Transcription of the human thyrotropin-releasing hormone receptor gene - Analysis of basal promoter elements and glucocorticoid response elements

The gene for the human thyrotropin-releasing hormone receptor (TRHR) spans 35 kb and contains three exons and two introns (Matre et at (1999) J. Neuro chem, 72, 1-11). Despite a reported transcription start site (TSS) mapped t o position -885 upstream of the translation initiation codon (Iwasaki et al . (1996) J. Biol. Chem. 271, 22183-8), we found cell type specific promoter activity directed by a fragment downstream of this site (-770 to +1). To e lucidate the basis for this unexpected activity, we analyzed basal promoter elements in this region of the gene. One divergent TATA box, TTTAAA in pos ition -759, was found by mutational analysis to be critical for promoter ac tivity, providing a likely explanation for the basal activity observed. Thi s proximal region apparently contains several promoter elements, including Pit-1 binding sequences within the first intron of the TRHR gene as previou sly reported. Here we describe the analysis of two putative glucocorticoid response elements (GREs) that we identified in this region, one (distal) ha lf site overlapping the proposed TSS at -885 and one (proximal) full site w ithin the first intron at position -624, Accordingly, stimulation of rat pi tuitary GH, and GH(4)C(1) cells with dexamethasone strongly enhanced transc ription activity of a reporter construct containing the distal GIB half sit e and the proximal GRE site. Both sites bound the glucocorticoid receptor ( GR) in a specific manner, Deletion of the distal GRE half site abolished th e dexamethasone induction of CAT transcription, as did mutations in the pro ximal site. We therefore conclude that both sites are necessary for regulat ion of the TRHR gene transcription by glucocorticoids. (C) 1999 Academic Pr ess.