REGULATION OF EXPRESION OF THE HUMAN THYROTROPIN RECEPTOR AND FUNCTIONAL ANALYSIS OF THE PROMOTER OF THE GENE

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The actions of human thyrotropin (TSH) as well as anti-TSH receptor (TSHR) antibodies are thought to be mediated through their receptor. We have previously shown the expression of TSHR is regulated at the level of mRNA by dibutyryl-cAMP and TSH in a time and dose-dependent manner. To elucidate the mechanism of transcriptional activity of TSHR gene, we isolated a 4-kilobase pair (kbp) genomic fragment of the human TSHR gene and characterized 1.2-kbp of the 5'-flanking region.A transcription initiation site located 101 bp upstream of the ATG translation initiation codon has been identified by primer extention. The promoter region of TSHR gene is extremely GC-rich and lacks typical TATA or CCAAT box but the sequence encompassing the transcription initiation site shows high homology to the initiator sequence. Transfection of thyroid cells or CHO-K1 cells with recombinant plasmids containing a series of the successively 5'-truncated and mutated segment of a 1.2-kbp of the promoter region li … More nked to the luciferase reporter gene revealed that the region located between -10 and -117 bp relative to the transcriptional site sufficient for the basal promoter activity, cell-specific expression and negative regulation by cAMP and TSH signal. Sequences within this segment comprised two elements, a cAMP response element (CRE) and a binding site for the thyroid-specific transcription factor (TTF-1), which is highly conserved in the rat and human genes. Loss of basal promoter activity and response to cAMP was localized to a 20 bp section located 21 bp 5' to the transcription start site. A binding site for the TTF-1 located 117 bp 5' to the transcriptional site dictates thyroid-specific expression and negative regulation of the gene by cAMP and TSH with CRE. Gel mobility shift and mutational analysis suggest that enhancer region represents the binding site for a complex transcriptional activating domains. Additional enhancer element on the expression of the luciferase chimeric gene and multiple potential binding sites of Sp1 and AP2, were identified 5' to the core promoter region.Moreover, the cell-specific presence and activity of the enhancer element was evaluated in several cell types with varying capabilities to synthesized TSHR, including human thyroid cells, adipose cells and fibroblastic cells. Although binding activity was presented in all cell types studied, enhancer activities was demonstrated only in thyroid cells. Additional transcriptional control resided in a tissue-specific promoter, which supported transcription only in thyroid cells.These results indicate that the complex pattern of cell-specific regulation of TSHR occurs, which derives from an cooperative interactions between the enhancer element and proximal promoter of TSHR promoter. Less