Abstract
We investigated the cis-acting elements and trans-acting proteins required for the transcriptional inhibition of the gonadotropin-releasing hormone (GnRH) gene by human chorionic gonadotropin (hCG) in GT1-7 neurons. Transient transfection of GT1-7 neurons with the 5'-flanking region of the rat GnRH gene-luciferase fusion constructs revealed that a 53-base pair (bp) sequence between -126 and -73 bp is required for the hCG inhibition. Nuclear extracts from GT1-7 neurons contained 110- and 95-kDa proteins that formed two complexes with the 53-bp sequence. These proteins are not related to Fos, cAMP response element-binding protein, Oct-1, or progesterone receptors, and hCG treatment selectively increased the 95-kDa protein. DNase I footprinting with GT1-7 cell nuclear extracts protected the -99 to -79-bp region, which contained a so-called imperfect AP-1 site (-99 to -94 bp) and two AT-rich palindromic sequences (-91 to -87 bp and -85 to -81 bp). The mutagenesis of the AT-rich regions, but not the AP-1 site, resulted in a loss of DNA binding of the 95-kDa protein and the inhibitory effect of hCG. In summary, our results are consistent with hCG inducing a 95-kDa trans-acting protein, which binds to -91- to -81-bp AT-rich sequences ...Continue Reading
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