Cloning and Characterization of the 4.2kb Region of the Rat Thyrotropin Receptor Promoter.

Abstract

We previously identified an approximately 200 bp "minimal promoter" of the rat TSH receptor (TSHR) gene which is essential for the promoter activity. In the present study, we have cloned and characterized an upstream region of the TSHR promoter to disclose additional functional element(s). We screened a rat genomic library and obtained a DNA fragment which contained a 4.2 kb 5'-flanking region. This fragment was 2.5 kb longer than that we previously studied (1.7 kb). To assess the promoter activity, chimeric plasmids containing the 4.2 kb promoter and its 5'-deletions ligated to a chloramphenicol acetyltransferase gene were transfected into thyroid and non-thyroid cells. These plasmids expressed significant promoter activity in FRTL-5 and FRT thyroid cells, but not in BRL liver cells. The strongest promoter activity was expressed by the -199 bp promoter, and the longer promoter expressed rather decreased activity. Co-expression of thyroid transcription factor-1 (TTF-1) increased the activity of the promoter region from -3187 to -199 bp, which encompassed one or two TTF-1 binding sites we previously identified, but not the -4206 bp promoter. In addition, FRTL-5 stable transfectants each having a chimeric construct were cultured in the presence or absence of TSH. All transfectants expressed higher promoter activity in the absence of TSH than in the presence of TSH, in particular, the -3187 bp plasmid expressed significantly higher activity by comparison to the -2617 and -4206 bp constructs. This result indicates that the region between -3187 and -2617 bp may contribute to TSH/cAMP-induced suppression and also suggests that the region between -4206 and -3187 bp involves the element(s) for constitutive suppression of the promoter activity. These results not only suggest that the 4.2 kb upstream region of the TSHR gene possibly contains some elements for the regulation of the gene expression, but also emphasize the importance of the minimal promoter region which we previously identified for the efficient expression of the gene.