Modulation of endogenous GATA-4 activity reveals its dual contribution to Müllerian inhibiting substance gene transcription in Sertoli cells.

Secretion of Müllerian inhibiting substance by fetal Sertoli cells is essential for normal male sex differentiation since it induces regression of the Müllerian ducts in the developing male embryo. Proper spatiotemporal expression of the MIS gene requires a specific combination of transcription factors, including the zinc finger factor GATA-4 and the nuclear receptor steroidogenic factor-1, which both colocalize with Müllerian inhibiting substance in Sertoli cells. To establish the molecular mechanisms through which GATA-4 contributes to MIS transcription, we have generated and characterized novel GATA-4 dominant negative competitors. The first one, which consisted solely of the GATA-4 zinc finger DNA-binding domain, was an efficient competitor of GATA transcription mediated both by direct GATA binding to DNA and protein-protein interactions involving GATA factors. The second type of competitor consisted of the same GATA-4 zinc finger DNA-binding domain but harboring mutations that prevented DNA binding. This second class of competitors repressed GATA-dependent transactivation by specifically competing for GATA protein-protein interactions without affecting the DNA-binding activity of endogenous GATA factors. These competitors, along with the GATA-4 cofactor FOG-2 (friend of GATA-2), were used to specifically modulate endogenous GATA-4 activity in Sertoli cells. Our results indicate that GATA-4 contributes to MIS promoter activity through two distinct mechanisms. Moreover, the GATA competitors described here should provide invaluable in vitro and in vivo tools for the study of GATA- dependent transcription and the identification of new target genes.