dorsal-lip-specific early response gene XFD-1 in Xenopus

S.Rastegar and H.Paul contributed equally to this work Ectopic expression of the ventralizing morphogen BMP-4 (bone morphogenetic protein-4) in the dorsal lip (Spemann organizer) of Xenopus embryos blocks transcription of dorsal-lip-specific early response genes. We investigated the molecular mechanism underlying the BMP-4-induced inhibition of the fork head gene XFD-1. The promoter of this gene contains a BMP-triggered inhibitory element (BIE) which prevents activation of this gene at the ventral/vegetal side of the embryo in vivo. In the present study, we show that BMP-4-induced inhibition is not direct but indirect, and is mediated by Xvent homeobox proteins. Micro-injections of Xvent-1 RNA and XFD-1 promoter dele-tion mutants demonstrate that Xvent-1 mimics the effect of BMP-4 signalling not only by suppression of the XFD-1 gene, but also by utilizing the BIE. Suppression could be reverted using a dominant-nega-tive Xvent-1 mutant. The repressor domain was local-ized to the N-terminal region of the protein. Gel-shift and footprint analyses prove that Xvent-1 binds to the BIE. Moreover, PCR-based target-site selection for the Xvent-1 homeodomain confirms distinct motifs within the BIE as preferential binding sites. Thus, biological and molecular data suggest that Xvent-1 acts as direct repressor for XFD-1 transcription and mediates BMP-4-induced inhibition