title: A binding site for steroidogenic factor-1 is part of a complex enhancer that mediates expression of the murine gonadotropin-releasing hormone receptor gene.
abstract: 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.
authors.affiliation: Department of Physiology, College of Veterinary Medicine, Colorado State University, Fort Collins 80523, USA.
authors.forename: D L
authors.initials: DL
authors.lastname: Duval
authors.forename: S E
authors.initials: SE
authors.lastname: Nelson
authors.forename: C M
authors.initials: CM
authors.lastname: Clay
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D051717:Fushi Tarazu Transcription Factors; D015870:Gene Expression; D018398:Homeodomain Proteins; D051379:Mice; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D018160:Receptors, Cytoplasmic and Nuclear; D011966:Receptors, LHRH / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D054339:Steroidogenic Factor 1; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection
chemical_list: D004268:DNA-Binding Proteins; D051717:Fushi Tarazu Transcription Factors; D018398:Homeodomain Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011966:Receptors, LHRH; D054339:Steroidogenic Factor 1; D014157:Transcription Factors; C515058:steroidogenic factor 1, mouse; D004247:DNA