Transcriptional Regulation

DPP strictly requires the Punt and Thick veins receptors to transduce the signal across the membrane. Genetic epistasis experiments indicate that shn functions downstream of the DPP signal and its receptors. The SHN protein acts as a nuclear target in the DPP signaling pathway directly regulating the expression of DPP-responsive genes (Arora, 1995 and Grieder, 1995).

Targets of Activity

The expression of DPP target genes is reduced in shn mutants (Frasch, 1995 and Staehling-Hampton, 1994). Genes whose expression depends on dpp activity include bagpipe, Ultrabithorax, wingless and dpp itself (Grieder, 1995 and Arora, 1994). Schnurri is also an essential component in the response of endodermal cells to the DPP signal. Labial expression in endodermal cells is absent in schnurri mutants (Greider, 1995). schnurri is required for cell determination and differentiation in dorsal ectodermal cells. The pannier gene, required for dorsal closure, is expressed in the dorsal ectoderm. Although normally expressed during early stages of gastrulation, Pannier is absent in dorsal cells after the beginning of germ-band retraction in schnurri mutants (Greider, 1995).

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).

Shn contains a total of seven Cys2-His2 zinc finger motifs organized as two widely separated pairs and a triad of fingers at the carboxy-terminal. The first two sets of fingers share 67% and 78% identity, respectively, with the corresponding paired finger domains in the human MBP-1 and MBP-2 proteins. The carboxy-terminal triad of fingers is not represented in vertebrate homologs of Shn and has limited identity with known zinc finger proteins. In the human protein both sets of paired fingers can bind DNA independently and recognize related but distinct sequence motifs (Fan, 1990; van't Veer, 1992). Therefore the binding specificity of the two paired DNA-binding domains (Shn DBD1 and Shn DBD2) were characterized individually, rather than as part of a single protein. A PCR-based binding site selection assay was used to determine the optimal binding sites recognized by each set of Shn zinc finger domains. Sequence data from a set of 16 clones bound by Shn DBD1 reveals that each clone contains a related 12-bp sequence 59-GGGA(A/T)CGTTCCC-39 or its complement. The consensus sequence is essentially palindromic in nature and consists of a central 6-nucleotide core flanked by GGG at the 59 location and CCC at the 39 end. Analysis of the sequences bound by Shn DBD2 has identified an optimal binding site 59-GGGGA(A/C)(A/T)TCCC-39 closely related to that recognized by Shn DBD1. One difference between the two consensus sequences is the spacing between the flanking GGG(Nn)CCC bases, with n=6 for DBD1 and n=5 for DBD2. The optimal Shn-binding sites are similar to those recognized by its vertebrate orthologs. The bacterially produced proteins were tested for their ability to recognize the sequence bound by human MBP-1 (Baldwin, 1990; Fan, 1990; Rustgi, 1990). Affinity-purified Shn DBD1 and DBD2 specifically bind oligos containing the MBP-1 site (GGGGATTCCCC) in gel-shift assays. This binding is resistant to the addition of 100-fold excess of nonspecific competitor DNA, but only a 10-fold excess of specific competitor prevents the formation of a DNA protein complex (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).

The homeobox gene tinman plays a key role in the specification of Drosophila heart progenitors and the visceral mesoderm of the midgut, both of which arise at defined positions within dorsal areas of the mesoderm. In addition to the heart and midgut visceral mesoderm, tinman is also required for the specification of all dorsal body wall muscles. Thus it appears that the precursors of the heart, visceral musculature, and dorsal somatic muscles are all specified within the same broad domain of dorsal mesodermal tinman expression. Because of the crucial role of dpp in inducing dorsal mesodermal tinman expression and the specification of dorsal mesodermal tissues, it is of interest to determine whether other components known to function in dpp-mediated signaling events during blastoderm are also required for mesoderm induction. Screw, a second BMP2/4-related gene product, Tolloid, a BMP1-related protein, and the zinc finger-containing protein Schnurri, are all shown to be required to allow full levels of tinman induction during this process. screw, which encodes a secod BMP2/4-related molecule, has been proposed to act synergistically with dpp to specify dorsal ectoderm and amnioserosa. Similarly, it has been shown that tolloid, which encodes a BMP1-related metalloproteinase, acts to enhance the activity of the dpp gene product during mesoderm induction. Both scw and tolloid are shown to be required for normal induction of tinman expression in the dorsal mesoderm, and in the absence of either gene activity, tinman expression in the dorsal mesoderm is reduced and segmentally interrupted. Thus scw and tld are necessary for achieving full levels of tinman induction, whereas dpp is obligatory for this event. In addition, unlike dpp mutants, mutants for scw or tld form some residual visceral mesoderm. However, heart formation is more sensitive to the activities of scw and tld and is disrupted to a similar extent as in dpp mutants. schnurri is also necessary for tinman induction in the dorsal mesoderm. The dorsal tinman domain is clearly reduced, as compared to wild-type embryos, although the levels of Tinman mRNA are close to normal. Therefore, shn may be required to enhance dpp signaling during tin induction, but significant levels of tin activation can still occur in the absence of its activity (Yin, 1998).

Targets of Activity

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

Protein Interactions

Schnurri and Mad can interact directly through discrete domains. Smads characteristically contain an amino-terminal Mad homology region 1 (MH1) and a carboxy-terminal Mad homology region 2 (MH2) separated by a poorly conserved linker region. Mad and Shn interactions were assayed using the yeast two-hybrid assay. Prey plasmids were generated that express full-length Mad (FL), Mad MH1, or the MH2 domain along with the linker (MH21L). In the two-hybrid assay, interaction of the bait and prey allows yeast expressing both proteins to grow on selective media and activate transcription of a beta-galactosidase reporter. Based on both these criteria, it was observed that Shn associates with Mad FL and Mad MH21L, but not with Mad MH1. The failure of Mad MH1 to interact with Shn cannot be ascribed to lower levels of expression of this domain, since antisera directed against an HA epitope present in each prey fusion detect comparable levels of all three Mad polypeptides on Western blots (Dai, 2000).

In order to delineate the domains in Shn that interact with Mad, GST pull-down assays were used. Mad MH21L domain was expressed as a glutathione S-transferase (GST) fusion protein. Sixteen overlapping subclones encompassing the entire Shn coding region were used to generate 35Smethionine-labeled protein fragments using coupled in vitro transcription and translation. The GST-Mad affinity matrix was tested for its ability to retain the 35S-labeled Shn peptides. Mad MH21L interacts most strongly with two overlapping regions, Shn 1069-1776 and Shn 1463-2318. Further deletions have allowed the interaction to be narrowed to Shn 1441-1635, a region of 194 residues preceding the second set of paired zinc fingers. Two additional nonoverlapping fragments, Shn 341-1069 and Shn 1776-2529, show a moderate interaction with Mad. Subdivision of these fragments significantly reduce their association with Mad. Thus, a total of one strong and two weaker Mad interaction domains (MIDs) has been detected in Shn. Shn 1441-1776 shows strong binding to itself with an affinity comparable to that displayed for Mad MH21L. In addition significant binding was detected to Shn 341-1069 and Shn 1776-2529, fragments that contain the other MIDs. The binding of Shn 1441-1776 is specific since it fails to bind either Shn 1-968 (a fragment that lacks a MID). Thus these results suggest that regions of the protein that are involved in association with Mad may also mediate homomeric Shn interactions (Dai, 2000).

Signals of Dpp are transmitted from the cell membrane to the nucleus by Medea and Mad, both belonging to the Smad protein family. Mad has been shown to bind to the Dpp-responsive element in genes such as vestigial, labial, and Ultrabithorax. The DNA binding affinity of Smad proteins is relatively low, and requires other nuclear factor(s) to form stable DNA binding complexes. schnurri (shn) was identified as a candidate gene acting downstream of Dpp receptors, but its relevance to Mad has remained unknown. The biochemical functions of Shn have been characterized in this study. Shn forms homo-oligomers. Shn is localized in the nucleus, and is likely to have multiple nuclear localizing signals. Shn interacts with Mad in a Dpp-dependent manner. The present results argue that Shn may act as a nuclear component of the Dpp signaling pathway through direct interaction with Mad (Udagawa, 2000).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The Drosophila Smad cofactor Schnurri engages in redundant and synergistic interactions with multiple corepressors

In Drosophila a large zinc finger protein, Schnurri, functions as a Smad cofactor required for repression of brinker and other negative targets in response to signaling by the transforming growth factor beta ligand, Decapentaplegic. Schnurri binds to the silencer-bound Smads through a cluster of zinc fingers located near its carboxy-terminus and silences via a separate repression domain adjacent to this zinc-finger cluster. This study shows that this repression domain functions through interaction with two corepressors, CtBP and Sin3A, and that either interaction is sufficient for repression. Schnurri contains additional repression domains that function through interaction with CtBP, Groucho, Sin3A and SMRTER. By testing for the ability to rescue a shn RNAi phenotype evidence is provided that these diverse repression domains are both cooperative and partially redundant. In addition Shn harbors a region capable of transcriptional activation, consistent with evidence that Schnurri can function as an activator as well as a repressor (Cai, 2009).

CTCF-dependent co-localization of canonical Smad signaling factors at architectural protein binding sites in D. melanogaster

The transforming growth factor beta (TGF-beta) and bone morphogenic protein (BMP) pathways transduce extracellular signals into tissue-specific transcriptional responses. During this process, signaling effector Smad proteins translocate into the nucleus to direct changes in transcription, but how and where they localize to DNA remain important questions. This study has mapped Drosophila TGF-beta signaling factors Mad, dSmad2, Medea and Schnurri genome-wide in Kc cells and find that numerous sites for these factors overlap with the architectural protein CTCF Depletion of CTCF by RNAi results in the disappearance of a subset of Smad sites, suggesting Smad proteins localize to CTCF binding sites in a CTCF-dependent manner. Sensitive Smad binding sites are enriched at low occupancy CTCF peaks within topological domains, rather than at the physical domain boundaries where CTCF may function as an insulator. In response to Decapentaplegic, CTCF binding is not significantly altered, whereas Mad, Medea, and Schnurri are redirected from CTCF to non-CTCF binding sites. These results suggest that CTCF participates in the recruitment of Smad proteins to a subset of genomic sites and in the redistribution of these proteins in response to BMP signaling (Van Bortle, 2015).

TGF-β effector proteins have been shown to co-localize with mammalian CTCF in a CTCF-dependent manner at just 2 individual loci. This observation has been extended to Drosophila using a genome-wide approach, providing evidence that architectural protein CTCF and canonical Smad signaling proteins, both highly conserved from fly to humans, co-localize on a global scale. Context-specific features were uncovered in which Smad localization is dependent or independent of CTCF binding. Interestingly, genome-wide analysis identifies Mad, dSmad2, Medea, and Schnurri binding to previously characterized response elements even in the absence of DPP ligand, in which levels of phosphorylated Mad are undetectable. This signal-independent clustering of signaling proteins suggests that the genomic TGF-β signaling response is not as simple as regulating binary 'off vs. on' states, dependent on phosphorylated Mad. However, attempts to map the genomic landscape of phosphorylated-Mad before and after DPP stimulation were unsuccessful, likely due to issues with currently available p-Mad antibodies. Though it was not possible to determine the role of phosphorylation as a determinant in Mad localization, it is conceivable that phosphorylation of Mad might play a role in regulating the resident time of DNA-binding, the recruitment of additional regulatory partners, or the ability to establish functional long-range interactions (Van Bortle, 2015).

Smad co-binding at dCTCF sites is sensitive to dCTCF depletion at low occupancy dCTCF target sequences for which Smad consensus sequences are depleted, whereas high occupancy dCTCF binding sites co-bound by additional architectural proteins remain unaffected. The dCTCF-independent recruitment of Smads to high occupancy APBSs suggests that additional architectural proteins may redundantly recruit Smads, or simply provide an accessible chromatin landscape to which Mad, Medea, and dSmad2 can associate. Nevertheless, dCTCF-dependent localization of Smad proteins to specific low occupancy elements is consistent with the CTCF-dependent nature of Smad binding at both the APP and H19 promoters in humans. It is speculated that dCTCF-dependent Smad localization to low occupancy APBSs within topological domains may represent regulatory elements involved in enhancer-promoter interactions, whereas dCTCF-independent high occupancy APBSs are involved in establishing higher-order chromosome organization. What role Smads might play in establishing or maintaining such long-range interactions relevant to chromosome architecture, or whether Smads and other transcription factors simply localize to high occupancy APBSs due to chromatin accessibility, remains difficult to address. However, it has been recently shown that high occupancy APBSs are distinct from analogous transcription factor hotspots, suggesting some level of specificity, most likely governed by protein-protein interactions, decides which factors can associate and where. Alternatively, the enrichment of ChIP-seq signal at high occupancy APBSs may, to some degree, reflect indirect association via long-range interactions with regulatory elements directly bound by Smad proteins. This possibility raises a potential explanation for why Smad ChIP signal is independent of dCTCF binding at high occupancy APBSs (Van Bortle, 2015).

Surprisingly, DPP-activated phosphorylation of Mad does not lead to significant changes in dCTCF binding, whereas Mad, Medea, and Schnurri levels increase at regulatory elements away from dCTCF. These results suggest that TGF-β signaling in Kc167 cells redirects Smad binding to genomic loci independent of architectural proteins, and that architectural proteins may facilitate binding of nuclear Smad proteins in the absence of signaling. The complete loss of Smad ChIP signal at numerous dCTCF binding sites enriched for the core dCTCF consensus sequence nevertheless provides compelling evidence that recruitment of Smad proteins is directly governed by Drosophila CTCF at a subset of binding sites. These results establish CTCF as an important determinant of Smad localization and, depending on the cell-type specific binding patterns of CTCF, suggest that CTCF might also influence the tissue-specific localization of Smad proteins analogous to master regulatory transcription factors in multi-potent stem cells (Van Bortle, 2015).

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

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