Structure of the rat gonadotropin releasing hormone (rGnRH) gene promoter and functional analysis in hypothalamic cells

Abstract

The gonadotropin releasing hormone (GnRH) gene encodes a protein which plays a critical role in mammalian reproductive physiology. Its expression is predominantly restricted to the hypothalamus although it has also been described in the placenta. To begin to determine the promoter elements important for tissue specific expression and to examine the mechanisms of developmental and hormonal regulation of the rat GnRH (rGnRH) gene, we cloned the rGnRH gene from a rat liver genomic DNA library. The nucleotide sequence of greater than 3 kb of 5'-flanking region was determined. The transcriptional initiation site in rat hypothalamic tissue and a mouse hypothalamic cell line were mapped by primer extension analysis and found to be different. In addition, transient transfection studies demonstrated that multiple regions of the distal promoter are important for tissue specific and basal promoter activity in hypothalamic cells. Furthermore, in these cells a potent activation region resides between -3026 and -1031 bp and suppressor region between -1031 and -903 bp upstream of the transcriptional start site. We conclude that different portions of the 5'-flanking region, which are activating and suppressing in nature, are critical for hypothalamic expression of the rGnRH gene.

Liganded Androgen Receptor Interaction with β-Catenin

“…Cells were also transfected with 0.1 g of RSV␤gal to control for transfection efficiency. Cells were harvested 16 -18 h post-transfection and assayed for luciferase and ␤-galactosidase activities as described previously (37). Statistical Analysis-Comparisons between ethanol control, and 5␣-DHT-treated samples were analyzed using unpaired t test using GraphPad InStat version 3.02 for Windows, GraphPad Software, San Diego, CA.…”

Adhesion-related Kinase Repression of Gonadotropin-releasing Hormone Gene Expression Requires Rac Activation of the Extracellular Signal-regulated Kinase Pathway

“…A HindIII fragment containing nucleotides Ϫ3026 to ϩ116 of the rat GnRH promoter was ligated into the HindIII site of pA 3 LUC (rGnRH-LUC), placing the GnRH promoter upstream from the LUC coding region (10). PRK5-Ark contains the mouse Ark cDNA and was provided by Paola Bellosta (New York University) (17).…”

“…The total amount of plasmid was maintained constant at 20 -25 g with the inclusion of the appropriate empty vector. Cells were harvested 16 -18 h posttransfection, and the lysate was assayed for LUCand ␤-gal activities as described previously (10). For the MEK1-R4F, MEK1-8E, MEK5(D), and MEK5(A) transfections, 3-10 g of plasmid was added to the assay.…”

“…Although GnRH expression is tightly controlled during development, puberty, and in the adult, the underlying mechanisms involved in its regulation remain unknown. We and others have used immortalized GnRH neuronal cell lines that retain properties of in vivo GnRH neurons to study GnRH neuronal physiology (5)(6)(7)(8)(9)(10). Two types of GnRH neuronal cell line exist, migratory and postmigratory.…”

Heterogeneous nuclear ribonucleoprotein A/B and G inhibits the transcription of gonadotropin-releasing-hormone 1

“…Using immortalized GnRH1 releasing cell lines (GT1-7) (Liposits et al, 1991;Mellon et al, 1990;Weiner et al, 1992;Wetsel, 1995), a proximal promoter Kepa et al, 1992) and distal enhancer regions (Kepa et al, 1996;Lawson et al, 1996;Whyte et al, 1995) critical for mammalian GnRH1 gene regulation have been identified. The proximal promoter is important for basal GnRH1 gene expression and the distal promoter for GnRH1 neuron-specific expression because of an enhancer region.…”