Interactive Fly, Drosophila

The coexpression of Pannier and Tin in the embryo results in a synergistic activation of cardiac gene expression and the ectopic induction of heart-like cells. To investigate a possible physical interaction of the two proteins, mammalian CV-1 cells were cotransfected with Pnr and Tin expression vectors and the cellular distribution of the factors was monitored by confocal microscopy and coimmunoprecipitation analysis. Cells expressing GFP-tagged Tin and Myc-tagged Pnr exhibit colocalization of the proteins in the nucleus. Furthermore, analysis of nuclear extracts by immunoprecipitation followed by Western blot reveals that Tin is coimmunoprecipitated with Pnr. Conversely, Pnr is also detected in the Tin immunoprecipitate. These results indicate that the two proteins form a physical complex in the cultured cells, consistent with their ability to work combinatorially in the regulation of cardiac gene expression (Gajewski, 2001).

In support of this in vivo data, regions of Tin that are able to bind to the Pnr protein were delineated. Eight GST-Tin fusion proteins containing all or part of the cardiogenic factor were used in in vitro pull-down assays with 35S-labeled full-length Pnr. The strongest interactions were observed with either wild-type Tin or truncated versions that contained the homeodomain region (Tin A1, A2, A23, and A31). Additionally, weaker but discernible binding of Tin and Pnr was observed with truncated proteins that contained the functionally defined 111 to 151 region (Tin A4, A5, and A34). These molecular results are consistent with the requirement of this internal domain for the functional synergism of Tin with Pnr in the activation of the D-mef2 cardiac enhancer in the CNS (Gajewski, 2001).

tinman encodes an NK-2 class homeodomain transcription factor that is required for development of the Drosophila dorsal mesoderm, including heart. Genetic evidence suggests its important role in mesoderm subdivision, yet the properties of Tinman as a transcriptional regulator and the mechanism of gene transcription by Tinman are not completely understood. Tinman can activate or repress target genes in cultured cells, based on evaluation of functional domains that are conserved between the tinman genes of Drosophila melanogaster and Drosophila virilis. Using GAL4-tinman fusion constructs, a transcriptional activation domain (amino acids 1-110) and repression domains (amino acids 111-188 and the homeodomain) have been mapped and an inhibitory function for the homeodomain has been found upon transactivation by Tinman (Choi, 1999).

Tinman is regulated by Twist and autoregulates its own promoter. The properties of Tin as a transcription factor were assessed using tinman P1 and P1E2m promoters and truncated forms of the Tin expression vectors (d8 and d6). The P1 reporter contains Tin-responsive elements (the E2 cluster) and is activated by Tin. The P1E2m reporter contains mutated Tin binding sites but otherwise is exactly the same as the wild-type P1 reporter, which also contains several weak Tin binding sites. Tin can activate the P1 reporter (6-fold activation). In contrast, Tin down-regulates the P1E2m reporter gene (3-fold repression). In this case, Tin binds to weak binding sites and represses the P1E2m reporter gene. These results indicate that Tin can act either as a transcriptional activator or repressor, depending on the context of the reporters (P1 or P1E2m). These phenomena are dependent on the functional domains of Tin. For example, deletion of the amino-terminal region of Tin abrogates activation of the P1 reporter, indicating that the amino terminus (aa 1-110) of Tin is required for transcriptional activation. Indeed, this Tin mutant (d8) represses gene expression of both the P1 and the P1E2m reporter. Further deletion of Tin (construct d6) relieves this repression, irrespective of the reporter gene used. These results suggest that the region following the amino terminus of Tin (aa 111-188) is required for the repressor activity of Tin. Taken together, these results indicate that, depending on the context of the target genes (for example P1 or P1E2m), Tin can act as either a transcriptional activator or repressor and that these different transcriptional activities are dependent on functional domains of Tin (Choi, 1999).

Tinman-dependent transactivation is augmented by the p300 coactivator; Tinman physically interacts with p300 via the activation domain. In addition, cotransfection experiments indicate that the repressor activity of Tinman is strongly enhanced by the Groucho corepressor. Using immunoprecipitation and in vitro pull-down assays, Tinman is shown to directly interact with the Groucho corepressor, for which the homeodomain is required. Together, these results indicate that Tinman can act as either a transcriptional activator or repressor. The first evidence of Tinman interactions with the p300 coactivator and the Groucho corepressor is provided (Choi, 1999).

Barbu (Bbu), an alternative name applied to Twin of m4 (Tom), can antagonize Notch signaling activity during Drosophila development. In this study Barbu/Tom was isolated in a search for proteins that physically interact with Tinman. It is uncertain whether this physical interaction is of biological significance, but it is clear that this gene functions to antagonize Notch signaling, as does E(spl) region transcript m4. Although there is no formal proof that the Bbu and Tinman interaction is relevant in vivo, several lines of evidence support its specificity, including the fact that three independent clones of Bbu cDNAs were isolated in the yeast two-hybrid screen and the observation that GST-Bbu fusion protein specifically associates with radiolabeled Tinman protein in in vitro binding assays. The observed co-expression of Bbu and Tinman in the early mesoderm would also favor the possibility of interactions of the two proteins in vivo. If this interaction were to occur, it would imply a nuclear function of Bbu, presumably as a cofactor of Tin in the mesoderm and additional transcription factors in other tissues. In this hypothesis, Bbu would interfere with transcriptional outputs of the Notch signaling cascade that require tissue-specific transcription factors such as Tinman. For example, it is possible that activated Notch/Su(H) complexes need to bind to enhancer sequences of certain target genes together with Tinman to activate their expression in the mesoderm, and that the binding of Bbu to Tin interferes with this cooperative activity. Indeed, tin-dependent specification events in heart and somatic muscle development also involve Notch signaling. While the predominantly cytoplasmic localization of Bbu protein seems to argue against an interaction with Tin in vivo, it is still possible that the nuclei contain low levels of Bbu protein that cannot be detected above background levels with antibody stainings (Zaffran, 2000).

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