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Expression of the GnRH receptor (GnRH-R) gene in the anterior pituitary gland, as with the genes encoding the gonadotropin subunits, is restricted to gonadotrophs. Thus, it is conceivable that a common mechanism is involved in activating cell-specific expression of these genes. In fact, expression of the alpha- and LHbeta-subunit genes appears to require binding of the nuclear orphan receptor, steroidogenic factor-1 (SF-1). Here we have used DNA protein-binding assays to identify a high-affinity binding site for SF-1 in the proximal promoter of the murine GnRH-R gene. Southwestern blot analysis using this site as a radiolabeled probe revealed binding to a 53-kDa protein in alphaT3-1 cell extracts. Furthermore, mutation of this site led to a 58% reduction in promoter activity in the gonadotroph-derived alphaT3-1 cell line. Thus, SF-1 may represent at least one common pathway for gonadotroph-specific gene expression. In addition, we used block-replacement mutagenesis to functionally scan approximately 100 base pairs (bp) in a region that we had previously identified as critical for cell-specific promoter activity. Mutation of a partial palindrome located at -393 bp relative to the start of translation led to a 63% loss of promoter activity. Finally, a region containing both the SF-1 binding site and the -393 site was sufficient to stimulate cell-specific expression from a heterologous, minimal promoter. Thus, we suggest that a complex enhancer that includes a binding site for SF-1 mediates cell-specific expression of the GnRH-R gene.

biology of reproduction

A Binding Site for Steroidogenic Factor-1 is Part of a Complex Enhancer that Mediates Expression of the Murine Gonadotropin-Releasing Hormone Receptor Gene1