Competition for cofactor-dependent DNA binding underlies Hox phenotypic suppression

Abstract

Hox transcription factors exhibit an evolutionarily conserved functional hierarchy, termed phenotypic suppression, in which the activity of posterior Hox proteins dominates over more anterior Hox proteins. Using directly regulated Hox targeted reporter genes in Drosophila, we show that posterior Hox proteins suppress the activities of anterior ones by competing for cofactor-dependent DNA binding. Furthermore, we map a motif in the posterior Hox protein Abdominal-A (AbdA) that is required for phenotypic suppression and facilitates cooperative DNA binding with the Hox cofactor Extradenticle (Exd). Together, these results suggest that Hox-specific motifs endow posterior Hox proteins with the ability to dominate over more anterior ones via a cofactor-dependent DNA-binding mechanism.

Figure 1.

AbdA dominance over Scr relies on an Exd-dependent DNA-binding mechanism. (A) Embryos carrying fkh250-lacZ (left panels) or fkh250^CON-lacZ (right panels) stained for β-galactosidase (β-gal). In wild-type embryos, endogenous Scr activates fkh250-lacZ in PS2 (arrowhead), while fkh250^CON-lacZ is activated by Scr, Antp, and Ubx from PS2 to PS6 (arrows) and is repressed by AbdA in the abdominal segments. Ectopic expression of Scr throughout the embryo, alone or in combination with AbdA, results in widespread activation of fkh250-lacZ. In contrast, fkh250^CON-lacZ is repressed when Scr and AbdA are both ectopically expressed. (B) Representative in vitro saturation binding experiments and dissociation constant (K_d in nanomolar) fits are shown for Scr–Exd (left) and AbdA–Exd (right) binding to fkh250^CON. AbdA–Exd dimers bound more tightly to fkh250^CON than Scr–Exd did, supporting a model in which AbdA dominance depends on cofactor-dependent DNA binding. All assays were performed in the presence of Exd–Hth^HM. The reported K_ds represent the averages and standard error of the means of repeated measurements (n = 5 for AbdA, and n = 6 for Scr) (see the Materials and Methods).

Figure 2.

AbdA dominance over Scr depends on its C-terminal UR motif. (A) Wild-type expression of fkh250^CON-lacZ (stained for β-gal). (B) fkh250^CON-lacZ activation in the context of ubiquitous expression of Scr. AbdA variants (AbdA^mut) (diagrammed at left) were ectopically expressed in combination with Scr to define motifs in AbdA necessary to suppress Scr activation of fkh250^CON-lacZ (stained for β-gal) (right). Shown are representative images for each AbdA variant, which are ordered according to their ability to repress fkh250^CON-lacZ from strongest (C, wild-type AbdA) to weakest (I, no C terminus: ΔC¹⁹⁷). (D,E) Wild-type-repressive activity was observed for an AbdA variant in which the YPWM and TDWM motifs were mutated to alanines (2W^Ala), or when part of the C terminus was deleted (ΔC²⁶³), suggesting that these motifs are not necessary for repression of this target. (I) Deletion of the entire C-tail of AbdA (ΔC¹⁹⁷) abolishes repression of fkh250^CON-lacZ. (F) Repression is significantly rescued by the addition of the UR motif adjacent to the homeodomain (HD) (ΔC²²⁰). (G) Consistently, an internal deletion of UR (Δ^200–220) partially impairs AbdA's ability to suppress activation of fkh250^CON-lacZ by Scr. (H) A variant in which both the YPWM and TDWM motifs are mutated in combination with this internal deletion (2W^AlaΔ^200–220) displayed no additional loss of repressive ability, suggesting that the UR motif of AbdA is critical for posterior dominance. In the schematics of the AbdA variants, the N-terminal YPWM and TDWM motifs are indicated by white bars (colored black when mutated to alanines), the HD is indicated, the C-terminal UR motif is in grey, and Q indicates a glutamine-rich region.

Figure 3.

The C-terminal UR motif of AbdA mediates cooperative binding with Exd on fkh250^CON. Variants of AbdA missing the UR motif (ΔC¹⁹⁷, Δ^200–220, and 2W^AlaΔ^200–220) do not form cooperative complexes with Exd on fkh250^CON in vitro, as analyzed by electrophoretic mobility shift assays (EMSAs). Cooperative complexes between AbdA variants and Exd are indicated by asterisks. EMSAs were performed in the presence of Exd–Hth^HM as indicated (see the Materials and Methods).

Figure 4.

The UR motif endows AbdA with the ability to dominate over Antp in patterning of the larval epidermis. Ectopic expression of Antp or AbdA transforms the T1 segment toward a T2 (T1 → T2) or an A2 (T1 → A2) fate, respectively. (A) Same-magnification representative images of wild-type and transformed T1 segments are shown. (B) When misexpressed in combination, wild-type AbdA dominates over Antp, generating an A2-like epidermal phenotype. AbdA variants (AbdA^mut) missing the UR motif (ΔC¹⁹⁷, Δ^200–220, and 2W^AlaΔ^200–220) resulted in T2-like phenotypes, suggesting that they were unable to overcome the activity of Antp. No such defect in dominance was observed when only the YPWM and TDWM motifs were mutated (2W^Ala), suggesting that the UR motif is used by AbdA to dominate over Antp as well as Scr. The percentages of T1 → A2 cuticles are normalized by the fraction of AbdA-containing embryos, according to the specific genotypes (see the Materials and Methods).