The human gonadotropin-releasing hormone gene (hGnRH) is synthesized in the hypothalamus, and to a lesser extent in the placenta, gonads, and the lactating mammary glands, and plays a key role in the control of reproduction through its release of pituitary gonadotropins. However, the molecular mechanisms responsible for hGnRH expression and regulation in man are largely unknown. Thus, the purpose of the proposed studies will be to elucidate several important aspects of hGnRH expression and regulation. First, the molecular mechanisms responsible for cell-specific hGnRH expression will be defined in vitro using DNA transfection studies of a GnRH-secreting hypothalamic cell line, (GN, developed in our laboratory), and a human placental cell line, JEG-3 as well as DNase I footprinting studies of the hGnRH gene utilizing nuclear extracts from these cell lines. Second, a direct role for estradiol regulation of the hGnRH gene will be studied in vitro by transfecting chimeric hGnRH gene constructs, containing a putative estrogen DNA response element (ERE), into placental and hypothalamic cell lines. The effect of various concentrations of the estrogen receptor (ER) in mediating this response will also be evaluated by co-transfecting an expression vector containing a viral promoter driving the expression of the human ER cDNA. Third, in animals and presumably man, the hGNRH neuron migrates from the olfactory placode to the hypothalamus. A chimeric construct containing the 1.1 kb of the hGnRH promoter driving the expression of the protein coding region of the simian virus 40 T antigen was used to neoplastically transform the GNRH neuron in transgenic mice. Neoplastic transformation of the GnRH neuron has interfered with its developmental migration from the olfactory placode. The cell line derived from the neoplastic transformation, GN, will be used to study cell-specific expression and estradiol regulation in vitro. Further studies to define cis-acting elements responsible for neuronal and placental-specific expression in vivo are planned utilizing 5'deletions of the hGnRH promoter fused to the luciferase reporter gene. Thus, these studies will provide new insights into the Molecular mechanisms of GNRH expression in the hypothalamus and placenta, as well as evidence that estrogen directly regulates GNRH gene expression in the hypothalamus. In addition, these studies will begin to define cis-acting elements and trans-acting factors that mediate neuronal and placental-specific expression of the hGnRH gene in man.
