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

Expression and activation of follicle-stimulating hormone receptor (FSHR) i n the granulosa and Sertoli cells are required for normal development of th e ovarian follicles and germ cells. However, little is known regarding the mechanisms by which FSHR expression is regulated. We fused an ovine FSHR pr omoter to a luciferase gene to understand the promoter regulation in two go nadal cell lines. Deletion studies revealed that the strongest promoter was at -200 to +163 relative to the transcription start site. One of cis-eleme nts protected from DNase I digestion was mapped to between +32 and +54 of t he 174-base pair (bp) minimal promoter. Electrophoretic mobility shift assa y 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-prot ein complex. Southwestern analysis detected a 43-kDa protein bound to the 2 6-bp probe. Cel supershift with upstream stimulatory factor 1 and 2 (USF-1/ 2) antibodies revealed that the DNA-protein complex contained these two tra nscription factors. Mutation within the E-box of the promoter abolished the sequence-specific binding and the minimal promoter activity but also great ly reduced the transcription of the proximal promoters by 49%-70%. These da ta suggest that the USF-1/2 binding to the promoter is required for the exp ression of the ovine FSHR in the gonadal cells.