Analysis of the thyroid transcription factor-1(TTF-1) gene expression in thyroid neoplastic tissues.

研究了甲状腺转录因子-1（TTF-1）基因在甲状腺中表达的调控。我们发现了一个新的转录起始位点，位于核苷酸（ut）-1917处，在先前描述的位点上游-1700 bp处，并且该区域编码从nt -1242到-14作为第一内含子。用FRTL-5细胞mRNA进行的北方印迹分析显示，靶向外显子2的探针识别3.7和2.7 kb的转录本，而靶向未翻译的外显子1和第二内含子的两种探针仅检测到3.7 kb的转录本，这表明3.7 kb的mRNA是从nt-1917转录的，并且它包含sacond内含子。氯霉素乙酰转移酶（CAT）报告基因检测表明，该基因的5 '侧翼区在FRTL-5细胞中具有启动子活性，而在大鼠肝细胞中没有，表明该区域具有甲状腺特异性表达的特性。两个共识TTF-1的结合基序检测在这个启动子区域，电泳迁移率变动分析表明，寡核苷酸探针，每个含有这些基序之一，形成了一个复合物与重组TTF-1同源结构域。此外，重组TTF-1增加了缺乏TTF-1的细胞的转录活性。这些结果表明，转录从新确定的TTF-1基因的起始位点是正调控TTF-1在甲状腺。

Regulation of the thyroid transcription factor-1(TTF-1) gene expression in the thyroid was investigated. We identified a new transcription start site as nucleotide (ut)-1917, -1700 bp upstream of previously described site, and the region encom passing nt -1242 to -14 as the first intron. Northern blot analysis with FRTL-5 cell mRNA revealed that a probe targeted to exon 2 recognized 3.7 and 2.7 kb-transcripts, whereas both probes targeted to the untranslated exon 1 and the second intron deteced only the 3.7 kb-transcript, suggesting that the 3.7 kb-mRNA is transcribed from nt -1917 and that it contains the sacond intron. Chloramphenicol acetyltransferase (CAT) reporter gene assays demonstrated that the 5'-flanking region exhibited promoter activity in FRTL-5 cells but not in rat liver cells, indicating that this region confers the property of thyroid-specific exprression. Two consensus TTF-1 binding motifs were detected in this promoter region, and electrophoretic mobility-shift assays showed that oligonucleotide probes, each containing one of these motifs, formed a complex with the recombinant TTF-1 homeodomain. Moreover, recombinant TTF-1 increased the transcriptional activity in cells lacking TTF-1. These results suggest that transcriptional from the newly identified start site in the TTF-1 gene is positively regulated by TTF-1 in the thyroid.