Cross-regulatory protein–protein interactions between Hox and Pax transcription factors

Abstract

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...

Function and specificity of synthetic Hox transcription factors in vivo

“…Such interactions have been demonstrated to take place between two HDs or a HD and a PAIRED domain (PD), resulting in mutual inhibition of DNA-binding properties, which leads to defects that resemble mutant phenotypes, caused by absence of either gene product. A phenotypic manifestation of such a negative posttranslational regulatory inhibition is the eye reduction caused by ectopic expression of various Hox proteins in the eye-antennal disc, shown to be triggered by binding of the HD of Hox proteins to the PD of eyeless (ey) (30,31). In accordance with these findings ectopic expression of Scr-HD wt and Scr-HD AA using dpp blink -Gal4 exhibited strong eye phenotypes ( Fig.…”

“…In accordance with these findings ectopic expression of Scr-HD wt and Scr-HD AA using dpp blink -Gal4 exhibited strong eye phenotypes ( Fig. 2A-D) as compared to the full-length peptide (30), ranging from reduction to the complete absence of eyes. No abnormal eye phenotype was observed with Scr-HD DD (Fig.…”

“…Our findings suggest that these mutations also abolish the capacity of Scr-HD DD to participate in HD-PD or HD-HD interactions. Although mutation of glutamate 19 to glycine abolished the dimerization capacity of Antp in vivo (30), in the case of Scr other residues of the HD, in addition to glutamate 19, might participate in protein-protein interactions, or, alternatively, the negative charge introduced by the aspartates might be responsible for abolishing these interactions through electrostatic repulsion. The fact that Scr-HD AA exhibited similar phenotypes as Scr-HD wt (compare Fig.…”

“…This transformation is DNA-binding-dependent for Antp (30), because residues that impair binding of the HD to the minor groove (47) also abolish its transformation capacity in vivo. This suggested a similar requirement for Scr and thus for synthetic Scr peptides.…”

Hox Genes: Master Regulators of the Animal Bodyplan

“…For example, the thoracic Antennapedia, when ubiquitously expressed, suppresses Hox genes of the head, resulting in posterior transformation of head segments towards a thoracic identity while not affecting the abdomen -here, the effect of Antp is phenotypically suppressed by bithorax-complex genes such as Ubx (Gonzalez-Reyes et al, 1990 Gibson andGehring, 1988 ). However, posterior prevalence occurs not only postranslationally (Plaza et al, 2008) but also at the levels of transcription (Beachy et al, 1988, Hafen et al, 1984, Appel and Sakonju, 1993, Struhl and White, 1985 and posttranscriptional regulation of mRNA abundance (Yekta et al, 2004, 2008, Woltering and Durston, 2007, Ronshaugen et al,2005 (Text box 3). It can thus also potentially regulate the mRNA expression of Hox genes.…”