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 homeobo x 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 tran scriptional activation upon ectopic expression of Smad1 and Smad4. Electrop horetic mobility shift assays with total cellular extracts demonstrated tha t a DNA fragment encompassing this region was competent in the formation of a BMP-4-induced protein-DNA complex containing Smad1. Two different Smad b inding regions were localized, a distal binding region for Smad1 containing two GCAT motifs and proximal AGNC binding sites for Smad4, the latter bein g conserved in other transforming growth factor-beta-responsive elements. M utation of the Smad4 binding motif completely abolished transcriptional act ivation, whereas mutation or deletion of the Smad1 recognition sequence inh ibited Smad1/Smad4 responsiveness. These results provide a functional chara cterization and identification of a vertebrate Smad1/Smad4 DNA response ele ment induced by BMP-4 signaling and offers insight into the transcriptional regulation of a component essential for dorsoventral patterning in Xenopus embryos.