen, 2007).

A major function of Ind/Gsh-type transcription factors is the restriction of the expression domains of proneural genes to distinct subsets of progenitors. The proneural gene, ac, is ectopically expressed in ind mutants, and this ectopic expression of ac expression leads to the loss of intermediate neuroblasts. This study shows that over-expression of ind causes down-regulation of ac in both ventral and lateral neuroblasts. Similarly, the proneural genes, neurogenenin 1 and 2, are ectopically expressed in gsh2 mutants. Moreover, just as Gsh2 represses Pax 6 in an adjacent domain, it was similarly found that Ind can repress eyeless, the Drosophila Pax 6 homologue. Ind and its vertebrate homologues differ however in their capacity to repress msh/msx genes. Whereas, the ability of ind to repress msh expression is critical to maintaining the tri-columnar organization of the neuroectoderm in Drosophila , Msx 1 expression is unaffected in gsh1; gsh2 double mutants, and the expression domains of these two proteins overlap. Thus, Ind shares many common properties with its vertebrate homologues, but also has repression targets that are not evolutionarily conserved. The non-conserved repression domains identified in Ind, additional to the Eh1 domain, may explain the divergence in the capacity of Ind/Gsh homeodomain proteins to repress Msx-msh gene expression. Further work is required to address whether the secondary repression domains in Ind are functionally significant in the embryo. In addition, whether primary protein structure alone accounts for some of the divergent activities of ind and gsh1 or gsh2 needs to be addressed, by determining whether ind's vertebrate homologues can functionally substitute for ind function in the Drosophila embryo (Van Ohlen, 2007).

Groucho interaction with Lozenge

Runx proteins have been implicated in acute myeloid leukemia, cleidocranial dysplasia, and stomach cancer. These proteins control key developmental processes in which they function as both transcriptional activators and repressors. How these opposing regulatory modes can be accomplished in the in vivo context of a cell has not been clear. The developing cone cell in the Drosophila visual system was used to elucidate the mechanism of positive and negative regulation by the Runx protein Lozenge (Lz). A regulatory circuit is described in which Lz causes transcriptional activation of the homeodomain protein Cut, which can then stabilize a Lz repressor complex in the same cell. Whether a gene is activated or repressed is determined by whether the Lz activator or the repressor complex binds to its upstream sequence. This study provides a mechanistic basis for the dual function of Runx proteins that is likely to be conserved in mammalian systems (Canon, 2003).

To understand negative regulation by the Lz protein, regulation of the deadpan (dpn) gene was investigated. In wild-type eyes, Dpn is expressed in photoreceptors R3/R4 and R7. In lz mutants, dpn is also ectopically activated in cone cells, suggesting that Lz either directly or indirectly represses dpn in these cells. Dpn was therefore used as a marker to investigate negative regulation by Lz (Canon, 2003).

The presence of two perfect consensus Runx protein-binding sites (5'-RACCRCA-3') upstream of the dpn-coding region suggested possible direct negative regulation by Lz. Gel-shift experiments showed that Lz specifically binds to both sites. To determine whether these sequences are required for proper dpn regulation, lacZ reporter constructs were made driven by dpn upstream and intronic fragments, and these were transformed into flies. A 4667-bp upstream fragment plus intron I (227 bp) caused expression of lacZ in R3/R4 and R7 faithfully recapitulating the pattern of wild-type dpn expression in the eye. This site is therefore referred to as the dpn eye enhancer (DEE). When the two Lz-binding sites (LBS) in the DEE were mutated (to 5'-RAAARCA-3'; DEE-MutLBS), lacZ expression was also seen in cone cells. Therefore, lack of Lz binding to this enhancer will cause its derepression in cone cells, establishing that Lz directly represses transcription of dpn in cone cells (Canon, 2003).

Like all Runx proteins, Lz contains the conserved C-terminal pentapeptide motif VWRPY, which binds the global corepressor Groucho (Gro). Gro does not bind DNA on its own, but functions as a repressor for sequence-specific DNA-binding factors. Gro is expressed ubiquitously and has early pleiotropic roles in eye development, such as mediating repression by bHLH proteins, making it difficult to study possible involvement of Gro in cone cell development in loss-of-function mutant clones in the eye. Therefore the Gro-interaction domain at the C terminus of Lz was altered from VWRPY to VWEAA, a change that abrogates Gro binding to bHLH proteins. Lz-EAA protein was then expressed under the control of the endogenous eye-specific lz enhancer and its ability to repress dpn was tested in vivo. Whereas a wild-type lz+ transgene efficiently represses dpn in cone cells, Lz-EAA was unable to keep dpn off in these same cells. Neuronal differentiation occurs normally in both cases as determined by the neural marker Elav. This shows that the C terminus of Lz, a known Gro-interaction domain, is required for Lz-mediated repression of dpn. The activation function of Lz-EAA, as determined by its ability to activate D-Pax2 expression, remains intact. Therefore, Gro mediates repression by Lz as it does for other Runx proteins. It still remained unclear, however, why in the same cell Lz represses dpn transcription while it directly activates D-Pax2. Clearly, the presence of Gro alone does not cause Lz to become a dedicated repressor in the cone cell (Canon, 2003).

Hairy-related proteins constitutively bind Gro through the conserved sequence WRPW, and function as dedicated repressors. To further address the significance of the C terminus of Lz, the C-terminal amino acids of Lz were changed from WRPY to WRPW to resemble Hairy-related repressors. As a correlate, a Lz-VP16 fusion was made, with the potent activation domain of VP16 fused onto the C terminus of Lz. The ability of Lz-WRPW and Lz-VP16 to regulate Lz targets was tested in vivo. Lz-WRPW efficiently represses dpn in cone cells like the wild-type Lz+ but was unable to activate expression of D-Pax2. In contrast, Lz-VP16 failed to repress dpn in cone cells but effectively activates D-Pax2 in cone cells. Therefore, Lz-WRPW functions as a dedicated repressor, and Lz-VP16 as a constitutive activator. These results suggest that Runx-Gro interactions are regulated, because wild-type Runx proteins function as both activators and repressors (Canon, 2003).

These observations suggest that Gro binds proteins with a WRPW motif in a stable manner and causes constitutive repression as seen for both Lz-WRPW and Hairy-related proteins that contain the WRPW motif. In contrast, Gro interaction with the WRPY motif in Runx proteins requires a cofactor, such as Cut, for stabilization. Therefore, repression is regulated as Runx forms a functional repressor complex with Gro only in the presence of the cofactor Cut. This hypothesis was tested in immunoprecipitation (IP) experiments. On its own, Lz weakly interacts with Gro. In the presence of Cut, however, the Lz-Gro interaction is dramatically increased. As expected, Lz-WEAA did not coimmunoprecipitate with Gro, with or without Cut, and Lz-WRPW interacted strongly with Gro, in both the presence and absence of Cut. These results are entirely consistent with all of the in vivo observations: (1) Lz functions as a repressor only in the cells that express the Cut protein; (2) Lz-WRPW, which functions as a constitutive repressor, can repress DEE-MutAT, in spite of the mutant AT-sites and absence of Cut binding; (3) wild-type Lz does not repress DEE-MutAT because Cut cannot bind, and therefore the Lz-Gro complex is not stabilized (Canon, 2003).

Groucho interaction with Odd-skipped

Drosophila Groucho, like its vertebrate Transducin-like Enhancer-of-split homologues, is a corepressor that silences gene expression in numerous developmental settings. Groucho itself does not bind DNA but is recruited to target promoters by associating with a large number of DNA-binding negative transcriptional regulators. These repressors tether Groucho via short conserved polypeptide sequences, of which two have been defined: (1) WRPW and related tetrapeptide motifs have been well characterized in several repressors; (2) a motif termed Engrailed homology 1 (eh1) has been found predominantly in homeodomain-containing transcription factors. A yeast two-hybrid screen is described that uncovered physical interactions between Groucho and transcription factors, containing eh1 motifs, with different types of DNA-binding domains. One of these, the zinc finger protein Odd-skipped, requires its eh1-like sequence for repressing specific target genes in segmentation. Comparison between diverse eh1 motifs reveals a bias for the phosphoacceptor amino acids serine and threonine at a fixed position, and a mutational analysis of Odd-skipped indicates that these residues are critical for efficient interactions with Groucho and for repression in vivo. These data suggest that phosphorylation of these phosphomeric residues, if it occurs, will down-regulate Groucho binding and therefore repression, providing a mechanism for posttranslational control of Groucho-mediated repression (Goldstein, 2005).

The eh1 Gro recruitment domain was originally defined as a heptapeptide motif that is conserved in members of the En family of homeodomain proteins and their vertebrate homologues. More recently, eh1-dependent binding to Gro has also been demonstrated in vitro for various other Drosophila and mammalian proteins, nearly all of which contain homeodomains. Given that Bowl and Odd, two non-homeodomain ZnF transcription factors, contain this motif and interact with Gro, the possibility was explored that eh1 motifs are prevalent among additional non-homeodomain transcription factor families. Indeed, an unbiased yeast screen for Gro-interacting proteins selected two additional transcriptional regulators that contain eh1-like motifs, namely, Sloppy-paired (Slp; Forkhead related) and Dorsocross (Doc; T box). Alignment of the eh1-like sequences of Bowl, Odd, Slp, and Doc with those of En and Gsc revealed three conserved amino acids: phenylalanine-x-isoleucine-x-x-isoleucine (Phe-x-Ile-x-x-Ile, where x is any amino acid). Subsequent database searches for presumptive Drosophila transcription factors containing this minimal peptide sequence identified a wide range of potential negative regulators belonging to different superfamilies as classified by their distinct DNA-binding domain types. Remarkably, eh1-related motifs have been preserved in many human homologues of these fly proteins, indicating that the ability to bind Gro/TLE has been evolutionarily conserved in human transcriptional regulators and that this sequence may have been widely adopted throughout the proteome as a Gro recruitment domain (Goldstein, 2005).

Several representatives, corresponding to different transcription factor families, were tested for the ability to bind Gro in biochemical assays. Where possible, full-length expressed sequence tags encoding these proteins were obtained; otherwise, single exons containing the eh1-like sequence were PCR amplified from genomic DNA. Each polypeptide was assessed for the ability to pull down radiolabeled Gro in vitro. GST-tagged Slp and Doc (amino acids 254 to 391) readily retain Gro, as do Eyes absent (Eya) and the homeodomain proteins Ventral nervous system defective (Vnd, 1 to 465), Bagpipe (Bap, 1 to 129), BarH1, and Empty spiracles (Ems, 1 to 360), as well as the orphan nuclear hormone receptor DHR96. To confirm that these interactions rely on intact eh1-related sequences, the eh1 motif of one of these, BarH1, was mutated by substituting glutamic acid for Phe at position 1, finding that its binding to Gro is reduced by >60% (Goldstein, 2005).

Based on these data, as well as on previous studies on En and Gsc, it is concluded that the eh1 peptide sequence, found in various proteins that belong to a wide range of distinct transcription factor families, is a good predictor of Gro-binding capability in vitro. Moreover, in vivo analysis of Odd's eh1 motif indicates that the above eh1 sequences impart Gro-mediated repression to a multitude of transcription factors (Goldstein, 2005).

The amino acid threonine is an essential element of Odd-skipped's eh1 domain. Comparison of more than 80 different eh1 regions revealed several recurring features, such as the prevalence of negatively charged amino acids at positions 4 and 5 between the two Ile residues, as well as a striking bias (>60%) toward Ser/Thr residues in position 2 adjacent to the invariant Phe residue. Given that phosphorylation of transcription factors has been well documented as a mechanism for regulating transcriptional outcomes, the importance of these potential phosphoacceptor amino acids for binding to Gro was examined by replacing the Thr residue in Odd's eh1 motif (T385) with other amino acids. The C-terminal 57-amino-acid portion of Odd binds Gro in a GST pulldown assay, whereas changes to alanine (T385A), methionine (T385M), or histidine (T385H) markedly reduce this interaction. In fact, the only construct tested that retains full binding to Gro is one in which Thr was changed to Ser (T385S). Thus, the Odd-Gro interactions appear highly sensitive to amino acid modifications at residue T385 (Goldstein, 2005).

To test if the above alterations affect Odd's repressor activity, flies were generated carrying Odd derivatives harboring modifications in T385. These were expressed throughout embryos under heat shock control and tested for the ability to repress two Odd targets, namely, eve stripe 1 and the secondary stripes of prd, both of which are fully repressed in close to 100% of embryos expressing native Odd. For simplicity of quantification, target gene repression was classified as 'full', 'partial', or 'none', with results depicted as the percentage of embryos displaying the corresponding expression pattern. Expression of the Odd T385S transgene was found to lead to strong repression of both targets, consistent with this variant's ability to bind Gro in vitro. In contrast, the T385A and T385M alterations brought about a significant loss of repressor activity, particularly of eve and less dramatically of prd. Thus, consistent with the biochemical protein interaction assay, a Ser/Thr residue appears to be a crucial element of the eh1 Gro recruitment domain for Odd, and presumably for other repressors as well (Goldstein, 2005).

The effect phosphorylation might have on Odd's repressor function was examined by introducing a negative charge at this site through the exchange of Odd's Thr residue for Asp (T385D). The C-terminal portion of Odd, containing the T385D alteration, does not associate with Gro in a GST pulldown assay. Furthermore, this pseudophosphorylated form of Odd is a much weaker repressor of eve stripe 1 and secondary prd stripes than T385M or T385A, being as ineffective as OddDeltaeh1. This suggests that if the Thr residue in the eh1 domain of Odd is subjected to phosphorylation, this modification would probably block binding to Gro and transcriptional repression (Goldstein, 2005).

Gro recruitment domains are not simply interchangeable. Whether the eh1- and WRPW-like Gro recruitment domains are interchangeable in vivo was tested by exchanging the eh1 sequence in Odd for a WRPW motif (OddDeltaeh1/WRPW). The WRPW motif has been shown to confer repressor potential on a number of transcription factors, and in line with this, the OddDeltaeh1/WRPW variant binds Gro vigorously in vitro. Unexpectedly, however, when misexpressed throughout the embryo, this transgene represses Odd target genes rather weakly. This result suggests that, at least with regard to Odd, the eh1 and WRPW motifs are not simply interchangeable. It is surmised that Odd's eh1-like sequence necessitates a particular structural and/or conformational context, or the cooperation of other distinct domains within Odd, for presenting Gro in a way that will mediate efficient transcriptional repression in vivo (Goldstein, 2005).

Groucho mediated repression: the EH1 motif in metazoan transcription factors

The Engrailed Homology 1 (EH1) motif is a small region, believed to have evolved convergently in homeobox and forkhead containing proteins, that interacts with the Drosophila protein Groucho (C. elegans unc-37, Human Transducin-like Enhancers of Split). The small size of the motif makes its reliable identification by computational means difficult. The predicted proteomes of Drosophila, C. elegans and human have been systematically searched for further instances of the motif. Using motif identification methods and database searching techniques, which homeobox and forkhead domain containing proteins also have likely EH1 motifs was examined. Despite low database search scores, there is a significant association of the motif with transcription factor function. Likely EH1 motifs are found in combination with T-Box, Zinc Finger and Doublesex domains as well as discussing other plausible candidate associations. Strong candidate EH1 motifs have been identified in basal metazoan phyla. Candidate EH1 motifs exist in combination with a variety of transcription factor domains, suggesting that these proteins have repressor functions. The distribution of the EH1 motif is suggestive of convergent evolution, although in many cases, the motif has been conserved throughout bilaterian orthologs. Groucho mediated repression was established prior to the evolution of bilateria (Copley, 2005).

Sequence motifs were sought in homeobox containing transcription factors taken from the proteins of human, Drosophila and C. elegans, by first masking known Pfam domains, and then using the expectation maximization algorithm implemented in the meme program. The first non-subfamily specific motif identified corresponded to previously known examples and new instances of, the EH1 motif, in 100 sites, with an E-value of < 10-126. The same approach was applied to Forkhead containing transcription factors, identifying 25 sites with a combined E-value of < 10-31. These motifs also appeared to conform to the consensus of the EH1 motif (Copley, 2005).

To further investigate the significance of this similarity, hidden Markov models (HMM) were constructed of the motif (EH1hox & EH1fh) which were then searched against the complete set of predicted proteins from human, D. melanogaster and C. elegans. The highest scoring non homeobox containing domain match of EH1hox was a Forkhead protein (human FOXL1), and the second highest scoring non-Forkhead containing match of EH1fh was to a homeobox containing protein (Drosophila Invected). In both cases, nearly all the high scoring hits were to proteins containing domains with transcription factor function. Among the best scoring matches of the EH1hox searches were several T-box (TBOX), Doublesex Motif (DM), Zinc finger (ZnF_C2H2) and ETS containing proteins (Copley, 2005).

The presence of EH1 motifs within various homeobox, and to a lesser extent, forkhead-containing proteins has been widely reported, although not systematically studied. EH1-like motifs co-occurring with 3 major groupings of homeobox sub-types were found: the extended-hox class, typified by Drosophila Engrailed; the paired class, including Drosophila Goosecoid, and the NK class, including Drosophila Tinman. Related to the paired class homeobox domains, a number of genes containing PAIRED domains only were also found to contain EH1-like motifs. With only a few exceptions, the EH1-like motif occurs N-terminal to the homeobox domain and C-terminal to the PAIRED domain when present. A number of these proteins have been shown to interact with Groucho or its orthologs, e. g., C. elegans cog-1, Drosophila Engrailed and Goosecoid, and in high throughput assays Drosophila Invected and Ladybird late (Copley, 2005).

A handful of EH1-like motifs are found C-terminal to homeobox domains. Of these, the best characterized is C. elegans unc-4, which has been shown to interact with the groucho ortholog unc-37; the Drosophila ortholog unc-4 also interacts with groucho in high throughput experiments. The C-terminal EH1-like motif is conserved in the closely related Drosophila paralog OdsH. The gene prediction for the human ortholog of unc-4 appears to be artefactually truncated, but the mouse ortholog (Uncx4.1) and corrected human gene models, contain EH1-like motifs both N- and C-terminal to the homeobox domain. Taken together with the fact that in the majority of related homeobox containing proteins the EH1-like motifs are N-terminal, this suggests that the N-terminal motif has been lost in Drosophila and C. elegans unc-4 orthologs (Copley, 2005).

EH1-like motifs also occur N- and C-terminal to Forkhead domains. The N-terminal class consists of the Sloppy-paired genes of Drosophila and orthologous or closely related sequences: human FOXG1, and Drosophila CG9571; the C. elegans ortholog fkh-2 contains an EH1-like motif although a cysteine residue causes a low score. The C-terminal class consists of an apparent clade including the human FOXA, FOXB, FOXC and FOXD genes, although if the EH1 motif was present in the common ancestor of this clade, multiple losses must have later occurred. The situation is complicated somewhat by an EH1-like motif at the N-terminus of C. elegans unc-130, i. e., in the FOXD like family. The EH1 motif in slp1 has been shown to interact with groucho, and FOXA type genes have been shown to interact with human groucho orthologs (Copley, 2005).

Likely EH1 motifs co-occurring with T-Box domains in two distinct contexts. The motif occurs C-terminal to the T-box in the Drosophila Dorsocross proteins Doc1, Doc2 and Doc3. It is found N-terminal to the T-box in 11 proteins including mls-1 and mab-9 from C. elegans; H15, Mid/Nmr2 and Bi/Omd from Drosophila; in humans there are strong matches to TBX18, TBX20 and TBX22 and more marginal matches to TBX3 and TBX2. As far as is known, none of these proteins has been shown to interact with groucho or its orthologs, although several are known to act as transcriptional repressors: for instance, in murine heart development, Tbx20 represses Tbx2 which in turn represses Nmyc; the Dorsocross genes from Drosophila repress wingless and ladybird, and Doc itself is repressed by mid/nmr2. The human proteins TBX1 and TBX10, and Drosophila Org-1 (which are all closely related to those above) do not appear to contain EH1 motifs. The human T (brachyury) protein contains a motif broadly similar to the EH1 consensus: LQYRVDHLLSA in a comparable N-terminal location to those found in other T-box containing proteins. Although this motif scores poorly against EH1hox, the homologous regions from other T orthologs provide a more persuasive case for the presence of a functioning EH1 motif in these proteins (Copley, 2005).

The highest scoring match of EH1hox to a C2H2 zinc finger containing protein, was ces-1 from C. elegans ; this protein interacts with the groucho ortholog unc-37 and can act as a repressor. The putative EH1 motif is at the N-terminal end of ces-1. In contrast, the Drosophila proteins Bowl and Odd have EH1-like motifs at their C-terminal ends. In neither case is there direct evidence from high throughput studies of an interaction with Groucho, but both can function as repressors. The human protein ZNF312 (bit score 8.6) is the ortholog of zebrafish Fezl, which contains an EH1 motif essential for repressor activity -- this motif is conserved in the human paralog and likely Drosophila ortholog CG31670 (Copley, 2005).

The Doublesex Motif (DM) was first found in proteins controlling sexual differentiation in Drosophila. Two DM containing proteins were confidently predicted to contain EH1-like motifs -- human DMRT2, and Drosophila dmrt11e. These are likely orthologs; a C. elegans protein, C27C12.6 contained a weaker match. The molecular function of these proteins is unknown (Copley, 2005).

The EH1 motif is found N- and C-terminal to homeobox, forkhead, T-box and Zn finger protein domains. Clearly, since the locations of the EH1 motif are non-homologous, the N- and C-terminal associations must have occurred independently. The short size of the motif makes it tempting to speculate that the motif itself may have arisen independently (i.e. in repeated cases it may have evolved within sequence that was already part of the gene, rather than via a recombination event). The strongest evidence for this is that, in general, the majority of domain combinations occur in a fixed N to C orientation, suggesting that recombination events combining domains are relatively rare. The fact that there have been many such events suggests that the alternative hypothesis of independent invention is more appropriate (Copley, 2005).

Groucho is orthologous to the C. elegans unc-37 gene, and the four human paralogs TLE1-4 (Transducin Like Enhancer of split). An ortholog is also found in the cnidarian Hydra mangipapillata (e. g., the EST with gi 47137860), and certain cnidarian homeobox containing genes also contain an EH1-like motif, suggesting groucho/EH1 mediated repression pre-dates the split between diplobasts and triplobasts; indeed, a sponge Bar/Bsh like homeobox containing protein also contains an EH1-like motif, as does paxb from the non-bilaterian placozoan Trichoplax adhaerens and a Tlx-like protein from a ctenophore, suggesting the repression system was in place in the earliest animals. High scoring EH1-like motifs are found in Forkhead domain containing proteins from sponges, cnidarians and ctenophores, in both the C-terminal (FOXA-D clade) and N-terminal (FOXG, sloppy paired clade) varieties. The presumed ortholog of 'T' from the Trichoplax adhaerens contains an EH1-like motif. These results suggest that groucho mediated repression using a variety of transcription factors was widespread in the last common ancestor of the metazoa. The EH1 motif is suggestive of a number of instances of convergent evolution, although in many cases the motif has been conserved throughout bilaterian orthologs. Together with the existence of a cnidarian Groucho ortholog, this leads to the conclusion that EH1/Groucho mediated repression was established prior to the evolution of bilateria (Copley, 2005).

Vnd represses target gene expression in conjunction with Groucho

The transcription factor, Vnd, is a dual regulator that specifies ventral neuroblast identity in Drosophila by both repressing and activating target genes. Vnd and its homologs have a conserved amino acid sequence, the Nk-2 box or Nk specific domain, as well a conserved DNA-binding homeodomain and an EhI-type Groucho interaction domain. However, the function of the conserved Nk-2 box has not been fully defined. To explore its function, the Nk-2 box was deleted and the regulatory activity of mutant Vnd in transgenic over-expression assays was compared to that of the wild-type protein. No regulatory activity could be assigned to the Nk-2 box using an over-expression assay, because the mutant protein activated expression of endogenous Vnd, masking a requirement for the Nk-2 box. However, in transgenic rescue assays, Vnd lacking the Nk-2 box repressed ind expression at 30% lower levels than the wild-type protein. Moreover, in transient transfection assays using Gal4 DNA-binding domain-Vnd chimeras, the repression activity of Vnd lacking the Nk-2 box was compromised. Because Vnd represses target gene expression in conjunction with Groucho, it was asked whether the Nk-2 box affects Vnd’s ability to interact with this co-repressor. Vnd lacking the Nk-2 box binds Groucho 30% less efficiently than wild-type Vnd in co-immunoprecipitations. These data suggest that the Nk-2 box contributes to the repression activity of Vnd by stabilizing its interaction with the co-repressor, Groucho (Uhler, 2007).

The conserved Eh1 domain, characterized by the consensus amino acid sequence, FxIxxIL, is essential for the interaction of non Hairy-type transcription factors with the co-repressor, Groucho. It has been shown that GST fusion protein including the candidate Vnd Eh1 domain pulled down Groucho, and mutation of this domain interfered with Vnd’s capacity to repress reporter expression in transgenic embryos. The data presented in this study indicate that a secondary domain, the Nk-2 box, also modulates Vnd's capacity to interact with Groucho, since Vnd’s interaction with this co-repressor is compromised when the Nk-2 box was deleted. The Nk-2 box deletion results in functional readouts including reduced ability to rescue the repression of ind expression in transgenic assays and reduced repression of reporter expression in a heterologous transfection assay (Uhler, 2007).

The Nk-2 box is not the first intramolecular domain identified that modulates Groucho binding to a transcription factor. It has been reported that deletion of the carboxyl terminal of Vnd, including both the homeodomain and the Nk-2 box, interferes with Groucho binding to this transcription factor. However, the domain that was deleted was relatively large, covering 200 amino acids. Another study found that the Eh1 domain of the Caenorhabditis elegans Unc-4 is insufficient for interaction with Unc-37, the worm Groucho. Mutant Unc-4 with the Eh1 domain intact, but lacking sequences amino terminal to the Eh1 domain, is deficient in in vivo repressor activity and fails to interact with Unc-37 in two hybrid interaction assays. The fact that a secondary domain, the Nk-2 box, is involved in modulating Groucho recruitment to the Eh1 domain is not altogether unexpected, because of the complexity of events that results from stable Groucho binding to Vnd-type transcription factors in the context of a target gene enhancer. These include the recruitment of repressosome components, the deacytlation of histones on target genes, chromatin condensation, and gene silencing (Uhler, 2007).

Vnd is one of a number of transcription factors that functions as a dual function regulator. Of the dual-function transcription factors characterized, the ability of the Rel-type transcription factor, Dorsal, to repress target gene expression is best understood. Dorsal activates genes in ventral regions and represses transcription of dorsal fate-determining genes, in the early Drosophila embryo. Groucho is dispensable for Dorsal-directed activation, but is essential for Dorsal-mediated ventral repression. Available evidence suggests that the context of a particular Dorsal-binding site determines whether Dorsal activates or represses target gene expression. Dorsal-dependent ventral silencers contain other elements that are required for Dorsal-dependent ventral repression in addition to Dorsal-binding sites. Mutagenesis of these additional elements [to which the transcription factors, Cut and Dead Ringer (Dri), bind], converts Dorsal from a repressor into an activator. In vitro-binding assays indicate that Dri and Dorsal work together to recruit Groucho to the template synergistically. While Dorsal-mediated repression requires Groucho, Dorsal-mediated activation depends on the co-activators, CBP (CREB-binding protein) and/or certain TAFs (TBP-associated factors) (Uhler, 2007 and references therein).

The conflicting abilities of Vnd to both activate and repress target gene expression are in part modulated by Vnd’s selective interaction with the co-activator, Dichaete, and the co-repressor, Groucho. Surprisingly, Vnd’s two activation domains map directly at the carboxyl side of the Eh1 domain and the Nk-2 box that mediate repression. The significance of the positioning of domains with opposite affects adjacent to each other is currently not well understood. Vnd’s secondary structure likely affects its interaction with Groucho, since the Eh1 domain and the Nk-2 box are at opposite ends of Vnd, separated by over 400 amino acids. Further analyses of Vnd mutant protein function in vivo and further characterization of vnd-dependent enhancers will help clarify which intramolecular interactions facilitate Vnd interacting with co-regulators that mediate opposite effects on target gene expression (Uhler, 2007).

The Him gene reveals a balance of inputs controlling muscle differentiation in Drosophila

Tissue development requires the controlled regulation of cell-differentiation programs. In muscle, the Mef2 transcription factor binds to and activates the expression of many genes and has a major positive role in the orchestration of differentiation. However, little is known about how Mef2 activity is regulated in vivo during development. This study characterized a gene, Holes in muscle (Him; Flybase name meso18E), which is part of this control in Drosophila. Him expression rapidly declines as embryonic muscle differentiates, and consistent with this, Him overexpression inhibits muscle differentiation. This inhibitory effect is suppressed by mef2, implicating Him in the mef2 pathway. Him downregulates the transcriptional activity of Mef2 in both cell culture and in vivo. Furthermore, Him protein binds Groucho, a conserved, transcriptional corepressor, through a WRPW motif and requires this motif and groucho function to inhibit both muscle differentiation and Mef2 activity during development. Together, these results identify a mechanism that can inhibit muscle differentiation in vivo. It is concluded that a balance of positive and negative inputs, including Mef2, Him, and Groucho, controls muscle differentiation during Drosophila development and suggest that one outcome is to hold developing muscle cells in a state with differentiation genes poised to be expressed (Liotta, 2007).

Analysis of mef2 function during Drosophila muscle development has shown that a major aspect of its role is in the differentiation pathway downstream of the genes that specify muscle. However, Mef2 protein expression precedes muscle differentiation. It is first expressed in the mesoderm at gastrulation, approximately 3 hr after egg laying (AEL). This is approximately 7 hr before the activation at stage 13 (10 hr AEL) of the expression of many known Mef2 target genes, e.g., Mhc, Mlc1, and wupA. This delay implies that the activity of Mef2 is restrained and that other regulatory proteins operate in the control of muscle differentiation during this period. However, little is known about these other proteins nor about how the gene expression at stage 13 is coordinated. This study addresses these unanswered questions through an analysis of the Him gene in muscle differentiation. Him was described in a computational screen, and it was isolated separately in an expression screen, but its function has not been analyzed. This study shows that Him is an inhibitor of Mef2 activity and muscle differentiation, and on the basis of this phenotype, it was called Holes in muscle (Him) (Liotta, 2007).

Him has a striking, transient pattern of expression during Drosophila embryogenesis. It is first expressed broadly in the mesoderm during stage 9. This expression then refines, and at stage 12 it is specifically expressed in the precursors of the somatic musculature and of the heart. Him expression then rapidly declines in the somatic mesoderm, such that in 90 min it has disappeared from the differentiating somatic muscle (stage 13). However, it persists in the adult muscle precursors (AMPs), which are set aside in the somatic mesoderm and which remain undifferentiated at this stage, and also in the developing heart. Him protein expression closely resembles that of Him RNA. The disappearance of Him coincides with the expression of Myosin, a classic marker of muscle differentiation. Double labeling with a Him-GFP fusion gene demonstrates that Myosin is expressed only after Him disappears from the developing muscle. The expression of Him in the progenitors of the somatic muscle and its disappearance from differentiating muscle are consistent with a role for Him as an inhibitor of muscle differentiation (Liotta, 2007).

To test whether Him is an inhibitor of muscle differentiation, it was overexpressed in the developing mesoderm by using the Gal4/UAS system. This induced a dramatic reduction in the number of Myosin-expressing cells and thereby produced large gaps or holes in the musculature. It was then asked when in muscle development Him has this effect. Up to stage 13 (10 hr AEL) muscle development proceeds similarly to that of the wild-type. At this stage, developing muscles are seen in the wild-type as small syncytia, which express founder cell markers, e.g., Kruppel, and Mef2, surrounded by Mef2-expressing myoblasts. When Him is overexpressed, the expression of these markers is similar. Subsequently, immunostaining for Mef2 reveals disrupted differentiation at stage 15, and there is increased cell death at stage 16. Together, these findings demonstrate that Him inhibits the differentiation phase, of muscle development, that occurs from stage 13 onward and that produces the morphologically distinct muscles of the functional musculature by the end of embryogenesis. To explore this function further, Him was knocked down by using RNAi from a splice-activated UAS hairpin vector. Although the musculature develops similarly to that of the wild-type, in the knockdown there is impaired muscle differentiation as revealed by disrupted muscle morphology (Liotta, 2007).

Overexpression of Him during muscle development phenocopies the mef2113 hypomorphic allele. Development of the musculature is inhibited similarly, and many of the residual muscles have a similar, abnormal morphology. This suggests that the two genes function in a common pathway. Consistent with this, Him and Mef2 are coexpressed in somatic muscle progenitors at stage 12, prior to the activation of muscle-differentiation markers such as Myosin. To test whether Him and mef2 genetically interact, both genes were overexpressed together. Strikingly, the inhibition of muscle differentiation caused by Him is rescued toward the wild-type by mef2. Furthermore, overexpression of Him alone induces lethality, and under the conditions of this experiment only 18% survive. This lethality is suppressed by mef2, and there are more than twice as many survivors. Together, the phenotypic analysis and genetic interaction findings indicate that Him functions in the Mef2 pathway that controls muscle differentiation (Liotta, 2007).

The Him protein sequence includes a putative bipartite nuclear localization signal (NLS). Consistent with this, colocalization with the transcription factor Twist in the AMP nuclei shows that Him is predominantly nuclear. The Him protein also has a WRPW motif at its C terminus. This tetrapeptide in this position is found in the Hairy group of transcriptional repressors and mediates their interaction with the corepressor Groucho (Gro). A pulldown assay was used to show that Him can also bind Gro. Moreover, this interaction requires the WRPW motif because Him with the WRPW motif deleted (HimΔWRPW) cannot bind Gro. To investigate the importance of the WRPW for Him function, HimΔWRPW was overexpressed in embryos and it was found that there was no dramatic loss of muscles, in contrast to the effect of full-length Him. Together, these results show that Him can bind Gro through its WRPW tetrapeptide and that this motif is required to inhibit muscle differentiation (Liotta, 2007).

The significance of the Him/Gro interaction in vivo during embryonic muscle development was investigated by overexpressing Him in a gro mutant background. Strikingly, the loss of gro function suppresses the inhibitory effect of Him, showing that Him requires gro to inhibit muscle differentiation. This result, together with the finding that mef2 can suppress the inhibitory effect of Him, indicates that Drosophila muscle differentiation in vivo is controlled by a balance between the activities of Him and Gro on the one hand and Mef2 on the other. The effect of overexpression of Him can be balanced by a reduction in Gro or by an increase in Mef2 (Liotta, 2007).

To further investigate the mechanism of action of Him, it was asked whether Him could inhibit Mef2 activity in cell culture in a direct Mef2-dependent gene-expression assay. When mef2 was transfected into S2 cells, it stimulated the expression of a Mef2-responsive luciferase reporter, and this effect was inhibited by cotransfection with Him. Then whether Him could also inhibit Mef2 activity was tested in the context of muscle development. The effect of Him overexpression on the expression of Mef2 and of β3-tubulin, which is a direct Mef2 target gene in somatic muscle, was analyzed. β3-tubulin expression is strongly reduced in the somatic mesoderm, whereas Mef2 protein expression is similar to that of the wild-type. This indicates that Him can downregulate Mef2 activity in vivo during embryonic development. It was further shown that Him with the Gro-interacting WRPW motif deleted does not affect β3-tubulin expression, nor does full-length Him in a groucho mutant background (Liotta, 2007).

Taken together, this combination of in vitro and in vivo assays reveals key features of Him's mechanism of action. They demonstrate that Him is found in the nucleus and requires its Gro-binding WRPW motif and gro function to inhibit both Mef2 activity and muscle differentiation during development. The previously characterized Drosophila proteins that have a C-terminal Gro-interacting WRPW motif are the Hairy group of HLH domain DNA-binding transcriptional repressors. However, Him is novel and does not have an HLH domain, suggesting that it does not bind DNA directly. Its mechanism of action may have parallels with Ripply1, which functions in vertebrate somitogenesis. Ripply1 also appears not to be an HLH protein and yet contains a functional Gro-interacting WRPW motif, although in this case near the N-terminus of the protein. Like Ripply1, Him may be part of a transcriptional-repressor protein complex. The precise mechanism by which Him targets Mef2 awaits analysis of this putative complex and the protein partners within it (Liotta, 2007).

Despite considerable progress, much remains to be learned about the regulation of muscle differentiation during animal development. Although studies in cell culture indicate that this control might include negative mechanisms, little is known about the identity and mode of action of specific molecules that inhibit muscle differentiation in vivo during development. This study identified and analyzed the targeting of Mef2 by Him. The inhibitory action of Him, coupled to its transient expression in developing muscle cells, is an explanation for the observation that Mef2 is present significantly before overt differentiation. It also offers an explanation for how a burst of expression of many Mef2 target genes at a specific phase (stage 13) of the differentiation program is coordinated. It is suggested that the rapid decrease in the expression of Him will lead to a concomitant increase in the activity of Mef2 and the ability to activate a cohort of these genes. Further studies will determine whether this will link reports that the ability of Mef2 to bind DNA is temporally regulated (Liotta, 2007).

The results also indicate that the inhibition of Mef2 activity by endogenous levels of Him is incomplete prior to stage 13. Thus, in normal muscle development, the Mef2 target gene β3-tubulin is expressed at stage 12, even though it was found that overexpression of Him can downregulate its expression then. This implies that in the wild-type embryo, there is some Mef2 activity at stage 12, and such activity is sufficient for β3-tubulin expression. This is consistent with other work that indicates that Mef2 regulates some gene expression at this stage and earlier and suggests that Him can provide one level of control of Mef2 activity during the muscle-differentiation program. Taken together, these results move the molecular analysis of muscle differentiation on from a simple model in which the key events are expression of pivotal positive regulators, for example Mef2. Rather they indicate that muscle differentiation in vivo is controlled by a balance of positive and negative regulators, including Him, Gro, and Mef2, that governs whether muscle precursors differentiate. In this model, one can think of Him and Gro as part of a mechanism holding the cells in a committed, but undifferentiated, state in which a cohort of muscle-differentiation genes is poised to be expressed. This might be a widespread strategy for coordinated gene expression in cell-differentiation programs. For example, it can be compared with melanocyte stem cell differentiation, where cells are primed to rapidly express terminal differentiation markers once Pax3/Groucho-mediated repression is relieved (Liotta, 2007).

Phosphorylation by the DHIPK2 protein kinase modulates the corepressor activity of Groucho

Groucho function is essential for Drosophila development, acting as a corepressor for specific transcription factors that are downstream targets of various signaling pathways. Evidence is provided that Groucho is phosphorylated by the DHIPK2 protein kinase. Phosphorylation modulates Groucho corepressor activity by attenuating its protein-protein interaction with a DNA-bound transcription factor. During eye development, DHIPK2 modifies Groucho activity, and eye phenotypes generated by overexpression of Groucho differ depending on its phosphorylation state. Moreover, analysis of nuclear extracts fractionated by column chromatography further shows that phospho-Groucho associates poorly with the corepressor complex, whereas the unphosphorylated form binds tightly. It is proposed that Groucho phosphorylation by DHIPK2 and its subsequent dissociation from the corepressor complex play a key role in relieving the transcriptional repression of target genes regulated by Groucho, thereby controlling cell fate determination during development (Choi, 2005. Full text of article).

A Myc-Groucho complex integrates EGF and Notch signaling to regulate neural development

Integration of patterning cues via transcriptional networks to coordinate gene expression is critical during morphogenesis and misregulated in cancer. Using DNA adenine methyltransferase (Dam)ID chromatin profiling, protein-protein interaction between the Drosophila Myc oncogene and the Groucho corepressor was identified that regulates a subset of direct dMyc targets. Most of these shared targets affect fate or mitosis particularly during neurogenesis, suggesting the dMyc-Groucho complex may coordinate fate acquisition with mitotic capacity during development. An antagonistic relationship was found between dMyc and Groucho that mimics the antagonistic interactions found for EGF and Notch signaling: dMyc is required to specify neuronal fate and enhance neuroblast mitosis, whereas Groucho is required to maintain epithelial fate and inhibit mitosis. The results suggest that the dMyc-Groucho complex defines a previously undescribed mechanism of Myc function and may serve as the transcriptional unit that integrates EGF and Notch inputs to regulate early neuronal development (Orian, 2007).

Gro is a downstream transducer of several signaling pathways and was placed at the crossroads of the Notch and EGF signaling pathways during patterning of the Drosophila nervous system, where EGF-induced site-specific phosphorylation of Gro attenuates it repression activity. During embryonic stage 9, the CNS matures in three bilaterally symmetrical longitudinal rows of neuroblasts, with the homeobox transcription factors, Vnd, Ind, and Msh, specifying the medial (ventral), intermediate, and lateral rows, respectively. EGF regulates the expression of both Vnd and Ind and is thus required for the formation of the ventral and intermediate rows. Interestingly, both Vnd and Ind are among the 38 dMyc-Gro shared targets identified in this study. Gro and dMyc, but not dMnt, are expressed in neuroblasts of stage 9 embryos. Because dMyc-Gro targets are associated with both neuroblast fate and mitosis, it is hypothesized that EGF and Notch coregulate cell fate and mitosis within the developing neuroectoderm via dMyc-Gro antagonism. Vnd expression (a shared Myc-Gro target whose expression overlaps with and is required for establishment of S1 neuroblasts), the overall number of neuroblasts, and mitotic activity in wild-type embryos were compared to groe47 loss-of-function (LOF) mutants (in which the maternal contribution of Gro is removed), Egfr2, or Notch55e11 [note that dMyc LOF embryos cannot be generated]. These parameters were also evaluated in embryos overexpressing either dMyc or Gro using the conditional Gal4/upstream activating sequence (UAS) expression system. Vnd expression is stronger and expanded in both Notch and gro LOF embryos, as well as in embryos overexpressing dMyc when compared with wild type. These mutants also show neuroblast hyperplasia and elevated mitotic activity. Furthermore, Egfr LOF or Gro-overexpressing embryos show reduced Vnd expression, neuronal hypoplasia, and reduced mitotic activity, consistent with the molecular nature of the dMyc-Gro common targets (Orian, 2007).

Myc proteins are required for both cell growth/size and cell proliferation. The model in which Myc functions are mediated by heterodimerization with Max and antagonized by Mxd (Mad/Mnt) proteins has been well established. However, recent studies suggest that a set of interactions outside the canonical Myc/Max/Mxd network also regulate some of Myc's functions. Interestingly, the current studies point to a subset of dMyc direct targets that are not shared with either dMax or dMnt. Furthermore, dMnt-Dam and dMax-Dam were not recruited to these dMyc targets even in experiments where the Dam fusions were coexpressed in the presence of high levels of dMax or dMyc, respectively, suggesting that previously uncharacterized mechanisms may mediate Myc's recruitment to DNA, and proteins other than dMnt may antagonize its transcriptional activity on this set of targets. This study reports the identification of Gro as the first component in a pathway that antagonizes dMyc function independent of dMnt and operates during Drosophila neurogenesis (Orian, 2007).

Transcriptionally, dMyc was found to be positively required for the expression of dMyc-Gro targets, activity that is antagonized by Gro. Importantly, dMyc is not a Gro target, and reducing Gro levels does not affect dMyc protein levels. Furthermore, Gro antagonism is limited only to the dMyc-Gro subset of shared targets and does not involve dMnt: there is no overlap between genes bound by dMnt or Gro, dMnt is not expressed in cells where the dMyc-Gro interaction is observed, RNAi to dMnt does not affect Myc-Gro shared target expression, and overexpression of dMnt does affect PNS development (Orian, 2007).

Although the possibility that dMyc-Gro targets are coregulated by individual dMyc and Gro complexes cannot be excluded, the results suggest that dMyc and Gro are part of a single larger protein complex. First, the observation that RNAi to dMyc results in reduction of target expression and is restored by coreducing Gro suggests that other activators coregulate shared target expression along with dMyc. Second, biochemical purification, binding data, and DNA adenine methyltransferase (Dam)ID Southern analyses support the idea that both proteins physically interact with one another yet associate with DNA through distinct binding sites. Third, Gro does not bind directly to DNA but must be recruited to targets by sequence-specific DNA-binding transcription factors. Fourth, most of the dMyc-Gro targets lack E-box sequences associated with canonical Myc network targets, suggesting that dMyc and Gro may be recruited to shared targets via a novel mechanism or by other protein(s) yet to be identified. Candidates for recruiting Gro may be the E(spl) proteins that convey the Notch signal, antagonize the EGF pathway, interact with Gro, and exhibit similar phenotypes. Thus, the identification of the entire dMyc-Gro complex and its regulation will be an important next step (Orian, 2007).

Gro's role as a downstream transducer of Notch signaling during neurogenesis is well documented, and mounting evidence supports Myc as a key player in progenitor cell proliferation. This study has identified a previously undescribed role for dMyc, together with Gro, during Drosophila early neuronal development. dMyc and Gro are required to directly regulate key fate controlling genes such as the homeodomain proteins vnd and ind that are downstream targets of EGF signaling. Because Vnd was identified as a regulator of the proneural gene complex, the differential regulation of vnd by dMyc and Gro implicates them as antagonistic regulators upstream of proneural genes. Thus, it is proposed that dMyc is transiently required within the neuroectoderm, where it promotes specific fate acquisition and allows mitotic expansion of committed neuronal cells (Orian, 2007).

Phenotypically, it was observed that, similar to EGF, dMyc promotes neurogenesis both in the PNS and CNS, whereas Gro and Notch inhibit neuroblast formation and mitosis. This is a different role than that previously ascribed to dMyc, because it is usually associated with regulation of cell size and organismal growth, functions that are antagonized by dMnt. Consistent with this, a recent study identified EGF-induced phosphorylation of c-Myc, Max, and TLE proteins in mammalian cells. The antagonistic relationship of Myc/EGF to Gro/Notch is likely to be highly dependent on the developmental context and the specific progenitor niche. For example, in cellular contexts in which Notch promotes proliferation, such as during the development of T cells in acute leukemia, Myc is a direct target of mutated Notch1 and is required for T cell proliferation and development. The current findings also fit well with observations that N-Myc is required during mouse progenitor development, and that the fly tumor suppressor Brat regulates dMyc levels posttranscriptionally in larval neuroblasts resulting in a 'tumorous' phenotype (Orian, 2007).

Taken together, the snapshot provided by DamID data leads to the suggestion of a model in which changes in neuronal progenitor fate and mitosis are determined by the balance between EGF and Notch signaling that is likely transcriptionally mediated by the dMyc-Gro complex. During epithelial development, Notch, like Gro, is required to specify and maintain epithelial fate. It is proposed that Gro sequesters dMyc in an inactive multiprotein complex formed by associating with dMyc, preventing the activation of dMyc-Gro shared targets. Upon EGF signaling, a molecular switch takes place whereby Gro is phosphorylated, and its repression is attenuated. dMyc, as part of an as-yet-to-be-identified activation complex, is then liberated to activate zygotic transcription of a subset of targets that determines neuronal fate and enhances mitosis. One of these targets is dMax, which is specifically expressed in the neuroectoderm. Activation of dMax would be expected to establish a feed-forward loop required for the subsequent activation of (E box-containing) Myc targets to promote cell growth. As development progresses, the dMnt gene would be induced, and dMnt-dMax complexes would replace dMyc-Max complexes, thereby promoting cellular differentiation (Orian, 2007).

Finally, both EGF/dMyc and Notch/Gro misregulation and mutation are intimately involved in hematological, epithelial, and neuroectodermal cancers. Thus, identification of a dMyc-Gro complex that could serve as a molecular junction to integrate EGF and Notch signaling inputs is highly relevant for both developmental biology and cancer (Orian, 2007).

Spreading of a corepressor linked to action of long-range repressor hairy

Transcriptional repressor proteins play key roles in the control of gene expression in development. For the Drosophila embryo, the following two functional classes of repressors have been described: short-range repressors such as Knirps that locally inhibit the activity of enhancers and long-range repressors such as Hairy that can dominantly inhibit distal elements. Several long-range repressors interact with Groucho, a conserved corepressor that is homologous to mammalian TLE proteins. Groucho interacts with histone deacetylases and histone proteins, suggesting that it may effect repression by means of chromatin modification; however, it is not known how long-range effects are mediated. Using embryo chromatin immunoprecipitation, a Hairy-repressible gene in the embryo was analyzed during activation and repression. When inactivated, repressors, activators, and coactivators cooccupy the promoter, suggesting that repression is not accomplished by the displacement of activators or coactivators. Strikingly, the Groucho corepressor is found to be recruited to the transcribed region of the gene, contacting a region of several kilobases, concomitant with a loss of histone H3 and H4 acetylation. Groucho has been shown to form higher-order complexes in vitro; thus, the observations suggest that long-range effects may be mediated by a 'spreading' mechanism, modifying chromatin over extensive regions to inhibit transcription (Martinez, 2008).

In support of the spreading model, Groucho binds hypoacetylated histone H3 and H4 tails, and mutations in its N-terminal oligomerization domain block its repression activity in vivo. The recruitment of histone deacetylases may thus enhance Groucho binding to adjacent histones in a positive-feedback loop. A similar mechanism has been suggested for Tup1, the Groucho homolog in yeast. As with Groucho, histone deacetylases have been shown to be crucial for Tup1 repression. Moreover, Tup1 also has an affinity for hypoacetylated amino-terminal histone tails, and mutations or deletions of the tails cause the derepression of Tup1 targets. As with the Hairy repressor here, Tup1 does not change the methylation status of target genes, and the deletion of histone methyltransferases does not affect Tup1-mediated repression. This suggests that methylation marks may not need to be reversed to achieve repression but they may facilitate ready reactivation seen upon the depletion of Hairy. Regarding the extent of association of Tup1 with target genes in yeast, chromatin immunoprecipitation studies have yielded conflicting pictures. Tup1 has been reported to interact with the a-cell-specific STE6 gene only at the promoter or over a distance from 1 to 3.5 kbp, encompassing the entire gene. This discrepancy may be due to differences in cross-linking or immunoprecipitation conditions, reflecting the difficulty in analyzing indirectly bound factors. Indeed, in the current study, promoter interactions by Groucho were observed only with the use of a double-cross-linking protocol (Martinez, 2008).

Models of transcriptional repression include direct competition, local 'quenching' (displacement or interference with activators), and interactions with the basal machinery. The finding that activators and coactivators are still present under conditions in which the gene is repressed by Hairy suggests that the third model might apply here. The Gal4 activator might represent a particularly stably bound protein, as it does not show the high rate of exchange noted for other transcriptional activators. Thus, it is possible that Hairy-mediated repression does interfere with the binding of some activators on endogenous loci. However, repression can be quite effective even in the absence of activator displacement, perhaps by targeting the basal machinery, similarly to Tup1-mediator interactions seen in yeast. The promoter-proximal location of the repressor in the current system might bias the system to such interactions, but this arrangement is physiologically relevant, since Hairy is found in such proximal locations on endogenous genes. In addition, the LexA-Hairy repressor is also active when bound at -2 kbp, indicating that promoter proximity is not required for activity. Interestingly, a recent chromatin immunoprecipitation survey of enhancers targeted by the Snail short-range repressor suggests that this repressor can be bound to inactive enhancers simultaneously with activators, raising the possibility that short-range repression might also involve direct interactions with the basal machinery (Martinez, 2008).

The extensive contacts of Groucho over the repressed locus are strongly reminiscent of the extended nucleoprotein structures deposited on regions repressed by stable, heritably acting systems such as Polycomb group (PcG) proteins in animals and Sir proteins in silent-mating-type loci and subtelomeric regions of yeast. There, chromatin regions are modified and inhibited for the formation of productive transcription complexes. Indeed, the association of activators and components of the transcriptional machinery with repressed loci in these systems mirrors the continued binding of activators and coactivators in the system that was studied here, suggesting that the limiting factor for transcription occurs at a later stage. What sort of inhibitory interaction might be involved in this case? A number of recent reports have raised the possibility that repressed, or nonactivated, promoters feature RNA polymerase II that is blocked for elongation, similar to the paused polymerase found at the hsp70 locus under noninducing conditions. It has been found that RNA polymerase II is not displaced from the slp1 gene upon repression with Runt, a Groucho-binding protein (Wang, 2007). It is possible that Groucho itself, through contacts with histone proteins and/or the recruitment of deacetylases such as Rpd3, establishes a chromatin environment that is inhibitory for transcriptional elongation. However, no reliable signals were obtained for RNA polymerase II at this promoter, precluding a definitive statement about polymerase occupancy in activated and repressed states (Martinez, 2008).

A difference between the repression complexes assembled by the Hairy repression domain and by these other proteins is the transience of the effect; while PcG regulation is linked to epigenetic modifications that allow repression to persist for an extended time when PcG proteins are depleted, the regulation that is seen in this study is readily reversed upon the loss of the LexA-Hairy repressor. Similar effects are observed with elements regulated by the endogenous Hairy protein; enhancers bearing Dorsal and Twist activator sites that are repressed by Hairy in the blastoderm embryo are reactivated minutes later in the germ band extended stage. Thus, Hairy appears to be designed for highly effective but readily reversible repression, which may be useful in particular developmental settings (Martinez, 2008).

In contrast to a model of linear spreading, an alternative picture of Groucho interaction that is consistent with the current observations is that the corepressor may be tethered to the promoter region, forming larger multimeric complexes around which proximal and distal portions of the gene are wrapped (the 'turban' model). Groucho would indirectly contact the promoter region via Hairy binding and make direct histone contacts with more-distal regions. The selective cross-linking of Groucho to promoter regions treated only with the additional DSP cross-linker is consistent with such a picture. This model may also explain why downstream interactions, albeit weak ones, are often detected of the LexA-Hairy repressor, particularly when employing the more extensive double-cross-linking protocol. In either case, Groucho itself may be important for interfering with activities of transcription factors or the transcription of distant loci. Both of these models suggest that the extensive spread or extensive contacts of Groucho is mechanistically linked to transcriptional repression; however, it is possible that Groucho's extensive contacts with downstream regions are not the main effector of Hairy-mediated repression. Promoter-proximal activities of Groucho, or of other Hairy corepressor proteins, may play the decisive role in dictating long-range effects. Extensive experimental evidence indicates that Groucho plays a key role in the repression mediated by Hairy; therefore, it seems parsimonious to assume that Groucho activity on the repressed gene is important for repression. In support of a 'turban' model of repression, a recent study of the human Groucho homolog Grg3 showed that the recruitment of Grg3 to chromatin induces the formation of a highly condensed structure in vitro (Sekiya, 2007). In addition, in vivo recruitment of Grg3 by FoxA resulted in Grg3 being detected by chromatin immunoprecipitation analysis up to distances of 1 kbp from the FoxA binding site (Martinez, 2008).

The current study demonstrates that repression by Hairy is associated with histone deacetylation, which is certainly consistent with the nature of cofactors associating with this protein. Interestingly, this modification appears to be restricted to regions close to the repressor binding sites, which places them close to the transcriptional initiation site. How might this be related to the long-range effects mediated by Hairy? One possibility is that Hairy, regardless of where it is bound, induces characteristic changes on chromatin close to the transcriptional start site, that would induce a dominant (and hence long-range) effect on target genes. Alternatively, the local chromatin deacetylation may reflect the reversal of promoter-localized histone acetylases (e.g., SAGA), and acetylation levels on other portions of the gene are already too low to show robust deacetylation. A third possibility is that other Hairy-induced chromatin modifications, not assayed in this study, are more extensive than the deacetylation (Martinez, 2008).

This study strongly supports a model for Hairy repression that involves contacts between the Groucho corepressor and extended regions of the silenced gene, providing a basis for the long-range repression observed for this protein that is independent of activator displacement. Interesting questions for future studies are how Groucho spreading is limited and whether specific chromatin signals modulate this activity. In addition, such extended repression complexes might be specific to subsets of Hairy targets. A recent study identified a mutation in gro that blocks multimerization but not repression of some genes, suggesting that this cofactor is likely to employ distinct activities at different genes (Jennings, 2008). In addition, genomic surveys indicate that Hairy is likely to associate with distinct cofactors at different loci. Future work will focus on identifying the roles of individual cofactors of this repressor at genes that represent the diversity of Hairy targets in Drosophila (Martinez, 2008).

Hipk is an essential protein that promotes Notch signal transduction in the Drosophila eye by inhibition of the global co-repressor Groucho

Homeodomain interacting protein kinase (Hipk) is a member of a novel family of serine/threonine kinases. Extensive biochemical studies of vertebrate homologs, particularly Hipk2, have identified a growing list of interactors, including proteins involved in transcriptional regulation, chromatin remodeling and essential signaling pathways such as Wnt and TGFβ. To gain insight into the in vivo functions of the single Drosophila Hipk loss of function alleles were characterized, that revealed an essential requirement for hipk. In the developing eye, hipk promotes the Notch pathway. Notch signaling acts at multiple points in eye development to promote growth, proliferation and patterning. Hipk stimulates the early function of Notch in promotion of global growth of the eye disc. It has been shown in the Drosophila eye that Hipk interferes with the repressive activity of the global co-repressor, Groucho (Gro). This paper proposes that Hipk antagonizes Gro to promote the transmission of the Notch signal, indicating that Hipk plays numerous roles in regulating gene expression through interference with the formation of Gro-containing co-repressor complexes (Lee, 2009).

To study hipk function in vivo, deletions were generated at the hipk locus through imprecise excision of a transposable element. Two excisions (hipk2 and hipk3) result in homozygous pupal lethality (with rare escaper adults) and trans-heterozygosity for any of these alleles and a deficiency removing hipk (Df(3L)ED4177) leads to lethality. A fourth allele, hipk4, was generated through targeted deletion of the DNA between two transposable elements flanking the locus and this allele causes lethality prior to the 3rd larval instar. Interallelic crosses reveal an allelic series in the order of weakest to strongest: hipk2 < hipk1 < hipk3 < hipk4. These findings demonstrate that the single hipk gene in Drosophila is essential. Indeed, the loss of both maternal and zygotic hipk results in embryonic lethality (Lee, 2009).

hipk mutants consistently displayed small, rough eyes. Dissection of pharate adults from pupal cases revealed that 42% of hipk3 homozygotes displayed a preferential loss in the ventral region, leading to a small round eye. Additional eye phenotypes include the appearance of non-retinal tissue in 25% of hipk3 homozygotes (Lee, 2009).

Staining of neuronal cells in 3rd instar eye imaginal discs with the neural anti-Elav antibody revealed that 25% of hipk3 homozygotes display a loss of photoreceptors. This loss was most prominent in the lateral poles of the eye disc as the Elav-positive cells did not extend to the dorsal and ventral margins of the eye disc as is seen in wildtype. The loss of photoreceptors likely correlates with the loss of eye structure in adults. Further reduction of Hipk activity by generating loss of function somatic clones with a stronger allele, hipk4, also led to a decrease of Elav staining. Under such conditions, neural differentiation is most sensitive to the loss of hipk near the MF. hipk4 clones proximal to the MF displayed diminished Elav staining. This effect is not restricted to the lateral poles, as was observed in hipk3 homozygotes. While photoreceptors in clones located posterior to the MF appeared to differentiate correctly, the spacing of these cells was reduced and irregular, suggesting hipk is also required for patterning of cells posterior to the MF. It was found that the loss of photoreceptors is likely not attributed to a defect in eye specification, since Ey expression is not diminished in hipk4 somatic clones (Lee, 2009).

It was next determined if the loss of photoreceptors observed in hipk clones could be a secondary effect of altered cell cycle regulation or cell death during retinal specification. No apparent changes were observed in discs stained to visualize cell proliferation, using anti-phospho histone 3 antibody, or levels of apoptosis, as visualized by staining for the activated Drosophila ICE caspase (drICE). Hence it appears that loss of photoreceptors in hipk mutant cells may be linked to a modification in early eye development, rather than altered cell death (Lee, 2009).

Consistent with loss of function analyses that implicate a role for hipk in eye patterning, it was found that ectopic expression of UAS-hipk also affected eye development. Using ey-Gal4 to drive wild type Hipk expression throughout larval eye development caused abnormal rough eyes, of which 33% also displayed cuticle-like structures. In these flies, a novel role was observed for hipk as a regulator of organ size. 39% of ey > hipk flies showed overgrown eyes that are likely caused during larval development, since overgrowths were also observed in imaginal discs. Thus Hipk plays a role in the patterning of the eye, although the underlying mechanism is still unknown (Lee, 2009).

During eye development, hipk is expressed in a dynamic pattern throughout the eye disc. Antisense RNA in situ hybridization revealed that in the late second larval instar (L2) hipk is enriched in the medial domain of the visual primordium including the D/V boundary of the eye disc and this localization persists into early third instar. Beginning in mid 3rd instar larval stage, hipk expression is enriched in the anterior folds of the eye discs and becomes broadly expressed in the anterior region of the eye disc ahead of the MF. Later in late third instar, the localization varies between discs and likely reflects very dynamic changes in expression. In these disc, hipk is further refined to a narrow stripe covering much of the width of the disc. Using a combination of fluorescent in situ hybridization (FISH) and antibody staining, co-localization was observed of hipk and the retinal determination factor Dachshund (Dac) at the anterior-most edge of the Dac expression domain. This edge of Dac expression delimits the anterior boundary of the cells entering the neural program. This dynamic pattern of expression shows that hipk is expressed at the D/V organizing center early and later in undifferentiated cells anterior to the MF (Lee, 2009).

N signaling controls many aspects of eye development such as proliferation and the establishment of the eye field. Loss of N signaling causes a small eye phenotype and gain-of-function mutants leads to an overproliferation of the eye. In addition, the dorsal and ventral eye regions are asymmetrically regulated, as the loss of the Ser regulator Lobe results in preferential loss of the ventral eye domain. The loss of eye tissue in hipk homozygous mutants and the overgrowth defects in ey > hipk resemble those observed with modulated activity of N and suggest a potential role for Hipk as a mediator of N-regulated growth processes (Lee, 2009).

Genetic interaction studies were undertaken to investigate the interaction between the N pathway and hipk. Heterozygosity for the N ligand Dl enhances the small eye phenotype of hipk3 mutants. 30% of these small eyes are half the normal size and more dramatically, 20% were a quarter of the normal eye size. In contrast, in hipk3 homozygotes, only 4% of eyes were reduced to half the size and 2% were a quarter of the normal size, respectively. These phenotypes were much more severe than those observed with the hipk3 homozygous mutation alone and suggested a potential synergy between Dl and hipk. This interaction was also observed with the hipk2 allele. Similarly, the overproliferation defect observed in ey > Dl was enhanced by the co-expression of Hipk (Lee, 2009).

Most strikingly, expression of dominant negative N with ey-Gal4 led to a dramatic loss of the eye which was suppressed by co-expression of Hipk. Such a rescue of reduced N signaling strongly suggests Hipk acts to promote N signaling downstream of the receptor. Further support for this model is seen upon examining imaginal disc phenotypes. Overexpression of the constitutively active NICD with ey-Gal4 leads to severely abnormal eye discs with dramatic overgrowths and reduced number of photoreceptors as a result of increased lateral inhibition. Decreasing hipk in these discs restored the population of photoreceptors. These findings suggest hipk likely regulates a subset of N-mediated processes (Lee, 2009).

These analyses suggested that hipk cooperates with the N pathway. To assess whether Hipk is required to promote the transduction of this cascade, N activity was measured in hipk mutant cells by examining the expression of the products of the E(spl) complex, direct targets of Su(H). Using an antibody that recognizes 4 of 7 products of the E(spl) complex, a decrease was observed in E(spl) expression in mutant cells, most evident in cells located near the furrow. Therefore, hipk is required for the efficient transduction of the N signal and hipk mutant cells have reduced N signaling activity (Lee, 2009).

Intriguingly, clones located in the posterior of the eye disc display slightly elevated expression of E(spl), suggesting additional mechanisms through which hipk patterns the eye. These findings, and the complexity of the hipk phenotype, demonstrate that hipk plays multiple roles during eye development in addition to its role as a positive regulator of the N signal (Lee, 2009).

Hairless (H) is an antagonist of N that functions as an adaptor to bridge Gro and Su(H) to form a repressor complex. This mechanism is utilized to inhibit N signaling in multiple developmental processes. It was shown that Hipk phosphorylation could antagonize Gro function by promoting the disassembly of the repressor complex, so it was investigated whether this may be the route through which Hipk promotes N activity. It was hypothesized that Hipk may serve as a general antagonist of Gro and consequently promote Su(H)-mediated transcription by inhibiting the interactions between the repressor complex and Su(H). If such a mechanism exists, expression of a phospho-mimetic form of Gro, in which Hipk phosphorylation sites are mutated to glutamic acid residues, should exert effects reminiscent of those observed by expressing wildtype Hipk (Lee, 2009).

To test this model, biochemical studies were performed to characterize the interaction between Hipk and Gro. Kinase assays were performed using purified Gro (full length and derivatives) from bacterial lysates in the presence of GST-Hipk. Hipk specifically phosphorylated the SP domain of Gro. Further analyses using synthetic Gro decapeptides identified two Hipk target residues, namely amino acids S297 and T300. These sites were mutated to alanine (GroAA) to test Hipk's specificity in a kinase assay. While full length Gro was phosphorylated by Hipk, the GroAA variant was resistant to phosphorylation, confirming S297 and T300 as Hipk target sites. These residues are also conserved in human Hipk2 (Lee, 2009).

To generate a phospho-mimetic variant, these target residues were mutated to glutamic acid (GroEE). If Hipk can indeed repress Gro activity, then this form of Gro should be constitutively inhibited, while the GroAA variant should display constitutive activity. To test the properties of these Gro variants in vivo, transgenic fly strains expressing groAA and groEE, under control of the UAS promoter were generated. Expression of GroAA with ey-Gal4 produced a similar loss of eye phenotype to that seen in ey > groWT flies. Such phenotypic similarities suggested that GroAA is functionally equivalent to wild type Gro. ey > groEE flies displayed a much less severe phenotype upon misexpression than groWT, suggesting the activity of GroEE is compromised (Lee, 2009).

Misexpression of Hipk can suppress the loss of eye phenotype caused by ey > groWT. Whether this rescue occurs by inhibiting Gro's repressive activity was examined. In contrast to the suppression of groWT, phenotypes induced by both groAA and groEE were less sensitive to elevated levels of Hipk. Co-expression of groAA or groEE with hipk showed a phenotype most similar to the Gro derivatives alone, indicating that these forms are not sensitive to the regulation by Hipk compared with the sensitivity seen with GroWT (Lee, 2009).

To investigate whether Hipk can promote N activity via its regulation of Gro, a series of genetic interaction assays were carried out involving groWT, groEE and groAA in conjunction with the N antagonist H. Both inhibition of N or ectopic groWT led to loss of eye structures. Co-expression of Hipk can rescue the effect of dominant negative N. The model predicts that if the rescue of N signaling by Hipk is mediated through direct inhibition of Gro through phosphorylation, then a similar rescue should be observed with the phospho-mimetic form GroEE. However, it is expected that the Hipk-resistant form GroAA will phenocopy the effects of GroWT. Decreasing Notch activity via expression of the antagonist H caused a complete loss of eye, similar to that caused by expression of NDN. Expression of hipk at this temperature only induced a mild rough eye. Co-expression of hipk and H partially restored the development of retinal tissue as observed by the presence of a small eye. Furthermore, morphological defects in the head, including ocellar defects in the dorsal head, were also rescued, suggesting Hipk may also regulate other N-dependent processes (Lee, 2009).

Next it was determined whether the mutated Gro forms could suppress the ey > H phenotype by mimicking the regulation seen with co-expression of Hipk. Consistent with the prediction, misexpressing groEE with ey-Gal4 was capable of restoring eye structures in the ey > H background, phenocopying the rescue by hipk. Conversely, co-expression of groAA led to a dramatic enhancement of the ey > H phenotype. In these flies, the eye fails to develop and head defects are magnified as indicated by the presence of only the dorsal vertex of the head in 41%, or more severely, the entire head is lost in 29% of the flies. As expected, similar interactions were also observed with groWT. Taken together, these results support a model in which Hipk phosphorylates and inhibits Gro to facilitate the N pathway (Lee, 2009).

To further confirm that these effects on transduction of the Notch signal were indeed a consequence of decreasing Gro's repressive activity, it was asked if the addition of Hipk could modify the interactions between the Gro derivatives and H. It was predicted that introducing Hipk would modify the effects of GroWT on H, but not those induced by the Gro derivatives, since these mutations would render Gro less sensitive to elevated levels of Hipk. The results suggest that these mutations bypass the regulation of Gro by Hipk, since it was observed that co-expression of hipk, H and groWT led to a slight rescue of the very abnormal head structures seen with ey > H, GroWT, and suppressed the enhancement of the ey > H phenotype incurred by introducing gro. This indicates that Hipk can inhibit Gro activity, thereby quenching its antagonistic effect on the N signal. However, co-expression of H and hipk in the presence of groEE or groAA did not detectably modify the phenotype seen with the combination of H and the Gro derivatives alone. These observations support the model that Hipk contributes to the propagation of the N signal by inhibiting Gro's repressive activity through phosphorylation of S297 and T300 (Lee, 2009).

These data support a model in which Hipk can promote N signaling during eye development through its repression of Gro. A previous report demonstrated that Hipk could promote the in vitro transcriptional activation activity of Eyeless by inhibiting Gro. In vivo data showed that Hipk could modify Gro activity and the resulting eye phenotypes were attributed to changes in Eyeless activity. Promotion of Ey activity would reflect a role solely in eye specification. The phenotypic consequences of modifying Hipk activity and hipk's dynamic expression profile both clearly suggest additional requirements for Hipk other than eye specification. To further clarify the mechanism underlying the genetic observations, it was examined whether the same phenotypic rescue of ey > H seen with Hipk could be seen with the misexpression of Ey. If the only function of Hipk were to promote Ey activity, then a similar rescue would be expected with elevated levels of Ey. However, it was found that ectopic ey failed to rescue the ey > H phenotype, and moreover, greatly enhanced it. Furthermore, it was also found that concomitant misexpression of hipk mildly modified ey > ey phenotype, rather than a potent synergistic modification. These genetic interactions suggest that the ability of Hipk to rescue diminished N signaling activity is independent of the ascribed role in promoting Ey activity. These findings are consistent with the genetic interaction studies and analyses of N target genes that indicate that Hipk acts to promote N signaling (Lee, 2009).

N is a recurring player in eye development, a feat that is accomplished through its unique interplay with members of the Pax6 family of transcriptional regulators. For example, N-controlled eye growth is specifically mediated via Eyg. Reducing N signaling activity induces an eye-loss phenotype, which is caused by a deregulation of organ growth through Eyg, rather than a reliance on the eye specification players Ey or Toy. Overexpression of Eyg but not Ey nor Toy reliably restored eye development in N deficient flies (Lee, 2009).

Several lines of evidence strongly suggested that Hipk promotes N-mediated eye growth. First, ey > hipk phenocopies the overgrowths seen with elevated levels of the N signal. Second, as was seen with Eyg, simultaneous misexpression of Hipk rescues the loss of eye phenotype in a dominant negative N background. Furthermore, hipk is expressed in a region encompassing the D/V organizing growth center where N acts to specifically control the global growth of the eye. The genetic examination were extended to confirm the model by further characterizing the interaction between Hipk and H. Specifically, it was addressed whether the ey > H phenotype was correlated with a defect in organ growth, rather than eye specification. Consistent with such a model, overexpression of the growth regulator eyg, but not ey, restored the eye in ey > H adults. These suppressive effects are identical to those observed with both hipk and groEE transgenes (Lee, 2009).

Similar to what is seen with components of the N pathway, Hipk induces pleiotropic effects throughout eye development. Attempts were made to confirm that the genetic rescues were not attributed to a secondary effect of Hipk-mediated processes unrelated to growth. To address this, the consequences were examined of modifying Hipk levels on the phenotype induced upon misexpression of Fringe (Fng). The small eye phenotype seen in ey > Fng flies is attributed solely to a defect in eye growth. Thus any observed modification in this sensitized genetic background would validate a requirement for Hipk in eye growth. As predicted by the model, overexpression of hipk or groEE partially rescues the ey > fng phenotype. More strikingly, the eye fails to form when hipk activity is reduced, a phenotype similar to what is seen with eyg or Dl mutants in an ey > Fng background. Taken together, these genetic interactions demonstrate that Hipk promotes N-mediated eye growth (Lee, 2009). The complementary expression domains of the N ligands Dl and Ser in the dorsal and ventral compartments, respectively, ensures that the N pathway is activated at the D/V boundary of the developing 2nd instar eye disc. N establishes the organizing center to mediate global growth by regulating eyg expression along the length of the D/V boundary. The expression of Ser and Dl appear normal in hipk4 clones suggesting that the D/V center is established normally in hipk mutant cells. However, it was observed that Eyg expression is autonomously reduced in hipk mutant clones. Such an effect on eyg expression is also observed in clones mutant for either Su(H) or Dl and Ser. Conversely, ey > hipk third instar eye discs display an expanded expression domain of Eyg. These observations indicate that Hipk is required for activation of normal Eyg expression, and loss of hipk induces a growth defect (Lee, 2009).

In conclusion, the lethality of hipk mutant alleles demonstrates that Hipk is an essential kinase in Drosophila that plays a critical role during eye development. Eye patterning and growth defects are observed in both hipk homozygous mutants and somatic clones. hipk mutant clones display reduced N signaling activity, as measured by the diminished expression of the N targets, E(spl) and Eyg. Therefore, these studies implicate Hipk in the positive regulation of the N signaling cascade during eye development. Although these data demonstrate that Hipk regulates N-mediated eye growth, the neural patterning defects are less severe than previously published N mutant phenotypes. The possibility cannot be excluded that these neuronal defects are due to a secondary consequence of Hipk's requirement in earlier phases of eye development or a role for Hipk in modulating additional eye patterning pathways other than the N pathway (Lee, 2009).

In vitro and in vivo data support a model in which Hipk phosphorylates Gro, and consequently relieves its repressive activity on the Su(H) transcriptional complex. Overexpression of the N antagonist H results in loss of eye. Hipk-mediated phosphorylation of Gro at S297 and T300 is necessary to rescue the phenotype caused by reduced N signaling. Indeed, genetic misexpression analyses clearly demonstrate that this phosphorylation event is necessary to relieve Gro's inhibitory effect on N, thereby permitting activation of downstream N targets. Since Gro is a global co-repressor, this interaction may represent a global mechanism through which Hipk can regulate gene expression during development (Lee, 2009).

This study has identified Hipk as a key player in modulating growth in the eye. Given that a similar role for Hipk in promoting growth was observed in additional tissues, this likely represents a general role for Hipk in organ and tissue growth. Although Hipk can induce outgrowths in the wing, it does so via a Notch-independent mechanism. Future studies will reveal to what extent Hipk can integrate multiple signaling inputs or regulate transcriptional complexes (Lee, 2009).

Differential in vivo requirements for oligomerization during Groucho-mediated repression

The Groucho (Gro)/transducin-like enhancer of split family of transcriptional corepressors are implicated in many signalling pathways that are important in development and disease, including those mediated by Notch, Wnt and Hedgehog. This study describes a genetic screen in Drosophila that yielded 50 new gro alleles, including the first protein-null allele; two mutations were obtained in the conserved Q oligomerization domain that has been proposed to have an essential role in corepressor activity. One of these latter mutations, encoding an amino-terminal protein truncation that lacks part of the Q domain, abolishes oligomerization in vitro and renders the protein unstable in vivo. Nevertheless, the mutation is not a null: maternal mutant embryos have intermediate segmentation phenotypes and relatively normal terminal patterning suggesting that the mutant protein retains partial corepressor activity. These results show that homo-oligomerization of Gro is not obligatory for its action in vivo, and that Gro represses transcription through more than one molecular mechanism (Jennings, 2008).

In vivo, Gro can mediate 'dominant' repression, causing the silencing of all linked enhancers to a gene. Gro has also been described as a 'long-range' repressor that can inhibit transcriptional initiation while bound to a distant (>1 kb away) enhancer element. These observations, together with that of Gro oligomerization through the Q domain, have fuelled the predominant 'spreading' model for Gro function, in which Gro oligomerizes along the DNA through the Q domain and thereby directs heterochromatic silencing and epigenetic changes in chromatin structure (Jennings, 2008).

However, Gro-dependent repression does not always cause the dominant silencing of linked enhancers within a complex cis-regulatory region. Moreover, Gro-mediated repression during animal development is frequently dynamic and rapidly reversible. Striped expression of Drosophila segmentation genes such as hairy and ftz evolves and decays within a period of approximately 30 min, serial production of Drosophila embryonic neuroblasts relies on five short pulses of E(spl)-mediated repression that occur within 4 h, and cyclic repression by Hes proteins during zebrafish somitogenesis has a periodicity of 20-30 min (Jennings, 2008 and references therein).

Although the results of this study indicate that simple Q-domain-directed oligomerization of Gro is not obligatory for its activity in vivo, the effects of overexpressing Gro in wing imaginal disks depend on oligomerization. The current results can be reconciled if, as seems possible, Gro mediates repression through more than one distinct molecular mechanism, with varying requirements for an intact Q domain according to the different transcription factor complexes assembled at different promoters. Local repression might predominate in dynamic developmental contexts that make use of rapidly reversible transcriptional inhibition (Jennings, 2008).

Hairless induces cell death by downregulation of EGFR signalling activity

Overexpression of the Notch antagonist Hairless (H) during imaginal development in Drosophila is correlated with tissue loss and cell death. Together with the co-repressors Groucho (Gro) and C-terminal binding protein (CtBP), H assembles a repression complex on Notch target genes, thereby downregulating Notch signalling activity. This study investigated the mechanisms underlying H-mediated cell death in S2 cell culture and in vivo during imaginal development in Drosophila. First, the domains within the H protein that are required for apoptosis induction in cell culture were mapped. These include the binding sites for the co-repressors, both of which are essential for H-mediated cell death during fly development. Hence, the underlying cause of H-mediated apoptosis seems to be a transcriptional downregulation of Notch target genes involved in cell survival. In a search for potential targets, transcriptional downregulation of rho-lacZ and EGFR signalling output were noted. Moreover, the EGFR antagonists lozenge, klumpfuss and argos were all activated upon H overexpression. This result conforms to the proapoptotic activity of H, as these factors are known to be involved in apoptosis induction. Together, the results indicate that H induces apoptosis by downregulation of EGFR signalling activity. This highlights the importance of a coordinated interplay of Notch and EGFR signalling pathways for cell survival during Drosophila development (Protzer, 2008).

This work allows two important conclusions: that overexpression of H induces cell-autonomous apoptosis, and that H requires the co-repressors Gro and CtBP for its proapoptotic activity. It is known that H assembles a repression complex together with the two co-repressors, resulting in transcriptional downregulation of Notch target genes. Hence, the ability of H to induce cell death is most likely a consequence of the repression of Notch target genes that are involved in cell survival. It is noted, however, that not every cell that receives an overdose of H dies. One simple explanation for this observation is that the only cells that die are those in which the relevant Notch target genes are normally active, as these cells require a Notch signal for survival. As H results in a repression of Notch activity, these cells would be driven into cell death, whereas those cells that do not depend on higher Notch levels for survival would be resistant to an H overdose. How is this effect of H realised at the molecular level? So far, it has not been possible to narrow down the analyses towards one target gene, the repression of which by the H repressor complex induces apoptosis. The most straightforward idea, repression of the anti-apoptotic protein Diap1, is not supported by the data. Instead, it was found that EGFR signalling activity is downregulated as a consequence of the upregulation of several negative regulators of EGFR (Protzer, 2008).

The existence of a densely woven network of genetic interactions between the EGFR and Notch signalling pathways is well established. This intensive cross-talk harmonises many developmental processes, such as proliferation, differentiation, cell fate specification, morphogenesis and programmed cell death. Still, the molecular basis of this genetic interplay remains largely obscure. So far, few molecular intersections between the Notch and EGFR pathways have been revealed. For example, EGFR signalling causes phosphorylation of the co-repressor Gro, thereby negatively modulating the transcriptional outputs of Notch signalling via the Enhancer of split [E(spl)] genes. Conversely, a myc-Gro complex was shown to inhibit EGFR signalling during neural development in the Drosophila embryo. Although mutual antagonism is probably the most prominent relationship in EGFR-Notch interactions, in some developmental situations both pathways cooperate to potentiate each other's signalling activities. One such example with regard to cell survival has been described in the retina of rugose mutant flies, where cell type-specific cell death could be reversed by an increase in Notch or EGFR signalling activity, indicating that both pathways adopt an anti-apoptotic function in this developmental context. Also, R7 photoreceptor cell specification requires the combined input of both Notch and EGFR signals. Moreover, Notch defines the scope of rho expression in the Drosophila embryo, thereby activating the EGFR pathway required for early ectodermal patterning. Also, during the development of mouse embryonic fibroblast, the Notch receptor-processing γ-secretase presenilin acts as a positive regulator of ERK basal level activity (Protzer, 2008).

A significant decrease was observed in the levels of activated MAPK (diP-ERK), which provides a good assessment of EGFR pathway activation, upon induction of H. Activated MAPK directly phosphorylates two transcription factors, Aop (Yan) and Pointed (Pntp2). Phosphorylation inactivates Aop, which in the unmodified state, represses EGFR targets. At the same time, phosphorylation activates Pointed, which then causes EGFR target gene transcription. As H is a well-defined transcriptional repressor of Notch target genes, it is most unlikely that it impedes EGFR activity at the level of phosphorylation. Moreover, it is not thought that H acts at the level of transcriptional regulation of EGFR target genes, even though combinatorial and antagonistic activities of the nuclear effectors of the EGFR and Notch signalling pathways have been described during eye development. Instead, the hypothesis is favored that H represses the transcription of EGFR activators, or might indirectly provoke the activation of EGFR repressors that affect, for example, the production of EGFR ligands or signal transduction (Protzer, 2008).

Rho activity is required for a timely and spatially regulated release of EGFR ligands. Accordingly, the expression of rho is highly dynamic during Drosophila development, and precedes the appearance of EGFR-induced activated MAPK. Hence, downregulation of rho by H would eventually result in lower levels of activated MAPK (diP-Erk). In contrast to other components of the EGFR signalling pathway, ectopic expression of rho results in EGFR activation in a wide range of tissues, indicating that Rho is an essential and limiting factor. So far, transcriptional control is the only known means of rho regulation. The complex array of enhancers regulating rho expression reflects the dynamic pattern of EGFR activation throughout Drosophila development (Protzer, 2008).

Interestingly, a transcriptional repression of rho-lacZ was observed in H gain-of-function clones that was dependent on the co-repressors Gro and CtBP. This effect might very well be direct, because it was shown previously that rho transcription is regulated by Su(H) in the neuroectoderm as well as in the gut of the Drosophila embryo. As mentioned above, Notch signalling has also been shown to regulate rho expression in the embryonic ectoderm. Moreover, during egg development, a band of Notch activity establishes the boundary between the two dorsal appendage tube cell types, whereby Notch levels are high in rho-expressing cells. In accordance with this, potential Su(H)-binding sites are present in the regulatory regions of rho1 and rho3, making a direct regulation of rho during eye development via the Notch-Su(H)-H complex very likely. It is noted, however, that the downregulation of rho-lacZ and of activated MAPK were focussed at the morphogenetic furrow, where primary photoreceptor cells are specified and ommatidia are founded. Regulation of rho by H would then be expected to interfere with photoreceptor formation rather than with cell survival, which is in agreement with the disturbed cellular architecture of H gain-of-function flies (Protzer, 2008).

Most interestingly, upon H overexpression, ectopic induction of lz, klu and aos was observed. All three genes are known to be involved in cell death induction during pupal eye development. There it was shown that the Runx protein Lz binds to the regulatory regions of klu and aos, resulting in the direct transcriptional activation of these target genes. Therefore, one might speculate that H executes its effect on klu and aos activity via the activation of lz. Moreover, as klu and aos are well-known inhibitors of EGFR signalling activity, this in itself suggests that H impedes EGFR signalling activity via these factors. This interpretation helps to explain why aos expression is induced in H gain-of-function clones, although it is well known that aos is triggered by EGFR signalling, thereby forming an inhibitory loop that acts on EGFR activity. The high levels of Lz still activate aos in H gain-of-function clones, keeping activity of the EGFR pathway low. Alternatively, aos and klu levels might be increased as a consequence of the downregulation, by H, of an as yet unknown repressor. Since H behaves as a kind of 'multi-adaptor protein', which not only recruits the transcriptional silencers Gro and CtBP to Notch targets but also binds other proteins such as Pros26.4, it is also possible that H interacts with positive regulators of lz, klu and aos (Protzer, 2008).

However, a model is favored whereby H influences EGFR signalling activity on two levels. On the one hand, through transcriptional repression of rho, H causes a loss of EGFR signalling output that interferes with cell specification. On the other hand, by interfering with their repressor(s), H relieves the restriction on lz, klu and aos expression, causing their accumulation. In consequence, the survival/death balance is tipped towards apoptosis in those cells that are susceptible to the effects of a lowered EGFR signal. Those cells that do not depend on high Notch and EGFR activity levels for survival would be resistant to an H overdose (Protzer, 2008).

Finally, one can envisage that a downregulation of Notch and EGFR signalling activities, resulting from the overexpression of H, might leave a cell in a state of 'uncertainty' that does not allow any further differentiation towards a certain cell type, but leaves the cell vulnerable to the apoptotic programme (Protzer, 2008).

Lines is required for normal operation of Wingless, Hedgehog and Notch pathways during wing development

The regulatory Lines/Drumstick/Bowl gene network is implicated in the integration of patterning information at several stages during development. This study shows that during Drosophila wing development, Lines prevents Bowl accumulation in the wing primordium, confining its expression to the peripodial epithelium. In cells that lack lines or over-expressing Drumstick, Bowl stabilization is responsible for alterations such as dramatic overgrowths and cell identity changes in the proximodistal patterning owing to aberrant responses to signaling pathways. The complex phenotypes are explained by Bowl repressing the Wingless pathway, the earliest effect seen. In addition, Bowl sequesters the general co-repressor Groucho from repressor complexes functioning in the Notch pathway and in Hedgehog expression, leading to ectopic activity of their targets. Supporting this model, elimination of the Groucho interaction domain in Bowl prevents the activation of the Notch and Hedgehog pathways, although not the repression of the Wingless pathway. Similarly, the effects of ectopic Bowl are partially rescued by co-expression of either Hairless or Master of thickveins, co-repressors that act with Groucho in the Notch and Hedgehog pathways, respectively. It is concluded that by preventing Bowl accumulation in the wing, primordial Lines permits the correct balance of nuclear co-repressors that control the activity of the Wingless, Notch and Hedgehog pathways (Benítez, 2009).

The Drosophila wing is a discrete organ that has been used to study the coordination of signaling pathways during development. The developing wing disc is a sac-like structure composed of the columnar epithelium or disc proper cells (DP), the cuboidal marginal cells (MC) and the overlying squamous cells (SC); MC and SC constitute the peripodial epithelium (PE). During larval development, imaginal cells proliferate extensively and are patterned. After metamorphosis, the DP cells differentiate into the cuticle that forms the adult wing and notum, whereas PE cells contribute little to these structures (Benítez, 2009).

The Lin/Drm/Bowl cassette is emerging as an important molecular mechanism with which to coordinate various pathways in different developmental contexts. In all cases, the steady-state accumulation of Bowl is regulated by the relative levels of Drm and Lin proteins. High levels of Drm impede binding of Lin to Bowl and, thus, this transcriptional repressor becomes stabilized in the nucleus. In this study it was found that regulatory interaction Lin/Drm/Bowl also functions during wing development. In lin- or Drm GOF cause ectopic expression of Bowl and dramatic overgrowths within the wing disc. These overgrowths frequently showed altered cell identity, resembling more proximal disc margin cells. Some of the effects can be explained by the ability of Bowl to interact with Gro co-repressor through the eh-1 motif, forming a complex that sequesters Gro from other repressors complexes such as Su(H)/H/Gro and Mtv/Gro (Benítez, 2009).

Although Bowl is ubiquitously transcribed in the wing disc, Bowl protein is present only in the SC and MC, being normally absent from the DP cells. The spatial distribution of nuclear Bowl is dependent on Drm, which causes Lin to relocalize to the cytoplasm. Drm is absent from most of the DP cells and, therefore, Lin turns down the steady-state accumulation of Bowl protein in these cells. In the absence of Lin, Bowl accumulates in the DP cell nuclei and elicits the dramatic alterations observed in lin- mutant cells. Therefore, the main function of Lin is to prevent Bowl accumulation in the DP cells, restricting Bowl protein to MC and SC of the PE (Benítez, 2009).

The main alterations in lin-, Drm GOF or Bowl GOF clones can be classified according to the signaling pathways temporally affected. The earliest defect observed is the repression of Wg pathway responses and the evidence suggests that Bowl functions as a repressor of the Wg pathway. However, activated forms of nuclear Wg pathway components, such as ArmS10 or dTcf, cannot restore the expression of the proximal-distal markers owing to repression of the Wg targets in lin-, indicating that Bowl must act in parallel to or downstream of Arm and dTcf (Benítez, 2009).

Bowl is a zinc-finger protein that can interact with the co-repressor Gro directly through the eh-1 motif. The results indicate that this mechanism is also important under conditions where Bowl accumulates in the wing disc. Most of the alterations observed in lin- or Drm GOF clones can be explained by Bowl sequestering Gro from other repression complexes (causing activation of N targets and Hh). Several results support this model. First, the strong genetic interaction between lin and gro alleles, where trans-heterozygous combinations between lin and gro alleles result in dramatic phenotypes, argue that Gro is a limiting factor. Second, removal of eh-1 motif that recruits Gro, eliminates the effects of Bowl on the Hh and N pathways. Third, ectopic expression of Gro, H or Mtv partially suppress the phenotypes of ectopic Drm or Bowl. These observations imply a 'tug of war' between Bowl, H and Mtv for Gro. Increased H or Mtv would shift the balance back in favor of N target repression and Hh repression (Benítez, 2009).

By contrast, the repression of Wg pathway observed in lin- cells appears to involve a different mechanism. Although the effect is Bowl dependent, repression of Wg targets also occurs with Bowleh1-, indicating that Gro sequestration is not required. Similarly, co-expression of Bowl with H or Mtv cannot re-establish the repression of the Wg targets. These results show that Bowl is able to repress Wg targets independently of Gro and the observation that Bowleh1- VP16 can cause some ectopic expression of Sens suggests that this may involve a direct effect of Bowl on Wg targets (Benítez, 2009).

Wnt/Wg, N and Hh signaling represent major conserved signaling channels to control cell identity and behavior during development. An antagonistic interaction between the Wg and Hh has also been described in the embryo and at the intersection of the D/V and A/P compartment borders of the wing disc. Similarly, Wnt/Wg and N activities are closely entangled in many different systems. Mutual dependent interactions between N and Wnt signaling have been observed in vertebrate skin precursors, in rhombomere patterning and in somitogenesis. It has also been reported that orthologues of the Odd-skipped family, Osr1 and Osr2, function as transcriptional repressors during kidney formation. It is possible therefore that Lin/Bowl/Gro interaction is evolutionary conserved and it will be interesting to discover whether lin is an important regulatory factor in other systems (Benítez, 2009).

By analyzing lin- clones in the wing primordium, this study has uncovered the consequences of stabilizing Bowl in the DP cells. There are, however, two regions where Bowl accumulates normally, in the MC and SC within the PE. Removal of Bowl in the PE might lead to ectopic Wg protein and thus to ectopic activity of the Wg signaling to transform PE from squamous to columnar cells. In this context, recently, it has shown that Bowl inhibition by ectopic expression of Lin results in the replacement of the PE by a mirror image duplication of the DP cells. However, not much alteration has been observed in cell morphology nor in the expression of markers such as Ubx or Hth when Bowl was depleted in PE cells (bowl- clones and UAS-BowlRNAi). It could be that the recovered bowl- clones were not induced early enough or that the levels of Bowl-RNAi were not sufficient to completely eliminate the Bowl function in these cells. Nevertheless, these manipulations revealed that bowl- phenotypes in the proximal wing and notum are consistent with a functional role in MC. Therefore, it is concluded that Lin has an important role in restricting Bowl to the MC (and PE), delimiting a Bowl-free territory that forms the DP cells and enables their responsiveness to key signaling pathways such as Wg (Benítez, 2009).

The Drosophila Nkx6 homeodomain protein has both activation and repression domains and can activate target gene expression

Consistent with the common role of Nkx6 family members in specifying motor neuron identity, this study shows that over-expression of Drosophila Nkx6 results in an increase in the number of Fasiclin II expressing motor neurons in the intersegmental nerve B branch. Dissection of the regulatory domains of Nkx6 using chimeric cell culture assays revealed the presence of two repression domains and a single activation domain within this transcription factor. As well as its conserved homeodomain, Nkx6 also has a candidate Engrailed homology 1 (Eh1) domain that is conserved among all NKx6 family members, through which vertebrate NKx6-type proteins bind the co-repressor Groucho. Paralleling previous reports that the Eh1 domain of Vnd and Ind are ineffective in Gal4 chimeric assays, this study found that the Eh1 domain of Nkx6 did not significantly enhance repression in Gal4 chimeric assays. However, when co-immunoprecipitation analyses was performed, it was found that Nkx6 can bind Groucho and that binding of Nkx6 to this co-repressor is modulated intra-molecularly. Full length Nkx6 interacted with Groucho poorly, because sequences at the carboxyl terminal of NKx6 interfere with Groucho binding, despite the presence of the Eh1 domain. In contrast, a carboxyl terminal Nkx6 deletion bound Groucho strongly. In keeping with the presence of an activation domain within Nkx6, it is also reported that Nkx6 can activate reporter expression driven by an Nkx6.1 enhancer that mediates auto-activation in transient transfection assays. The presence of multiple repression domains in Nkx6 supports Nkx6's role as a repressor, potentially using both Groucho-dependent and independent mechanisms. Thus, Nkx6 likely functions as a dual regulator in embryos (Syu, 2009).

The Drosophila Smad cofactor Schnurri engages in redundant and synergistic interactions with multiple corepressors

In Drosophila a large zinc finger protein, Schnurri, functions as a Smad cofactor required for repression of brinker and other negative targets in response to signaling by the transforming growth factor beta ligand, Decapentaplegic. Schnurri binds to the silencer-bound Smads through a cluster of zinc fingers located near its carboxy-terminus and silences via a separate repression domain adjacent to this zinc-finger cluster. This study shows that this repression domain functions through interaction with two corepressors, CtBP and Sin3A, and that either interaction is sufficient for repression. Schnurri contains additional repression domains that function through interaction with CtBP, Groucho, Sin3A and SMRTER. By testing for the ability to rescue a shn RNAi phenotype evidence is provided that these diverse repression domains are both cooperative and partially redundant. In addition Shn harbors a region capable of transcriptional activation, consistent with evidence that Schnurri can function as an activator as well as a repressor (Cai, 2009).

Degringolade, a SUMO-targeted ubiquitin ligase, inhibits Hairy/Groucho-mediated repression

Transcriptional cofactors are essential for proper embryonic development. One such cofactor in Drosophila, Degringolade (Dgrn), encodes a RING finger/E3 ubiquitin ligase. Dgrn and its mammalian ortholog RNF4 are SUMO-targeted ubiquitin ligases (STUbLs; see Model for SUMO-directed ubiquitination by the conserved STUbL family) (see SUMO). STUbLs bind to SUMOylated proteins via their SUMO interaction motif (SIM) domains and facilitate substrate ubiquitylation. This study shows that Dgrn is a negative regulator of the repressor Hairy and its corepressor Groucho [Gro/transducin-like enhancer (TLE)] during embryonic segmentation and neurogenesis, as dgrn heterozygosity suppresses Hairy mutant phenotypes and embryonic lethality. Mechanistically Dgrn functions as a molecular selector: it targets Hairy for SUMO-independent ubiquitylation that inhibits the recruitment of its corepressor Gro, without affecting the recruitment of its other cofactors or the stability of Hairy. Concomitantly, Dgrn specifically targets SUMOylated Gro for sequestration and antagonizes Gro functions in vivo. These findings suggest that by targeting SUMOylated Gro, Dgrn serves as a molecular switch that regulates cofactor recruitment and function during development. As Gro/TLE proteins are conserved universal corepressors, this may be a general paradigm used to regulate the Gro/TLE corepressors in other developmental processes (Abed, 2011).

Transcriptional cofactors are essential for the function of sequence-specific transcription factors and are part of the machinery required to execute temporally coordinated gene expression programs. Regulation of cofactor recruitment and activity is emerging as a major level of gene expression regulation. For example, Hairy/Enhancer of split/Deadpan (HES) family repressors are the primary transducers of the Notch signalling pathway that has a central role in patterning, stem cell development, and is misregulated in cancers. A well-studied case is the Drosophila repressor Hairy, a typical HES family member, which encodes a basic helix-loop-helix (bHLH) Orange repressor required for embryonic segmentation and adult peripheral nervous system (PNS) specification. Hairy-mediated repression is dependent on its ability to recruit cofactors. For example, Hairy recruits the corepressor Groucho (Gro) through it C-terminal WRPW domain, an interaction that is essential for periodic repression of fushi tarazu (ftz). In addition, Hairy recruits dCtBP and dSir2 through its PLSLV and basic domains, respectively. While these cofactors are required for Hairy-mediated repression, they exhibit context-dependent recruitment and function. Interestingly, some cofactors enhance Hairy-mediated repression (e.g., Gro and dSir2), whereas others are required to refine Hairy's function (e.g., dCtBP and dTopors). Consistent with this, it was found that most of the genomic loci bound by Hairy in the context of Kc cells exhibit corecruitment of dSir2 and dCtBP, but are not co-bound by Gro. However, the mechanisms that regulate context-selective cofactor association with Hairy or that may regulate cofactor activities are largely unknown (Abed, 2011).

A possible mechanism is that post-translational modification of Hairy regulates its association with a given cofactor and determines its overall function. One such modification is ubiquitylation that in many cases regulates the stability of transcription factors. However, ubiquitylation can also serve as a regulatory modification that does not lead to degradation, but affects protein-protein interaction or intracellular localization. Similarly, SUMOylation is a post-transcriptional modification that is involved in the regulation of gene expression and is mediated by the SUMO-specific E1-, E2-, and E3-SUMO ligase enzymes. Both ubiquitin and SUMO modifications are highly regulated. These two modifications can also be connected through proteins collectively termed SUMO-targeted ubiquitin ligases (STUbLs). STUbLs are RING proteins that bind non-covalently to the SUMO moiety of SUMOylated proteins via their N-terminal SUMO interaction motif (SIM) domains, and subsequently target the SUMOylated protein for ubiquitylation via their RING domain. Thus, STUbLs are able to 'sense' SUMOylated targets and modify them by ubiquitylation. The observation that STUbLs are associated with transcription complexes suggests that their function is directly linked to regulation of gene expression. For example, the STUbL protein RNF4 was found to be a positive regulator of steroid hormone transcription. Importantly, STUbLs are structurally and functionally conserved, as the mouse and human RNF4 proteins can substitute for their yeast orthologs in functional assays. STUbLs are required for the correct assembly of kinetochores, for the cell's ability to cope with genotoxic stress, and for genome stability. RNF4 is highly expressed in the stem cell compartment of the developing gonads and brain, and its expression is enriched in progenitor cells, likely representing its role in 'stemness'. Recently, RNF4 was shown to regulate the SUMO- and ubiquitin-mediated degradation of PML and PML-RAR. However, the role of STUbL proteins in transcription during development of higher eukaryotes is largely unknown (Abed, 2011).

This study shows that Degringolade (Dgrn), the only Drosophila STUbL protein, physically and genetically interacts with Hairy and its cofactor Gro and antagonizes Hairy/Gro-mediated repression during segmentation and neurogenesis. Ubiquitylation of Hairy by Dgrn affects choice of cofactor by preventing Gro, but not dCtBP, from binding to Hairy. It was also found that Dgrn specifically targets SUMOylated Gro, alleviates Gro-dependent transcriptional repression, and suppresses Gro functions in vivo throughout development. DamID chromatin profiling experiments revealed that the antagonism between Dgrn and Gro is aimed at a broad array of genomic loci, suggesting that Gro-Dgrn antagonism is of general importance beyond Dgrn's interaction with Hairy (Abed, 2011).

Dgrn binds directly to Hairy and is capable of ubiquitylating Hairy in a reconstituted system and in cells. The recognition motif for Dgrn within Hairy maps to Hairy's basic region and requires a specific positive charge (Arg33). This motif is transferable and functionally conserved, not only in Hey and other HES proteins (e.g., E(spl)m8 and Dpn), but also in dMyc and other bHLH proteins including the activator Sc. Therefore, it may reflect a general property of bHLH recognition by STUbL proteins. No evidence was found for direct SUMOylation of the HES and bHLH proteins: bacterially purified Hairy and Dgrn proteins interact, anti-SUMO antibodies fail to detect SUMOylated Hairy, Hairy's mobility in SDS-PAGE is not altered upon incubation with the dUlp1 SUMO peptidase, and mutating putative SUMOylation sites within Hairy does not alter its recognition or ubiquitylation by Dgrn. Accordingly, this study found that Dgrn's interaction with Hairy is mediated through Dgrn's RING motif independent of the SIM domains. Similarly, the yeast STUbL Slx5-Slx8 recognizes the MATα2 repressor independent of SUMOylation (Xie, 2010). Hairy recognition by Dgrn/RNF4 is also different from its recognition of substrates, such as GST-SUMO or PML, that involves direct SUMOylation of the targeted protein and requires the Dgrn/RNF4 SIM domains (Abed, 2011 and references therein).

Importantly, SUMOylation and the SIM motifs are necessary for Dgrn to target SUMOylated Gro and for Dgrn's suppression of HES/Gro repression in vivo, it is likely that the SIM domains interact with the poly-SUMO chain itself (Geoffroy, 2010). Dgrn possessing two separate recognition modules is reminiscent of the dual recognition properties described for the RING protein UBR1 (E3alpha). As the current dogma is that STUBLs recognize (via their SIM domains) poly SUMO chain(s) rather than the substrate, the dual recognition mechanism observed with Dgrn may further substantiate substrate recognition and specificity (Abed, 2011).

The contribution of each SIM domain is additive, and a Dgrn mutant harbouring a single SIM domain is capable of binding to GST-SUMO, as well as conjugating Hairy, although to a lesser extent than wild-type Dgrn. Correspondingly, it was found that elevated levels of SUMOylated proteins are detected in dgrn null embryos (Barry, 2011; Abed, 2011).

As an ubiquitin ligase, Dgrn catalyses the formation of mixed poly-ubiquitin chains on Hairy. This ubiquitylation does not map to Hairy's basic region, its putative SUMOylation sites, or to a single Lys residue. Importantly, this poly-site ubiquitylation does not affect Hairy protein stability or integrity, but rather selectively inhibits Gro binding to Hairy. Furthermore, in cells in which Dgrn protein levels are reduced via RNAi, Hairy protein levels are also decreased compared with control cells, suggesting that Dgrn is likely required for Hairy expression. This is different from dTopors, a Hairy-associated PHD-RING finger protein, which catalyses Lys48-linked chains and regulates Hairy turnover. Further work will be required to determine the exact molecular events and the role that specific ubiquitin chain linkage has in Dgrn's ability to inhibit Gro from binding to Hairy in vivo (Abed, 2011).

Despite extensive efforts, ubiquitylated Gro forms were not identifed in this study. Nonetheless, the data suggest that Dgrn specifically targets the SUMO chains on Gro, which likely serve as a signal for Gro sequestration by as yet to be identified machinery (Abed, 2011).

In transcription assays, Dgrn is a potent activator of ac and Sxl transcription, a function that requires its catalytic activity. Dgrn antagonizes Hairy-, Dpn-, and Gro-mediated repression in vivo. Dgrn specifically targets SUMOylated Gro, Dgrn function inversely correlates with SUMOylation, and a reduction in SUMO levels impairs Dgrn's ability to fully alleviate repression. Thus, Dgrn's activity suppresses the local repressive chromatin structure generated by repressors, their associated cofactors, and the SUMO pathway. It was also found that expression of DgrnHC/AA can inhibit the activation mediated by Da/Sc, suggesting that Dgrn is required to alleviate repression by endogenous repressors and/or corepressors. This fits well with the observation that reduction in Dgrn protein levels via RNAi impairs Da/Sc-mediated activation. While this study focused on Dgrn's effects on the repressive machinery, it is also possible that part of Dgrn ligase activity enhances the function of activators and/or coactivators. For example, Dgrn efficiently ubiquitylates the pro-neural activator Sc, and significant activation of the ac or Sxl promoters requires only Dgrn along with either Da or Sc (Abed, 2011).

These data suggest that part of Dgrn's activity is aimed specifically at the Gro corepressor that is shared by all HES proteins. First, Dgrn-mediated ubiquitylation of Hairy prevents Gro recruitment to Hairy. Second, Dgrn specifically targets SUMOylated Gro and its associated Gro oligomers for sequestration. Specifically, it was found that the detected level of Gro protein is dependent on Dgrn and the method of protein extraction. For example, in embryos that lack Dgrn (dgrnDK) and when protein extracts are made in RIPA buffer, the detected levels of Dgrn in dgrnDK embryos is higher compared with that of wild type. However, if the extraction is performed in 4% SDS buffer, the detected levels of Gro protein in wild-type and dgrnDK embryo extracts is equal. Likewise, the signal detected for Gro using immunostaining in embryos is highly complementary to the milder RIPA extraction. dgrnDK embryos show an increased signal compared with wild-type embryos (as in the absence of Dgrn, less Gro is sequestered and more Gro molecules are available for detection by the antibody). The majority of Gro appears to be sequestered. Since only 90% of Gro can be recovered after co-transfection of Dgrn using SDS extraction, the possibility cannot be ruled out that a fraction of the SUMOylated Gro is degraded. All together, these data suggest that Dgrn is required for Gro sequestration and that loss of Dgrn 'liberates' sequestered Gro (Abed, 2011).

While the data support a model in which Dgrn targets SUMOylated Gro for sequestration, Dgrn may also regulate the molecular machinery that is required for Gro SUMOylation and subsequently sequestration. Furthermore, while it is established that STUbL targets SUMOylated proteins for ubiquitylation and degradation, it is also possible that Dgrn has an impact on the SUMO pathway and SUMO isopeptidases (Abed, 2011).

Gro and its mammalian orthologs, the transducin-like enhancers of split (TLE1-4) proteins, repress transcription via several mechanisms, including oligomerization to generate local repressive chromatin structures, and are negatively regulated by phosphorylation. This study found that site-specific phosphorylation used by RTK signalling to inactivate Gro is not a prerequisite for Dgrn activity. However, the details surrounding other phosphorylations, the role of site-specific SUMOylation of Gro, and the molecular machinery mediating sequestration, as well as Dgrn's effects on specific Gro-dependent repressive mechanisms await further studies (Abed, 2011).

In vivo, it was found that Dgrn antagonism of Gro is highly relevant for embryonic segmentation, PNS development, and sex determination, processes that are regulated by Gro (Barry, 2011). Indeed, Dgrn can suppress the gain-of-function phenotypes of Gro, as well as rescue the phenotypes associated with tissue-specific inactivation of Gro using RNAi transgenes. The genomic targets of Gro and Dgrn are distinct from that of dCtBP or dSir2, and that 38% of Gro direct targets are shared with Dgrn. Thus, it is predicted that Dgrn will be involved in other HES-independent, but Gro-regulated, processes as well. It is likely that both proteins have unique regulatory roles during early development. This notion stems from observations that each of the factors has exclusive, non-overlapping, genomic binding sites, and that neither of the two genes can functionally rescue the embryonic lethality associated with mutants of the other protein (i.e., Gro cannot rescue the female sterility associated with dgrn null females, and reducing the dose of Dgrn does not rescue the lethality associated with the groE48 mutant) (Abed, 2011).

Finally, an open question is how can the activity of a general corepressor be temporally and spatially regulated during development. The data to date suggest a model in which Dgrn has a regulatory role. Since it is suggested that SUMOylation enhances Gro-mediated repression, one can imagine that ATP-dependent SUMOylation of Gro within the repressor complex will result in local augmented repression. However, concomitantly, SUMOylation will promote Dgrn recruitment, and subsequent inactivation of the repression complex on chromatin or in its vicinity, ensuring that local SUMO-augmented repression is limited in time and space. It is speculated that this type of transcriptional regulation will be instrumental to define and sharpen patterning borders throughout development (Abed, 2011).

The Drosophila STUbL protein Degringolade limits HES functions during embryogenesis

Degringolade encodes a Drosophila SUMO-targeted ubiquitin ligase (STUbL) protein similar to that of mammalian RNF4. Dgrn facilitates the ubiquitylation of the HES protein Hairy, which disrupts the repressive activity of Hairy by inhibiting the recruitment of its cofactor Groucho. This study shows that Hey and all HES family members, except Her, interact with Dgrn and are substrates for its E3 ubiquitin ligase activity. Dgrn displays dynamic subcellular localization, accumulates in the nucleus at times when HES family members are active and limits Hey and HES family activity during sex determination, segmentation and neurogenesis. Dgrn interacts with the Notch signaling pathway by antagonizing the activity of E(spl)-C proteins. dgrn null mutants are female sterile, producing embryos that arrest development after two or three nuclear divisions. These mutant embryos exhibit fragmented or decondensed nuclei and accumulate higher levels of SUMO-conjugated proteins, suggesting a role for Dgrn in genome stability (Barry, 2011).

A common theme among DNA-bound transcriptional regulators is the recruitment of co-activators and/or co-repressors to carry out their function. An important aspect of all HES family regulation is their recruitment of the co-repressor Groucho. Abed (2011) has shown that the ubiquitylation of Hairy does not lead to its degradation, but rather interferes with the ability of Hairy to recruit Gro, thereby antagonizing Hairy's repressive activity. This study found that dgrn mutant embryos show defects in segmentation. It is suggested that, similar to Dgrn's interaction with Hairy in segmentation, the ubiquitylation of other HES family members or Hey leads to their inability to recruit the cofactor Gro and thus antagonizes the repressor activity of this protein family. Consistent with this, it was found that loss of Dgrn function affects known Hey and HES family early functions, including sex determination and nervous system development (Barry, 2011).

It was surprising that Dgrn is female sterile rather than exhibiting zygotic lethality. Another, as yet unidentified, STUbL protein might function redundantly to Dgrn postzygotically. As the early Drosophila embryo develops essentially as a closed system running on maternally provided mRNA and proteins, the early syncytial embryo relies heavily on translational and post-translational modifications to control protein activity. Both Dgrn and Gro are maternally contributed and ubiquitously distributed. Thus, Dgrn might be recruited to the nucleus at different times during these early developmental stages to attenuate Gro's ability to be a potent co-repressor in the absence of active transcription, thereby modulating Hey and HES family activity (Barry, 2011).

Dgrn's human homolog is the transcriptional cofactor and STUbL protein RNF4. Indeed, human RNF4 acts as a functional homolog of Dgrn. RNF4 has also been shown to be a functional ortholog of the Rfp1/Rfp2-Slx8 heterodimer (from now on referred to as Rfp-Slx8) in S. pombe and the Slx5-Slx8 heterodimer in S. cerevisiae. RNF4 and the yeast Rfp-Slx8 and Slx5-Slx8 heterodimers have been shown to be important for DNA repair, kinetochore assembly and genome stability, with the loss of these proteins leading to fragmented chromosomes, elongated nuclei, asymmetric positioning of the nuclei and an accumulation of SUMOylated proteins (Barry, 2011 and references therein).

The budding yeast Slx5-Sx8 proteins were identified as a complex of proteins required for the viability of SGS1 (a gene encoding the only RecQ helicase involved in genomic integrity in S. cerevisiae) mutant cells. In Drosophila, loss of RecQ5 function leads to the loss of synchronous divisions in the syncytial embryo, an increased number of double strand breaks and a slight increase in the number of abnormal nuclei falling from the surface of the embryo. Mutations of the RecQ family member DmBlm (mus309 -- FlyBase; the Drosophila ortholog of human BLM, which leads to the human disorder Bloom Syndrome when mutated) are female sterile with severe defects in embryogenesis: syncytial embryos frequently include anaphase bridges, gaps in the normally uniform monolayer of nuclei and asynchronous mitoses (Barry, 2011 and references therein).

Recently, smt3 (SUMO) mutant embryos were shown to display embryonic nuclear cycle defects, including irregular size and distribution of nuclei, chromosome clustering, chromosome bridges, fragmentation and reduced number of nuclei in relation to the centrosome pairs. Several cell cycle factors were identified that are substrates for SUMOylation, it was and proposed that SUMOylation of these factors is important for controlling the cell cycle. The fragmented and de-condensed nuclei observed in the early arrest phenotypes of dgrnDK null embryos are reminiscent of the Slx8-Slx5 mutant phenotypes, fly RecQ mutant phenotypes and smt3 mutant phenotypes, suggesting that the role of STUbL proteins in genome stability and DNA repair might be a conserved function (Barry, 2011).

Alternatively, mutations in actin cytoskeleton and cell cycle checkpoint components in Drosophila have also been shown to exhibit nuclear arrest phenotypes. Defects in cell cycle checkpoint proteins, including Pan Gu, Plutonium and Giant Nuclei affect the S-phase checkpoint in the early embryo such that mutation of any of these genes leads to unregulated S-phase, resulting in giant polyploid nuclei. Disruption of the actin cytoskeleton can also lead to nuclear division abnormalities of cortical nuclei. For example, the scrambled and nuclear fallout mutants exhibit severe abnormalities in the appearance and localization of cortical nuclei. Further experiments will be needed to determine the molecular mechanism(s) underlying Dgrn's early arrest phenotype. However, regardless of the mechanism, this represents a new function for Hey or HES family proteins or a function for Dgrn that is not HES-dependent (Barry, 2011).

Interestingly, nuclear cycles during which Dgrn accumulates in the nucleus correspond to times when HES family members are active, which would be necessary for Dgrn to interact physically with HES proteins and subsequently affect their functions. One exception to this is Dgrn nuclear localization at nuclear cycle 9. There are no known HES family activities at nuclear cycle 9; however, several HES family members are yet to be characterized molecularly and genetically. Dgrn also exhibits a novel accumulation pattern during the gastrulation stages where it prefigures morphogenetic furrows, suggesting a possible role for Hey or HES family members in morphogenesis. Chromatin profiling experiments identifying direct transcriptional targets of Hairy identified a number of targets important for morphogenesis, suggesting that Hairy might play a role in morphogenesis. Consistent with this, a new hairy allele (h674) has been reported to affect the early stages of salivary gland morphogenesis. Thus, although Dgrn might work with Hey, Hairy and/or other HES family members during these times, it also remains possible that these Dgrn activities are Hey- and HES-independent (Barry, 2011).

Dpn is a negative regulator of Sxl. dpn mutants have a modest effect on Sxl in males leading to ectopic expression from the Sxl-Pe promoter that is sufficient to induce the inappropriate female fate in some cells. The Hey and HES family co-repressor Gro has also been shown to act as a negative regulator of Sxl; the loss of maternal Gro results in severe misexpression of Sxl in males leading to female fate. The relatively mild effect of Dpn on Sxl regulation compared with Gro led to a search for additional HES family proteins involved in Sxl regulation. Hey was identified as a maternal repressor of Sxl-Pe, albeit in a spatially variable pattern in males. Unlike the mammalian homologs of Hey, which are unable to bind Gro presumably owing to its C-terminal YRPW domain, this study found that Drosophila Hey binds Gro in GST pulldown assays. The data suggests that Dgrn is an important player in sex determination where it interferes with the repressive activities of Dpn and Gro; both Dpn and Hey are substrates for Dgrn's E3 ubiquitin ligase activity. In addition, Sxl protein staining and in vitro transcription assays demonstrate that Dgrn antagonizes the repression of Sxl. As proposed for the interaction of Dgrn with Hairy during segmentation (Abed, 2011), this study found that Dgrn provides a new level of control over the activity of Hey and the HES family members in sex determination. This control is mediated by ubiquitylation that probably disrupts the ability of these repressors to recruit Gro, thereby antagonizing their ability to repress transcription of Sxl-Pe in males (Barry, 2011).

It has been proposed that sex in Drosophila is not determined by the ratio of X-chromosomes to sets of autosomes (X:A ratio), but rather by X chromosome dose. It was speculated that a feedback mechanism in females is caused by the acetylation of chromatin, which inhibits Gro-mediated repression. Interestingly, the finding that Dgrn antagonizes Gro activity via the ubiquitylation of Hey and HES family repressors and targets SUMOylated Gro for sequestration provides an alternate scenario for this feedback mechanism in females (Barry, 2011).

Notch (N), through the E(spl) proteins (its downstream targets), heads one of the major developmental signaling pathways that functions in progenitor cell fate determination and differentiation. Recently, Sxl has been shown to inhibit Notch RNA translation and to negatively regulate the Notch signaling pathway in females. The notched wing phenotype of N was shown to be sensitive to Sxl, such that reducing the dose of Sxl suppressed the lethal effects of N hypomorphic alleles. This study found that reducing the dose of dgrn can also partially rescue the lethal effects of N hypomorphic alleles suggesting that Dgrn antagonizes N signaling. More specifically, it is hypothesized that the rescue of N1 male lethality is due to a decrease in Sxl expression. Dgrn heterozygosity also suppresses the vein patterning phenotype associated with NAX1682, suggesting that it is required for N signaling in this context also. Interestingly, Dgrn could be antagonizing N by two distinct mechanisms (or a combination of the two): the first an indirect antagonization of N signaling through Dgrn's control of Sxl expression, and the second by direct inhibition of the repressor activities of the E(spl)-C protein by ubiquitylation, thus blocking the repressive arm of the N pathway. The second mechanism has implications in regulating crosstalk between N and EGFR signaling pathways. Further studies will be required to determine the role of Dgrn's STUbL activity and whether Dgrn's activity on E(spl)-C proteins is redundant to EGFR signaling or whether both of these activities are required to antagonize Notch signaling (Barry, 2011).

Groucho corepressor functions as a cofactor for the Knirps short-range transcriptional repressor

Despite the pervasive roles for repressors in transcriptional control, the range of action of these proteins on cis regulatory elements remains poorly understood. Knirps has essential roles in patterning the Drosophila embryo by means of short-range repression, an activity that is essential for proper regulation of complex transcriptional control elements. Short-range repressors function in a local fashion to interfere with the activity of activators or basal promoters within approximately 100 bp. In contrast, long-range repressors such as Hairy act over distances >1 kb. The functional distinction between these two classes of repressors has been suggested to stem from the differential recruitment of the CtBP corepressor to short-range repressors and Groucho to long-range repressors. Contrary to this differential recruitment model, this study reports that Groucho is a functional part of the Knirps short-range repression complex. The corepressor interaction is mediated via an eh-1 like motif present in the N terminus and a conserved region present in the central portion of Knirps. This interaction is important for the CtBP-independent repression activity of Knirps and is required for regulation of even-skipped. This study uncovers a previously uncharacterized interaction between proteins previously thought to function in distinct repression pathways, and indicates that the Groucho corepressor can be differentially harnessed to execute short- and long-range repression (Payankaulam, 2009).

Groucho mediates the CtBP-independent repression activity of Knirps. The essential logic of Drosophila blastoderm transcription cascade is reliant on the short range of gap repressors proteins such as Knirps, Kruppel, and Giant acting on modular enhancers. Thus, the functional features of these repressors, which set them apart from long-range acting proteins such as Hairy, have been of special interest. Earlier studies suggested that the distinction between these classes of repressors may be attributed to differential recruitment of the CtBP corepressor to short-range repressor and Groucho to long-range repressors. The genetic and physical interactions of CtBP and Hairy were contradictory to this simple model, but further work has indicated that CtBP may not in fact serve as a Hairy corepressor, but as an antagonist of Groucho. In another case, the Brinker transcription factor can interact with Groucho and CtBP in vitro, but appears to rely on Groucho for repression of many target genes, whereas CtBP has a minor role. Significantly, the range of Brinker repression has never been elucidated. Foreshadowing this study, it has been shown that Slp1 acts as a gap type regulator of pair-rule genes in the early embryo. The short-range nature of this regulation was apparent on eve, hairy, run, ftz, prd, and odd when the Slp1 protein was expressed in a ventral pattern. Consistent with a role for Groucho, a mutant form of the Slp1 protein lacking the eh1 motif was reported to be inactive, but this assay was complicated by the brief temporal window of repression. This study shows that the well characterized short-range repressor Knirps physically and functionally interacts with Groucho, and this interaction is pivotal for the CtBP-independent repression potential of Knirps. These findings are definitely not consistent with the differential recruitment model of short- and long-range repression. Instead the results suggest an alternative model, that Groucho functions distinctly in the context of short- and long-range repression (Payankaulam, 2009).

One possible explanation for the diverse function of Groucho may involve oligomerization. Recent studies have shown that Groucho and its homolog can form oligomeric structures that have been proposed to spread along DNA. Mutations that block Groucho oligomerization in vitro compromise the activity of this protein in vivo in the imaginal disc. Thus, Groucho oligomerization has been assumed to be critical for its function and potentially related to the long-range activity of repressors such as Hairy. However, it seems likely that in the context of Knirps repressor complex, Groucho does not spread, because repression effects are clearly short range. Possibly the mode of recruitment dictates whether Groucho oligomerizes or not. It is hypothesized that the distinct eh1-like repression motifs in Knirps interact with Groucho in a unique conformation to restrain Groucho from spreading and, thus, from mediating long-range repression. Crystal structures of the WD domain of human Groucho homolog TLE1 bound to either WRPW or eh1 peptide revealed that these peptides adopt different conformations on the corepressor binding surface. Such differences may affect the ability of Groucho to oligomerize. Other components in the Knirps corepressor complex may also control Groucho oligomerization. An 'optional oligomerization' by Groucho model may explain earlier studies that found Hairy does not always cause dominant silencing of nearby enhancers. Also, hypomorphic alleles of Groucho have been identified that appear to compromise oligomerization but still retain some activity (Payankaulam, 2009).

What role might Groucho have in Knirps-mediated repression? As shown previously, the CtBP-independent repression activity of Knirps is critical for full activity on some endogenous enhancers, underscoring the importance of Knirps-Groucho association. The histone deacetylase Rpd3 is recruited by Groucho, and is also a part of the Knirps repression complex. CtBP proteins are known to interact with histone deacetylases; thus, both CtBP and Groucho may recruit Rpd3 cooperatively. The deacetylase activity may then augment Groucho-histone interactions, bringing about local modification of the chromatin, resulting in enhanced repressor output. Consistent with the cooperative recruitment of Rpd3, the purification of Knirps complexes indicated that Rpd3 associates preferentially with the full-length protein, and not the CtBP-independent domain alone. Therefore, in the context of Kni 1-330, Groucho may use another HDAC protein or rely on its HDAC-independent repression activity. The functional importance of this association may be to achieve quantitatively correct levels of Knirps activity, suggesting a similarity of function of these two corepressors. For example, in the context of the composite eve promoter, both of these activities can have roles in repressing enhancers of differential sensitivity (Payankaulam, 2009).

In conclusion, this study provides compelling evidence that Groucho can mediate short-range repression; thus, the long- and short-range effects of transcriptional repression do not appear to be a simple function of differential recruitment of distinct corepressors. Not only does this change the perspective of Groucho, it changes the perspective of different repressor proteins. It appears that long-range repressors such as Hairy and short-range repressors such as Knirps may function as modulators of the repression range of common machinery (Payankaulam, 2009). Interestingly, Knirps protein sequences from different insect genomes indicate that Groucho binding by Knirps may be an ancestral trait, because the Groucho-binding eh1-like motif is present in Drosophila species as well as Tribolium and Apis. In contrast, the critical CtBP-interacting residues are present only in Drosophila, suggesting that the acquisition of an additional corepressor may be a derived trait, possibly as a part of the remodeling of embryonic gene circuitry associated with the unique syncytial environment (Payankaulam, 2009).

CoREST acts as a positive regulator of Notch signaling in the follicle cells of Drosophila melanogaster

The Notch signaling pathway plays important roles in a variety of developmental events. The context-dependent activities of positive and negative modulators dramatically increase the diversity of cellular responses to Notch signaling. In a screen for mutations affecting the Drosophila follicular epithelium, a mutation was isolated in CoREST that disrupts the Notch-dependent mitotic-to-endocycle switch of follicle cells at stage 6 of oogenesis. Drosophila CoREST positively regulates Notch signaling, acting downstream of the proteolytic cleavage of Notch but upstream of Hindsight activity; the Hindsight gene is a Notch target that coordinates responses in the follicle cells. CoREST genetically interacts with components of the Notch repressor complex, Hairless, C-terminal Binding Protein and Groucho. In addition, it was demonstrated that levels of H3K27me3 and H4K16 acetylation are dramatically increased in CoREST mutant follicle cells. The data indicate that CoREST acts as a positive modulator of the Notch pathway in the follicular epithelium as well as in wing tissue, and suggests a previously unidentified role for CoREST in the regulation of Notch signaling. Given its high degree of conservation among species, CoREST probably also functions as a regulator of Notch-dependent cellular events in other organisms (Domanitskaya, 2012).

The highly conserved Notch signaling pathway plays a crucial role in a broad array of developmental events, including the maintenance of stem cells, cell fate specification, control of proliferation and apoptosis. Misregulation of the Notch pathway is associated with a number of diseases, including different types of cancer. The binding of the transmembrane ligands DSL (Delta, Serrate, LAG-2) to the extracellular domain of Notch, exposed on a neighboring cell, activates the signaling cascade by triggering a sequence of proteolytic cleavages of Notch protein. Extracellular cleavage (S2) leads to the formation of an intermediate membrane-bound C-terminal fragment of Notch, called NEXT. This event is followed by an intramembranous cleavage (S3) by the γ-secretase complex. The intracellular domain of Notch (NICD) then translocates to the nucleus and binds to a transcription factor of the CSL family [CBF-1, Su(H), LAG-1], converting it from a transcriptional repressor to an activator. In the canonical Notch pathway, Su(H) directly activates Notch target genes in response to signaling. Despite the relative simplicity of the Notch transduction pathway, the presence of a large number of proteins that positively or negatively influence Notch signaling dramatically increases the complexity of the Notch pathway and its cellular responses. For instance, extracellular modulators, such as Fringe, alter ligand-specific Notch activation, whereas cytoplasmic modulators, such as Numb, restrict signal transduction. Nuclear modulators, for instance Mastermind, influence the transcriptional activity of the NICD-containing complex. In addition, there is increasing evidence of the importance of the epigenetic regulation of Notch targets, which can cause differential cellular responses upon Notch activation (Domanitskaya, 2012).

Drosophila serves as an excellent model system to dissect the regulation of the Notch pathway. The Drosophila genome contains only a single Notch protein and two ligands [Delta (Dl) and Serrate (Ser)]. The Notch pathway is involved in several aspects of Drosophila development. The role of Notch in lateral inhibition during neurogenesis has been extensively studied; it restricts neural cell fates in the embryo, and leads to restriction of sensory-organ formation and induction of boundary formation in the wing discs. Notch activity is also required for many aspects of oogenesis, such as the establishment of egg chamber polarity, polar cell formation, control of follicle cell (FC) proliferation, differentiation, cell fate specification and morphogenesis. The Drosophila FCs are somatically derived epithelial cells that form a monolayer covering the germline cells during oogenesis. FCs divide mitotically from stage 2 to stage 6 of oogenesis, followed by the switch from the mitotic cycle to the endocycle (the M/E transition). Endocycles take place from stage 7 to stage 10A of oogenesis and include three rounds of DNA duplication without subsequent cell division. The M/E switch is triggered upon Notch pathway activation. Dl produced in the germline binds to its receptor Notch, expressed in the FCs, and induces activation of the canonical Notch signaling pathway. Removal of Dl from germline cells, or of Notch from FCs, maintains follicle cells in the mitotic cycle throughout oogenesis. NICD complexed with Su(H) activates transcription of downstream target genes required for the M/E switch, such as Hindsight (Hnt). Hnt then mediates the Notch-dependent downregulation of Cut, String (Stg) and Hedgehog (Hh) signaling in the FCs, thus promoting the M/E switch (Domanitskaya, 2012).

This study describes the identification of the transcriptional cofactor Corepressor for element-1-silencing transcription factor (CoREST) as a positive modulator of Notch signaling in the FCs and during wing development. CoREST is required for the promotion of the M/E switch during oogenesis. CoREST acts downstream of NICD release but upstream of Hnt activity, and it is a previously unidentified modulator of the Notch pathway. The genetic interactions between CoREST and Hairless (H), CtBP and Groucho (Gro), members of the Notch repressor complex, suggest that CoREST might influence the activity of either Notch transcriptional repressor or activator complexes. In addition, CoREST specifically affects tri-methylation of lysine 27 of histone 3 (H3K27) and acetylation of H4K16 in FCs, because these chromatin modifications show elevated levels in the CoREST mutant cells. These findings point to a possible role of CoREST in regulation of the activity of the Notch repressor-activator complexes and/or epigenetic regulation of the components of the repressor-activator complexes or of factors involved in the transduction of the signaling or directly of target genes of the Notch signaling pathway (Domanitskaya, 2012).

Initially, CoREST was identified in humans as a corepressor with REST (RE1 silencing transcription factor) in mediating repression of the proneuronal genes, and thus as an important factor in the establishment of non-neural cell specificity. Subsequently, CoREST was identified in a variety of vertebrate and invertebrate species, and was shown to play a functionally conserved role in neurogenesis. Recent studies show that CoREST regulates a very broad range of genes by both REST-dependent and REST-independent means, including genes encoding members of key neural developmental signaling pathways, such as BMP, SHH, Notch, RA, FGF, EGF and WNT. Analysis of CoREST downstream target genes and their developmental expression profiles suggested that the liberation of CoREST from gene promoters is associated with both gene repression and activation depending on the cell context. In the work reported in this study, a lethal allele of Drosophila CoREST was isolated, and the contribution of CoREST to the development of FCs, a process that involves cell proliferation and differentiation, was analyzed. This study has implicated CoRESTin the regulation of Notch signaling, and acts as a positive modulator of the Notch pathway in Drosophila FCs (Domanitskaya, 2012).

This study has identified a role for CoREST in the Notch-mediated regulation of the M/E switch during stage 6 of oogenesis. Loss of CoREST activity in FCs primarily disrupts the Notch signaling pathway. We further demonstrated that CoREST regulates the Notch pathway downstream of NICD release and upstream of Hnt. The misexpression of Hnt in the CoREST mutant clones rescues the failure in the M/E switch. Furthermore, the role of CoREST in Notch pathway regulation is not restricted to FCs: CoREST also interacts with Notch during wing development. Interestingly, CoREST was identified as a negative modulator of Notch signaling in Caenorhabditis elegans in a genetic screen for suppressors of the developmental defects in sel-12 presenilin mutants. Presenilin is a component of the γ-secretase complex that performs the S3 cleavage of Notch. Mutations in spr-1, the C. elegans homolog of CoREST, suppress the developmental defects observed in sel-12 animals by derepressing the transcription of the other functionally redundant presenilin gene, hop-1. Therefore, CoREST acts as a negative regulator of the γ-secretase complex in C. elegans, and hence proteolytic cleavage of Notch and release of NICD. By contrast, Drosophila CoREST does not affect the processing of the Notch receptor in the follicle cells, and instead acts as a positive modulator of the Notch pathway functioning downstream of NICD release (Domanitskaya, 2012).

CoREST plays transcriptional and epigenetic regulatory roles: it can promote gene activation in addition to repression, as well as being able to modify the epigenetic status of target gene loci distinct from its effects on transcription. Several possible scenarios of how CoREST could be involved in the regulation of Notch signaling are discussed, based on the previous knowledge about CoREST and considering the current data (Domanitskaya, 2012).

hnt, the downstream target gene of Notch signaling in FCs, fails to be properly upregulated upon Notch activation in the CoREST mutant cells. CoREST might therefore act as a transcriptional repressor for an unknown factor, which is in turn involved in the transcriptional repression of hnt. Alternatively, CoREST could be directly involved in the transcriptional regulation of hnt and act as an activator. hnt was shown to be a putative direct target of Notch signaling in DmD8 cells from the analysis of genes for which mRNA levels increase within 30 minutes of Notch activation, and which contain regions occupied by Su(H). If hnt is a direct target of Notch in FCs, its transcription would be regulated by the balance between Notch repressor and activator complexes, and CoREST might be involved in the regulation of stability or activity of either of these. Interestingly, CoREST was shown to interact with CtBP1 in mammals (Kuppuswamy, 2008), and to bind to the SIRT1-LSD1-CtBP1 complex, which is required for the repression of certain Notch target genes (Mulligan, 2011). Thus, Drosophila CoREST might similarly directly bind to the repressor complex containing CtBP and modify its activity or destabilize it. However, CoREST could be involved in the transcriptional regulation of the components of Notch repressor or activator complexes. In this scenario, in CoREST mutant FCs, upregulation of negative regulator(s) would lead to greater activity of negative than positive regulators, resulting in disruption of Notch signaling. Both suggested models of the direct and indirect transcriptional role of CoREST are consistent with the current results, given that the CoREST mutant phenotype could be suppressed by removal of one copy of H, CtBP or Gro, components of the Notch repressor complex (Domanitskaya, 2012).

More recently, epigenetic mechanisms have emerged as an important interface regulating context-dependent and stage-specific gene regulation. Mammalian CoREST acts as a scaffold for recruitment of transcriptional regulators such as REST, and epigenetic factors such as the enzymes HDAC1, HDAC2 and LSD1. In Drosophila, using two-hybrid interaction, CoREST was also shown to interact with Su(VAR)3-3 (Drosophila homolog of LSD1) and Rpd3 (HDAC1). This study has shown that the levels of H3K27me3 and H4K16 acetylation are significantly and specifically increased in the CoREST mutant FCs. Recently, the H3K27me3 demethylase UTX was shown to act as a suppressor of Notch- and Rb-dependent tumors in Drosophila eyes, and in addition to increased level of H3K27me3 staining, an excessive activation of Notch was detected in Utx mutant eye discs. The observation of increased levels of H3K27me3 coupled to cell overproliferation and modified Notch signaling in both of these cases suggests that the increased H3K27me3 results in epigenetic regulation of genes involved in Notch signaling and/or of Notch target genes. However, in the eye tumor system, this increase in H3K27me3 promotes Notch signaling, whereas in the follicle cells, it reduces Notch signaling. This indicates a strong context-dependent effect on Notch signaling by certain chromatin modifications. Thus, these chromatin modifications might be involved in cell-context-dependent Notch target gene silencing and/or activation. Interestingly, many Notch-regulated genes are highly enriched in a characteristic chromatin modification pattern, termed a bivalent domain, consisting of regions of H3K4me3, a marker for actively expressed genes, and H3K27me3, a marker for stably repressed genes; and Notch signaling could be involved in resolving these domains, leading to gene expression (Schwanbeck, 2011). Therefore, the increased level of H3K27me3 in CoREST mutant FCs might lead to a repression of certain Notch target genes, for instance hnt (Domanitskaya, 2012).

To further understand the function of the Drosophila CoREST in Notch pathway regulation, identification of other CoREST essential and specific binding partners would be useful. One previously identified partner for CoREST is Chn (Tsuda, 2006). Given that wild-type expression of Hnt and Cut was observed in chn mutant cells, this factor does not appear to partner CoREST in regulation of Notch signaling in FCs. Using yeast two-hybrid analyses and an embryonic cDNA fusion protein library, it was shown that all three splice variants of Drosophila CoREST interact with the unique C-terminus of Tramtrack88 (Ttk88), a known repressor without homology to REST. In addition, a Ttk69 splice variant can form a complex with CoREST and Ttk88. However, Ttk88 was not detected in the ovary by immunofluorescence or western blot analysis, and disruption of Ttk88 does not have any impact on oogenesis. Conversely, Ttk69 is steadily expressed in FCs before stage 10 and it is required for the M/E transition. However, in contrast to CoREST, which acts upstream of Hnt, Hnt expression is not affected in ttk1e11 mutant FCs, indicating a role of Ttk69 downstream of Hnt in the control of the M/E switch. Additionally, Ttk69 is not required for cell differentiation, as expression of FasIII, a cell fate marker for immature follicle cells, is normal in ttk1e11 mutant FCs. From these important phenotypic differences between Ttk69, Ttk88 and CoREST, it appears that CoREST plays a Ttk-independent role in Notch pathway regulation in the FCs. Future work to identify transcription regulators that act as binding partners of CoREST will help in determining the precise biochemical role of CoREST in modulating Notch signaling (Domanitskaya, 2012).

These results demonstrate an unexpected role for CoREST in positively regulating Notch signaling. The effect of the loss of CoREST is particularly strong in the PFCs and relatively mild in the lateral and anterior follicle cells. This implies that CoREST is crucially required in cells that are more sensitive to loss of Notch signaling. The difference between the PFCs and the other follicle cells is established at approximately stages 6-7 of oogenesis by EGF receptor activation in response to Gurken produced by the oocyte. EGF signaling, therefore, is active around the same time as the Notch pathway and hence it is probable that downstream effector(s) of EGFR signaling result in the increased sensitivity of PFCs to the loss of CoREST. In the model of CoREST negatively affecting a repressor of Notch signaling, EGFR signaling would be expected to act positively to enhance expression and/or activity of a Notch repressor. Thus, loss of CoREST from the PFCs would occur in a cell type where repressor activity is already augmented, which would explain the observation of differential loss of Notch signaling in the PFCs (Domanitskaya, 2012).

In summary this study has shown that CoREST, a component of transcriptional repressor complexes, acts positively in Notch signaling in the ovarian follicle cells of Drosophila. The results also show that different cell types are differentially sensitive to loss of this repressor. Future identification of partners and targets of CoREST in the follicle cells should further elucidate how activity of EGFR and other signaling pathways are integrated in this process (Domanitskaya, 2012).

Brinker possesses multiple mechanisms for repression because its primary co-repressor, Groucho, may be unavailable in some cell types

Transcriptional repressors function primarily by recruiting co-repressors, which are accessory proteins that antagonize transcription by modifying chromatin structure. Although a repressor could function by recruiting just a single co-repressor, many can recruit more than one, with Drosophila Brinker (Brk) recruiting the co-repressors CtBP and Groucho (Gro), in addition to possessing a third repression domain, 3R. Previous studies indicated that Gro is sufficient for Brk to repress targets in the wing, questioning why it should need to recruit CtBP, a short-range co-repressor, when Gro is known to be able to function over longer distances. To resolve this, genomic engineering was used to generate a series of brk mutants that are unable to recruit Gro, CtBP and/or have 3R deleted. These reveal that although the recruitment of Gro is necessary and can be sufficient for Brk to make an almost morphologically wild-type fly, it is insufficient during oogenesis, where Brk must utilize CtBP and 3R to pattern the egg shell appropriately. Gro insufficiency during oogenesis can be explained by its downregulation in Brk-expressing cells through phosphorylation downstream of EGFR signaling (Upadhyai, 2013).

A structure/function analysis of the transcriptional repressor Brk has been performed by replacing the endogenous brk gene with a ΦC31 bacteriophage attP site into which mutant forms of brk were introduced by integrase-mediated transgenesis. The goal was to generate mutations that disrupted the ability of Brk to recruit the CoRs Gro and CtBP and/or that deleted the less well characterized 3R repression domain and to test their activity in different tissues at different times of development to determine if and why they are required by Brk to repress transcription. Previous studies with Brk and other TFs that can recruit both CoRs indicated that Gro recruitment is essential for at least some of the activities of these TFs, but the reason for recruiting CtBP has proven more elusive. This study has confirmed that Gro recruitment is essential for Brk activity, but have also showed that Brk needs to recruit CtBP and to possess the 3R domain for full activity in some tissues, in particular during oogenesis (Upadhyai, 2013).

Lethality of the brkGM mutant reveals Gro recruitment is necessary for Brk activity. The brkΔ3RCM mutant, which utilizes Gro as its sole repressive activity, can progress from fertilization to an almost morphologically wild-type adult, indicating that Gro is close to sufficiency in this regard. However, brkΔ3RCM mutants often die as embryos and show defective oogenesis, with eggs having aberrant egg shell pattering, a characteristic of brk null mutants. The single mutants, brkΔ3R and brkCM, show less severe egg shell defects and reduced fertility, the latter probably relating to a defective micropyle, the structure through which sperm normally enter. The apparent inactivity of BrkΔ3RCM protein in follicle cells appears to be explained by active, unphosphorylated Gro being reduced there. The egg shell is patterned by the surrounding follicle cells, where Brk is expressed at high levels in the dorsal anterior. This coincides with high levels of EGFR signaling and previous studies have shown that Gro activity is attenuated following phosphorylation by MAPK downstream of EGFR signaling. As expected, lower levels of unphosphorylated or active Gro were found in the dorsal-anterior follicle cells. Consistent with the activity of BrkΔ3RCM being compromised by EGFR-dependent downregulation of Gro activity, upregulation of EGFR signaling in the wing disc of brkCM mutants results in derepression of the targets salE1 and ombZ (Upadhyai, 2013).

EGFR signaling also probably reduces the levels of active Gro available for Brk in other tissues, including the ventral ectoderm where Brk activity is required to ensure proper patterning of the denticle belts and where EGFR signaling is known play a key role. Many brkΔ3RCM mutants do not survive embryogenesis and demonstrate defects in denticle patterning similar to, but weaker than, those of null mutants. In addition, the VDB phenotype of brkGM mutants is less severe than in brkKO or brk3M mutants. Thus, CtBP and 3R appear to provide repressive activity in the ventral ectoderm (Upadhyai, 2013).

No Brk targets have been characterized in the follicle cells, but these would be expected to be partially derepressed in both brkCM and brkΔ3R mutants and possibly completely derepressed in brkΔ3RCM mutants based on the egg shell phenotypes, although there might be some differences between brkCM and brkΔ3R given the differences between CtBP and 3R just discussed. However, again, this would not imply that these targets are CtBP/3R specific, because the inability of Gro to participate in their repression is presumed to be due to its unavailability. Thus, although studies have indicated that TFs that have the ability to recruit both Gro and CtBP may only recruit one or other at specific targets, this might not reflect a CoR specificity for individual targets, but rather a cell-specific availability of CoRs (Upadhyai, 2013).

It is possible that if Gro were available in all cells then the CiM and 3R domain would be dispensable and so, at least for Brk, downregulation of Gro by MAPK phosphorylation could be considered inconvenient. This might be true for other TFs, including Hairy, Hairless and Knirps, which also function in multiple tissues, many of which are exposed to RTK signaling, and might explain why these TFs need to resort to recruiting CtBP as well as Gro. It should also be noted that Gro activity can be downregulated in other ways, including phosphorylation by Homeodomain-interacting protein kinase. This downregulation of Gro activity has been explained in terms of reducing the activity of specific repressors in specific tissues, such as E(Spl) factors during wing vein formation. This appears to be a somewhat illogical way to downregulate the activity of specific repressors, as there are almost certainly many other TFs utilizing Gro in the same cells and in other tissues exposed to RTK signaling and their activity might be compromised. There are no data indicating whether the downregulation of Gro activity in follicle cells serves any purpose and could simply be a consequence of the decision to downregulate Gro activity by this means in other tissues. However, this has serious implications for Brk and has required Brk to be versatile in its mechanisms of repression. Of course, the possibility has not been ruled out that downregulation of Gro activity does serve a purpose for Brk in follicle cells; for example, if Gro were available here it might provide Brk with too much activity or allow it to inappropriately repress a target that CtBP or 3R cannot. This might be tested by assessing egg shell phenotypes after driving unphosphorylatable Gro at physiological levels in a brkΔ3RCM mutant, but currently this is technically challenging (Upadhyai, 2013).

The idea that repressors need to be versatile in their repressive mechanisms because of variable CoR availability presumably extends beyond Brk and Hairless, Hairy and Knirps. In fact, other repressors in Drosophila possess both CtBP- and Gro-interaction motifs, including Snail. This might not be simply related to downregulation of Gro activity, as CtBP activity can also be modulated; for example, SUMOylation and acetylation of mammalian CtBPs is implicated in regulating their nuclear localization. In addition, other CoRs might similarly be available only in some cells; MAPK activity has been shown to phosphorylate and lead to the nuclear export and inactivation of the SMRT CoR complex. Finally, a further consideration raised by the present study is that care should be taken in assuming that a TF requires and can use a specific CoR to repress its targets in a particular tissue simply because it possesses an interaction motif for that CoR (Upadhyai, 2013).

Constitutive scaffolding of multiple Wnt enhanceosome components by Legless/BCL9

Wnt/β-catenin signaling elicits context-dependent transcription switches that determine normal development and oncogenesis. These are mediated by the Wnt enhanceosome, a multiprotein complex binding to the Pygo chromatin reader and acting through TCF/LEF-responsive enhancers. Pygo renders this complex Wnt-responsive, by capturing β-catenin via the Legless/BCL9 adaptor. This study used CRISPR/Cas9 genome engineering of Drosophila legless (lgs) and human BCL9 and B9L to show that the C-terminus downstream of their adaptor elements is crucial for Wnt responses. BioID proximity labeling revealed that BCL9 and B9L, like PYGO2, are constitutive components of the Wnt enhanceosome. Wnt-dependent docking of β-catenin to the enhanceosome apparently causes a rearrangement that apposes the BCL9/B9L C-terminus to TCF. This C-terminus binds to the Groucho/TLE co-repressor, and also to the Chip/LDB1-SSDP enhanceosome core complex via an evolutionary conserved element. An unexpected link between BCL9/B9L, PYGO2 and nuclear co-receptor complexes suggests that these β-catenin co-factors may coordinate Wnt and nuclear hormone responses (van Tienen, 2017).

The Wnt/β-catenin signaling cascade is an ancient cell communication pathway that operates context-dependent transcriptional switches to control animal development and tissue homeostasis. Deregulation of the pathway in adult tissues can lead to many different cancers, most notably colorectal cancer. Wnt-induced transcription is mediated by T cell factors (TCF1/3/4, LEF1) bound to Wnt-responsive enhancers, but their activity depends on the co-activator β-catenin (Armadillo in Drosophila), which is rapidly degraded in unstimulated cells. Absence of β-catenin thus defines the OFF state of these enhancers, which are silenced by Groucho/TLE co-repressors bound to TCF via their Q domain. This domain tetramerizes to promote transcriptional repression (Chodaparambil, 2014), which leads to chromatin compaction apparently assisted by the interaction between Groucho/TLE and histone deacetylases (HDACs) (van Tienen, 2017).

Wnt signaling relieves this repression by blocking the degradation of β-catenin, which thus accumulates and binds to TCF, converting the Wnt-responsive enhancers into the ON state. This involves the binding of β-catenin to various transcriptional co-activators via its C-terminus, most notably to the CREB-binding protein (CBP) histone acetyltransferase or its p300 paralog, resulting in the transcription of the linked Wnt target genes. Subsequent reversion to the OFF state (for example, by negative feedback from high Wnt signaling levels near Wnt-producing cells, or upon cessation of signaling) involves Groucho/TLE-dependent silencing, but also requires the Osa/ARID1 subunit of the BAF (also known as SWI/SNF) chromatin remodeling complex which binds to β-catenin through its BRG/BRM subunit. Cancer genome sequencing has uncovered a widespread tumor suppressor role of the BAF complex, which guards against numerous cancers including colorectal cancer, with >20% of all cancers exhibiting at least one inactivating mutation in one of its subunits, most notably in ARID1A. Thus, it appears that failure of Wnt-inducible enhancers to respond to negative feedback imposed by the BAF complex strongly predisposes to cancer (van Tienen, 2017).

How β-catenin overcomes Groucho/TLE-dependent repression remains unclear, especially since β-catenin and TLE bind to TCF simultaneously (Chodaparambil, 2014). Therefore, the simplest model envisaging competition between β-catenin and TLE cannot explain this switch, which implies that co-factors are required. One of these is Pygo, a chromatin reader binding to histone H3 tail methylated at lysine 4 (H3K4m) via its C-terminal PHD finger (Fiedler, 2008). In Drosophila where Pygo was discovered as an essential co-factor for activated Armadillo, its main function appears to be to assist Armadillo in overcoming Groucho-dependent repression. It has been discovered recently that Pygo associates with TCF enhancers via its highly conserved N-terminal NPF motif that binds directly to the ChiLS complex, composed of a dimer of Chip/LDB (LIM domain-binding protein) and a tetramer of SSDP (single-stranded DNA-binding protein, also known as SSBP). Notably, ChiLS also binds to other enhancer-bound NPF factors such as Osa/ARID1 and RUNX, and to the C-terminal WD40 domain of Groucho/TLE, and thus forms the core module of a multiprotein complex termed 'Wnt enhanceosome' (Fiedler, 2015). This study proposed that Pygo renders this complex Wnt-responsive by capturing Armadillo/β-catenin through the Legless adaptor (whose orthologs in humans are BCL9 and B9L, also known as BCL9-2). The salient feature of this model is that the Wnt enhanceosome keeps TCF target genes repressed prior to Wnt signaling while at the same time priming them for subsequent Wnt induction, and for timely shut-down via negative feedback depending on Osa/ARID1 (Fiedler, 2015; van Tienen, 2017 and references therein).

This study assessed the function of Legless and BCL9/B9L within the Wnt enhanceosome. Using a proximity-labeling proteomics approach (called BioID) in human embryonic kidney (HEK293) cells, a compelling association was uncovered between BCL9/B9L and the core Wnt enhanceosome components, regardless of Wnt signaling. Co-immunoprecipitation (coIP) and in vitro binding assays based on Nuclear Magnetic Resonance (NMR) revealed that BCL9 and B9L associate with TLE3 through their C-termini, and that they bind directly to Chip/LDB-SSDP via their evolutionary conserved homology domain 3 (HD3). These elements are outside the sequences mediating the adaptor function between Pygo and Armadillo/β-catenin, but they are similarly important for Wnt responses during Drosophila development and in human cells, as is shown by CRISPR/Cas9-based genome editing. The results consolidate and refine the Wnt enhanceosome model, indicating a constitutive scaffolding function of BCL9/B9L within this complex. The evidence further suggests that BCL9/B9L but not Pygo undergoes a β-catenin-dependent rearrangement within the enhanceosome upon Wnt signaling (see Model of the Wnt enhanceosome), gaining proximity to TCF, which might trigger enhanceosome switching (van Tienen, 2017).

This study has uncovered genetic and physical interactions between two constitutive core components of the Wnt enhanceosome and the C-terminus of Legless/BCL9. The first of these is ChiLS, the core module of the Wnt enhanceosome (Fiedler, 2015): ChiLS is a direct and specific ligand of the α-helical HD3 element of B9L and, likely, of other Legless/BCL9 orthologs, given the strong sequence conservation of this α-helix. The physiological relevance of this interaction with ChiLS is underscored by genetic analysis in flies. The evidence thus implicates HD3 as an evolutionary conserved contact point between Legless/BCL9 and ChiLS, although the primary link between these two proteins appears to be provided by Pygo (van Tienen, 2017).

A second link between the Legless/BCL9 C-terminus and the Wnt enhanceosome is mediated by the WD40 domain of TLE/Groucho. Given evidence from RIME, this link is also likely to be direct although, for technical reasons, it has not been possible to prove this. The function of the C-terminus of Legless/BCL9 for transducing Wnt signals was revealed by the wg-like phenotypes in Drosophila larvae and flies and by their defective transcriptional Wg responses, and by the loss of transcriptional Wnt responses in BCL9/B9L-deleted human cells. The evidence indicates that Legless/BCL9 undergoes three separate functionally relevant interactions with distinct components of the Wnt enhanceosomewith Pygo, ChiLS and Groucho/TLE. Importantly, BioID revealed that these interactions are constitutive, preceding Wnt signaling, and that they hardly change upon Wnt stimulation. Taken together with its multivalent interactions with the Wnt enhanceosome, this is consistent with Legless/BCL9 being a core component of this complex, providing a scaffolding function that facilitates its assembly and/or maintains its cohesion (van Tienen, 2017).

Following Wnt stimulation, Legless/BCL9 undergoes an additional physiologically relevant interaction, by binding to (stabilized) Armadillo/β-catenin via HD2. Legless/BCL9 thus confers Wnt-responsiveness on the Wnt enhanceosome through its ability to capture Armadillo/β-catenin. In other words, in addition to scaffolding the enhanceosome, Legless/BCL9 also earmarks this complex for Wnt responses. Intriguingly, the BioID data indicated that the capture of β-catenin by Legless/BCL9 triggers its rearrangement within the complex, apposing its C-terminus to TCF. This apparent β-catenin-dependent apposition is consistent with structural data showing that BCL9/B9L HD2 is closely apposed to TCF when in a ternary complex with β-catenin. The evidence supports the notion of Legless/BCL9 acting as an Armadillo loading factor, facilitating access of Armadillo/β-catenin to TCF, but argues against the original co-activator hypothesis which posited that Legless/BCL9 is recruited to TCF by Armadillo/β-catenin exclusively in Wnt-stimulated cells. Whatever the case, the β-catenin-dependent apposition of the Legless/BCL9 C-terminus to TCF is likely to trigger Wnt enhanceosome switching from OFF to ON, resulting in the relief of Groucho/TLE-dependent repression and culminating in the Wnt-dependent transcriptional activation of linked target genes (van Tienen, 2017).

This transition of the Wnt enhanceosome from OFF to ON is accompanied by a proximity gain between Legless/BCL9 and CBP/p300, likely to reflect at least in part its de novo binding to Armadillo/β-catenin. However, the evidence indicates that CBP/p300 is associated with the Wnt enhanceosome prior to Wnt signaling, possibly via direct binding to B9L as suggested by RIME, and that the docking of Armadillo/β-catenin to the Wnt enhanceosome strengthens its association with CBP/p300, and/or directs the histone acetyltransferase activity of CBP/p300 towards its substrates, primarily the histone tails. By acetylating these tails, CBP/p300 appears to promote Wnt-dependent transcription in flies and human cells. Indeed, CBP-dependent histone acetylation has been observed at Wg target enhancers in Drosophila although, interestingly, this preceded transcriptional activation. This is consistent with BioID data, indicating constitutive association of CBP/p300 with the Wnt enhanceosome (van Tienen, 2017).

It seems plausible that histone acetylation at Wnt target enhancers is instrumental in antagonizing the compaction of their chromatin imposed by Groucho/TLE, which depends on its tetramerization via its Q domain as well as its binding to HDACs. Indeed, HDACs were found near the bottom of the BioID lists, and one of the top hits identified by B9L was GSE1, a subunit of the BRAF-HDAC complex. However, CBP/p300 also has non-histone substrates within the Wnt enhanceosome, including dTCF in Drosophila whose Armadillo-binding site can be acetylated by dCBP, which thus blocks the binding between the two proteins and antagonizes Wg responses. It thus regulates Wnt-dependent transcription positively as well as negatively, similarly to Groucho/TLE which not only silences Wnt target genes but also earmarks them for Wnt inducibility, as a core component of the Wnt enhanceosome. It is intriguing that both bimodal regulators are associated constitutively with this complex. A corollary is that the docking of Armadillo/β-catenin to the Wnt enhanceosome changes their substrate specificities and/or activities (van Tienen, 2017).

An important refinement of the initial enhanceosome model is with regard to the BAF complex, which appears to be a constitutive component of the Wnt enhanceosome, as indicated by BioID data. This complex is highly conserved from yeast to humans, and it contains 15 subunits in human cells (Kadoch, 2015), including the DNA-binding Osa/ARID1 subunit. A wealth of evidence from studies in flies and mammals indicates that this complex primarily antagonizes Polycomb-mediated silencing of genes, most notably of the INK4A locus which encodes an anti-proliferative factor, which could explain why the BAF complex functions as a tumor suppressor in many tissues. However, recall that this complex also specifically antagonizes Armadillo/β-catenin-mediated transcription, likely via its BRG/BRM subunit which directly binds to β-catenin. Evidence from studies in Drosophila of Wg-responsive enhancers suggests that this complex mediates a negative feedback from high Wg signaling levels near Wg-producing cells which results in re-repression, imposed by the Brinker homeodomain repressor and its Armadillo-binding Teashirt co-repressor. The same factors may also install silencing on Wnt-responsive enhancers upon cessation of Wnt signaling. Notably, mammals do not have a Brinker ortholog, which could explain some of the apparent functional differences between flies and mammals with regard to the BAF complex (Kadoch, 2015). However, the closest mammalian relatives of Teashirt are the Homothorax/MEIS proteins, a family of homeodomain proteins whose expression can be Wnt-inducible. They are thus candidates for Wnt-induced repressors that confer BAF-dependent feedback inhibition (van Tienen, 2017).

Notably, none of BioID lists contained RUNX proteins. Based on functional evidence from Drosophila midgut enhancers, it is proposed that these proteins (which bind to both enhancers and Groucho/TLE) are pivotal for initial assembly of the Wnt enhanceosome at Wnt-responsive enhancers during early embryonic development, or in uncommitted progenitor cells of specific cell lineages (Fiedler, 2015). However, HEK293 cells are epithelial cells and may thus not express any RUNX factors. In any case, the negative BioID results suggest that RUNX factors function in a hit-and-run fashion. Evidently, the Wnt enhanceosome complex, once assembled at Wnt-responsive enhancers, can switch between ON and OFF states without RUNX (van Tienen, 2017).

In summary, this study has uncovered a fundamental role to Legless/BCL9 as a scaffold of the Wnt enhanceosome, far beyond its role in linking Armadillo/β-catenin to Pygo. Indeed, the function of Legless/BCL9 may extend beyond transcriptional Wnt responses, as indicated by the unexpected discovery of its strong association with nuclear co-receptor complexes. Potentially, these associations underlie the observed cross-talk between Wnt/β-catenin and nuclear hormone receptor signaling, documented extensively in the literature, including evidence for direct activation of the androgen receptor by β-catenin. Furthermore, a strong association between TLE1 and the estrogen receptor has been discovered in breast cancer cells, where TLE1 assists the estrogen receptor in its interaction with chromatin and its proliferation-promoting function. This is reminiscent of the role of Groucho/TLE as a cornerstone of the Wnt enhanceosome, proposed to earmark TCF enhancers for subsequent β-catenin docking and transcriptional Wnt responses (Fiedler, 2015). It will be interesting to test experimentally the putative roles of BCL9/B9L and Pygo in enabling cross-talk between β-catenin and nuclear hormone receptor signaling, both during normal development and in cancer (van Tienen, 2017).

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

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