STEROID-HORMONE REGULATION AND TISSUE-SPECIFIC EXPRESSION OF THE HUMAN GNRH GENE IN CELL-CULTURE AND TRANSGENIC ANIMALS

In order to study the molecular mechanisms involved in the control of GnRH. gene expression, the human GnRH gene was cloned and characterize d. The gene was expressed in cells obtained from CNS tumors in transge nic mice generated utilizing 1131 bp of 5' flanking GnRH DNA fused to the simian virus 40 large T antigen. We have shown a stimulatory estro gen response element in the human GnRH gene by transient transfection studies. DNase I footprinting and an avidinbiotin DNA binding assay de monstrated that the human GnRH gene bound ER. The GN cell line was fou nd to have nuclear ERs utilizing an I-125 estradiol binding study and by in situ hybridization histochemistry. In order to study GnRH expres sion in vivo, either 5000 or 484 bp of GnRH flanking DNA was fused to the luciferase (Luc) reporter gene, and transgenic mice generated. Exp ression in the transgenic animals was found in the hypothalamus of ani mals bearing the -5000Luc transgene, but not in animals bearing the -4 84Luc transgene. The transgenic mice expressing the -5000Luc gene were gonadectomized resulting in a 20-30% increase in hypothalamic Luc exp ression in the males and a 65% increase in females, while mice who wer e gonadectomized and replaced with testosterone (males) or E(2) (femal es) showed a 50% decrease in Luc expression over control levels. Thus, these studies present in vitro evidence of E(2) modulation of GnRH ge ne expression and an in vivo model in which sensitive studies of GnRH regulation and expression can be performed. (C) 1994 Academic Press, I nc.