Identification of an upstream promoter in the human gonadotropin-releasinghormone receptor gene

Analysis of the human gonadotropin-releasing hormone receptor (hGnRHR) gene 5' flanking region revealed the presence of multiple TATA, CCAAT, and tran scription start sites. In addition, at least three different transcripts (5 .0, 2.5, and 1.5 kb) were detected by Northern blot analysis. Taken togethe r, these data indicated the existence of multiple promoter elements in the hGnRHR gene, and these promoters are responsible for the multiplicity of re gulation of human reproductive functions. In this report, by progressive 5' and 3' deletion (-2197 to -1351, relative to the ATG) and NotI linker scan ning mutagenesis coupled to transient transfection into the mouse gonadotro pe-derived alpha T3-1 cell, a distal promoter element was identified at -17 05/-1674. The promoter was located immediately 5' to a previously identifie d CAP site at -1673 in human pituitary and it drove a 17.6- +/- 1.0-fold in crease in reporter gene activity. Within the promoter, a pyrimidine-rich in itiator element (Inr) (-1682) and a CCAAT box (-1702) were found and mutati on of these elements abrogated both protein bindings and promoter activitie s. By 1- and 2-D South-Western blot assays, multiple nuclear factors (40 to 54 kDa) were found to interact specifically with this promoter element. Th ese nuclear factors were also present in other cells, including COS-7, JEG- 3, and SKOV-3 cells, and these findings were consistent with functional stu dies which showed that the promoter is also active in these cells. (C) 2000 Academic Press.