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Fig. 4. Characterization of BMPRE gel shift binding sites. (A) 200 bp BMPRE is shown with numbered horizontal lines of selected double-stranded oligonucleotides shifted by embryonic extracts (see Fig. 3 and text). Likely binding sites for embryonic gel shifts are boxed (orange for Site B, red for C and C', blue for Site D. Nucleotide changes made in mutant oligos are shown in red under their cognate site and are also highlighted in either white or red on the 200 bp BMPRE sequence. (B) Gel shifts of putative GATA-binding sites. Oligomers 3, 5 and 7, containing Sites B, C and C', respectively, were used in EMSA experiments with nuclear extracts prepared from control COS cells, or COS cells expressing GATA4, GATA5 and GATA6. All three oligomers bind all cardiac GATAs (lanes 2-4, site B; lanes 10-12, site C; lanes 18-20, site C') and binding is greatly reduced or eliminated upon mutation of consensus GATA-binding sites within the oligomers (lanes 6-8, mutant site B; lanes 14-16, mutant site C; lanes 22-24, mutant site C'). (C) Gel shifts and antibody supershifts of either wild-type (WT) (lanes 1-3, 7-9, 13-15 and 19) or mutant (mut) (lanes 4-6, 10-12, 16-18 and 20) site B, C, C' and D oligomers with day 3 heart whole cell extracts. Supershifts for sites B (lanes 1-6), C (lanes 7-12) and C' (lanes 13-18) were performed with either control mouse Ig (lanes 1, 4, 7, 10, 13 and 16), anti-GATA4 (lanes 2, 5, 8, 11, 14 and 17) or anti-GATA-6 (lanes 3, 6, 9, 12, 15 and 18) rabbit polyclonal antibodies as indicated. (D) BMP responsiveness of either wild-type Nkx2.5-lux-CAR3 reporter or similar reporters bearing mutations in either sites B, C/C' or D (as diagrammed in A). (E-L) Representative X-gal stained F0 transient transgenic mice embryos containing either wild type or mutant Nkx2.5-lacZ-CAR3 reporters. Embryonic stages are shown in the bottom left-hand corner. Results shown are representative of the following numbers of transgenic embryos: mut Site B, 11/11 embryos; mut site C/C', 14/15 embryos (1/15 E8.5 embryos displayed residual heart staining); mut Site D, 7/7 embryos. Abbreviations are as in previous figures.





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