SOX7 and GATA-4 are competitive activators of Fgf-3 transcription.

Fgf-3 is expressed in a dynamic and complex spatiotemporal pattern during mouse development. Previous studies identified GATA-4 as a transcription factor that binds the key regulatory element PS4A of the Fgf-3 promoter and stimulates transcription. Here we show that members of the SOX family of transcription factors also bind PS4A and differentially modulate transcription. At least five SOX genes, Sox2, Sox6, Sox7, Sox13, and Sox17, were expressed in F9 cells, and of these, Sox7 and Sox17 were dramatically induced in parallel with Fgf-3 following differentiation into parietal endoderm-like cells with retinoic acid and dibutyryl cAMP. Complexes could be detected on PS4A with SOX2, SOX7, and SOX17 by using nuclear extracts from differentiated F9 cells. However, only Sox7 expression markedly activated the Fgf-3 promoter in these cells. By contrast, SOX2 was a poor activator of Fgf-3 transcription, and when Sox2 was coexpressed with Gata4, it negatively modulated the strong activation mediated by GATA-4. More detailed analyses showed that SOX7 competes with GATA-4 for PS4A occupancy and to activate the Fgf-3 promoter. In situ hybridization analysis showed that Sox7 is co-expressed with Fgf-3 and Gata4 in the parietal endoderm of E7.5 mouse embryos. In culture, GATA-4-deficient embryonal stem cells were shown to express Fgf-3 upon differentiation into embryoid bodies, although at lower levels than were found in wild type embryonal stem cells. This Fgf-3 expression was virtually abolished when Sox7 expression was suppressed by RNA interference. These results show that SOX7 is a potent activator of Fgf-3 transcription.

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.