Ubx regulation: Table of contents

Promoter Structure

Ultrabithorax midgut enhancer

Decapentaplegic (Dpp) is an extracellular signal of the transforming growth factor-ß family with multiple functions during Drosophila development. For example, it plays a key role in the embryo during endoderm induction. During this process, Dpp stimulates transcription of the homeotic genes Ultrabithorax (in the visceral mesoderm) and labial (in the subjacent endoderm). A cAMP response element (CRE) from an Ultrabithorax enhancer mediates Dpp-responsive transcription in the embryonic midgut, and endoderm expression from a labial enhancer depends on multiple CREs. The enhancer, termed Ubx B confers Wingless- and Decapentaplegic-dependent expression in the visceral mesoderm. Staining mediated by Ubx B is in two stripes of cells in the visceral mesoderm, a wide prominent one in parasegments 6-9 and a narrow weak one in parasegment 3. The Drosophila CRE-binding protein dCREB-2 binds to the Ultrabithorax CRE. Binding is at a palindromic sequence TGGCGTCA that resembles a typical cAMP response element (CRE) (TGACGTCA). Mutation of this site results in the elimination of response to Dpp, but a maintenance of response to Wg. This residual expression is in parasegment 8 and 9 coinciding with the main source of wg expression in the middle midgut. The Ubx CRE can also mediate response to Dpp signaling in the endoderm. Other transcription factors act through the Ubx B enhancer to confer its tissue-specific response to Dpp in the visceral mesoderm. CRE needs to cooperate with an LEF-1 binding site to respond to the Dpp signal in the visceral mesoderm. Schnurri, a transcription factor implicated in Dpp signaling, fails to interact with Ubx B. Adjacent to the CRE is another palindromic sequence that antagonizes the activating effects of Dpp and Wg signaling on the Ubx B enhancer. Ubiquitous expression of a dominant-negative form of dCREB-2 suppresses CRE-mediated reporter gene expression and reduces labial expression in the endoderm. Therefore, a dCREB-2 protein may act as a nuclear target, or as a partner of a nuclear target, for Dpp signaling in the embryonic midgut (Eresh, 1997).

Examination of the Ultrabithorax midgut enhancer, for which the Dpp response element has been localized to the 95 bp DI-DII interval, and the labial endoderm enhancer reveals a Mothers against Dpp binding-site consensus of GCCGnCGC. The two sites of highest affinity match the MAD consensus perfectly, and three lower-affinity sites contain mismatches in one or as many as three positions (Kim, 1997).

LEF-1, a nuclear factor coordinating signaling inputs from wingless and decapentaplegic

The murine transcription factor lymphocyte enhancer binding factor 1 (LEF-1) recognizes a minimal wingless response sequence in the midgut enhancer of Ultrabithorax. This visceral mesoderm enhancer, located 2.9 kb from the Ubx start site contains adjacent elements that respond to wg and dpp signaling. The DPP response sequence within this enhancer is a cAMP-response element (CRE). Wingless and DPP act independently but synergistically through this enhancer to stimulate Ubx expression in the midgut. The LEF-1-binding site contains an excellent match to the LEF-1 binding site first identified in the T cell receptor alpha chain enhancer. LEF-1 binds the Ubx wingless response sequence (WRS) with high affinity and specificity (Riese, 1997).

Mouse LEF-1 was used in these experiments because the endogenous protein (now known to be Pangolin) had not yet been identified. The WRS is recognized by LEF-1 in a ternary complex with Armadillo protein. Expressing LEF-1 throughout the mesoderm results in an anterior expansion of Ubx expression in the visceral mesoderm. A similar anterior expansion is observed after the expression of arm throughout the visceral mesoderm. Under these circumstances the second midgut constriction appears precociously and tends to form as a double constriction. LEF-1 activity depends on arm, since LEF-1 fails to stimulate Ubx transcription in arm mutants. In contrast, LEF-1 expressing wg mutants show a moderate level of Ubx transcription in LEF-1 expressing embryos. This implies that LEF-1, perhaps by virtue of being overexpressed, bypasses the need for Wingless stimulation (Riese, 1997).

If overexpressing mouse LEF-1 functions independently of wingless signaling, why are its effects localized to certain regions of the midgut? One possibility is that LEF-1 activity may be restricted by the dpp signal, which itself is localized to the middle midgut region where most LEF-1 activity is seen. When LEF-1 and DPP are coexpressed in the mesoderm, UBX staining stretches through the visceral mesoderm, whereas the effects of LEF-1 or of DPP by themselves are regionally restricted. There is, in fact very little effect of mesodermally expressed LEF-1 in dpp mutants embryos (Riese, 1997).

LEF-1 ovexpression has phenotypic effects in other developmental contexts in which WG and DPP operate. LEF-1 was expressed in the wing disc. LEF-1 overexpression leads to many extra bristles on the notum, a derivative of the wing disc. This LEF-1 effect resembles the phenotype of shaggy mutant clones. The wings of these flies look highly abnormal. In strong LEF-1 transformants, the wings are rudimentary, but they frequently show tufts of bristles as observed in shaggy mutant clones. These bristle tufts are localized to the tip of the wing. The effects of LEF-1 in these wings mimic loss of wingless function, such as missing wing margin bristles and gaps in the wing margin itself. Apparently, mouse LEF-1 overexpression in the margin primordium somehow interferes with the function of the endogenous wingless target transcription factor in a dominant negative way (Riese, 1997).

The requirement for both wingless and dpp signaling in the midgut is a prime example of how two genes acting together can result in a highly localized emergent structure. Clearly, the signaling inputs from wg and dpp are independent and separate until they reach their final target, the Ubx enhancer, where they converge at neighboring response elements. Neither signal response element on its own is sufficient, to stimulate transcription in the visceral mesoderm (Riese, 1997).

Transcriptional repression due to high levels of Wingless signalling

Extracellular signals can act at different threshold levels to elicit distinct transcriptional and cellular responses. The transcriptional regulation of the Wingless target gene Ultrabithorax has been examined in the embryonic midgut of Drosophila. Ubx transcription is stimulated in this tissue by Dpp and by low levels of Wingless signaling. High levels of Wingless signaling can repress Ubx transcription. The response sequence within the Ubx midgut enhancer required for this repression coincides with a motif required for transcriptional stimulation by Dpp, namely a tandem array of binding sites for the Dpp-tranducing protein, Mad. Indeed, Wingless-mediated repression depends on low levels of Dpp, although apparently not on Mad itself. In contrast, high levels of Dpp signaling antagonize Wingless-mediated repression. This suggests that transcriptional activation of Ubx is subject to competition between Dpp-activated Mad and another Smad whose function as a transcriptional repressor depends on high Wg signaling. Wingless can repress its own expression via an autorepressive feedback loop that results in a change of the Wingless signaling profile during development (Yu, 1998).

Dpp and Wg signaling synergize in the visceral mesoderm to stimulate Ubx transcription, targeting distinct, albeit adjacent, response sequences in the Ubx midgut enhancer. Therefore, efficient stimulation of Ubx transcription by Wg depends on dpp. Wg-mediated repression also depends on dpp, but, remarkably in this case, the response sequence for Wg-mediated repression within the Ubx enhancer coincides with that for Dpp-mediated stimulation. Indeed, the WRS-R/DRS (Wingless response sequence mediating repression and Dpp response sequence) functions in two antipodal responses: it mediates efficient transcriptional stimulation when the signaling levels of Dpp are high and those of Wg are low, but it is also required for transcriptional repression when the Wg signaling levels are high and those of Dpp are low. This raises the possibility that the same factor may confer the two antipodal responses. However, this is unlikely to be the case since Mad itself, which binds to the DRS to mediate the positive response to Dpp, is apparently not required for the Wg-mediated repression (Yu, 1998).

Thus it is proposed that the two antipodal responses are conferred by two distinct factors: by Mad and by a hypothetical protein WR. It is further proposed that WR is a Mad-related protein, i.e. a Smad, since WR acts through Mad-binding sites and since its function as a repressor depends on dpp. It is envisaged that WR, like Mad itself and other Smads, is activated by Dpp signaling through phosphorylation by ligand-bound membrane receptors, an event that promotes their subsequent translocation to the nucleus. In this scenario, Dpp enables WR (which also needs to be activated by high Wg signaling) to occupy the Mad-binding sites within the Ubx enhancer. Once bound to this enhancer, WR dominantly represses Ubx transcription, overriding the activating function of Arm-Pangolin and other transcriptional activators bound to the same enhancer (Yu, 1998).

How is WR's repressor function activated by high Wg levels? It is presumed that high Wg signaling regulates, directly or indirectly, the availability of WR as an enhancer-binding protein: either high Wg signaling controls a post-transcriptional event (e.g. it may promote WR's association with Armadillo, or WR's translocation into the nucleus), or it simply activates transcription of the WR gene. The latter possibility of indirect regulation, which involves transcriptional coupling, is favored because it accomodates readily the dependence of Wg-mediated repression on arm and Pangolin. Whatever the case, it is emphasized that high Wg signaling controls the activity of the protein WR (possibly a Smad), which also requires Dpp signaling. Thus, WR is a common target for two signaling pathways and represents a point of convergence between them (Yu, 1998).

This model readily explains how high Dpp levels antagonize WR, namely by promoting maximal levels of nuclear Mad which now competes with WR for binding to the Ubx enhancer. The outcome of this competition is the transcriptional activation or repression of target genes, depending on the prevalence of Mad or WR. This may illustrate a general principle, namely that the response sequence for the positive effect of one signal is also the response sequence for the negative effect of an antagonistic signal. Such a layout provides a sharp flipping of the response from positive to negative in an area where cells are experiencing increasingly more of one signal and increasingly less of the antagonizing one (Yu, 1998).

Medea is the Smad4 homolog that is known to be the common oligomerization partner for pathway-specific Smads. Furthermore, Medea binds to the same DNA sequences as Mad. This raises the possibility that Medea is an oligomerization partner of WR: while Medea, together with Mad, is expected to activate transcription, together with WR it may repress transcription. A precedent for this scenario is the Myc/Mad/Max system, in which Mad (a bHLH protein that happens to have the same name as the Dpp transducer Mad) is a common dimerization partner for either Myc, a transcriptional activator, or Max, a transcriptional repressor. In addition to antagonism, there is also synergy between Wg and Dpp in the embryonic midgut. This synergy apparently results from cooperation between the nuclear target factors activated by the two signals, i.e. between Arm-Pangolin and Mad/CRE-binding proteins. Other examples of apparent synergy between Wg and Dpp are the leg and wing imaginal discs, where these signals act together in central disc regions to stimulate expression of homeobox genes. But the two signals also antagonize each other in leg discs, as well as in eye discs. Although it is conceivable that the developmental context determines the synergy or antagonism between Dpp and Wg, the situation in the midgut suggests that the decisive factor in each case may be the levels of signaling (Yu, 1998 and references).

It is interesting that Wg signaling can repress its own expression when signaling levels reach a critically high level. This indicates a negative feedback loop, which could account for two observations: (1) Wg signaling shifts its own expression towards the anterior over time. It is not known at present whether this shift has any biological significance. (2) Wg has the potential for switching itself off over time. This is actually observed, since Wg expression becomes undetectable by the end of embryogenesis. Clearly, Wg's negative feedback loop is capable of changing the Wg signaling profile as development procedes. There are negative feedback loops for other signaling pathways in Drosophila. For example, the epidermal growth factor (EGF) receptor inhibits itself eventually, after signaling has reached a critical level, by switching on expression of an inhibitory ligand, Argos. In the ovary, this negative feedback loop causes splitting of a single signaling peak into twin peaks. Furthermore, Hedgehog signaling in the eye imaginal disc is repressive at high Hedgehog levels, but stimulatory in cells, further away from the signaling source, which experience lower Hedgehog levels. Perhaps such 'hard-wired' negative feedback loops in signaling pathways are fairly universal, and serve to stop these pathways from escalating out of control. If so, this would be akin to feedback inhibition of metabolic pathways, which provides homeostatic control (Yu, 1998 and references).

A function of CBP as a transcriptional co-activator during Dpp signalling

In the visceral mesoderm, dpp is expressed in parasegment (ps) 7 under the control of the homeotic gene Ultrabithorax (Ubx). In this cell layer, dpp stimulates its own expression and the expression of Ubx. dpp also stimulates the expression of wingless (wg), an extracellular signaling molecule of the Wnt family, in the neighbouring ps8. wg in turn feeds back to stimulate Ubx and dpp expression in ps7. Thus, dpp is part of a parautocrine feedback loop by which Ubx maintains its own expression indirectly through controlling dpp and wg. Dpp also diffuses from its mesodermal source through the endodermal cell layer of the embyonic midgut, where it stimulates the expression of D-Fos and of the homeotic gene labial. These inductive steps ultimately specify the differentiation of distinct cell types in the larval midgut epithelium. In order to understand the mechanism by which dpp stimulates transcription, a short enhancer fragment of Ubx, called Ubx B, has been characterized that contains response sequences for dpp and wg signaling in the embryonic midgut. The dpp response sequence of this enhancer is bipartite, consisting of a tandem repeat of Mad binding sites and a cAMP response element (CRE). The presence of the latter raised the question whether the co-activator CBP (CREB-binding protein, binding to CREs) might participate in Dpp-induced transcriptional activation (Waltzer, 1999).

Drosophila CBP loss-of-function mutants show specific defects that mimic those seen in mutants that lack the extracellular signal Dpp or its effector Mad. CBP loss severely compromises the ability of Dpp target enhancers to respond to endogenous or exogenous Dpp. CBP binds to the C-terminal domain of Mad. These results provide evidence that CBP functions as a co-activator during Dpp signaling, and they suggest that Mad may recruit CBP to effect the transcriptional activation of Dpp-responsive genes during development (Waltzer, 1999).

The embryonic midgut of nejire (nej) mutants (whose CBP function is reduced) show phenotypes related to wg gain-of-function phenotypes: increased labial expression in the endoderm, and derepression of the Ubx B enhancer in the visceral mesoderm. These phenotypes do not resemble those seen in dpp or Mad mutants: in Mad mutants, labial expression is strongly reduced, and so is the beta-galactosidase (lacZ) staining mediated by the Ubx B enhancer in the middle midgut. However, the narrow band of lacZ staining normally visible in the visceral mesoderm of the gastric caeca (in ps3) is absent in nej mutant embryos. Indeed, closer inspection reveals that the gastric caeca frequently fail to elongate in these mutants. A similar phenotype is observed in Mad and in dpp mutants. Thus nej, like dpp, is required for the formation of the gastric caeca, and also for the activity of the Ubx B enhancer in the caecal primordia. The activity of this enhancer in these primordia coincides with Dpp expression and depends on dpp function. The formation of the first midgut constriction is often impeded. While this could reflect overactive Wg signaling, it also mimics loss of glass bottom boat (gbb) signaling: Gbb is a Dpp homolog expressed in the visceral mesoderm and whose function is required for the formation of the first midgut constriction (Waltzer, 1999).

The hypothesis that CBP is a co-activator of dpp-induced transcription was tested by examining the Dpp response of the Ubx enhancer in nej mutants. Because it was expected that the repressive effect of CBP on this enhancer would mask a possible activating effect of CBP in cells in which the enhancer is stimulated by Wg signaling, a mutant version of Ubx B, called B4, was used whose positive response to Wg is abolished. B4 activity in the midgut is reduced compared with the wild-type enhancer; however, B4 still contains a fully functional dpp-response sequence and can be efficiently stimulated by ectopic Dpp. B4 can thus be used to selectively monitor the stimulation of Ubx by Dpp in the visceral mesoderm. The activity of Ubx B4 is significantly reduced in nej mutants. LacZ staining is particularly weak in ps6/7 (near the Dpp source), but also in ps10, and is barely detectable in the gastric caeca. Furthermore, in nej mutant embryos derived from nej mutant germlines (nej), lacZ staining mediated by B4 is even weaker than in the zygotic nej mutants: although these nej GLC embryos are somewhat variable in terms of their phenotypes the most severely mutant embryos show lacZ staining in only a few cells in the ps8 region. Similarly, in Mad12 mutant embryos, lacZ staining is much reduced, with some staining remaining in ps6 and ps8. This implies that CBP, like Mad, is required for the Dpp response of the Ubx B4 enhancer (Waltzer, 1999).

The response of B4 to GAL4-mediated ectopic Dpp was examined in nej mutant embryos. If Dpp is expressed throughout the mesoderm, B4-mediated lacZ staining is increased and detectable throughout the midgut mesoderm. In nej mutants, this response of B4 to ectopic Dpp is strikingly disabled: there is barely any lacZ staining in the anterior midgut, and only a moderate increase of lacZ staining in the ps8/9 region, indicating a residual Dpp response in this region. These results strongly support the conclusion that CBP is required for the transcriptional response of the Ubx enhancer to Dpp signalling. They argue that CBP functions downstream of the Dpp signal (Waltzer, 1999).

The zinc finger protein Schnurri acts as a Smad partner in mediating the transcriptional response to Decapentaplegic

Genetic analysis has implicated Schnurri (Shn), a zinc finger protein that shares homology with mammalian transcription factors, in the Dpp signal transduction pathway. However, a direct role for Shn in regulating the transcriptional response to Dpp has not been demonstrated. In this study it is shown that Shn acts as a DNA-binding Mad cofactor in the nuclear response to Dpp. Shn can bind DNA in a sequence-specific manner and recognizes sites within a well-characterized Dpp-responsive promoter element, the B enhancer of the Ultrabithorax (Ubx) gene. The Shn-binding sites are relevant for in vivo expression, since mutations in these sites affect the ability of the enhancer to respond to Dpp. Furthermore Shn and Mad can interact directly through discrete domains. To examine the relative contribution of the two proteins in the regulation of endogenous Dpp target genes a cell culture assay was developed and it has been shown that Shn and Mad act synergistically to induce transcription. These results suggest that cooperative interactions between these two transcription factors could play an important role in the regulation of Dpp target genes. This is the first evidence that Dpp/BMP signaling in flies requires the direct interaction of Mad with a partner transcription factor (Dai, 2000).

The Ubx B element, a discrete 269-bp enhancer from the Ubx promoter, mediates dpp- and wg-dependent reporter gene expression in the midgut visceral mesoderm. Recent studies have identified two Mad-binding sites within Ubx B. Mutations in these sites significantly reduce (but do not eliminate) the ability of the enhancer to respond to Dpp, indicating that these sites are required in vivo. In order to determine whether shn plays a role in Ubx B transcription, the enhancer was examined for the presence of Shn-binding sites using DNaseI footprinting assays. In these experiments the ability of the bacterially produced protein to bind an MBP1 oligo that resembles the optimal Shn sites, was examined as a control for activity. Shn DBD2 protects two regions within the Ubx B fragment. The S1 region that extends from nucleotides 458 to 487 shows a slightly higher affinity for Shn, compared to the second binding site (S2) that spans nucleotides 524 to 538. Both protected areas contain sites with a 6/7 match to the GGGG and CCC motifs in the consensus Shn binding sites. The central portion of the sites show a poor match to the consensus, indicating that these nucleotides may be less critical for binding. Shn DBD1 also shows protection of the same regions in Ubx B, consistent with the finding in site selection experiments that both sets of fingers recognize closely related sequences (Dai, 2000).

dpp expression in parasegment 3 (ps3) and ps6-7 of the visceral mesoderm is required for induction of Ubx expression and transcription of the endogenous gene is lost in shn mutants. While the number of visceral mesodermal cells is reduced in embryos lacking shn activity, this is unlikely to be the cause for loss of Ubx expression, since other genes such as Sex combs reduced that are expressed in the visceral mesoderm can be detected in shn mutant embryos. Whether the Ubx B reporter also requires shn function for dpp-responsive expression was assessed. In wild-type embryos Ubx B-LacZ is expressed in a narrow anterior stripe in the visceral mesoderm in ps3 and in a broader posterior stripe encompassing ps6-9. The broader posterior stripe represents a response to both Dpp signaling in ps6-7 and Wg signaling in ps8-9. In embryos lacking shn protein, Ubx B expression is lost at all sites, suggesting that Shn is required to mediate the transcriptional response to Dpp. However, it has been established that dpp expression is maintained through an indirect autoregulatory loop that involves positive feedback from Ubx expression in the same cells. In addition, dpp expression in ps7 is required to maintain wg expression in the adjacent cells of ps8. Mutations in shn disrupt these autoregulatory interactions and result in loss of dpp expression in the midgut. The absence of reporter gene expression in ps8-9 in shn mutants may also be attributed to the loss of dpp expression and its effect on maintenance of wg transcription. These observations raise the possibility that loss of Ubx B-LacZ in shn mutants could be an indirect consequence of the loss of dpp transcription. To test this, Dpp was provided exogenously in shn mutant embryos using a heat shock promoter and reporter gene expression was assayed. In a wild-type background, ectopic Dpp results in induction of Ubx B in an expanded domain. However in shn mutants, a complete lack of LacZ staining was observed in the visceral mesoderm; i. e., reporter gene expression is not recovered even when Dpp is exogenously supplied. These results indicate that Shn acts downstream of Dpp in the visceral mesoderm and is obligately required for Ubx B transcription (Dai, 2000).

In order to examine the contribution of Shn binding to Ubx B expression, PCR-based mutagenesis was used to introduce base substitutions in the Shn sites S1 and S2. The Ubx BS1S2 fragment was tested by footprint analysis and does not show protection by Shn over the range of concentrations that bind wild-type Ubx B. Transgenic flies carrying a reporter mutant for both sites were generated by germline transformation and analyzed. Mutation of the Shn sites significantly reduces the domain of expression in ps3 and lowers the level of transgene expression in ps6-9. Similar results have been obtained with a construct that contains mutant S1 sites but is deleted for S2. Transcription of the reporter in the gastric cecae (ps3) appears more sensitive to the loss of Shn sites, suggesting that these cells may require higher levels of Dpp signaling. This is consistent with the loss of Ubx expression in ps3 but not in ps7 in weak dpp alleles as well as in embryos lacking zygotic Mad. Taken together these results provide in vivo evidence that the Shn-binding sites in Ubx B are important for Dpp responsiveness (Dai, 2000).

Although mutations in the binding sites for Mad result in more severe loss of Ubx B expression compared to that caused by mutations in the Shn sites, in neither case is the expression abolished, raising the possibility that inputs from both proteins contribute to the regulation of Ubx B. There is increasing evidence that protein-protein interactions between Smads and accessory transcriptional factors can result in cooperative binding and synergistic transcription of reporter genes. The fact that a Ubx B reporter that lacks Shn-binding sites (BS1S2) shows residual staining, while Ubx B expression is completely absent in shn mutant embryos, suggests that loss of Shn protein has more severe consequences than loss of Shn-binding sites. In order to determine whether protein-protein interactions as well as DNA-binding contribute to activation of Ubx B by Shn and Mad, an assay was developed to study the nuclear response to Dpp signaling. The B enhancer was cloned upstream of a minimal promoter driving expression of the luciferase gene (Ubx B-Luc) and its activity was examined in cultured cells. This reporter shows very low levels of basal expression in the BMP-responsive C3H10T1/2 cells. Cotransfection with Mad/ Medea results in only a slight elevation of luciferase activity. However coexpression of Mad/Medea with constitutively activated TkvA results in a dramatic 25-fold increase in promoter activity relative to the basal response. In other words, coexpression of all three components causes a 5-fold stronger stimulation than expression of either Mad/Medea or TkvA alone. The response to TkvA is dependent on Mad and Medea since transfection with the receptor alone leads to only a small increase in transcription over basal levels, perhaps due to phosphorylation of endogenous BMP-specific Smads (Dai, 2000).

Whether coexpression of Shn with Mad and Medea could enhance transcriptional activation of Ubx B-Luc was examined. Expression of Shn or Mad/Medea alone elicits a weak transcriptional response. However coexpression of all three proteins results in a 32-fold induction of reporter gene activity relative to the basal response. This is a 6-fold increase over the response to either Shn or Mad/Medea alone. More strikingly this induction is 3-fold greater than the expected additive response to expression of the individual proteins. To test the importance of Mad and Shn DNA-binding to synergistic activation, a luciferase reporter construct was generated lacking both Mad sites known to be required for expression in the embryo (Ubx BM2). As anticipated, it was found that the response of Ubx BM2-Luc to stimulation by TkvA and Mad is significantly reduced when compared to wild-type Ubx B. Interestingly, however, deletion of the Mad-binding sites in BM2 does not affect the induction of reporter activity by Mad/Medea in the presence of Shn. In analogous experiments using a Ubx BS1S2-Luc reporter, loss of the Shn-binding sites only marginally affects the cooperative response to Shn and Mad/Medea. These results could indicate that synergistic transcriptional activation by overexpression of Shn and Mad/Medea does not depend entirely on their ability to bind DNA, but involves cooperative protein-protein interactions (Dai, 2000).

To test this, a reporter was constructed that lacks both Mad as well as Shn-binding sites (Ubx BM2S1S2-luc). The response of Ubx B to overexpression of Mad/Medea and Shn is strongly reduced in the double mutant. It is concluded that binding sites for either Mad or Shn are sufficient to mediate synergistic activation of the Ubx B reporter. However, when neither protein can bind the enhancer, it is no longer possible to elicit a transcriptional response. While the data may be interpreted as redundancy for Mad/Medea and Shn in stimulating UbxB transcription, this view is contradicted by the fact that expression of either protein alone clearly does not stimulate maximal response of the UbxB reporter. Taken together these data indicate that Shn can act as a transcriptional coactivator with Mad to regulate the expression of the Ubx B enhancer (Dai, 2000).

Osa-containing Brahma chromatin remodeling complexes are required for the repression of Wingless target genes

The Wingless signaling pathway directs many developmental processes in Drosophila by regulating the expression of specific downstream target genes. The product of the trithorax group gene osa is required to repress such genes in the absence of the Wingless signal. The Wingless-regulated genes nubbin, Distal-less, and decapentaplegic and a minimal enhancer from the Ultrabithorax gene are misexpressed in osa mutants and repressed by ectopic Osa. Osa-mediated repression occurs downstream of the up-regulation of Armadillo but is sensitive both to the relative levels of activating Armadillo/Pangolin and repressing Groucho/Pangolin complexes that are present, and to the responsiveness of the promoter to Wingless. Osa functions as a component of the Brahma chromatin-remodeling complex; other components of this complex are likewise required to repress Wingless target genes. These results suggest that altering the conformation of chromatin is an important mechanism by which Wingless signaling activates gene expression (Collins, 2000).

To test the requirements for Osa to repress the expression of Wg target genes, the expression of a lacZ reporter gene driven by a well-characterized wg-responsive enhancer was examined. The midgut enhancer (UbxB) of the Ultrabithorax (Ubx) promoter drives lacZ expression in the embryonic midgut in a pattern that is dependent on both wg and decapentaplegic (dpp). In wild-type embryos UbxB-lacZ is expressed primarily in parasegment (ps) 6, 7, and 8, with weaker expression in ps 3. This expression is de-repressed in embryos lacking the maternal contribution of osa, such that the expression of lacZ expands anteriorly as far as ps 3. Similarly expanded expression is induced by ectopic expression of wg in the mesoderm using 24B-Gal4. Conversely, expression of UAS-Osa or UAS-DN-Pan in the mesoderm represses the expression of UbxB-lacZ. However, neither wg nor dpp is ectopically expressed in the midgut in embryos lacking maternal osa (Collins, 2000).

When the dpp response element in UbxB is mutated (UbxBC), the expression of the lacZ reporter is severely reduced; only weak levels of lacZ expression are detectable in ps 8. Expression of UbxBC-lacZ is unchanged in the absence of maternal osa, suggesting that the dpp response element is still required for the expression of the reporter construct in the absence of Osa. When one of the two wg response elements in UbxB is mutated (UbxB4), the expression of lacZ is reduced in wild-type embryos. However, removal of maternal osa allows an expansion of UbxB4-lacZ expression. This suggests that lack of osa can compensate for a reduction in the responsiveness of the promoter to Wg but not to Dpp. Furthermore, the expression of wild-type UbxB-lacZ is also de-repressed in embryos lacking maternal osa even in the presence of DN-Pan. These data argue that Osa functions specifically to repress the activation of the UbxB enhancer by the Wg pathway (Collins, 2000).

Repression of Dpp targets by binding of Brinker to Mad sites

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The transcriptional repressor Brinker antagonizes Wingless signaling

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

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

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

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

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

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

Regulation of Ubx expression by epigenetic enhancer silencing in response to Ubx levels and genetic variation

For gene products that must be present in cells at defined concentrations, expression levels must be tightly controlled to ensure robustness against environmental, genetic, and developmental noise. By studying the regulation of the concentration-sensitive Drosophila melanogaster Hox gene Ultrabithorax, this study found that Ubx enhancer activities respond to both increases in Ubx levels and genetic background. Large, transient increases in Ubx levels are capable of silencing all enhancer input into Ubx transcription, resulting in the complete silencing of this gene. Small increases in Ubx levels, brought about by duplications of the Ubx locus, cause sporadic silencing of subsets of Ubx enhancers. Ubx enhancer silencing can also be induced by outcrossing laboratory stocks to D. melanogaster strains established from wild flies from around the world. These results suggest that enhancer activities are not rigidly determined, but instead are sensitive to genetic background. Together, these findings suggest that enhancer silencing may be used to maintain gene product levels within the correct range in response to natural genetic variation (Crickmore, 2009).

Together, these results demonstrate that Ubx enhancer silencing is triggered when Ubx is present at higher than normal levels. When Ubx concentration is especially high (when Ubx is ectopically expressed via Gal4 or heat-shock promoters) all enhancer input into Ubx can be silenced, resulting in the complete absence of Ubx expression and haltere-to-wing transformations. Although such high levels of Ubx are not physiological, it was also found that Ubx enhancer silencing can be triggered by additional copies of Ubx+, which in principle results in less than double the amount of Ubx protein. In this case, it was found that the expression of some Ubx enhancer traps is clonally silenced (e.g. Ubx-Gal4lac1), while the expression of other enhancer traps (e.g., Ubx-lacZ166) is reduced. Thus, different Ubx enhancers are differentially sensitive to negative autoregulation; some are shut off by relatively low Ubx levels, while others require high Ubx levels to be silenced (Crickmore, 2009).

Most remarkably, this study found that enhancer silencing can occur simply by varying the genetic background. In Drosophila melanogaster, due in part to its large population size, the frequency of DNA polymorphisms between individuals in the wild is estimated to be as high as 1 in 100 basepairs. Due to these polymorphisms, it is imagined that different strains of D. melanogaster, when kept in isolation from each other, may have subtly different ways of regulating Ubx. These may be due to strain-specific differences in the Ubx cis-regulatory elements, in the trans regulators of Ubx expression, or both. Consistent with this idea, it is of interest that gene expression levels, when assayed across entire genomes, show a lot of variability in natural populations. Although it was found that the final Ubx expression pattern and levels are very similar between lab and wild D. melanogaster strains, when two strains are bred together genetic differences may result in fluctuations in the initial Ubx levels. The silencing system described in this stud may function to compensate for these fluctuations and thus ensure that the correct Ubx levels are produced throughout the haltere (Crickmore, 2009).

In the crosses to wild D. melanogaster strains, this study found that the expression of genetically marked Ubx alleles varied tremendously, depending on the genetic background. Extrapolating from these results suggests that there is a lot of previously undetected variability in enhancer activities at the Ubx locus in wild files that would not have been detected using traditional assays. Thus, these results challenge the standard view that a given transcriptional enhancer integrates the same inputs and produces the same outputs, regardless of genetic background. Instead, due to natural genetic variation, the activity of a particular enhancer may vary widely between individuals in wild populations. Additionally, the results show that the activity of an enhancer can even vary among the cells within its expression domain (e.g., the haltere) in a single individual. It is suggested that plasticity in enhancer activities is essential to compensate for genetic and perhaps environmental variation. Moreover, given that many genes may have multiple, partially redundant enhancers, enhancer silencing may be essential to buffer gene expression levels so that they remain within a narrow, biologically tolerable range. In contrast, small differences in enhancer activities in flies in the wild may serve as a potential source of phenotypic variation that can be acted upon by natural selection. Since population genetic theory predicts that selection differentials of a small fraction of a percent are seen in natural populations with the effective population size of Drosophila, it is plausible that this variation is functionally significant, perhaps through a subtle influence of haltere morphology on flight performance (Crickmore, 2009).

Ubx regulation: Table of contents

Ultrabithorax: Biological Overview | Evolutionary Homologs | Targets of activity | Protein Interactions | Posttranscriptional regulation | Developmental Biology | Effects of Mutation | References

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