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

Ursula B Kaiser,Elena Sabbagh,Marian T Chen,William W Chin,Brian D Saunders

doi:10.1074/jbc.273.21.12943

Ursula B Kaiser, Elena Sabbagh + Show 3 more

Open Access

https://doi.org/10.1074/jbc.273.21.12943

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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 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.

Highlights

The pituitary gonadotropins luteinizing hormone (LH)1 and follicle-stimulating hormone (FSH) play integral roles in the regulation of normal reproductive development and function
Using oligonucleotides corresponding to A1– A5, we performed Electrophoretic Mobility Shift Assay (EMSA) to ascertain whether GGH3-1Ј nuclear proteins were able to bind to these sequences (Fig. 1B)
LH and FSH production are highly regulated, and much of this regulation occurs at the level of gene transcription

Summary

EXPERIMENTAL PROCEDURES

Materials—The GnRH agonist des-Gly10, [D-Ala6]GnRH ethylamide (GnRHAg) was purchased from Sigma. Oligonucleotides corresponding to sense and antisense strands containing dimers of each of these five regions were synthesized, incorporating BamHI/BglII restriction enzyme sites at the ends These five oligonucleotides were each subcloned into BamHI/BglII polylinker restriction sites upstream of the rat growth hormone gene minimal promoter in GH50-pXP1, to generate LHA12GH50LUC– LHA52GH50LUC. The mutagenic primer M1 spanned region Ϫ457/Ϫ418 of the rat LH␤ gene promoter and generated a BamHI restriction site in addition to introducing the desired mutations. The mutagenic primer M2 spanned region Ϫ417/Ϫ376 of the rat LH␤ gene promoter and generated an XhoI restriction site in addition to introducing the desired mutations (see Fig. 7A). Antibody (1 ␮l) was added to the EMSA reaction samples after 30 min and incubated at 4 °C for an additional 2 h prior to gel electrophoresis.

The three arrows indicate three specific

RESULTS

DISCUSSION