Transcription of the Human Thyrotropin-Releasing Hormone Receptor Gene—Analysis of Basal Promoter Elements and Glucocorticoid Response Elements

Abstract

The gene for the human thyrotropin-releasing hormone receptor (TRHR) spans 35 kb and contains three exons and two introns (Matreet al.(1999)J. Neurochem.72, 1–11). Despite a reported transcription start site (TSS) mapped to position −885 upstream of the translation initiation codon (Iwasakiet 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 elucidate the basis for this unexpected activity, we analyzed basal promoter elements in this region of the gene. One divergent TATA box, TTTAAA in position −759, was found by mutational analysis to be critical for promoter activity, providing a likely explanation for the basal activity observed. This proximal region apparently contains several promoter elements, including Pit-1 binding sequences within the first intron of the TRHR gene as previously reported. Here we describe the analysis of two putative glucocorticoid response elements (GREs) that we identified in this region, one (distal) half site overlapping the proposed TSS at −885 and one (proximal) full site within the first intron at position −624. Accordingly, stimulation of rat pituitary GH3and GH4C1cells with dexamethasone strongly enhanced transcription activity of a reporter construct containing the distal GRE half site and the proximal GRE site. Both sites bound the glucocorticoid receptor (GR) in a specific manner. Deletion of the distal GRE half site abolished the dexamethasone induction of CAT transcription, as did mutations in the proximal site. We therefore conclude that both sites are necessary for regulation of the TRHR gene transcription by glucocorticoids.