Fig. 5. SMAD-dependent BMP response is associated with site D activity. (A) BMPRE sequence is displayed. Colored horizontal lines show extent of double-stranded oligonucleotides used to create multimerized enhancers encompassing SBE1 plus GATA6 and GATA4/5 binding sites (SMAD region 1 or SR1, orange line), SBE2 plus GATA4-binding sites (SMAD region 2 or SR2, red line) and SBE3 plus binding site D (SMAD region 3 or SR3, blue line). Site D and SBE3 mutations are displayed below sequence of BMPRE in red, and corresponding altered nucleotides are displayed as white. (B) BMP response of Nkx2.5-lux driven by either the 2 kb CAR3, the 200 bp BMPRE, or 5x-multimerized repeats of SR1, 2 or 3. Reporters are shown on the left, with colored arrowheads representing each repeat and its orientation. Cognate BMP response is shown to the immediate right of the construct schematic, and is expressed as fold activation over basal activity. (C) BMP induction of SR3 requires both binding site D and SBE3. BMP response of Nkx2.5-lux driven by either 4x-multimerized repeats of SR3-WT, or multimers of SR3 bearing mutations in either binding site D or associated SMAD-binding element 3 (SBE3) (shown in A). (D) SMAD4 dependence of SR3-mediated BMP response. BMP4 response of Nkx2.5-lux-4xSR3 was assayed in the SMAD4-deficient MDA-MB468 cell line in the absence or presence of co-transfected SMAD1 and SMAD4 expression vehicles. Activation is seen only in the presence of both co-expressed SMAD proteins and BMP4 (bottom lanes).