Variable motif utilization in homeotic selector (Hox)-cofactor complex formation controls specificity

Abstract

Homeotic selector (Hox) proteins often bind DNA cooperatively with cofactors such as Extradenticle (Exd) and Homothorax (Hth) to achieve functional specificity in vivo. Previous studies identified the Hox YPWM motif as an important Exd interaction motif. Using a comparative approach, we characterize the contribution of this and additional conserved sequence motifs to the regulation of specific target genes for three Drosophila Hox proteins. We find that Sex combs reduced (Scr) uses a simple interaction mechanism, where a single tryptophan-containing motif is necessary for Exd-dependent DNA-binding and in vivo functions. Abdominal-A (AbdA) is more complex, using multiple conserved motifs in a context-dependent manner. Lastly, Ultrabithorax (Ubx) is the most flexible, in that it uses multiple conserved motifs that function in parallel to regulate target genes in vivo. We propose that using different binding mechanisms with the same cofactor may be one strategy to achieve functional specificity in vivo.

Fig. 1.

Scr requires the W motif for Exd-dependent functions. (A) EMSA of Scr proteins with Exd-Hth^HM on fkh250. Hth^HM is a homeodomainless isoform of Hth that is sufficient for fkh250-lacZ regulation in vivo (12). Complexes are indicated with arrows. (B) Schematics of WT and mutant Scr proteins. The homeodomain (HD) is shown in black. Blue designates the W-motif. Red indicates residues mutated to alanines (YPWM→AAAA). (C–E) fkh250-lacZ expression. AG11-Gal4 is a ubiquitous Gal4 driver controlled by armadillo. Embryos were stained for β-galactosidase to monitor fkh250-lacZ expression (white). Yellow arrowheads indicate areas of WT lacZ expression. Brackets indicate induction of ectopic lacZ expression. Images are approximately 480 μm wide. (F) Phase contrast image of a WT T1 ventral denticle pattern normally controlled by Scr. Phase contrast images depicting ventral T2 cuticle patterns for WT larvae (G) or animals ectopically expressing WT Scr (H) or Scr-W1A (I) using the AG11-Gal4 driver. Cuticle images are approximately 140 μm wide.

Fig. 2.

AbdA has multiple motifs that mediate cooperative complex formation with Exd/Hth. (A) Schematics of WT and mutant AbdA proteins. Diagrams are drawn approximately to scale. The homeodomain (HD) is shown in black. Blue designates W-motifs. Green designates the UbdA motif (U). Purple designates the RRDR motif (R). Red indicates residues mutated to alanines (YPWM→AAAA, TDWM→AAAA, KEINEQ→AAAAAA, and RRDR→AAAA). (B) Average binding of different AbdA mutants to the DMX-R1 probe. The bar graph represents the mean of n ≥ 3 ratios from independent experiments for each mutant. Error bars represent the SEM. To determine if the difference in cooperative binding is significant for a subset of mutants, t tests were used (**P = 0.004; *P = 0.031).

Fig. 3.

AbdA uses conserved motifs in a context-dependent manner. (A–K) Thoracic region of a WT embryo (A) or embryos expressing AbdA proteins in T2 via the prd-Gal4 driver, stained for Dll (white or red) and myc-AbdA (green). The protein variant used is indicated on the left. Images are approximately 140 μm wide. Phase contrast images depicting ventral T2 cuticle patterns for WT larvae (L) or animals ectopically expressing WT AbdA (M) or mutant variants (N–V) using the AG11-Gal4 driver. (W) WT A2 ventral denticle pattern normally controlled by AbdA. Cuticle images are approximately 140 μm wide.

Fig. 4.

Ubx does not require C-terminal or W motifs for in vivo functions. (A) Schematics of WT and mutant Ubx proteins. Diagrams are drawn approximately to scale. Blue designates W-motifs. Green designates the UbdA motif (U). Purple designates the QA motif (Q). Red indicates residues mutated to alanines (PDWL→AAAA, YPWM→AAAA, TAWN→TAAN, and DIWN→DIAN). (B–G) Thoracic region of a WT embryo (B) or embryos expressing Ubx proteins in T2 via the prd-Gal4 driver, stained for Dll (white; here and in Fig. 5 the ectopic expression of Hox proteins was robust and nuclear but the images were omitted due to space constraints). The protein variant is indicated on the left. Images are approximately 140 μm wide. (H–M) Visceral mesoderm region of a WT embryo (H) or embryos expressing Ubx proteins via the AG11-Gal4 driver, stained for β-galactosidase to monitor dpp674-lacZ expression (white). Yellow brackets indicate ectopic lacZ activation. Images are approximately 270 μm wide. Phase contrast images depicting ventral T2 cuticle patterns for WT larvae (N) or animals ectopically expressing WT Ubx (O) or mutant Ubx variants (P–S). (T) WT A1 ventral denticle pattern normally specified by Ubx. Cuticle images are approximately 140 μm wide.

Fig. 5.

C-terminal motifs are sufficient for Ubx function. (A) Schematics of NTΔ Ubx proteins. Diagrams are drawn approximately to scale. Red indicates residues mutated to alanines or valines (KELNEQ→AALVAV and EKQAQAQK→AAVAVAVA). (B) Average binding of Ubx NTΔ mutants to the DMX-R probe. The bar graph represents the mean of n ≥ 3 ratios from independent experiments for each mutant. Error bars represent the SEM. To determine if the cooperative binding is significantly different from 1.0, t tests were used (***P = 0.0005; *P = 0.043). Because of cleavage of the protein in bacteria, the NTΔ;UA;QA mutant could not be purified and analyzed by EMSA. (C–H) Thoracic region of a WT embryo (A) or embryos expressing Ubx proteins in T2 via the prd-Gal4 driver, stained for Dll (white). The protein variant is indicated on the left. Images are approximately 140 μm wide. (I–N) Visceral mesoderm region of a WT embryo (I) or embryos expressing Ubx proteins via the AG11-Gal4 driver, stained for β-galactosidase to monitor dpp674-lacZ expression (white). Yellow brackets indicate ectopic lacZ activation. Images are approximately 270 μm wide. Phase contrast images depicting ventral T2 cuticle patterns for WT larvae (O) or animals ectopically expressing WT Ubx (P) or mutant variants (Q–T). (U) WT A1 ventral denticle pattern normally specified by Ubx. Cuticle images are approximately 140 μm wide.

Fig. 6.

UbdA motif mediates interaction with En. (A and B) EMSAs of AbdA and Ubx proteins with En on DMX-R1. The positions of Hox–En cooperative complexes are indicated with arrows. (A) Truncations include the homeodomain (HD) and/or the UbdA motif (U).