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J Biol Chem, Vol. 273, Issue 21, 12943-12951, May 22, 1998

Sp1 Binds to the Rat Luteinizing Hormone  (LH) Gene Promoter and Mediates Gonadotropin-releasing Hormone-stimulated Expression of the LH Subunit Gene

From the Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115

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  (LH) 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 LH gene 5'-flanking sequence and the first 5 base pairs of the 5'-untranslated region fused to a luciferase reporter (797/+5LHLUC) 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 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 T3-1 nuclear extracts. Mutations of the Sp1 binding sites, which block binding of Sp1, blunt the stimulation of the LH gene promoter by GnRH. These data define GnRH-responsive elements in the LH 5'-flanking sequence and suggest that Sp1 plays an important role in conferring GnRH responsiveness to the LH subunit gene.

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