Fig. 7. The Tbx3 promoter is directly activated by Bmp Smads.

(A) Bmp Smads bind to the Tbx3 promoter in vivo. (Top) A comparison of the human and mouse sequences showing a conserved consensus Smad binding site approximately 1.3 kb upstream of the ATG site within the Tbx3 promoter. Two other conserved elements (GAGC sequences and GC' sequences), previously found to be required for Smad activation of the Id1 promoter (Korchynskyi et al., 2002), were identified within the Tbx3 promoter region. (Bottom) In vivo ChIP analysis utilizing extracts from embryonic hindlimb and an antibody specific for Bmp Smads demonstrated specific binding of Bmp Smads to a region containing the Smad binding element (SBE; 2). Control primers flanking an unrelated region demonstrated no specific binding with Bmp Smads (A; 1). No recruitment was found with IgG. (B) Transient transfection analysis in P19 cells. A 2 kb fragment of the Tbx3 promoter region driving luciferase expression was cotransfected into P19 cells with control vector alone (pCDNA3), or expression constructs for Smad1, Smad4, or both Smad1 and Smad4. The Tbx3 promoter exhibited a significant increase in relative luciferase activity in response to Smad1/Smad4. ^**P<0.01, ^*P<0.05, paired t-test.