Figure 8. GATA4 directly regulates the Vegfa gene promoter.

(A) Schematic representation of the mouse VEGF promoter showing 3 putative GATA binding sites. The number of base pairs upstream of the transcription start site are denoted by –240 and –1300. (B) VEGF promoter activity shown as fold increase with Ad-GATA4 infection with Ad–β-gal infection set to 1. pGL2-basic is the backbone luciferase reporter vector with the VEGF promoter, while pGL2-control contains the SV40 promoter and is not induced by Ad-GATA4 infection. Results from a representative experiment are shown (n = 3 per promoter construct). The experiment was repeated twice with similar results. (C) Results from a similar experiment to that shown in B, except that Ad-ΔCnA (activated calcineurin) infection was used instead of Ad-GATA4, and a NFAT-luciferase reporter control adenovirus was used to show the effectiveness of Ad-ΔCnA. (D) EMSA to detect GATA4 binding to the 3 putative GATA binding sites in the Vegfa gene promoter. Oligonucleotides were incubated with unprogrammed or GATA4-programmed reticulocyte lysate. (E) Schematic of a portion of the Vegfa gene locus showing the position of the 2 primer pairs used for ChIP (arrows). (F) ChIP from GATA4 DTG hearts with GATA4 antibody or a nonspecific IgG to the promoter region or exon 8. (G) Schematic of the Vegfa gene promoter with sites 1 and 3 mutated. (H) Relative luciferase activity from cardiomyocytes transfected with the WT VEGF-1300 luciferase reporter or an identical reporter containing mutations in GATA sites 1 and 3. Myocytes were infected 24 hours prior with Ad–β-gal or Ad-GATA4. *P < 0.01 versus WT Ad–β-gal; ^#P < 0.01 versus WT Ad-GATA4.