Direct transcriptional control of the Dpp target omb by the DNA binding protein Brinker

Abstract

The gradient morphogen Decapentaplegic (Dpp) organizes pattern by inducing the transcription of different target genes at distinct threshold concentrations during Drosophila development. An important, albeit indirect, mode by which Dpp controls the spatial extent of its targets is via the graded downregulation of brinker, whose product in turn negatively regulates the expression of these targets. Here we report the molecular dissection of the cis-regulatory sequences of optomotor-blind (omb), a Dpp target gene in the wing. We identify a minimal 284 bp Dpp response element and demonstrate that it is subject to Brinker (Brk) repression. Using this omb wing enhancer, we show that Brk is a sequence-specific DNA binding protein. Mutations in the high-affinity Brk binding site abolish responsiveness of this omb enhancer to Brk and also compromise the input of an unknown transcriptional activator. Our results therefore identify Brk as a novel transcription factor antagonizing Dpp signalling by directly binding target genes and repressing their expression.

Opposing inputs by Hedgehog and Brinker define a stripe ofhairyexpression in theDrosophilaleg imaginal disc

“…Prepared proteins were incubated on ice for 30 minutes with ~50,000 c.p.m. of labeled probes in binding buffers as described (Hepker et al, 1999;Kim et al, 1997;Sivasankaran et al, 2000). In some samples, unlabelled wild-type oligos (specific competitor) and mutant oligos (nonspecific competitor) were included.…”

“…A potential candidate, the transcriptional repressor of Dpp target genes, Brk (Campbell and Tomlinson, 1999;Jazwinska et al, 1999;Kirkpatrick et al, 2001;Minami et al, 1999;Rushlow et al, 2001;Saller and Bienz, 2001;Sivasankaran et al, 2000;Zhang et al, 2001), is suggested by evidence indicating that Dpp is required to override REPE function. In the wing and leg imaginal discs, brk expression is repressed by and is roughly reciprocal to Dpp signaling.…”

“…If so, we might expect the REPE to contain one or more functional Brk-binding sites. Hence, we searched the REPE for the Brk consensus binding site, GGCG(C/T)(C/T) (Kirkpatrick et al, 2001;Sivasankaran et al, 2000;Zhang et al, 2001), and identified a potential Brk binding site which overlaps two sequences similar to a consensus binding sites for Mad, GCCGNCGC (Kim et al, 1997), and a sequence similar to a cAMP response element (CRE), TGACGTCA (Montminy et al, 1986). The sequence of overlapping CRE, Brk and Mad sites was designated the CMB element (see Fig.…”

“…However, more recent studies indicate that Dpp also regulates its target genes by blocking expression of a repressor. In imaginal discs, Mad/Med and the zinc-finger protein Shn, repress expression of the brinker (brk) gene (Marty et al, 2000;Muller et al, 2003;Torres-Vazquez et al, 2000), which encodes a direct repressor of Dpp target genes (Campbell and Tomlinson, 1999;Jazwinska et al, 1999;Kirkpatrick et al, 2001;Minami et al, 1999;Rushlow et al, 2001;Saller and Bienz, 2001;Sivasankaran et al, 2000;Zhang et al, 2001). The Brk repressor is distributed in a gradient reciprocal to Dpp signaling and functions to delimit the domains of Dpp target gene expression.…”

bantam microRNA is a negative regulator of the Drosophila decapentaplegic pathway

“…omb is a Drosophila T-box gene positively regulated by Dpp [48,55] and is expressed in a broad region in the middle of the wing disc (Figure 1I). X-Gal staining was used to monitor the expression levels of omb-lacZ in wing discs, the enhancer trap lines for omb.…”