rhomboid

Promoter Structure

The promoter has three potential TATA boxes (Bier, 1990).

A 300-bp region of the RHO promoter (the NEE), which is sufficient for neuroectoderm expression, contains a cluster of Dorsal and b-HLH activator sites that are closely linked to SNA repressor sites. The NEE is located from 1500 to 3200 kb upstream from the proximal promoter. Krüppel and Snail can mediate either quenching or direct repression of the transcription complex, depending on the location of repressor sites. Local quenching and dominant repression require close linkage (less than 100 base pairs) of the repressor within either upstream activators or in a proximal promoter adjacent to the the transcription complex. SNA acts on the 300bp rhomboid neuroectodermal enhancer, acting in a competition mechanism to prevent Dorsal activation, but SNA fails to prevent activation when SNA repressor sites are moved away from the closest activators. Likewise KR can repress DL activation of a rhomboid enhancer by a locally acting quenching mechanism (Gray, 1996).

Dorsal protein acts in concert with basic helix-loop-helix (b-HLH) proteins, possibly including Twist, to activate rhomboid in both lateral and ventral regions. Expression is blocked in ventral regions (the presumptive mesoderm) by Snail, which is also a direct target of the DL morphogen. Disruption of SNA-binding sites causes a derepression of the pattern throughout ventral regions, providing evidence that SNA is directly responsible for establishing the mesoderm/neuroectoderm boundary before gastrulation. (Ip, 1992).

Drosophila dorsoventral patterning and mammalian hematopoiesis are regulated by related signaling pathways (Toll, interleukin-1) and transcription factors (Dorsal, Nuclear factor-kappa B). These factors interact with related enhancers, such as the rhomboid NEE and kappa light chain enhancer, that contain similar arrangements of activator and repressor binding sites. The kappa enhancer can generate lateral stripes of gene expression in transgenic Drosophila embryos in a pattern similar to that directed by the rhomboid NEE. Drosophila DV determinants direct these stripes through the corresponding mammalian cis regulatory elements in the kappa enhancer, including the kappa B site and kappa E boxes. These results suggest that enhancers can couple conserved signaling pathways to divergent gene functions (Gonzalez-Crespo, 1994).

Binding the the TFIID complex to a target promoter depends on at least three different core promoter elements located within a 50- to 60-base pair sequence flanking the transcription start site, the TATA box, the initiator element (Inr), and the downstream promoter element (Dpe). In general, promoters that lack a TATA sequence must possess conserved copies of the Inr and/or Dpe. Conversely, promoters containing optimal TATA sequences do not require Inr and Dpe elements for the binding of TFIID. The presences of these three elements define two common types of promoters: type I promoters contain a TATA box, whereas type II promoters contain Inr and Dpe sequences. There are numerous examples of shared enhancers interacting with just a subset of target promoters. These "shared enhancer" type of interactions are contrasted with a "competitive interaction" type. In some cases, specific enhancer-promoter interactions depend on promoter competition, whereby the activation of a preferred target promoter precludes expression of linked genes. A transgenic embryo assay was used to obtain evidence that promoter selection is influenced by the TATA element. Both the AE1 (located between Sex combs reduced and fushi tarazu) enhancer from the Drosophila Antennapedia gene complex (ANT-C) and the IAB5 enhancer (which selectively activates Abdominal-B, not abdominal-A) from the Bithorax complex (BX-C) preferentially activate the type I, TATA-containing, promoters when challenged with linked TATA-less promoters. The AE1 autoregulatory element in the ANT-C specifically interacts with the ftz promoter, but does not activate the equidistant Sex combs reduced gene. AE1 and IAB5 exhibit a competitive type of interaction. In contrast, the rho neuroectoderm enhancer (NEE) does not discriminate between type I and type II classes of promoters and exhibit a shared enhancer type of interaction. Thus, certain upstream activators, such as Ftz, prefer TATA-containing promoters, whereas other activators, including Dorsal, work equally well on both classes of promoters (Ohtsuki, 1998).

Related artifically constructed core promoter sequences were initially used for the analysis of AE1. ftz and eve contain optimal TATA sequences, but lack Inr (INIT) and Dpe (DPE) elements. AE1 also activates white and Tp promoters. white and Tp each contain conserved copies of the INIT and DPE sequences, but lack a TATA sequence (white) or contains a suboptimal TATA (Tp). AE1 can simultaneously activate linked TATA-containing promoters or linked INIT/DPE-containing promoters. In spite of AE1's ability to activate type I and type II promoters, promoter competition can be demonstrated. There is a substantial reduction in white expression when the Tp promoter is replaced with the core eve promoter sequence. This AE1-eve interaction appears to block the expression of the linked white gene. In the absence of eve, white is fully active. These observations are compatible with a promoter-competition mechanism whereby AE1-eve interactions inhibit white (Ohtsuki, 1998).

Similarly, IAB5 prefers the eve promoter. The 1-kb IAB5 enhancer exhibits a preference for TATA-containing promoters. IAB5 was placed downstream of an eve/lacZ fusion gene; the linked CAT reporter gene was placed under the control of the mini-white promoter. There is strong expression of the lacZ reporter gene in the presumptive abdomen, whereas CAT is not expressed above background levels. This result suggests that IAB5 prefers the eve promoter over white. An eve-white chimeric promoter was analyzed in an effort to assess the importance of the core elements, particularly the TATA sequence. An ~20-bp region of the eve sequence (the TATA region) was replaced with the corresponding region of white. This modified eve promoter (evewhite) is attenuated and mediates only weak expression of lacZ in the presumptive abdomen. In contrast, the linked white promoter directs strong expression of CAT. These results suggest that the removal of the eve TATA releases the IAB5 enhancer so that it can now interact with the white promoter (Ohtsuki, 1998).

The 300-bp rhomboid NEE is equally effective in activating the two classes of promoters. Additional experiments were done to determine whether the targeting of IAB5 to eve influences the activities of the nonspecific rho NEE. The latter enhancer is activated by the maternal gradient of Dorsal transcription factor in lateral stripes within the neurogenic ectoderm. A synthetic gene complex was prepared that contains both the NEE and IAB5 enhancers. white and CAT reporter genes were attached to the mini-white promoter, whereas lacZ is driven by eve. The rho NEE activates all three reporter genes, so that white, CAT, and lacZ are all expressed in lateral stripes. In contrast, IAB5 primarily activates the eve promoter, so that only lacZ exhibits strong expression within the presumptive abdomen. These results suggest that IAB5-eve interactions do not influence the nonspecific activities of the rho NEE (Ohtsuki, 1998).

It has been suggested that TATA-containing promoters are intrinsically stronger than TATA-less promoters, possibly because of higher affinity interactions with the TFIID complex. The divergent activities of the IAB5 and NEE enhancers, however, are most easily interpreted on the basis of qualitative, not quantitative, differences in type I and type II core promoter sequences. For example, the insertion of a TATA sequence in the white promoter allows it to compete with a linked eve promoter, whereas the removal of TATA from eve permits activation of white. These alterations in the white and eve promoters, the insertion and removal of TATA, dramatically alter the activities of IAB5, but have virtually no effect on the NEE enhancer. NEE is equally effective in activating the eve, white, evewhite, and whiteTATA promoters, and thereby serves as an internal control for normal promoter function (Ohtsuki, 1998).

These results suggest that the IAB5 and AE1 activators, particularly Ftz, prefer type I promoters. NEE activators, including Dorsal (dl) and bHLH proteins, appear to be promiscuous and work equally well on both classes of core promoters. The authors propose that the TFIID complex adopts different conformations on type I and type II promoters. Basal targets for the Ftz activator may be displayed in a more accessible conformation when TFIID binds TATA. In contrast, basal targets for the Dorsal and bHLH activators may be equally accessible whether TFIID binds TATA or Inr/Dpe elements (Ohtsuki, 1998).

Neurogenesis depends on a family of proneural transcriptional activator proteins, but the 'proneural' function of these factors is poorly understood, in part because the ensemble of genes they activate, directly or indirectly, has not been identified systematically. A direct approach to this problem has been undertaken in Drosophila. Fluorescence-activated cell sorting was used to recover a purified population of the cells that comprise the 'proneural clusters' from which sensory organ precursors of the peripheral nervous system (PNS) arise. Whole-genome microarray analysis and in situ hybridization was then used to identify and verify a set of genes that are preferentially expressed in proneural cluster cells. Genes in this set encode proteins with a diverse array of implied functions, and loss-of-function analysis of two candidate genes shows that they are indeed required for normal PNS development. Bioinformatic and reporter gene studies further illuminate the cis-regulatory codes that direct expression in proneural clusters (Reeves, 2005).

Patterned expression of the proneural genes ac and sc defines the PNCs for most external sensory bristles in adult Drosophila, and ac-sc function is required for PNC and SOP gene expression, as well as for specification of the SOP cell fate. Fifteen of the genes identified by the combined cell sorting/microarray approach also require proneural gene function for their expression. In an ac⁻ sc⁻ proneural mutant background, transcript accumulation from members of both the PNC (CG11798, CG32434/loner, edl, PFE) and SOP (CG3227, CG30492, CG32150, CG32392, Men, qua) classes is lost from PNCs that require ac-sc function. This result is further evidence that the approach has identified bona fide PNC genes, and it demonstrates that expression of these ten genes is, directly or indirectly, downstream of the bHLH activators encoded by ac and sc. The data further show that the PNC-specific imaginal disc expression of the previously studied genes mira, phyl, rho, Spn43Aa, and Traf1 is likewise downstream of proneural gene function (Reeves, 2005).

The identification of sets of genes comprising the genetic programs deployed in PNCs and SOPs by the action of proneural proteins offers a powerful opportunity to investigate the regulatory organization of these programs. Specifically, it was of interest to find out (1) which genes are directly activated by proneural regulators, and which indirectly, and (2) the nature of the cis-regulatory sequences and their cognate transcription factors that distinguish PNC- versus SOP-specific target gene expression. This analysis was initiated by examining potential regulatory sequences of several of the genes that have been identified for the presence of conserved, high-affinity proneural protein binding sites of the form RCAGSTG. The initial approach was to ask whether evolutionarily conserved clusters of these binding sites identify cis-regulatory modules of the appropriate specificity. To date, this strategy has proven very successful. Genomic DNA fragments bearing proneural protein binding site clusters associated with CG11798, edl, Traf1, CG32434/loner, and rho confer PNC-specific activity on a heterologous promoter, while similar modules from CG32150, mira, and PFE drive SOP-specific expression. In three cases, double labeling with the SOP marker anti-Hindsight (Hnt) reveals that PNC-specific expression of the reporter gene includes the SOP as well as the non-SOP cells. Mutation of the proneural protein binding sites in four of the enhancer-bearing fragments severely reduces (CG11798) or abolishes (CG32150, edl, Traf1) reporter gene expression in PNCs/SOPs. Such results indicate that these genes are indeed direct targets of activation by proneural proteins in vivo (Reeves, 2005).

A regulatory code involving Dorsal, Twist and Su(H) regulate rhomboid

Bioinformatics methods have identified enhancers that mediate restricted expression in the Drosophila embryo. However, only a small fraction of the predicted enhancers actually work when tested in vivo. In the present study, co-regulated neurogenic enhancers that are activated by intermediate levels of the Dorsal regulatory gradient are shown to contain several shared sequence motifs. These motifs permit the identification of new neurogenic enhancers with high precision: five out of seven predicted enhancers direct restricted expression within ventral regions of the neurogenic ectoderm. Mutations in some of the shared motifs disrupt enhancer function, and evidence is presented that the Twist and Su(H) regulatory proteins are essential for the specification of the ventral neurogenic ectoderm prior to gastrulation. The regulatory model of neurogenic gene expression defined in this study permitted the identification of a neurogenic enhancer in the distant Anopheles genome. The prospects for deciphering regulatory codes that link primary DNA sequence information with predicted patterns of gene expression are discussed (Markstein, 2004).

Previous studies identified two enhancers, from the rho and vnd genes, that are activated by intermediate levels of the Dorsal gradient in ventral regions of the neurogenic ectoderm. The present study identified a third such enhancer from the brk gene. This newly identified brk enhancer corresponds to one of the 15 optimal Dorsal-binding clusters described in a previous survey of the Drosophila genome. Although one of these 15 clusters has been shown to define an intronic enhancer in the short gastrulation (sog) gene, the activities of the remaining 14 clusters were not tested. Genomic DNA fragments corresponding to these 14 clusters were placed 5' of a minimal eve-lacZ reporter gene, and separately expressed in transgenic embryos using P-element germline transformation. Four of the 14 genomic DNA fragments were found to direct restricted patterns of lacZ expression across the dorsoventral axis that are similar to the expression patterns seen for the associated endogenous genes (Markstein, 2004).

The four enhancers respond to different levels of the Dorsal nuclear gradient. Two direct expression within the presumptive mesoderm where there are high levels of the gradient. These are associated with the Phm and Ady43A genes. The third enhancer maps ~10 kb 5' of brk, and is activated by intermediate levels of the Dorsal gradient, similar to the vnd and rho enhancers. Finally, the fourth enhancer maps over 15 kb 5' of the predicted start site of the CG12443 gene, and directs broad lateral stripes throughout the neurogenic ectoderm in response to low levels of the Dorsal gradient. In terms of the dorsoventral limits, this staining pattern is similar to that produced by the sog intronic enhancer (Markstein, 2004).

The remaining ten clusters failed to direct robust patterns of expression and are thus referred to as 'false-positives'. Since analysis of spacing and orientation of the Dorsal sites alone did not reveal features that could discriminate between the false positives and the enhancers, whether additional sequence motifs could aid in this distinction was examined. A program called MERmaid was developed that identifies motifs over-represented in specified sets of sequences. MERmaid analysis identified a group of motifs, which was largely specific to the brk, vnd and rho enhancers, suggesting that the regulation of these coordinately expressed genes is distinct from the regulation of genes that respond to different levels of nuclear Dorsal (Markstein, 2004).

The rho, vnd and brk enhancers direct similar patterns of gene expression. The rho and vnd enhancers were previously shown to contain multiple copies of two different sequence motifs: CTGNCCY and CACATGT. A three-way comparison of minimal rho, vnd and brk enhancers permitted a more refined definition of the CTGNCCY motif (CTGWCCY), and also allowed for the identification of a third motif, YGTGDGAA. The CACATGT and YGTGDGAA motifs bind the known transcription factors, Twist and Suppressor of Hairless [Su(H)], respectively. All three motifs are over-represented in authentic Dorsal target enhancers directing expression in the ventral neurogenic ectoderm, as compared with the 10 false-positive Dorsal-binding clusters. Some of the false-positive clusters contain motifs matching either Twist or CTGWCCY; however, none of the false-positive clusters contain representatives of both of these motifs. The rho enhancer is repressed in the ventral mesoderm by the zinc-finger Snail protein. The four Snail-binding sites contained in the rho enhancer share the consensus sequence, MMMCWTGY; the vnd and brk enhancers contain multiple copies of this motif and are probably repressed by Snail as well (Markstein, 2004).

The functional significance of the shared sequence motifs was assessed by mutagenizing the sites in the context of otherwise normal lacZ transgenes. Previous studies have suggested that bHLH activators are important for the activation of rho expression, since rho-lacZ fusion genes containing point mutations in several different E-box motifs (CANNTG) exhibited severely impaired expression in transgenic embryos. However, it was not obvious that the CACATGT motif was particularly significant since it represents only one of five E-boxes contained in the rho enhancer. Yet, only this particular E-box motif is significantly over-represented in the rho, vnd and brk enhancers. vnd-lacZ and brk-lacZ fusion genes were mutagenized to eliminate each CACATGT motif, and analyzed in transgenic embryos. The loss of these sites causes a narrowing in the expression pattern of an otherwise normal vnd-lacZ fusion gene. By contrast, the brk pattern is narrower in central and posterior regions, but relatively unaffected in anterior regions. The brk enhancer contains two copies of an optimal Bicoid-binding site, and it is possible that the Bicoid activator can compensate for the loss of the CACATGT motifs in anterior regions (Markstein, 2004).

Similar experiments were performed to assess the activities of the Su(H)-binding sites (YGTGDGAA) and the CTGWCCY motif. Mutations in the latter sequence cause only a slight reduction and irregularity in the activity of the vnd enhancer, whereas similar mutations nearly abolish expression from the brk enhancer. Thus, CTGWCCY appears to be an essential regulatory element in the brk enhancer, but not in the vnd enhancer. Mutations in both Su(H) sites in the brk enhancer caused reduced staining of the lacZ reporter gene, suggesting that Su(H) normally activates expression. Further evidence that Su(H) mediates transcriptional activation was obtained by analyzing the endogenous rho expression pattern in transgenic embryos carrying an eve stripe 2 transgene with a constitutively activated form of the Notch receptor (Notch^IC). rho expression is augmented and slightly expanded in the vicinity of the stripe2-Notch^IC transgene. A similar expansion is observed for the sim expression pattern (Markstein, 2004).

To determine whether the shared motifs would help identify additional ventral neurogenic enhancers, the genome was surveyed for 250 bp regions containing an average density of one site per 50 bp and at least one occurrence of each of the four motifs for Dorsal, Twist, Su(H) and CTGWCCY. In total, only seven clusters were identified. Three of the seven clusters correspond to the rho, vnd and brk enhancers. Two of the remaining clusters are associated with genes that are known to be expressed in ventral regions of the neurogenic ectoderm: vein and sim. Both clusters were tested for enhancer activity by attaching appropriate genomic DNA fragments to a lacZ reporter gene and then analyzing lacZ expression in transgenic embryos. The cluster associated with vein is located in the first intron, about 7 kb downstream of the transcription start site. The vein cluster (497 bp) directs robust expression in the neurogenic ectoderm, similar to the pattern of the endogenous gene. The cluster located in the 5' flanking region of the sim gene (631 bp) directs expression in single lines of cells in the mesectoderm (the ventral-most region of the neurogenic ectoderm), just like the endogenous expression pattern. These results indicate that the computational methods define an accurate regulatory model for gene expression in ventral regions of the neurogenic ectoderm of D. melanogaster (Markstein, 2004).

To assay the generality of these findings, genomic regions encompassing putative sim orthologs from the distantly related dipteran Anopheles gambiae were scanned for clustering of Dorsal, Twist, Su(H), CTGWCCY and Snail motifs. One cluster located 865 bp 5' of a putative sim ortholog contains one putative Dorsal binding site, two Su(H) sites, three CTGWCCY motifs (or close matches to this motif), a CACATG E-box and several copies of the Snail repressor sequence MMMCWTGY. A genomic DNA fragment encompassing these sites (976 bp) was attached to a minimal eve-lacZ reporter gene and expressed in transgenic Drosophila embryos. The Anopheles enhancer directs weak lateral lines of lacZ expression that are similar to those obtained with the Drosophila sim enhancer. These results suggest that the clustering of Dorsal, Twist, Su(H) and CTGWCCY motifs constitutes an ancient and conserved code for neurogenic gene expression (Markstein, 2004).

This study defines a specific and predictive model for the activation of gene expression by intermediate levels of the Dorsal gradient in ventral regions of the neurogenic ectoderm. The model identified new enhancers for sim and vein in the Drosophila genome, as well as a sim enhancer in the distant Anopheles genome. Five of the seven composite Dorsal-Twist-Su(H)-CTGWCCY clusters in the Drosophila genome correspond to authentic enhancers that direct similar patterns of gene expression. This hit rate represents the highest precision so far obtained for the computational identification of Drosophila enhancers based on the clustering of regulatory elements. Nevertheless, it is still not a perfect code (Markstein, 2004).

Two of the seven composite clusters are likely to be false-positives: they are associated with genes that are not known to exhibit localized expression across the dorsoventral axis. It is possible that the order, spacing and/or orientation of the identified binding sites accounts for the distinction between authentic enhancers and false-positive clusters. For example, there is tight linkage of Dorsal and Twist sites in each of the five neurogenic enhancers. This linkage might reflect Dorsal-Twist protein-protein interactions that promote their cooperative binding and synergistic activities. Previous studies identified particularly strong interactions between Dorsal and Twist-Daughterless (Da) heterodimers. Da is ubiquitously expressed in the early embryo and is related to the E12/E47 bHLH proteins in mammals. Dorsal-Twist linkage is not seen in one of the two false-positive binding clusters (Markstein, 2004).

The regulatory model defined by this study probably fails to identify all enhancers responsive to intermediate levels of the Dorsal gradient. There are at least 30 Dorsal target enhancers in the Drosophila genome, and it is possible that 10 respond to intermediate levels of the Dorsal gradient. Thus, half of all such target enhancers might have been missed. Perhaps the present study defined just one of several 'codes' for neurogenic gene expression (Markstein, 2004).

The possibility of multiple codes is suggested by the different contributions of the same regulatory elements to the activities of the vnd and brk enhancers. Mutations in the CTGWCCY motifs nearly abolish the activity of the brk enhancer, but have virtually no effect on the vnd enhancer. Future studies will determine whether there are distinct codes for Dorsal target enhancers that respond to either high or low levels of the Dorsal gradient. Indeed, it is somewhat surprising that the sog and CG12443 enhancers essentially lack Twist, Su(H) and CTGWCCY motifs, even though they direct lateral stripes of gene expression that are quite similar (albeit broader) to those seen for the rho, vnd and brk enhancers (Markstein, 2004).

This study provides direct evidence that Twist and Su(H) are essential for the specification of the neurogenic ectoderm in early embryos. The Twist protein is transiently expressed at low levels in ventral regions of the neurogenic ectoderm. SELEX assays indicate that Twist binds the CACATGT motif quite well. The presence of this motif in the vnd, brk and sim enhancers, and the fact that it functions as an essential element in the vnd and brk enhancers, strongly suggests that Twist is not a dedicated mesoderm determinant, but that it is also required for the differentiation of the neurogenic ectoderm. However, it is currently unclear whether the CACATGT motif binds Twist-Twist homodimers, Twist-Da heterodimers or additional bHLH complexes in vivo. Su(H) is the sequence-specific transcriptional effector of Notch signaling. The restricted activation of sim expression within the mesectoderm depends on Notch signaling; however, the rho, vnd and brk enhancers direct expression in more lateral regions where Notch signaling has not been demonstrated. Nonetheless, mutations in the two Su(H) sites contained in the brk enhancer cause a severe impairment in its activity. This observation raises the possibility that Su(H) can function as an activator, at least in certain contexts, in the absence of an obvious Notch signal (Markstein, 2004).

The Dorsal gradient produces three distinct patterns of gene expression within the presumptive neurogenic ectoderm. It is proposed that these patterns arise from the differential usage of the Su(H) and Dorsal activators. Enhancers that direct progressively broader patterns of expression become increasingly more dependent on Dorsal and less dependent on Su(H). The sog and CG12443 enhancers mediate expression in both ventral and dorsal regions of the neurogenic ectoderm, and contain several optimal Dorsal sites but no Su(H) sites. By contrast, the sim enhancer is active only in the ventral-most regions of the neurogenic ectoderm, and contains just one high-affinity Dorsal site but five optimal Su(H) sites. The reliance of sim on Dorsal might be atypical for genes expressed in the mesectoderm. For example, the m8 gene within the Enhancer of split complex may be regulated solely by Su(H). The Anopheles sim enhancer might represent an intermediate between the Drosophila sim and m8 enhancers, since it contains optimal Su(H) sites but only one weak Dorsal site. This trend may reflect an evolutionary conversion of Su(H) sites to Dorsal sites, and the concomitant use of the Dorsal gradient to specify different neurogenic cell types. A testable prediction of this model is that basal arthropods use Dorsal solely for the specification of the mesoderm and Su(H) for the patterning of the ventral neurogenic ectoderm (Markstein, 2004).

Deciphering a transcriptional regulatory code: modeling short-range repression in the Drosophila embryo

Systems biology seeks a genomic-level interpretation of transcriptional regulatory information represented by patterns of protein-binding sites. Obtaining this information without direct experimentation is challenging; minor alterations in binding sites can have profound effects on gene expression, and underlie important aspects of disease and evolution. Quantitative modeling offers an alternative path to develop a global understanding of the transcriptional regulatory code. Recent studies have focused on endogenous regulatory sequences; however, distinct enhancers differ in many features, making it difficult to generalize to other cis-regulatory elements. This study applied a systematic approach to simpler elements and presents the first quantitative analysis of short-range transcriptional repressors, which have central functions in metazoan development. Fractional occupancy-based modeling uncovered unexpected features of these proteins' activity that allow accurate predictions of regulation by the Giant, Knirps, Krüppel, and Snail repressors, including modeling of an endogenous enhancer. This study provides essential elements of a transcriptional regulatory code that will allow extensive analysis of genomic information in Drosophila melanogaster and related organisms (Fakhouri, 2010).

In this study, by using a reductionist analysis of short-range repression, a relatively untouched, yet central aspect of gene regulation was explored in Drosophila. Earlier qualitative studies highlighted the extreme distance dependence of short-range repressors, and comparative analysis has shown many instances of evolutionary plasticity of regulatory regions controlled by these proteins. Knowing that transcription factors influence each other in a local manner permitted the identification of novel enhancers, based on the clustering of binding sites. Yet, clustering studies alone do not provide the basis for predicting evolutionary changes that reshape transcriptional output, or predicting activity of coregulated enhancers. For example, the original hypothesis that the affinity and or number of Bicoid-binding sites dictates the output of regulated genes has been replaced by an understanding that other, as-yet unknown features, seem to have more decisive functions (Fakhouri, 2010).

Earlier modeling studies focused on endogenous enhancers, which have complex arrangements of transcription factor-binding sites. The curret studies focused on detecting quantitative differences resulting from subtle differences in binding sites, allowing modeling with a tractable number of parameters. A common block of Dorsal and Twist activator sites was used, allowing a focus on changes made in the number and arrangement of repressor sites; clearly, differences in affinity, number, and arrangement of activator sites also have decisive functions in dictating transcriptional output; thus, future modeling efforts will need to integrate these elements as well. The tight focus on short-range repressors with the analysis of a relatively small number of reporter genes provided sufficient data for robust estimation of important parameters. From the comparison of repression by other short-range repressors, it is likely that the analysis of Giant can guide studies of other similarly acting repressors, including Krüppel, Knirps, and Snail (Fakhouri, 2010).

Relating to transcriptional regulatory code, this study uncovered specific quantitative features that seem to apply to short-range repressors in a general context. A complex non-linear quenching relationship was found that suggests that within the range of activity, Giant, and probably other short-range repressors, have an optimum distance of action that may reflect steric constraints. Multiple formulations of the model generated very similar predictions, suggesting that this non-linear distance function is a real feature of the system. Consistent with this notion, an earlier study of transcription factor-binding sites in Drosophila enhancers discovered an overall preference of Krüppel sites to be found 17 bp from Bicoid activator sites, which may be an indication that other short-range repressors also have preferred distances for optimal activity (Fakhouri, 2010).

The similar quenching efficiencies for repressors acting adjacent to Dorsal or Twist activator sites were an additional significant finding. The similar effect on disparate activator proteins indicates that the effects of short-range repression are general, and are likely to be translatable to distinct contexts. Earlier empirical tests had already pointed in this direction; for example, insertion of ectopic-binding sites for Knirps and Krüppel into rho NEE sequences is sufficient to induce repression, although these proteins do not usually cross-regulate. In addition, short-range repressors can counteract a variety of transcriptional activation domains with similar efficiency, suggesting that specific protein-protein contacts are not essential. In one area quantitative differences were found between parameters derived from the synthetic gene modules and the endogenous regulatory regions. The importance of homotypic cooperativity predicted for Snail sites in the context of the rho NEE was overall much higher than that found for Giant, Krüppel, and Knirps sites acting on the synthetic gene constructs; this might be an example in which the individual proteins do exhibit different context dependencies perhaps because the proteins differ in level of stickiness. Alternatively, the distance between the Snail sites in question, 23 bp, might facilitate cooperative interactions much more than the closely apposed spacing used in the genes genes used in this study, in which steric interference may have an opposing function (Fakhouri, 2010).

In modeling mutant forms of the endogenous rho NEE, several important features of the architecture of this regulatory region were uncovered. This enhancer seems to use redundancy in use of Snail to mediate repression; based on earlier experiments, it seems that even a single Snail site is sufficient to mediate repression. Such redundancy may provide the correct dynamical response, with a swift repression of rho at an early enough time in which Snail levels are still low, or it may ensure that gene output is robust to environmental and genetic noise (Fakhouri, 2010).

The rho NEE modeling also highlighted features of transcriptional activators. Activator-scaling factors for Dorsal were reproducibly lower than those of Twist, and this was apparent for several different assumptions of expression level. The relative differences in contribution to activation can be explained by examination of the structure of the enhancer; contribution by the low intrinsic values of Dorsal is amplified by strong cooperativity with Twist, setting up a chain of interacting weak sites that together are highly active. Experimental evidence bears out these conclusions: isolated Dorsal sites tested on reporter genes mediate relatively weak activation, and a rho NEE lacking Twist sites, but containing four Dorsal sites, is similarly compromised (Fakhouri, 2010).

Earlier studies suggested that many developmental enhancers, including those regulated by short-range repressors, may possess a flexible 'billboard' design, in which individual factors or small groups of proteins would independently communicate with the promoter region, so that the net output of an enhancer would reflect the cumulative set of contacts over a short time period. Such a view of enhancers would account for the evolutionary plasticity observed in regulatory sequences. No DNA-scaffolded superstructure, reflecting the formation of a unique three-dimensional complex, would be necessary in this scenario. Yet, the modeling suggests that the rho NEE might involve communication between relatively distant-binding sites, through sets of cooperative interactions. In this case, it is possible that such distant interactions might be compatible with a flexible structure, if many distinct configurations of binding sites provide such a cooperative network. Current studies have indeed highlighted potential frameworks involving Dorsal and interacting factors on same classes of enhancer. Application of a transcriptional regulatory code integrating activities of activators and repressors is a critical next step to illuminate enhancer design and evolution (Fakhouri, 2010).

Soma-dependent modulations contribute to divergence of rhomboid expression during evolution of Drosophila eggshell morphology

Patterning of the respiratory dorsal appendages (DAs) on the Drosophila melanogaster eggshell is tightly regulated by epidermal growth factor receptor (EGFR) signaling. Variation in the DA number is observed among Drosophila species; D. melanogaster has two DAs and D. virilis has four. Diversification in the expression pattern of rhomboid (rho), which activates EGFR signaling in somatic follicle cells, could cause the evolutionary divergence of DA numbers. Here we identified a cis-regulatory element of D. virilis rho. A comparison with D. melanogaster rho enhancer and activity studies in homologous and heterologous species suggested that these rho enhancers did not functionally diverge significantly during the evolution of these species. Experiments using chimeric eggs composed of a D. virilis oocyte and D. melanogaster follicle cells showed the evolution of DA number was not attributable to germline Gurken (Grk) signaling, but to divergence in events downstream of Grk signaling affecting the rho enhancer activity in somatic follicle cells. A transcription factor, Mirror, which activates rho, could be one of these downstream factors. Thus, evolution of the trans-regulatory environment that controls rho expression in somatic follicle cells could be a major contributor to the evolutionary changes in DA number (Nakamura, 2007).

Changes in gene expression patterns during evolution can be attributed to two distinct mechanisms. First, alterations in the cis-regulatory sequence of a gene can be responsible for the divergence of its expression pattern. Second, changes in trans-regulatory factors can cause gene expression patterns to diverge, even if the cis-regulatory elements of these genes are conserved during evolution. Diversification in enhancer elements is known to contribute predominantly to the evolution of animal morphology. The gain and loss of cis-acting elements have played central roles in the divergence of the expression patterns of genes that play crucial roles in the generation of specific characteristics in different species. This study investigated the contribution of these two processes to the evolutionary diversification of DA numbers in D. virilis and D. melanogaster. In addition to the importance of cis-regulatory elements, the current findings suggest that the landscape of trans-regulatory factors could also change and affect morphological divergence during evolution (Nakamura, 2007).

In D. melanogaster, rho expression has an instructive role in defining the pattern of DA precursor cell formation. In addition, it has been demonstrated that the expression patterns of rho diverged and were correlated with the position and number of DAs in D. virilis and D. melanogaster (Nakamura, 2003). Therefore, in this study, focused was placed on the enhancers of rho in these species. To distinguish whether divergence in the trans-regulatory landscape or the cis-regulatory elements is important for the evolutionary change in rho expression patterns between D. melanogaster and D. virilis, reporter constructs of Dvir rho^4.2 and Dmel rho^2.2 were introduced into these two species. Phylogenic analyses of Drosophila species suggest that the four DAs are an ancestral characteristic, and that the flies with two DAs evolved from four-DA ancestors. Thus, the characteristics of Dmel rho^2.2 were probably derived from the ancestral Dvir rho^4.2 enhancer. It was found that Dvir rho^4.2 and Dmel rho^2.2 adopted the expression pattern of the endogenous rho of the heterologous species. These results suggest that Dvir rho^4.2 and Dmel rho^2.2 did not diverge in terms of their ability to respond to the trans-acting factors in follicle cells. Therefore, it is speculated that changes in the cis-regulatory elements from Dvir rho^4.2 to Dmel rho^2.2 were not the main cause for divergence in the activation patterns of these enhancers in their homologous species (Nakamura, 2007).

Although the DNA sequences of Dvir rho^4.2 and Dmel rho^2.2 diverged drastically, several putative binding sites for transcription factors, such as ETS, Su(H) and BR-C, were common to both, which could explain the conserved function of the two enhancers. Recently, it was reported that BR-C represses the activity of Dmel rho^2.2 in a cell-autonomous manner during DA patterning (Ward, 2006), and this repression allows the enhancer to be activated in the L-shaped region. These two rho enhancers share five overlapping binding sites for BR-C. Thus, these BR-C-binding sites might serve as cis-regulatory elements to transmit the conserved functions of these two enhancers. Notch (N) signaling also regulates Dmel rho^2.2 (Ward, 2006), and it was found that one binding site for Su(H) is conserved among all six Drosophila species examined. Conservation of the binding sites for these various transcription factors and their possible involvement in the evolution of DA patterning suggest that rho expression is controlled by complex responses to multiple transcription factors, instead of by a simple EGFR-signal feedback system (Nakamura, 2007).

Mirr was identified as a candidate for the difference in the landscape of trans-regulatory factors between D. melanogaster and D. virilis. The distribution of the mirr transcript was significantly different between these species. mirr induces rho expression, and regulates N signaling by repressing fringe, probably thereby regulating rho. Although whether or not Mirr function is also involved in the regulation of rho transcription in D. virilis remains to be tested, it is conceivable that changes in the expression patterns of mirr may account, at least in part, for the divergence in the activation patterns of Dvir rho^4.2 and Dmel rho^2.2 in D. melanogaster and D. virilis (Nakamura, 2007).

In D. melanogaster, rho is expressed in a saddle-shaped pattern at stage 10A. This study analyzed the genomic region within 26.2-kb upstream and 11.8-kb downstream of the transcription initiation site of rho, but failed to identify an enhancer element responsible for this early expression pattern. The function of this early rho expression in DA formation has not yet been studied. Therefore, the possibility could not be excluded that an enhancer that regulates the early expression of rho is involved in the diversification of the rho expression pattern. However, it is speculated that this early expression of rho does not play a significant role in determining the number of DAs, because D. pseudoobscura and D. melanica have eggs with two DAs, but the saddle-shaped pattern of rho expression was not detected in these species. Therefore, the subsequent expression of rho is probably what plays a crucial role in determining the DA number (Nakamura, 2007).

The present analysis revealed that the functions of Dvir rho^4.2 and Dmel rho^2.2 are largely conserved. However, it was also found that Dmel rho^2.2 had evolved a novel trait during its diversification from Dvir rho^4.2. In D. melanogaster, both Dvir rho^4.2 and Dmel rho^2.2 were activated in the L-shaped pattern at stage 10B. However, Dvir rho^4.2 was activated in one or two extra rows of cells posterior to the single row of cells where Dmel rho^2.2 was active at this stage. At stage 12, Dvir rho^4.2 was activated much more posteriorly, although Dmel rho^2.2 was still active only in the single row of cells. Given that Dvir rho^4.2 is ancestral to Dmel rho^2.2, it is speculated that Dmel rho^2.2 lost a cis-acting element capable of being activated in this posterior region, or gained a cis-acting element that suppresses its activity in this region at stage 12. Indeed, it is likely that this posterior activation of rho is an ancestral characteristic, because the endogenous expression of rho in this region is found in D. virilis but not D. melanogaster (Nakamura, 2007).

For the formation of DAs, the patterning of EGFR signaling activity in the follicle cells plays crucial roles in D. melanogaster. Two major events are involved in the regulation of EGFR signaling activity in these cells: (1) Grk specifically localizes to the dorsal anterior part of the oocyte and activates EGFR in the overlying follicle cells; (2) in the follicle cells, positive and negative feedback loops elaborate the pattern of EGFR signaling activity that ultimately determines the number of DAs. Thus, the first and second events are germ- and soma-derived events, respectively (Nakamura, 2007).

As predicted from the above model, the intensity of Grk expression and the width of its expression domain in the oocyte are thought to define the number of DAs. A mathematical study predicted that changes in the amount and distribution of Grk protein in the oocyte can account for the evolution of eggshells with zero to four DAs in Drosophila species. However, the current experiments involving a chimeric egg chamber suggest that changes in the follicle cells, but not in the oocyte, have an instructive role in determining the number of DAs. These results suggest that the change in Grk signaling did not contribute to the evolution of DA numbers in these species. This is consistent with a previous finding that the distribution and amount of grk mRNA do not show a significant difference between D. melanogaster and D. virilis. However, the current results do not exclude the possibility that changes in Grk signaling play major roles in the diversification of DA numbers during the evolution of other Drosophila species (Nakamura, 2007).

Hox and senseless antagonism functions as a molecular switch to regulate EGF secretion in the Drosophila PNS

Hox factors are key regulators of distinct cells, tissues, and organs along the body plan. However, little is known about how Hox factors regulate cell-specific gene expression to pattern diverse tissues. This study shows an unexpected Hox transcriptional mechanism: the permissive regulation of EGF secretion, and thereby cell specification, by antagonizing the Senseless transcription factor in the peripheral nervous system. rhomboid expression in a subset of sensory cells stimulates EGF secretion to induce hepatocyte-like cell development. A rhomboid enhancer was identified that is active in these cells; an abdominal Hox complex directly competes with Senseless for enhancer binding, with the transcriptional outcome dependent upon their relative binding activities. Thus, Hox-Senseless antagonism forms a molecular switch that integrates neural and anterior-posterior positional information. As the vertebrate Senseless homolog is essential for neural development as well as hematopoiesis, it is proposed Hox-Senseless antagonism will broadly control cell fate decisions (Li-Kroeger, 2008).

Hox genes have long been known to specify distinct cell types along the body axes of both vertebrates and invertebrates. However, it has remained elusive how Hox factors regulate transcription in a tissue- or cell-specific manner. In this study, a Hox-regulated enhancer (Rho654) active within a subset of PNS cells was identified. Rho654 drives gene expression in abdominal C1-SOP cells to induce oenocytes, and an Exd/Hth/Abd-A complex stimulates gene expression by directly competing with Sens for this enhancer. These findings have three main implications: (1) They demonstrate how a Hox selector gene integrates A-P positional information with a PNS factor to differentially regulate gene expression along the body plan. (2) They uncover a permissive rather than instructive role for Hox factors in regulating transcription. (3) As Hox and Sens binding sites share a common core sequence, they suggest that additional target genes will be regulated through this mechanism. Moreover, genetic studies in mice have linked Gfi1 and Hox factors to both neural and blood cell development, and this study found that vertebrate Hox and Gfi1 factors compete for binding sites in blood cells (Li-Kroeger, 2008).

Sensory organs within the fly head, thorax, and abdomen require sens for their development. However, the type, location, and number of sensory organs that form in different body regions are regulated, at least in part, by Hox factors. The results provide new insight into how Hox factors provide positional information to modify gene expression in sensory cells. A series of point mutations was used to demonstrate that Hox-Sens competition forms a molecular switch whose outcome correlates with the binding activity of each factor. Intrinsic to this model is the following prediction: If Hox factors differ in their ability to interact with composite sites, then A-P differences in Hox-Sens target expression will be observed. Previous biochemical studies revealed that posterior Hox factors have higher affinity for DNA when bound with Pbx (Exd) than anterior Hox proteins (LaRonde-LeBlanc, 2003). Consistent with these results, this study found that a posterior Hox complex (Abd-A/Hth/Exd) that stimulates Rho654 binds 5-fold more RhoA than an anterior Hox complex (Antp/Hth/Exd) that fails to stimulate Rho654. Thus, differences in binding activities between Hox factors for Hox-Sens composite sites result in the differential regulation of gene expression along the A-P axis of the sensory system (Li-Kroeger, 2008).

Hox proteins instructively regulate gene expression by either activating and/or repressing transcription. In fact, the same Hox factor can perform both functions. Abd-A directly binds regulatory elements to activate wingless (wg) and repress decapentaplegic (dpp) in the same cells of the visceral mesoderm. So what determines if a Hox factor activates or represses transcription? Two recent studies revealed that the transcriptional outcome depends upon the binding of additional transcription factors (Gebelein, 2004; Walsh, 2007). The repression of Distal-less (Dll) by the Abd-A and Ultrabithorax (Ubx) Hox factors requires the binding of two transcription factors in addition to Exd and Hth. In posterior compartment cells, the Engrailed (En) protein collaborates with Abd-A/Exd/Hth to bind DNA and repress Dll. In anterior compartment cells, the Sloppy-paired (Slp) protein binds DNA near the Hox complex to repress Dll (Gebelein, 2004). As both En and Slp interact with the Groucho (Gro) corepressor, their recruitment by Hox factors suggests a mechanism to repress transcription. Similarly, Walsh and Carroll found that Ubx and Smad binding are required to repress spalt-major (salm) in the wing. In this case, the Smad proteins recruit the Schnurri corepressor to inhibit transcription. Thus, Hox factors collaborate with additional factors to determine the transcriptional outcome (Li-Kroeger, 2008).

Studies on Abd-A stimulation of a rho enhancer reveal an unexpected mechanism by which Hox factors control gene expression: through competition with the Sens repressor for DNA binding sites. Sens binds RhoA to repress thoracic gene expression, whereas in the abdomen Exd/Hth/Abd-A is permissive for activation by out-competing Sens. Importantly, mutations that disrupt both Sens and Hox binding to RhoA (SensM/HoxM) are expressed in the thorax and abdomen, revealing that Exd/Hth/Abd-A binding is not required to activate gene expression. In addition, coexpression of Exd, Hth, and Abd-A in cultured cells failed to stimulate Rho654- or RhoAAA-luciferase unless Abd-A is fused to a potent activation domain. Thus, unlike other Hox target genes, Hox complexes on RhoA are permissive rather than instructive and stimulate Rho654 by interfering with the binding of a transcriptional repressor (Li-Kroeger, 2008).

A comparison of consensus Sens, Hox/Exd, and Exd/Hth sites reveal a shared core sequence, suggesting that additional target genes will be regulated through Hox-Sens antagonism. In fact, bioinformatics reveals many Hox-Sens composite sites throughout the Drosophila and mammalian genomes. However, both the Sens and Hox sites extend beyond this core sequence, indicating that only a subset of target genes will comprise composite sites. Thus, three types of target genes for those factors are proposed: (1) those regulated by only Hox factors, (2) those regulated by only Sens/Gfi1, and (3) those regulated by both Hox and Sens/Gfi1. For example, many of the previously characterized Hox target genes in the Drosophila embryo are controlled in tissues that do not express Sens, suggesting they are only regulated by Hox genes. However, the Hox and Sens/Gfi1 factors are coexpressed in many neural cells of the developing PNS in both flies and vertebrates, indicating that similarly to rho regulation in abdominal SOP cells, additional targets will be coregulated by Hox and Sens (Li-Kroeger, 2008).

Like Hox genes, the Sens gene family is conserved in C. elegans (Pag-3), Drosophila, and vertebrates (Gfi1 and Gfi1b). These zinc finger transcription factors are essential for nervous system development in all three organisms. In addition, Gfi1 plays a critical role in hematopoiesis, where it participates in regulating stem cell renewal as well as specific blood cell lineages. Interestingly, Hox factors also regulate blood cell differentiation, proliferation, and stem cell renewal. HoxA9, for example, is required for normal hematopoiesis in mice, and alterations in HoxA9 expression have been implicated in acute myeloid leukemia (AML). In fact, a study analyzing the expression profile of 6817 genes in AML patients who either responded or did not respond to treatment found the highest correlated gene associated with poor prognosis is HoxA9. To determine if the Hox-Sens mechanism uncovered in Drosophila is conserved in mammals, in vitro DNA binding assays were used to show that HoxA9 forms a complex with Pbx and Meis that competes with Gfi1 for common binding sites. Moreover, mouse genetic studies support the hypothesis that Hox-Gfi1 factors antagonize each other to regulate gene expression and blood cell development. Thus, Hox-Sens/Gfi1 competition for composite binding sites is likely a conserved mechanism for the regulation of gene expression in organisms from flies to humans (Li-Kroeger, 2008).

Atonal, senseless, and abdominal-A regulate rhomboid enhancer activity in abdominal sensory organ precursors

The atonal (ato) proneural gene specifies different numbers of sensory organ precursor (SOP) cells within distinct regions of the Drosophila embryo in an epidermal growth factor-dependent manner through the activation of the rhomboid (rho) protease. How ato activates rho, and why it does so in only a limited number of sensory cells remains unclear. A rho enhancer (RhoBAD) has been identified that is active within a subset of abdominal SOP cells to induce larval oenocytes and it has been shown that RhoBAD is regulated by an Abdominal-A (Abd-A) Hox complex and the Senseless (Sens) transcription factor (Li-Kroeger, 2008). This study shows that ato is also required for proper RhoBAD activity and oenocyte formation. Transgenic reporter assays reveal RhoBAD contains two conserved regions that drive SOP gene expression: RhoD mediates low levels of expression in both thoracic and abdominal SOP cells, whereas RhoA drives strong expression within abdominal SOP cells. Ato indirectly stimulates both elements and enhances RhoA reporter activity by interfering with the ability of the Sens repressor to bind DNA. As RhoA is also directly regulated by Abd-A, a model is proposed for how the Ato and Sens proneural factors are integrated with an abdominal Hox factor to regulate region-specific SOP gene expression (Witt, 2010).

This study found that the Atonal proneural factor is required for both normal rho enhancer function and the proper specification of abdominal oenocytes. In addition, it was determined that two distinct regions of the RhoBAD enhancer contribute to gene activity within the C1 SOP cells. The RhoA element preferentially drives gene expression within abdominal SOP cells, whereas RhoD drives weaker gene expression within the C1 SOP cells of both the thoracic and abdominal segments. Using a combination of genetic and biochemical analyses, it was found that the Ato, Sens, and Abd-A inputs contribute to proper rho enhancer activity. In particular, it was shown that RhoA, but not RhoD, is directly responsive to the Abd-A Hox factor. In addition, Ato was found to indirectly stimulate RhoBAD activity through both the RhoA and RhoD elements. Although it is currently not understood how Ato stimulates RhoD, it was found that Ato limits the DNA binding activity of the Sens repressor protein to RhoA. Coupled with other recent findings on proneural gene function, these results have two major implications: 1) A model is described for how Ato and Sens inputs are integrated to differentially regulate gene expression during SOP cell lineage development, and 2) How proneural input (Ato) and a Hox factor (Abd-A) cooperate to regulate Rho enhancer activity, at least in part, by limiting Sens-mediated repression is discussed (Witt, 2010).

Sens and the proneural factors are intricately linked during PNS development in Drosophila. Loss-of-function mutations in proneural genes disrupt sens expression resulting in a decrease in sensory organ formation and sens mutations result in decreased proneural gene expression and widespread sensory organ deficits. While both encode transcription factors required for PNS development, they have opposite effects on gene expression when bound to DNA. Proneural factors bind E-box DNA sequences with Daughterless to activate gene expression, whereas Sens binds a distinct DNA sequence to repress gene expression. However, recent data revealed that proneural proteins can convert Sens from a transcriptional repressor to a co-activator. Three different proneural factors (Ac, Sc, and Ato) interact with Sens in GST-pulldown and/or co-immunoprecipitation assays. In addition, cell culture assays showed that Sens stimulates the activation potential of proneural factors bound to E-Box sequences. Thus, Sens is a transcriptional repressor when directly bound to DNA through its zinc finger motifs whereas it is a potent co-activator when recruited to DNA by proneural proteins (Witt, 2010).

This study provides two pieces of information that add to understanding of how Sens and proneural factors regulate gene expression. First, purified Sens and Ato/Da proteins were used to show that Ato decreases the ability of Sens to bind the RhoA enhancer element. As RhoA contains a relatively low affinity Sens site, a parallel experiment was performed using a high affinity Sens site (SensS), and it was found that Ato does not significantly alter Sens binding to an optimized site. This data reveals that Ato's ability to interfere with Sens binding to DNA is site-specific and dependent upon binding affinity. How might Ato interfere with Sens binding to DNA? It has been shown that Ato, Ac, and Sc all directly interact with Sens through the second and third Sens zinc finger motifs. Since Sens requires these motifs to bind DNA, it is likely that the proneural factors compete with DNA for the same zinc fingers. Thus, the following model is proposed: if the binding affinity of Sens to DNA is high, Ato cannot interfere with Sens-mediated repression. However, if the binding affinity of Sens to DNA is low, Ato binds Sens and interferes with its ability to repress gene expression (Witt, 2010).

Secondly, expression analysis revealed that cells of the C1 SOP lineage differentially express Ato and Sens during their maturation. The initial SOP cell (SOPI) expresses both Ato and Sens during sensory organ specification. However, Ato protein is rapidly extinguished and no longer detectable once the SOP cell divides, whereas Sens persists into the SOPII cells. The rapid loss of Ato, even when it is expressed using a Gal4 driver, is consistent with recent findings that proneural proteins activate an E3 ubiquitin ligase pathway to trigger their own degradation. Thus, these findings suggest that the early SOP cell expresses both Ato and Sens and that Ato can alter Sens function in two ways: 1) by recruiting Sens to E-Box sequences as a co-activator, and 2) by interfering with Sens's ability to bind low affinity DNA sites (Witt, 2010).

It has been reported that rho is initially weakly expressed in C1 SOP cells in both the thorax and abdomen, and is only up-regulated in the abdominal SOP cells by the Abd-A Hox factor. This study found that the RhoBAD-lacZ reporter is also expressed in this pattern; it is proposed that Ato is part of an initiator pathway that allows rho expression in early C1 SOP cells. Ato does so in two ways: 1) by inhibiting Sens binding to RhoA through direct protein-protein interactions, and 2) by indirectly stimulating RhoD through an unknown mechanism. In total, these interactions result in the initiation of rho expression in early C1 SOP cells of both thoracic and abdominal segments. Ato's subsequent degradation releases Sens to bind RhoA and repress gene expression in thoracic SOP cells. Consistent with this idea, mutations that abolish Sens binding (SensM) result in de-repression of Rho reporters in the thorax. In the abdomen, however, an Abd-A complex out-competes Sens for RhoA to allow continued rho expression, subsequent EGF signaling, and the specification of additional cell types. Thus, Ato cooperates with the Abd-A Hox factor to stimulate EGF signaling by up-regulating rho expression via interfering with Sens-mediated repression (Witt, 2010).

While these findings provide insight into how rho is up-regulated in abdominal SOP cells, they uncover an interesting question: why is rho activated at all within thoracic SOP cells? Currently, there is no known function for rho activity within the thorax as rho mutant embryos show no phenotypic defect in cells surrounding the thoracic SOPs. As the lack of oenocyte production within the thorax is solely due to insufficient Spi secretion (oenocytes form in the thorax if rho is ectopically expressed), these data suggest that Rho levels are too low to trigger enough Spi secretion to affect neighboring cell fate. Consistent with this prediction is that the levels of an activated kinase downstream of EGF signaling (phospho-ERK) are very low in cells neighboring the thoracic C1 SOP cells compared to the abdominal SOP cells. So, why is rho activated within the thorax if it has no functional consequences? One interpretation is that Ato may provide competency for rho expression so that an additional positional factor such as Abd-A can fully stimulate rho and trigger Spi secretion and EGF signaling. In support of this idea, the widespread expression of Abd-A within the thorax activates RhoBAD-lacZ expression only within the C1 SOP cells and oenocytes form only in close proximity to these thoracic SOP cells. Thus, weak rho expression downstream of ato may provide a flexible and responsive system for activating Spi secretion in different body regions (Witt, 2010).

Atonal, senseless, and abdominal-A regulate rhomboid enhancer activity in abdominal sensory organ precursors

The atonal (ato) proneural gene specifies different numbers of sensory organ precursor (SOP) cells within distinct regions of the Drosophila embryo in an epidermal growth factor-dependent manner through the activation of the rhomboid (rho) protease. How ato activates rho, and why it does so in only a limited number of sensory cells remains unclear. A rho enhancer (RhoBAD) has been identified that is active within a subset of abdominal SOP cells to induce larval oenocytes and it has been shown that RhoBAD is regulated by an Abdominal-A (Abd-A) Hox complex and the Senseless (Sens) transcription factor (Li-Kroeger, 2008). This study shows that ato is also required for proper RhoBAD activity and oenocyte formation. Transgenic reporter assays reveal RhoBAD contains two conserved regions that drive SOP gene expression: RhoD mediates low levels of expression in both thoracic and abdominal SOP cells, whereas RhoA drives strong expression within abdominal SOP cells. Ato indirectly stimulates both elements and enhances RhoA reporter activity by interfering with the ability of the Sens repressor to bind DNA. As RhoA is also directly regulated by Abd-A, a model is proposed for how the Ato and Sens proneural factors are integrated with an abdominal Hox factor to regulate region-specific SOP gene expression (Witt, 2010).

This study found that the Atonal proneural factor is required for both normal rho enhancer function and the proper specification of abdominal oenocytes. In addition, it was determined that two distinct regions of the RhoBAD enhancer contribute to gene activity within the C1 SOP cells. The RhoA element preferentially drives gene expression within abdominal SOP cells, whereas RhoD drives weaker gene expression within the C1 SOP cells of both the thoracic and abdominal segments. Using a combination of genetic and biochemical analyses, it was found that the Ato, Sens, and Abd-A inputs contribute to proper rho enhancer activity. In particular, it was shown that RhoA, but not RhoD, is directly responsive to the Abd-A Hox factor. In addition, Ato was found to indirectly stimulate RhoBAD activity through both the RhoA and RhoD elements. Although it is currently not understood how Ato stimulates RhoD, it was found that Ato limits the DNA binding activity of the Sens repressor protein to RhoA. Coupled with other recent findings on proneural gene function, these results have two major implications: 1) A model is described for how Ato and Sens inputs are integrated to differentially regulate gene expression during SOP cell lineage development, and 2) How proneural input (Ato) and a Hox factor (Abd-A) cooperate to regulate Rho enhancer activity, at least in part, by limiting Sens-mediated repression is discussed (Witt, 2010).

Sens and the proneural factors are intricately linked during PNS development in Drosophila. Loss-of-function mutations in proneural genes disrupt sens expression resulting in a decrease in sensory organ formation and sens mutations result in decreased proneural gene expression and widespread sensory organ deficits. While both encode transcription factors required for PNS development, they have opposite effects on gene expression when bound to DNA. Proneural factors bind E-box DNA sequences with Daughterless to activate gene expression, whereas Sens binds a distinct DNA sequence to repress gene expression. However, recent data revealed that proneural proteins can convert Sens from a transcriptional repressor to a co-activator. Three different proneural factors (Ac, Sc, and Ato) interact with Sens in GST-pulldown and/or co-immunoprecipitation assays. In addition, cell culture assays showed that Sens stimulates the activation potential of proneural factors bound to E-Box sequences. Thus, Sens is a transcriptional repressor when directly bound to DNA through its zinc finger motifs whereas it is a potent co-activator when recruited to DNA by proneural proteins (Witt, 2010).

This study provides two pieces of information that add to understanding of how Sens and proneural factors regulate gene expression. First, purified Sens and Ato/Da proteins were used to show that Ato decreases the ability of Sens to bind the RhoA enhancer element. As RhoA contains a relatively low affinity Sens site, a parallel experiment was performed using a high affinity Sens site (SensS), and it was found that Ato does not significantly alter Sens binding to an optimized site. This data reveals that Ato's ability to interfere with Sens binding to DNA is site-specific and dependent upon binding affinity. How might Ato interfere with Sens binding to DNA? It has been shown that Ato, Ac, and Sc all directly interact with Sens through the second and third Sens zinc finger motifs. Since Sens requires these motifs to bind DNA, it is likely that the proneural factors compete with DNA for the same zinc fingers. Thus, the following model is proposed: if the binding affinity of Sens to DNA is high, Ato cannot interfere with Sens-mediated repression. However, if the binding affinity of Sens to DNA is low, Ato binds Sens and interferes with its ability to repress gene expression (Witt, 2010).

Secondly, expression analysis revealed that cells of the C1 SOP lineage differentially express Ato and Sens during their maturation. The initial SOP cell (SOPI) expresses both Ato and Sens during sensory organ specification. However, Ato protein is rapidly extinguished and no longer detectable once the SOP cell divides, whereas Sens persists into the SOPII cells. The rapid loss of Ato, even when it is expressed using a Gal4 driver, is consistent with recent findings that proneural proteins activate an E3 ubiquitin ligase pathway to trigger their own degradation. Thus, these findings suggest that the early SOP cell expresses both Ato and Sens and that Ato can alter Sens function in two ways: 1) by recruiting Sens to E-Box sequences as a co-activator, and 2) by interfering with Sens's ability to bind low affinity DNA sites (Witt, 2010).

It has been reported that rho is initially weakly expressed in C1 SOP cells in both the thorax and abdomen, and is only up-regulated in the abdominal SOP cells by the Abd-A Hox factor. This study found that the RhoBAD-lacZ reporter is also expressed in this pattern; it is proposed that Ato is part of an initiator pathway that allows rho expression in early C1 SOP cells. Ato does so in two ways: 1) by inhibiting Sens binding to RhoA through direct protein-protein interactions, and 2) by indirectly stimulating RhoD through an unknown mechanism. In total, these interactions result in the initiation of rho expression in early C1 SOP cells of both thoracic and abdominal segments. Ato's subsequent degradation releases Sens to bind RhoA and repress gene expression in thoracic SOP cells. Consistent with this idea, mutations that abolish Sens binding (SensM) result in de-repression of Rho reporters in the thorax. In the abdomen, however, an Abd-A complex out-competes Sens for RhoA to allow continued rho expression, subsequent EGF signaling, and the specification of additional cell types. Thus, Ato cooperates with the Abd-A Hox factor to stimulate EGF signaling by up-regulating rho expression via interfering with Sens-mediated repression (Witt, 2010).

While these findings provide insight into how rho is up-regulated in abdominal SOP cells, they uncover an interesting question: why is rho activated at all within thoracic SOP cells? Currently, there is no known function for rho activity within the thorax as rho mutant embryos show no phenotypic defect in cells surrounding the thoracic SOPs. As the lack of oenocyte production within the thorax is solely due to insufficient Spi secretion (oenocytes form in the thorax if rho is ectopically expressed), these data suggest that Rho levels are too low to trigger enough Spi secretion to affect neighboring cell fate. Consistent with this prediction is that the levels of an activated kinase downstream of EGF signaling (phospho-ERK) are very low in cells neighboring the thoracic C1 SOP cells compared to the abdominal SOP cells. So, why is rho activated within the thorax if it has no functional consequences? One interpretation is that Ato may provide competency for rho expression so that an additional positional factor such as Abd-A can fully stimulate rho and trigger Spi secretion and EGF signaling. In support of this idea, the widespread expression of Abd-A within the thorax activates RhoBAD-lacZ expression only within the C1 SOP cells and oenocytes form only in close proximity to these thoracic SOP cells. Thus, weak rho expression downstream of ato may provide a flexible and responsive system for activating Spi secretion in different body regions (Witt, 2010).

Proneural and abdominal Hox inputs synergize to promote sensory organ formation in the Drosophila abdomen

The atonal (ato) proneural gene specifies a stereotypic number of sensory organ precursors (SOP) within each body segment of the Drosophila ectoderm. Surprisingly, the broad expression of Ato within the ectoderm results in only a modest increase in SOP formation, suggesting many cells are incompetent to become SOPs. This study shows that the SOP promoting activity of Ato can be greatly enhanced by three factors: the Senseless (Sens) zinc finger protein, the Abdominal-A (Abd-A) Hox factor, and the epidermal growth factor (EGF) pathway. First, it was shown that expression of either Ato alone or with Sens induces twice as many SOPs in the abdomen as in the thorax, and does so at the expense of an abdomen-specific cell fate: the larval oenocytes. Second, Ato was shown to stimulate abdominal SOP formation by synergizing with Abd-A to promote EGF ligand (Spitz) secretion and secondary SOP recruitment. However, it was also found that Ato and Sens selectively enhance abdominal SOP development in a Spitz-independent manner, suggesting additional genetic interactions between this proneural pathway and Abd-A. Altogether, these experiments reveal that genetic interactions between EGF-signaling, Abd-A, and Sens enhance the SOP-promoting activity of Ato to stimulate region-specific neurogenesis in the Drosophila abdomen (Gutzwiller, 2010).

How proneural pathways that specify sensory precursor cells throughout the body are integrated with region-specific patterning genes to yield the correct type and number of sensory organs is not well understood. This study shows that three factors enhance the ability of Ato to promote ch organ SOP cell fate in the Drosophila abdomen; the EGF pathway mediated by the Spi ligand, the Abd-A Hox factor, and the Sens zinc finger transcription factor (Gutzwiller, 2010).

EGF signaling is used reiteratively throughout development to specify the formation of distinct cell types along the body plan. In the embryonic Drosophila abdomen, EGF signaling initiated by the activation of rhomboid (rho) in a set of ch organ SOP cells induces the formation of both a cluster of abdomen-specific oenocytes as well as a set of 2° ch organ SOP cells. But how does the EGF-receiving cell know whether to become a larval oenocyte that is specialized to process lipids or a ch organ SOP cell that forms part of the peripheral nervous system? Previous studies have shown that oenocyte specification requires at least two inputs: (1) the reception of relatively high levels of EGF signaling and (2) the expression of the Spalt transcription factors. Hence, oenocytes develop in close proximity to the abdominal C1 SOP cells that lie within a Spalt expression domain and express high levels of rho. In contrast, 2° SOP cells require less EGF signaling and form if the receiving cells lack Spalt. Consistent with this model, genetic studies have shown that oenocytes fail to develop and one to two additional ch organ SOP cells are specified in Spalt mutant embryos, whereas ectopic Spalt expression in the ventral ectoderm inhibits the recruitment of 2° SOP cells. Thus, Spalt promotes oenocyte development and antagonizes 2° ch organ specification in the Drosophila embryo (Gutzwiller, 2010).

Evidence that ato has the opposite effect as Spalt: it promotes ch organ SOP cells at the expense of oenocyte specification. Witt (2010) showed that ato loss-of-function results in decreased expression of activity of the rho enhancer, RhoBAD (Witt, 2010), in C1 SOP cells and induces fewer oenocytes. These data are consistent with EGF signaling being compromised in ato mutant embryos and oenocyte specification being dependent upon the reception of high levels of Spi. This study shows that Ato gain-of-function stimulates RhoBAD expression yet results in the inhibition of oenocyte formation. Importantly, the loss of oenocytes is not due to decreased EGF signaling as similar whorls of phospho-ERK-positive cells and even extra phospho-ERK staining are observed in Ato-expressing segments compared with non-expressing segments. In addition, no difference was detected in cell death between Ato-expressing and non-Ato-expressing segments (using an anti-cleaved Caspase3 marker), indicating the oenocyte loss is not due to apoptosis. Instead, Ato promotes the formation of additional ch organ SOP cells in abdominal segments that normally form oenocytes. Moreover, while the broad activation of EGF signaling (PrdG4;UAS-Rho) induces many extra oenocytes and a few scolopodia, the co-expression of Ato and Rho induces many scolopodia and few oenocytes. These data suggest that if the Spi-receiving cell expresses high Ato relative to Salm then ch organ development occurs whereas if the Spi-receiving cell expresses high Salm relative to Ato then oenocytes are formed. Thus, Ato plays a role in both the Spi-secreting (induction of rho expression) and Spi-receiving cell to dictate the choice of cell fate (Gutzwiller, 2010).

The broad expression of Ato within the ectoderm revealed differences in sensory organ competency between the thorax and abdomen. In particular, it was found that Ato induced approximately twice as many ch organ SOP cells in the abdomen as in the thorax. Moreover, the co-expression of Ato with the Abd-A Hox factor induced significantly more ch organ cell formation than expression of either factor alone (none by Abd-A, four by Ato, and eight by Ato/Abd-A). These data suggest that Ato and Abd-A synergize to enhance ch organ SOP formation in the abdomen, an prompted an examination whethere these SOP cells are predominantly 1° or 2° cells. This problem was first addressed by first showing that the co-expression of Ato and Abd-A stimulates Rho enhancer activity (RhoAAA) within additional cells and results in enhanced phospho-ERK staining. Second, it was shown that Ato and Abd-A require the EGF pathway to enhance ch organ development as co-expression of both factors in a spi mutant embryo failed to promote more ch organs than expression of Ato alone. These data indicate that the co-expression of Ato and Abd-A enhances the ability of 1° ch organ SOP cells to activate rho, stimulates Spi secretion and, since the receiving cell expresses Ato, 2° SOPs form instead of oenocytes. The net result is that Ato and Abd-A synergize to activate the EGF pathway to promote region-specific neurogenesis within the Drosophila abdomen (Gutzwiller, 2010).

The Sens transcription factor is essential for the formation of much of the peripheral nervous system in Drosophila and previous studies revealed that Sens can stimulate the sensory bristle-forming activity of the Scute and Achaete proneural factors in the wing disc. Similarly, it was found that Sens stimulates the ability of Ato to generate internal stretch receptors in the embryo and that Ato and Sens promote more sensory organ development in the abdomen than in the thorax. In addition, while the overall number of ch organs formed by Ato and Sens co-expression is decreased in spi mutant embryos, significantly more ch organ SOP cells in the abdomen than in the thorax are observed in this EGF-compromised genetic background. Thus, Ato and Sens can stimulate abdominal ch organ SOP cell development in the presence or absence of Spi-mediated cell signaling (Gutzwiller, 2010).

So, what is the relationship between Ato, Sens, and Abd-A in regulating both EGF signaling and region-specific sensory organ formation? It was previously found that Ato, Sens, and Abd-A control EGF signaling through the regulation of a cis-regulatory element within the rhomboid (rho) locus (RhoBAD) (Li-Kroeger, 2008; Witt, 2010). RhoBAD acts in abdominal C1 SOP cells to induce oenocyte formation, and Ato and Abd-A both stimulate RhoBAD expression, at least in part, by limiting the ability of Sens to repress RhoBAD activity. Moreover, they do so using different mechanisms. An Abd-A Hox complex containing Extradenticle and Homothorax directly competes with the Sens repressor for overlapping binding sites in RhoBAD (Li-Kroeger, 2008). In contrast, Ato does not directly bind RhoBAD but does directly interact with Sens to limit its ability to bind and repress Rho enhancer activity (Witt, 2010). Consequently, SOPs that co-express Ato and Abd-A are likely to limit the ability of Sens to repress Rho and thereby increase the number of ch organ SOP cells that secrete Spi. Consistent with this prediction, the co-expression of Ato and Sens preferentially stimulates Rho enhancer activity within abdominal segments compared to thoracic segments. Each SOP cell that expresses rho would further enhance sensory organ development through the recruitment of 2° SOP cells via Spi-mediated signaling. Hence, the genetic removal of spi results in a significant decrease in the number of ch organ SOP cells that develop in response to Ato and Sens. Thus, the ato-sens genetic pathway, which is used throughout the body to promote SOP formation, interacts with an abdominal Hox factor to stimulate EGF signaling and promote additional cell fate specification in the abdomen (Gutzwiller, 2010).

While the above model fits well with most of the data, two unexpected findings were observed when comparing the ability of Ato-Sens co-expression to induce ch organ development in the presence and absence of spi function: First, it was predicted that Ato-Sens co-expression in the thoracic regions, which lack Abd-A, should predominantly induce the formation of 1° ch organ SOP cells that do not require EGF signaling for their development. However, it was found that significantly fewer ch organs form in the thorax of spi mutants, indicating that EGF signaling can enhance 2° sensory organ formation within thoracic segments that co-express Ato and Sens. Interestingly, previous studies have shown that both rho and the Rho enhancers are weakly active within thoracic C1 SOP cells, but their levels do not reach a high enough threshold to induce oenocyte formation. However, it is possible that the co-expression of Ato and Sens sufficiently sensitizes the receiving cells to respond to low levels of EGF signaling and become ch organ SOP cells. The second unanticipated finding is that Ato and Sens co-expression still induced significantly more ch organ development within the abdomen (5-6 extra SOP cells) relative to the thorax (1-2 extra SOP cells) in the absence of Spi-mediated signaling. This finding suggests that Ato and Sens can genetically interact with the Abd-A Hox factor to promote sensory organ development in an Spi-independent manner. Currently, it is not understood how Abd-A enhances the proneural activity of the Ato-Sens factors in the absence of Spi signaling. One possibility is that Abd-A and Ato use similar mechanisms to limit Sens-mediated repression of additional target genes besides rho to stimulate ch organ development. Alternatively, Abd-A could independently regulate other factors such as those involved in the Notch-Delta pathway to enhance the competency of the ectoderm to respond to the Ato-Sens pathway. Intriguingly, a Hox factor (lin-39) in C. elegans has been shown to directly regulate Notch signaling during vulval development, and the vertebrate Hoxb1 factor regulates neural stem cell progenitor proliferation and maintenance by modulating Notch signaling. Since differential Notch-Delta signaling is a key pathway in deciding neural versus non-neural cell fates, the ability of Hox factors to modify this pathway could result in segmental differences in neurogenesis (Gutzwiller, 2010).

Transcriptional Regulation

Continued: Rhomboid Transcriptional regulation part 2/3 | part 3/3

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