A unique Extradenticle recruitment mode in the Drosophila Hox protein Ultrabithorax

Abstract

Hox transcription factors are essential for shaping body morphology in development and evolution. The control of Hox protein activity in part arises from interaction with the PBC class of partners, pre-B cell transcription factor (Pbx) proteins in vertebrates and Extradenticle (Exd) in Drosophila. Characterized interactions occur through a single mode, involving a short hexapeptide motif in the Hox protein. This apparent uniqueness in Hox-PBC interaction provides little mechanistic insight in how the same cofactors endow Hox proteins with specific and diverse activities. Here, we identify in the Drosophila Ultrabithorax (Ubx) protein a short motif responsible for an alternative mode of Exd recruitment. Together with previous reports, this finding highlights that the Hox protein Ubx has multiple ways to interact with the Exd cofactor and suggests that flexibility in Hox-PBC contacts contributes to specify and diversify Hox protein function.

Fig. 1.

The UbdA motif mediates Exd/Hth recruitment. (A) Sequence alignment (ClustalW and Boxshade treatment) of AbdA, Ubx, and Antp protein sequences including and flanking the HD. Except for the HD and HX (blue bars), the UbdA motif (red bar) is the most conserved sequence between Ubx and AbdA. The UbdA motif is not found in any other Drosophila Hox protein, as illustrated here by its absence in Antp. (B) Structure of the Ubx variants used. Mutations, indicated as black boxes, are YPWM to YAAA for the HX and AIKELNEQ to AIVVLIVA for the UbdA motif. (C) EMSA shows that assembling the Ubx–Exd–Hth trimeric complex on DllR (lanes 4–7, red arrowhead) does not depend on HX (lanes 8–11) but on UbdA motif integrity (lanes 12–15). Mutation of both the HX and UbdA motifs (lanes 16–19) does not decrease Ubx–Exd–Hth complex formation compared with mutation of the UbdA motif alone. Six, 12, 25, and 50 ng of Ubx or its variant were used. Lanes 1–3 are, respectively, the DIIR probe alone, nonprogrammed, and Exd–Hth programmed lysate. The gray arrowhead indicates the position of Exd–Hth–DNA complexes.

Fig. 2.

The UbdA motif is required for Exd-dependent Dll repression. (A and B) DME activity, detected by an anti-β-galactosidase staining (green and highlighted by arrows), is in wild-type embryos restricted to thoracic segments where Ubx (detected by an anti-Ubx staining, red) is not expressed. (C–J) Ubiquitous expression of wild-type and Ubx variants was driven by arm-Gal4. Ubx (C and D) and Ubx^HX(E and F) repress DME activity, whereas Ubx^UbdA (G and H) and Ubx^HX,UbdA (I and J) do so only poorly. Quantification of the repressive activity of Ubx and its variants on DME reporter expression (K, see Materials and Methods). Embryos, anterior to the left and ventral side down, are at stage 11.

Fig. 3.

The UbdA motif confers Exd recruitment potential to Antp. (A) Structure of the Ubx/Antp variants. A color code identifies Ubx (red) and Antp (blue) sequences. Mutations are indicated as black boxes and are YPWM to YAAA for the HX and AIKELNEQ to AIVVLIVA for the UbdA motif. Antp and Ubx C termini are, respectively, from amino acids 360–378 and 358–389. (B) EMSA of the DllRcon probe, on which Antp recruits Exd in the absence of Hth (lanes 3 and 4, red arrowhead). Mutation of the HX (Antp^HX) impairs Exd recruitment (lanes 5 and 6). Note that the HX mutation increases Antp monomer binding, as observed previously for the HX mutation in the Lab protein (12). Recruitment is restored upon swapping Antp and Ubx C termini (Antp^HX:UC; lanes 7 and 8). Altering the integrity of the UbdA motif (Antp^HX:UC^UbdA) alleviates Exd recruitment (lanes 9 and 10). Lanes 1 and 2 are, respectively, the Dllcons probe alone and nonprogrammed lysate. The gray arrowhead indicates the position of the Hox–DNA complexes. Twenty-five nanograms of Antp or its variants was used. (C) A Hox–Exd–Hth trimeric complex forms in vitro on tsh cis-regulatory DNA (box2, ref. 28) using Antp (lanes 3–5, red arrowhead) or Antp^HX:UC (lanes 9–11) but not Antp^HX (lanes 6–8) or Antp^HX:UC^UbdA (lanes 12–14). The two first lanes are, respectively, the probe alone and nonprogrammed lysate. The gray arrowhead indicates the position of the Hox–DNA complexes. Twenty-five nanograms of Antp or its variants was used.

Fig. 4.

The UbdA motif confers Exd-dependent activity to Antp. (A and B) tsh transcripts (detected by in situ hybridization, green) and Antp protein (detected by an anti-Antp staining, red) are absent from wild-type head segments. (C–J) Ubiquitous expression driven by arm-Gal4 of Antp (C and D) or of Antp^HX:UC (G and H) induces ectopic tsh in the head (arrows in C and G), whereas that of Antp^HX (E and F) or of Antp^HX:UC^UbdA (I and J) does not. Embryos, anterior to the left and ventral side down, are at stage 11.

Fig. 5.

The HX and UbdA motifs are dispensable for Exd-dependent activation of dpp. Distribution of dpp transcripts (detected by in situ hybridization, green) and Ubx proteins (detected by an anti-Ubx staining, red) is shown. Dashed lines delineate ps3 and ps7 in the midgut of a wild-type embryo (A and B), and arrows point toward dpp ectopic transcription similarly induced by mesodermal ubiquitous expression (24B-Gal4) of Ubx (C and D), Ubx^HX (E and F), Ubx^UbdA (G and H), or of Ubx^HX,UbdA (I and J). Embryos, anterior to the left and ventral side down, are at stage 13.

Fig. 6.

Complexity in Ubx–Exd functional interactions. The scheme highlights the diversity of interactions underlying Ubx–Exd functional interactions. The motif mediating Exd recruitment varies according to the target gene regulated: UbdA for Dll down-regulation (A), HX for regulation of genes (gene X) involved in segment identity specification (B), or a third unidentified motif in the process of dpp activation (C). In the last case, functional interplay may also not rely on direct interaction between Exd and Ubx (D).