The cell-specific expression of gonadotropin-releasing hormone (GnRH) is essential for the coordinate regulation of the mammalian reproductive system. The relatively small numbers of GnRH neurons and their diffuse locations has made the study of GnRH difficult. The human GnRH gene has been cloned and it's transcriptional start site mapped in the hypothalamus and placenta. In this proposal, DNA sequences within the 5' flanking region of the human GnRH (hGnRH) gene important for cell- specific expression will be characterized. Trasgenic mice have been generated using gene constructs consisting of various deletions of the hGnRH promoter fused to the luciferase reporter gene (LUC), yielded a preliminary localization of a GnRH cell-specific element. Additional transgenic experiments will prove whether this element is sufficient for GnRH cell-specific expression. The GnRH cell-specific element will also be mapped in vitro using GnRH-expressing neuronal cell lines. Two high related cell lines NLT and Gn11, which express GnRH mRNA at widely different levels, are available for this purpose. DNAse I footprinting will be used to determine which sequences of the hGnRH promoter are bound by nuclear proteins from GnRH-expressing cellular extracts. To identity nuclear proteins bound to functionally important cis-acting elements, gel-shift analyses will be performed with antibodies directed at candidate proteins. Brain 2(Brn-2) mRNA has been identified in one GnRH cell line (NLT) expressing high amount of GnRH mRNA but not in another cell line (Gn11) expressing low amounts of GnRH mRNA, suggesting it may serve as a cell-specific activator of the GnRH gene. Brn-2 and other POU protein expression vectors will be transfected into Gn11 cells determine if they can activate the GnRH promoter via its cell-specific element. Finally, the presence of POU proteins in the GnRH neuron will be determined using in situ hybridization studies. Thus, cis-acting elements and transcriptional factors responsible for the control of GnRH gene expression in the hypothalamus will be isolated and characterized. These studies will provide important molecular insights into the control of human reproduction and suggest transcription factors which might be defective in certain pathological conditions of the reproductive axis.
