Melatonin activates membrane-bound G-protein-coupled receptors mt1 and MT2, but may also bind a family of orphan nuclear receptors, including RORα and RZRβ, representing another potential molecular mechanism of melatonin action. Recently, we demonstrated that melatonin downregulates gonadotropin-releasing hormone (GnRH) gene expression in the GT1–7 cell line, specifically at the level of the neuron-specific GnRH gene enhancer. In this study, we have examined the region located at –1736/–1728 of the GnRH enhancer shown to be involved in the repression of GnRH by melatonin. This region includes hexameric consensus binding sites for orphan nuclear receptors, including ROR/RZR and COUP-TFI, as well as other defined consensus binding sites for AP-1 and C/EBP. Using electrophoretic mobility shift analysis (EMSA), we have demonstrated that GT1–7 nuclear proteins bind specifically to this region of the GnRH enhancer to form 4 complexes. EMSA antibody supershift analysis indicates that the transcription factors COUP-TFI and C/EBP beta bind two specific complexes, but RORα, Oct-1, Pbx-1, c-fos, or c-jun antibodies failed to produce any detectable supershifts. These results provide the first evidence that melatonin may mediate its direct neuroendocrine control of GnRH gene expression through transcription factor binding at specific regions of the GnRH enhancer.

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