FIG. 3.

Hey factors downregulate the GATA4 promoter. (A) Dose-dependent repression of GATA4; 1 μg of the 10.6-kb GATA4 luciferase reporter was coexpressed in 293 cells with empty pCS2 plasmid or with increasing amounts (0.1, 0.25, 0.5, 0.75, and 1.0 μg) of pCS2-Hey1, -Hey2, or -HeyL expression vector. Inset: Equal protein expression levels of Hey1, Hey2, and HeyL in luciferase experiments were confirmed by Western blotting (B) Deletion analysis of the mouse GATA4 promoter. Hey factors are able to repress the minimal 0.5-kb promoter. (C) Mutation of the E-box CACGTG by introducing a 25-bp oligonucleotide in the 10.6-kb GATA4 promoter almost abolishes its basal activity. However, Hey1 can still repress this reporter construct. (D) Mutation analysis of the 0.5-kb GATA4 promoter. Destruction of the E-box by two point mutations does not prevent repression of the GATA4 promoter by Hey factors, indicating that Hey proteins act in a manner independent of interaction with their proposed DNA binding site. For all experiments, basal luciferase activity with empty pCS2 plasmid was given a value of 100%.