Homeodomain transcription factor Hesx1/Rpx occupies Prop-1 activation sites in porcine follicle stimulating hormone (FSH) β subunit promoter

Homeodomain repressor factor Hesx1/Rpx plays a crucial role in the formation of Rathke’s pouch at the start of pituitary organogenesis and represses the Prop-1-dependent expression of Pit-1 gene, which promotes the differentiation of Pit-1-dependent hormone producing cells. Recently, we discovered a novel function of Prop-1 by which it activates the porcine follicle stimulating hormone β subunit (FSHβ) gene through Fd2 region (−852/−746). The present study aimed to determine whether Hesx1 exerts its role in the Prop-1-dependent activation of FSHβ gene.Transient transfection assay for the porcine FSHβ promoter −985/+10, electrophoretic mobility shift assay (EMSA) and DNase I footprinting analysis for Fd2 region were carried out. Transfection assay in GH3 cells demonstrated that expression of Hesx1 alone does not change the promoter activity but the coexpression with Prop-1 represses the Prop-1-dependent activation of FSHβ promoter. Similar results were obtained for the mutant reporter vector deleting the region −745/−104 indicating that Fd2 region is a target site of Hesx1 as well as Prop-1. EMSA and DNase I footprinting analysis using recombinant Hesx1 and Prop-1 protein demonstrated that Hesx1 and Prop-1 certainly bind to the AT-rich regions in a different manner. These results suggest that Hesx1 blocks the advanced expression of FSHβ gene in the early stage of pituitary development, and Prop-1 thereafter appears and activates this gene.