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

人促甲状腺激素（TSH）以及抗TSH受体（TSHR）抗体的作用被认为是通过其受体介导的。我们以前已经表明TSHR的表达在mRNA水平上由二丁酰-cAMP和TSH以时间和剂量依赖性的方式调节。为了阐明TSHR基因的转录活性机制，我们从人TSHR基因组中分离了一个4-磷酸酶对（4-phosphase pair，kbp）的基因片段，并对该片段的5 '侧翼区进行了1.2-kbp的序列分析，通过引物延伸法确定了一个位于ATG翻译起始密码子上游101 bp处的转录起始位点。TSHR基因的启动子区富含GC，缺乏典型的TATA或CCAAT盒，但包含转录起始位点的序列与起始序列具有高度同源性。用重组质粒转染甲状腺细胞或CHO-K1细胞，所述重组质粒含有一系列1.2kbp的启动子区的5 ′-连续截短和突变片段， ...更多信息 与荧光素酶报告基因的连接显示，该区域位于相对于转录位点的-10至-117bp之间，足以进行基础启动子活性、细胞特异性表达以及cAMP和TSH信号的负调控。该片段内的序列包括两个元件，cAMP反应元件（CRE）和甲状腺特异性转录因子（TTF-1）的结合位点，其在大鼠和人类基因中高度保守。基础启动子活性的丧失和对cAMP的响应定位于位于转录起始位点5'端21 bp的20 bp区段。位于转录位点5'端117 bp处的TTF-1结合位点决定了甲状腺特异性表达以及cAMP和TSH与CRE对该基因的负调控。凝胶迁移率和突变分析表明，增强子区域代表了一个复杂的转录激活结构域的结合位点。在核心启动子区的5'端鉴定了荧光素酶嵌合基因表达的额外增强子元件和Sp1和AP 2的多个潜在结合位点。此外，在具有不同合成TSHR能力的几种细胞类型（包括人甲状腺细胞、脂肪细胞和成纤维细胞）中评估了增强子元件的细胞特异性存在和活性。虽然结合活性，在所有类型的细胞研究，增强子的活动，证明只有在甲状腺细胞。这些结果表明TSHR的细胞特异性调控是一种复杂的模式，它来源于TSHR启动子的增强子元件和近端启动子之间的协同作用。少

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