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.