E2F and GATA-1 are required for the Sertoli cell-specific promoter activity of the follicle-stimulating hormone receptor gene

The follicle-stimulating hormone receptor (FSHR) gene is expressed in Serto li cells in males and in granulosa cells in females. Cis-acting sequences a nd associated binding factors responsible for the transcription of the TATA -less FSHR gene in Sertoli cells were analyzed with dimethylsulfate (DMS) f ootprinting assays and electrophoretic mobility shift assay (EMSA). In vivo footprints in the core promoter using nuclear proteins from Sertoli cells identified several protected sequences, including an inverted GATA (TATC, - 88/-85), and an E2F (TTTCGCG, -45/-39) motif. EMSA showed the presence of o ne or more sequence-specific proteins interacting with these potential regu latory elements. Antibody-supershift assays as well as competition assays f urther revealed that testis-specific GATA-1 recognized the inverted GATA el ement. The functional role of the potential cis-acting elements was analyze d by transient transfection assays with and without mutations of the putati ve elements. The mutational analysis indicated that the GATA and E2F elemen ts were each required for optimal promoter activity, The effects of each of the promoter elements was examined in transfections in which mutations wer e made in each of the known regulatory sites, including the E box, GATA, an d E2F sites in various combinations. All of these sites contribute to the m aximum promoter activity such that mutations of the E box, GATA, and E2F si tes eliminated nearly all promoter activity.