Functional mapping of a placenta-specific upstream promoter for human gonadotropin-releasing hormone receptor gene

GnRH has been showed to regulate hCG expression and secretion from the plac enta through a GnRH receptor (GnRHR)-mediated process. Recently. we have re ported the isolation of human GnRHR full-length complementary DNA from the human placental cells including choriocarcinoma JEG-3 cells, immortalized e xtravillous trophoblasts, and primary cultures of trophoblasts. Despite the se observations. the molecular mechanism that controls the transcription re gulation of the GnRHR gene expression in the placenta remains unknown. Here we described the identification of an upstream placenta-specific promoter located between nucleotide (nt) -1737 and -1346 (relative to the translatio n start site) for the human GnRHR gene. Using transient transfection studie s, this upstream promoter has been shown to determine the placental cell-sp ecific expression of this gene. Primer extension studies further confirmed the utilization of this promoter in JEG-3 cells in vivo. By mutagenesis cou pled to functional studies, we have identified four putative transcription factor-binding sites, namely human glucocorticoid receptor (hGR)-Oct-1 (nt -1718 to -1710), hGR-cAMP response element (CRE; nt -1649 to -1641), hGR-GA TA (nt -1602 to -1597), and hGR-activating protein-1 (nt -1518 to -1511), t hat are essential to the expression of this gene. Mutations of these cis-ac ting motifs reduced the promoter activity. The CRE and GATA motifs were sub sequently shown to be placenta specific, as mutations of these motifs cause d a dramatic loss in promoter activities in the placental JEG-3 cells, but not in the ovarian carcinoma OVCAR-3, monkey kidney COS-1, and human embryo nic kidney 293 cells. Gel mobility assays confirmed the binding of nuclear proteins Oct-1, CRE-binding protein, GATA-2, GATA-3, c-Fos, and c-Jun from JEG-3 cells to these four elements.