Transcriptional Regulation

Capicua is involved in Dorsal-mediated repression of zerknullt expression in Drosophila embryo

The maternal transcription factor Dorsal (Dl) functions as both an activator and a repressor in a context-dependent manner to control dorsal-ventral patterning in the Drosophila embryo. Previous studies have suggested that Dl is an intrinsic activator and its repressive activity requires additional corepressors that bind corepressor-binding sites near Dl-binding sites. However, the molecular identities of the corepressors have yet to be identified. This study presents evidence that Capicua (Cic) is involved in Dl-mediated repression in the zerknullt (zen) ventral repression element (VRE). Computational and genetic analyses indicate that a DNA-binding consensus sequence of Cic is highly analogous with previously identified corepressor-binding sequences and that Dl failed to repress zen expression in lateral regions of cic mutant embryos. Furthermore, electrophoretic mobility shift assay (EMSA) shows that Cic directly interacts with several corepressor-binding sites in the zen VRE. These results suggest that Cic may function as a corepressor by binding the VRE (Shin, 2014).

Targets of Activity

Pannier, a GATA family transcription factor, that is expressed in the dorsal portion of the embryo just after cellularization, lies downstream of decapentaplegic and zerknüllt. In embryos null for dpp, no pannier is expressed (Winick, 1993).

Dorsal represses zerknüllt. zen expression requires dpp and tolloid. Null mutations cause a ventralization phenotype. Early broad expression of zen is normal in dpp mutants, but during cellularization, absence of dpp has an effect on zen maintenance (Rushlow, 1990).

Race, a putative angiotensin-converting enzyme, might be a target of zerknüllt. Soon after zen expression is restricted to the dorsal-most regions of the embryonic ectoderm, Race is activated in a coincident pattern and becomes associated with the amnioserosa during germ band elongation, shortening and heart morphogenesis. After germ band elongation, Race is also expressed in both the anterior and posterior midgut, where it persists throughout embryogenesis. Race expression is lost from the dorsal ectoderm in either zen- or dpp- mutants, although gut expression is unaffected (Tatei, 1995).

DPP target gene zerknüllt (zen) activates the amnioserosa-specific expression of a downstream target gene, Race (Related to angiotensin converting enzyme), the earliest known marker gene for the amnioserosa. Two TGF-beta growth factors, dpp and screw, function synergistically to subdivide the dorsal ectoderm into two embryonic tissues, the amnioserosa and dorsal epidermis. Previous studies have shown that peak dpp activity is required for the localized expression of zen. ZEN in turn directly activates the amnioserosa-specific expression of a downstream target gene, Race. A 533 bp enhancer from the Race promoter region is shown to mediate selective expression in the amnioserosa, as well as the anterior and posterior midgut rudiments. This enhancer contains three ZEN protein binding sites, and mutations in these sites virtually abolish the expression of an otherwise normal Race-lacZ fusion gene in the amnioserosa, but not in the gut. Genetic epistasis experiments suggest that ZEN is not the sole activator of Race, although a hyperactivated form of ZEN (a zen-VP16 fusion protein) can partially complement reduced levels of dpp activity. These results suggest that dpp regulates multiple transcription factors, which function synergistically to specify the amnioserosa. It is unknown whether the dpp pathway leads to the post-translational modification of ZEN or whether an unknown transcription factor serves as a DPP substrate and then participates in the activation of zen (Rusch, 1997).

The proliferating cell nuclear antigen promoter function resides within a 192-bp region (-168 to +24 with respect to the transcription initiation site). Cotransfection with a zerknüllt (zen)-expressing plasmid specifically repressed CAT expression. Since ZEN does not actually bind the promoter, it is thought that zen indirectly represses PCNA gene expression, probably by regulating the expression of some transcription factor(s) that binds to the PCNA gene promoter (Yamaguchi, 1991).

The Drosophila DNA polymerase alpha gene is repressed by Zerknüllt. The expression of zen results in reduction of the abundance of mRNA, both DNA polymerase alpha and PCNA. A positive cis-acting element found in both DNA polymerase alpha and PCNA genes is responsible for repression by ZEN protein or downstream of ZEN action. The nuclear extract of tissue culture cells transfected by a zen-expressing plasmid contains lesser amounts of a DNA replication-binding factor (DREF) than that of untransfected or mutant zen-transfected cells (Hirose, 1994).

DREF, a transcription regulatory factor that specifically binds to the promoter-activating element DRE (DNA replication-related element) of DNA replication-related genes, was purified to homogeneity from nuclear extracts of Drosophila Kc cells. DREF is a polypeptide of 701 amino acid residues that contains three characteristic regions: one rich in basic amino acids, another rich in proline, and the third in acidic amino acids. A part of the N-terminal basic amino acid region (16-115 amino acids) is responsible for the specific binding to DRE. Antibodies against DREF specifically inhibit the transcription of the DNA polymerase alpha promoter in vitro. Overproduction of DREF protein overcomes the repression of the proliferating cell nuclear antigen gene promoter by the zerknüllt gene product. DREF is a trans-activating factor for DNA replication-related genes. DREF polypeptide is present in nuclei after the eighth nuclear division cycle, suggesting that nuclear accumulation of DREF is important for the coordinate zygotic expression of DNA replication-related genes carrying DRE sequences (Hirose, 1996).

The DRE/DREF system plays an important role in transcription of DNA replication genes, such as those encoding the 180 and 73 kDa subunits of DNA polymerase alpha as well as the gene that encodes PCNA. Two sequences were found homologous to DNA replication-related element (DRE; 5'-TATCGATA) in the 5'-flanking region (-370 to -357 with respect to the transcription initiation site) of the D-raf gene. Transcriptional activity was confirmed through gel mobility shift assays, transient CAT assays, and spatial patterns of lacZ expression in transgenic larval tissues carrying D-raf and lacZ fusion genes. The D-raf gene was found to be another target of the Zerknullt (Zen) protein with the observation of D-raf repression by Zen protein in cultured cells and its ectopic expression in the dorsal region of the homozygous zen mutant embryo. The evidence of DRE/DREF involvement in regulation of the D-raf gene strongly supports the idea that the DRE/DREF system is responsible for the coordinated regulation of cell proliferation-related genes in Drosophila (Ryu, 1997).

Signals from the BMP family member Decapentaplegic (Dpp) play a role in establishing a variety of positional cell identities in dorsal and lateral areas of the early Drosophila embryo, including the extra-embryonic amnioserosa as well as different ectodermal and mesodermal cell types. Although a reasonably clear picture is available of how Dpp signaling activity is modulated spatially and temporally during these processes, a better understanding of how these signals are executed requires the identification and characterization of a collection of downstream genes that uniquely respond to these signals. Three novel genes, Dorsocross1, Dorsocross2 and Dorsocross3, referred to collectively as Dorsocross, are described that are expressed downstream of Dpp in the presumptive and definitive amnioserosa, dorsal ectoderm and dorsal mesoderm. These genes are good candidates for being direct targets of the Dpp signaling cascade. Dorsocross expression in the dorsal ectoderm and mesoderm is metameric and requires a combination of Dpp and Wingless signals. In addition, a transverse stripe of expression in dorsoanterior areas of early embryos is independent of Dpp. The Dorsocross genes encode closely related proteins of the T-box domain family of transcription factors. All three genes are arranged in a gene cluster, are expressed in identical patterns in embryos, and appear to be genetically redundant. By generating mutants with a loss of all three Dorsocross genes, it has been demonstrated that Dorsocross gene activity is crucial for the completion of differentiation, cell proliferation arrest, and survival of amnioserosa cells. In addition, the Dorsocross genes are required for normal patterning of the dorsolateral ectoderm and, in particular, the repression of wingless and the ladybird homeobox genes within this area of the germ band. These findings extend knowledge of the regulatory pathways during amnioserosa development and the patterning of the dorsolateral embryonic germ band in response to Dpp signals (Reim, 2003).

Robust and stable induction of Doc expression in a dorsal stripe requires the activity of the homeodomain protein Zen as a co-activator of Dpp signals. The zen gene features an early, broad expression domain along the dorsal embryonic circumference, which is initially Dpp independent but subsequently requires Dpp for it to be maintained. Thereafter, its expression refines into a narrow dorsal domain in a process that requires peak levels of Dpp. The activation of Doc expression occurs at the same time as the refinement of zen expression and within the same narrow domain, which also coincides with high phospho-Mad levels. Although the maintenance and refinement of zen by Dpp is zen independent, it is proposed that Zen synergizes with peak signals of Dpp to trigger Doc gene expression in a dorsal stripe. The requirement for this proposed interaction between zen and dpp would explain the failure of zen to activate Doc genes in an early, broad domain as well as the observed low levels of residual Doc expression in zen mutant embryos, that may be due to inputs from Dpp alone. Formally, this proposed mechanism would be analogous to previously described inductive events in the early dorsal mesoderm, where the synergistic activities of the homeodomain protein Tinman and activated Smads induce the expression of downstream targets such as even-skipped. The identification of functional binding sites for Zen and Smads in Doc enhancer element(s) will be necessary for demonstrating that an analogous mechanism is active during induction of Doc gene expression in a dorsal stripe. In the absence of such data, it cannot be completely ruled out that dorsal Doc expression is controlled indirectly by Dpp, possibly via the combinatorial activities of zen and another high-level target gene of Dpp. Since mutations in several other genes that are expressed in the early amnioserosa, including pannier (pnr), hnt, srp, tup and ush, do not affect Doc expression until at least stage 12, these genes can be excluded as candidates for early upstream regulators of Doc (Reim, 2003).

Unlike zen, which is expressed only transiently, Doc expression is maintained throughout amnioserosa development. Hence, the Doc genes provide a functional link between the early patterning and specification events in dorsal areas of the blastoderm embryo and the subsequent events of amnioserosa differentiation. The activity of zen is required for all aspects of amnioserosa development that have been examined to date, including normal activation of C15. By contrast, the data demonstrate that the Doc genes execute only a subset of the functions of zen, which include the activation of Kr and hnt, but not that of C15 and early race, in amnioserosa cells. This interpretation is consistent with the failure to obtain a significant increase of amnioserosa cells upon ectopic expression of any of the Doc genes in the ectoderm or throughout the early embryo (using e22c and nanos-GAL4 drivers, respectively). The residual expression of hnt in some amnioserosa cells of Doc mutant embryos could be due to direct inputs from zen itself or from a yet undefined zen downstream gene acting in parallel with Doc. Nonetheless, the strong reduction of hnt expression in Doc mutant embryos could largely account for their amnioserosa-related phenotypes, including the absence of Kr expression, the decline of race expression, premature apoptosis and failure of germ band retraction. All of these phenotypes have also been observed in hnt mutant embryos. However, it is likely that Doc gene activity is required for the activation not only of hnt but also of additional genes of the u-shaped group and that Doc genes exert some of their functions in parallel with hnt. Some evidence for this notion is derived from the observation that loss of Doc activity has a stronger effect on Kr expression than loss of hnt activity (Reim, 2003).

One of the hallmarks of amnioserosa development is that the cells of this tissue never resume mitotic divisions after the blastoderm divisions. To a large extent, this cell cycle arrest is due to the absence of expression of cdc25/string in the prospective amnioserosa: this absence prevents the cells from entering M-phase and leads to G2 arrest. In addition, the expression of the Cdk inhibitor p21/Dacapo in the early amnioserosa is thought to contribute to the cell cycle arrest. Although a detailed description of the regulation of string and dacapo expression in dorsal embryonic areas is lacking, it has been reported that zen is required for repressing dorsal string expression -- this repression is expected to prevent further cell divisions. Notably, the observation that amnioserosa cells re-enter the cell cycle in Doc mutant embryos demonstrates that Doc genes are required for the cell cycle block in addition to zen. Whereas zen mutant embryos already feature ectopic cell divisions in dorsal areas from stage 8 onwards, in Doc mutants the amnioserosa cells resume mitosis only during and after stage 10, which is shortly after Zen protein disappears. Thus, it is hypothesized that the Doc genes take over the function of zen in repressing string and prevent cell divisions at later stages of amnioserosa development when Zen is no longer present. Overall, the phenotype of Doc mutant embryos suggests that amnioserosa differentiation, including cell cycle arrest and the development of squamous epithelial features, initiates in the absence of Doc activity but is not maintained beyond stage 11. Thereafter, cell division resumes and there is a reversal of the partially differentiated state. Apoptotic events are not observed prior to stage 11 in Doc mutants. However at later stages, many amnioserosa cells die prematurely and the remaining cells are difficult to distinguish morphologically from dorsal ectodermal cells (Reim, 2003).

Peak levels of BMP in the Drosophila embryo control target genes by a feed-forward mechanism involving Zen and Mad

Gradients of morphogens determine cell fates by specifying discrete thresholds of gene activities. In the Drosophila embryo, a BMP gradient subdivides the dorsal ectoderm into amnioserosa and dorsal epidermis, and also inhibits neuroectoderm formation. A number of genes are differentially expressed in response to the gradient, but how their borders of expression are established is not well understood. Evidence is presented that the BMP gradient, via the Smads, provides a two-fold input in regulating the amnioserosa-specific target genes such as Race. Peak levels of Smads in the presumptive amnioserosa set the expression domain of zen, and then Smads act in combination with Zen to directly activate Race. This situation resembles a feed-forward mechanism of transcriptional regulation. In addition, ectopically expressed Zen can activate targets like Race in the presence of low level Smads, indicating that the role of the highest activity of the BMP gradient is to activate zen (Xu, 2005).

Specific activation or repression of transcription by a combination of transcription factors is a common theme in the regulation of developmentally important genes. The results from the genetic analysis and the molecular dissection of the Race enhancer clearly show that Race is activated by the combined action of Smads and Zen. Although Smads can single handedly activate Race when overexpressed, under normal circumstances concurrent Zen activity is required. Why are both Smads and Zen necessary (Xu, 2005)?

Zen may act to restrict target gene expression specifically to the presumptive amnioserosa. Since the Dpp pathway is used repeatedly during development, other factors must function in combination with Dpp to ensure tissue specificity. Ectopic expression studies support this idea. In normal embryos, Race is activated only in regions where there are peak levels of PMad and Zen. In embryos where Zen is ubiquitously expressed, Race can now be activated in regions where there are lower levels of PMad, indicating that high level PMad is not the determining factor for amnioserosa tissue specificity. Rather PMad allows expression, and Zen determines the border of expression. The overexpression studies where Dpp can activate Race alone are interpreted to be situations where there are such high levels of Smads that Race and hindsight (hnt/pebbled) become activated promiscuously, and hence differential regulation is lost. In normal embryos, the combination of Smads and Zen ensures that the high level target genes are activated only in the presumptive amnioserosa (Xu, 2005).

In contrast, why the need for Smads? One role for Smads is suggested from the observation that Smads facilitate the binding of Zen to the Race enhancer. It is well established that Hox proteins often require co-factors for DNA binding to target enhancers. For example, composite sites that also bind the co-factor Extradenticle (Exd) ensure a greater selectivity for binding over the higher frequency Hox core site such as TAAT. In other examples, binding sites for signaling pathway effectors lie close to Hox/Selector-binding sites. The closely apposed Zen- and Smad-binding sites in the Race enhancer is one such scenario, since Zen can be thought of as a Selector gene. The current studies add to this idea of Smads and Selector cooperativity by demonstrating enhanced binding of Zen in the presence of Smads. Though the observed enhancement observed in in vitro assays is not dramatic, it is possible that in the embryo a moderate enhancement is functionally significant as is the twofold doubling of the dpp dose (Xu, 2005).

Another potential role of Smads was suggested from previous overexpression studies. Zen was able to activate Race only in the absence of Dpp if fused to a strong activation domain derived from VP16. This suggests that Smads provide a transactivation function different from that of Zen. The Smad MH2 domain has been shown to interact with the transcriptional co-activators CBP and p300. Zen has not yet been analyzed for interaction with transcriptional co-activators, however, the activation domain of Zen lies within the C-terminal 119 amino acids, and does not overlap with the homeodomain or the Mad interaction domain. Mechanistically, the difference in the activation potential between Zen and Smads could be due to their ability to recruit different co-activators to the transcriptional machinery (Xu, 2005).

Gradients of morphogens provide positional information to the cells by activating different genes at different threshold concentrations. In early Drosophila embryos, the transcriptional threshold responses to the Bicoid and Dorsal (Dl) morphogens have been extensively studied. The major mechanisms by which thresholds are established exploits the DNA-binding affinities of Bcd and Dl to their operator sites, as well as synergistic interactions with other transcription factors bound to the cis-regulatory sequences (Xu, 2005).

The BMP morphogen gradient also elicits different threshold responses from its targets, and a combinatorial mechanism is used to activate Race, a high level Dpp target. The genetic results indicate that Race, and also another high level target hnt, are activated only when a specific threshold of Zen and Smad activities are reached. In sog mutant embryos, Zen and Smad concentrations are relatively high, though below peak levels, and there is just enough of their combined activity to weakly activate Race. By contrast, in the double heterozygous embryos dpphr4/+; zenw36/+, Race is not activated because Zen and Smad concentrations are below the threshold levels required for activation (Xu, 2005).

A simple way to explain these results is if the Race enhancer has low affinity to Zen and Smad proteins in vivo. To transcribe Race effectively would then require relatively high concentrations of the proteins, which are indeed reached in the dorsalmost cells. It has been known for some time that the enhancers of the high level Dl targets contain binding sites with lower affinity for Dl compared with genes responding to lower levels of Dl. Recently, it has been shown that increasing the affinities of Smad-binding sites in the Race enhancer broadens the Race expression domain, which argues that the affinities of the Smad-binding sites in this high level Dpp target gene enhancer are low. The results suggest that cooperative binding between Smads and Zen, which is dependent on their physical interaction, should increase their binding to the Race enhancer. It is possible that interacting with Smads at the protein level either increases the binding affinity of Zen or effectively increases the local concentration of Zen when Smads bind the adjacent sites. This in turn leads to a robust transcriptional response of Race. The overexpression results are consistent with such a model. Ectopic Zen can only activate Race if some detectable level of PMad is present, and in addition Zen must contain the Smad interaction domain (Xu, 2005).

How are the lower level target genes activated? u-shaped (ush) and rhomboid are expressed in a broader domain, the border coinciding exactly with that of low level PMad staining. The Zen domains, however, do not; refined zen is not broad enough, while early Zen is too broad encompassing the entire dorsal domain, though Zen could possibly be graded in this region. Thus, it is possible that this class of target genes relies on a mechanism that uses numerous high-affinity Smad sites, and/or synergistic action of Smads with other co-factor(s) besides Zen. Such a mechanism resembles the activation of target genes in the neurogenic ectoderm of the embryo by Dorsal. It has been shown that the threshold responses from these genes depend on high-affinity Dl-binding sites, as well as synergistic interactions of Dl with bHLH transcription factors (i.e., dorsal/twist interactions) (Xu, 2005).

The pnr expression domain, which is about three times broader than ush, may represent a third threshold of Dpp activity. However, pnr is a different type of target gene compared with the prior classes, in that it is repressed by Brk, which is present in a reverse gradient to Dpp. In brk mutants, pnr expands into the ventral region, while Race and ush, for example, are unchanged. It is expected that the pnr gene enhancer contains Brk-binding sites, whereas the Race enhancer does not. Brk binding sites often overlap with GNCN sites, and it is possible that in the embryo, as in the wing disc, a concentration-dependent competition between Smads and Brk establishes the expression domains of the target genes regulated by both inputs. However, whether direct competition for binding can generate threshold responses remains to be seen. In summary, it appears that different classes of Dpp target genes are regulated by different combinations of transcription factors (Xu, 2005).

One of the simple regulatory motifs used in transcriptional networks is the feed-forward or self-enabling mechanism, whereby one regulator controls a second regulator and then both bind a common target gene. It has been shown both in prokaryotes and yeasts that this mode of regulation appears relatively frequently and is favored over others, e.g., autoregulation motifs, single input motifs in which one regulator controls several genes, or regulator chain motifs whereby one gene regulates a second which regulates a third, and so on. Such an over-representation of the feed-forward motif is probably due to its potential to provide enhanced sensitivity and temporal control to the transcriptional response. The feed-forward loop is especially suitable for eliciting precise threshold responses of morphogen targets because it allows a strong response of the target gene to small changes in the activity of the regulator that initiates the loop (Dpp), due to the combined action with the second regulator (Zen). In fact Bcd and Dl use mechanisms that are reminiscent of the feed-forward loop to activate their high level targets. Bcd regulates zygotic hunchback (hb) and together Bcd and Hb activate the downstream target even-skipped (eve) stripe 2, and Dl activates sna with the help of Twi. It is striking that the three morphogen gradients involved in specifying the Drosophila embryonic axes use the feed-forward strategy to regulate downstream target genes (Xu, 2005).

An unexpected implication from these results concerns the role of the high concentration end of the BMP morphogen gradient. In Drosophila embryos, the refined zen domain depends on peak levels of BMP activity, and Zen can activate high level targets as long as there is some level of PMad present to facilitate DNA binding. It can be then concluded that, for the high level targets, the role of Dpp is twofold: to set the domain of zen, which can be referred to as a primary target gene; and then to act in combination with Zen to activate the other, secondary, target genes such as Race and hnt. In addition, with respect to the BMP gradient in the Drosophila embryo, it is further proposed that the sole purpose of the peak of the gradient is to set up the zen domain (Xu, 2005).

scylla and charybde are transcriptionally regulated targets of Dpp/Zen-mediated signal transduction

Robotic methods and the whole-genome sequence of Drosophila melanogaster were used to facilitate a large-scale expression screen for spatially restricted transcripts in Drosophila embryos. In this screen, scylla (scyl) and charybde (chrb), which code for dorsal transcripts in early Drosophila embryos and are homologous to the human apoptotic gene RTP801, were identified. In Drosophila, both gene products are transcriptionally regulated targets of Dpp/Zen-mediated signal transduction and appear more generally to be downstream targets of homeobox regulation. Gene disruption studies revealed the functional redundancy of scyl and chrb, as well as their requirement for embryonic head involution. From the perspective of functional genomics, these studies demonstrate that global surveys of gene expression can complement traditional genetic screening methods for the identification of genes essential for development: beginning from their spatio-temporal expression profiles and extending to their downstream placement relative to dpp and zen, these studies reveal roles for the scyl and chrb gene products as links between patterning and cell death (Scuderi, 2006).

The foundation for the current study was a survey of RNA expression patterns by automated whole-mount RNA hybridization in situ. This screening protocol led to the identification of scyl as a dorsally restricted transcript in blastoderm stage embryos. Based on its spatial and temporal expression properties, which represent a subset of the dpp expression pattern, it was postulated that scyl is a transcriptionally regulated target of the Dpp signaling cascade that specifies early embryonic dorsal fates. To test this hypothesis, scyl transcript distributions were compared in wild-type and dorsoventral patterning mutant embryos. Fate determining genes functioning downstream of the ventral morphogen Dorsal (Dl) and/or the dorsal morphogen Dpp are expected to exhibit altered expression patterns in Dl-deficient/Dpp-constitutive and Dl-constitutive/Dpp-deficient mutant embryos. Indeed, this was found to be the case for the scyl transcript. Transcription of scyl in wild-type and mutant embryos was similar to that of zen, the best characterized target of Dpp, indicating that Dpp is both necessary and sufficient for scyl transcription. In blastoderm stage embryos, zen is expressed at the posterior pole and in the dorsal-most 40% of the developing embryo. In like fashion, scyl is expressed at the posterior pole and is dorsally restricted. scyl transcripts, however, are confined to the subset of zen-expressing cells that correspond to the dorsal-most 10% of the developing embryo. Both scyl and zen are ubiquitously expressed in dorsalized embryos derived from dl mutant females and in which the dorsal morphogen Dpp is ubiquitously expressed. Neither scyl nor zen is expressed in the abdominal regions of ventralized embryos, which are derived from cactus (cact) mutant females and which lack zygotic Dpp (Scuderi, 2006).

Two observations that led to an examination of the regulation of scyl and chrb by downstream components of the Dpp signaling cascade are: (1) scyl, like zen, is a transcriptionally regulated target of Dpp-mediated signaling and (2) scyl, chrb and zen are expressed in overlapping dorsal fields. The gene encoding the divergent homeobox transcription factor Zen is itself activated by Dpp-mediated signaling in dorsal domains of the blastoderm stage embryo. In zen mutant embryos, dorsally restricted scyl and chrb transcripts are lost, placing both genes downstream of the Zen transcriptional effector of Dpp-mediated signal transduction (Scuderi, 2006).

In addition to the dorsal field of scyl and chrb expression in early embryos, segmental expression along the anteroposterior axis suggests that scyl and chrb may also be sensitive to regulation by homeobox genes other than zen. Both scyl and chrb transcripts are localized in anteroposteriorly segmented patterns in ventral regions of the blastoderm and in the three thoracic segments of stage 13 embryos. In stage 13 embryos, genes of the bithorax complex (BX-C) repress expression of target genes in abdominal segments, restricting their expression to the thorax. The expression of scyl and chrb was examined in BX-C mutant embryos: expansion was observed of thoracic expression into abdominal segments of mutant embryos, thereby placing scyl and chrb downstream of the BX-C homeobox transcription factors, Ubx, Abd-A and/or Abd-B. Consistent with this observation is the finding that Ubx binds to regulatory regions of both scyl and chrb (Scuderi, 2006).

Finally, as an extension of the observation that scyl and chrb are downstream targets of homeobox genes acting in distinct signaling pathways, bioinformatic tools were used to identify conserved elements of the scyl and chrb promoters in D. melanogaster and D. pseudoobscura. In a computational cross-genome comparison utilizing algorithms based on both Gibbs sampling and Artificial Neural Networks, one 24-nucleotide motif and three 16-nucleotide motifs were identified that are conserved in the promoter regions of both genes in both species. Motif 1 was found much more frequently than expected for a random sequence, suggestive of its role as a generic transcription factor binding site or regulatory element. Motifs 2-4 were observed much less frequently, and this statistic is interpreted to be indicative of more specific roles for motifs 2-4 in the co-regulation of scyl and chrb. With respect to the identification of defined binding sites, neither motif 1 nor motif 2 corresponds to any canonical transcription factor binding sites listed in the TRANSFAC transcription factor database. Motif 3, however, is GC-rich and contains canonical binding sites for two widely used transcriptional activators: SP1 (GCCCGCCCCCC) and AP2 (GCCCGCGGC). More notable, however, is the characterization of motif 4, which was found only twice in each of the Drosophila genomes (in the promoter regions of scyl and chrb). In scans of the TRANSFAC database, it was found that motif 4 harbors a 10-bp canonical binding site for Zen (ATTTAAATGA) (Scuderi, 2006).

zerknüllt: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.