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

Abstract

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 β (LHβ) gene transcription, the mechanisms by which this occurs remain to be elucidated. We have shown previously that GH3 cells transfected with the rat GnRH receptor cDNA (GGH3-1' cells) support the expression of a cotransfected fusion gene composed of 797 base pairs of rat LHβ gene 5'-flanking sequence and the first 5 base pairs of the 5'- untranslated region fused to a luciferase reporter (-797/+5LHβLUC) 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 LHβ gene. We have now identified two putative binding sites for Sp1, a three-zinc-finger transcription factor, within this region. Using electrophoretic mobility shift assay, DNase I footprinting, and methylation interference assays, we demonstrate that Sp1 can bind to these sites and that Sp1 is responsible for DNA-protein complexes formed using GGH3-1' and αT3-1 nuclear extracts. Mutations of the Sp1 binding sites, which block binding of Sp1, blunt the stimulation of the LHβ gene promoter by GnRH. These data define GnRH-responsive elements in the LHβ 5'-flanking sequence and suggest that Sp1 plays an important role in conferring GnRH responsiveness to the LHβ subunit gene.