Transcriptional Regulation

Even-skipped functions to repress odd-skipped. Thus odd-skipped could be a true secondary pair-rule gene whose range of expression is determined by EVE (Manoukian, 1993).

Ectopic expression of the 69 kDa Tramtrack protein significantly represses even-skipped, odd-skipped, hairy and runt. The 88 kDa form does not similarly repress these genes (Brown, 1993).

The response kinetics of known and putative target genes of Fushi tarazu has been examined in order to distinguish between direct and indirect Ftz targets. This kinetic analysis was achieved by providing a brief pulse of Ftz expression and measuring the time required for genes to respond. The time required for Ftz to bind and regulate its own enhancer, a well-documented interaction, is used as a standard for other direct interactions. Surprisingly, both positively and negatively regulated target genes respond to Ftz with the same kinetics as autoregulation. The rate-limiting step between successive interactions (less than 10 minutes) is the time required for regulatory proteins to either enter or be cleared from the nucleus, indicating that protein synthesis and degradation rates are closely matched for all of the proteins studied. The matching of these two processes is likely to be important for the rapid and synchronous progression from one class of segmentation genes to the next. In total, 11 putative Ftz target genes have been analyzed, and the data provide a substantially revised view of Ftz roles and activities within the segmentation hierarchy (Nasiadka, 1999).

To determine the regulatory relationships between Ftz and the other non-primary pair-rule genes, the expression of odd-skipped (odd) and sloppy-paired (slp) was examined in HSFtz embryos fixed 20 and 35 minutes post Ftz induction. Expression patterns of these genes were also examined in ftz mutant embryos to obtain genetic confirmation of the interactions observed. Like ftz, en and prd, odd appears to be directly activated by Ftz. In stage 5 embryos, ectopic expression of Ftz causes rapid expansion of odd, from its initiating pattern of six stripes, to near homogeneous expression across the germband. In stage 6 embryos, odd is normally expressed in 14 evenly expressed stripes. Ectopic Ftz causes an intensification of the primary odd stripes at this stage. These stripes are derived from the original 7 stripes that overlap ftz stripes. In stage 7 embryos, these primary stripes are not only intensified, but expand anteriorly as well (from about 1 cell wide to 2 cells wide). The percentage of embryos responding to ectopic Ftz, at all stages tested, is about the same as the percentage of embryos that show ftz autoregulation, en and prd activation and wg repression. Thus, Ftz appears to be an activator of odd at all stages tested. This positive relationship between Ftz and odd is consistent with the differences in odd expression observed in ftz mutant embryos. Stripes of odd appear to be diminished in intensity in stage 5 embryos, and primary stripes are weak or missing in stage 6 and 7 embryos (Nasiadka, 1999).

Unlike prd and odd, the pair-rule gene slp is negatively regulated by Ftz: ectopic expression of Ftz results in the differential repression of secondary slp stripes. Again, the penetrance of repression at the 20 minute recovery time was about 60%, as has been measured for the other genes exhibiting direct responses. As might be expected, slp stripes expand in ftz mutant embryos, filling the regions where Ftz is normally expressed. Thus, as with wg, Ftz appears to act as a direct repressor of slp. This effect is likely exerted through the response elements or trans-acting factors that regulate secondary stripe expression (Nasiadka, 1999).

Working out the effects of Rpd3 on segmentation gene expression requires the scientific equivalent of investigative journalism. The Berkeley Drosophila Genome Project identified a P-induced lethal mutation (l(3)04556) that maps 47 bp downstream of the Rpd3 putative transcription start site. Previous work by Perrimon (1996) has shown that embryos derived from l(3)04556 homozygous germline clones exhibit pair-rule patterning defects that are similar to those observed in fushi tarazu mutants. It was first established that most repressors are active in Rpd3 mutant embryos. These results suggest that the Rpd3 mutation might impair expression of Fushi tarazu or Ftz-F1 proteins, known to be gene activators, because these are required for the expression of the even-numbered engrailed stripes. Alternatively, the loss of Rpd3 might lead to a change in the expression pattern of a repressor, which in turn inhibits Ftz activity (Mannervik, 1999).

To distinguish between these possibilities, an examination was made of the expression of odd-skipped, a known repressor of en. odd is initially expressed in a pair-rule pattern of seven stripes, but during gastrulation seven additional secondary stripes are formed to generate a 14-stripe expression pattern. In normal embryos, these stripes are evenly spaced, whereas in Rpd3 mutants they are not. In the mutant embryos there is a partial pair-wise alignment of adjacent odd stripes. A similar change is observed in eve embryos. Previous studies suggest that both ftz and odd stripes are under the control of the Eve repressor. Differential repression of ftz and odd resolves the two patterns, so that each ftz stripe is normally shifted anterior to each odd-numbered odd stripe. In Rpd3 mutants, the ftz and odd patterns fail to resolve, so that odd-numbered odd stripes mostly coincide with the ftz stripes. It is suggested that this failure in ftz-odd resolution is responsible for the pair-rule phenotype observed in Rpd3 mutant embryos. A prediction of this proposal is that eve mutants should exhibit similar alterations in the ftz and odd expression patterns. Double staining assays reveal that eve mutant embryos exhibit a similar failure to resolve the ftz and odd expression patterns (Mannervik, 1999).

There are several possible explanations for impaired Ftz function in Rpd3 mutants. It is conceivable that the Rpd3 mutation disrupts Ftz-mediated activation. However, the idea that Rpd3 functions as a corepressor of Eve is favored. The similarities in the Rpd3 and ftz mutant phenotypes may be caused by the coincident odd and ftz expression patterns observed in embryos derived from l(3)04556 germline clones. The Odd repressor is thought to block Ftz-mediated activation of en. Evidence is presented that this expansion in Odd might result from an inability of Eve to repress odd expression in Rpd3 mutant embryos. Consistent with this proposal, in vitro translated Eve is shown to interact with a glutathione S-transferase-Rpd3 fusion protein. Because the Eve repressor is required for both the odd- and even-numbered en stripes, it would appear that the Rpd3 mutation does not cause a general loss of Eve function. It would therefore appear that Eve fails to repress certain promoters (e.g., odd and possibly ftz) in Rpd3 mutant embryos, but retains repressor function on other promoters (e.g., paired and sloppy-paired). This selectivity in the regulation of different target promoters is consistent with the notion that Eve mediates repression through multiple mechanisms, including the recruitment of corepressors and direct interactions with TBP. Multiple modes of repression may be mediated by other transcriptional repressors, such as Hairy, which appears to interact with different classes of corepressors (Mannervik, 1999 and references).

The possession of segmented appendages is a defining characteristic of the arthropods. By analyzing both loss-of-function and ectopic expression experiments, the Notch signaling pathway has been shown to play a fundamental role in the segmentation and growth of the Drosophila leg. Local activation of Notch is necessary and sufficient to promote the formation of joints between segments. This segmentation process requires the participation of the Notch ligands, Serrate and Delta, as well as Fringe. These three proteins are each expressed in the developing leg and antennal imaginal discs in a segmentally repeated pattern that is regulated downstream of the action of Wingless and Decapentaplegic. While Dl expression overlaps fngand Ser, in some cases, it appears to extend into regions of the disc where neither fng nor Ser is expressed (Rauskolb, 1999).

While the requirements for odd-skipped (odd) function during leg development have not yet been described, this gene is of special interest because it is required for embryonic segmentation in Drosophila and is expressed in a segmentally repeated pattern both in the embryo and in leg discs. odd expression, like nub, is induced within clones of cells expressing activated Notch in many regions of the leg disc, though not in ta1-ta4. The observation that three different genes expressed in segmentally repeated patterns all respond to Notch signaling, together with the severe effects of Notch mutant clones, indicates that Notch acts at a crucial step in a leg segmentation hierarchy. Together, these observations outline a regulatory hierarchy for the segmentation and growth of the leg. The Notch pathway is also deployed for segmentation during vertebrate somitogenesis, which raises the possibility of a common origin for the segmentation of these distinct tissues (Rauskolb, 1999).

Homeodomain proteins are DNA-binding transcription factors that control major developmental patterning events. Although DNA binding is mediated by the homeodomain, interactions with other transcription factors play an unusually important role in the selection and regulation of target genes. A major question in the field is whether these cofactor interactions select target genes by modulating DNA binding site specificity (selective binding model), transcriptional activity (activity regulation model) or both. A related issue is whether the number of target genes bound and regulated is a small or large percentage of genes in the genome. These issues have been addressed using a chimeric protein that contains the strong activation domain of the viral VP16 protein fused to the Drosophila homeodomain-containing protein Fushi tarazu. Genes previously thought not to be direct targets of Ftz remain unaffected by FtzVP16. Addition of the VP16 activation domain to Ftz does, however, allow it to regulate previously identified target genes at times and in regions that Ftz alone cannot. It also changes Ftz into an activator of two genes that it normally represses. Taken together, the results suggest that Ftz binds and regulates a relatively limited number of target genes, and that cofactors affect target gene specificity primarily by controlling binding site selection (Nasiadka, 2000).

Activity regulation plays an important role in Ftz function, but this role is mainly to refine the temporal and spatial windows of target gene regulation and to modulate levels of expression. This conclusion is supported by the following observations. Five of the genes tested (ftz, odd, slp, en and wg) could be activated ectopically by FtzVP16 in regions and at times that Ftz could not induce a response. This shows that Ftz has the ability to bind to these promoters, but that it must be bound in an inactive state. For Ftz to function in these cells, it probably requires the addition of requisite cofactors, the removal of repressors or both. For the five genes listed above, the VP16 activation domain is able to overcome some of these limitations. The regulation by Ftz of en is a good example of this type of temporal and spatial refinement in activity. Results with FtzVP16 show that Ftz can bind to the en promoter during the time that ftz autoregulation and odd activation are well under way. However, the ability of Ftz to activate en is normally delayed until cellularization is completed (approx. 45 minutes). This delay may be necessary to allow other en regulators to resolve into the complex patterns of expression that are required for en to initiate in 14 narrow stripes. Like most homeodomain proteins, Ftz has the ability to function as both a transcriptional activator and repressor. This dual capacity suggests a requirement for distinct activity-regulating cofactors. However, differential activity can also be achieved, at least in part, by binding to different sites on different genes. For example, the response elements required for repression of the Distalless gene by Ubx and activation by Dfd are different. This also appears to be the case for activation of the dpp gene by Ubx and its repression by Abd-A. The different cofactors that help recruit the three proteins to these sites may also be partly responsible for their differences in transcriptional activity. For example, Exd is thought to generally work as a coactivator, acting in part to alter Hox protein conformation. Other factors bound in the vicinity of these sites are also likely to play a major role in activity regulation (Nasiadka, 2000).

In addition to showing that positively acting cofactors are important for Ftz specificity, these data implicate the actions of powerful negative regulators that limit the gene's temporal and spatial domains of activity. The strength and diversity of these negative regulators were emphasized by their ability to suppress the actions of the fused VP16 activation domain despite its previously reported reputation of strength and autonomy. It may be the low DNA binding specificity of the homeodomain that has necessitated this need for diverse mechanisms of repression, since low DNA specificity provides the potential to regulate a large number of inappropriate target genes. Indeed, a rapidly growing number of homeodomain proteins have been shown to be capable of functioning as oncogenes or proto-oncogenes, and oncogenicity can be conferred by fusions to other transcriptional activators. Further studies will be required to identify many of the cofactors and inhibitors that modulate Ftz activity and to determine how they do so (Nasiadka, 2000).

How to make stripes: deciphering the transition from non-periodic to periodic patterns in Drosophila segmentation.

The generation of metameric body plans is a key process in development. In Drosophila segmentation, periodicity is established rapidly through the complex transcriptional regulation of the pair-rule genes. The 'primary' pair-rule genes generate their 7-stripe expression through stripe-specific cis-regulatory elements controlled by the preceding non-periodic maternal and gap gene patterns, whereas 'secondary' pair-rule genes are thought to rely on 7-stripe elements that read off the already periodic primary pair-rule patterns. Using a combination of computational and experimental approaches, a comprehensive systems-level examination was conducted of the regulatory architecture underlying pair-rule stripe formation. runt (run), fushi tarazu (ftz) and odd skipped (odd) were found to establish most of their pattern through stripe-specific elements, arguing for a reclassification of ftz and odd as primary pair-rule genes. In the case of run, long-range cis-regulation was observed across multiple intervening genes. The 7-stripe elements of run, ftz and odd are active concurrently with the stripe-specific elements, indicating that maternal/gap-mediated control and pair-rule gene cross-regulation are closely integrated. Stripe-specific elements fall into three distinct classes based on their principal repressive gap factor input; stripe positions along the gap gradients correlate with the strength of predicted input. The prevalence of cis-elements that generate two stripes and their genomic organization suggest that single-stripe elements arose by splitting and subfunctionalization of ancestral dual-stripe elements. Overall, this study provides a greatly improved understanding of how periodic patterns are established in the Drosophila embryo (Schroeder, 2011).

The transition from non-periodic to periodic gene expression patterns is a key step in the establishment of the segmented body plan of the Drosophila embryo. The pair-rule genes that lie at the heart of this process have been the subject of much investigation, but important questions, in particular regarding the organization of cis-regulation, have remained unresolved. We have revisited the issue in a comprehensive fashion by combining computational and experimental cis-dissection with mutant analysis and a detailed characterization of expression dynamics, both of endogenous genes and of cis-regulatory elements. This systems-level analysis under uniform conditions reveals important insights and gives rise to a refined and in some respects substantially revised view of how periodic patterns are generated (Schroeder, 2011).

The most important finding of this study is that ftz and odd, which had been regarded as secondary pair-rule genes, closely resemble eve, h and run in nearly all respects and should thus be co-classified with them as primary pair-rule genes. Expression dynamics clearly subdivide the pair-rule genes into two distinct groups, with eve, h, run, ftz and odd all showing an early and non-synchronous appearance of most stripes, whereas the 7-stripe patterns of prd and slp1 arise late and synchronously. The early expression of stripes is associated with the existence of stripe-specific cis-elements that respond to positional cues provided by the maternal and gap genes. Unlike previously thought, maternal and gap input is used pervasively in the initial patterning not only of eve, h and run, but also of ftz and odd. Aided by computational predictions, eight new stripe-specific cis-elements were identifed for ftz, odd, and run. Although molecular epistasis experiments reveal a stronger role for eve, h and run in pair-rule gene cross-regulation, odd clearly affects the blastoderm patterning of other primary factors as well and ftz affects the patterning of odd (Schroeder, 2011).

The revised grouping brings the classification of pair-rule genes in line with their role in transmitting positional information to the subsequent tiers in the segmentation hierarchy. By the end of cellularization, the expression patterns of h, eve, run and ftz/odd are offset against each other to produce neatly tiled overlaps of four-nuclei-wide stripes. In combination, these patterns define a unique expression code for each nucleus within the two-segment unit that specifies both position and polarity in the segment and is read off by the segment-polarity genes. ftz (as an activator of odd) and odd together represent the fourth essential component of this positional code and are thus functionally equivalent to h, eve and run. Placing all components of this code under the same direct control of the preceding tier of regulators presumably increases both the speed and the robustness of the process (Schroeder, 2011).

Although the results support a subdivision between primary and secondary pair-rule genes, they also reveal surprising complexities in the regulatory architecture that are not captured by any rigid dichotomy. The five primary pair-rule genes all have different cis-regulatory repertoires: stripe formation in h appears to rely solely on stripe-specific cis-elements, whereas eve employs a handoff from stripe-specific elements to a late-acting 7-stripe element. In run, ftz and odd, by contrast, the emergence of the full 7-stripe pattern is the result of joint action by stripe-specific elements and early-acting 7-stripe elements, with ftz and odd requiring the 7-stripe element to generate a subset of stripes. This difference in the importance of 7-stripe elements in stripe formation is supported by the results of rescue experiments: whereas the 7-stripe elements of both run and ftz provide partial rescue of the blastoderm expression pattern when the stripe-specific elements are missing, this is not the case for eve. Interestingly, run appears to occupy a unique and particularly crucial position among the five genes: it has both a full complement of stripe-specific elements and an early-acting 7-stripe element, and thus serves as an early integration point of maternal/gap input and pair-rule cross-regulation. Its regulatory region is particularly large and complex, with cis-elements acting over long distances across intervening genes and with partial redundancy between elements. Moreover, the removal of run has the most severe effects on pair-rule stripe positioning among the five genes. Another complexity lies in the fact that the early anterior expression of slp1 and prd, which otherwise exhibit all the characteristics of secondary pair-rule genes, has a clear role in patterning the anteriormost stripes of the primary pair-rule genes. In fact, evidence is provided that the most severe defect observed in the primary pair-rule gene mutants, namely the loss of pair-rule gene stripes 1 or 2 under eve loss-of-function conditions, is an indirect effect attributable to the regulation of these stripes by slp1 (Schroeder, 2011).

This investigation provides important new insight regarding the relative roles of maternal/gap input and pair-rule gene cross-regulation in stripe formation. As described, nearly all stripes of the primary pair-rule genes are initially formed through stripe-specific cis-elements. Cross-regulatory interactions between the pair-rule genes then refine these patterns by ensuring the proper spacing, width and intensity of stripes, as revealed by mutant analysis. However, these two aspects or layers of regulation are not as clearly separated as has often been thought. For example, 7-stripe elements have been regarded primarily as conduits of pair-rule cross-regulation; by contrast, this study found that the 7-stripe elements of run, ftz and odd are activefrom phase 1 onwards and contain significant input from the maternal and gap genes, resulting in modulated or partial 7-stripe expression early. In the case of the KR binding sites predicted within the run 7-stripe element, mutational analysis showed that this input is indeed functional. Conversely, stripe-specific cis-elements receive significant regulatory input from pair-rule genes. This is particularly clear in the case of eve and h: expression dynamics indicate that their 7-stripe patterns become properly resolved without the involvement of 7-stripe elements, and the defects in the h and eve expression patterns that were observe in pair-rule gene mutants occur at a time when only stripe-specific elements are active. In a few cases, pair-rule input into stripe-specific elements has been demonstrated directly, but the comparative analysis of expression dynamics underscores that it is indeed a pervasive feature of pair-rule cis-regulation (Schroeder, 2011).

An important question arising from these findings is how the different types of regulatory input are integrated, both at the level of individual cis-elements and at the level of the locus as a whole. Binding sites for maternal and gap factors typically form tight clusters, supporting a modular organization of cis-regulation. Such modularity is crucial for the 'regional' expression of individual stripes, which can be achieved only if repressive gap inputs can be properly separated between cis-elements. By contrast, binding sites for the pair-rule factors appear to be more dispersed across the cis-regulatory regions and not tightly co-clustered with the maternal and gap inputs. Unlike the gap factors, which are thought to act as short-range repressors, with a range of roughly 100 bp, most pair-rule genes act as long-range repressors, with an effective range of at least 2 kb. Pair-rule factor inputs may therefore influence expression outcomes at greater distances along the DNA, which would allow a single cluster of binding sites to affect multiple cis-elements. Remarkably, the 7-stripe elements of ftz, run, eve, prd and odd all combine inputs from promoter-proximal and more distal sequence over distances of at least 5 kb, and, is seen in the case of odd, the combination can involve non-additive effects. Thus, the refinement of expression patterns through pair-rule cross-regulation might rely on interactions over greater distances, consistent with the 'global' role of pair-rule genes as regulators of the entire 7-stripe pattern. How such interactions are realized at the molecular level and how the inputs from stripe-specific and 7-stripe elements are combined to produce a defined transcriptional outcome is currently unknown (Schroeder, 2011).

Taken together with previous studies, these data support a coherent and conceptually straightforward model of how stripes are made in theDrosophila blastoderm. In the trunk region of the embryo, the maternal activators BCD and CAD form two overlapping but anti-correlated gradients; they coarsely specify the expression domains of region-specific gap genes, which become refined by cross-repressive interactions among the gap factors. The result is a tiled array of overlapping gap factor gradients, centered around the bilaterally symmetric domains of KR and KNI, which are flanked on either side by the bimodal domains of GT and HB. The same basic principles are used again in the next step: following the initial positioning of stripes by maternal and gap factor input, cross-repressive interactions among the primary pair-rule genes serve to refine the pattern and ensure the uniform spacing and width of expression domains. The significant correlations that were observe between the strength of KR/KNI input and the position of the resulting stripes relative to the respective gradients supports the notion that these factors function as repressive morphogens in defining the proximal borders of the nascent pair-rule stripes. Importantly, owing to the symmetry of the KR and KNI gradients, combined with the largely symmetric positioning of the flanking GT and HB gradients, the same regulatory input can be used to specify two distinct positions, one on either slope of the gradient; this is exploited in a systematic fashion by dual-stripe cis-elements, which account for the majority of stripe-generating elements. Thus, the key to stripe formation is the translation of transcription factor gradients into an array of narrower, partially overlapping expression domains that are stabilized by cross-repressive interactions; the iteration of this process, combined with the duplication of position through bilateral gradient symmetry, creates a periodic array of stripes that is sufficient to impart a unique identity to each nucleus in the trunk region of the embryo (Schroeder, 2011).

With the exception of the anteriormost pair-rule gene stripes, which are subject to more complex regulation (as evidenced by the crucial role of slp1), this model accounts for most of the stripe formation process. Particularly striking is the similarity between the central gap factors and the primary pair-rule factors with respect to regulatory interactions and expression domain positions. The offset arrangement of two pairs of mutually exclusive gap domains, KNI/HB and KR/GT, is mirrored at the pair-rule gene level, with the anti-correlated and mutually repressive stripes of HAIRY and RUN phase-shifted against the similarly anti-correlated expression patterns of EVE and ODD. The parallel suggests that this regulatory geometry is particularly suited to robustly specify a multiplicity of positions. However, such a circuitry of cross-repressive relationships is compatible with a range of potentially stable expression states and thus is insufficient by itself to uniquely define position along the anterior-posterior axis. Therefore, the initial priming of position by the preceding tier of the regulatory hierarchy is crucial. At the level of the pair-rule genes, the extensive repertoire of maternal/gap-driven cis-elements that initiate stripe expression for all four components of the array is thus necessary to ensure that the cross-regulatory dynamics will drive the correct overall pattern. Finally, to achieve a smooth transition between the tasks of transmitting spatial information from the preceding tier of the hierarchy and refinement/stabilization of the pattern, the two layers of regulation need to be closely integrated. This is likely to be facilitated by the fact that in each of the mutually repressive pairs, one gene generates stripes purely through stripe-specific elements (h, eve), whereas the other has an early-acting 7-stripe element that mediates pair-rule cross-repression and acts concurrently with stripe-specific elements (run, odd) (Schroeder, 2011).

Stripe positioning is not always symmetric around the central gap factor gradients. In such cases, the conflicting needs for appropriate regulatory input into the relevant cis-elements appear to have driven the separation of ancestral dual-stripe elements into more specialized elements optimized for generating a single stripe. The co-localization of the cis-elements that generate stripes 1 and 5 within all pair-rule gene regulatory regions, be it in the form of dual-stripe elements or of adjacent but separable single-stripe elements, provides strong evidence that this is indeed a key mechanism underlying the emergence of single-stripe cis-elements. Given the evolutionary plasticity of regulatory sequence, it is not difficult to envision such a separation and subfunctionalization of cis-elements. How the crucial transition from a non-periodic to periodic pattern is achieved in other insects is a fascinating question. Intriguingly, most of the molecular players and general features of their expression patterns are well conserved beyond Diptera, and it is tempting to speculate that dual-stripe cis-regulation might have arisen through co-option as the blastoderm fate map shifted to include more posterior positions. It will be interesting to see to what extent the mechanisms and principles of stripe formation that we have outlined here apply in other species (Schroeder, 2011).

Targets of Activity

Odd-skipped is a negative regulator of engrailed. The runt gene is required to limit the domains of engrailed expression in the odd numbered parasegments, while the odd-skipped gene is required to limit the domains of en expression in the even-numbered parasegments. Activation of en at the anterior margins of both sets of parasegments requires the repression of runt and odd by the product of the eve gene (Manoukian, 1993). In addition, ODD represses expression of fushi tarazu, a known activator of engrailed (Mullen, 1995).

wingless expression is a regulated by both odd-skipped and the pair-rule gene paired. Odd-skipped represses wg expression, while Paired restricts the domain of expression of odd-skipped .

Although many of the genes that pattern the segmented body plan of the Drosophila embryo are known, there remains much to learn in terms of how these genes and their products interact with one another. Like many of these gene products, the protein encoded by the pair-rule gene odd-skipped (Odd) is a DNA-binding transcription factor. Genetic experiments have suggested several candidate target genes for Odd, all of which appear to be negatively regulated. Pulses of ectopic Odd expression have been used to test the response of these and other segmentation genes. Three different phenotypes are generated in embryos in which odd is expressed from a heat shock promoter: head defects only, a pair-rule phenotype and a pair-rule phenotype restricted to the dorsal half of the embryo. The head defects only phenotype prevails when Odd is induced between 2:10 and 2:30 hours after egg laying (AEL). The second phenotype is generated when Odd is induced between 2:30 and 2:50 AEL, while the third phenotype prevails when heat shocks are administered between 2:50 and 3:10 AEL. The results are complex, indicating that Odd is capable of repressing some genes wherever and whenever Odd is expressed, while the ability to repress others is temporally or spatially restricted (Dréan, 1998).

Two of the seven pair-rule genes tested do not show significant changes in expression at the stages examined. These include the genes odd-paired (opa) and, surprisingly, ftz. In odd minus embryos, ftz stripes do not resolve properly, remaining about 3 cells wide until well into the process of germ band extension. This suggests that Odd may be a repressor of ftz. However ectopic Odd does not repress ftz expression. Also unexpected was the fact that ectopic Odd has effects on all three of the ‘primary’ pair-rule genes. These were previously thought not to be regulated by Odd. In stage 5 embryos, stripe 1 of hairy is efficiently repressed by ectopic Odd. The first stripe of eve is also repressed at this stage. Repression of h stripe 1 continues in older embryos and is accompanied by weaker repression of stripes 2-6. These effects of Odd on h correlate with what appears to be a modest broadening of h stripes in odd-minus embryos, particularly stripe 1. Early repression of the first stripes of h and eve likely accounts for the cuticular head defects that arise from early pulses of ectopic Odd expression. Interestingly, in odd-minus embryos, the entire 7-stripe pattern of h appears to expand, both anteriorly and posteriorly. This is also true of eve and runt stripes. These data provide no explanation for this, but it may explain the fairly consistent spacing of h stripes, despite their apparent broadening (Dréan, 1998).

One target gene, fushi tarazu, is both repressed and activated by Odd, the outcome depending upon the stage of development. Rather than negatively regulating ftz, ectopic Odd causes a rapid expansion of all 7 ftz stripes. In some embryos, interstripe regions are difficult to discern. Since this activation is observed within 20-25 minutes of Odd induction, it likely reflects a direct interaction between the two genes. Consistent with this positive relationship, initiating ftz stripes are irregular in width and intensity in odd mutant embryos. Stripes 3-6 are the most strongly affected, particularly stripe 4. Odd does appear to be able to repress the ftz gene, but only in the middle region of the ftz parasegment, and only after gastrulation. When Odd switches from an activator to a repressor of ftz, its ability to repress ftz is excluded from the anterior-most cells of each ftz stripe. The inability of Odd to repress ftz in these cells indicates that either a necessary cofactor for Odd is missing in these cells, or that an overriding factor is present. Another possibility is that the levels of Odd required to repress ftz are higher than those that were induced. Previous studies could not establish unambiguously whether Odd acts as a direct or indirect repressor of the en and wg genes. The data presented here show that during gastrulation Odd appears to regulate both genes, not only directly, but indirectly as well. Indirect repression is mediated by selective repression of the en and wg activators: ftz, prd, eve and slp. The result of these interactions in hs-odd embryos is first the loss of all fourteen en and wg stripes due to direct repression and then failure of certain stripes to reinitiate. These results indicate that the activity of Odd is highly dependent on the presence of cofactors and/or overriding inhibitors. Based on these results, and the segmental phenotypes generated by ectopic Odd, a number of new roles for Odd in the patterning of embryonic segments are suggested. These include gap-, pair rule- and segment polarity-type functions (Dréan, 1998).

The early expression of the Drosophila segment polarity gene gooseberry is under the control of the pair-rule genes. A 514-bp enhancer, -5.3 to -4.8 kb interval (called fragment IV), has been identified that reproduces the early gsb expression pattern in transgenic flies. The transcription factor Paired (Prd) is the main activator of this enhancer in all parasegments of the embryo. It binds to paired-and homeodomain-binding sites, which are segregated on the enhancer. Using site-directed mutagenesis, sites critical for Prd activity have been identified. Negative regulation of this enhancer is mediated by the Even-skipped protein (Eve) in the odd-numbered parasegments and by the combination of Fushi-tarazu (Ftz) and Odd-skipped proteins in the even-numbered parasegments. The organization of the Prd-binding sites, as well as the necessity for intact DNA binding sites for both the paired- and homeodomain-binding sites, suggests a molecular model whereby the two DNA-binding domains of the Prd protein cooperate in transcriptional activation of gsb. This positive activity appears to be in competition with Eve and Ftz on Prd homeodomain-binding sites (Bouchard, 2000).

The establishment of the posterior border of gsb in the even-numbered parasegments requires an efficient mechanism of repression, since Prd is present throughout all all even-numbered parasegments at the time of gsb initiation. The expression of transgenic line IV-LacZ is derepressed in ftz and odd mutant embryos. Moreover, Prd activity is directly competed by Ftz and Odd in tissue cultured cells. These data identify Ftz and Odd proteins as being responsible for the establishment of gsb expression borders in the even-numbered parasegments (Bouchard, 2000).

In the genetic analysis of fragment IV, it was observed that neither ftz nor odd mutant embryos show a complete derepression in the even-numbered parasegments. An odd embryo shows an anterior widening of the odd-numbered gsb stripes, suggesting a more important role for Odd in the region of low Ftz concentration. This result also indicates that Ftz is a potent repressor in the embryo since it is still able to partially repress gsb, even though Prd levels remain high in the central part of the even-numbered parasegments in an odd embryo, as opposed to its gradual repression in this region in a wild-type background. In a ftz embryo, a posterior widening of two to three cells in the even-numbered stripes is observed. This limited expansion can be explained by the action of Odd in the posteriormost portion of the parasegment combined with the fact that Prd is fading exclusively in this region in a ftz mutant embryo. The true repressor effect of Odd on fragment IV is possibly masked in these genetic experiments by the fact that, in an odd mutant embryo, Ftz is not properly repressed in the posterior portion of the parasegment. In such an embryo, Ftz is thus compensating for the absence of Odd. At the molecular level, the mechanism of action of Odd is unclear. It is possible that Odd binds directly to fragment IV via its zinc-finger domain, but this interaction would have been missed due to insufficient binding activity in vitro. Alternatively, Odd could bind Prd via protein-protein interaction and thereby interfere with its transactivation properties (Bouchard, 2000).

Protein Interactions

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).

odd-skipped: Biological Overview | Evolutionary homologs | Developmental Biology | Effects of Mutation | References

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