GATA-4 interacts distinctively with negative and positive regulatory elements in the Fgf-3 promoter

GATA-4 binds two sites in the Fgf-3 promoter, PS4A and PS13, which function as positive and negative regulatory elements, respectively. In spite of their opposite functions, both PS4A and PS13 acted as potent enhancer elements when three copies of each were appended to a minimal tk promoter. Mutational analysis showed that the negative regulatory activity of PS13 was dependent on its close proximity to the major transcription initiation site (P3), since it was a stronger repressor when moved closer to P3, but had no significant activity when moved to more distal positions. While only the C-terminal zinc finger and the basic domain of GATA-4 were required for binding to PS13, this was insufficient for binding at PS4A. In addition to the PS4A GATA site, the presence of sequences located 10-12 bp distant was required for efficient binding. Both the sequence and location of this second site was crucial for binding and enhancer activity. Truncation deletions of GATA-4 showed that efficient binding to PS4A was dependent on both zinc fingers and the basic domain, suggesting a direct interaction between one zinc finger domain and a possible second site (AGACAA) that shows some similarity to a GATA motif. GATA-4 binding to PS4A through both zinc finger domains was essential for Fgf-3 promoter activity. The substitution in PS4A of a GATA-binding sequence similar to PS13, which only requires a single zinc finger domain, bound GATA-4 efficiently but did not activate the Fgf-3 promoter. These differences in GATA-4 binding were also reflected in DNA bending assays that suggested clear conformational differences between complexes formed on PS4A and PS13.