Sp1 binds to the rat luteinizing hormone β (LHβ) gene promoter and mediates gonadotropin-releasing hormone-stimulated expression of the LHβ subunit gene

The hypothalamic hormone gonadotropin-releasing hormone (GnRH) plays a critical role in reproductive function by regulating the biosynthesis and secretion of the pituitary gonadotropins. Although it is known that GnRH induces luteinizing hormone beta (LH beta) gene transcription, the mechanisms by which this occurs remain to be elucidated. We have shown previously that GH(3) cells transfected with the rat GnRH receptor cDNA (GGH(3)-1' cells) support the expression of a cotransfected fusion gene composed of 797 base pairs of rat LH beta gene 5'-flanking sequence and the first 5 base pairs of the 5'-untranslated region fused to a luciferase reporter (-797/+5LH beta LUC) and respond to a GnRH agonist with a 10-fold stimulation of activity. Furthermore, we have shown that DNA sequences at -490/-352 confer GnRH responsiveness to the rat LH beta gene. We have now identified two putative binding sites for Spl, a three-zinc-finger transcription factor, within this region. Using electrophoretic mobility shift assay, DNase I footprinting, and methylation interference assays, we demonstrate that Spl can bind to these sites and that Spl is responsible for DNA-protein complexes formed using GGH(3)-1' and alpha T3-1 nuclear extracts. Mutations of the Spl binding sites, which block binding of Spl, blunt the stimulation of the LH beta gene promoter by GnRH, These data define GnRH-responsive elements in the LH beta 5'-flanking sequence and suggest that Spl plays an important role in conferring GnRH responsiveness to the LH beta subunit gene.