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）和抗TSH受体（TSHR）抗体的作用被认为是通过它们的受体介导的。我们之前的研究表明，TSHR的表达在mRNA水平上受二丁基camp和TSH的调控，并呈时间和剂量依赖性。为了阐明TSHR基因的转录活性机制，我们分离了人类TSHR基因的4千碱基对（kbp）基因组片段，并对其5'侧区1.2 kbp进行了表征。通过引物延伸鉴定出位于ATG翻译起始密码子上游101 bp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，但转录起始位点周围的序列与启动子序列具有高度的同源性。用含有一系列与荧光素酶报告基因相关的启动子区域的1.2 kbp的5'连续截断和突变片段的重组质粒转染甲状腺细胞或CHO-K1细胞，发现该区域相对于转录位点位于-10和-117 bp之间，足以进行基础启动子活性，细胞特异性表达和cAMP和TSH信号的负调控。该片段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，TTF-1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的反应定位于转录起始位点21 bp 5'处的20 bp区域。TTF-1的结合位点位于转录位点的117 bp 5'处，决定了cAMP和TSH与CRE的甲状腺特异性表达和负调控。凝胶迁移转移和突变分析表明，增强子区代表了一个复杂的转录激活域的结合位点。在核心启动子区5′处发现了荧光素酶嵌合基因表达的额外增强子元件和Sp1和AP2的多个潜在结合位点。此外，在几种具有不同合成TSHR能力的细胞类型中，包括人甲状腺细胞、脂肪细胞和成纤维细胞，对增强子元件的细胞特异性存在和活性进行了评估。虽然在研究的所有细胞类型中都有结合活性，但增强子仅在甲状腺细胞中表现出活性。额外的转录控制存在于组织特异性启动子中，该启动子仅在甲状腺细胞中支持转录。这些结果表明，TSHR的细胞特异性调控发生了复杂的模式，这源于增强子元件与TSHR启动子的近端启动子之间的合作相互作用。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