Direct competition between Brinker and Drosophila Mad in Dpp target gene transcription

Abstract

Brinker is a nuclear protein that antagonizes Dpp signalling in Drosophila. Its expression is negatively regulated by Dpp. Here, we show that Brinker represses Ultrabithorax (Ubx) in the embryonic midgut, a HOX gene that activates, and responds to, the localized expression of Dpp during endoderm induction. We find that the functional target for Brinker repression coincides with the Dpp response sequence in the Ubx midgut enhancer, namely a tandem of binding sites for the Dpp effector Mad. We show that Brinker efficiently competes with Mad in vitro, preventing the latter from binding to these sites. Brinker also competes with activated Mad in vivo, blocking the stimulation of the Ubx enhancer in response to simultaneous Dpp signalling. These results indicate how Brinker acts as a dominant repressor of Dpp target genes, and explain why Brinker is a potent antagonist of Dpp.

Fig. 1. Gene expression in the midgut and signal-responsive sequence from Ubx. (A) Expression of HOX proteins (top), Wg (Wg) and Dpp (Bienz, 1996) in the midgut mesoderm in relation to estimated domains of brk expression (Jazwinska et al., 1999b); midgut limits, positions of the gastric caeca (in ps3) and of the gut constrictions are indicated above. Critical regulatory interactions between the genes in the middle midgut are shown (arrows, stimulatory; barred line, repressive). (B) Top, signal-responsive sequence from Ubx B; underneath, mutant enhancer and L-CRE sequences, as indicated. Three perfect matches to the Brk consensus binding site are given at the bottom, and their presence in the mutant enhancers is indicated on the right. The ability of Brinker to bind to, or to repress, a mutant enhancer correlates with the presence of Brk bs1 or 2 (highlighted in yellow). nd, not done.

Fig. 1. Gene expression in the midgut and signal-responsive sequence from Ubx. (A) Expression of HOX proteins (top), Wg (Wg) and Dpp (Bienz, 1996) in the midgut mesoderm in relation to estimated domains of brk expression (Jazwinska et al., 1999b); midgut limits, positions of the gastric caeca (in ps3) and of the gut constrictions are indicated above. Critical regulatory interactions between the genes in the middle midgut are shown (arrows, stimulatory; barred line, repressive). (B) Top, signal-responsive sequence from Ubx B; underneath, mutant enhancer and L-CRE sequences, as indicated. Three perfect matches to the Brk consensus binding site are given at the bottom, and their presence in the mutant enhancers is indicated on the right. The ability of Brinker to bind to, or to repress, a mutant enhancer correlates with the presence of Brk bs1 or 2 (highlighted in yellow). nd, not done.

Fig. 2. Expression of Ubx and Ubx reporters in brk mutants. Dorsolateral views of 12–14-hour-old wild-type and brk mutant embryos, bearing Ubx reporter genes as indicated, stained with α-Ubx (A and B) or α-lacZ antibody (C–N). Arrows point to derepression in the anterior and posterior midgut of brk mutants. Anterior to the left, dorsal up.

Fig. 3. Direct binding of Brinker to the MadA binding site in Ubx B. (A) Map of Brinker, with DNA binding domain (black) and CtBP-recruiting motif PMDLS indicated (see Jawinska et al., 1999a). (B–D) Gel shift assays with GST–Brinker fusion proteins as indicated (B; 10 µg per assay), or with the DNA binding domain of Brinker (Brk44–99; 1 µg) (C and D). Wild-type (B and D) or mutant sequences (C) as indicated above lanes were used as probes; in (D), an excess of unlabelled wild-type or BM2 mutant probe was used as competitor. (E) Gel shift assays, with 15 µg of the DNA binding domain of Mad (MH1+L) per assay; competitors as in (D). (F) Competition between Mad and Brinker; 15 µg of Mad (MH1+L) and increasing amounts of Brk44-99 (0, 0.001, 0.003, 0.01, 0.03, 0.1 and 0.3 µg) were used. (G) Quantitative evaluation of two independent experiments [(F) and not shown] showing competition between Brinker and Mad.

Fig. 4. Ectopic Brinker represses Ubx and Ubx reporters. Dorsolateral views of 12–14-hour-old embryos, bearing Ubx reporters as indicated, stained with α-Ubx (A and B) or α-lacZ antibody (C–H); note that the GAL4 driver in (H) was 48Y.GAL4 instead of 24B.GAL4. Open triangles point to missing (B and D) or reduced expression (F); arrowhead points to endodermal bulge in the Brinker-overexpressing embryo (B and D). These also lack the first and second gut constriction [(F); vertical bars in (E) mark constrictions in the wild type]. The staining marked by an arrow in (H) reflects full activity of L-CRE in the visceral mesoderm, while its endodermal activity is undetectable. Anterior to the left, dorsal up.

Fig. 5. Competition between Brinker and activated Mad in vivo. Dorsolateral views of 12–14-hour-old embryos, bearing Ubx B or the dpp reporter BMPX as indicated, stained with α-lacZ antibody. Lack of lacZ staining and endodermal bulges due to ectopic Brinker are marked by open triangles and arrowheads, respectively; vertical bars point to the gut constrictions that form under the various conditions.