HOX genes specify segment identity along the anteroposterior axis of the embryo. They code for transcription factors harbouring the highly conserved homeodomain and a YPWM motif, situated amino terminally to it. Despite their highly diverse functions in vivo, HOX proteins display similar biochemical properties in vitro, raising the question of how this specificity is achieved. In our study, we investigated the importance of the Antennapedia (Antp) YPWM motif for homeotic transformations in adult Drosophila. By ectopic overexpression, the head structures of the fly can be transformed into structures of the second thoracic segment, such as antenna into second leg, head capsule into thorax (notum) and eye into wing. We found that the YPWM motif is absolutely required for the eye-to-wing transformation. Using the yeast two-hybrid system, we were able to identify a novel ANTP-interacting protein, Bric-à-brac interacting protein 2 (BIP2), that specifically interacts with the YPWM motif of ANTP in vitro, as well as in vivo, transforming eye to wing tissue. BIP2 is a TATA-binding protein associated factor (also known as dTAFII3) that links ANTP to the basal transcriptional machinery.

Hox genes encoding homeodomain transcriptional regulators are known to specify the body plan of multicellular organisms and are able to induce body plan transformations when misexpressed. These findings led to the hypothesis that duplication events and misexpression of Hox genes during evolution have been necessary for generating the observed morphological diversity found in metazoans. It is known that overexpressing Antennapedia (Antp) in the head induces antenna-to-leg as well as head-to-thorax transformation and eye reduction. At present, little is known about the exact molecular mechanism causing these phenotypes. The aim of this study is to understand the basis of inhibition of eye development. We demonstrate that Antp represses the activity of the eye regulatory cascade. By ectopic expression, we show that Antp antagonizes the activity of the eye selector gene eyeless. Using both in vitro and in vivo experiments, we demonstrate that this inhibitory mechanism involves direct protein-protein interactions between the DNA-binding domains of EY and ANTP, resulting in mutual inhibition.

Homeotic Hox selector genes encode highly conserved transcriptional regulators involved in the differentiation of multicellular organisms. Ectopic expression of the Antennapedia (ANTP) homeodomain protein in Drosophila imaginal discs induces distinct phenotypes, including an antenna-to-leg transformation and eye reduction. We have proposed that the eye loss phenotype is a consequence of a negative posttranslational control mechanism because of direct protein-protein interactions between ANTP and Eyeless (EY). In the present work, we analyzed the effect of various ANTP homeodomain mutations for their interaction with EY and for head development. Contrasting with the eye loss phenotype, we provide evidence that the antenna-to-leg transformation involves ANTP DNA-binding activity. In a complementary genetic screen performed in yeast, we isolated mutations located in the N terminus of the ANTP homeodomain that inhibit direct interactions with EY without abolishing DNA binding in vitro and in vivo. In a bimolecular fluorescence complementation assay, we detected the ANTP-EY interaction in vivo, these interactions occurring through the paired domain and/or the homeodomain of EY. These results demonstrate that the homeodomain supports multiple molecular regulatory functions in addition to protein-DNA and protein-RNA interactions; it is also involved in protein-protein interactions. Drosophila ͉ antagonism ͉ regulation H omeotic Hox genes are selector genes that generate morphological diversity along the antero-posterior body axis during animal development (1). They encode highly conserved transcription factors defining various cellular identities in the body segments along the antero-posterior axis of the embryo (2, 3). Hox genes share a common sequence element of 180 bp, the homeobox, first isolated in Drosophila (4, 5), encoding a 60-aa homeodomain (HD) DNA-binding domain. The HD presents a stereotypical three ␣-helical structure, and its mode of interaction with DNA is largely invariant. The amino acid 50, a glutamine signature for the Hox proteins, plays a fundamental role in DNA-binding specificity (6). Hox factors have very similar DNA-binding properties (7). Therefore, the limited sequence selectivity of the HD is not sufficient to explain the diversity of cell types and the batteries of downstream genes under the control of Hox proteins. Consequently, elucidating the mechanisms of Hox protein function is critical for understanding development and the diversification of serially homologous structures. Several studies have indicated that Hox factors cooperate with signaling pathways and act with cofactors that modify DNA binding and activation/repression properties (8-10). To date, the best-known Hox cofactors are the Drosophila extradenticle (EXD/PBX) and homothorax (HTH/Meis) proteins. EXD was shown to modulate the DNA-binding specificity and the activity of Hox proteins (11-13), whereas HTH promotes EXD translocation into the nucleus where they participate in a DNA-bound HOX/EXD/HTH ternary complex (14). Altho...