cis-Regulatory Sequences and Functions

Genome-wide analysis of clustered Dorsal binding sites was used to examine the distribution of Dorsal recognition sequences in the Drosophila genome. The homeobox gene zerknullt (zen) is repressed directly by Dorsal, and this repression is mediated by a 600-bp silencer, the ventral repression element (VRE), which contains four optimal Dorsal binding sites. The arrangement and sequence of the Dorsal recognition sequences in the VRE were used to develop a computational algorithm to search the Drosophila genome for clusters of optimal Dorsal binding sites. There are 15 regions in the genome that contain three or more optimal sites within a span of 400 bp or less. Three of these regions are associated with known Dorsal target genes: sog, zen, and Brinker. The Dorsal binding cluster in sog is shown to mediate lateral stripes of gene expression in response to low levels of the Dorsal gradient. Two of the remaining 12 clusters associated with genes that exhibit asymmetric patterns of expression across the dorsoventral axis. These results suggest that bioinformatics can be used to identify novel target genes and associated regulatory DNAs in a gene network (Markstein, 2002).

zen is an immediate target of the maternal Dl gradient. The gene is activated initially at nuclear cleavage cycle 11-12 within 1 h after the Dl gradient is formed. zen initially exhibits a broad pattern of expression in the presumptive dorsal ectoderm and at the termini. High and low levels of the Dl gradient keep zen off in ventral and lateral regions. sog exhibits a complementary pattern of expression because it is activated by Dl, whereas zen is repressed. As seen for zen, sog expression is detected shortly after the formation of the Dl gradient (Markstein, 2002).

The zen VRE contains four optimal Dl recognition sequences within a span of 400 bp. Three of the four Dl binding sites contained within the zen VRE conform to the following consensus sequence for high-affinity Dl binding sites: GGG(W)nCCM (where W = A or T, M = C or A, and n corresponds to either four or five W residues). The fourth recognition sequence (binding site 3 within the VRE) contains a G residue in the AT-rich central region and is represented by the optimal consensus sequence GGGWDWWWCCM (where D = A, T, or G). To determine whether a similar density of optimal Dl sites might account for the regulation of sog, the entire Drosophila genome was scanned for clusters of any of the 208 unique Dl sequences that conform (either directly or by reverse complement) to two degenerate sequences: GGG(W)4CCM and GGGWDWWWCCM (Markstein, 2002).

The genome was scanned for clusters of Dl binding sites in windows of 400 bp, the interval within which the sites are clustered in the zen VRE, and also for clustering in windows of 1,000 bp because the operational size of enhancers can generally be thought of as about 1,000 bp. Although the genome-wide occurrence of 676 clusters of two or more optimal Dl sites in 1,000 bp is not statistically significant, the occurrence of 55 clusters with at least three sites and of eight clusters containing four sites is enriched beyond what one would expect from random chance. However, none of the clusters within 1,000 bp identified known Dl targets that were missed by the more stringent screen for clustering within 400 bp. Therefore, this study focussed on the results from the more stringent screen (Markstein, 2002).

As expected, the occurrence of 400-bp windows containing at least two sites (327 clusters) is much greater than the occurrence of 400-bp windows containing at least three sites (15 clusters) or four sites (3 clusters). However, the statistical significance of the clusters increases with their rarity. For example, the occurrence of 15 clusters with three or more Dl sites is 6 standard deviations from expected, making the probability of finding 15 clusters by random chance less than one in a million. The probability of finding three 400-bp clusters with at least four Dl sites is less than 10-49. Remarkably, two of the clusters in this rarest class are associated with the sog and zen genes, which exhibit the most sensitive response to the Dl gradient. Of the remaining 13 clusters containing three or more Dl sites, one is associated with the Brinker gene, which is expressed in lateral stripes and probably is a direct target of the Dl gradient. The Brinker site is located ~10 kb 5' of the transcription start site. Brinker probably is a direct target of the Dl gradient in that it exhibits lateral stripes of expression that are similar to those observed for rhomboid. The other remaining 12 clusters were found to neighbor genes that were not known previously to be involved in dorsoventral patterning (Markstein, 2002).

Morphogen gradients control body pattern by differentially regulating cellular behavior. Molecular events underlying the primary response to the Dpp/BMP morphogen have been analyzed in Drosophila. Throughout development, Dpp transduction causes the graded transcriptional downregulation of the brinker (brk) gene. Significance for the brk expression gradient is provided by showing that different Brk levels repress distinct combinations of wing genes expressed at different distances from Dpp-secreting cells. The brk regulatory region has been dissected and two separable elements have been identified with opposite properties, a constitutive enhancer and a Dpp morphogen-regulated silencer. Furthermore, genetic and biochemical evidence is presented that the brk silencer serves as a direct target for a protein complex consisting of the Smad homologs Mad/Medea and the zinc finger protein Schnurri. Together, these results provide the molecular framework for a mechanism by which the extracellular Dpp/BMP morphogen establishes a finely tuned, graded read-out of transcriptional repression (Müller, 2003).

The Dpp signaling system shapes an inverse profile of Brk expression, which serves as a mold for casting the spatial domains of Dpp target genes. Thus, the question of how the Dpp morphogen gradient is converted into transcriptional outputs can be largely reduced to the question of how Dpp generates an inverse transcriptional gradient of brk expression. An unbiased approach was applied to this problem by isolating the regulatory elements of brk. A protein complex has been identified and characterized that binds to and regulates the activity of these elements in a Dpp dose-dependent manner (Müller, 2003).

Dissection of the brk locus reveals two separable elements with opposite properties: a constitutive enhancer and a morphogen-regulated silencer. Both elements have a direct effect on the level of brk expression, and it is the net sum of their opposing forces that dictates the transcriptional activity of brk in any given cell. In this sense, expression of the brk gene behaves like a spring that is compressed by Dpp signaling. Its silencer and enhancer embody the variable compressing and constant restoring forces, respectively. As stated by Hooke's law, an increased elastic constant (e.g., two copies of the constitutive enhancer) either shifts the brk levels toward those normally present at more lateral positions or necessitates a correspondingly higher compressing force (e.g., more silencer elements or higher levels of Dpp signaling). Given the central role Brk plays in controlling growth and pattern together with the direct impact of the two regulatory elements on brk levels, it appears inevitable that their quantitative properties must exhibit a fine-tuned evolutionary relationship with each other and with those of the Dpp transduction system. It appears, furthermore, that both the brk enhancer as well as the brk silencer elements represent ideal substrates for evolutionary changes in morphology (Müller, 2003).

Based on combined genetic and biochemical analysis, it is proposed that upon Dpp signaling the following key players meet at the brk silencer elements to execute repression: the Smad proteins Mad and Med and the zinc finger protein Shn. The role of Shn must be to direct the signaling input provided by Mad and Med into transcriptional silencing. In principle, two scenarios can be envisaged by which Shn fulfills this task. Shn could possess repressor activity (presumably via recruitment of corepressors) but lack the ability to bind the brk silencer and, hence, depend on Mad/Med for being targeted to its site of action. Alternatively, Shn could be prebound to the silencer, but only be capable of recruiting corepressors upon interaction with Mad/Med. Based on the observation that a Shn/DNA complex cannot be detected in the absence of Mad/Med, the first of these two possibilities is favored. The molecular architecture of the protein complex binding to the brk silencer as well as the DNA sequences providing the specificity for the local setup of this complex remain to be determined in detail (Müller, 2003).

An additional protein, which appears to influence the events at the brk silencer, is Brk itself. Genetic experiments indicate that Brk negatively modulates its own expression, forming a short regulatory loop that contributes to the final shape of the Brk gradient. This autoregulatory action occurs also via the brk silencer element, suggesting that Brk directly participates in the protein-protein or protein-DNA interactions at this site (Müller, 2003).

Most regulatory events ascribed to Smad proteins to date concern signaling-induced activation of target gene transcription. In the case of the brk silencer Shn could be regarded as a 'switch factor' that converts an inherently activating property of Smad proteins into transcriptional repression activity. Indeed, it has been shown that Smad proteins have the ability to recruit general coactivators with histone acetyl transferase activity. However, in an alternative and more general view, Smad proteins per se may provide no bias toward activation or repression. Their main function may be to assemble transcriptional regulatory complexes involving other DNA binding proteins and endow these complexes with additional DNA binding capacity. Such associated DNA binding factors would not only determine target site specificity, but, by their recruitment of either coactivator or corepressor proteins, also define the kind of regulatory influence exerted on nearby promoters. Since Shn directs Mad/Med activity toward repression, the existence of at least one other such Mad/Med partner in Drosophila is hypothesized to account for Mad/Med-mediated activation of gene expression. Such Mad/Med-mediated activation appears to be required for peak levels of sal and vg transcription, as well as for defining gene expression patterns in domains where brk expression is completely repressed, e.g., close to the Dpp source of the dorsal embryonic ectoderm (Müller, 2003).

At the heart of the model is the direct causal relationship between the formation of a Shn/Mad/Med/brk-silencer complex and the silencing of brk gene transcription. Although the two observations have been derived from different experimental data sets (biochemical versus genetic, respectively), there is a firm correlation between the requirements for either event to occur. brk is not repressed when either (1) the brk silencer elements are lacking or mutated; (2) or when Dpp input is prevented (and hence Mad is neither phosphorylated, nor nuclearly localized, nor associated with Med), or when (3) Shn is not present or is deprived of its C-terminal zinc fingers. The same set of requirements was observed for the formation of the Shn/Mad/Med/brk complex. Moreover, it is the concurrence of all three of these conditions that appears to provide the exquisite specificity to the Dpp-regulated silencing of gene transcription. (1) It only occurs in conjunction with a functional brk silencer, or an equivalent element. (2) There is an absolute requirement for Dpp input in Shn-mediated silencing. Not even a partial repressor activity of Shn was observed in cells that do not receive Dpp signal (e.g., loss of shn function in cells situated in lateral-most positions of the wing disc does not cause a further upregulation of brk transcription). (3) Shn represents only one of several zinc finger proteins expressed in Dpp receiving cells, yet none of the other proteins is able to substitute for Dpp-mediated repression. A major determinant for the specificity with which Shn engages in the signaling-dependent protein/DNA complex appears to be the triple zinc-finger motif. Although it is likely that this structural feature is required for contacting specific nucleotides on the brk silencer, the possibility cannot be not excluded that some of the zinc fingers mediate protein-protein interactions between Shn and Mad, Med or other cofactors (Müller, 2003).

While all of the above-discussed elements contribute to the specificity of signaling-regulated repression, it is important to emphasize that one possibility for specificity has not been exploited. The brk repression element does not specifically impinge upon the constitutive brk enhancer but promiscuously diminishes transcriptional activation by heterologous enhancers. It is likely, therefore, that the brk repression element interferes directly with events at the promoter, a property that may permit it to function as a bona fide silencer (Müller, 2003).

A fundamental characteristic of any morphogen system is that cells at different positions in the concentration gradient respond in qualitatively different ways. Cells must be able to activate different sets of genes at different threshold concentrations. The simplest way by which cells could produce two distinct responses at different threshold concentrations would be the employment of two kinds of receptors of different affinity for the morphogen. This mechanism does not appear to apply for the Dpp morphogen gradient, where Tkv and Punt appear to mediate both low- and high-threshold responses. Thresholds could also be imposed at any downstream event in the signal transduction cascade. Surprisingly, it appears that in the case of the Dpp morphogen, no such gates are in place, and the transcription of the brk gene is a negative image of the Dpp gradient. Thus, while these findings provide mechanistic insights into how an extracellular protein gradient is converted into a nuclear gradient of gene activity, they pass the burden of generating threshold effects on to downstream events. Several morphogen gradients operating in the early syncytial embryo, however, have been sufficiently well studied to explain the mechanistic principles of how a gradient of transcriptional activity can specify thresholds of gene activity and tissue differentiation (Müller, 2003).

A key difference between such embryonic transcriptional gradients and that of brk concerns the nature of their outputs: while all of them affect cellular patterns, Brk also controls growth. Flattening the brk gradient during development has catastrophic effects: reducing its high end causes overgrowth, and increasing its low end causes growth arrest. It may be this fundamental role in growth control that prohibits a discontinuous conversion of the Dpp morphogen gradient into its first transcriptional output. The identification of the elusive growth target(s) controlled by the Brk gradient represents one of the major challenges in the field (Müller, 2003).

Multiple modular promoter elements drive graded brinker expression in response to the Dpp morphogen gradient

Morphogen gradients play fundamental roles in patterning and cell specification during development by eliciting differential transcriptional responses in target cells. In Drosophila, Decapentaplegic (Dpp), the BMP2/4 homolog, downregulates transcription of the nuclear repressor brinker (brk) in a concentration-dependent manner to generate an inverse graded distribution. Both Dpp and Brk are crucial for directing Dpp target gene expression in defined domains and the consequent execution of distinct developmental programs. Thus, determining the mechanism by which the brk promoter interprets the Dpp activity gradient is essential for understanding both Dpp-dependent patterning and how graded signaling activity can generate different responses through transcriptional repression. This study uncovered key features of the brk promoter that suggest it uses a complex enhancer logic not represented in current models. First, it was found that the regulatory region contains multiple compact modules that can independently drive brk-like expression patterns. Second, each module contains binding sites for the Schnurri/Mad/Medea (SMM) complex, which mediates Dpp-dependent repression, linked to regions that direct activation. Third, the SMM repression complex acts through a distance-dependent mechanism that probably uses the canonical co-repressor C-terminal Binding Protein (CtBP). Finally, these data suggest that inputs from multiple regulatory modules are integrated to generate the final pattern. This unusual promoter organization may be necessary for brk to respond to the Dpp gradient in a precise and robust fashion (Yao, 2008).

The brk gene is unique in that eleven SMM sites are present in its regulatory region: no other locus in the genome has more than three sites. The SMM sites are characterized by a GRCGNC(N5)GTCTG motif. The GRCGNC sequence is bound by Mad while Med binds GTCTG, and the five-nucleotide spacer is crucial for recruitment of Shn to the complex. These sites are widely dispersed over 16 kb and separated from each other by 0.35 to 5.5 kb, with the exception of sites 7/8/9, which are clustered in a 183 bp region. For seven of the eleven SMM sites (3, 4, 5, 7/8/9 and 10), sequences that mediate transcriptional activation are located within ~380 bp of the SMM sites. These SMM sites and linked activator sequences can independently generate brk-like expression patterns, suggesting that they function as autonomous modules. The fact that the L1 transgene, which contains a single SMM site (#1), also drives a brk-like pattern, strongly argues for a sixth module in addition to the five that have been demonstrated. Thus, the 11 SMM sites in the brk regulatory region probably correspond to a total of 9 or 10 distinct modules, depending on whether the 7/8/9 cluster represents one or more modules. The evolutionary conservation of this unusual promoter organization provides additional support for its functional importance. Analysis of brk flanking regions in D. pseudoobscura and D. virilis, which are 30 and 40 million years distant from D. melanogaster, identified 12 and 11 SMM sites, respectively, arranged with a similar spacing relative to the basal promoter. Furthermore, 11 sites are found upstream of the brk-coding region in the mosquito Anopheles gambiae, which is separated from Drosophila by ~200 million years (Yao, 2008).

How does the brk promoter read the pMad gradient and generate a complementary graded expression pattern, and what benefit could the presence of multiple modular enhancers confer in generating the Brk gradient? This work, as well as previous studies, indicates that SMM sites act as sensors for Dpp signaling by binding a repressor complex that antagonizes broadly expressed activators in a dose-dependent manner. The data that SMM-mediated repression has a limited range suggests that each module can autonomously generate an output representing the balance between activation and signaling-dependent repression within that module. The patterns produced by individual modules probably reflect variation in SMM site sequence and affinity, the distance between SMM and activator sites, activator site sequence and number, as well as whether sites for additional transcription factors are present (Yao, 2008).

The endogenous brk pattern does not appear to reflect the activity of a single 'dominant' module, but rather is a composite pattern resulting from integration of multiple modular inputs. This can be inferred from the fact that large promoter fragments containing more than one module (e.g. L2 and L6) drive patterns that resemble, but are not identical to, those of their constituent modules. Furthermore, the additive effect of module multimerization on expression levels in regions of low Dpp activity is also consistent with integration across modules. Finally, strong support for this idea comes from the buffering capacity of multimodular promoter fragments (Yao, 2008).

A significant feature of the brk promoter is the remarkable ability of intact modules to override medial activation by mutant modules that are Dpp insensitive. This is apparent from the data that activators uncoupled from Dpp-dependent repression drive strikingly different expression patterns in isolation than they do in the context of larger fragments containing additional wild-type modules. Thus, disruption of the SMM sites in module 3 (L12M3), module 4 (L14M4) and module 7/8/9 (L15M7/8/9) caused derepression throughout the center of the wing disc. However, the same mutations in a larger fragment containing several additional modules (L13M3, L13M4 and L13M7/8/9) resulted in no derepression in the center of the disc. These results are inconsistent with a simple model in which only modules unbound by SMM complexes contribute to the transcriptional output of the promoter. If this were the case, in cells at the AP boundary, high levels of Dpp signaling would repress all intact modules in the L13M variants, leaving the constitutively active mutant module(s) free to interact with the transcriptional machinery. As a consequence, L13M variants would be expected to upregulate expression throughout the medial region of the disc. One potential explanation for the ability of wild-type modules to dampen expression from mutant modules could be that activators from SMM-repressed modules may compete disproportionately with activators from unrepressed modules for access to the transcriptional machinery, thus diluting the effect of the mutant modules. Alternatively, the SMM repressor complexes bound at multiple modules could act cooperatively (perhaps by modifying chromatin structure), thus reducing the output from adjacent mutant modules. In both cases, the absence of any expression in the medial region even with two Dpp-insensitive modules present, argues that repressed modules make a significant contribution to the transcriptional output compared with the derepressed modules. Such an integrative mechanism also provides a framework for understanding how poorly resolved patterns like those generated by module 4, could be refined to generate the wild-type brk pattern. An important consequence of this promoter logic is that although individual SMM repression complexes act locally, modules in aggregate can, nevertheless, exert a long-range/global effect on promoter activity (Yao, 2008).

The specialized architecture of the brk promoter may provide a mechanism to respond to Dpp signaling in a uniquely precise and robust fashion. Multiple modules allow simultaneous parallel reads of the pMad gradient, thus increasing the precision with which the brk promoter detects Dpp morphogen levels. Integration would also be predicted to increase the fidelity of the brk promoter response by making it less sensitive to fluctuations at any individual module. This fidelity would be further enhanced by a disproportionate contribution from repressed as opposed to active modules. This buffering ability of the brk promoter is likely to be important in preventing stochastic fluctuations or transcriptional noise in wild-type animals, as well as in rendering brk transcription more resistant to mutational insults (Yao, 2008).

Several developmentally important genes have modular promoters consisting of multiple non-overlapping enhancers that function autonomously to generate a composite expression pattern. The segmentation gene eve provides an archetypal example, with five enhancers that drive expression in seven discrete stripes in the embryonic blastoderm. Although brk resembles eve in its modular promoter organization and the ability of individual modules to function independently, three key differences make brk unique. First, individual eve elements are bound by different combinations of activators and repressors, and thus drive expression in distinct stripes in the embryo. By contrast, individual brk modules respond to a common set of repressive cues and drive expression in largely overlapping domains. Second, in any given region of the embryo, the eve pattern represents the output of a single enhancer. By contrast, multiple brk modules are active in each cell and contribute collectively to the final expression pattern. A final crucial difference is that in eve short-range repression prevents crosstalk between enhancers that drive expression in different stripes, while in brk the outputs of modules that appear to respond autonomously to the Dpp gradient are integrated. Why do brk and eve cis-regulatory elements display different properties, even though both use the CtBP co-repressor? One potential explanation arises from the fact that CtBP functions as part of a complex that includes histone deacetylases, histone methylase/demethylases and SUMO E2/E3 ligases. CtBP complexed with SMM on the brk promoter may recruit a different subset of activities from a CtBP-gap gene complex on eve enhancers. In addition, the SMM complex itself may recruit unique activities to the brk promoter. Furthermore, as the activators that mediate brk and eve expression are likely to be distinct, they may be affected by CtBP differentially (Yao, 2008).

Two lines of evidence argue that SMM activity is distance dependent: Shn interacts directly with the co-repressor dCtBP, and there is a functional requirement for close linkage of SMM sites and activator sequences. Short-range repression appears to be a property of the SMM complex in other contexts as well, as an SMM site located ~89 bp from a germ cell-specific enhancer in the bag of marbles (bam) gene fails to mediate repression when this spacing is increased. Furthermore, an SMM site and activator sequences are closely linked in a compact 514 bp Dpp-dependent enhancer in the gooseberry (gsb) promoter. Loss of dCtBP binding strongly reduces repression by ShnCTM, demonstrating that this interaction is relevant in vivo. However, ShnCTM still retains residual ability to repress brk-LacZ, and brk is not ectopically expressed in dCtBP clones in the wing disc. This could indicate that the dCtBP interaction motif actually has a different function in vivo. Alternatively, Shn may employ redundant repression strategies, consistent with the current view that Shn proteins act as scaffolds for co-repressors, and indeed co-activators and other modulators, enabling the Smad complex to elicit different transcriptional responses dependent on cellular context (Yao, 2008).

The identity of the activator(s) targeted by the SMM repression complex remain to be determined, as do the precise sequences to which it binds. It is possible that different brk modules incorporate inputs from distinct activators, and that some of these activators are spatially or temporally restricted. In addition to inputs from the SMM complex and the activator, there is genetic evidence that brk negatively autoregulates its own expression, most prominently in the mediolateral regions of the wing disc. Consistent with this, the brk promoter contains multiple sites that match the Brk consensus and may mediate autoregulation (Yao, 2008).

Regulation of Brinker involves a regulatory code involving Dorsal, Twist and Su(H)

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 (NotchIC). rho expression is augmented and slightly expanded in the vicinity of the stripe2-NotchIC 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).

Analysis of a brinker enhancer reveals that a simple molecular complex mediates widespread BMP-induced repression during Drosophila development

The spatial and temporal control of gene expression during the development of multicellular organisms is regulated to a large degree by cell-cell signaling. A simple mechanism has been uncovered through which Dpp, a TGFß/BMP superfamily member in Drosophila, represses many key developmental genes in different tissues. A short DNA sequence, a Dpp-dependent silencer element, is sufficient to confer repression of gene transcription upon Dpp receptor activation and nuclear translocation of Mad and Medea. Transcriptional repression does not require the cooperative action of cell type-specific transcription factors but relies solely on the capacity of the silencer element to interact with Mad and Medea and to subsequently recruit the zinc finger-containing repressor protein Schnurri. These findings demonstrate how the Dpp pathway can repress key targets in a simple and tissue-unrestricted manner in vivo and hence provide a paradigm for the inherent capacity of a signaling system to repress transcription upon pathway activation (Pyrowolakis, 2004).

One of the primary events controlled by the Dpp morphogen gradient during growth and patterning of imaginal discs is the establishment of an inverse gradient of brk expression. brk expression is controlled by two opposing activities, a ubiquitous enhancer and a Dpp-dependent silencer. The minimal requirements for a functional silencer complex, both at the DNA and at the protein level, have been determined. Importantly, it has been demonstrated that the minimal element functions in vivo when assayed in the vicinity of a strong enhancer (the brk enhancer) or when present in a single copy in chimeric transgenes (brk enhancer-bamSE fusions) or from within an endogenous gene (gsb-enhancer lacZ fusions). The minimal functional silencer contains a distinct, single binding site for each of the two signal mediators, Mad and Med. Med binds to a GTCTG site, previously recognized as a high-affinity site for Smad binding. Mad binds to a different, GC-rich sequence. Upon binding of Mad and Med, the zinc finger protein Shn is recruited to the protein-DNA complex, bringing along a highly effective repression domain. Although ShnCT contains three essential zinc fingers, it does not bind the silencer element in the absence of Mad and Med. These data suggest that even in the triple protein complex, Shn might bind DNA with moderate sequence specificity, since only a single nucleotide position was identified that is essential for Shn recruitment. However, a number of other cis-regulatory elements that bind Mad and Med (derived from the vestigial, labial tinman, and ubx genes failed to recruit Shn, demonstrating the exquisite selectivity of the element defined in this study (Pyrowolakis, 2004).

Part of this selectivity is accounted for by the specific spacing and orientation of the Mad and Med binding sites in the silencer. Deletion and insertion of single base pairs between the two sites abolish Shn recruitment in vitro and Dpp-dependent repression in vivo, although such alterations still allow the efficient formation of a Mad/Med complex. These findings suggest that Shn recruitment requires a specific steric positioning of amino acid residues in the Smad signal mediators. Strikingly, GTCTG- and GC-rich elements were also found to be crucial for the activation of the Id gene by BMP signaling, but in this case the spacing between the GTCTG- and the GC-rich sites is much larger, and additional factors might be involved in the signal-dependent activation of the Id gene. A more recent study also links these two elements to transcriptional activation of the BMP4 synexpression group in Xenopus. It is tempting to speculate that simple sequence elements similar to the one identified here in several Drosophila genes might be involved in the repression of genes by BMP signaling. Interestingly, human Smad1/5 and Smad4 do form a complex with ShnCT on the Drosophila silencer element from brk; however, a mammalian protein sharing clear homology with Shn in the C-terminal three zinc fingers has not been identified (Pyrowolakis, 2004).

The Dpp-dependent SE allows cells in the developing organism to read out the state of the Dpp signaling pathway. This readout is relatively straightforward because the SE participates in a single switch decision, that is, either to repress (bind Mad/Med and recruit Shn along with its repression domain) or not to repress (not bind Mad/Med, thus failing to recruit Shn). This decision is critically dependent upon one major parameter: the amount of available nuclear Smad complex. For the SE to be functional in vivo, it only needs to interact with a Mad/Med heteromer in those regions of the genome that are actively transcribed; genes that are not active in a given tissue do not need to be repressed by Dpp signaling. This might be one of the main characteristics explaining why such a simple sequence element can have operator-like function in vivo; the element only needs to be recognized by the relevant trans-acting factors in open and active chromatin regions (Pyrowolakis, 2004).

A minimal Dpp-dependent silencer element derived from the brk gene has been identified and demonstrated to function in vivo in a single copy. Its interaction with relevant trans-acting factors have been identified. Based on the results of this analysis, it was possible to derive a consensus sequence, GRCGNCN(5)GTCTG, which allowed scanning of the entire Drosophila genome for potential additional elements. Approximately 350 sites were identified, that, when assayed using transgenic approaches in vivo or in cell culture, should function in a manner analogous to the SEs isolated from the brk regulatory region. Strikingly, and likely significantly, in silico search revealed that the brk gene contains a total of ten SEs, three of them in regions that have been shown to respond to Dpp-dependent repression. Since brk transcription responds to (or can respond to) Dpp signaling in all tissues examined so far, brk might require a SE in the vicinity of each of the different enhancers driving expression in distinct tissues. Alternatively, the readout of the Dpp morphogen gradient might require several SEs, each contributing to the graded repression by Dpp signaling (Pyrowolakis, 2004).

Interestingly, subsequent analysis of two genes containing such Dpp-dependent SEs has demonstrated that these elements function in these transcription units the same way as they do in the brk regulatory region. Therefore, the same molecular principle underlies morphogen readout (brk repression), germline stem cell maintenance (bam repression), and restriction of gene expression to the ventral side of the developing embryo (gsb repression). When the SEs from these three genes are aligned, all the parameters determined to be important for complex formation and for repression are conserved; at all other positions, different base pairs were found in different SEs. In addition, several genes harboring silencer elements are expressed in the wing imaginal disc in a pattern similar to brk or are known to be repressed by Dpp signaling. In contrast, SEs were not found in the vicinity of enhancers known to be activated by Dpp signaling (Pyrowolakis, 2004).

Clearly, these findings implicate that Dpp-induced, Shn-dependent repression via SE elements is a key aspect of development. The readout of the brk gradient contributes to growth and patterning of appendages, and the repression of bam in the germline is essential for the maintenance of germline stem cells. To what extent the repression of gsb contributes to proper cell fate determination along the dorsoventral axis will have to be determined by rescuing the gsb phenotype with a transgene lacking the gsbSE. However, it has been observed that wingless (wg) expression expands from ventral positions to the dorsal side in shn mutant embryos. Since gsb activates wg transcription, the expansion of gsb (in the absence of the gsbSE) possibly leads to the expansion of wg and subsequently to the alteration of dorsoventral cell fate assignments (Pyrowolakis, 2004).

It is important to note that genes repressed by a signaling pathway will not easily be identified in genetic screens because the loss-of-signaling phenotype does not correspond to the loss-of-function phenotype of a repressed gene; in the absence of the signal, such genes are ectopically expressed, leading to a locally restricted gain-of-function phenotype of the corresponding gene. Moreover, since these specific, local patterns of misexpression are likely to result in different phenotypes than widespread overexpression would, simple gain-of-function screens for candidate targets of signal-mediated repression are unlikely to offer straightforward results. Since the target sequence of Dpp/Shn-mediated repression have been identified, the genome can now be scanned and potential target genes can be identified by expression studies and enhancer dissection. It is likely that additional Dpp-repressed genes will be identified using this approach, and this will allow the painting of a much clearer picture of the gene network controlled by Dpp signaling (Pyrowolakis, 2004).

Only a few cases of signal-induced repression have been studied at the molecular level. In most of these cases, repression relies on cooperative action of cell type-specific transcription factors with nuclear signal mediators. The DNA elements that have been demonstrated to mediate repression of particular genes have not been demonstrated to be important for the regulation of other genes, and genome-wide identification of potential target genes using a bioinformatic approach might therefore be difficult, if not impossible (Pyrowolakis, 2004).

The Dpp-dependent repression system identified in this study relies on the organization of Smad binding motifs into Smad/Shn complex-recruiting SEs. The simplicity of these SEs and their capacity to repress transcription in different tissues argues that they function in the absence of tissue-restricted factors. The simple consensus sequence of the SE provides a signature for Dpp-dependent repression, allowing for a genome-wide analysis of potential target genes. Confirmed Dpp-repressed target genes can then be expressed ectopically under the control of the appropriate SE-mutated enhancers to assess the biological importance of repression in a given tissue (Pyrowolakis, 2004).

Conservation of enhancer location in divergent insects

Dorsoventral (DV) patterning of the Drosophila embryo is controlled by a concentration gradient of Dorsal, a sequence-specific transcription factor related to mammalian NF-kappaB. The Dorsal gradient generates at least 3 distinct thresholds of gene activity and tissue specification by the differential regulation of target enhancers containing distinctive combinations of binding sites for Dorsal, Twist, Snail, and other DV determinants. To understand the evolution of DV patterning mechanisms, Dorsal target enhancers from the mosquito Anopheles gambiae and the flour beetle Tribolium castaneum were identified and characterized. Putative orthologous enhancers are located in similar positions relative to the target genes they control, even though they lack sequence conservation and sometimes produce divergent patterns of gene expression. The most dramatic example of this conservation is seen for the 'shadow' enhancer regulating brinker: It is conserved within the intron of the neighboring Atg5 locus of both flies and mosquitoes. These results suggest that, like exons, an enhancer position might be subject to constraint. Thus, novel patterns of gene expression might arise from the modification of conserved enhancers rather than the invention of new ones. It is proposed that this enhancer constancy might be a general property of regulatory evolution, and should facilitate enhancer discovery in nonmodel organisms (Cande, 2009).

This study identified 5 different DV enhancers in A. gambiae and T. castaneum, representing a broad spectrum of patterning responses to the Dorsal gradient. These enhancers embody the largest collection of functionally defined regulatory DNAs engaged in a common process in divergent insects. Despite extensive differences in the DV regulatory networks of flies, mosquitoes, and beetles, the enhancers are located in similar positions relative to the promoters of the target genes they control. A constrained position is also observed for the previously identified vnd enhancer in Anopheles. Altogether, these 6 enhancers are located in all possible orientations, including the immediate 5' flanking region (twist, sim), remote 5' flanking region (sog), intron 1 (vnd), 3' intron (cactus), and within a neighboring gene (brinker) (Cande, 2009).

Given the rapid divergence of noncoding sequences due to the constant turnover of individual transcription-factor binding sites within enhancer, there are no arrangements of sites or even individual sites that can be thought of as orthologous in any of the pairs of enhancers described in this study. The most closely related pair of species examined in this study, Anopheles and Drosophila, last shared a common ancestor >200 million years ago. The 2 genomes have been rearranged to such an extent that only an estimated 34% of orthologous genes can be sorted into microsyntenic blocks. In light of this, it is somewhat surprising to see such conservation in enhancer positions. There are at least 2 possible explanations. First, the different enhancers identified in Anopheles and Tribolium might be orthologous, that is, they might derive from a common enhancer in the last shared ancestor. The position of the enhancer within the locus is apparently unchanged simply because no genomic events that would perturb its position have occurred. According to this view, selection might depend on the modification of preexisting enhancers rather than the creation of new ones, similar to the evolution of protein coding sequences. A nonexclusive alternative explanation is that the enhancer position is functionally constrained within a genomic locus. For example, enhancers might be able to communicate with the target promoter only when located in particular positions within the higher-order structure of a complex genetic locus. Thus, enhancers might be in a constant flux of death and birth, but de novo enhancers work only when located in particular positions. The Hox gene complexes represent extreme examples of constrained enhancer organization. Perhaps simpler loci are subject to similar, but somewhat less stringent, organizational constraints (Cande, 2009).

The conservation of enhancer location observed in this study applies to developmental control genes. It is certainly conceivable that enhancer turnover alters the locations of regulatory DNAs, particularly in the case of housekeeping genes. An interesting example is seen for the spec genes in the sea urchin Strongylocentrotus purpuratus, which have coopted repetitive elements to function as enhancers. In contrast, regulatory genes engaged in interlocking network interactions, as seen for the genes considered in this study, might tend to retain old enhancers rather than invent new ones. An implication of this study is that the evolution of novel patterns of gene expression depends on the modification of existing enhancers rather than the invention of new ones. This has already been documented for the evolution of the sim expression pattern and ventral midline of divergent insects. It is proposed that the modification of constrained or orthologous enhancers will prove to be a general mechanism for the evolution of gene expression patterns (Cande, 2009).

Ancestral and conserved cis-regulatory architectures in developmental control genes

Among developmental control genes, transcription factor-target gene 'linkages'-- the direct connections between target genes and the factors that control their patterns of expression--can show remarkable evolutionary stability. However, the specific binding sites that mediate and define these regulatory connections are themselves often subject to rapid turnover. This paper describes several instances in which particular transcription factor binding motif combinations have evidently been conserved upstream of orthologous target genes for extraordinarily long evolutionary periods. This occurs against a backdrop in which other binding sites for the same factors are coming and going rapidly. These examples include a particular Dpp Silencer Element upstream of insect brinker genes, in combination with a novel motif referred to as the Downstream Element; combinations of a Suppressor of Hairless Paired Site (SPS) and a specific proneural protein binding site associated with arthropod Notch pathway target genes; and a three-motif combination, also including an SPS, upstream of deuterostome Hes repressor genes, which are also Notch targets. It is proposed that these stable motif architectures have been conserved intact from a deep ancestor, in part because they mediate a special mode of regulation that cannot be supplied by the other, unstable motif instances (Rebeiz, 2012).

Previous studies described the phylogenetically widespread occurrence of single, high-affinity bHLH repressor (R) binding sites (a consensus GGCACGCGCC, with variants in the last two bases) upstream of bilaterian proneural genes (Rebeiz, 2005). The possibility could not be ruled out that only the 'linkage' (direct transcription factor-target gene relationship) has been maintained, and that the binding site itself has been replaced repeatedly in the course of animal evolution. However, several lines of evidence suggest that these R sites have been conserved from a deep common ancestor. These included the stability of the precise 10-bp sequence of the site over very long intervals, and the strong conservation of both the motif and flanking sequences in some instances, clearly suggesting that the sites are indeed orthologous (Rebeiz, 2012).

The present report substantially expands the inventory of such apparently ancient and conserved cis-regulatory motifs in developmental control genes. This study describes five additional cases in which specific motif combinations have evidently been retained over hundreds of millions of years of evolution. With the exception of two novel elements [the insect brk Downstream Element (DE) and the deuterostome Hes XE], these motifs represent high-affinity binding sites for known transcription factors. The retention of these specific motif instances is especially striking when considered against the background of rapid appearance and disappearance of other binding sites for the same factors (Rebeiz, 2012).

The conservation of the distinctive SE + DE motif (SE: GRCGNCN5GTCTG) combination upstream of insect brk genes extends over perhaps 270-300 My, reflecting the fact that the brk gene itself is found only in insects. A similar (minimum) age can be assigned to the P + SPS architecture found upstream of insect bHLH repressor genes, while the E + SPS + P combination associated with arthropod BFM genes is even older, in excess of 400 My, in view of its occurrence in the crustacean D. pulex. Finally, it is likely that the X + R + SPS ensemble upstream of deuterostome Hes1 genes was present in the common ancestor, over 500 My ago. It is also possible that an SPS element was associated with an ancestral bilaterian Hes repressor gene, which would make this feature close to 600 My old (Rebeiz, 2012).

This analyses do not permit the discerning of the population genetic/microevolutionary processes by which the distinctive cis-regulatory architectures first arose and became fixed in an ancestral population. However, it is believed that some useful insights can be offered into why these architectures have endured over such lengthy timescales (Rebeiz, 2012).

What characteristics of ancient and conserved motifs drive their long-term preservation by selection, even as other binding sites for the same factors come and go rapidly in evolution? An earlier proposal is first reiterated that such deeply conserved motifs mediate abstract or generic regulatory functions of fundamental utility to all or most members of an ancient clade (Rebeiz, 2005). It is certainly plausible that, once established, the capacity to repress brk transcription in response to a Dpp signal remained of great utility to all the descendants of the common insect ancestor, as diverse as they became. Similarly, the abstract ability to activate a Hes repressor gene via Notch signaling would remain of exceptional utility to descendants of a bilaterian (or earlier) ancestor that had evolved it. Finally, a generic capability for autorepression of a Hes bHLH repressor gene might very well be retained by descendants of a deuterostome ancestor (Rebeiz, 2012).

But it is certainly sensible to argue that, to retain such abstract and valuable regulatory capabilities, it would suffice to preserve only the linkage between the appropriate transcription factors and their targets. In this view, individual factor-binding motifs need not be retained; they would be free to turn over during evolution. However, the examples described in this study suggest a second important reason for the long-term evolutionary retention of particular motifs or motif combinations. It is proposed that these conserved sequence elements mediate a distinctive regulatory capability not conferred by other instances of the same motif or motifs. In the case of the SPS element, considerable confident can be had that this perspective is correct. The SPS has been shown to mediate cooperative binding of two Su(H)/Mam/NICD trimers, thus conferring on the associated target gene unusually high sensitivity to Notch signaling. While two 'lone' Su(H) sites are indeed able to contribute to a target gene's response to activated Notch, they would not do so in a cooperative manner. In a similar vein, it seems plausible to suggest that while all SE motifs may be able to participate in signal-dependent repression of brk, the SE + DE combination offers a unique and valuable version of this capability (e.g., greater signal sensitivity), possibly conferring a fitness advantage. It is hypothesized that in both cases, once the specialized motif architecture (SPS or SE + DE) had evolved to confer a distinctive capacity, it would be selectively retained in evolution. As is known, other instances of the SE or Su(H) binding motifs do arise and become fixed in individual clades, but these would not be expected to exhibit the same durability, since (according to the hypothesis) they confer no unique capability. The foregoing interpretation is particularly supported by the frequent observation that if only one element mediating a particular response [either SE or Su(H) site] is present upstream of an orthologous gene in a given species, it is of the 'special' type (SE + DE or SPS). Examples include the SE + DE combination in T. castaneum brk and the SPS motifs in the A. gambiae bHLHR1 gene, the A. mellifera BFM gene, and H. sapiens HES1 (Rebeiz, 2012).

Another factor that may contribute to the long-term evolutionary conservation of the specialized motif architectures this study has considered is their very complexity. Both the SE + DE unit and the SPS represent unusually extended and constrained motif combinations. While in principle this does not prevent them from turning over by duplication/degeneration, they are unlikely to evolve de novo (Rebeiz, 2012).

Finally, an intriguing feature is noted of the conserved motif architectures described in this study that involve the SPS: the apparently conserved order and even orientation of the individual sequence elements. The arthropod BFM genes are associated with a 'lower-strand' E motif followed by an SPS followed by a 'lower-strand' P site; insect Hes repressor genes bear an 'upper-strand' P site followed by an SPS; and deuterostome Hes1 genes have an 'upper-strand' X site followed by an 'upper-strand' R site followed by an SPS, which also has fixed orientation. Inter-site distances are often not conserved; consider the varying separation of the SPS and the P site in the BFM genes, or the different distances between the X + R combination and the SPS in the deuterostome Hes1 genes. Evidently, the motif order and orientation of these architectures have functional significance, consistent with an 'enhanceosome' model for the structure of these regions. Alternatively, these features may suggest the existence of a 'scanning' mechanism for optimal enhancer-promoter interaction. Such a property might be a particular characteristic of promoter-proximal cis-regulatory modules such as these, as contrasted with more distal enhancers. In the latter case, interaction with the promoter by 'looping' may impose fewer architectural constraints (Rebeiz, 2012).

This study has proposed that the distinctive cis-regulatory architectures ancient ones that have been conserved from a deep ancestor. However, it also seems likely that, because of their very complexity, they may not represent the 'original' version of their respective regulatory linkages. These two realizations can be reconciled via the following general evolutionary scenario (Rebeiz, 2012).

The direct linkage of an ancestral Hes gene to Su(H) and the Notch pathway evidently originated in a deep metazoan ancestor, and was very likely mediated by a lone Su(H) binding site or sites. The genome of the demosponge A. queenslandica includes one member of the closely related Hey repressor family, but no Hes genes; this Amphimedon Hey gene has one high-affinity Su(H) site 600 bp upstream of the transcription start site. The placozoan T. adhaerens has one Hey ortholog, one Hey-related gene, and one Hes gene. The Hey ortholog has three high-affinity Su(H) sites in the first 800 bp upstream of the ATG start codon, while the Hes gene includes a single such site within 500 bp of its ATG. The genome of the cnidarian N. vectensis (sea anemone) is endowed with a large paralogous family of 11 Hes genes, many of them with multiple lone Su(H) sites immediately upstream. Likewise, the Nematostella Hey ortholog has two upstream Su(H) sites. The SPS evidently did not appear upstream of a Hey/Hes gene until after the cnidarian-bilaterian divergence, but this association is now widespread among both protostomes and deuterostomes (Rebeiz, 2012).

It is suggested, then, that what appeared first was the simple capacity to regulate a Hey/Hes gene directly by Su(H) (presumably linked to the Notch pathway), via one or more lone Su(H) binding sites. Then, in a bilaterian ancestor, an SPS came into being upstream of an individual Hes gene, making possible a cooperative and thus highly sensitive response to Notch-activated Su(H). Once this novel regulatory capacity was established, it bestowed a sufficient selective advantage to ensure its subsequent retention in a wide variety of bilaterian taxa. Such a scenario can account for the phylogenetic distribution of the SPS-containing cis-regulatory architectures described. More complex histories cannot be ruled out, including the possibility that the SPS arose independently more than once in association with Hes genes (Rebeiz, 2012).

It is important to note the finding that, in the case of target genes that are part of paralogous families (Hes repressor and BFMs), only one particular paralog in a given species is typically associated with the conserved motif architectures described. This is true even if other paralogs make use of the same overall cis-regulatory 'code' (combination of transcription factor binding sites) to direct a similar expression specificity. For example, of the seven unambiguous Hes repressor paralogs in H. sapiens, only HES1 bears the X + R + SPS motif combination, though four others have upstream S sites and two of these also have upstream R sites. Likewise, the D. melanogaster genome includes nine BFM genes, most of which employ the S + P code, but only one, E(spl)m4, is associated with an SPS + P combination. It seems likely that, while the distinctive regulatory capability conferred by an ancient and conserved motif combination is of long-term selective value, it suffices for a single paralog in the genome to retain it (Rebeiz, 2012).

This observation is consistent with a duplication-divergence model for the evolution of Hes and BFM paralogs. The special cis-regulatory architectures this study has described, along with the associated protein coding sequences, comprise functional units that have been conserved from deep common ancestors because of the unique regulatory capabilities they confer. Paralogous genes that arise by duplication within various taxa (this is a widespread phenomenon in the case of Hes genes) would not be subject to the same stringent constraints on their cis-regulatory architecture, since the ancestral gene would be present to provide the distinctive capabilities. The paralogs would thus be free to evolve their cis-regulatory motifs according to other selective pressures or genetic drift, yielding the many variations on a basic theme (e.g., S + P) that is observed within a single species today (Rebeiz, 2012).

Repression of Dpp targets in the Drosophila wing by Brinker

Patterning along developing body axes is regulated by gradients of transcription factors, which activate or repress different genes above distinct thresholds. Understanding differential threshold responses requires knowledge of how these factors regulate transcription. In the Drosophila wing, expression of genes such as omb and sal along the anteroposterior axis is restricted by lateral-to-medial gradients of the transcriptional repressor Brinker (Brk). omb is less sensitive to repression by Brk than sal and is consequently expressed more laterally. Contrary to previous suggestions, it has been shown that Brk cannot repress simply by competing with activators, but requires specific repression domains along with its DNA-binding domain. Brk possesses at least three repression domains, but these are not equivalent; one, 3R, is sufficient to repress omb but not sal. Thus, although sal and omb show quantitative differences in their response to Brk, there are qualitative differences in the mechanisms that Brk uses to repress them (Winter, 2004).

The simplest method of transcriptional repression involves competition with an activator, and can operate at the level of DNA if the activator and the repressor have the same, or overlapping, binding sites in an enhancer. In theory, assuming a transcription factor is nuclear, it should only require a DNA-binding domain to act in this fashion. Brk has been shown to possess an N-terminal sequence-specific DNA-binding domain (DBD), and this study has identified several mutations in this domain that either completely inactivate or reduce the activity of the protein, indicating that this region is essential for Brk activity (Winter, 2004).

Previous studies suggested that Brk could function by competition, more specifically, by competing with Mad for overlapping binding sites in vitro. However, a nuclear localized Brk protein consisting primarily of the DBD, BrkNLS, cannot repress any Brk target in vivo, including the embryonic UbxB reporter, which has been shown to possess overlapping Brk and Mad binding sites that Brk and Mad can compete for in vitro. It is possible that BrkNLS cannot bind to DNA in vivo. However, a modified protein, BrkNLSW, which is identical to BrkNLS apart from the addition of the four amino acids WRPW that recruit the co-repressor Gro, can repress targets, indicating that BrkNLS should also be capable of binding to these targets in vivo (Winter, 2004).

Competition has been proposed as a mechanism for many transcriptional repressors. However, direct in vivo support for or against such proposals is rare, at least of the sort presented here, i.e., testing, in vivo, the ability of a protein consisting largely of a functional DBD, which has access to the nucleus, to repress a target for which there is in vitro evidence for overlapping binding sites with an activator. There is some in vivo evidence that the Drosophila embryonic repressor Kruppel can repress a synthetic enhancer containing overlapping binding sites with the activators Dorsal and Bicoid. However, although this repression is CtBP-independent, and further studies are required to rule out additional domains outside of the DBD being required in a similar fashion to the 3R domain in Brk. The paucity of good examples of binding-site competition in vivo in eukaryotes is in stark contrast to that in prokaryotes, and raises the question of how common this phenomenon really is in eukaryotes (Winter, 2004).

If Brk cannot repress by competition it must possess repression domains/motifs, and previous studies identified interaction motifs for the co-repressors CtBP and Gro (CiM and GiM). However, repression of at least one Brk target, omb, has been shown shown not to require CtBP or Gro. This is consistent with the demonstration that the protein produced by the endogenous mutant brkF138, which is truncated before the CiM and GiM, can still repress omb. Truncated proteins that lack the CiM and GiM, BrkStop1, BrkEC and BrkA, can also repress omb, but only if they contain a specific region between the DBD and CiM that has been classified as a third repression domain, 3R. Further studies are required to determine if 3R is a true autonomous repression domain, i.e., if it can function outside of Brk, or if it is more specific (for example, antagonizing activators such as Mad), and to determine what its specific properties are (for example, how close do Brk sites have to be to activator sites for 3R to be effective?) (Winter, 2004).

The three repression domains/motifs of Brk are not equivalent. Wild-type Brk and proteins containing only a GiM, BrkNLSW, or only a CiM, BrkStop1NAC, can repress both sal and omb, and they are more effective at repressing sal than omb. Analysis of gro and CtBP single and double mutant clones reveals that Gro is required for normal repression of sal in wing discs, and that CtBP can provide some, but not always complete, activity for the repression of sal in the absence of Gro. By contrast, Gro and CtBP are not required for repression of omb (Winter, 2004).

The 3R domain (the region between the DBD and the CiM) is sufficient for Brk to repress omb and the UbxB enhancer in embryos, but is deficient for the repression of sal. Furthermore, misexpression of proteins possessing only the 3R domain (plus the DBD) are much more effective at repressing omb than sal, i.e., the converse of wild-type Brk or Brk possessing only a GiM or a CiM. Although some results suggested that 3R may confer a limited ability to repress sal, this is probably indirect, because a previous study demonstrated that sal requires Omb to be expressed, and if omb is repressed directly, sal will be lost also. However, the possibility cannot be ruled out that high levels of proteins possessing only the 3R domain can repress sal directly (Winter, 2004).

Contradictory results were obtained regarding the ability of 3R to repress the vg-QE. Expression of the vg-QE did show expansion in some brkF138 clones, indicating that the truncated protein produced in this mutant (which only has the 3R repression domain) cannot efficiently repress this enhancer. However, similar in vitro truncated proteins, such as BrkStop1, could efficiently repress vg-QE expression when misexpressed using the UAS/Gal4 system. Such a difference could simply be a reflection of the high levels of expression achieved with the Gal4/UAS system, and that, at physiological levels, the 3R domain is not sufficient for complete repression of the vg-QE (Winter, 2004).

Whether a single repression domain is sufficient for Brk to repress a particular target may depend upon the positioning of Brk sites in relation to activator sites (or possibly the promoter) at that target. The UbxB reporter has overlapping Brk and activator (Mad) sites. Analysis of an omb enhancer revealed that an important Brk site may also overlap with an activator. Conversely, analysis of the cis-regulatory elements of the sal gene indicate that activator and Brk sites are separated. Proteins possessing only 3R can repress UbxB and omb, but not sal, suggesting that 3R may only be sufficient for the repression of genes in which the Brk sites are situated very close to activator sites (Winter, 2004).

Why does Brk possess at least three, probably four, independent repression domains/motifs? There are two obvious answers: qualitative, different repression domains/motifs are required for the repression of different targets; quantitative, more domains/motifs provide greater repressor activity. Other transcription factors have multiple repression domains and there is evidence that they have these for either qualitative or quantitative reasons, and, in some cases, both. For example, in the Drosophila embryo, the pair-rule protein Runt requires Gro for the repression of one stripe of the pair-rule genes, even skipped (eve) and hairy, but not for the repression of engrailed. The gap protein Knirps represses different stripes of eve; for stripes 4 and 6 it requires CtBP, but for stripes 3 and 7, it does not. However, this appears to be a quantitative difference, because increasing the levels of Knirps allows it to repress stripes 4 and 6 even in the absence of CtBP. Similarly, Gro appears to increase the repressor activity of the Eve protein (Winter, 2004).

There is some difference in the ability of the three domains/motifs in Brk to repress different targets. For example, the 3R domain is sufficient for the normal repression of omb but not sal. However, either the CiM or GiM appear to be sufficient for the repression of both sal and omb, so why does Brk need the 3R domain? In the absence of Gro and CtBP, the Brk protein appears fully active in its ability to repress omb, and recruiting Gro and CtBP does not seem to increase its activity towards omb; otherwise, the width of the omb domain would be expected to shift in brkF138 mutant cells, which have no CiM or GiM, or in CtBP gro double mutant cells, but Brk protein does not. It is possible that, in regard to omb, the 3R domain is more efficient than either of the other two and provides Brk with sufficient activity to establish the omb domain in the correct position (Winter, 2004).

Brk needs to recruit either CtBP or Gro for the repression of some targets, including sal and brk itself, or just Gro for some others, including the vg-QE. Consequently, why does Brk need to recruit CtBP? Mutation of the CiM alone, in common with mutation or deletion of just the GiM and 3R, does reduce activity of Brk, as judged by its effect when misexpressed. However, there is no evidence that CtBP is required specifically for the repression of any Brk target in the wing, because CtBP mutant clones have no effect on the expression of any known Brk target in the wing. The CtBP and Gro motifs in Brk have been conserved over millions of years, and thus, recruiting CtBP is presumably important for Brk activity. It is possible that CtBP is required outside of the wing -- for example in the embryo -- or for some other, as yet, uncharacterized targets in the wing (Winter, 2004).

Recruiting both CtBP and Gro does appear to be a little illogical from what is known about their basic properties, CtBP acting only over a short range, while Gro acts over much longer ranges. It might be assumed that different transcription factors would use either Gro or CtBP, because the primary advantage of recruiting CtBP is that it would allow a transcription factor to repress one enhancer without disrupting the activity of one nearby, which would be repressed if Gro was recruited, although this simple model does not always hold. Consequently, most transcription factors do recruit only one of these co-repressors. However, there are two other exceptions, Hairy and Hairless. In Hairy it appears that CtBP may actually be functioning to antagonize Gro activity and not in its standard role as a co-repressor. There is no evidence that it does this in Brk, where it can provide repressor activity. For Hairless, there is genetic evidence that both CtBP and Gro provide repressor activity to the protein, although it is not clear if CtBP is required to increase the general activity of Hairless, or for repression of specific targets that cannot be repressed adequately by Gro (Winter, 2004).

With the exception of the brkF138 mutant, this analysis has been limited to analyzing the effects of misexpressing modified Brk proteins in positions where the endogenous protein is not found. Consequently, further insights into the precise roles of individual repression domains will require replacing the endogenous gene with one in which only one or two repressions domain have been mutated or deleted (Winter, 2004).

To conclude, it is often assumed that the sensitivity of one enhancer to a transcription factor compared with that of another enhancer is based largely upon the number or the affinity of the binding sites for that transcription factor in each enhancer. However, other factors are also important; for example, the ability of the Giant transcription factor to repress a promoter is related to how closely it binds. This study has shown that the two best characterized outputs of the Dpp morphogen gradient, sal and omb, appear to be regulated differently by Brk. Consequently, simply counting binding sites and measuring their affinity will not reveal why one is more sensitive to Brk than the other, and it is necessary to factor in what specific repressive mechanisms are being used, and the relative efficiencies of each (Winter, 2004).

Decapentaplegic-responsive silencers contain overlapping mad-binding sites

Smad proteins regulate transcription in response to transforming growth factor-β signaling pathways by binding to two distinct types of DNA sites. The sequence GTCT is recognized by all receptor-activated Smads and by Smad4. The subset of Smads that responds to bone morphogenetic protein signaling recognizes a distinct class of GC-rich sites in addition to GTCT. Recent work has shown that Drosophila Mad protein, the homologue of bone morphogenetic protein rSmads, binds to GRCGNC sites through the same MH1 domain β-hairpin interface used to contact GTCT sites. However, binding to GRCGNC requires base-specific contact by two Mad proteins, and this study provides evidence that this is achieved by contact of the two Mad subunits that overlap across the two central base pairs of the site. This topology is supported by results indicating that His-93, which is located at the tip of the Mad β-hairpin, is in close proximity to base pairs 2 and 5. Also consistent with the model is disruption of binding by mutation of Glu-39 and Glu-40, which are predicted to lie at the interface of the two overlapping Mad MH1 domains. As predicted from the overlapping model, binding is disrupted by insertion of 1 bp in the middle of the site, whereas insertion of 2 bp creates abutting sites that can be bound by the Mad-Medea heterotrimer without requiring Glu-39 and Glu-40. Overlapping Mad sites predominate in Decapentaplegic response elements, consistent with a high degree of specificity in response to signaling (Gao, 2006).

DNA contact by Smad proteins has been shown to play an important role in many instances of target regulation by TGFβ pathways. For the consensus Smad3/Smad4-binding site, GTCT can also be bound by Mad- and BMP-specific Smad1, but Smad3 does not bind to GC-rich Mad/Smad1-binding sites, leaving open the question of whether such sites are contacted by a different mechanism. Recent work had shown that Mad-binding sites within the brk and bam silencers are bound by two Mad subunits and that in each case two Mad MH1 domains simultaneously contact a single 6-bp site using the same three β-hairpin residues that are responsible for base-specific contact by Smad3. By using mutational analysis and directly measuring binding, this study provides evidence that two Mad MH1 domains bind to the 6-bp site by overlapping across the two central base pairs. Smad1-binding sites match this 6-bp motif, a likely indication that overlap is also a feature of BMP-response elements (Gao, 2006).

The overlapping structure of Mad sites explains the seeming discrepancy between the Mad consensus and that of Smad3/Smad4. Smad3 differs from Mad at two positions that influence binding to brkS. Arg-58 at the C terminus of helix 2 is absolutely essential for binding to brkS; in Smad3 this position is a threonine, whereas the adjacent Lys-59 of Mad is absent in Smad3. Glu-39 at the N terminus of helix 2 contributes substantially to binding affinity for brkS, and Smad3 has instead a glutamine at this position. In addition, the loop between helices 1 and 2 is three residues shorter in Mad than in Smad3, a difference that modeling suggests will affect the structure of the alpha-carbon backbone and side chains near the N terminus of helix 2. Each of these differences is conserved between Smad3 and Smad2 and between Mad and the vertebrate BMP-specific rSmads (Gao, 2006).

Mutational analysis indicated the optimal sequence for an overlapping Mad site is GGCGCC, meaning each Mad MH1 prefers GGCG in the context of overlap. However, even when the two Mad sites are spaced such that they do not overlap, GGCG is still bound by Mad with about the same affinity as GTCT. The structural basis for this compatibility with two distinct sites remains to be determined, but the differential effects of helix 2 alanine substitutions suggest distinct docking geometries. Individual GGCG motifs occur in Dpp and BMP-response elements, and the results indicate that these are likely sites for contact by Mad and Smad1 (Gao, 2006).

Although the natural brkS element was specific for the Mad-Medea heterotrimer, changing the Mad site to abutting SBEs allowed binding by Mad alone or by Medea alone. The ability of such a site to be bound by Medea oligomers (putatively homotrimers) without Dpp signaling seemingly would make it ill-suited to function as a Dpp-response element, although signaling-induced activation was observed by reporter analysis (perhaps an indication that Medea alone is a poor activator). However, the brkS derivative with abutting GGCG sites (i.e. GGCGCGCC) shows little or no Medea binding in the absence of active Mad, is able to recruit Shn, and causes repression in response to signaling. Similar sites in the Dpp-response element of Race (GACGCGAC), which does not respond to repression by Brk protein, and in a BMP-response element of Smad7 (GGCGCGCC) appear to be examples of functional nonoverlapping Mad/Smad1 sites. In Drosophila a potentially significant difference between overlapping and nonoverlapping Mad sites is that the overlapping motif allows for competitive binding by the Brinker protein and thus dual control of Dpp targets, whereas the nonoverlapping motif does not. This may account for the predominance of overlapping Mad sites in Drosophila. The predominance of overlapping sites in BMP-response elements may reflect specificity for Smad1 but not Smad3 (Gao, 2006).

Autoregulatory Feedback Controls Sequential Action of cis-Regulatory Modules at the brinker Locus

cis-regulatory modules (CRMs) act sequentially to regulate temporal expression of genes, but how the switch from one to the next is accomplished is not well understood. To provide insight, here this study investigate the cis-regulatory system controlling ()brinker (brk) expression in the Drosophila embryo. Two distally located CRMs support expression at different times, while a promoter-proximal element (PPE) is required to support their action. In the absence of Brk protein itself or upon mutagenesis of Brk binding sites within the PPE, the late-acting CRM, specifically, is delayed. This block to late-acting CRM function appears to be removed when the early-acting CRM is also deleted. These results demonstrate that autoregulatory feedback is necessary for the early-acting CRM to disengage from the promoter so that the late-acting CRM may act. Autoregulation may be a commonly used mechanism to control sequential CRM action necessary for dynamic gene expression throughout the course of development (Danipace, 2013).

Collectively, these results show that (1) two CRMs control spatially and temporally distinct patterns of brk expression; (2) the switch from one CRM to the next requires a promoter-proximal sequence; and (3) levels of Brk protein influence the switch from early enhancer to late-acting enhancer in the early embryo. Although the 5' CRM is the primary acting module precellularization, at the onset of cellularization (mid-stage 5) competition between the 5' and 3' CRMs for access to the promoter complex is likely. This competition is affected by Brk protein and the PPE. In the presence of these two factors, the 3' CRM is able to outcompete the 5' CRM for access to the promoter. In the absence of Brk protein, or when the PPE is not intact, the 5' CRM remains active and blocks the activity of the 3' CRM. The results of mutagenesis of Brk binding sites within the PPE provide strong evidence for a role for Brk at the PPE; however, the 3C experiments suggest that large-scale changes in chromatin conformation do not necessarily accompany the switch between 5' CRM and 3' CRM action. A model is favored in which Brk acts through the PPE to modulate the local 3D chromatin environment to bias 3' versus 5' CRM action and thereby catalyze the switch between CRMs. The general implication of this study is that autoregulatory feedback may afford one CRM a positive advantage in competition with other CRMs for engagement with the promoter. Whether CRM competition is acting to control temporal expression of other genes remains to be determined, but it is suggested to be likely. The current view is that important developmental regulators that control large numbers of genes will be autoregulated, because their levels of expression must be tightly controlled. Autoregulatory control may therefore be a common and effective mechanism used to control temporal gene expression through regulation of sequential activation of CRMs. Once the amounts of a factor rise to a particular level that supports autoregulation, then the timing may be right to switch to a subsequently acting CRM. What better cue to support timing of CRM switch than the factor itself (Danipace, 2013).

Manipulating the sensitivity of signal-induced repression: quantification and consequences of altered Brinker gradients

Traditionally, the analysis of gene regulatory regions suffered from the caveat that it was restricted to artificial contexts (e.g. reporter constructs of limited size). With the advent of the BAC recombineering technique, genomic constructs can now be generated to test regulatory elements in their endogenous environment. The expression of the transcriptional repressor brinker (brk) is negatively regulated by Dpp signaling. Repression is mediated by small sequence motifs, the silencer elements (SEs), that are present in multiple copies in the regulatory region of brk. This study manipulated the SEs in the brk locus. The effects of the individual SEs on the Brk gradient in the wing disc were precisely quantified by employing a 1D data extraction method, followed by the quantification of the data with reference to an internal control. It was found that mutating the SEs results in an expansion of the brk expression domain. However, even after mutating all predicted SEs, repression could still be observed in regions of maximal Dpp levels. Thus, the data point to the presence of additional, low affinity binding sites in the brk locus (Gafner, 2013).

Making use of genomic constructs featuring between zero and 13 functional consensus SEs in their endogenous context as well as a differently labeled internal control as a reference, this study developed a quantification method that allowed very precise quantification of the effect of single SEs or SE combinations on the Brk gradient. Consistent with the idea that the SEs seem incapable of exerting repression over long distances it was found that in a genomic context, a single functional SE is not sufficient to reproduce the endogenous brk expression pattern. In other words, a single functional SE is not able to repress the enhancers that are dispersed over the brk locus. The results indicate that the presence of functional SE3&4 has a significantly stronger effect on target gene repression than the presence of functional SE1&2. Furthermore, SE10 has a stronger effect than the combination of SE1&2. Along the same lines, SE4 causes a more potent repression than SE10. SE4 is also stronger than SE3, as the presence of SE4 alone causes more or less similar repression as the combination of SE3&4. SE3 might be exceptionally weak and therefore does not detectably add to the effect of SE4 (Gafner, 2013).

The effect of the different SEs on brk repression may be influenced by a combination of factors, including the strength of individual SEs and the strength of the enhancers. Both the proximity of the SEs to the enhancers as well as the proximity of the SEs and enhancers to brk's transcriptional start may play a role. In the future, it will be interesting to analyze more in detail the enhancers in the brk locus. Furthermore, it is conceivable that individual enhancer-SE combinations may show some tissue specific responsiveness to Dpp signaling. SE13, which does not seem to contribute to brk repression in the wing imaginal disc, may play a role in other tissues (G. Pyrowolakis, unpublished) (Gafner, 2013).

In comparison to Drosophila melanogaster, which features a relatively high number of SEs, other insect species contain significantly less such regulatory elements in the brk locus. This interesting evolutionary observation might offer nice models for future investigations of the architecture of SEs and enhancers on a simpler level (Gafner, 2013).

Mutating all the consensus SEs in the brk locus results in incomplete derepression. The residual medial repression seems to be still mediated by Dpp signaling. The mechanism underlying this residual repression is still unclear, although the results suggest that it is not post-transcriptional regulation via, for example, a miRNA. Another explanation for the remaining medial repression is the existence of a novel type of Shn-dependent repressor element; or repression might also happen indirectly as a secondary effect, for example via target genes of the Dpp pathway that might repress brk in medial parts of the wing disc. Alternatively, residual repression could depend on the presence of additional SEs which might have been missed simply because the definition of the SEs has been too strict (Gafner, 2013).

Indeed, the results indicate that the residual medial repression is (at least partly) due to the presence of SE motifs with a more degenerate sequence than previously anticipated, although it cannot be excluded at this stage that there are additional regulatory elements of completely different nature that also contribute to brk regulation. Focusing on the fragment covering SE1&2mut, two degenerate SE sequences were additionally mutated. These are two of only three degenerate SE sequences present in the locus that showed a single bp substitution at position one or three, compared to the perfect consensus (the third one, m3-2, is located in an intron of atg5, downstream of the brk coding sequence. Mutating these sites led to an expansion of the readout, proving their functionality. If the relaxed consensus that were used in this study are combined with these additional relaxations at the two newly identified positions, 20 additional putative SEs are uncovered: 12 SEs with the consensus GNNGNC (N)5 GNCTN and eight with the consensus NNCGNC (N)5 GNCTN and some of these are located in the fragments covering SE3-8 and SE9-12. Clearly, this consensus might be too relaxed and not all of the sites will have an effect in vivo. Indeed, two of these proposed novel SEs, which are based on a combination of the relaxed consensus with a wobble at either position one or three of the consensus, showed no complex formation when randomly picked for in vitro testing. However, any future dissection of the elements regulating brk expression will need to take potential additional sites into account (Gafner, 2013).

Previous studies had used reductionist approaches to elucidate the mechanisms underpinning brk expression. Taking advantage of technological advances, this study analyzed the role of the SEs in the context of the entire locus rather than in an isolated fragment. While on the whole supporting the existing model, the results indicate that it is necessary to revise the notion of the SE as a motif with a strict consensus. The obvious explanation for the observations of this study would be that in regions of high signaling, components of the repression complex (pMad-Medea-Shn) bind to sites that do not have the optimal sequence, although it remains to be shown that other regions of the brk regulatory region also contain such degenerate SEs (obviously, ChIP with Shn would be an experiment to test this, although it is not a straightforward approach and beyond the scope of this work). It may even be a general biological phenomenon that sites diverging from the perfect consensus are bound by the pathway mediators in regions of maximal signaling activity and that this will affect the expression of target genes. This has implications for the interpretation of large scale CHIP-on-CHIP and CHIP-seq datasets and serves as a note of caution against assuming that a consensus sequence can have no wobble (Gafner, 2013).

Zelda potentiates morphogen activity by increasing chromatin accessibility

Zygotic genome activation (ZGA) is a major genome programming event whereby the cells of the embryo begin to adopt specified fates. Experiments in Drosophila and zebrafish have revealed that ZGA depends on transcription factors that provide large-scale control of gene expression by direct and specific binding to gene regulatory sequences. Zelda (Zld) plays such a role in the Drosophila embryo, where it has been shown to control the action of patterning signals; however, the mechanisms underlying this effect remain largely unclear. A recent model proposed that Zld binding sites act as quantitative regulators of the spatiotemporal expression of genes activated by Dorsal (Dl), the morphogen that patterns the dorsoventral axis. This study tested this model experimentally, using enhancers of brinker (brk) and short gastrulation (sog), both of which are directly activated by Dl, but at different concentration thresholds. In agreement with the model, it was shown that there is a clear positive correlation between the number of Zld binding sites and the spatial domain of enhancer activity. Likewise, the timing of expression could be advanced or delayed. Evidence is presented that Zld facilitates binding of Dl to regulatory DNA, and that this is associated with increased chromatin accessibility. Importantly, the change in chromatin accessibility is strongly correlated with the change in Zld binding, but not Dl. It is proposed that the ability of genome activators to facilitate readout of transcriptional input is key to widespread transcriptional induction during ZGA (Foo, 2014).

In blastoderm embryos, brinker (brk) is activated in an eight- to ten-cell-wide domain that develops into the ventral neurogenic ectoderm (NE), whereas short gastrulation (sog) is expressed in a broader band of 16-18 cells encompassing the entire NE. Both genes have the same ventral expression boundary due to repression by Snail (Sna) in the presumptive mesoderm. The dorsal borders of their domains lie in regions of the Dorsal (Dl) gradient where amounts are low and change little, raising the question of how their enhancers can interpret small differences in Dl concentrations (Foo, 2014).

sog and brk each have two reported cis-regulatory modules (enhancers) that are active in early embryos. The sog intronic lateral stripe enhancer (LSE) is less well conserved and drives a slightly narrower stripe of expression relative to the sog shadow enhancer, also known as the neurogenic ectoderm enhancer (NEE), which recapitulates the broad endogenous sog pattern. The brk 5′ and 3′ enhancers both support lateral stripes similar to endogenous brk; however, the brk 3′ enhancer drives a more dynamic pattern that broadens at cellularization. Thus, this study focused on the brk 5′ enhancer to avoid confounding dynamic change of width (Foo, 2014).

The sog 426 bp NEE contains three CAGGTAG heptamer sites for optimal Zelda (Zld) binding. However, the brk 498 bp 5′ enhancer does not have any canonical Zld binding sites (also known as TAGteam sites). To explain its Zld dependence, electrophoretic mobility shift assays were used to look for Zld binding sites in the brk 5′ enhancer. Three CAGGTCA sequences and a tandem GAGGCACAGGCAC sequence were identified that promote very weak Zld binding, which was abolished upon mutation of the sites (Foo, 2014).

To test whether altering the number of Zld binding sites in the NE enhancers can affect the expression they drive, mutant forms of the brk and sog enhancers were created. The sog NEE drives a lacZ reporter expression pattern identical to endogenous sog. Mutation of all three CAGGTAG sites dramatically reduced the expression width (sog). Similar changes were also observed by a previous study when the CAGGTAG sites were mutated in the sog LSE. Costaining of lacZ and endogenous sog illustrates that the narrowed lacZ domain resulted from a collapse of the dorsal, not the ventral, border. It is inferred that without Zld, sog is unable to be activated by the lower levels of Dl in the dorsal neuroectoderm region. In embryos lacking maternal Zld (referred to as zld-), both the endogenous sog and sog wt domains shrink and become sporadic. This is not due to an indirect effect on the Dl concentration gradient because it is unchanged in zld-. Thus, loss of Zld in trans, or Zld binding sites in cis, has the same effect on NEE activity, indicating a direct modulation of sog by Zld (Foo, 2014).

Next the opposite experiment was performed by introducing three CAGGTAG sites into the brk 5′ enhancer. This modified enhancer (brk+3a) drives a considerably expanded expression domain compared to brkwt. A second form of the brk enhancer with CAGGTAG sites added to different locations (brk+3b) also drives the same expanded expression domain, arguing against the requirement of precise motif grammar in Zld’s regulation of NE genes (Foo, 2014).

To rule out the possibility that the expansion in domain width of brk+3a is caused by inadvertent disruption of a repressor binding site rather than addition of Zld binding sites, the three added CAGGTAG sequences were mutated in brk+3a into 7-mers that are neither the original sequence nor Zld binding sites. Mutation of these sites reduced the expanded domain of brk+3a back to a width similar to brk wt. When each of the brk+3a , brk+3b , and brk+3m transgenic enhancers was placed into a zld- background, narrow and sporadic expression resulted resembling that of endogenous brk in zld-, again supporting that the CAGGTAG-driven broadened expression is Zld dependent. Moreover, mutation of the newly found weak Zld binding sites led to a narrowed and weakened stripe of expression, identical to the pattern of brk wt in zld- (Foo, 2014).

To better correlate the number of Zld sites with the extent of reporter expression, six different forms of the sog NEE were constructed containing either one or two of the three CAGGTAG sites). The width of expression correlated moderately to the number of Zld sites in the enhancer. However, some sites appear to be more important than others in contributing to the expression width, indicating a context dependency for Zld binding sites. From these results and others’ work demonstrating weakened NE gene expression upon removal of Zld or Zld sites, it is evident that Zld is indispensable for the proper expression of NE genes (Foo, 2014).

It was next asked whether the number of Zld binding sites also influences the timing of Dl target expression, since previous reports have implicated Zld as a developmental timer. A correlation has been observed between the onset of zygotic gene expression and strength of Zld binding at nuclear cycle 8. Besides that, when the enhancer region of zen, which contains four Zld binding sites, was multimerized, it drove precocious activation of reporter expression. And finally, it has been shown that the expression of many patterning genes is delayed in zld- embryos, including sog and brk. It was reasoned that since Dl nuclear concentrations increase from nuclear cycles 10 to 14, the lower levels of Dl present in earlier cycles would no longer be adequate to activate target genes without Zld’s input, resulting in delayed activation of sog and brk (Foo, 2014).

To measure the onset of transcription, it was determined when the four transgenic enhancers (sogwt, sog0, brkwt, and brk+3a) could activate an intron-containing yellow reporter gene, which allows detection of nascent transcripts. Reporter expression driven by the sog wt enhancer was first detectable in nuclear cycle 10 embryos, whereas no reporter activity was observed for the sog0 enhancer until nuclear cycle 11. Even in nuclear cycle 12, the expression driven by sog0 is more sporadic compared to sog wt. Unlike in nuclear cycle 14 embryos, reporter expression can be seen in ventral nuclei of nuclear cycle 11 and nuclear cycle 12 embryos because the Sna repressor has not yet accumulated to high levels. Adding three Zld sites to the brk enhancer resulted in advanced initiation of reporter activity from nuclear cycle 11 to nuclear cycle 10, and reporter expression also became more robust, in terms of both the proportion of nuclei showing expression and the ratio of embryos with expressing nuclei. These results clearly illustrate that by manipulating Zld binding sites, the timing of NE gene activation can be altered. Temporal regulation by transcription factor binding sites has also been shown in Ciona where the number of Brachyury binding sites governs the timing of notochord gene expression (Foo, 2014).

It is believed that Zld regulates the temporal and spatial expression of NE genes by promoting Dl activity, rather than acting independently, because nuclear Dl is absolutely required for the activation of brk and sog, which exhibit no expression in genetic backgrounds lacking nuclear Dl. One possible mechanism may involve cooperativity at the level of DNA binding. To test the hypothesis that the extent of Zld binding impacts Dl binding at target enhancers, chromatin immunoprecipitation was performed followed by quantitative PCR (ChIP-qPCR) to measure Zld and Dl binding to the different transgenic enhancers (Foo, 2014).

The sog0 enhancer without Zld sites has diminished Zld binding when compared to sog wt. Dl binding is also much reduced. As an internal control, Zld and Dl binding to the endogenous sog locus showed no significant difference between the lines. On the other hand, introduction of Zld sites into the brk transgenic enhancer led to higher Zld binding and Dl binding, while Zld and Dl binding to the endogenous locus remained similar between lines. These results illustrate that changing the number of Zld sites, and therefore changing the amount of Zld binding to the NE enhancers, influences the level of Dl binding to its target sites in vivo (Foo, 2014).

The results from reporter expression analyses and ChIP experiments suggest that Zld promotes transcriptional output by facilitating Dl DNA binding. Zld might directly interact with Dl, leading to cooperative DNA binding as in the Dl-Twist (Twi) interaction. Alternatively, Zld might assist factor binding by interacting with common coactivators or by changing the local chromatin accessibility. The latter possibility is favored for several reasons: (1) Zld binding greatly overlaps with that of many other transcription factors such as Bcd, Hunchback, Dl, Twi, Sna, and Mothers against Dpp (Mad); (2) Zld helps the binding of Twi and Bcd to target DNA; (3) the presence of Zld binding sites is associated with high levels of transcription factor binding; and (4) the Zld site (CAGGTA; [2]) is the most enriched motif in transcription factor binding 'HOT regions,' which were seen to correlate with decreased nucleosome density. Hence, it is more likely that Zld plays a more general role, such as 'opening' the underlying chromatin, than that it interacts specifically with multiple other factors (Foo, 2014).

The hypothesis was addressed that Zld facilitates the binding of Dl by making the local chromatin more accessible. DNase I’s preferential digestion of nucleosome-depleted DNA in the genome can be used to map active regulatory regions accessible for transcription factor binding. DNase I hypersensitivity assays followed by qPCR (DNase I-qPCR) were performed to measure the chromatin 'openness' of transgenic enhancers carrying varying numbers of Zld sites. The sog transgenic enhancer region had significant reduction of chromatin accessibility when Zld sites were mutated, while adding Zld sites to the brk transgenic enhancer increased sensitivity to DNase I digestion. The DNase I hypersensitivity assessed on endogenous brk and sog loci were comparable between transgenic lines, serving as a control for embryo staging between transgenic lines and the DNase I digestion procedure (Foo, 2014).

These results suggest that the presence of Zld sites, and thus Zld binding, makes the local chromatin more accessible for Dl, and potentially other transcription factors. However, it is feasible that the total number of factor binding sites influences chromatin accessibility rather than the number of Zld sites in particular. Therefore, the DNase I hypersensitivity of a transgenic brk enhancer was assayed that lacks all Dl binding sites and shows no reporter expression. Dl binding decreased nearly to background levels compared to brk wt, but the Zld binding and DNase I hypersensitivity showed only slight decreases, which is not comparable to the effects seen upon manipulation of Zld sites on the brk and sog enhancers. It was reasoned that the binding of each transcription factor may contribute to the DNase I hypersensitivity to a certain extent but that the major influence comes from Zld binding. To further evaluate the contribution of Zld versus Dl sites to chromatin accessibility, the fold change in Zld and Dl binding for sog0, brk+3a , and brk0Dl was calculated relative to their corresponding wt transgenic enhancers and then correlated the fold change in factor binding with the change in DNase I hypersensitivity. A strong correlation was found between the change in Zld binding and DNase I hypersensitivity, whereas the change in Dl binding and DNase I hypersensitivity do not correlate. These results support the idea that the number of Zld sites rather than Dl sites is important in determining chromatin accessibility (Foo, 2014).

Using Zld's coregulation of NE genes as a case in point, this study has shed light on how Zld functions as a zygotic genome activator. The data reveal that Zld works in combination with Dl and regulates Dl target genes by binding differentially to their regulatory sequences. Changing the number of Zld sites on Dl target gene enhancers has a pronounced effect on their expression both temporally and spatially. As a uniformly distributed factor, Zld supplies positional information by promoting Dl binding to target enhancers, thereby increasing the 'apparent dosage' of Dl. Zld's input is especially important where the level of morphogen is low and likely plays a similar role for other key factors in the blastoderm embryo, such as Twi, Bcd, and Mad. Uniform factors have been found to act in combination with Sonic Hedgehog in neural tube differentiation, and the current findings on how Zld potentiates morphogen activity will be relevant to vertebrate systems (Foo, 2014).

Although the results do not rule out other possible mechanisms, they strongly support the idea that Zld binding increases chromatin accessibility, which is thought to contribute greatly to how it activates such a wide range of targets. In this model, the amount of Zld binding on a region would determine how open and therefore how active it is. At the center of this property is Zld’s ability to occupy a large fraction of its recognition sites in early embryos. Besides that, Zld is present in nuclei as early as nuclear cycle 2, which is considerably earlier than other factors. Therefore, Zld may act as a pioneer factor as previously suggested, but whether Zld binds to its sites in nucleosomes and repositions them, or whether it recruits histone modifiers that in turn affect binding of other factors like Dl, awaits further investigation. Interestingly, this idea may extend beyond flies, since newly discovered genome activators in zebrafish zygotic genome activation have been seen to cooperate with developmental regulators and prime the genome for subsequent activation. Thus, it seems that developmental control of zygotic genome activation is highly similar in flies and fish (Foo, 2014).

Transcriptional Regulation

brk expression in the imaginal discs is not uniform but shows complementarity to regions of Decapentaplegic (Dpp) signaling. In wing discs, brk is highly expressed in lateral regions that are distant from the Dpp source in the center of the disc. In leg discs, brk expression is lowest in the dorsal compartment, which is specified by high levels of Dpp signaling. Double stainings for brk-lacZ and Omb protein demonstrate the complementarity between high levels of brk transcription and the expression of a low-threshold target gene of Dpp in wing and leg imaginal discs. They also reveal a narrow zone of overlap between low brk levels and omb expression in the wing pouch, suggesting that brk expression extends into regions of low-level Dpp signaling. In this region of overlap between Omb and brk, brk levels are declining in a graded fashion and become undetectable at positions where Sal expression starts. The complementarity between brk expression and regions of Dpp signaling may reflect a negative regulation of brk by Dpp. Consistent with this view, clones of mutant cells missing the Dpp receptor Tkv express high levels of brk, irrespective of their location within the wing pouch. Thus, brk expression would occur evenly throughout the wing pouch in the absence of a Dpp gradient emanating from the center of the disc. An important function of Dpp signaling in the wing disc might be to generate the asymmetric distribution of a repressor (such as brk) of Dpp's target genes (Jazwinska, 1999a).

brk could be a transcription factor based both on its epistatic position in the pathway and on some features of the protein sequence. If brk specifically represses only the promoters of low- and intermediate-level target genes of Dpp, then loss of brk would lead to the activation of these genes at ectopic positions. At these positions, structures would form that correspond to low or intermediate levels of Dpp signaling, not because signaling has occurred, but instead because a specific subset of target genes had been activated in a signaling-independent way. If it is assumed that brk is a target gene-specific transcriptional regulator, then two models can be envisaged describing how Dpp regulates the target genes controlled by brk. In both models, the transcriptional control of brk by Dpp plays an important role. Dpp signaling is a potent repressor of brk transcription and seems to be required throughout wing development. As soon as Dpp signaling is abolished, strong brk expression can be seen at any position in the wing pouch. If brk is ectopically expressed in the center of the wing, then induction of omb and sal is suppressed even in regions of high Dpp signaling. All these observations suggest that Dpp signaling, at least in part, counteracts brk repression by reducing the amount of repressor. The promoter regions responsible for omb and low-level sal expression might even have only Brk-binding sites, so that their activation would be completely dependent on downregulation of brk expression. Alternatively, these promoters might integrate both the activation by SMAD proteins and repression by Brk (Jazwinska, 1999a).

The ventrolateral expression of brk in early embryos suggests that brk, like sog and rho, is a target gene of the maternal Dorsal protein gradient. In support of this notion, brk expression is completely abolished in maternally dorsalized embryos. Conversely, in maternally ventralized embryos derived from Toll 9Q heterozygous mothers, brk expression is initiated along the entire embryonic circumference except in the presumptive mesoderm. In sna twi mutant embryos and in sna single mutants brk expression is uniform at the ventral side. Thus, as is known for rho, sna might be a ventral repressor of brk transcription. The complementarity between brk expression and regions of Dpp signaling in the embryo might arise if brk is itself negatively regulated by Dpp, as occurs in imaginal discs (Campbell, 1999; Jazwinska, 1999, and Minami, 1999). To test this idea, brk expression was examined in dpp mutant embryos. Here, brk expression is normal before the onset of gastrulation, but subsequently expands toward the dorsal side of the embryo so that brk becomes uniformly expressed in the entire ectoderm. The opposite phenotype results if dpp expression expands into the ventrolateral region, as in a sog mutant embryo with extra wild-type copies of dpp. These embryos exhibit a strong repression of brk transcription in the ventrolateral region although a small domain of brk expression is maintained close to the border of the mesoderm. This residual expression might be responsible for the narrow stripe of neuroblasts that still forms in sog embryos with four copies of dpp+. Does the expansion of brk expression in dpp mutants require the previous Dl-dependent activation of brk transcription? In dl;dpp double mutant embryos, brk is initially not expressed; nevertheless, uniform brk expression is initiated during gastrulation. Thus, absence of dpp leads to derepression of brk irrespective of whether Dl is present, indicating that other mechanisms of transcriptional activation of brk exist that are normally counteracted by Dpp signaling (Jazwinska, 1999b).

The role of the zinc finger transcription factor Schnurri (Shn) in mediating the nuclear response to Dpp during adult patterning has been investigated. Using clonal analysis, it has been shown that wing imaginal disc cells mutant for shn fail to transcribe the genes spalt, optomotor blind, vestigial, and Dad, that are known to be induced by dpp signaling. shn clones also ectopically express brinker, a gene that is downregulated in response to dpp, thus implicating Shn in both activation and repression of Dpp target genes. Loss of shn activity affects anterior-posterior patterning and cell proliferation in the wing blade, in a manner that reflects the graded requirement for Dpp in these processes. Furthermore, shn is expressed in the pupal wing and plays a distinct role in mediating dpp-dependent vein differentiation at this stage. The absence of shn activity results in defects that are similar in nature and severity to those caused by elimination of Mad, suggesting that Shn has an essential role in dpp signal transduction in the developing wing. These data are consistent with a model in which Shn acts as a cofactor for Mad (Torres-Vazquez, 2000).

Several essential components of the Dpp signaling pathway have been identified, including the Dpp receptors Punt and Thick veins (Tkv) as well as the cytoplasmic mediators Mad and Medea. For target genes to be activated, Dpp signaling must suppress transcription of a repressor encoded by the brinker (brk) gene. Schnurri (Shn), a large zinc-finger protein, is essential for Dpp-mediated repression of brk transcription; in contrast, Shn is not required for target-gene activation. Thus, the Dpp signaling pathway bifurcates, downstream of the signal-mediating SMAD proteins, into a Shn-dependent pathway leading to brk repression and a Shn-independent pathway leading to gene activation. The existence of several Shn-like proteins in vertebrates and the observation that Brk functions in BMP signaling in Xenopus indicates that a similar regulatory cascade may be conserved in higher organisms (Marty, 2000).

Decapentaplegic (Dpp), a homolog of vertebrate bone morphogenic protein 2/4, is crucial for embryonic patterning and cell fate specification in Drosophila. Dpp signaling triggers nuclear accumulation of the Smads Mad and Medea, which affect gene expression through two distinct mechanisms: direct activation of target genes and relief of repression by the nuclear protein Brinker (Brk). The zinc-finger transcription factor Schnurri (Shn) has been implicated as a co-factor for Mad, based on its DNA-binding ability and evidence of signaling dependent interactions between the two proteins. A key question is whether Shn contributes to both repression of brk as well as to activation of target genes. During embryogenesis, brk expression is derepressed in shn mutants. However, while Mad is essential for Dpp-mediated repression of brk, the requirement for shn is stage specific. Analysis of brk;shn double mutants reveals that upregulation of brk does not account for all aspects of the shn mutant phenotype. Several Dpp target genes are also expressed at intermediate levels in double mutant embryos, demonstrating that shn also provides a brk-independent positive input to gene activation. Shn-mediated relief of brk repression establishes broad domains of gene activation, while the brk-independent input from Shn is crucial for defining the precise limits and levels of Dpp target gene expression in the embryo (Torres-Vazquez, 2001).

Genetic evidence implicates both Shn and Mad in dpp-dependent repression of brk. In the wing disc, cells that lack Mad or shn ectopically express brk and fail to activate the Dpp-responsive genes optomotor-blind, vestigial, spalt and Dad. Abolition of shn or Mad activity results in upregulation of brk in the embryo and in the absence of shn ectopic Dpp cannot suppress brk expression. Since Shn and Mad interact directly, an attractive hypothesis is that a Shn/Mad complex is involved in the Dpp-dependent repression of brk. It has recently been suggested that Dpp signaling bifurcates downstream of Mad/Med into a Shn-dependent pathway, leading to brk repression and a Shn-independent pathway that triggers gene activation. According to this model, Shn acts primarily as a dedicated repressor that switches Mad from a transcriptional activator to a transcriptional repressor on the brk promoter. However several lines of evidence from this study are incompatible with such an interpretation (Torres-Vazquez, 2001).

A strong argument that shn has additional roles beyond brk repression comes from the fact that simultaneous loss of brk and shn activity results in a phenotype that is distinct from that of brk-null animals. If the sole function of shn is to mediate brk repression, then shn activity should be redundant in a brk mutant background. However, both at the overt phenotypic level as well as in the regulation of individual target genes, brk;shn double mutants display defects consistent with lower levels of Dpp signaling, compared with embryos that lack brk alone. These results indicate that shn participates in gene activation through brk-independent mechanisms as well. The finding that Shn is not obligately required to suppress brk transcription prior to germband elongation, while Mad is essential in this process, also argues against an exclusive role for Shn as a Mad co-repressor. In dpp- and Mad-null embryos, brk is upregulated at stage 8, while in embryos lacking shn function, derepression occurs approximately 3 hours later than the transition of brk regulation from maternal to zygotic control. Thus, brk transcription is insensitive to the absence of shn function at a time when it is responsive to Dpp and Mad. This idea is reinforced by the fact that ectopic Dpp signaling (through a constitutively activated form of Tkv called TkvA) can repress brk transcription at stage 5/6 in both wild-type and shn- animals, but not in Mad-null embryos. Collectively these data provide compelling evidence that refutes a model in which all aspects of the shn mutant phenotype result from derepression of brk transcription (Torres-Vazquez, 2001).

The unexpected result that at high levels TkvA mediates activation of brk promoter, while at low levels it causes repression reveals a possible mechanism by which Shn contributes to Mad activity. One explanation for these concentration-dependent effects of TkvA could be that the default mode of Mad action is transcriptional activation, and interaction with a co-repressor (perhaps present in limiting amounts) is crucial to bring about repression. Cells that receive very high levels of signaling could experience 'squelching', owing to excess nuclear Mad that binds to the brk promoter without recruitment of the co-repressor, thus promoting activation rather than repression. Supporting this idea, injection of TkvA into embryos that lack Mad does not induce either brk activation or repression. The increased frequency of ectopic brk expression in shn- embryos could indicate that Shn stabilizes a Mad/co-repressor complex on the brk promoter. It is worth bearing in mind that even in shn- embryos, ectopic activation did not occur independent of brk repression in the peripheral cells. Thus, it appears that Shn does not determine whether Mad acts as an activator or as a repressor, but may promote its interaction with other factors that determine the polarity of Mad transcriptional activity (Torres-Vazquez, 2001).

Analysis of Dpp-responsive gene expression in brk; shn double mutants has allowed an assessment the brk-independent input from shn to gene activation at different developmental stages in a range of tissues. Although it has not been demonstrated that each of these markers is a direct target of Dpp signaling, three categories of responses can be distinguished based on these studies. In the first group (class A), exemplified by dpp in the leading edge of the dorsal ectoderm, expression in the double mutant is indistinguishable from that in brk- embryos. Thus, shn contributes to class A gene expression primarily by relief of brk repression. Promoters belonging to class B include Dad and pnr in the dorsal ectoderm during germband extension. Expression of class B genes is downregulated in the double mutant compared with brk- embryos, but is equivalent to wild-type levels. It is inferred from this result that in the absence of Brk and Shn, Mad-mediated activation may be sufficient for expression within the normal domain, but cannot sustain the lateral expansion encountered in brk mutants. A third category of responses (class C) includes dpp and Ubx in the midgut, and sna in the primordia of the wing/haltere imaginal discs. Genes in this class show significantly reduced levels of expression in the double mutant, not only relative to brk- but also compared with wild-type animals. Class C promoters incorporate a brk-independent positive input from shn that is necessary for wild-type levels of expression. The inability of ectopic Dpp to induce sna expression in shn mutants demonstrates the essential nature of the requirement for Shn in activation of class C genes (Torres-Vazquez, 2001).

It is evident that repression of brk is crucial for expression of all three classes of genes described, and as such accounts for a significant part of the positive input from shn to gene activation. In addition, the data suggest that Mad and Shn contribute equally to repression of brk and regulation of class A genes. However, the fact that brk activity is only partially epistatic with respect to class B and C promoters, indicates that the majority of genes examined in this study integrate positive inputs from shn, as well as negative inputs from brk. The near wild-type expression of class B genes in double mutant embryos suggests that the brk-independent input from shn may be crucial at the margins of the expression domains and may be less significant in regions of the embryo that receive moderate to high levels of Dpp signaling. In contrast, the positive input from shn to class C targets appears to be important throughout the domain of expression. The observation that genes such as dCreb-A and Scr, which are repressed by dpp signaling, and which are also sensitive to loss of brk, raises the possibility that Dpp regulates their expression indirectly. In this event, the dpp target genes that mediate repression of dCreb-A and Scr would belong to classes A and C, respectively (Torres-Vazquez, 2001).

The partial restoration of dpp target gene expression in the double mutants relative to shn- embryos provides a basis for interpreting the cuticle phenotype. Homozygous brk;shn animals as well as brk;tkv mutants have an intermediate phenotype in that they show rescue of the dorsal closure defect observed in shn and tkv mutants, but they also display a reduced dorsal epidermis compared with brk-null embryos. Both dpp and pnr have been implicated in dorsal closure, which results from movement of the epidermal cells over the amnioserosa and their suturing at the midline. In light of this, the recovery of their expression in the dorsalmost ectodermal cells in the double mutants correlates well with the restoration of dorsal closure. Likewise, the compromised expression of dorsal ectodermal markers such as Dad and pnr in brk;shn embryos relative to brk null animals, provides molecular correlates for the ventralization observed in the double mutants (Torres-Vazquez, 2001).

The data presented in this study indicate that Shn can mediate both gene activation and brk repression in response to Dpp signaling. An important question is whether Shn has a Mad-independent role in activation. Shn contains a potential activation domain, and the human ortholog of Shn (PRDII-BF1) can elicit a 10-fold increase in gene expression in transfection assays. However, a Shn-Gal4 fusion protein does not activate transcription in yeast, and Shn is only marginally effective in stimulating a Dpp-responsive reporter in the absence of Mad in cell culture assays. Taken together these results suggest that Shn acts by promoting Mad binding to DNA and/or its interactions with the transcriptional machinery. There is ample precedent for such a mechanism, since several vertebrate DNA-binding Smad partners such as FAST1, OAZ, Mixer and Milk, do not have an innate ability to stimulate transcription, but potentiate gene activation by Smads in a pathway specific manner. A prediction from this data is that promoters of class B and class C genes are likely to contain binding sites for Shn as well as Mad, and that Shn increases Mad specificity by recruiting it to a subset of promoters that contain binding sites for both proteins. Analysis of gene expression in brk;tkv mutants demonstrates that for class B and class C genes Mad provides a greater brk-independent input compared with shn, consistent with the idea that Mad plays a primary role in Dpp-dependent gene activation and that shn facilitates Mad activity. Further support comes from the observation that deletion of Mad sites in the Ubx midgut enhancer had a more profound effect than abolition of Shn binding (Torres-Vazquez, 2001).

It has been shown that Mad interacts with Nejire (Nej), the Drosophila homolog of the co-activator p300/CREB binding protein (CBP). Reduction in nej activity affects the expression of ush, pnr and Ubx, and disrupts events that are dpp and shn-dependent, like tracheal migration and imaginal disc patterning. It is interesting to speculate that Shn may interact directly with Nej and stabilize complex formation between Mad/Medea and Nej (Torres-Vazquez, 2001 and references therein).

The requirement for Shn and Mad in both aspects of Dpp signaling suggests that Shn does not confer the ability to activate or repress transcription. It appears more likely that the activity of the Mad/Shn complex is modulated in a promoter specific fashion analogous to the mechanisms that convert Dl from an activator to a repressor. Similarly, the presence of binding sites for factors that bring co-repressors into proximity with Mad/Shn could permit inhibition of transcription at the brk promoter while target genes that lack these sites could be activated in the same cells. It has been shown that Smad4 interacts with the co-repressor TGIF and the co-activator CBP in a mutually exclusive manner. Thus, the ability to recruit co-activators as opposed to Smad co-repressors (such as cSki and SnoN), or more general transcriptional repressors like Groucho or CtBP, would be crucial to determining whether Dpp stimulation resulted in activation or repression of the target gene (Torres-Vazquez, 2001).

It is conceivable that in addition to repressing brk transcription, Shn and Mad could prevent residual Brk protein in the nucleus from binding to target gene promoters through steric hindrance or direct competition for common binding sites. Related anti-repression mechanisms have been postulated for Smad1 and Smad2 that interact with the transcriptional repressors Hoxc-8 and SIP1, respectively, triggering their dissociation from the osteopontin and X-Bra promoters. Although such a mechanism could potentially enhance the efficiency with which Shn and Mad antagonize brk activity, it does not account for the brk-independent input from shn observed in brk;shn embryos, since there is no Brk protein in the double mutants.

Despite the fact that shn transcripts are present from the precellular blastoderm stage onwards, loss of shn activity does not affect either brk repression or the expression of Dpp target genes until germband extension. Germline clonal analysis and ds-RNAi experiments indicate that the insensitivity of Dpp target gene expression to loss of shn during early embryogenesis is unlikely to result from perdurance of maternal message. Thus, the 'weakness' of the shn mutant phenotype may reflect a limited temporal requirement for shn in dpp signaling, rather than a lesser requirement for shn activity throughout development. The functional redundancy of shn during early patterning could be due to the presence of another protein that contributes a Shn-like activity to Dpp signal transduction. Alternatively, Mad activity alone could be sufficient for induction of early D/V patterning genes if they contain promoter elements that are more sensitive to Mad. It is also possible that the higher levels of nuclear Mad resulting from the synergy between Scw and Dpp in early embryogenesis renders the potentiation of Mad by Shn unnecessary. Finally, given the conserved nature of the BMP signal transduction pathway and the identification of Shn homologs in humans, frogs and worms, it is possible that Shn-like proteins in other systems potentiate Smad activity in an analogous manner (Torres-Vazquez, 2001).

Mitosis-associated repression in development

Transcriptional repression is a pervasive feature of animal development. This study employed live-imaging methods to visualize the Snail repressor, which establishes the boundary between the presumptive mesoderm and neurogenic ectoderm of early Drosophila embryos. Snail target enhancers were attached to an MS2 reporter gene, permitting detection of nascent transcripts in living embryos. The transgenes exhibit initially broad patterns of transcription but are refined by repression in the mesoderm following mitosis. These observations reveal a correlation between mitotic silencing and Snail repression. The study proposes that mitosis and other inherent discontinuities in transcription boost the activities of sequence-specific repressors, such as Snail (Esposito, 2016).

The enhancers tested in this study were derived from two different dorsal-ventral patterning genes: brk and sog; both encode inhibitors of BMP signaling. The brk enhancer is located ~10 kb upstream of the transcription start site, while the sog enhancer is located within the first intron of the transcription unit, ~1.5 kb downstream from the start site. Each enhancer was placed immediately upstream of its cognate promoter and attached to a yellow reporter gene containing 24 MS2 stem-loops within the 5' untranslated region (UTR). Nascent transcripts were visualized in living embryos using a maternally expressed MCP-GFP fusion protein (Esposito, 2016).

Both transgenes recapitulate the expression profiles of the endogenous genes; namely, they are activated throughout the presumptive mesoderm and neurogenic ectoderm and then repressed in the mesoderm. Prior studies with fixed embryos suggest that these enhancers respond to different levels of the Snail repressor (e.g., see Bothma et al. 2011). The brk 5' enhancer appears to be more efficiently repressed by Snail as compared with the sog intronic enhancer. Both transgenes were examined in living embryos to determine whether they exhibit distinctive repression dynamics (Esposito, 2016).

The btk>MS2 transgene exhibits an expression profile that is similar to that seen for the endogenous locus based on conventional in situ hybridization methods. The main difference is that the transgene produces a slightly narrower pattern due to the absence of the 3' 'shadow' enhancer (Esposito, 2016).

There is broad activation of the btk>MS2 transgene in both ventral and lateral regions during nuclear cleavage cycles 10-13 (nc10-nc13). btk>MS2 nascent transcripts are lost during the general silencing of transcription at each mitosis. Interestingly, upon reactivation of the transgene at the onset of nc14, a sudden loss of de novo transcription was observed in the mesoderm. Transcripts are restricted to the neurogenic ectoderm, suggesting that the mature brk expression pattern is established immediately following mitosis (Esposito, 2016).

In an effort to quantify the dynamics of this repression, individual nuclei were partitioned within the presumptive mesoderm and ectoderm and the fraction of active nuclei was calculated in these regions throughout nc13-nc14. Active nuclei are defined as those exhibiting nascent RNA signals in at least one z-series (20 sec). No significant variation was observed in the fraction of active nuclei between the mesoderm and lateral ectoderm during nc13. However, at the onset of nc14, ~90% of the nuclei in the mesoderm are silenced, while expression persists in the lateral ectoderm (Esposito, 2016).

To obtain more detailed information on the dynamics of repression in the ventral mesoderm, the fluorescence of individual transcription foci was quantified, since previous studies have shown that it scales with the number of RNA polymerase II (Pol II) complexes engaged in active transcription. Quantitative analysis of the btk>MS2 transgene reveals that the ventral-most nuclei in the mesoderm exhibit ~25% reduction in signal intensity during nc13 as compared with active nuclei in the lateral ectoderm. The majority of nuclei that display nascent transcripts at the onset of nc14 is located in the ectoderm, and the few active nuclei in the mesoderm show a substantial reduction in signal intensity (~60% reduction). Thus, Snail begins to attenuate the brk 5' enhancer during nc13, and this repression appears to be strongly reinforced during mitosis. It is suggested that mitotic silencing augments the activity of the localized Snail repressor (Esposito, 2016).

sog is regulated by two enhancers with overlapping activities: a distal enhancer located ~20 kb upstream of the sog promoter and an intronic enhancer located ~1.5 kb downstream from the transcription start site. The distal 5' enhancer contains high-affinity Snail-binding sites and exhibits repression dynamics similar to that of the btk>MS2 transgene following mitosis. The intronic enhancer contains weak Snail-binding sites and shows modest repression in the ventral mesoderm of wild-type embryos. Most nuclei are reactivated following mitotic silencing but display a significant reduction (more than twofold) in expression (Esposito, 2016).

To explore the dynamics of sog repression, embryos carrying three copies of the snail locus were examined. They exhibit significantly more complete repression of the sog>MS2 transgene during the onset of nc14 as compared with wild-type embryos. Fewer than half the nuclei in the mesoderm reactivate sog>MS2 expression at the onset of nc14 (Esposito, 2016).

The preceding results suggest a clear correlation between mitotic silencing and repression of the btk>MS2 transgene and attenuation of sog>MS2. There is an approximately twofold reduction in the levels of sog>MS2 expression in the mesoderm following mitotic silencing in wild-type embryos and a substantial reduction in the number of nuclei that reactivate the transgene at the onset of nc14 in embryos containing three copies of snail. Nonetheless, it is not clear whether the ~8-min interval during mitosis is a more effective period of Snail-mediated repression than a comparable interphase period (Esposito, 2016).

Additional support stems from the analysis of a rare haploid embryo. Following fertilization, the paternal and maternal haploid pronuclei sometimes fail to fuse, and development proceeds with successive divisions of the maternal pronucleus. Due to the reduced amount of DNA, these haploid nuclei undergo an additional, 14th mitotic division. As expected, the resulting nc15 nuclei are half the volume of normal nc14 diploid nuclei due to the extra division cycle. Strikingly, the sog>MS2 transgene exhibits a dramatic loss of expression in mesoderm nuclei at the onset of nc15. There is only a modest loss in the number of nuclei that exhibit repression in the mesoderm during nc14 (~10%). This is followed by an ~10-fold reduction in the number of nuclei that reactivate the transgene at the onset of nc15. This loss in expression following mitosis is similar to that seen for the btk>MS2 transgene in wild-type embryos. Unfortunately, given the scarcity of spontaneous haploid embryos, it was not possible to perform these measurements on only one embryo (Esposito, 2016).

These observations reinforce the correlation between mitosis and repression. Additional evidence was obtained by extending the normal period of mitosis by lowering the temperature of developing embryos. A temperature-controlled microfluidic chamber was used to produce a transient reduction in temperature-from 22°C to 17°C during the 13th mitosis while maintaining the temperature at 22°C during interphases 13 and 14. This treatment diminishes the rate of embryonic development and extends the time of mitosis from ~8 to ~15 min. There is a more pervasive loss of expression in the mesoderm upon reactivation of the sog>MS2 transgene at the onset of nc14. Less than half the mesoderm nuclei exhibit expression of the sog>MS2 transgene upon reactivation at the onset of nc14 (Esposito, 2016).

It is not believed that the correlation between mitosis and repression is a peculiarity of the Snail repressor. Mitotic silencing might influence gap repressors such as Kruppel, since there is a substantial refinement in the eve stripe-2 expression pattern at the onset of nc14 (Esposito, 2016).

The preceding analyses employed heterozygous embryos carrying a single copy of the brk or sog transgenes. Next homozygous embryos were examined to determine whether the two alleles of a locus display coordinated or uncoupled patterns of repression. The btk>MS2 transgene exhibits a sharp transition from active to inactive mesoderm nuclei following mitosis. This study therefore focused analysis on the regulation of sog>MS2 homozygotes (Esposito, 2016).

There is no detectable repression of either sog>MS2 allele during nc13. At the onset of nc14, most of the daughter nuclei reactivate expression following mitotic silencing but produce fewer transcripts than the nuclei in the ectoderm. Moreover, mesoderm nuclei exhibit asymmetric reactivation of the two alleles. One of the alleles exhibits expression that is comparable with each allele in the ectoderm, whereas the other allele is either silent or exhibits very weak and transient expression. These observations suggest that the diminished levels of expression that are observed for sog>MS2 at the onset of nc14 are mainly due to the repression of one of the alleles. Thus, Snail represses sog expression in the mesoderm one allele at a time. Instead of diminishing the levels of both alleles, there is a clear trend to silence one of the alleles. The basis for this 'digital' mode of repression is uncertain, but it is possible that it reflects the exact time when each homolog is silenced and then decondensed following mitosis (Esposito, 2016).

This study presents evidence that transcriptional repression is intimately linked to the cell cycle, and it is proposed that Snail exploits the general silencing of transcription that occurs during mitosis. Mitotic silencing offers an opportunity to reset the balance between transcriptional activators and repressors. It is possible that Snail outcompetes the Dorsal activator during mitosis. Indeed, immunohistochemical localization assays suggest that the Snail repressor remains associated with the apical cytoplasm during mitosis, whereas Dorsal becomes distributed throughout the cytoplasm. This might give Snail 'the jump' on Dorsal after the completion of mitosis. It is also possible that the balance between the Dorsal activator and the Snail repressor is influenced by 'titration' of Dorsal due to increases in chromosomal templates arising from replication (Esposito, 2016).

It is proposed that Snail and other developmental repressors exploit natural discontinuities in transcription. In addition to mitotic silencing, many genes exhibit transcriptional bursts. It is possible that repressors like Snail get the upper hand during the refractory periods between bursts. Indeed, inhibition between successive bursts of sog>MS2 expression was observed in the mesoderm following mitotic silencing. Approximately 80% of mesoderm nuclei exhibit a single burst of expression before falling silent, and a similar trend in the repression of the btk>MS2 transgene was observed. The Snail repressor may be more effective in maintaining the off state following a burst (or mitotic silencing) than inhibiting a gene at the peak of its activity. Mitotic silencing and transcriptional bursting might represent intrinsic mechanisms that foster dynamic repression of gene expression during development (Esposito, 2016).

Targets of Activity

To test whether the spatial regulation of brk is indeed important for normal Dpp function, brk was ectopically expressed in the center of the wing pouch. This misexpression did not interfere with the transcription of endogenous dpp. Despite this, brk expression in the optomotor blind (omb) domain causes a strong reduction of wing size and the corresponding third instar larval discs do not have the normal folded morphology and contain fewer cells than wild-type discs. omb and spalt (sal) expression are strongly reduced in the wing pouch region of such discs and can be seen only in some centrally located scattered groups of cells. Although the stochastic aspect of the omb and sal pattern in these discs cannot be explained, ectopic brk expression clearly leads to the repression of both Dpp target genes in most of the cells in which they would normally be expressed. Interestingly, brk affects omb and sal expression even in regions of high Dpp signaling close to the compartment boundary. These data suggest that brk expression is a powerful antagonist of Dpp signaling and must be tightly controlled to avoid interference with normal Dpp function (Jazwinska, 1999a).

brk could be considered to function as a corepressor with Dorsal in regulating dpp, tld and early zen. To test whether brk can in fact act as repressor in the absence of Dl, UAS-brk was expressed under the control of a maternally expressed GAL4 driver to achieve uniformly high-level expression of brk in early wild-type embryos. This completely suppresses the formation of the dorsal dpp domain. It also abolishes terminal expression of dpp, which is not subject to regulation by Dl. The resulting embryos secrete only cuticle with ventral denticle belts and resemble dpp mutant embryos. Thus, early uniform expression of brk can completely block all dpp expression. BRK mRNA was injected into dl mutant embryos that uniformly express dpp. At the site of injection dpp is repressed and the injected embryos form cuticle with ventral denticles that resembles the cuticle from dl;dpp double mutants. However, since dpp is not uniformly repressed, cuticles often show a transition from ventral epidermis to dorsal epidermis with increasing distance from the site of injection. In the region of transition, dorsolateral structures are formed, such as Filzkörper. This indicates that different amounts of repression by brk might lead to different ectodermal cell fates. Thus, the ectopic expression data show that brk repression of dpp is independent of prior repression by Dl and therefore brk functions in early embryos, as in imaginal discs, as a regulatory component of the Dpp signaling pathway (Jazwinska, 1999b).

The dorsal ectoderm of the Drosophila embryo is subdivided into different cell types by an activity gradient of two TGFbeta signaling molecules, Decapentaplegic and Screw. Patterning responses to this gradient depend on a secreted inhibitor, Short gastrulation and a newly identified transcriptional repressor, Brinker, which are expressed in neurogenic regions that abut the dorsal ectoderm. The expression of a number of Dpp target genes has been examined in transgenic embryos that contain ectopic stripes of Dpp, Sog and Brk expression. These studies suggest that the Dpp/Scw activity gradient directly specifies at least three distinct thresholds of gene expression in the dorsal ectoderm of gastrulating embryos. Brk was found to repress two target genes, tailup/islet (tup) and pannier, that exhibit different limits of expression within the dorsal ectoderm. These results suggest that the Sog inhibitor and Brk repressor work in concert to establish sharp dorsolateral limits of gene expression. Evidence is provided that the activation of Dpp/Scw target genes depends on the Drosophila homolog of the CBP histone acetyltransferase (Ashe, 2000).

The dpp stripe results in an expansion in both the hnt and ush expression patterns. The broadening of these patterns is particularly evident in anterior regions in the vicinity of the eve stripe. Increases in dpp+ gene dose do not expand the pnr expression pattern. For example, four copies of dpp+ result in augmented levels of pnr expression, but the dorsoventral limits of expression are essentially normal. The stripe2-dpp transgene has no obvious effect on the early sog and brk expression patterns (Ashe, 2000).

Previous studies have shown that the pnr expression pattern expands into neurogenic regions in brk- mutant embryos. No such expansion was observed for other Dpp/Scw target genes, including ush. To test the role of the Brk repressor in establishing the different responses of Dpp target genes, the brk-coding sequence was attached to the eve stripe 2 enhancer. Transgenic embryos carrying the stripe2-brk transgene exhibit both the normal pattern (lateral stripes) in the neurogenic ectoderm as well as an ectopic stripe of expression. pnr is normally expressed in a series of 5 stripes in the dorsal ectoderm. The anteriormost stripe is lost in transgenic embryos carrying the stripe2-brk fusion gene. This result suggests that Brk is sufficient to repress pnr in an ectopic location in the embryo (Ashe, 2000).

Additional Dpp/Scw target genes were examined for repression by the stripe2-brk transgene. Those showing altered patterns of expression include tup, rho, hnt and Race. The normal tup expression pattern encompasses both the presumptive amnioserosa and dorsal regions of the dorsal epidermis. In transgenic embryos, there is a gap in the pattern in regions where the stripe2-brk fusion gene is expressed. These results suggest that Brk represses tup, even though it appears to respond to a different threshold of Dpp/Scw signaling than pnr. Additional experiments were done to determine whether Brk directly represses tup expression, or works indirectly by inhibiting Dpp signaling (Ashe, 2000).

To examine the relative contributions of the Sog inhibitor and the Brk repressor in establishing different thresholds of Dpp/Scw signaling activity, target genes were analyzed in gastrulation defective (gd) mutants that express either a stripe2-sog or stripe2-brk transgene. Mutant embryos collected from gd-/gd - females lack a Dl nuclear gradient and therefore lack ventral tissues such as the mesoderm and neurogenic ectoderm. All tissues along the dorsoventral axis form derivatives of the dorsal ectoderm, mainly dorsal epidermis. Hereafter, such embryos are referred to as gd-. These mutants lack endogenous sog and brk products, so that the stripe2 transgenes represent the only source of expression. Although the stripe2-sog transgene inhibits Dpp signaling, it does not cause activation of brk. The pnr and tup expression patterns are derepressed in gd- mutants, and exhibit uniform staining in both dorsal and ventral regions. These expanded patterns correlate with the expanded expression of dpp, which is normally repressed in ventral and lateral regions by the Dl gradient. As seen in wild-type embryos, the stripe2-brk transgene represses the anterior portion of the pnr expression pattern. In contrast, the stripe2-sog transgene has virtually no effect on the pattern. These observations suggest that Brk is the key determinant in establishing the lateral limits of the pnr pattern at the boundary between the dorsal ectoderm and neurogenic ectoderm. The failure of stripe2-sog to inhibit pnr expression might be due to redundancy in the action of the Dpp and Scw ligands. Perhaps either Scw alone or just one copy of dpp+ is sufficient to activate pnr. This would be consistent with the observation that the initial pnr expression pattern is essentially normal in dpp-/dpp- and scw-/scw- mutant embryos (Ashe, 2000).

The limits of the tup expression pattern seem to depend on both Sog and Brk. The introduction of the stripe2-brk transgene leads to a clear gap in the tup expression pattern, although there is a narrow stripe of repression in gd- mutants lacking the transgene. The stripe2-sog transgene causes a more extensive gap in the tup pattern. The stripe2-brk transgene was also found to repress Race, hnt and rho in this assay (Ashe, 2000).

In principle, the gap in the tup pattern caused by the stripe2-brk transgene could be indirect, and caused by the repression of dpp. Previous studies have shown that the early dpp expression pattern expands into the ventral ectoderm in brk- mutant embryos. To investigate this possibility, tup expression was monitored in brk- embryos, and in wild-type embryos carrying both the stripe2-brk and stripe2-dpp transgenes. The tup expression pattern exhibits a transient expansion in brk- mutant embryos. However, this expansion is only seen in early embryos, prior to the completion of cellularization. By the onset of gastrulation, the pattern is essentially normal. The stripe2-brk transgene creates an early gap in the normal dpp expression pattern in wild-type embryos. This observation raises the possibility that the repression of tup and rho is indirectly mediated by the inhibition of Dpp signaling. To test this, the tup pattern was examined in embryos carrying both the stripe2-brk and stripe2-dpp transgenes. As expected, the stripe2-dpp transgene alone causes a local expansion of the tup pattern in the vicinity of the stripe 2 pattern. However, the simultaneous expression of both stripe2-dpp and stripe2-brk leads to a clear gap in the tup expression pattern. Thus, it would appear that Brk can repress tup even in regions containing high levels of Dpp signaling. Similar assays suggest that Race, hnt and rho are not directly repressed by Brk (Ashe, 2000).

A summary is presented of Dpp signaling thresholds in the embryo. The Dpp/Scw activity gradient presumably leads to a broad nuclear gradient of Mad and Medea across the dorsal ectoderm of early embryos. It is conceivable that the early lateral stripes of brk expression lead to the formation of an opposing Brk repressor gradient through the limited diffusion of the protein in the precellular embryo. Peak levels of Dpp and Scw activity lead to the activation of Race and hnt at the dorsal midline. The tup and ush patterns represent another threshold of gene activity. The similar patterns might involve different mechanisms of Dpp signaling since tup is repressed by Brk, whereas ush is not. Finally, the broad pnr pattern represents another threshold of gene activity. It is not inhibited by Sog but is repressed by Brk. It is possible that tup and pnr are differentially repressed by a Brk gradient. Low levels of Brk might be sufficient to direct the lateral limits of the tup pattern, whereas high levels may be required to repress pnr (Ashe, 2000).

Direct transcriptional control of the Dpp target omb by the DNA binding protein Brinker

The gradient morphogen Decapentaplegic (Dpp) organizes pattern by inducing the transcription of different target genes at distinct threshold concentrations during Drosophila development. An important, albeit indirect, mode by which Dpp controls the spatial extent of its targets is via the graded downregulation of brinker, whose product in turn negatively regulates the expression of these targets. The molecular dissection is reported of the cis-regulatory sequences of optomotor-blind (omb), a Dpp target gene in the wing. A minimal 284 bp Dpp response element is described and it is demonstrated that this element is subject to Brinker (Brk) repression. Using this omb wing enhancer, it has been shown that Brk is a sequence-specific DNA binding protein. Brk contains a DNA binding domain, which is located within its N-terminal 175 amino acids. Mutations in the high-affinity Brk binding site abolish responsiveness of this omb enhancer to Brk and also compromise the input of an unknown transcriptional activator. These results therefore identify Brk as a novel transcription factor antagonizing Dpp signaling by directly binding target genes and repressing their expression (Sivasankaran, 2000).

To identify a consensus sequence for Brk target sites, a Dpp-responsive enhancer, lab550, was analyzed using the same footprinting strategy used on the omb enhancer. A consensus sequence, GGCGC/TC/T, for high-affinity Brk binding was obtained from both the lab and omb enhancers. To assess the importance of each nucleotide, the WF12 Brk binding site of the omb enhancer was scanned with single point mutations and the ability of these mutants to bind in vitro synthesized full-length Brk protein was tested. Mutations in the five central nucleotides of the Brk site abolish Brk binding, indicating that these residues form the core recognition motif and that Brk is a DNA binding protein with a high target site specificity (Sivasankaran, 2000).

Loss of Brk activity in vivo results in ectopic activation of the WF12 wing enhancer, so one would predict that mutations in WF12 that abolish Brk binding should also lead to ectopic enhancer activity. To test this hypothesis, the core of the Brk binding site from GGCGCC to GATATC was mutated. As expected, this change completely abolishes Brk binding in EMSA analysis. The same 4 bp mutation was introduced into the WF12-lacZ reporter transgene. Unexpectedly, this mutation completely abolishes lacZ expression rather than expanding it. This result is interpreted as an indication that the Brk site overlaps with that of an activating input. Single base pair mutations that interfere with Brk binding were introduced into WF12-lacZ, anticipating that at least some of these mutations would still allow the unknown activator to bind, resulting in an uncoupling of the two inputs (Sivasankaran, 2000).

Two of the five point mutations that prevent Brk binding, mut271 and mut272, completely abolish WF12 enhancer activity as observed with the 4 bp mutation. However, mutations mut273, mut274 and mut275 still express lacZ, albeit only in a narrow stripe along the dorsoventral boundary. This pattern unravels a hitherto masked, strong input into WF12 from a dorsoventral patterning system. Although these mutant enhancers show a reduced extent of expression along the dorsoventral axis, all of them exhibit a clear expansion in expression along the anteroposterior axis. This latter property corresponds to the behavior expected from the loss of a functional Brk binding site in the WF12 enhancer. These observations are interpreted to indicate that mut273-mut275 represent mutations that completely abolish Brk binding but only partially prevent input by the activator. Mutations flanking the Brk binding site (mut269, mut270, mut276 and mut277) abolish expression of WF12-lacZ, indicating that the binding site of the activator extends beyond that of Brk (Sivasankaran, 2000).

Attempts were made to validate the assumption that mut273-mut275 exhibit an extended expression along the dorsoventral boundary due to loss of Brk-mediated repression. Both the wild-type WF12 enhancer and the mutant derivatives were examined in cells that ectopically express Brk protein from a tub>CD2>brk flip-out transgene. While Brk potently represses the transcriptional activity of WF12, it does not repress the mutant enhancers. Thus, the ability to bind Brk in vitro, the lateral repression by endogenous Brk and the responsiveness to ectopic Brk in vivo all correlate with single nucleotide exchanges in the Brk core binding site. Together, these results are taken as evidence that the wild-type omb WF12 enhancer is a direct target of Brk repression (Sivasankaran, 2000).

Direct competition between Brinker and Drosophila Mad in Dpp target gene transcription

Brinker is a nuclear protein that antagonizes Dpp signalling in Drosophila. Ultrabithorax (Ubx) is a HOX gene that activates, and responds to, the localized expression of Dpp during endoderm induction. Ubx expression is negatively regulated by Dpp. Brinker represses Ubx in the embryonic midgut. The functional target for Brinker repression coincides with the Dpp response sequence in the Ubx midgut enhancer, namely a tandem of binding sites for the Dpp effector Mad. Brinker efficiently competes with Mad in vitro, preventing the latter from binding to these sites. Brinker also competes with activated Mad in vivo, blocking the stimulation of the Ubx enhancer in response to simultaneous Dpp signalling. These results indicate how Brinker acts as a dominant repressor of Dpp target genes, and explain why Brinker is a potent antagonist of Dpp (Saller, 2001).

The control of Ubx by Dpp and Wg signalling has been studied by functional dissection of a minimal midgut enhancer called Ubx B. This enhancer directs expression of a linked ß-galactosidase (lacZ) gene in parasegments (ps) 6-9, and also in ps3, of the midgut mesoderm as a result of stimulation by Dpp and Wg, which are expressed in or near these regions. This stimulation requires distinct Dpp and Wg response sequences (DRS and WRS) within Ubx B. In addition, Ubx B is repressible by high Wg levels near the Wg signalling source, and is also repressed in the absence of Wg signalling in cells remote from the source. The former repression is mediated by the WRS-R, a sequence coinciding with the Mad binding sites within the DRS, the latter by the WRS, a binding site for Pangolin, the Drosophila T cell factor (Saller, 2001).

Since Ubx is a Dpp target gene in the embryonic midgut, it was asked whether this HOX gene might be under brk control. Thus, brk mutant embryos were stained with an antibody against Ubx and weak ectopic Ubx staining was found in the posterior midgut mesoderm of these mutants. Normally, the HOX protein Abdominal-A represses Ubx in the posterior midgut, but evidently this is not sufficient to keep Ubx repressed in the absence of brk. However, no Ubx derepression was observed in the anterior midgut of brk mutants, probably because of the silencing of Ubx in this region by Polycomb. But derepression was found in the anterior and posterior midgut of brk mutant embryos when examining lacZ expression conferred by an extensive Ubx midgut enhancer called RP9, the expression of which closely resembles Ubx expression in the midgut. These stainings show that brk represses Ubx in the embryonic midgut (Saller, 2001).

Next, a series of mutant versions of Ubx B was tested that carry nested point mutations. Most of these are still derepressed in brk mutants, e.g. BM1, which has a mutated MadB site. However, three mutant enhancers were no longer derepressed: B4, which has a mutated Pangolin binding site; B4R8, which carries a mutation in a conserved sequence motif; and BM2, in which both Mad binding sites are mutated. Formally, each of these mutations could define a target for Brinker repression. Alternatively, they define sequences that are essential for enhancer activation, in particular for ectopic activation at the midgut ends. This is a clear possibility since B4, BM2 and B4R8 are each considerably less active than Ubx B and other mutant enhancers such as BM1 (Saller, 2001).

Full-length and various fragments of Brinker were expressed as glutathione S-transferase (GST) fusion proteins in bacteria, in order to test whether these fusion proteins can bind to the signal-responsive sequence from Ubx B in gel shift assays. This revealed that full-length Brinker, or its N-terminus alone, can bind to this sequence, whereas the C-terminus cannot. This is consistent with the suggestion that the N-terminus contains a putative DNA binding domain similar to the homeodomain. Indeed, a minimal fragment spanning this domain (BRK44-99) binds to the probe as well as full-length Brinker (Saller, 2001).

Next, Brinker binding to mutant DNA probes was tested. Of these, BM2 and BM0 are the only mutants that no longer show any binding to Brinker. Likewise, Brinker binding to DNA can be competed with an excess of unlabelled wild-type probe, but not with mutant BM2 probe. This shows that Brinker binds to Ubx B in a sequence-specific manner, and that the residues mutated in BM2 and BM0 are critical for Brinker binding (Saller, 2001).

Three perfect matches to a consensus site for Brinker binding, GGCG C/T C/T, are found in Ubx B. These are adjacent to one another, and each of them is mutated in BM2. The results with BM1 indicate that the first of these matches (Brk bs1) is sufficient for Brinker function in vivo and in vitro. However, Brk bs3 alone is unlikely to be sufficient for function, given that Brinker cannot bind to the mutant probe BM0. Finally, the results indicate that Brk bs2 (perhaps together with bs3) can substitute for Brk bs1 and provide full function: BC2 is repressible by brk in vivo, and Brinker binds to BC2, BC and BM01 mutant probes, all of which lack Brk bs1 (Saller, 2001).

Interestingly, the three Brinker binding sites completely overlap the two Mad binding sites within the DRS. Indeed, the Dpp response critically depends on MadA; MadA fully overlaps Brk bs1, which is sufficient for Brinker function in vitro and in vivo. It was thus asked whether Brinker might be able to compete with Mad for DNA binding. Competitive DNA binding experiments were performed using bacterially expressed DNA binding domains of Brinker and Mad. This revealed that the former is capable of competing successfully with the latter for DNA binding at a molar ratio of 1:150, and Brinker almost completely blocks Mad binding at a ratio of 1:15. Note that full-length Mad binds to DNA less efficiently than its isolated DNA binding domain, indicating that Brinker would be able to compete even more successfully with full-length Mad. Thus, Brinker can block Mad binding to DNA in vitro in the presence of a considerable molar excess of Mad (Saller, 2001).

To confirm that the above Brinker binding sites within Ubx are functional targets in vivo, Brinker was expressed throughout the midgut mesoderm with the GAL4 system. This revealed that expression of Ubx in the middle midgut is nearly eliminated in Brinker-overexpressing embryos. Instead, many of these embryos show an endodermal bulge in the middle midgut that is also observed in Ubx mutants. Furthermore, the first and second midgut constrictions are rudimentary at best, and often missing altogether. Again, loss of the second constriction is indicative of mutations of Ubx and dpp, while loss of the first may reflect mutation of the dpp-related gene gbb. Finally, ectopic Brinker also drastically reduces dpp and wg expression in the middle midgut, which is expected since their expression depends on Ubx. This indicates that Brinker, by virtue of repressing Ubx, is capable of blocking the whole process of endoderm induction that depends on this HOX gene (Saller, 2001).

These results indicate that Brinker is a direct repressor of Ubx, and thus a potent antagonist of the Dpp-dependent process of endoderm induction. It is noted that Brinker is expressed in 'signal-free' zones bordering the anterior and posterior limits of the midgut. Its presence in these zones may have a barrier function, helping to block the spread of the Dpp response beyond the midgut limits (Saller, 2001).

Interestingly, the critical Brinker target site within Ubx B overlaps MadA, a functional Mad binding site that is required for the stimulation of this enhancer by Dpp signalling. Furthermore, Brinker competes effectively with Mad in binding to this site in vitro, and blocks activated Mad from stimulating Ubx B in vivo. This indicates that the mechanism by which Brinker repression dominates over stimulatory Dpp inputs is based on direct competition for binding to Dpp target enhancers. Given that most, if not all, Dpp signalling is mediated by Mad, it seems likely that this competition-based mechanism of Brinker repression is widespread and extends to genes that are Dpp targets in other developmental contexts (Saller, 2001).

Notably, MadA is also the target sequence for repression of Ubx B in response to high Wg levels in the middle midgut. MadA is thus a pivotal enhancer sequence that gauges and integrates positive inputs from Dpp and negative inputs from Brinker and Wg. Wg-mediated repression in the middle midgut is mediated by the zinc finger protein Teashirt and can be overriden by simultaneous Dpp stimulation. In contrast, Brinker-mediated repression dominates over simultaneous Dpp stimulation. It thus appears that Brinker is a more potent repressor than Teashirt, and is designed to function as a signal-antagonist even in the presence of high levels of Dpp signalling (Saller, 2001).

Brinker contains the sequence PMDLS, which resembles the P-DLS motif through which a number of transcription factors recruit the co-repressor dCtBP. Indeed, using in vitro pull-down assays, it was found that dCtBP binds to full-length Brinker as well as to an N-terminal Brinker fragment that contains the PMDLS motif. This suggests that Brinker may recruit dCtBP to repress Dpp target genes in the embryo. Interestingly, dCtBP assists various transcription factors, such as Knirps, Snail and Krüppel, that act at short-range to repress their target genes. These short-range repressors bind to autonomous enhancers to quench nearby bound transcriptional activators, which has prompted the suggestion that dCtBP may be specifically designed to quench. Therefore, this quenching ability of dCtBP could enable Brinker to not only compete efficiently with activated Mad in the binding of DNA, but also out-compete the activity of nearby transcription factors such as activated dTCF (Saller, 2001).

Transcriptional regulation of the Drosophila gene zen by competing Smad and Brinker inputs

In both Drosophila and Xenopus embryos, gradients of Dpp/BMP activity are established that are responsible for patterning along the dorsoventral axis. Dpp activity has its highest levels along the dorsal midline of the cellular blastoderm embryo and declines toward more lateral regions where it is inhibited by the product of the short gastrulation (sog) gene. The high levels determine the cell fate of the amnioserosa in the dorsal-most cells, whereas lower levels specify aspects of the dorsal epidermis in dorsolateral cells. The absence of Dpp activity in ventrolateral regions permits the formation of the neurogenic ectoderm, which gives rise to both the ventral epidermis and the central nervous system (Rushlow, 2001).

How does Dpp specify cell fate in a concentration-dependent manner? It is thought that Dpp signaling in the early embryo regulates the transcription of downstream target genes that are expressed in nested domains centered around the dorsal midline. High-level Dpp targets such as Race and u-shaped (ush) are expressed in the presumptive amnioserosa. pannier (pnr) is expressed in a broader domain that spans the amnioserosa and part of the dorsal ectoderm. Thus, it requires lower levels of Dpp. Finally, low-level targets such as early zerknullt (zen) and dpp are expressed in an even broader domain that abuts the ventral ectoderm. A possible molecular mechanism to explain the threshold responses of Dpp target genes is that their promoters have different affinities to Smads and therefore can be induced by different levels of nuclear Smads, similar to the mechanism of differential activation by the Drosophila morphogens Dorsal (Dl) and Bicoid (Bcd). The fact that an additional mechanism is involved came from the characterization of the brinker (brk) gene. brk negatively regulates low-level and intermediate-level target genes. Study of the response elements of these target genes can therefore provide clues about the mechanisms of threshold responses to the Dpp morphogen, as well as the interplay of positive and negative inputs in the expression of target genes (Rushlow, 2001 and references therein). zen has a dynamic pattern of expression in the early embryo. During precellular nuclear division cycles 11-13 and during early cellularization (nuclear cycle 14), zen is expressed in a broad dorsal-on/ventral-off pattern. This pattern is thought to be activated by an unknown ubiquitous activator present throughout the embryo and repressed by the Dl morphogen localized in ventral regions. It is Dpp-independent because early zen expression is normal in dpp null mutants. However, slightly later, during early to mid-cellularization, maintenance of the zen pattern becomes dependent on Dpp because zen transcripts fade away suddenly in dpp null mutants. It also becomes dependent on Brk repression because zen transcripts expand into the ventral ectoderm in brk mutants. Thus, the broad pattern of zen is maintained by Dpp in the dorsal region and repressed by Brk in ventral regions. During mid- to late-cellularization, this pattern undergoes a process of refinement in which zen transcripts are lost from the lateral regions and become restricted to a narrow domain of the dorsal-most cells. Brk plays no role in refinement because in brk mutants, although zen expands ventrally, it refines normally (Rushlow, 2001 and references therein).

zen expression is directed by 1.6 kb of 5' flanking DNA sequences referred to as the zen promoter. The distal part of the promoter between 1.2 and 1.4 kb is responsible for Dl-dependent ventral repression. Sequences required for the initiation, maintenance, and refined expression of zen are located in the proximal 0.7 kb of the promoter, but they are not well-characterized (Rushlow, 2001 and references therein).

The regulation of zen during cellular blastoderm formation has been analyzed. Low levels of the Dpp signal transducer p-Mad (phosphorylated Mad), together with Brinker, define the spatial limits of zen transcription in a broad dorsal-on/ventral-off domain. The subsequent refinement of this pattern to the dorsal-most cells, however, correlates with high levels of p-Mad that accumulate in the same region during late blastoderm. Examination of the zen regulatory sequences reveals the presence of multiple Mad and Brk binding sites, and these results indicate that a full occupancy of the Mad sites due to high concentrations of nuclear Mad is the primary mechanism for refinement of zen. Interestingly, several Mad and Brk binding sites overlap, and it has been shown that Mad and Brk cannot bind simultaneously to such sites. A model is proposed whereby competition between Mad and Brk determines spatially restricted domains of expression of Dpp target genes (Rushlow, 2001).

The experiments presented here show that Mad/Medea and Brk regulate zen by binding to separated and overlapping DNA binding sites. There are 10 Mad/Medea and 6 Brk binding sites in the zen promoter, 5 of which are shared, indicating duality in their function. Indeed, the results from mutagenesis of the zen promoter show that the shared sites mediate both Brk and Mad/Medea functions. Five Brk and nine Smad binding sites are clustered in the zen proximal regulatory element over about 600 bp with spacing not exceeding 120 bp. This organization is similar to that of several well-studied enhancers from Drosophila. These enhancers are activated by a variety of transcriptional activators and repressed by short-range repressors such as Snail (Sna), Knirps (Kni), and Krüppel (Kr). All three of these repressors are DNA-binding proteins that can inhibit activator function when they are bound not further than 150 bp away from the activator binding site. It has been shown that they all contain a short stretch of amino acids, P-DLS-K, that is required for recruitment of the corepressor dCtBP. Analysis of zen regulation indicates that Brk also may be a short-range repressor. It is a DNA-binding protein and contains a PMDLSG domain. Preliminary in vitro experiments showed that Brk interacts with dCtBP; however, embryos devoid of dCtBP activity do not ectopically express zen and dpp, indicating that dCtBP is dispensable for Brk repression and other corepressors interact with Brk, or that Brk repression of these targets does not require additional factors (Rushlow, 2001).

What remains illusive is the identity of the ubiquitous transcriptional activator that activates zen in the dorsal ectoderm during precellular stages and early cellularization. It is possible that this activator interacts with Smads to enhance transcription of zen at a time when p-Mad levels are low. Also, Brk represses the ubiquitous activator, because zen becomes ectopically expressed in brk mutants. Thus far, deletion analysis of the zen promoter has not uncovered any sequences that might interact with this putative activator. It is possible that these sequences are redundant and scattered over the entire promoter and may in fact overlap with Smad and/or Brk binding sites (Rushlow, 2001).

In the cellularizing embryo, Dpp and Brk activities overlap in the lateral-most region. Here Dpp and Brk function to set thresholds of response for target genes such as zen and pnr. In this same region, Dpp signaling negatively regulates brk expression. Similarly, in the wing disc, the Brk expression domain overlaps with that of the Dpp target gene omb in the region where activated p-Mad is present. It has been proposed that a dual mechanism whereby Dpp can simultaneously down-regulate Brk repressor levels and antagonize its function on target gene promoters would be very efficient in establishing sharp threshold responses. Based on the experiments described here, a molecular model is proposed to explain mechanistically the antagonizing activities of Brk and Smads. It is proposed that they are involved in direct competition for binding to shared binding sites on target promoters. Thus, it is the balance of their opposing activities that determines the transcriptional state of the target genes. Two sets of experiments support this model: (1) ectopic expression of Brk in eve-stripe 2 abolishes zen expression in those cells. The elevated level of Brk in the stripe was therefore sufficient to repress the zen promoter even in the presence of activated Smads. The possibility that zen is repressed indirectly through Brk-mediated repression of dpp is highly unlikely because there was no delay in zen repression. (2) In vitro competition experiments also support the model. Especially revealing is the fact that the outcome of competition depends on the relative concentrations of both proteins and their binding affinities. Competitive mechanisms have been proposed to operate on many promoters where mutually exclusive DNA-binding factors are involved, and, in some instances, DNA-binding assays similar to the ones used in this study were used to show competition for binding between activator and repressor proteins. For example, bHLH proteins compete with a zinc-finger repressor for E-box binding in the immunoglobulin heavy chain enhancer (Rushlow, 2001 and references therein).

The findings presented here provide a framework for further study of the mechanisms of regulation of Dpp morphogen targets. zen is the only one of the known Dpp target genes that responds to two threshold activities: low (during early to mid-cellularization) and high (during late cellularization). Based on the results presented here and the proposed competition mechanism for activation and repression of the zen promoter, predictions can be made about the organization of the regulatory elements of the other Dpp target genes. High-level targets such as ush strongly depend on high levels of Smads, and their regulatory elements may have many, and possibly closely packed, Smad binding sites. Low-level targets such as omb in the wing imaginal disc may be repressed by Brk binding to their regulatory sequences. The spatial domains of expression of the intermediate targets such as pnr in the embryo and sal in the wing disc, which are dependent on both Dpp signaling and Brk repression, might be determined by the net balance of positive and negative inputs. Interestingly, this type of mechanism can result in expression domains that vary largely in size and may result in even broader domains than the low-level targets. An example is the vg gene. In third-instar imaginal wing discs, vg is expressed in a broader domain than omb. Its expression along the anterior-posterior boundary in the wing pouch is activated by the quadrant enhancer that contains Mad binding sites essential for activation. At the same time, vg is repressed by Brk. However, the essential Smad binding sites do not match the Brk binding sites, like many of the Smad sites in the zen promoter, suggesting that they will have no or low affinity for the Brk protein. Neither are there strong zen-like Brk binding sites in the quadrant enhancer. Its broad expression domain could then be explained if the positive inputs from Smads, enhanced by signals from the dorsoventral boundary, are able to overcome Brk repression far from the Dpp source (Rushlow, 2001).

Further studies of the arrangement, affinities, and numbers of repressor and activator sites in Dpp target promoters will determine to what extent the different thresholds of responses to the Dpp morphogen activity are shaped by a simple balance of positive and negative transcriptional inputs (Rushlow, 2001).

Repression of Dpp targets by binding of Brinker to Mad sites

Signaling by Dpp activates targets such as vestigial indirectly through negative regulation of brinker. The Brk protein functions as a repressor by binding to Dpp response elements. The Brk DNA binding activity is found in an amino-terminal region containing a putative homeodomain. Brk binds to a Dpp response element of the Ultrabithorax (Ubx) midgut enhancer at a sequence that overlaps a binding site for Mad. Furthermore, Brk is able to compete with Mad for occupancy of this binding site. This recognition of overlapping binding sites provides a potential explanation for why the G/C-rich Mad binding site consensus differs from the Smad3/Smad4 binding site consensus. The Dpp response element from Ubx is more sensitive to repression by Brk than is the vg quadrant enhancer. This difference correlates with short-range activation of Ubx by Dpp in the visceral mesoderm, whereas vg exhibits a long-range response to Dpp in the wing imaginal disc, indicating that Brk binding sites may play a critical role in limiting thresholds for activation by Dpp. Evidence suggests that Brk is capable of functioning as an active repressor. Thus, whereas Brk and Mad compete for regulation of Ubx and vg, Brk may regulate other Dpp targets without direct involvement of Mad (Kirkpatrick, 2001).

Binding of Brk to the Ubx and vg probes generates multiple bands, possibly indicating that Brk binds to more than one site. The Ubx element contains an inverted repeat of GGCGCT that overlaps a previously identified Mad binding site. Whereas the Mad site embedded in this repeat resembles the vg Mad site, the repeat as a whole is only matched at 7 of 12 positions in vg. Brk was tested for the ability to bind one copy of this sequence in a DNA probe that was otherwise divergent in sequence from the Ubx element. Brk binds to the GGCGCT probe with affinity that is similar to its affinity for the Ubx probe and yields a single major shifted band at about the same position as the lower most band observed with the Ubx probe. Although two weak upper bands are also observed with the GGCGCT probe, overall, these results are consistent with high affinity interaction of Brk with just one site in the GGCGCT probe (Kirkpatrick, 2001).

To investigate the specificity of Brk for the GGCGCT sequence, the effects of single base pair substitutions were determined. This was done measuring the ability of unlabeled 'wildtype' (GGCGCT) and mutant DNAs to compete with the labeled GGCGCT probe. In all, five mutants exhibited an ~20-fold reduction in the binding affinity, whereas the least critical position contributed as much as 3-fold to binding affinity. These results indicate that Brk makes base-specific contacts across the entire GGCGCT sequence (Kirkpatrick, 2001).

The GGCGCT repeat in the Ubx element overlaps a Mad binding site that can be modeled to consist of two degenerate Smad boxes, suggesting that Brk may compete with Mad for binding. This could not be determined unequivocally using the Ubx probe because Mad and Brk complexes have nearly identical mobilities in the band shift assay. However, the GGCGCT probe forms a complex with Mad that is easily resolved from the main complex formed with Brk; this probe makes clear that formation of Brk complexes correlates with reduced binding of Mad. In contrast, the same amount of Brk did not reduce binding of Mad to the M7 probe, evidence that Brk reduces the level of Mad binding by competition rather than by sequence-independent inhibition (Kirkpatrick, 2001).

To determine whether the Brk binding sites identified using the band shift assay are actually required for repression, the Ubx element was mutated to disrupt Brk binding. Each of three GGCG(C/T) sequences was changed to GTCG or to GGCGA, both of which dramatically reduce Brk binding but still allow Mad to bind. Introduction of the same triple-substitutions into the 2×Ubx-lacZ reporter result in an ~100-fold decrease in sensitivity to repression by cotransfected Brk. These results demonstrate that Brk binding sites are required for repression and confirm that the sequence specificity characterized in band shift experiments is also observed in cells (Kirkpatrick, 2001).

The overlap of Mad and Brk binding sites in the Ubx midgut element suggests that Brk might repress Dpp targets by simply competing with Mad for occupancy of an enhancer element. However, repressors generally function by quenching the activating potential of transcription factors bound nearby or by means of long range interfering effects on the general transcription machinery. To determine whether Brk is capable of functioning as an active repressor, Brk binding sites were positioned adjacent to sites for the unrelated Notch-responsive activator, Suppressor of Hairless [Su(H)] and reporter expression was monitored in response to cotransfected Brk, Su(H), and activated Notch effector plasmids. Brk completely prevents activation by Su(H), whereas a control reporter containing only Su(H) sites was repressed only 2-4-fold, an effect that may have been caused by the presence of a single Brk binding site adjacent to the hsp70 TATA box. Given this ability of Brk to function as a generic active repressor, it is reasonable to speculate that Brk might control a subset of Dpp targets without the direct involvement of Mad (Kirkpatrick, 2001).

Opposing inputs by Hedgehog and Brinker define a stripe of hairy expression in the Drosophila leg imaginal disc

The sensory organs of the Drosophila adult leg provide a simple model system with which to investigate pattern-forming mechanisms. In the leg, a group of small mechanosensory bristles is organized into a series of longitudinal rows, a pattern that depends on periodic expression of the hairy gene and the proneural genes achaete and scute. Expression of ac in longitudinal stripes in prepupal leg discs defines the positions of the mechanosensory bristle rows. The ac/sc expression domains are delimited by the Hairy repressor, which is itself periodically expressed. In order to gain insight into the molecular mechanisms involved in leg sensory organ patterning, a Hedgehog (Hh)- and Decapentaplegic (Dpp)-responsive enhancer of the h gene, which directs expression of h in a narrow stripe in the dorsal leg imaginal disc (the D-h stripe) has been examined. These studies suggest that the domain of D-h expression is defined by the overlap of Hh and high-level Dpp signaling. The D-h enhancer consists of a Hh-responsive activation element (HHRE) and a repression element (REPE), which responds to the transcriptional repressor Brinker (Brk). The HHRE directs expression of h in a broad stripe along the anteroposterior (AP) compartment boundary. HHRE-directed expression is refined along the AP and dorsoventral axes by Brk1, acting through the REPE. In D-h-expressing cells, Dpp signaling is required to block Brk-mediated repression. This study elucidates a molecular mechanism for integration of the Hh and Dpp signals, and identifies a novel function for Brk as a repressor of Hh-target genes (Kwon, 2004).

The D-h and V-h stripes are regulated by separate enhancers, which map between 32-38 kb 3' to the h transcription unit. ac stripes are not expressed until 6 hours after puparium formation (APF). The flanking narrow D-h stripe is positioned a few cells anterior to the compartment boundary, allowing expression of two dorsal ac stripes in the anterior compartment. V-h, however, is expressed directly adjacent to the AP boundary so that there is only one ventral ac stripe in the anterior compartment. Expression of each h stripe in its proper register is essential for positioning of the ac stripes and consequently for sensory bristle patterning in the adult leg. Focus was placed on the mechanisms that lead to expression of the D-h stripe in its precise register near the AP boundary (Kwon, 2004).

A question is raised regarding the identity of the repressor(s) that acts through the REPE to refine HHRE-directed expression. A potential candidate, the transcriptional repressor of Dpp target genes, Brk, is suggested by evidence indicating that Dpp is required to override REPE function. In the wing and leg imaginal discs, brk expression is repressed by and is roughly reciprocal to Dpp signaling. Hence, in the leg disc, brk expression is lowest in dorsal-most leg cells. D-h-GFP is expressed within the region of low-level brk expression in leg discs. Furthermore, brk expression expands dorsally in dppd6/dppd12 legs, in which D-h expression is severely reduced (Kwon, 2004).

To determine whether Brk functions as a repressor of D-h expression, D-h-GFP expression was examined in clones lacking brk function. Loss of brk function results in ectopic expression of D-h-GFP on either side of the D-h-GFP stripe. Ectopic expression is observed in clones anterior to the D-h-GFP stripe. However, the expansion is confined to a region two or three cells wide, directly juxtaposed to D-h expression, which presumably corresponds to the HHRE-responsive zone. In addition, ectopic expression is observed in ventral clones. Overexpression of brk along the AP boundary drastically reduces D-h-GFP expression but does not affect HHRE-GFP expression, indicating that Brk acts through the REPE to repress D-h expression. Since D-h expression is activated primarily by the Hh-responsive HHRE, these observations identify Brk as repressor of Hh as well as Dpp target genes (Kwon, 2004).

Genetic data support a hypothesis in which Brk acts through the REPE of the D-h enhancer to modulate activity of the HHRE. If so, it might be expected that the REPE would contain one or more functional Brk-binding sites. Hence, the REPE was examined for the Brk consensus binding site, GGCG(C/T)(C/T), and a potential Brk binding site was identified that overlaps two sequences similar to a consensus binding sites for Mad: GCCGNCGC, and a sequence similar to a cAMP response element (CRE), TGACGTCA. The sequence of overlapping CRE, Brk and Mad sites was designated the CMB element. Site directed mutational studies are consistent with the hypotheses that Brk acts through the CMB to repress D-h expression (Kwon, 2004).

A short sequence in the REPE, the CMB, has been identified that functions to restrict HHRE expression to a narrow dorsal domain. In this study, evidence is provided for the hypothesis that the transcriptional repressor Brk acts through the CMB to repress D-h expression. Although previous studies have shown that brk expression is very low or undetectable in cells near the Dpp source, a genetic requirement has been demonstrated for brk in repression of D-h in this region. In addition, overexpression of brk results in a dramatic reduction of D-h-GFP expression, but only mildly affects expression from a D-h-GFP transgene with a compromised Brk binding site (Kwon, 2004).

Dpp acts through the REPE to block Brk-mediated repression. It is proposed that high-level Dpp signaling defines the domain of D-h expression within the HHRE-response zone. This idea is supported by the observations that D-h-GFP but not HHRE-GFP expression is dependent on Dpp, indicating that Dpp signals through the REPE, and that elevation of Dpp signaling results in expansion of D-h expression along the AP and DV axes, within the domain of HHRE activity. Current studies suggest that the function of Dpp in regulation of D-h expression may be limited to repression of brk. Yet, the presence of Mad-binding sites in the CMB suggests a potentially more direct role for activated Mad (act-Mad), the transcriptional mediator of Dpp signaling. Brk has been shown to be a potent competitor of Mad in vitro for binding to overlapping binding sites in Dpp target enhancers. Hence, a potential role for Mad would be to prevent Brk from binding the CMB, thereby blocking Brk repression in cells receiving high-level Dpp signaling. If this model is correct, one might have expected the Mad1/Mad2 (MM) mutation to compromise D-h expression, which was not the case. However, the destabilization of Brk binding to the MM mutant might have masked a requirement for the Mad sites in blocking Brk repression (Kwon, 2004).

It has recently been shown that an act-Mad/Shn complex represses brk expression by binding a silencer element. Therefore, since mutation of the Mad sites expands D-h expression, it is possible that Mad acts in concert with Brk through the CMB to repress D-h expression. This notion is not inconsistent with genetic evidence, indicating a requirement for Mad in D-h expression, since loss of Mad function elevates Brk levels, which can overcome the requirement for CMB-sequences other than the Brk site. However, if this were the case, a more severe expansion phenotype might be expected with the MM mutant, in which both Brk and Mad binding are compromised. Further analysis is required to determine the role, if any, of the CMB-Mad-binding sites in D-h expression (Kwon, 2004).

Given the genetic evidence that Brk represses D-h expression and that Brk binds the CMB element in vitro, the most straightforward hypothesis is that Brk acts directly through the CMB in vivo to repress D-h expression. However, since mutation of the CRE also causes loss of repression, it is formally possible that the CRE rather than the Brk site is important for repression. A potential explanation for this observation is that mutation of the CRE lowers the affinity of this element for binding to Brk, even though the Brk binding site is intact in the CMB-C mutant. Because the levels of Brk in the dorsal leg are limiting, altered affinity could have a significant effect on the level of Brk occupancy of the CMB. However, through EMSA analysis it has been observed that the CRE mutant CMB binds Brk with an affinity greater than that of the wild-type element (Kwon, 2004).

Since it was not possible to mutate the Brk site without affecting the CRE, the CRE was altered in the BM2 and MBM mutants such that it more closely resembles a canonical CRE. Nevertheless, this change in the CRE may have affected its function. If so, this would be consistent with a model in which the CRE mediates repression of D-h expression, and Brk acts indirectly through the CRE rather than the Brk site. However, the finding that Brk overexpression drastically reduces D-h-C-GFP but not D-h-CB-GFP expression suggests that Brk can act directly through the Brk site, independent of the CRE (Kwon, 2004).

The requirement for CMB-sequences outside the Brk binding site suggests that the context of the Brk site within the CMB is important for repression. A plausible explanation for the requirement of the CRE is that it is bound by a factor, X, which functions to facilitate recruitment of Brk under conditions where Brk levels are limiting, such as in the dorsal leg. Consistent with this hypothesis is the observation that overexpression of Brk greatly reduces D-h-C-GFP expression, suggesting that the requirement for the CRE can be bypassed if the levels of Brk are high enough. However, when Brk levels are limiting, the CRE might contribute more to D-h repression than the Brk site. For example, in the dorsal leg, Factor X might bind the CMB and then form a complex with Brk, relieving the necessity for Brk to bind the CMB directly. This model could explain why D-h expression appears to be significantly more sensitive to Brk-mediated repression than other Brk targets in imaginal discs, such as vestigial (vg) and optomotor-blind (omb). vg and omb are each expressed in broad domains across the center of the wing disc and are repressed by higher levels of Brk than is D-h. Perhaps, the CRE and/or other sequences in the REPE mediate heightened response to Brk. It will be of interest to determine whether other Brk-target genes, such as spalt, which are also repressed by very low levels of Brk, are similarly regulated (Kwon, 2004).

A second potential function for a CRE-binding factor X is to act in concert with Brk to mediate D-h repression. Several lines of evidence suggest that Brk is a versatile repressor, which can inhibit transcription by competing with activators for binding to a common site or by active repression. Active repressors can act either at short range, by inhibiting activity of activators bound to nearby elements (150 bp away or less), or at long range by interfering with activators bound at a greater distances. Brk can mediate active repression, and binds the co-repressors dCtBP and Groucho (Gro), which mediate short- and long-range repression, respectively. Brk requires Gro and/or dCtBP function for repression of a subset of its target genes, whereas neither is required for repression of others. In the D-h enhancer, the CMB is positioned about 1 kb from the HHRE, suggesting that CMB-binding repressor(s) act at long range to repress HHRE-directed expression. Although Brk directly binds Gro, factor X could facilitate recruitment of Gro or other co-factors required for long-range repression (Kwon, 2004).

This study has identified a novel function for Brk as repressor of Hh-target gene expression. Brk was originally identified as a repressor of Dpp-target genes and a recent study indicates that Brk can block Wg-mediated transcription as well. Brk was shown to antagonize function of a Wg-responsive element in the midgut enhancer of the Ultrabithorax (Ubx). The Ubx midgut enhancer drives Ubx expression in parasegment (ps) 7 of the embryonic midgut. Two elements, one of which is Wg responsive (the WRS) and another Dpp responsive (the DRS), function synergistically to activate Ubx expression in ps 7 expression. In the adjacent ps8, however, Brk binds to the DRS and blocks the activity of the WRS. Curiously, the D-h-CMB and the Ubx-DRS are similarly organized in that each consists of overlapping CRE/Mad and Brk sites. The Ubx-DRS appears to mediate two modes of signal integration which involve: (1) synergistic activation, in which Mad/Med and dTCF act together to activate expression; and (2) activation and refinement, in which there is Wg mediated activation combined with Brk repression, which is blocked by Dpp. In the D-h enhancer, however, the CMB appears to be a component of a dedicated repression element, which appears to mediate only the second mode of signal integration: activation and refinement. The similar organization of the CMB and DRS suggests that it may be possible to predict the structure of enhancers known to be Brk responsive and which integrate Dpp and a second signal (Kwon, 2004).

Despite the similarities, there are important distinctions between the D-h and Ubx-midgut enhancers, suggesting that the mechanisms of Brk-mediated repression might differ in each case. In the Ubx-midgut enhancer, the DRS and WRS are separated by 10 bp, suggesting that Brk acts at short range to inhibit WRS activity. In the D-h enhancer, however, the CMB is positioned at least 1 kb from the HHRE, implying a long-range effect for this element. Furthermore, Brk repression of the WRS depends on Teashirt (Tsh), which binds Brk and acts as a co-repressor. Tsh is unlikely to be required for D-h repression because it is only expressed in proximal leg segments. The current studies suggest the requirement for a second DNA-bound factor, which binds the CRE, in addition to Brk for repression. The DRS-CRE, however, is required in addition to the Mad-binding sites for activation of Ubx in ps 7 (Kwon, 2004).

Together, these observations are consistent with a model in which Ci, acting through the HHRE, activates D-h expression. The domain of HHRE activity can be divided into two zones, 1 and 2. The HHRE has the potential to direct expression in both zones 1 and 2, but its activity is restricted to zone 1 by Brk and perhaps a second factor, X, which binds the CRE. In zone 2 cells, Brk would bind to the CMB and repress HHRE-directed expression. It is proposed that zone 1 is defined by the overlap of Hh and high-level Dpp signaling. Dpp promotes D-h expression by repressing brk expression in zone 1. However, the presence of Mad-binding sites in the CMB suggests the potential for a more direct role for Mad in D-h regulation, perhaps in competing with Brk for binding to the CMB, or in directly mediating repression. Confirmation of a role for the Mad sites awaits further analysis of the D-h enhancer (Kwon, 2004).

A combinatorial enhancer recognized by Mad, TCF and Brinker first activates then represses dpp expression in the posterior spiracles of Drosophila

Analysis of a reporter gene carrying a 375-bp region from a dpp intron (dppMX-lacZ) revealed that the Wingless and Dpp pathways are required to activate dpp expression in posterior spiracle formation. Within the dppMX region there is an enhancer with binding sites for TCF and Mad that are essential for activating dppMX expression in posterior spiracles. There is also a binding site for Brinker likely employed to repress dppMX expression. This combinatorial enhancer may be the first identified with the ability to integrate temporally distinct positive (TCF/Pangolin and Mad) and negative (Brinker) inputs in the same cells. Cuticle studies on a unique dpp mutant lacking this enhancer showed that it is required for viability and that the Filzkorper are U-shaped rather than straight. Together with gene expression data from these mutants and from brk mutants, the results suggest that there are two rounds of Dpp signaling in posterior spiracle development. The first round is associated with dorsal-ventral patterning and is necessary for designating the posterior spiracle field. The second is governed by the combinatorial enhancer and begins during germ band retraction. The second round appears necessary for proper spiracle internal morphology and fusion with the remainder of the tracheal system. Intriguingly, several aspects of dpp posterior spiracle expression and function are similar to demonstrated roles for Wnt and BMP signaling in proximal-distal outgrowth of the mammalian embryonic lung (Takaesu, 2008).

These data show that within the dppMX region there is a combinatorial enhancer that contains binding sites recognized by TCF and Mad that are essential for activating dpp expression in the spiracular chambers, in the spiracular branches and in the dorsal trunk branches. There is also a binding site recognized by Brinker that is likely employed to repress dpp expression late in spiracle development (Takaesu, 2008).

What makes this enhancer different from other enhancers in Drosophila also capable of integrating three inputs in the same cells. These enhancers integrate only positive signals. In all cases, PointedP2 binding displaces the Yan repressor that is constitutively bound to the enhancer in the absence of PointedP2. The difference is that the dppMX enhancer is actively repressed by Brk binding after being stimulated by positive input from the Dpp and Wg pathways. What makes this enhancer different from other enhancers in Drosophila that integrate positive and negative signals such as the enhancer of Ultrabithorax where positive input from TCF is associated with a competition between Mad (positive) and Brk (negative) inputs. The difference is that in the same cells the dppMX enhancer responds sequentially to positive combinatorial input from TCF and Mad and then to negative input from Brinker. The Ultrabithorax enhancer responds simultaneously to positive input from TCF and Mad in parasegment seven and to negative input from Brinker in the adjacent cells of parasegment 8 (Takaesu, 2008).

If combinatorial signaling by the Dpp and Wg pathways, via TCF and Mad, turn on the dppMX enhancer in posterior spiracle primordia of the dorsal ectoderm at stage 13, then where do the Dpp signals originate? One possibility is that Dpp signals derive from the adjacent region of the dorsal ectoderm -- leading edge cells located just anterior to the posterior spiracle primordia. In leading edge cells of the dorsal ectoderm, dpp expression is activated at stage 8. dpp leading edge expression is activated by enhancers distinct from the dppMX enhancer, and the leading edge enhancers are themselves stimulated, in part, by dpp blastoderm expression that sets up the embryonic dorsal/ventral axis. In this scenario, the activation of the dppMX enhancer in posterior spiracles by Dpp leading edge signaling represents the last step in a cascade, covering nearly all of embryogenesis, of increasingly spatially restricted rounds of Dpp dorsal ectoderm signaling (Takaesu, 2008).

The most likely the source of the Wg signal is a small group of cells in the spiracular chamber. wg expression in the spiracular chamber becomes visible at stage 11 and is present through the remainder of embryogenesis. This group of Wg expressing cells is required for the maintenance of Cut and Spalt expressions, genes shown in this study to be independent of Dpp signaling. The involvement of Wg in spiracle cell fate determination and dpp activation results in more severe spiracle defects in wg mutants than in brkF124 embryos or dpp null embryos with two copies of the dpp-ΔKX rescue construct (Takaesu, 2008).

The source of the signal that activates brk in the posterior spiracles is less easy to identify. However, one possibility is suggested by the mutant phenotype generated by ubiquitous expression of unpaired (a ligand of the Jak/Stat pathway with a role in posterior spiracle formation. These embryos display a U-shaped Filzkorper similar to brkF124 embryos and dpp null embryos with two copies of the dpp-ΔKX rescue construct (Takaesu,2008)

The data advance understanding of posterior spiracle development and the role that Dpp signaling plays in this process in three areas: (1) that dpp activity in dorsal/ventral patterning is genetically separable, in part, from its activity in posterior spiracle development; (2) that dpp signaling does not appear to influence posterior spiracle cell fate or external morphology but instead regulates spiracle internal morphology; and (3) that a functioning posterior spiracle is necessary for viability prior to hatching (Takaesu, 2008).

Regarding the separability of dpp dorsal-ventral patterning and posterior spiracle functions, this view contrasts with the prevailing wisdom that all dpp posterior spiracle defects are downstream consequences of dorsal-ventral patterning defects. Instead, the results suggest that there are two rounds of Dpp signaling in posterior spiracle development. The first round is necessary for setting up the posterior spiracle field in association with dorsal-ventral patterning at the blastoderm stage. The second begins during germ band retraction and appears to regulate the internal morphology of the spiracles. One possible explanation for why these distinct aspects of dpp function have been connected in the conventional wisdom is that the dppMX enhancer is located in an intron alongside dorsal/ventral patterning enhancers and is deleted in several widely studied dppHin alleles (Takaesu, 2008).

This two-round model for dpp signaling in posterior spiracle development fits well with analysis of Dpp signaling in heart development. Here, there is a second round of Dpp dorsal ectoderm to mesoderm signaling late in development that maintains the boundary between pericardial cells and the adjacent dorsal muscle cells. The second round of Dpp signaling in heart development is autoactivated by Dpp signals that also likely derive from dpp leading edge expression. Thus, in heart development, there is also evidence of a multi-step cascade of increasingly spatially restricted rounds of Dpp dorsal ectoderm signaling (Takaesu, 2008).

Regarding the function of the second round of Dpp signaling in posterior spiracle development, the data show that the expression of three transcription factors essential for cell fate determination in the spiracles is independent of Dpp signaling. In addition, pMad data show that the lumen of the spiracular chamber forms normally suggesting that spiracle external morphology and invagination, under the control of Rho signaling, is also independent of Dpp (Takaesu, 2008).

Cuticle data indicate that the primary defect in dpp posterior spiracle mutants is fully differentiated but U-shaped Filzkorper that do not appear to connect to the dorsal trunk branches. This phenotype plus the fact that dpp mRNA and pMad expressions normally span the spiracular chamber, spiracular branches and dorsal trunk branches suggests the hypothesis that Dpp regulates the internal morphology of the spiracles. Given the mutant phenotype and gene expression patterns, it is tempting to speculate that Dpp signaling via pMad directs the anterior outgrowth of the spiracles, the posterior outgrowth of the dorsal trunk branches and their eventual fusion into a coherent tracheal system (Takaesu, 2008).

Regarding posterior spiracle function in embryos, the fact that dpp posterior spiracle mutants do not hatch suggests that gas exchange through the posterior spiracles and the spiracular branches begins and is required to sustain the individual prior to hatching. This is an advance in the understanding of Drosophila embryonic and larval respiration (Takaesu, 2008).

Molecular integration of Wingless, Decapentaplegic, and autoregulatory inputs into Distalless during Drosophila leg development

The development of the Drosophila leg requires both Decapentaplegic (Dpp) and Wingless (Wg), two signals that establish the proximo-distal (PD) axis by activating target genes such as Distalless (Dll). Dll expression in the leg depends on a Dpp- and Wg-dependent phase and a maintenance phase that is independent of these signals. This study shows that accurate Dll expression in the leg results from the synergistic interaction between two cis-regulatory elements. The Leg Trigger (LT) element directly integrates Wg and Dpp inputs and is active only in cells receiving high levels of both signals. The Maintenance (M) element is able to maintain Wg- and Dpp-independent expression, but only when in cis to LT. M, which includes the native Dll promoter, functions as an autoregulatory element by directly binding Dll. The 'trigger-maintenance' model describes a mechanism by which secreted morphogens act combinatorially to induce the stable expression of target genes (Estella, 2008).

This study provides evidence that Dll expression during Drosophila leg development is controlled by separate, synergistically interacting cis-regulatory elements. The first element, LT, activates transcription only in response to high levels of Wg and Dpp signaling. The second element, M, includes the Dll promoter and has the ability to activate transcription in a Wg- and Dpp-independent manner, but only when in cis to LT. Together, these results fit well with previous genetic experiments showing that the Wg and Dpp inputs into Dll are only required transiently, prior to ~60 hr AEL. Based on the data, it is hypothesized that LT, and perhaps other elements with similar properties, is responsible for activating the Wg- and Dpp-dependent phase of Dll expression. Further, the data suggest that the combination of LT+M executes the Wg- and Dpp-independent phase of Dll expression. The existence of a two-component cis-regulatory system for Dll expression has several interesting implications and provides a mechanistic understanding of how Wg, Dpp, and Dll inputs are integrated into Dll expression (Estella, 2008).

The requirement for multiple inputs for gene activation is a common theme in transcriptional regulation. Enhancer elements can be thought of as 'logic integrators' that are only active in the presence of the correct activators and in the absence of repressors. The LT element defined here behaves as such a logic integrator. To be active, at least three conditions must be met. (1) LT must be bound to a transcriptionally active form of Tcf, a condition which indicates high levels of Wg signaling. (2) LT must be bound to a transcriptionally active form of Mad, and, (3) LT must not be bound to Brk. The second and third of these three conditions both indicate high levels of Dpp signaling. This combination of inputs ensures that LT is triggered only only where Wg and Dpp signaling are both active. In addition, it is hypothesized that there must be another input that restricts LT's activity to the ventral discs (e.g., it is not active in other tissues where Wg and Dpp signaling intersect such as the wing disc). Such a ventral-specific input could be Dll itself, which is expressed before LT is active via the Dll304 enhancer, and/or another ventral-specific factor such as buttonhead (btd), which is also required for Dll expression. Consistent with this idea, LT-lacZ is lost in Dll clones and in Dll hypomorphic discs, suggesting that Dll input, in addition to Wg and Dpp, is required for its activity (data not shown) (Estella, 2008).

As noted above, Dpp signaling uses two mechanisms (Mad binding and absence of Brk) to control LT's activity. Because Brk, a transcriptional repressor, binds directly to LT, it restricts the domain in which Wg signaling can activate this element. This conclusion is best supported by the expression pattern of the LT reporter gene in which the Brk-binding sites were mutated. Specifically, the expression of this reporter (LTBrk–-lacZ) was expanded ventrally, indicating its potential to be activated more broadly by Wg signaling in the absence of this repressor. Thus, it is suggested that the primary role of Brk is to provide spatial information to LT activation. The absence of Brk, however, is apparently not sufficient for LT activation; Mad input into LT appears also to be essential. Several experiments support this conclusion. Most informatively, LT-lacZ was not expressed in Mad; brk clones, and LT-lacZ reporter genes with either Mad site mutated were not expressed in brk clones. Thus, even in the absence of Brk, LT requires Mad input. It is suggested that in contrast to providing spatial information, the Mad input into LT is important for boosting the level of its activation, together with Tcf, by providing an additional potent transcriptional activator. Further, LT is unlikely to be the only Dll cis-regulatory element that integrates Wg plus Dpp signaling during leg development. Although LT was the only fragment within the 14 kb of 5' DNA that drove strong expression in the leg disc in a standard reporter gene assay, thus allowing the dissection of Wg and Dpp signal integration, a second fragment was identified that was able to synergize with M to produce a Dll-like expression pattern. In summary, these data suggest that during the Wg- and Dpp-dependent stage, Dll expression is regulated by the direct binding of Tcf, Mad, and Brk to LT and, perhaps, additional regulatory elements (Estella, 2008).

As is the case for Dll, there are examples of other genes that have separable initiation and maintenance phases of expression. For many of these examples, expression is maintained by the trxG and PcG of epigenetic regulators. There are also examples of genes that require enhancer-promoter communication for maintenance. For example, a regulatory element from the Hoxb4 gene requires sequences from its own promoter for stable expression in the mouse hindbrain. In this case, a key input into the promoter-proximal sequences is the PcG protein, YY1. Dll expression is unaffected in trx mutant clones but is lost in a subset of Pc and Scm clones, raising the possibility that PcG functions play a role in maintenance. However, PcG functions are more typically associated with maintaining genes in a repressed state, not an expressed state. Moreover, because of PcG's widespread role in gene silencing, many genes are likely to be derepressed in these clones. In fact, the Hox gene Abd-B is derepressed in these clones, and Abd-B has the ability to repress Dll. Thus, on balance, it seems more likely that the loss of Dll expression observed in some Pc clones is an indirect effect. In contrast, the results strongly argue that positive autoregulation, by direct binding of Dll to the M element, plays an important role in Dll maintenance (Estella, 2008).

One conclusion drawn from these observation that both LT and M are required for maintenance is that LT requires the Dll promoter to be fully active. Such promoter-specific enhancer activation has been observed previously and is generally thought to be important for remote enhancers to stimulate transcription from the correct promoter in gene-dense regions of the genome. The LT+M synergy described in this study is distinct from these other examples. In this case, although enhancer-promoter compatibility may be part of the reason that LT works better with M (and over large distances), the results show that the combination of the two has properties that are not exhibited by either element on its own. Specifically, while M-lacZ is very weakly expressed in leg discs, and LT-lacZ requires continuous Wg and Dpp inputs, the combination of LT+M allows Dll autoregulation to occur in a Wg- and Dpp-independent manner. Moreover, LT+M is not simply a Dll autoregulatory element: even though Dll is expressed in the wing disc, transcriptional activation by LT+M remains restricted to the ventral imaginal discs. This observation implies that the Dll input into LT+M can only occur in cells where LT was activated, which itself only happens in ventral discs. Thus, LT+M is not only a two-component Dll autoregulatory element, but is an autoregulatory element that requires the prior Wg- and Dpp activation of LT (Estella, 2008).

These observations lead to the suggestion of two classes of models by which maintenance may occur. In one, an activated LT element changes the chromatin structure of M, for example, by changing the position of a repressive nucleosome so that it can function as an autoregulatory element. According to this model, the continued presence of LT is required to maintain this chromatin structure. A second model that would also accommodate these data is that the combination of LT plus M is required to increase the efficacy of transcriptional activation by, for example, providing additional Dll (or other activator) binding. According to this scenario, LT activation by Wg and Dpp triggers the initial interaction between the LT and M elements, which would then be stabilized in a Wg- and Dpp-independent manner. These models are not mutually exclusive and both can be tested by analyzing the chromatin status at the M and LT elements (Estella, 2008).

The results also raise the question of what purpose this two-step trigger-maintenance mechanism may serve. One possibility is that, by having only a transient requirement for Wg and Dpp, these morphogens are available for carrying out completely different tasks, without affecting Dll expression. In support of this idea, in addition to working together to create the PD axis, Wg and Dpp function independently to instruct ventral and dorsal leg fates, respectively. Some of these late Wg and Dpp patterning functions may also require Dll input. The trigger-maintenance logic described in this study in principle allows Wg and Dpp to execute functions in collaboration with their own downstream target, Dll (Estella, 2008).

It is also noteworthy that the transient nature of the Wg and Dpp inputs into Dll is not the typical way these morphogens regulate their target genes in other tissues. In the Drosophila wing, for example, Dpp and Wg are required to continuously activate their targets, such as vestigial, optormotor blind, and spalt. One signficant difference between the regulation of wing and leg target genes by these morphogens is that in the wing Wg and Dpp generally act independently, whereas in the leg they act combinatorially to activate PD genes. Specifically, although they are expressed in ventral and dorsal sectors, respectively, Wg and Dpp activate Dll and dac in circular or nearly circular domains whose centers are located where the Dpp and Wg expression domains touch, in the middle of the leg disc. The trigger-maintenance mechanism defined in this study avoids the need for target genes such as Dll to continuously integrate Wg and Dpp inputs as the disc grows in size, and provides a mechanism to generate circular domains of gene expression using dorsal and ventral morphogen inputs (Estella, 2008).

A conserved activation element in BMP signaling during Drosophila development

The TGF-β family member Decapentaplegic (Dpp) is a key regulator of patterning and growth in Drosophila development. Previous studies have identified a short DNA motif called the silencer element (SE), which recruits a trimeric Smad complex and the repressor Schnurri to downregulate target enhancers upon Dpp signaling. The minimal enhancer of the dad gene was isolated, and a short motif was discovered that was termed the activating element (AE). The AE is similar to the SE and recruits the Smad proteins via a conserved mechanism. However, the AE and SE differ at important nucleotide positions. As a consequence, the AE does not recruit Schnurri but rather integrates repressive input by the default repressor Brinker and activating input by the Smad signal transducers Mothers against Dpp (Mad) and Medea via competitive DNA binding. The AE allows the identification of hitherto unknown direct Dpp targets and is functionally conserved in vertebrates (Weiss, 2010).

A 520-bp fragment was discovered within the second intron of the dad gene. This fragment induces an expression pattern very similar to that of the endogenous dad gene in embryonic, larval and adult tissues and contains evolutionarily conserved and largely overlapping binding sites for Smad and Brk proteins within a short sequence element that was called activating element (AE). The Smad and Brk proteins bind in a competitive manner to the AE, a mechanism similarly proposed for zen and Ubx enhancer elements. By precise targeted mutations, Brk binding was selectively abolished, and it was possible to unlink Smad and Brk input. Notably, the AE assembles a high-affinity trimeric complex of full-length Mad and Medea proteins. In Drosophila, such complexes have so far only been demonstrated for a so-called silencer element (SE) (Pyrowolakis, 2004). Therefore, this study presents the first example of such complex formation on a short-sequence element in the context of a gene activated by Dpp (Weiss, 2010).

The AE very closely resembles the SE, but despite their analogy, AE and SE differ in several key aspects. Because of the arrangement of the Smad binding sites, they are both able to recruit a complex of Mad and Medea. However, only the SE includes the second thymidine, which is essential for the recruitment of the repressor Shn (Pyrowolakis, 2004). Furthermore, the AE identified in the dad enhancer is able to interact with the Brk repressor. Brk competes with Mad for binding to the AE, which fulfills the consensus sequence derived from analysis of the SE with regard to Mad binding (GRCGNC) as well as the sequence for Brk binding (TGGCGYY). In contrast, Brk does not bind to the SEs described (Pyrowolakis, 2004). Thus, the AE and SE use a very similar sequence to exert opposite effects. These results provide a striking molecular scenario for Dpp signaling readout, based on the assembly of a trimeric Smad complex and its recruitment of a corepressor (Shn) or its competition with a dedicated repressor of the pathway (Brk) (Weiss, 2010).

Inverse regulation of target genes at the brink of the BMP morphogen activity gradient

BMP-dependent patterning in the Drosophila melanogaster wing imaginal disc serves as a paradigm to understand how morphogens specify cell fates. Profile of the transcriptional response to the graded signal of BMP, relies upon two counter active gradients of pMad and Brinker (Brk). This patterning model is inadequate to explain the expression of target genes, like vestigial and spalt, in lateral regions of the wing disc where BMP signal decline and Brk levels peak. This study shows that in contrast to the reciprocal repressor gradient mechanism, where Brk represses BMP targets in medial regions, in lateral regions target expression is downregulated by BMP signaling and activated by Brk. Brk induces lateral expression indirectly apparently through repression of a negative regulator. These findings provide a model explaining how the expression of an established BMP target is differentially and inversely regulated along the anterior-posterior axis of the wing disc (Ziv, 2012).

During Drosophila wing disc development the BMP morphogenetic gradient established by the collective actions of the two BMP ligands, Dpp and Gbb, patterns the cellular field by modulating gene expression in a concentration-dependent manner. How is the BMP concentration gradient translated into coordinated target gene expression? Current model relies upon two opposing activity gradients of the transcriptional regulators pMad and Brk, established in response to the BMP gradient. This model has been particularly successful in elucidating the regulatory influence exerted by BMP signaling although most of the attention has been focused on wing pouch, a region proximal to the peak of the gradient. Moreover, it is assumed that the target gene expression, in lateral regions where the BMP activity gradient decline, is independent of the signaling influence. Data presented in this study demonstrate that this supposition is incorrect. Alterations in pMAD signaling even in the lateral regions of the wing disc indeed lead to changes in positive target gene expression such as vg and sal albeit in an unexpected manner. In a classic 'role reversal' mode, the expression of the same targets is positively regulated by Brk and negatively by BMP signaling. Thus, while the classical morphogen model assumes that morphogens pattern a homogeneous field of responding cells, this study shows that in the developing wing disc interpretation and response to the BMP morphogenetic signal qualitatively differ along the anterior-posterior axis. The fact that sal is already expressed in both medial and lateral regions of the wing disc during early second larval stage, implies that this subdivision occurs early in development disc (Ziv, 2012).

By comparing sal expression in the different regions of the wing disc, this study has uncovered a novel circuitry underlying inverse regulation of an archetypal BMP target gene in distant regions where the morphogen levels decline. How is this counter regulation of sal achieved? Using the enhancer fragment that drives sal expression just in the lateral regions of the wing disc, evidence is provided that Brk induces expression of sal at the periphery of the wing disc indirectly through repression of a negative regulator (NRS). In one case, NRS represses sal expression by binding to a cis-regulatory element that contains neither Brk nor pMad-Med-Shn complex binding sites. In another, NRS is itself negatively regulated by Brk. The experiments described in this paper provide an initial working model to explain how the expression pattern of an established BMP target like sal is in fact differentially and inversely regulated in different regions along the anterior-posterior axis of the wing disc disc (Ziv, 2012).

Phenotypic consequences of the loss of brk on sal expression are qualitatively distinct and the effects vary in a position dependent manner. Compromising brk function results in upregulation of sal in the central region shows no effect towards the edge of the wing pouch and leads to a loss of sal expression in the periphery of the disc disc (Ziv, 2012).

Brk is known to repress lateral expression of classic BMP responsive genes such as omb and dad through direct binding to specific sequences within their enhancers, indicating that it is highly active in lateral regions. Since the medial enhancer of sal also contains two Brk consensus-binding sequences, this raises the question as to why the medial enhancer does not function to repress sal in the lateral zone? While the precise mechanism underlying this position dependent-repression is not known, the fact that a P-lacZ reporter of the sal medial enhancer is induced in lateral regions upon removal of Brk, indicates that in isolation the medial enhancer responds to Brk repressive activity also in the periphery of the wing disc. Similarly, other studies have found that mutating the relevant Brk binding sites in the isolated medial enhancer of sal expanded the expression to lateral regions. Combined together, these observations suggest that in the context of the full-length endogenous promoter of sal, (yet unknown) trans-factors are differentially distributed along the A-P axis of the developing wing disc to prevent repression by Brk (via the medial enhancer) in lateral regions and thus to confer position-dependent expression (Ziv, 2012).

This analysis implies that Brk drives endogenous sal expression in region IV by repressing a negative regulator of sal (NRS), which targets the lateral enhancer. Brk levels, which decline medially, enable NRS to be active which in turn represses sal expression in lateral regions of the wing pouch (region III). By manipulating Brk levels and monitoring the activity of the AK-lacZ reporter, evidence is provided that NRS is normally expressed and active all along the wing pouch (regions I, II and III). This raises the question as to how endogenous sal in the center of wing pouch escapes repression mediated by NRS. In principle, high pMad activity in medial regions (I and II) could overcome the repressive function of NRS. While plausible, this is an unlikely scenario as in the absence of both brk and mad, sal is ectopically expressed in medial regions. It is therefore proposed that in the context of the endogenous promoter, the activity of NRS is antagonized specifically in center of the wing pouch (regions I and II) with the assistance from the localized, non-uniform distribution of trans-acting factors along the A-P axis of the wing disc to confer a position-dependent transcriptional response (Ziv, 2012).

In these experiments sal is ectopically induced near the edge of the wing pouch (region III) in tkv mutant clones. Even more perplexingly tkvQD overexpressing clones also behave in a similar manner. Both of these outcomes are difficult to reconcile with the current model describing how the BMP morphogen gradient is linearly interpreted as both extreme situations; either complete loss (tkv) or substantial gain (tkvQD) of endogenous pMad activity, results in ectopic expression of sal . However, this conundrum can be partially resolved by taking into account the activity of the newly invoked, additional component NRS into the BMP-dependent patterning system. In wild type wing disc, pMad activity in region III, although low, is still sufficient to downregulate Brk levels just enough to allow for concomitant rise in NRS levels ultimately resulting in repression of sal expression. Importantly, low levels of pMad in region III [acting through the medial enhancer (ME)] are inadequate to antagonize the repressor activity of NRS [mediated through the lateral enhancer; (LE)]. The absence of pMad activity in tkv mutant clones in region III increases the levels of Brk, which in turn represses NRS and thus de-represses sal expression. In the case of tkvQD overexpressing clones, the substantially elevated pMad activity represses brk expression leading to elevated NRS activity. In region III clones (periphery of the wing pouch) the high activity of pMad (acting from the ME) overcomes the repressing activity of NRS (acting from the LE), ultimately resulting in activation of sal. By contrast, in the vast majority of tkvQD overexpressing clones (57 out of 63 clones) located in region IV (periphery of the wing disc) endogenous expression of sal is either lost or diminished. Why in region IV (periphery of the wing disc) pMad inducing activity does not have an edge over NRS repressing activity, as is the case in region III? It is proposed that in the context of the endogenous promoter, the activity of pMad is antagonized specifically in region IV due to the activity of unevenly distributed trans-acting factors. The rare occasions, where in the tkvQD overexpressing clones located in region IV sal expression was upregulated could be due to a rare event leading to acquisition of wing pouch like identity by the cells at the wing disc periphery, presumably due to early exposure to high pMad activity. Nevertheless, differential transcriptional response behavior exhibited by the cells from regions III v/s IV supports the subdivision of the developing wing disc on the basis of distinct regional competence (Ziv, 2012).

How an exponentially decaying morphogen gradient of BMP gives rise to computable changes in gene expression ultimately leading to discreet morphological structures is a fascinating question. A steep slope of the BMP activity gradient near the peak allows sharp expression domains of target genes to be defined within the wing pouch area. However in the lateral regions of wing disc, the activity gradient of BMP dips considerably raising the question as to how small differences in signal strength provide discrete threshold responses. Indeed, it is believed that BMP/Brk patterning system does not regulate the lateral expression of sal. These data argue that not only the signaling is active in the lateral regions but the inverse regulatory mode adopted by the signaling circuitry is in fact responsible for generating distinct threshold responses (Ziv, 2012).

Bridging Decapentaplegic and Wingless signaling in Drosophila wings through repression of naked cuticle by Brinker

Wnts and bone morphogenetic proteins (BMPs) are signaling elements that are crucial for a variety of events in animal development. In Drosophila, Wingless (Wg, a Wnt ligand) and Decapentaplegic (Dpp, a BMP homolog) are thought to function through distinct signal transduction pathways and independently direct the patterning of the wing. However, recent studies suggest that Mothers against Dpp (Mad), the key transducer of Dpp signaling, might serve as a node for the crosstalk between these two pathways, and both positive and negative roles of Mad in Wg signaling have been suggested. This study describes a novel molecular mechanism by which Dpp signaling suppresses Wg outputs. Brinker (Brk), a transcriptional repressor that is downregulated by Dpp, directly represses naked cuticle (nkd), which encodes a feedback inhibitor of Wg signaling, in vitro and in vivo. Through genetic studies, this study demonstrates that Brk is required for Wg target gene expression in fly wing imaginal discs and that loss or gain of brk during wing development mimics loss or gain of Wg signaling, respectively. Finally, it was shown that Dpp positively regulates the expression of nkd and negatively regulates the Wg target gene Distal-less (Dll). These data support a model in which different signaling pathways interact via a negative-feedback mechanism. Such a mechanism might explain how organs coordinate inputs from multiple signaling cues (Yang, 2013).

This study has shown that Brk directly represses nkd expression. The direct repression of nkd by Brk is underscored by three observations. First, a Brk site was identified in the intronic region of nkd, which Brk physically occupies in vitro. Second, ChIP analysis shows that Brk binds a DNA region near this Brk site in embryos in a manner inversely related to Wg activity. Third, reporter analysis in Kc cells indicates that Brk represses Arm-dependent activation of an intronic WRE containing this Brk site, but only when the Brk site is intact. In addition, genetic analyses has shown that the repression of nkd by Brk is functionally significant. In the developing wing, it was found that the loss of brk de-represses nkd and downregulates Wg target proteins, such as Dll and Sens. Conversely, ectopic brk inhibits nkd expression and markedly enhances Dll expression. Furthermore, removal of nkd prevents the loss of Dll in brk clones whereas co-expression of nkd abolishes the expanded Dll caused by ectopic brk. In adult wing, the loss and gain of brk phenotypically resembles the loss and gain of Wg signaling, respectively. Consistent with a repressive role of Dpp cascade on brk, it was found that ectopic Dpp signaling enhances nkd and inhibits Wg signaling). These results support a model in which Dpp signaling increases the expression of Nkd, a Wg inhibitor, by the downregulation of Brk, and thereby inhibits the Wg outputs. In another words, nkd might fall into a class of Dpp targets, which are de-repressed upon the activation of Dpp signaling. This study has thus uncovered a previously unsuspected molecular mechanism underlying the interaction between Wg and Dpp signaling pathways in Drosophila wing development (Yang, 2013).

Until recently, little has been known about the cross-interaction between Wg and Dpp signaling in Drosophila wings, in spite of the fact that the fly wing has served as an excellent model system for the dissection of the molecular basis of these signaling transduction pathways. This is in contrast to Drosophila leg imaginal discs, in which mutual repression between Wg and Dpp signaling has long been suspected. However, several studies have indicated that manipulation of Dpp signaling levels in the wing sometimes leads to phenotypes resembling those caused by loss or gain of Wg activity. Notably, ectopic Dpp signaling increases notches in the wing, which is characteristic of reduced Wg signaling. However, the underlying mechanism for this effect of Dpp is not clear. Recently, independent research groups have suggested that Mad, the key effector of Dpp signaling, might play a role in the regulation of Wg target gene expression in fly wings. The molecular basis for their findings has mainly been the physical interaction between Mad and TCF, similar to the findings in mammals, in which several Smad proteins interact with members of the lymphoid enhancer binding factor 1/TCF family of DNA-binding HMG box transcription factor. It remains to be determined whether the role of Mad is direct or indirect because the reporter assays in these studies were performed with TOPFlash or similar constructs in mammalian cell culture, which might not always accurately represent the complicated situation of the in vivo regulation of Wg target genes. Furthermore, manipulation of Mad expression in wing discs influences Dll expression in different directions. Although these intriguing discrepancies can be explained by the physical interaction between Mad and TCF, the current model offers an alternative interpretation based on the negative regulation of nkd by Brk, which might suggest an indirect role of Mad in Wg signaling. For example, the current model could provide an explanation for the previous finding that ectopic Dpp signaling, caused by Mad, Medea, TkvQD, etc., results in notched wings (Yang, 2013).

The role of Brk in Wg signaling has been previously documented in Drosophila. It has been suggested that brk is able to antagonize Wg signaling based on the activity of a midgut-specific Ubx reporter gene in which physical interactions among Brk, Teashirt and CtBP have been described. In leg discs, Wg signaling may directly repress Dpp morphogen expression via an Arm-TCF-Brk complex, offering a direct model for the cross-talk between Wg and Dpp. However, the current studies have indicated a positive role for Brk in Wg signaling through an indirect action. In addition to the repression of Dpp targets, the roles of Brk in Wg signaling described in these different models exemplify the pleiotropic actions of brk throughout development and might provide the molecular basis for tissue-specific consequences of developmental signaling pathways (Yang, 2013).

nkd was first identified as a Drosophila segment-polarity gene, mutation of which gives rise to major deficits in fly embryonic development. Its expression appears to be universally induced by Wg in fly embryos and larval imaginal discs. It is interesting that although the loss of nkd in embryos has an effect similar to gain of wg, decreased nkd function in fly wings shows little impact. However, none of the nkd alleles used in these studies has been well characterized at the molecular level. Given the complexity of nkd transcriptional regulation, it could be that these mutant forms of nkd still possess residual function in the wing. Alternately, overexpression of nkd blocks ectopic Wg signaling in the eyes and generates PCP phenotypes in the wing through a direct interaction with Dsh. Consistent with these observations, this study found that loss of brk can cause a dramatic increase of nkd expression in certain areas of the wing imaginal disc, leading to wing notches and PCP defects. The current findings suggest that nkd may indeed play roles, at a certain level, in both canonical and noncanonical Wg signaling in fly wings. However, a closer examination of nkd function in fly wings is needed (Yang, 2013).

In conclusion, this study found that Brk influences Wg signaling by directly repressing nkd expression and could serve as a node for cross-talk between the Wg and Dpp signaling pathways. Wnt-BMP cross-interactions have been implicated in many developmental and disease processes). For example, a Wnt-BMP feedback circuit mechanism is important for inter-tissue signaling dynamics in tooth organogenesis in mouse. The findings may therefore add new insights into cell differentiation and human cancer (Yang, 2013).

Protein Interactions

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The transcriptional repressor Brinker antagonizes Wingless signaling

In the embryonic midgut of Drosophila, Wingless (Wg) signaling elicits threshold-specific transcriptional response, that is, low-signaling levels activate target genes, whereas high-signaling levels repress them. Wg-mediated repression of the HOX gene Ultrabithorax (Ubx) is conferred by a response sequence within the Ubx B midgut enhancer, called WRS-R. It further depends on the Teashirt (Tsh) repressor, which acts through the WRS-R without binding to it. Wg-mediated repression of Ubx B depends on Brinker, which binds to the WRS-R. Brinker binds to a site distinct from that occupied by the Wg effector, the Pangolin/Armadillo activator complex. Brinker thus acts at short range to block the activity of this complex. Furthermore, Brinker blocks transcriptional activation by ubiquitous Wg signaling. Brinker binds to Tsh in vitro, recruits Tsh to the WRS-R, and mutual physical interactions are found between Brinker, Tsh, and the corepressor dCtBP. This suggests that the three proteins may form a ternary repressor complex at the WRS-R to quench the activity of the nearby-bound Pangolin/Armadillo transcription complex. Finally, brinker and tsh produce similar mutant phenotypes in the ventral epidermis, and double mutants mimic overactive Wg signaling in this tissue. This suggests that Brinker, which was initially discovered as an antagonist of Dpp signaling, may have a widespread function in antagonizing Wg signaling (Saller, 2002).

Most likely, Brinker uses a mechanism called quenching to block Pangolin/Armadillo. Quenching involves interaction of repressors (and the corepressors they recruit) with activators bound to nearby sites. Brinker is known to be able to quench target genes by recruiting the corepressor Groucho, which is involved in multiple quenching processes. groucho antagonizes wg, and TCF factors can bind to Groucho proteins directly, so Pangolin may thus be able to recruit Groucho unassisted. However, these findings do not rule out the possibility that Pangolin relies on cooperation with Brinker to achieve Groucho recruitment (Saller, 2002 and references therein).

Brinker can bind to the corepressor dCtBP, so Brinker may recruit dCtBP instead of, or in addition to, Groucho. Recall that Tsh plays a critical role in the Wg-mediated repression in the midgut. Moreover, Tsh can bind to Brinker as well as to dCtBP, so it seems plausible that Tsh plays a pivotal role in assisting Brinker in the recruitment of dCtBP. Like Groucho, dCtBP is a corepressor with quenching activity. In addition, Tsh may itself be involved in the quenching process. It has been suggested that quenching may be based on obstruction of the interaction between the activation domain of a transcriptional activator and the general transcription machinery -- intriguingly, hypophosphorylated Tsh binds to the carboxy-terminal activation domain of Armadillo to modulate Wg signaling (Saller, 2002 and references therein).

The Drosophila midgut has provided a model system in which Wg signaling regulates gene transcription in a concentration-dependent manner; low signaling levels activate Wg target genes, whereas high levels repress the same genes. The discovery that Brinker confers transcriptional repression by Wg completes the picture of the DNA-binding proteins that interpret these different signaling thresholds. Pangolin confers Wg-induced stimulation of target genes, but its activity can be blocked by Brinker, which confers Wg-mediated repression of the same genes. Pangolin depends on Armadillo for its activity, whereas Brinker depends on Tsh to block the activity of the Pangolin/Armadillo complex. In turn, the availability of Armadillo depends directly on Wg signaling, which promotes its stabilization and nuclear translocation, whereas the availability of Tsh depends on transcription of its gene (which itself depends on wg). In other words, high Wg signaling induces locally the expression of the Tsh corepressor, which then cooperates with Brinker to repress Wg target genes in the same cells. One of these targets is wg itself, so Brinker and Tsh take part in the negative feedback loop of Wg signaling in the middle midgut (Saller, 2002).

Ubx B is not only a Wg-responsive enhancer, but it is also stimulated by Dpp signaling. Furthermore, Dpp signaling antagonizes Wg-mediated repression. This can be explained in two ways: (1) high levels of Dpp-activated Mad are expected to compete with Brinker for binding to the WRS-R; (2) the brinker gene itself may be down-regulated by Dpp signaling, since this is the case in other tissues, so Brinker may only be present at very low levels in cells within the Dpp-signaling domain. brinker expression cannot be detected in this domain, whereas low levels of expression are detectable in the neighboring Wg-signaling domain. In contrast, in the latter domain, in which the levels of activated Mad are expected to be low, Brinker successfully competes with Mad for binding to the WRS-R and, together with Tsh, which is present at high levels in this domain, blocks the activity of Pangolin/Armadillo. Note that Dpp signaling promotes this repression indirectly, by contributing to the stimulation of Tsh expression in ps8 (Saller, 2002).

Dpp and Wg signaling cooperate in multiple developmental contexts. In some contexts they synergize, whereas in other contexts, they antagonize each other. Given that most, if not all, Dpp target genes, and multiple Wg target genes, are repressible by Brinker, this suggests that Brinker may have a universal key role in this decision between synergy and antagonism: absence of Brinker allows synergy between Dpp and Wg, whereas presence of Brinker (and Tsh) mediates antagonism (Saller, 2002).

DNA recognition by the brinker repressor--an extreme case of coupling between binding and folding

The Brinker (Brk) nuclear repressor is a major element of the Drosophila Decapentaplegic morphogen signaling pathway. Its N-terminal part has weak homology to the Antennapedia homeodomain and binds to GC-rich DNA sequences. The conformation and dynamics of the N-terminal 101 amino acid residues of Brk were investigated in the absence and in the presence of cognate DNA by solution NMR spectroscopy. In the absence of DNA, Brk is unfolded and highly flexible throughout the entire backbone. Addition of cognate DNA induces the formation of a well-folded structure for residues R46 to R95. This structure consists of four helices forming a helix-turn-helix motif that differs from homeodomains, but has similarities to the Tc3 transposase, the Pax-6 Paired domain, and the human centromere-binding protein. The GC-rich DNA recognition can be explained by specific major groove hydrogen bonds from the N-terminal end of helix a3. The transition from a highly flexible, completely unfolded conformation in the absence of DNA to a well-formed structure in the complex presents a very extreme case of the 'coupling of binding and folding' phenomenon (Cordier, 2006).

The co-regulator dNAB interacts with Brinker to eliminate cells with reduced Dpp signaling

The proper development of tissues requires morphogen activity that dictates the appropriate growth and differentiation of each cell according to its position within a developing field. Elimination of underperforming cells that are less efficient in receiving/transducing the morphogenetic signal is thought to provide a general fail-safe mechanism to avoid developmental misspecification. In the developing Drosophila wing, the morphogen Dpp provides cells with growth and survival cues. Much of the regulation of transcriptional output by Dpp is mediated through repression of the transcriptional repressor Brinker (Brk), and thus through the activation of target genes. Mutant cells impaired for Dpp reception or transduction are lost from the wing epithelium. At the molecular level, reduced Dpp signaling results in Brk upregulation that triggers apoptosis through activation of the JNK pathway. This study shows that the transcriptional co-regulator dNAB is a Dpp target in the developing wing that interacts with Brk to eliminate cells with reduced Dpp signaling through the JNK pathway. Both dNAB and Brk are required for cell elimination induced by differential dMyc expression, a process that depends on reduced Dpp transduction in outcompeted cells. A novel mechanism is proposed whereby the morphogen Dpp regulates the responsiveness to its own survival signal by inversely controlling the expression of a repressor, Brk, and its co-repressor, dNAB (Ziv, 2009).

NAB proteins comprise a family of transcriptional co-regulators implicated in various developmental processes in different organisms. Drosophila NAB was found to be required for determining specific neuronal fates in the embryonic CNS and for wing hinge patterning. This study shows that dNAB induces cell elimination through induction of the JNK pathway, which in turn triggers Caspase-3-mediated apoptosis. dNAB acts as a co-repressor that physically interacts with Brk to induce apoptotic cell elimination. This conclusion is based on several lines of evidence. First, dNAB-induced apoptosis is completely nullified by removal of Brk. Second, epistatic analysis placed dNAB in the Dpp signaling pathway downstream of the receptor complex and of brk transcriptional repression and upstream of Brk. Third, dNAB physically associates with Brk through its NCD2 domain in vitro. Fourth, dNAB enhances the killing activity of Brk in the presumptive wing blade region and is required for elimination of Dad-overexpressing cells, a process that is completely dependent upon Brk function. Finally, ectopic expression of dNAB represses the expression of Dpp/Brk target genes (Ziv, 2009).

Competitive interactions occur between cells differing in their levels of dMyc, such that cells expressing more dMyc both outgrow neighboring cells and induce their death. This competitive behavior correlates with, and can be modulated by, the activation of the Dpp survival signaling pathway, showing that dMyc-induced cell competition relies on Dpp signaling. The fact that dNAB, similar to Brk, is crucial for dMyc-induced cell competition strongly supports a role for dNAB as an effector of cell elimination of underperforming cells with reduced Dpp signaling (Ziv, 2009).

Elimination of underperforming cells takes place only during early larval stages. Clones generated later, during the third instar larval stage, persist to adulthood. Consistently, using double staining of wing discs with antibodies directed against Brk and dNAB, it was found that the two do not overlap in the second instar larval stage [60 hours after egg laying (AEL)] and only slightly overlap during the third instar (80 hours AEL). These findings suggest that the Brk-dNAB complex is active in cell elimination only during early development. This might indicate that either another factor required for complex activity is present only during early development, or that a factor is present during later stages that inhibits the complex. Alternatively, intensive growth/proliferation might be required for the execution of the killing activity of the complex (Ziv, 2009).

The morphogen Dpp acts through a well-characterized transduction pathway to simultaneously regulate growth, survival and patterning. To a large extent, Dpp signaling acts through negative regulation of brk expression. This implies that a complete answer to how the Dpp signal directs different cellular and developmental processes requires an understanding of how Brk executes its transcriptional repression functions. The finding that dNAB is a Brk co-repressor is in accordance with recent results showing that overexpression of Brk forms that cannot bind either Gro or CtBP results in repression of sal, omb and vg, and that Brk contains additional co-repressor-binding domains. On contrast to Gro, a known co-repressor of Brk, the function of dNAB is not required for Dpp-dependent patterning. However, Gro does not play a similar role to that of dNAB in promoting JNK-mediated cell killing. These findings imply that the choice of Brk co-repressor determines the specificity of target gene repression, thereby modulating different Dpp outputs. Mechanistically, this could be achieved in a number of ways: for example, dNAB or Gro association might alter the DNA-binding specificity of Brk, or the promoters of Brk target genes might be differentially responsive to dNAB and Gro. In addition, the fact that Gro is ubiquitously expressed throughout the developing wing, and that Dpp induces dNAB expression in the center of the wing disc while restricting Brk expression to lateral regions, provide another means for differentially modulating Dpp outputs (Ziv, 2009).

Based on these findings, a molecular model is proposed to explain how the morphogen Dpp regulates the cellular response to its own survival signal in the developing wing by inversely controlling the expression of two key factors, Brk and dNAB. In the center of the wing disc, Dpp represses brk and induces dnab expression, so that in situations in which Dpp signaling activity is abnormally reduced, the resulting local increase in the levels of Brk, which complexes with dNAB, activates the apoptotic pathway. Thus, the Dpp signal sensitizes cells in the center of the wing disc to the apoptotic effect associated with reduced Dpp signaling by maintaining dNAB expression. In lateral regions of the wing disc, where Brk expression is normally higher, apoptotic cell elimination is attenuated, at least in part owing to a lack of dNAB. Thus, by invoking dNAB as a Dpp effector molecule that sensitizes cells to the levels of Brk, it can be at least in part explained why cells in the center of the wing disc, near the Dpp source, are more susceptible to cell elimination induced by reduced Dpp signaling, and why high levels of Brk in the periphery do not necessarily bring about apoptosis (Ziv, 2009).

Given that dNAB appears to play no role in Dpp-mediated patterning, it is proposed that dNAB functions in the wing to prevent developmental errors and discontinuities along the Dpp signaling gradient. This mechanism might be a general feature of morphogen gradients that functions to avoid the accumulation of detrimental developmental mistakes that would otherwise lead to embryonic malformation, and is potentially important in cancer, where tumor cells overexpressing oncogenes such as Myc may act as super-competitors. Thus, the molecular principles underlying such developmental fail-safe mechanisms are clearly of biomedical interest (Ziv, 2009).

brinker: Biological Overview | Developmental Biology | Effects of Mutation | References

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