Repression of Dpp targets by binding of brinker to mad sites

Signaling by decapentaplegic (Dpp), a Drosophila member of the transforming growth factor (TGF) beta superfamily of growth factors, has recently been shown to activate targets such as vestigial (vg) indirectly through negativ e regulation of brinker (brk), Here we show that the Brk protein functions as a repressor by binding to Dpp response elements. The Brk DNA binding act ivity was localized to an amino-terminal region containing a putative homeo domain. Brk bound to a Dpp response element of the Ultrabithorax: (Ubx) mid gut enhancer at a sequence that overlaps a binding site for the Smad protei n, Mothers Against Dpp (Mad). Furthermore, Brk was able to compete with Mad for occupancy of this binding site. This recognition of overlapping bindin g sites provides a potential explanation for why the G/C-rich Mad binding s ite consensus differs the Smad3/Smad4 binding site consensus. We also found that the Dpp response element from Ubx was more sensitive than the vg quad rant enhancer to repression by Brk. This difference correlates with short-r ange activation of Ubx by Dpp in the visceral mesoderm, whereas vg exhibits a long-range response to Dpp in the wing imaginal disc, indicating that Br k binding sites may play a critical role in limiting thresholds for activat ion by Dpp. Finally, we provide evidence that Brk is capable of functioning as an active repressor, Thus, whereas Brk and Mad compete for regulation o f Ubx and vg, Brk may regulate other Dpp targets without direct involvement of Mad.