A Drosophila model to study the functions of TWIST orthologs in apoptosis and proliferation.

The twist gene has been characterized for its role in myogenesis in several species. In addition, in mammalian cultured cells, it has been shown that twist is a potential oncogene antagonizing p53-dependent apoptosis. To study, in vivo, the role of twist in apoptosis and proliferation, we constructed transgenic Drosophila lines allowing ectopic expression of different twist orthologs. We report that: (i) Drosophila twist induces apoptosis and activates the reaper promoter, (ii) nematode twist induces arrest of proliferation without apoptosis, and (iii) human twist retains its potentialities observed in mammalian cultured cells and antagonizes Drosophila p53-dependent apoptosis. In addition, we show that human twist is able to induce cell proliferation in Drosophila. Data suggest that the pathway by which human twist antagonizes Drosophila p53 could be conserved. These transgenic lines thus constitute a powerful tool to identify targets and modifiers of human twist.

Truncated products of the vestigial proliferation gene induce apoptosis.

The vestigial (vg) gene in D. melanogaster, whose mutant phenotype is characterized by wing atrophy, encodes a novel nuclear protein involved in cell proliferation. The original vg mutant (vgBG) displays massive apoptosis in the wing imaginal disc. Here we tested the hypothesis that the vg mutant phenotype could be due: (i) to lack of cell proliferation in null mutants due to the absence of the Vg product and, (ii) to apoptosis in vgBG and other mutants due to the presence of a major Vg truncated product. In agreement with our hypothesis no cell death was observed in null vg mutants, and the anticell death baculovirus P35 product is unable to rescue the mutant phenotype caused by absence of the Vg product. In addition, expression of the antiproliferative gene dacapo, the homolog of p21, induces a mutant wing phenotype without inducing cell death. In contrast the wing phenotype of the original vg mutant could be reproduced by the ectopic expression of the reaper cell death gene when expressed by vg regulatory sequences. In agreement with the hypothesis, the classic vg mutant spontaneously displays an increase in reaper expression in the wing disc and its phenotype can be partially rescued by the P35 product. Finally, we showed that ectopic expression of a truncated Vg product is able on its own to induce ectopic cell death and reaper expression. Our results shed new light on the function of the vg gene, in particular, they suggest that the normal and truncated products affect vg target genes in different ways.

The Drosophila wing differentiation factor vestigial-scalloped is required for cell proliferation and cell survival at the dorso-ventral boundary of the wing imaginal disc.

Links between genes involved in development, proliferation and apoptosis have been difficult to establish. In the Drosophila wing disc, the vestigial (vg) and the scalloped (sd) gene products dimerize to form a functional transcription factor. Ectopic expression of vg in other imaginal discs induces outgrowth and wing tissue specification. We investigated the role of the VG-SD dimer in proliferation and showed that vg antagonizes the effect of dacapo, the cyclin-cdk inhibitor. Moreover, ectopic vg drives cell cycle progression and in HeLa cultured cells, the VG-SD dimer induces cell proliferation per se. In Drosophila, ectopic vg induces expression of dE2F1 and its targets dRNR2 and string. In addition vg, but not dE2F1, interacts with and induces expression of dihydrofolate reductase (DHFR). Moreover, a decrease in VG or addition of aminopterin, a specific DHFR inhibitor, shift the dorso-ventral boundary cells of the disc to a cell death sensitive state that is correlated with reaper induction and DIAP1 downregulation. This indicates that vg in interaction with dE2F1 and DHFR is a critical player for both cell proliferation and cell survival in the presumptive wing margin area.

TONDU (TDU), a novel human protein related to the product of vestigial (vg) gene of Drosophila melanogaster interacts with vertebrate TEF factors and substitutes for Vg function in wing formation.

The mammalian TEF and the Drosophila scalloped genes belong to a conserved family of transcriptional factors that possesses a TEA/ATTS DNA-binding domain. Transcriptional activation by these proteins likely requires interactions with specific coactivators. In Drosophila, Scalloped (Sd) interacts with Vestigial (Vg) to form a complex, which binds DNA through the Sd TEA/ATTS domain. The Sd-Vg heterodimer is a key regulator of wing development, which directly controls several target genes and is able to induce wing outgrowth when ectopically expressed. Here we show that Vg contains two distinct transcriptional activation domains, suggesting that the function of Vg is to mediate transcriptional activation by Sd. By expressing a chimeric GAL4-Sd protein in Drosophila, we found that the transcriptional activity of the Vg-Sd heterodimer is negatively regulated at the AP and DV boundary of the wing disc. We also identify a novel human protein, TONDU, which contains a short domain homologous to the domain of Vg required for interaction with Sd. We show that TONDU specifically interacts with a domain conserved in all the mammalian TEF factors. Expression of TDU in Drosophila by means of the UAS-GAL4 system shows that this human protein can substitute for Vg in wing formation. We propose that TDU is a specific coactivator for the mammalian TEFs.