Smooth muscle plays a prominent role in many fundamental processes and diseases, yet understanding of the transcriptional network regulating its development is very limited. The FoxF transcription factors are essential for visceral smooth muscle development in diverse species, although their direct regulatory role remains elusive. A transcriptional map of Biniou (a FoxF transcription factor) and Bagpipe (an Nkx factor) activity is presented as a first step to deciphering the developmental program regulating Drosophila visceral muscle development. A time course of chromatin immunoprecipitatation followed by microarray analysis (ChIP-on-chip) experiments and expression profiling of mutant embryos reveal a dynamic map of in vivo bound enhancers and direct target genes. While Biniou is broadly expressed, it regulates enhancers driving temporally and spatially restricted expression. In vivo reporter assays indicate that the timing of Biniou binding is a key trigger for the time span of enhancer activity. Although bagpipe and biniou mutants phenocopy each other, their regulatory potential is quite different. This network architecture was not apparent from genetic studies, and highlights Biniou as a universal regulator in all visceral muscle, regardless of its developmental origin or subsequent function. The regulatory connection of a number of Biniou target genes is conserved in mice, suggesting an ancient wiring of this developmental program (Jakobsen, 2007; full text of article).
The dynamic enhancer binding of Biniou suggested that the timing of Biniou occupancy is important for the timing of enhancer activity. To assess this in vivo, a number of regions from each of the three temporal clusters were linked to a GFP reporter. The timing of enhancer activity was assayed in vivo by in situ hybridization in transgenic embryos, to avoid time delays due to GFP protein folding and protein perdurance. All regions examined drive expression in a subset of Biniou-expressing cells and recapitulate all or part of the target genes' expression. This study focused on their temporal activity (Jakobsen, 2007).
The initiation of enhancer activity closely matches the first time point of Biniou binding for >90% of enhancers examined (10 of 11 CRMs). The early-bound enhancers (ttk, fd64a-e, lame duck (lmd), bap3) drive expression at stages 10-11, reflecting the binding of Biniou at these stages of development. Similarly, all four continuous-bound enhancers (HLH54F, otk, mib2, bap-FH) initiate expression at the first time period when Biniou binds. The two late-bound enhancers, in contrast, do not initiate expression at stages 10 or 11 of development, matching the lack of Biniou binding during these stages. Instead, the expression of the fd64a late enhancer initiates at stage 13, while the ken enhancer initiates VM expression at stage 14. This shift in the initiation of activity mirrors Biniou binding to these enhancers at stages 12-13 and 13-14, respectively. The only exception is the CG2330 enhancer, which initiates expression at stage 11, while Biniou enhancer binding was first detected at stage 13-14). As the expression of endogenous CG2330 does not initiate until stage 13, the apparent discrepancy in enhancer activity may simply reflect the exclusion of some regulatory motifs within the limits of the cloned region (Jakobsen, 2007).
Remarkably, the duration of enhancer activity is also tightly correlated with the time span of Biniou binding in 10 out of 11 CRMs examined. This is particularly striking in the early-bound enhancers: When Biniou ceases to bind to these CRMs (lmd, ttk, fd64a early, and bap3), their ability to regulate expression is lost. The converse is also true. Continuous Biniou binding correlates with continuous enhancer activity, specifically for bap-FH, HLH54F, and otk. The exception is the mib2 enhancer. In the context of this module Biniou binding it is not sufficient to maintain enhancer activity in the VM at late developmental time points (Jakobsen, 2007).
Taken together, these data indicate that the timing of Biniou enhancer binding is predictive for temporal enhancer activity in the large majority of cases (Jakobsen, 2007).
The subcellular localization of D-mib. Anti-D-mib antibodies were generated that specifically detected D-mib on Western blots and on fixed tissues. Using these antibodies, D-mib was detected in all imaginal disc cells. D-mib subcellular distribution was examined in epithelial cells located along the edge of the wing discs because cross-sectional imaging affords better resolution along the apical-basal axis. D-mib co-localizes with Ser, Dl, and N at the apical cortex. Dl and Ser are also detected in large intracellular vesicles that probably correspond to multivesicular bodies in that they also stained for hepatocyte growth factor-regulated tyrosine kinase substrate. The intracellular dots seen with the anti-D-mib antibodies are distinct from the Dl- and Ser-positive dots and appear to result from background staining. The reduced cytoplasmic staining seen in D-mib mutant cells suggests that D-mib is also present in the cytoplasm. A similar localization at the apical cortex and in the cytoplasm is seen for a functional yellow fluorescent protein (YFP)::D-mib fusion protein. These localization data suggest that D-mib may act at the apical cortex to regulate the activity of Dl and/or Ser (Le Borgne, 2005).
To test whether this specific increase in the level of Ser at the apical cortex results from reduced Ser endocytosis in D-mib mutant cells, the endocytosis of Ser was followed in living imaginal discs using an antibody uptake assay. Briefly, dissected wing discs were cultured for 15 min in the presence of antibodies that recognize the extracellular part of Ser or Dl, then washed, cultured for another 45 min in medium without antibodies, and then fixed. The uptake of anti-Ser and anti-Dl antibodies was then assessed using secondary antibodies. Using this assay, it was found that anti-Ser-and anti-Dl antibodies are internalized in wild-type epithelial cells. The complete loss of D-mib activity in D-mib1 wing discs does not significantly change the internalization of anti-Dl antibodies, indicating that D-mib is not required for Dl endocytosis in this tissue. However, the loss of D-mib activity strongly inhibits the endocytosis of anti-Ser. Moreover, high levels of anti-Ser antibodies were seen at the apical surface, confirming that D-mib mutant cells accumulate high levels of Ser at their surface. It is therefore concluded that D-mib is specifically required for the endocytosis of Ser in wing discs (Le Borgne, 2005).
Ubiquitin-mediated endocytosis is thought to depend on monoubiquitination. Thus, by analogy with the function of Mib in D. rerio (Chen, 2004), it is suggested that D-mib may directly monoubiquitinate Ser. Consistent with this hypothesis, it was shown that D-mib binds Ser. Moreover, a mutation in the C-terminal catalytic RING domain of D-mib abolished its ability to internalize Ser in transfected S2 cells, implying that the E3 ubiquitin ligase activity of D-mib is required for Ser internalization. Biochemical analysis of the ubiquitination events regulated by D-mib will be needed to further define the mechanism by which D-mib regulates the endocytosis of Ser in vivo (Le Borgne, 2005).
The regulation of Ser endocytosis by D-mib suggests that D-mib may regulate Ser signaling. Ser expression is restricted to dorsal cells in second instar wing imaginal discs. Ser in dorsal cells signals across the D-V boundary to activate N in ventral cells. If D-mib is required for Ser signaling during wing development, then loss of D-mib activity in dorsal cells should affect the specification of the wing margin in a non-autonomous manner. Loss of D-mib activity in large dorsal clones of D-mib2 mutant cells results in a loss of Cut expression at the D-V interface. The lack of Cut expression in wild-type ventral cells abutting the D-V boundary indicates that D-mib is required for Ser signaling by dorsal cells and acts in a non-autonomous manner to activate N in ventral cells. Conversely, loss of D-mib activity in large ventral clones does not disrupt margin specification, indicating that D-mib is not strictly required for Dl signaling by ventral cells. However, a narrowing of the Cut-positive margin is observed, suggesting that D-mib contributes to regulating the level of Dl signaling. Of note, ventral D-mib mutant cells express Cut, implying that D-mib is not required for N signal transduction (Le Borgne, 2005).
Next, whether expression of D-mib in dorsal cells is sufficient to rescue the D-mib wing phenotype was tested. D-mib was expressed in dorsal cells of D-mib2/D-mib3 mutant discs using Ser-GAL4. Similarly to the expression of the Ser gene, Ser-GAL4 expression is restricted to dorsal cells in second/early third instar larvae and is weakly expressed in ventral cells in mid/late third instar larvae, i.e., after margin cell specification. Expression of D-mib in dorsal cells is sufficient to rescue growth of the wing pouch and of the expression of Cut in margin cells in D-mib mutant discs. This result confirms that D-mib regulates Ser signaling by dorsal cells (Le Borgne, 2005).
A similar rescue was observed with a YFP::D-mib protein, indicating that YFP::D-mib is functional. YFP::D-mib localizes at the apical cortex and in the cytoplasm, as seen for endogenous D-mib. YFP::D-mib co-localizes with Dl and Ser at the apical cortex of cells expressing low levels of YFP::D-mib. However, cells expressing high levels of YFP::D-mib showed a strong reduction in the level of both Dl and Ser at the cortex, further indicating that D-mib down-regulates the levels of both Ser and Dl at the apical cortex (Le Borgne, 2005).
Loss- and gain-of-function analyses indicate that the major function of D-mib is to regulate Notch signal transduction. Since Delta is a bona fide substrate of zebrafish Mib, tests were performed for a physical association of D-mib and Delta by co-immunoprecipitation. Cultured cells were co-transfected with Delta and various D-mib expression vectors, and co-immunoprecipitation was performed in both directions. Although Delta did not successfully co-immunoprecipitate full-length D-mib, it did associate with all isoforms that contain the D-mib N terminus and lack the C-terminal RING finger (namely D-mib-N, D-mibDelta3RF and D-mibDeltaRF. Conversely, these same D-mib isoforms efficiently co-immunoprecipitate Delta; full-length D-mib also shows modest association with Delta in this direction. It was consistently observed that the presence of full-length D-mib reduces Delta levels, which might account for why this interaction is poorly detected. Notably, D-mib-N shows the strongest interaction with Delta. In fact, immunoprecipitated D-mib-N brings down both full-length Delta and cleaved DeltaIC, consistent with a direct interaction between the N terminus of D-mib and the intracellular domain of Delta. A truncated D-mib protein lacking the N-terminal domain (D-mib-C) shows no binding to Delta, demonstrating that this region is crucial for association with Delta (Lai, 2005).
Physical association between D-mib proteins and Serrate was tested. D-mib:Serrate interactions appear to be somewhat weaker than D-mib:Delta interactions; however, the overall profile of the different D-mib truncations in association with Serrate and Delta is identical. These findings lead to the conclusion that the N terminus of D-mib mediates physical association with both Drosophila DSL ligands. In addition, full-length D-mib similarly reduces the accumulation of Serrate, indicating that D-mib downregulates both DSL ligands (Lai, 2005).
In vitro data correlate well with in vivo studies, in that all RING-finger-deleted D-mib isoforms that retain the ability to associate with DSL ligands (D-mib-N, D-mibDeltaRF and D-mib3DeltaRF) have at least some ability to inhibit Notch signaling. However, full specificity and activity of D-mib requires inclusion of the ankyrin repeats and the two non-canonical RING fingers. Curiously, there is no significant similarity at the primary amino acid level between the intracellular domains of Delta and Serrate. In this regard, it is relevant to note that Xenopus Neur (X-Neur) robustly regulates Drosophila Delta in vivo, even though there is no significant similarity between the intracellular domains of Delta and X-Delta. D-mib and Neur may therefore recognize a more hidden, possibly structural, feature that is shared by DSL ligands (Lai, 2005).
Notch is the receptor in a signalling pathway that operates in a diverse spectrum of developmental processes. Its ligands (e.g. Serrate) are transmembrane proteins whose signalling competence is regulated by the endocytosis-promoting E3 ubiquitin ligases, Mindbomb1 and Neuralized. The ligands also inhibit Notch present in the same cell (cis-inhibition). This study identifies two conserved motifs in the intracellular domain of Serrate that are required for efficient endocytosis. The first, a dileucine motif, is dispensable for trans-activation and cis-inhibition despite the endocytic defect, demonstrating that signalling can be separated from bulk endocytosis. The second, a novel motif, is necessary for interactions with Mindbomb1/Neuralized and is strictly required for Serrate to trans-activate and internalise efficiently but not for it to inhibit Notch signalling. Cis-inhibition is compromised when an ER retention signal is added to Serrate, or when the levels of Neuralized are increased, and together these data indicate that cis-inhibitory interactions occur at the cell surface. The balance of ubiquitinated/unubiquitinated ligand will thus affect the signalling capacity of the cell at several levels (Glittenberg, 2006; full text of article).
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