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

人促甲状腺激素受体的表达调控及基因启动子的功能分析

• 批准号: 10671042
• 负责人: YOSHIDA Tadashi
• 金额: $ 1.54万
• 依托单位: HOSHI UNIVERSITY (2000-2001)Keio University (1998-1999)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10671042/
• 关键词: Thyrotropic receptor (TSHR) genes; Regulation of expression of TSHR; Promoter activity; G-protein coupled receptor; Mutated receptors; Transgenic mice

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

人促甲状腺素 (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。我们之前已经证明，TSHR 的表达在 mRNA 水平上受到二丁酰-cAMP 和 TSH 的调节，并具有时间和剂量依赖性。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 kbp 基因组片段，并表征了 5' 侧翼区域的 1.2 kbp。通过引物延伸鉴定了位于 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GC含量极高，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显示出高度同源性。用重组质粒转染甲状腺细胞或 CHO-K1 细胞，该重组质粒包含与荧光素酶报告基因连接的启动子区域 1.2-kbp 的一系列连续 5'-截短和突变片段，结果表明该区域相对于转录位点位于 -10 至 -117 bp 之间，足以保证基础启动子活性、细胞特异性表达和 cAMP 的负调节。 促甲状腺激素信号。该片段内的序列包含两个元件，一个 cAMP 反应元件 (CRE) 和一个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。基础启动子活性和对 cAMP 响应的丧失位于转录起始位点 5' 21 bp 的 20 bp 部分。 TTF-1 的结合位点位于转录位点 5' 117 bp 处，决定了甲状腺特异性表达以及 cAMP 和 TSH 与 CRE 对基因的负调节。凝胶迁移率变化和突变分析表明增强子区域代表复杂转录激活结构域的结合位点。荧光素酶嵌合基因表达的附加增强子元件以及 Sp1 和 AP2 的多个潜在结合位点在核心启动子区域 5' 处被鉴定。此外，在具有不同合成 TSHR 能力的几种细胞类型中评估了增强子元件的细胞特异性存在和活性，包括人甲状腺细胞、脂肪细胞和成纤维细胞。尽管在所研究的所有细胞类型中均存在结合活性，但增强子活性仅在甲状腺细胞中得到证实。额外的转录控制位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 发生细胞特异性调节的复杂模式，这源于 TSHR 启动子的增强子元件和近端启动子之间的协作相互作用。较少的

项目成果

吉田正, 他: "医学・薬学のための免疫学"東京化学同人(東京). 83-166 (2002)

Tadashi Yoshida 等人：“医学和药学免疫学”东京化学同人（东京）83-166（2002）。

吉田正 他: "医学・薬学のための免疫学"東京化学同人 (東京). 83-166 (2002)

Tadashi Yoshida 等：“医学和药学免疫学”东京化学同人（东京）83-166（2002）。

吉田正 他: "Association between platelet GP Iba genotype and ischemic cerebic-vascular disease"Stroke. 31. 493-497 (2000)

Tadashi Yoshida 等人：“血小板 GP Iba 基因型与缺血性脑血管疾病之间的关联” 中风。

吉田 正: "バセドウ病の診断・治療"クリニカルスタディー. 20(3). 11-18 (1999)

Tadashi Yoshida：“格雷夫斯病的诊断和治疗”临床研究 20(3) (1999)。

Sonoda A., Murata M., Ito D., Tanahashi N., Ohta A., Tada Y., Takeshita E., Yoshida T., Saito I., Yamamoto M., Ikeda Y., Fukuuchi Y., Watanabe K.: "Association between platelet glycoprotein Iba genotype and ischemic cerebrovascular disease"Stroke. 31. 493

Sonoda A.、Murata M.、Ito D.、Tanahashi N.、Ohta A.、Tada Y.、Takeshita E.、Yoshida T.、Saito I.、Yamamoto M.、Ikeda Y.、Fukuuchi Y.、Watanabe K