Characterization of regulatory elements of ovine follicle-stimulating hormone (FSH) receptor gene: The role of E-box in the regulation of ovine FSH receptor expression

Abstract

Expression and activation of follicle-stimulating hormone receptor (FSHR) in the granulosa and Sertoli cells are required for normal development of the ovarian follicles and germ cells. However, little is known regarding the mechanisms by which FSHR expression is regulated. We fused an ovine FSHR promoter to a luciferase gene to understand the promoter regulation in two gonadal cell lines. Deletion studies revealed that the strongest promoter was at -200 to + 163 relative to the transcription start site. One of cis-elements protected from DNase I digestion was mapped to between +32 and +54 of the 174-base pair (bp) minimal promoter. Electrophoretic mobility shift assay with a 26-bp probe (+32 to +57) and nuclear extracts from Sertoli (15P1) and granulosa (JC-410) cell lines demonstrated a sequence-specific DNA-protein complex. Southwestern analysis detected a 43-kDa protein bound to the 26-bp probe. Gel supershift with upstream stimulatory factor 1 and 2 (USF-1/2) antibodies revealed that the DNA-protein complex contained these two transcription factors. Mutation within the E-box of the promoter abolished the sequence-specific binding and the minimal promoter activity but also greatly reduced the transcription of the proximal promoters by 49%-70%. These data suggest that the USF-1/2 binding to the promoter is required for the expression of the ovine FSHR in the gonadal cells.