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First published online 12 September 2007
doi: 10.1242/dev.009522

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Collaboration between Smads and a Hox protein in target gene repression

Howard Hughes Medical Institute and Laboratory of Molecular Biology, University of Wisconsin, 1525 Linden Drive, Madison, WI 53706, USA.

View larger version (99K): [in this window] [in a new window]   Fig. 1. Mad, Med and shn are required to repress sal expression in the Drosophila haltere. The proteins visualized are indicated at the bottom left of each panel; in E-P, the mutant gene in clones is indicated at the top of each panel. The absence of GFP expression indicates the position of homozygous mutant clones. (A,B) Wild-type Sal expression in the wing (A) and haltere (B) imaginal discs. Sal is repressed in the haltere pouch (dashed outline). (C,D) Brk is expressed in both the wing (C) and haltere (D) imaginal discs. (E-H) Sal is not expressed in Mad homozygous mutant clones in the wing (E,F, arrowheads), owing, at least in part, to the repression of sal by the upregulation of Brk in clones (G, arrowhead). (H) Merged image of E-G. (I-P) Sal is derepressed in Mad (I-K, arrowheads), Med (M,N, arrowheads), and shn (O,P, arrowheads) mutant clones in the haltere. (L) Brk is expressed in Mad mutant clones but is insufficient to repress sal expression in the haltere. (K) Merged image of I,J. (N) Merged image of M and GFP channel (data not shown). (P) Merged image of O and GFP channel (data not shown).

View larger version (38K): [in this window] [in a new window]   Fig. 2. Mad and Med directly repress sal expression in the haltere through binding sites in the sal1.1 CRE. (A) Schematic of the Drosophila sal1.1 CRE with Ubx binding sites 1-7 (red circles). Putative Med (green box) and Mad (yellow box) binding sites are located between Ubx binding sites 5 and 6 (red boxes). Binding site mutations introduced into the M1 site are indicated below the wild-type sal1.1 sequence. The binding of proteins to the probes was quantitated with ImageQuant software to assess the effect of mutations on their affinity for specific sites. (B-I) The effect of mutations in the putative M1 site on Mad and Med binding in vitro and their effect on reporter gene expression in vivo are aligned in columns. (B) Electrophoretic mobility shift assays (EMSAs) with GST-MedMH1. In each set of lanes, the protein concentration increases from left to right. The point mutation at bp 808 (lanes 5-8) eliminates Med binding as compared with the wild-type M1 site (lanes 1-4). Mutations at positions 812 (lanes 9-12), 813 (lanes 13-16) and 814 (lanes 17-20) have little to no effect on Med binding. (C) EMSAs with GST-MadN on wild-type and mutant M1 probes. In each set of lanes, the protein concentration increases from left to right. Each point mutation (808, 812, 813 and 814) causes a decrease in the strength of Mad binding (2.2-, 2.7-, 2.6- and 3.6-fold, respectively) as compared with the wild-type sequence (compare lanes 25-29 with 30-49). Combining the four point mutations in kM1 causes an 8.6-fold reduction in Mad binding affinity (lanes 50-54). (D-I) Haltere imaginal discs of transgenic sal1.1 reporter lines that are either wild-type or that carry mutations in the M1 site, immunolabeled for lacZ expression.

View larger version (31K): [in this window] [in a new window]   Fig. 3. Discrete Ubx and Smad binding sites are closely juxtaposed, but independent of one another, in the sal1.1 CRE. Probes with Ubx binding sites 5 and 6 (red circles) and Med and Mad binding sites (green and yellow boxes, respectively) and mutated binding sites (marked with an `X') are depicted above each EMSA. Protein-DNA complexes are indicated by arrowheads. (A,B) EMSAs with either purified Ubx homeodomain (Ubx-HD) (A) or full-length Ubx1a (B) on wild-type and mutated sal probes. (A) Mutations in Ubx binding sites 5 and 6 cause a 10-fold decrease in Ubx-HD binding affinity for probe (compare lanes 1-7 with 9-14), but mutations in the kM1 site (lanes 16-21) have no effect on binding. (B) Mutations in Ubx binding sites 5 and 6 eliminate the binding of full-length Ubx protein to the probe (compare lanes 1-5 with 6-10), but mutations in the kM1 site (lanes 11-15) have no effect on binding. (C) Mutations in Ubx binding sites 5 and 6 do not affect Med binding affinity for its site, as compared with the wild-type probe (compare lanes 1-4 with 5-8). (D) Mutations in Ubx binding sites 5 and 6 do not affect Mad binding affinity for its site, as compared with the wild-type probe (compare lanes 9-13 with 14-18).

View larger version (45K): [in this window] [in a new window]   Fig. 4. Ubx and Mad/Med binding sites collaborate to repress sal expression in the haltere. Schematics above each panel highlight the relevant changes to CREs. (A,B) Haltere imaginal discs from transgenic Drosophila carrying sal1.1 reporter constructs with mutations either in Ubx binding sites 5 and 6 or in the M1 site. The level and pattern of derepression is identical whether Ubx sites 5 and 6 or the M1 site are mutated. (C) Mutations in Ubx binding sites 5 and 6 in a subelement of the sal1.1 CRE, sal328, derepress the reporter gene lacZ in haltere discs. (D) The addition of one copy of Ubx binding site 5 to both the 5' and 3' ends of sal328koU5&6 (sal328koU5&6+2-U5) is insufficient to repress reporter gene expression in the haltere. (E) The vgB CRE is expressed along the dorsal-ventral compartment boundary in both the wing (W) and haltere (H) imaginal discs. (F) The addition of two cassettes, each containing four copies of Ubx binding site 5, to the 5' and 3' ends of the vgB is not sufficient to impart Ubx-dependent repression in the haltere imaginal disc.

View larger version (22K): [in this window] [in a new window]   Fig. 5. The conserved topology of Ubx binding sites 5 and 6 and the M1 site in the sal1.1 CRE. Alignment of a deeply conserved block of 37 nucleotides (shaded blue, indicating 100% nucleotide conservation) containing Ubx binding sites 5 and 6 and the collaborating M1 site in the sal1.1 CRE from four species of Drosophila. Nucleotides conserved in three out of four species are shaded in gray.

View larger version (37K): [in this window] [in a new window]   Fig. 6. Collaboration between Ubx and Smads in the selective repression of the sal gene in the Drosophila haltere disc. (Left) A depiction of the wing and haltere imaginal discs of the second and third thoracic segments (T2 and T3, respectively). Sal is expressed (yellow) where high levels of Dpp signaling emanate from the anterior-posterior compartment boundary. Brk (purple) represses sal and restricts its expression to the center of the wing imaginal disc. Ubx (red stripes) is expressed in the haltere disc, where it regulates target genes such as sal. (Right) The genetic circuitry and molecular mechanisms allowing sal expression in the wing and repressing sal in the haltere. In the wing, Sal is expressed in cells where the Dpp-responsive Mad-Med-Shn repressor complex binds to a high-affinity Smad site in a brk CRE to directly repress Brk expression. The sal1.1 CRE contains a low-affinity Smad site but this is insufficient to repress sal in the wing. However, in the haltere, sal is repressed via the collaborative action of Ubx and Mad-Med-Shn binding to the sal1.1 CRE. Both the affinity and topology of repressor binding sites are critical to the selective repression of sal in the T3 flight appendage.