Sp1 binds to the rat luteinizing hormone beta (LHbeta) gene promoter and mediates gonadotropin-releasing hormone-stimulated expression of the LHbeta 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 (LHbeta) 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 LHbeta gene 5'-flanking sequence and the first 5 base pairs of the 5'-untranslated region fused to a luciferase reporter (-797/+5LHbetaLUC) 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 LHbeta 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 alphaT3-1 nuclear extracts. Mutations of the Sp1 binding sites, which block binding of Sp1, blunt the stimulation of the LHbeta gene promoter by GnRH. These data define GnRH-responsive elements in the LHbeta 5'-flanking sequence and suggest that Sp1 plays an important role in conferring GnRH responsiveness to the LHbeta subunit gene.
Kaiser
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UB
Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA. kaiser@rascal.med.harvard.edu
Sabbagh
E
E
Chen
M T
MT
Chin
W W
WW
Saunders
B D
BD
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D018983:DNA Footprinting; D005786:Gene Expression Regulation; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D007986:Luteinizing Hormone / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D011966:Receptors, LHRH / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000378:metabolism*
D011966:Receptors, LHRH; D016329:Sp1 Transcription Factor; D007987:Gonadotropin-Releasing Hormone; D007986:Luteinizing Hormone; D004247:DNA