Interactive Fly, Drosophila

Several different mechanisms have evolved for preventing gene transcription. One class of repressors function as transcriptional 'poisons', forming protein complexes with activators to retain them in the cytoplasm, rendering them unable to bind DNA. Other repressors act by competing for common or overlapping DNA target sites, thereby excluding activators from access to target promoters (Tailless, for example). A third class of negative transcriptional regulators also binds DNA but appears to act in a more instructive manner. Some such repressors (Snail, Kruppel, and Knirps for example) act at short range to mask adjacent, proximally bound activators. Others act over long-range, probably by directly interfering with the general transcriptional promoter sites. This latter class of DNA-bound repressors is likely to be assisted by general cofactors, by analogy to transcriptional activators that recruit co-activators to target gene promoters. Hairy and Enhancer of split proteins, and their corepressor Groucho, are included in this latter class of proteins (Paroush, 1997).

Tailless (Tll) acts as a repressor of Kruppel and knirps in the central domain of the recently fertilized embryo. Groucho acts throughout the embryo to repress the repressor of Kruppel and knirps, allowing the expression of these gap genes in the central domain of the embryo. Patterning of the non-segmental termini of the Drosophila embryo depends on signaling via the Torso receptor tyrosine kinase (RTK). Activation of Torso at the poles of the embryo triggers expression of the terminal zygotic gap genes tailless and huckebein (hkb). The Groucho (Gro) corepressor acts in this process to confine terminal gap gene expression to the embryonic termini. Embryos lacking maternal gro activity display ectopic tll and hkb transcription; in turn, tll then leads to lack of abdominal expression of the Kruppel and knirps gap genes. torso signaling permits terminal gap gene expression by antagonizing Gro-mediated repression. Groucho-mediated repression of tailless is relieved by the torso pathway suggesting that Groucho is the nuclear target for MAP kinase signaling. It is suggested that Groucho functions as a corepressor along with an unknown protein unrelated to Hairy, since Groucho mediated repression takes place in the absence of known Hairy-related bHLH proteins. Thus, the corepressor Gro is employed in diverse developmental contexts and, probably, by a variety of DNA-binding repressors (Paroush, 1997).

Maternal Groucho is essential for segmentation. groucho appears to be required for hairy and even-skipped dependent repression of fushi tarazu (Paroush, 1994). Sex-lethal is also a target of groucho. groucho-deadpan complexes act directly to repress early sex-lethal transcription (Paroush, 1994).

In normal growth of imaginal discs, posterior specific genes like hedgehog and engrailed are required to be restricted to the posterior compartment. Hedgehog has the potential to activate engrailed in the anterior compartment, but both hedgehog and engrailed are specifically repressed in anterior cells by the activity of neurogenic gene groucho. In groucho mutant discs, hh and en are expressed in the anterior compartment leading to ectopic activation of decapentaplegic and patched, and to a localized increase in cell growth associated with pattern duplications. This leads to a transformation of the anterior into the posterior (de Celis, 1995). Groucho also acts during embryonic development to restrict engrailed and hedgehog to the anterior parasegmental compartment (de Celis, 1995).

Transcriptional control of the Drosophila terminal gap gene huckebein (hkb) depends on Torso (Tor) receptor tyrosine kinase (RTK) signaling and the Rel/NFB homolog Dorsal (Dl). Dl acts as an intrinsic transcriptional activator in the ventral region of the embryo, but under certain conditions, such as when it is associated with the non-DNA-binding co-repressor Groucho (Gro), Dl is converted into a repressor. Gro is recruited to the enhancer element in the vicinity of Dl by sequence-specific transcription factors such as Dead Ringer (Dri). The interplay between Dl, Gro and Dri on the hkb enhancer was examined and it was shown that when acting over a distance, Gro abolishes rather than converts Dl activator function. However, reducing the distance between Dl- and Dri-binding sites switches Dl into a Gro-dependent repressor that overrides activation of transcription. Both of the distance-dependent regulatory options of Gro -- quenching and silencing of transcription -- are inhibited by RTK signaling. These data describe a newly identified mode of function for Gro when acting in concert with Dl. RTK signaling provides a way of modulating Dl function by interfering either with Gro activity or with Dri-dependent recruitment of Gro to the enhancer (Hader, 1999).

The cis-acting element has been identified that mediates expression of the Drosophila gene hkb, which is necessary for terminal pattern formation and to size the mesoderm anlage in the blastoderm embryo. Deletion analysis of this element reveals a 162 base pair (bp) sub-element that integrates the activities of the Tor-dependent RTK signaling cascade and the morphogen Dl. This element, termed hkb ventral element (VE), comprises a 112 bp ventral activator element (VAE) and a 50 bp ventral repressor element (VRE) (Hader, 1999).

The VAE contains a Dl-binding site, identified in vitro, and mediates gene activation along the ventral side of the embryo. VAE-mediated gene expression is absent in embryos lacking Dl activity and extends throughout Toll^10b mutants, in which Dl is present in all nuclei of the embryo. The expression pattern is not altered in embryos lacking snail and twist, the zygotic mediators of Dl. It is also not affected in embryos that lack Tor or express constitutively active Tor^Y9, which causes RTK signaling throughout the embryo. In contrast, the VE fails to activate in the absence of Tor and mediates broad ventral expression in tor^Y9 embryos not seen in the absence of Dl activity. This indicates that VAE mediates transcriptional activation by Dl, that the VRE, which by itself fails to activate transcription, is necessary to prevent Dl-dependent activation in the central region of the embryo, and that the activity of the unknown repressor, mediated by the VRE, is relieved by RTK signaling (Hader, 1999).

The evolutionarily conserved co-repressor Gro acts as a repressor of Dl activity, since both hkb expression and VE-driven gene expression expand along the ventral side of embryos lacking groucho (gro) activity. However, VAE-driven gene expression and the terminal expression domains of hkb are not significantly affected by lack of Gro. Thus, Gro functions as a repressor of VAE-directed, Dl-dependent transcriptional activation in the ventral region of the embryo and must act through the VRE (Hader, 1999).

Previous results have shown that Gro switches the transcriptional activator Dl into a potent silencer of transcription. This requires the formation of a multiprotein repressor complex of which Dl and Gro are obligatory components. Complex formation requires that Gro is recruited next to Dl by sequence-specific transcription factors such as Cut or Dri. Lack of either Gro or Dri activity results in VE-driven gene expression along the ventral axis of the embryo, indicating that both factors are necessary for repression of Dl-dependent activation. A single binding site has been identified for Dri in the VRE. Replacement of 5 bp in this site (VE-^DRI) results in loss of repression in the central region of the embryo, indicating that Dri is necessary for recruitment of Gro to the VE (Hader, 1999).

The VE differs from the cis-acting elements of the genes zerknullt (zen) and decapentaplegic (dpp), both of which mediate long-range Dl-dependent transcriptional silencing by Gro. In these elements, binding sites for Dri and Dl are directly adjacent, whereas in the VE they are some 90 bp apart. This distance suggested the possibility that Gro cannot associate with Dl on the VE, implying that Gro must prevent Dl-dependent activation by a means other than formation of a long-range silencing complex, for example, by short-range quenching. This proposal was tested by monitoring gene expression patterns directed by a cis-acting activator element of the gene knirps (kni-element) to which the VRE, the VAE, the VE or molecularly defined variants of the VE were fused (Hader, 1999).

The kni-element drives gene expression throughout the embryo except in the posterior pole region. It mediates activation in response to the transcriptional activators Bicoid (Bcd) and Caudal (Cad) and acts in a Dl-independent fashion. Addition of the VRE to the kni-element does not cause ventral repression, nor does addition of the VE or the VAE. This indicates that within the VE, Gro abolishes the activator function of Dl instead of converting Dl into a long-range repressor that interferes with transcriptional activation by Bcd and Cad (Hader, 1999).

To investigate whether this action of Gro on Dl is determined by the arrangement of Dri- and Dl-binding sites in the VE, the transcription patterns driven by a modified VE-kni-element were examined in which the normal distance of 91 bp between the binding sites was reduced to 45 bp. This reduction results in Dl-dependent repression along the ventral side of wild-type embryos. Repression is not observed in the absence of Gro or Dl or in embryos expressing the constitutively active Tor^Y9 protein. In contrast, the repression domain expands anteriorly in tor mutant embryos, which lack RTK signaling, and is found to be Dl-dependent. This suggests that the spatial arrangement of the Dl- and Dri-binding sites dictates the mechanism by which Gro and Dl act within the enhancer element. In one case, Dl is suppressed by Gro, in the other, Dl is converted into a potent silencer of transcription that can override activation by Bcd and Cad. Both modes of repression are controlled by Tor-dependent RTK signaling (Hader, 1999).

In the zen and dpp cis-acting elements, Gro causes Dl-mediated long-range silencing. Gro functions either by inhibiting the assembly and function of the core RNA polymerase II complex, by positioning nucleosomes over the core promoter and/or by recruiting the histone deacetylase Rpd3 to the template, where the enzyme can modulate local chromatin structure. However, in the VE, Gro only inhibits Dl-dependent activation without converting Dl into a repressor. The different modes of Gro function, that is, long-range silencing and short-range quenching, as shown here, are dependent on the distance between the Dl- and Dri-binding sites and/or their orientation on the enhancer, since shortening of the spacer distance converts the VE into a dpp- or zen-like element. This suggests that the way in which Gro regulates Dl activity depends on whether or not the two proteins can directly interact in vivo. Furthermore, both regulatory options of Gro on Dl are abolished by RTK signaling, a phenomenon that corresponds to the observation that Dl-dependent repression of dpp and zen is relieved by local Tor activity in the pole regions of the embryo. RTK-dependent phosphorylation may therefore interfere with the binding of Dri to the DNA template, the recruitment of Gro, or with both. Phosphorylation of the vertebrate Gro homolog TLE1 has been demonstrated, and many potential phosphorylation sites have been noted in Dri. Thus, local RTK-dependent phosphorylation may render one or both factors inactive, preventing Gro-dependent repression of Dl in the termini of the wild-type embryo (Hader, 1999).

These results establish that the cooperation between two maternal signaling systems, which determines the spatial limits of the Drosophila mesoderm anlage through hkb expression, is based on the management of the ubiquitously distributed factors Gro and Dri by local RTK signaling and that Gro can act through different modes on Dl. Lack of dead ringer (dri) activity does not result in an overt expansion of hkb expression on the ventral side of the embryo. However, as has been observed for VE-dependent gene expression, it causes only weak defects in mesoderm formation as compared with Gro-deficient embryos or embryos that express hkb under the control of the VAE. Thus, the interactions shown here represent only the Dri-dependent aspect of Gro's effect on hkb expression. The full picture of hkb control is likely to involve additional and redundantly acting factor(s) that recruit Gro to sites flanking the VE within the hkb control region (Hader, 1999).

ro^Dom is a dominant allele of rough (ro) that results in reduced eye size due to premature arrest in morphogenetic furrow (MF) progression. The ro^Dom stop-furrow phenotype is sensitive to the dosage of genes known to affect retinal differentiation, in particular members of the hedgehog (hh) signaling cascade. ro^Dom interferes with Hh's ability to induce the retina-specific proneural gene atonal (ato) in the MF and normal eye size can be restored by providing excess Ato protein. ro^Dom was used as a sensitive genetic background in which to identify mutations that affect hh signal transduction or regulation of ato expression. In addition to mutations in several unknown loci, multiple alleles of groucho (gro) and Hairless (H) were recovered. Analysis of their phenotypes in somatic clones suggests that both normally act to restrict neuronal cell fate in the retina, although they control different aspects of ato's complex expression pattern (Chanut, 2000).

Loss-of-function ro mutations cause eye roughness, due to mis-specification of photoreceptors R2 and R5, and the formation of ommatidia with more than one R8 photoreceptor. Repression of R8 cell fate has been attributed to inhibition of ato expression by the Ro homeodomain protein. In support of this proposal, Rough and Atonal proteins appear in complementary sets of cells behind the MF, and ato expression is expanded behind the MF in ro mutants. Generalized expression of ro under a heat-shock promoter (hs-ro) leads to loss of ato expression in the MF and eventually results in furrow arrest. Furrow arrest in ro^Dom is also accompanied by loss of ato expression in the MF. By analogy to the hs-ro phenotype, it is proposed that ro^Dom leads to excess Ro production, although that excess is not detectable by antibody staining (Chanut, 2000).

ro^Dom is very sensitive to alterations of ato gene dosage, since it is enhanced by loss-of-function ato alleles and almost completely rescued when high levels of ato expression are restored ahead of the MF. The ro^Dom phenotype therefore appears to result primarily from inhibition of ato expression due to excess Ro protein. On the basis of this understanding, the role of two of the strongest suppressors isolated in this screen, new alleles of gro and H, were analyzed on ato regulation and furrow progression (Chanut, 2000).

gro encodes a transcription inhibitor that combines with b-HLH genes of the E(spl) complex to inhibit expression of proneural genes such as achaete and scute. In gro mutant clones, expression of ato persists behind the MF longer than in wild-type tissue. This is consistent with a role for Gro in the N signaling events that lead to the refinement of ato expression behind the MF (Chanut, 2000).

However, Gro is also known to form inhibitory complexes with other transcription factors of the b-HLH class, such as Hairy, or of other classes, such as the c-Rel homolog Dorsal or the homeodomain, segment polarity regulator Engrailed. Association of Gro with Hairy deserves to be envisaged here, since Hairy has been implicated in inhibition of ato as well. It is found, however, that gro mutant clones expand ato expression posterior to the MF, whereas h inhibits ato expression anterior to the MF. Another intriguing possibility is that Gro associates with Ro to mediate inhibition of ato expression behind the MF. Although this hypothesis cannot be completely eliminated, it is unlikely, because complete loss-of-function phenotypes of ro and gro are different. While both of them lead to increased ato expression and imperfect R8 resolution, this effect is much more extensive and long lasting in gro mutant tissue than in ro mutant tissue. In addition, neuronal hyperplasia is not observed in ro mutant tissue, which suggests that at least this gro function must involve factors other than Ro. However, removal of E(spl) function results in neuronal hyperplasia and excess R8 development very similar to removal of gro function. Therefore, the hypothesis is favored that Gro restricts ato expression by combining with proteins of the E(spl) complex whose expression is induced by N signaling (Chanut, 2000).

Even in the complete absence of gro [or E(spl)] function, some refinement of ato expression still occurs, which indicates that factors independent of N and Gro play important roles in patterning Ato behind the MF. Candidates include Ro, the Egfr inhibitor Argos, and Hh. Moreover, outer photoreceptors differentiate in large excess between the R8 precursors and are the main cause of neuronal hyperplasia. Neuronal hyperplasia could occur as a direct consequence of the excess of R8 precursors in gro [and E(spl)] mutant tissue, which would, through the normal serial induction process, recruit an excess of neighboring cells into ommatidial clusters. However, differentiation of extra outer photoreceptors was observed with a hypomorphic gro allele in the absence of excess R8 differentiation. The excess of all photoreceptor types observed with a stronger gro allele may therefore reflect an involvement of gro in the restriction of cell fates at each step of ommatidial formation (Chanut, 2000).

gro mutant clones can also induce extensive overgrowth of head capsule and retinal tissues. In the wing, gro clones have been found to cause overgrowth via the induction of ectopic hh expression. Hh is also a powerful inducer of overgrowth in eye discs, when provided in excess or ectopically. However, overgrowth due to ectopic hh expression is accompanied by ectopic and premature photoreceptor differentiation, a phenotype not observed in overgrown gro mutant tissue. It is therefore unlikely that gro mutant clones cause ectopic hh expression in the eye. Besides, if gro mutations allowed increased Hh production, one would expect enhancement, rather than suppression, of the ro^Dom phenotype. While scenarios cannot be eliminated for ro^Dom in which a slight increase in cell proliferation allows the MF to progress further, it is more likely that gro mutations suppress ro^Dom by allowing Ato protein to persist longer in the MF (Chanut, 2000).

Finding that similar levels of suppression can be achieved by loss-of-function mutations in H and gro (which act in opposite directions in the N pathway) is not unique. A similar situation was encountered in another study where mutations in gro and H were both found to enhance the wing and bristle phenotypes associated with loss-of-function mutations in Egfr. The observation that mutations in both genes elevate ato expression in the vicinity of the MF, but at different stages of the differentiation process, helps resolve this paradox. The results also indicate that the exact timing (or location) of ato expression might not be crucial to MF progression, provided adequate levels are reached. This conclusion is supported by the finding that Ato supplied anterior to its normal expression domain, in the h expression domain, restores normal eye size in a ro^Dom background. Whether proper R8 spacing and ommatidial patterning can be achieved under these conditions remains to be shown (Chanut, 2000).

A function of Gro in imaginal development has been investigated, namely the repression of hedgehog in anterior wing pouch cells. hh is repressed in anterior compartments at least partly via Ci[rep], a form of the multifunctional transcription factor Cubitus interruptus (Ci). Cells in the wing primordium close to the AP boundary need gro activity to maintain repression of hh transcription, whereas in more anterior cells gro is dispensable. This repressive function of Gro does not appear to be mediated by Ci[rep]. Analysis of mutant gro transgenes has revealed that the Q and WD40 domains are both necessary for hh repression. Yet, deletion of the WD40 repeats does not always abolish Gro activity. These findings provide new insights both into the mechanisms of AP patterning of the wing and into the function of Gro (Apidianakis, 2001).

Although Ci[rep]-mediated repression can account for the lack of hh expression away from the AP boundary, it has not been firmly established that Ci[rep] is operational close to the AP boundary. These cells receive high Hh signal and as a result not only do they not process Ci to Ci[rep], but also they activate full-length Ci into a strong activator, Ci[act], by post-translational modification. There is indirect evidence that Hh-receiving cells do not contain sufficient Ci[rep] levels to repress hh: in posterior cells, ci is repressed by En; other than this, the cellular mechanism for Hh signal transduction is present. When full-length ci is provided by ectopic expression in the posterior compartment, hh-lacZ is not repressed. This suggests that these cells cannot produce appreciable amounts of Ci[rep], consistent with their responding to Hh signaling. That this is indeed the case was shown by the fact that ectopic expression of ci does repress posterior hh-lacZ in smo loss-of-function clones, where the Hh signal transduction has been disrupted. If anterior cells that are exposed to Hh behave similarly, then the lack of hh expression there cannot be attributed to Ci[rep]. It is proposed that a Gro-dependent repression complex supplies this function, since gro^- clones exhibit strong derepression of hh-lacZ near the AP boundary. The Gro complex is not required in anterior cells far from the boundary, because those receive no Hh signal and thus contain sufficient Ci[rep] to repress hh. Accordingly, by supplying increased levels of Ci[rep] near the AP boundary via the ci^Ce2 allele, the need for Gro-mediated hh repression is able to be largely abolished, with the exception of the DV boundary. Since Gro is a ubiquitous co-repressor, one has to postulate the existence of a DNA-tethering factor, which will be referred to as 'X' for the purpose of this discussion, and some process of spatial regulation of the X-Gro complex activity. The possibility that X is a form of Ci itself was tested and the answer was negative: using three different assays -- GST pulldowns, yeast two-hybrid and transfection colocalization -- no interaction between Gro and either form of Ci could be shown. Most importantly, the fact that Ci[rep] does not require Gro to repress hh in anterior cells away from the boundary supports a model where Ci and Gro repress hh independently of each other (Apidianakis, 2001).

The quantitative aspect of hh derepression in gro^- clones is intriguing: clones abutting the AP boundary (type I) express the highest hh-lacZ levels, which drop gradually as clones arise further from the P compartment. This might reflect the fact that Ci[rep]-dependent repression gradually increases away from the boundary, and this is independent of gro. This interpretation assumes that basal (unrepressed) hh transcription in the A compartment would be high and subject to the dual repressors (Ci and X-Gro). Alternatively, basal hh transcription could be low, but, in addition to the repression control, hh could display a positive response to Hh signaling at the AP boundary. The latter model is consistent with the fact that in ci^- cells, basal hh expression appears to be low. It also agrees with the behavior of large type I gro^- clones in the present study. In these clones, high levels of hh-lacZ could be observed throughout the clone, even at a distance from the AP boundary. This could be accounted for by Hh signaling, which, having risen over some threshold owing to hh derepression, further stimulates hh transcription to a high level. This effect would spread to the edge of the clone, beyond which activation of the X-Gro repressor would silence hh transcription. The putative inducer of hh by Hh signaling may be Ci[act], as with all other direct Hh target genes; alternatively, it may be another factor induced by Ci[act]. The hypothesis that Ci[act] itself can activate hh transcription is not unreasonable, since hh should contain a regulatory region(s) that bind(s) Ci[rep]. Ci[act] and Ci[rep] contain the same DNA-binding domain and recent work has shown that the two forms of Ci bind the same target sites, although some enhancers may be configured in such a way as to respond preferentially to either the activator or repressor form (Apidianakis, 2001).

For the sake of simplicity, the existence of a low level ubiquitous activator of hh (basal levels) with a stronger activator located in P cells is postulated to account for the high levels of hh expression in P cells. In A cells that do not receive the Hh signal, the basal activity of hh is repressed by Ci[rep] and gro is not required. In A cells close to the Hh source, the basal transcription of hh would be enhanced by positive autoregulation; however, the presence of the repressive X-Gro complex does not allow this activation to take place. Implicit in this model is that X is itself activated by Hh (e.g. transcriptionally induced via Ci[act]), so that it only functions in Hh-receiving cells. In addition X production/activity should be spatially limited to the A compartment (e.g. repressed by En), since ectopic expression of full-length ci in the posterior cannot induce X-Gro activity to repress endogenous hh. According to this model, ci^- clones close to the AP boundary express basal hh levels, since they lack both the X-Gro repressor (no activation of X in the absence of Ci[act]) and the activator of hh transcription (Ci[act] itself or a downstream target). By contrast, gro^- clones in the same region only lack the repressive X-Gro complex and thus actively transcribe hh in response to Ci[act]; the high levels of hh produced are sufficient to initiate Hh signaling, which can propagate this effect of hh derepression throughout the clone (Apidianakis, 2001).

gro^- clones near the DV boundary behave somewhat aberrantly. hh-lacZ derepression there is more efficient, observable in further anteriorly arising clones, compared with equivalent clones away from the DV boundary -- it even occurs in the presence of increased Ci[rep]. Although the mechanism remains to be discovered, one way to account for this special behavior, without invoking additional regulators, is that Ci[rep] is less active near the DV boundary and/or Ci[act] is more active, and this modulation of Ci activity in favor of the activator form allows high level hh expression at a greater distance from the Hh source and even in the ci^Ce2/+ background. Interestingly, ci^- clones show little or no hh-lacZ derepression at the DV boundary, consistent with Gro, rather than Ci[rep], being the major hh repressor there (Apidianakis, 2001).

The model put forward here is perhaps the simplest, but by no means the only one that fits the existing data. For example, Gro might interact with Ci[act] itself, switching it from an activator into a repressor, given the right enhancer context, much like the effect Gro has on other activators, such as Dorsal. This interaction may be weak and/or require additional factors, accounting for the inability to detect it. To resolve the mechanism of hh repression at the AP boundary will necessitate detailed molecular dissection of the hh regulatory regions and characterization of relevant trans acting factors. Whatever the mechanism, it appears that a Gro-containing complex is deployed in the wing to block the spread of hh expression anteriorly from the AP compartment boundary. This should ensure a spatially fixed organizer (dpp expression stripe), in contrast to a moving one, as found in the fly retina (Apidianakis, 2001).

Gro is the founder of a family of transcriptional co-repressors encountered in invertebrates and vertebrates. Gro proteins are multipurpose co-repressors, since they can interact with a good number of DNA-binding repressors. A number of Gro mutants were tested both for subcellular localization. Grocdc2^- and GroDeltaQ show the same nuclear accumulation as wild-type Gro. GroDeltaWD40 is also nuclear, but it shows a striking departure from the rather uniform wild-type pattern, since it localizes predominantly to a small number of subnuclear particles. GroNLS^- is both nuclear and cytoplasmic, whereas GroDeltaGCS is exclusively cytoplasmic. This suggests that the GP, CcN and SP domains contain at least two different regions needed for efficient nuclear accumulation, one of which is the canonical NLS. It can be speculated that other such regions might be those necessary for association with histones or with DNA-bound repressors, which might promote nuclear accumulation of Gro even in the absence of the NLS (Apidianakis, 2001).

In vivo activity was tested by assaying the ability of mutant Gro proteins to repress anterior hh-lacZ expression. GroDeltaQ and GroDeltaWD40 proteins were inactive in this assay. In contrast, Grocdc2^- was as active as wild-type Gro. The inability of GroDeltaQ to function as a co-repressor is expected, since the Q domain is the strongest repression domain and is needed both for tetramerization as well as for histone interaction. The inactivity of the GroDeltaWD40 mutant might be accounted by its inability to interact with the X-factor tether. Or one could suggest an alternative explanation based on the localization data: that GroDeltaWD40 is retained in subnuclear particles and as a result cannot gain access to target genetic loci. Whether the aberrant subnuclear localization of GroDeltaWD40 is a cause or a consequence of its inactivity is a matter for further study. Despite its aberrant localization, GroDeltaWD40 is as active as wild-type Gro and Grocdc2^- when overexpressed by omb-Gal4: all three transgenes results in abnormal leg development. Gro-DeltaQ, -NLS^- and -DeltaGCS did not have such an effect. This shows that GroDeltaWD40 retains some activity, although in the absence of data regarding the cause of defects in leg patterning, the function of the mutant protein is as yet unknown. 'Short' Gro family proteins that lack WD40 repeats exist in vertebrates. These, human AES and mouse Grg5, contain only Q and GP domains, thus they are not entirely equivalent to the DeltaWD40 mutant. It has been shown that these proteins are cytoplasmic, although they are readily transported to the nucleus upon interaction with a Tcf partner. Their role in transcription seems to be context dependent, since they can act as co-repressors in some cases, whereas in others they might counter repression by 'long' Gro proteins. One study suggests that this anti-repression effect is not necessarily due to the absence of the CcN/SP/WD40 domains, but rather due to the inability of the GP domain of the 'short' proteins to interact with HDAC1. In this study, GroDeltaWD40 was active in one assay and inactive in another. It will be interesting to determine its activity in additional biological contexts where Gro is required (Apidianakis, 2001).

Low-level ectopic expression of the Runt transcription factor blocks activation of the Drosophila melanogaster segmentation gene engrailed (en) in odd-numbered parasegments and is associated with a lethal phenotype. By using a genetic screen for maternal factors that contribute in a dose-dependent fashion to Runt-mediated repression, it is shown that there are two distinct steps in the repression of en by Runt. The initial establishment of repression is sensitive to the dosage of the zinc-finger transcription factor Tramtrack. By contrast, the co-repressor proteins Groucho and dCtBP, and the histone deacetylase Rpd3, do not affect establishment but instead maintain repression after the blastoderm stage. The distinction between establishment and maintenance is confirmed by experiments with Runt derivatives that are impaired specifically for either co-repressor interaction or DNA binding. Other transcription factors can also establish repression in Rpd3-deficient embryos: this indicates that the distinction between establishment and maintenance may be a general feature of eukaryotic transcriptional repression (Wheeler, 2002).

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