Smad1 and Smad4 are components of the bone morphogenetic protein-4 (BMP-4)-induced transcription complex of the Xvent-2B promoter

To study the mechanism of transcriptional activation of the Xenopus homeobox gene Xvent-2B, we have delineated the bone morphogenetic protein-4 (BMP-4)-responsive region between -275/-152 in the proximal pro meter. Consistent with the BMP-4 inductive nature of this region, this element exhibits transcriptional activation upon ectopic expression of Smad1 and Smad4. Electrophoretic mobility shift assays with total cellular extracts demonstrated that a DNA fragment encompassing this region was competent in the formation of a BMP-4-induced protein-DNA complex containing Smad1. Two different Smad binding regions were localized, a distal binding region for Smad1 containing two GCAT motifs and proximal AGNC binding sites for Smad4, the latter being conserved in other transforming growth factor-beta-responsive elements. Mutation of the Smad4 binding motif completely abolished transcriptional activation, whereas mutation or deletion of the Smad1 recognition sequence inhibited Smad1/Smad4 responsiveness. These results provide a functional characterization and identification of a vertebrate Smad1/Smad4 DNA response element induced by BMP-4 signaling and offers insight into the transcriptional regulation of a component essential for dorsoventral patterning in Xenopus embryos.