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).

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 (L12^M3), module 4 (L14^M4) and module 7/8/9 (L15^M7/8/9) caused derepression throughout the center of the wing disc. However, the same mutations in a larger fragment containing several additional modules (L13^M3, L13^M4 and L13^M7/8/9) 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 L13^M variants, leaving the constitutively active mutant module(s) free to interact with the transcriptional machinery. As a consequence, L13^M 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 ShnCT^M, demonstrating that this interaction is relevant in vivo. However, ShnCT^M 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).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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).

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).

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).

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, Brk^NLS, 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 Brk^NLS cannot bind to DNA in vivo. However, a modified protein, Brk^NLSW, which is identical to Brk^NLS apart from the addition of the four amino acids WRPW that recruit the co-repressor Gro, can repress targets, indicating that Brk^NLS 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 brk^F138, which is truncated before the CiM and GiM, can still repress omb. Truncated proteins that lack the CiM and GiM, Brk^Stop1, Brk^EC and Brk^A, 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, Brk^NLSW, or only a CiM, Brk^Stop1NAC, 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 brk^F138 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 Brk^Stop1, 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 brk^F138 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 brk^F138 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).

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).

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).

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).

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 brk^wt. 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 (sog^wt, sog⁰, brk^wt, 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 brk^0Dl 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).

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 gro^E48 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 (Brk^RD) readily retains [³⁵S]methionine-labelled Gro. To test further the specificity of this interaction, three mutant derivatives of the Brk^RD, fused to the GST moiety, were generated in which the Gro recruitment domain (Brk^RDmutG; FKPY to FEAY), the core of the CtBP-binding motif (Brk^RDmutC; DLS to AAA) or both (Brk^RDmutC/G) were altered. Brk's binding to Gro is impaired by the modifications in the FKPY motif. Significantly, however, Gro associates with the GST- Brk^RDmutC construct as strongly as it does with the native GST-Brk^RD fusion. GST-Brk^RD 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-Brk^RD 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-Brk^RDmutC, Sxl is substantially repressed, although not as effectively as by Hairy-Brk^RD. 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-Brk^RDmutC/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 gro^E48 (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 (Brk^mutG), but not with CtBP (Brk^mutC), 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 Brk^mutC, 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 Brk^mutG-expressing embryos, in comparison with wild-type embryos or embryos expressing Brk^mutC/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).

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).

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).