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

Abstract

Regulated synthesis of luteinizing hormone (LH) requires coordinated transcriptional control of the ␣ and LH␤ subunits in pituitary gonadotropes. Several cis-acting elements and trans-acting factors have been defined for control of the LH␤ promoter through heterologous cell culture models. In this report, we describe the identification of bipartite NF-Y (CBF/CP1) binding sites within the proximal bovine LH␤ promoter. When multimerized, one of these sites activates the heterologous, minimal HSV thymidine kinase promoter in the gonadotrope-derived cell line ␣T3-1. The functional role of the promoter-distal site in regulating the full-length bovine LH␤ promoter was assessed in vivo using transgenic mice harboring a mutant promoter linked to the chloramphenicol acetyltransferase reporter gene. While this element is important for conferring high level activity of the LH␤ promoter in pituitary, it does not appear to be essential for mediating gonadotropin-releasing hormone (GnRH) regulation. This is the first characterization of a cis-acting element within this GnRH-dependent promoter that is restricted to regulating basal expression and not GnRH-induced activity.Stimulation of gametogenesis and synthesis of sex steroids by luteinizing hormone (LH) 1 is essential for reproduction in all mammalian species. Thus, understanding the regulated synthesis and secretion of this hormone from the gonadotropes in the pituitary is paramount to understanding the reproductive process.LH is a member of the glycoprotein hormone family whose members are composed of a shared ␣ subunit that combines with unique, hormone-defining ␤ subunits. Synthesis and secretion of LH requires coordinated, gonadotrope-specific expression of the genes encoding both the ␣ and LH␤ subunits (1). Gonadotrope-specific expression of the ␣ subunit gene requires a number of cis-acting elements located within the promoterproximal 400 bp of the 5Ј-flanking region of the gene (2-7).Discernment of the essential cis-acting elements involved in expression of the LH␤ subunit has lagged behind that made for the ␣ subunit due to the lack of readily accessible, appropriate model systems.Five models have been used to uncover regulatory elements within the LH␤ promoter. These include cell lines corresponding to kidney fibroblasts (CV-1) (8, 9), modified somatotropes (GGH 3 -1Ј) (10, 11), primordial gonadotropes (␣T3-1) (12-14), and differentiated gonadotropes (L␤T2) (15, 16) as well as transgenic mice (17-21). 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). This configuration is con...

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

“…Thus, p8 may function in a similar fashion to activate the LH␤ promoter, forming an enhanceosome with factors known to be important for basal expression of LH␤. In fact, one of the numerous factors that HMGA has been shown to interact with is NF-Y (NF-YA subunit) (56), a factor that is known to be important for activation of LH␤ (27) and FSH␤ (57) gene expression. In this regard, determining the protein partners of p8 in L␤T2 cells may provide insight toward its function in LH␤ gene expression.…”

“…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.…”

Molecular Physiology of Pituitary Development: Signaling and Transcriptional Networks

“…The distal region of the bovine LH␤ promoter contains two NFY binding sites with different affinity. The more distal highaffinity NFY site is critical for conferring high basal activity of the LH␤ promoter (142).…”

Maximal Activity of the Luteinizing Hormoneβ-Subunit Gene Requires β-Catenin

“…The Ϫ779/ϩ10-bp bovine LHB reporter construct has been described (9,17,45). The Egr1 expression vector (CMVNGF1A) was kindly provided by Dr. Jeffery Milbrandt (Washington University Medical School, St. Louis, MO) (46); the murine Axin1 in a pcS2ϩMT expression vector was generously provided by Dr. Frank Costantini [Columbia University, New York, NY (30)]; and the murine ⌬ 90 ␤-catenin in the pUHD10-3 vector was provided by Dr. James Nelson (Stanford University of Medicine, Palo Alto, CA) (34).…”