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 ␤ (LH␤) 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␤ gene 5-flanking sequence and the first 5 base pairs of the 5-untranslated region fused to a luciferase reporter (؊797/؉5LH␤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␤ gene. We have now identified two putative binding sites for Sp1, a three-zincfinger 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 GGH 3 -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. The pituitary gonadotropins luteinizing hormone (LH)1 and follicle-stimulating hormone (FSH) play integral roles in the regulation of normal reproductive development and function. The biosynthesis and secretion of these pituitary glycoproteins are controlled by the complex interaction of multiple factors, among the most important of which is gonadotropin-releasing hormone (GnRH). Pulsatile GnRH stimulates the secretion of LH and FSH as well as transcription, steady-state mRNA levels, and biosynthesis of the gonadotropin subunits ␣, LH␤, and FSH␤ (1-4). This regulation is dependent on GnRH pulse amplitude and frequency, which varies with physiologic state, during puberty, during the rat estrous and human menstrual cycles, and during menopause (5, 6). An understanding of the mechanisms of regulation of LH␤ gene expression is an important first step in elucidating the mechanisms of physiologic differential regulation of LH and FSH by GnRH.Studies of the gonadotropin ␣-subunit gene have identified a number of DNA elements in the 5Ј-flanking region that mediate tissue-specific and regulated expression and their cognate binding factors. Recently, two transcription factors, steroidogenic factor-1 (SF-1) and early growth response-1 (Egr-1), have been recognized to be involved in expression of the LH␤ gene (7-9). Nevertheless, relatively little is known about transcription factors that direct gonadotrope-specific or hormonally regulated expression of the LH␤ and FSH␤ subunit genes. A systematic approach to identifying mechanisms of hormonal re...

The regulation of neuroendocrine function: Timing is everything

“…POMC (ACTH, MSH) No (Drouin et al, 1985) FSH No (Kumar, 1994-direct submission) GH No (Das et al, 1996) LH No (Kaiser et al, 1998) Prolactin No (Gubbins et al, 1980) TSH (beta subunit) Yes (3) (Croyle and Maurer, 1984) …”

Embryonic Expression of the Luteinizing Hormone β Gene Appears to Be Coupled to the Transient Appearance of p8, a High Mobility Group-related Transcription Factor

“…In this case, however, the elements consist of those that bind early growth response 1 (Egr1) (11)(12)(13)(14)(15)(16), steroidogenic factor 1 (SF-1) (11)(12)(13)(14)(15)(16)(17)(18)(19)(20), Pitx1 (21)(22)(23), and an as yet unidentified Otx-related homeodomain protein (24), as well as proteins that bind elements in the distal domain, such as nuclear factor-Y (NF-Y) and Sp1 (16,(25)(26)(27). Unlike ␣GSU, which is a marker of early pituitary development, expression of the LH␤ gene is one of the last steps that defines a mature gonadotrope.…”

An NF-Y Binding Site Is Important for Basal, but Not Gonadotropin-releasing Hormone-stimulated, Expression of the Luteinizing Hormone β Subunit Gene

“…These include cell lines corresponding to kidney fibroblasts (CV-1) (8,9), modified somatotropes (GGH 3 -1Ј) (10,11), primordial gonadotropes (␣T3-1) (12)(13)(14), and differentiated gonadotropes (L␤T2) (15,16) as well as transgenic mice (17)(18)(19)(20)(21). Using these approaches, four transcription factors have been identified that regulate expression of the LH␤ subunit gene through direct binding to its promoter (see Fig.…”

“…1A). These include the orphan nuclear receptor, SF-1 (8,15,21), an early growth response protein, Egr-1 (22,23), a bicoid-related homeodomain protein, Pitx1 (9), and the ubiquitous transcription factor, Sp1 (11). There are two Egr-1 and SF-1 binding sites that exist in a pairwise conformation interrupted by a Pitx1 binding site within the promoter-proximal 150 bp (24).…”

“…Rather, Pitx1 can interact directly with SF-1 and Egr-1 to induce expression (25). Sp1 regulates the rat LH␤ promoter by binding two elements that reside further upstream in the promoter at (Ϫ451/Ϫ428) and (Ϫ411/ Ϫ386) (11). This region is less conserved across species than the promoter-proximal sequences, and its importance in other species remains to be determined.…”