Smad2 and Smad3 differentially modulate chordin transcription via direct binding on the distal elements in gastrula Xenopus embryos

Transforming growth factor (TGF)β/activin superfamily regulates diverse biological processes including germ layer specification and axis patterning in vertebrates. TGFβ/activin leads to phosphorylation of Smad2 and Smad3, followed by regulation of their target genes. Activin treatment also induces the essential organizer gene chordin (chrd). The involvement of Smad2/3 in chrd expression has been unclear as to whether Smad2/3 involvement is direct or indirect and whether any cis-acting response elements for Smad2/3 are present in the proximal or distal regions of its promoter. In the present study, we isolated the −2250 bps portion of the chrd promoter, showing that it contained Smad2/3 direct binding sites at its distal portion, separate from the proximal locations of other organizer genes, goosecoid and cerberus. The pattern of transcription activation for the promoter (−2250 bps) was indistinguishable from that of the endogenous chrd in gastrula Xenopus embryos. Reporter gene assays and site-directed mutagenesis analysis of the chrd promoter mapped two active activin/Smad response elements (ARE1 and ARE2) for Smad2 and Smad3. For a differential chrd induction, Smad2 acted on both ARE1 and ARE2, but Smad3 was only active for ARE2. Collectively, the results demonstrate that the distal region of chrd promoter contains the direct binding cis-acting elements for Smad2 and Smad3, which differentially modulate chrd transcription in gastrula Xenopus embryos. •Chrd promoter contains two Activin/Smad2/3 response elements (ARE1 and ARE2).•Smad2 interacts with both AREs and Smad3 interacts with ARE2 only.•Chrd is a direct target of Smad2/3 in Xenopus.•Smad2/3 bound to AREs and activate chrd transcription.•AREs located in distal region of chrd promoter.