Endo T, Kaneshige M, Nakazato M, Ohmori M, Harii N, Onaya T
Third Department of Internal Medicine, Yamanashi Medical University, Tamaho, Japan.
Mol Endocrinol. 1997 Oct;11(11):1747-55. doi: 10.1210/mend.11.11.0012.
We have cloned 15 kbp of rat thyroid Na+/I- symporter gene from liver genomic library, which contains 6 kbp upstream sequence from the translation initiation site. Southern blot analysis of the genomic DNA from the liver has revealed that thyroid Na+/I- symporter gene is the single gene in the rat. To study the tissue-selective expression mechanism of the gene, we at first determined the transcriptional start site of the gene. Results of a rapid amplification of cDNA end procedure as well as that of primer extension analysis indicated that the transcriptional start sites clustered between -96, -95, and -93 bp of the gene (A in ATG is designated as +1). Chimeras containing 1.9 kbp (-1967 to -46 bp) of the 5'-flanking sequence of the Na+/I- symporter gene and luciferase gene expressed significant enzyme activity when transfected into a rat thyroid cell line, FRTL-5, but little activity was observed in BRL-3A rat liver cells. Deletion analysis of the constructs indicated that a minimal region, exhibiting promoter activity and cell specificity, is located between -291 and -134 bp of the gene. Deoxyribonuclease I footprinting shows that nuclear extracts from FRTL-5, but not BRL-3A, cells protect a region between -245 and -230 bp. Electrophoretic mobility shift assays have demonstrated that nuclear extracts from FRTL-5 cells formed a specific DNA-protein complex with an oligonucleotide probe corresponding to -250 to -211 bp of the gene, but that from BRL-3A cells did not, suggesting that thyrocyte-selective nuclear factors bind to the region. When the nuclear extracts from FRTL-5 cells were preincubated with antibody against thyroid transcription factor-1 (TTF-1), homeodomain containing nuclear protein, formation of the complex was abolished and the band was supershifted. We also found that the probe formed a DNA-protein complex with the recombinant TTF-1 homeodomain, and mutations of the binding site eliminated factor binding. When pRc/CMV-TTF-1 was cotransfected with the minimal promoter fragment of thyroid Na+/I- symporter gene into FRT cells, which express no TTF-1, it caused a significant increase in the transcriptional activity of the reporter construct, but not of the construct having mutated TTF-1-binding element. These results suggest that TTF-1 confers the cell-selective expression of Na+/I- symporter gene in thyrocytes.
我们从肝脏基因组文库中克隆了大鼠甲状腺钠/碘同向转运体基因的15kbp片段，该片段包含翻译起始位点上游6kbp的序列。对肝脏基因组DNA进行的Southern印迹分析表明，甲状腺钠/碘同向转运体基因是大鼠中的单拷贝基因。为了研究该基因的组织选择性表达机制，我们首先确定了该基因的转录起始位点。cDNA末端快速扩增以及引物延伸分析的结果表明，转录起始位点集中在该基因的-96、-95和-93bp之间（ATG中的A被指定为+1）。含有钠/碘同向转运体基因5'侧翼序列1.9kbp（-1967至-46bp）和荧光素酶基因的嵌合体在转染到大鼠甲状腺细胞系FRTL-5中时表达出显著的酶活性，但在BRL-3A大鼠肝细胞中观察到的活性很低。对构建体的缺失分析表明，一个显示启动子活性和细胞特异性的最小区域位于该基因的-291至-134bp之间。脱氧核糖核酸酶I足迹分析表明，FRTL-5细胞而非BRL-3A细胞的核提取物可保护-245至-230bp之间的区域。电泳迁移率变动分析表明，FRTL-5细胞的核提取物与对应于该基因-250至-211bp的寡核苷酸探针形成了特异性的DNA-蛋白质复合物，而BRL-3A细胞的核提取物则没有，这表明甲状腺细胞选择性核因子与该区域结合。当FRTL-5细胞的核提取物与抗甲状腺转录因子-1（TTF-1，一种含同源结构域的核蛋白）的抗体预孵育时，复合物的形成被消除，条带发生超迁移。我们还发现该探针与重组TTF-1同源结构域形成了DNA-蛋白质复合物，结合位点的突变消除了因子结合。当将pRc/CMV-TTF-1与甲状腺钠/碘同向转运体基因的最小启动子片段共转染到不表达TTF-1的FRT细胞中时，它导致报告基因构建体的转录活性显著增加，但对具有突变TTF-1结合元件的构建体则没有影响。这些结果表明，TTF-1赋予了甲状腺细胞中钠/碘同向转运体基因的细胞选择性表达。
