Espinoza C R, Schmitt T L, Loos U
Department of Internal Medicine I, University Clinic of Ulm, D-89081 Ulm, Germany.
J Mol Endocrinol. 2001 Aug;27(1):59-67. doi: 10.1677/jme.0.0270059.
Thyroglobulin (Tg) is an essential thyroid-specific protein, which serves as the matrix for thyroid hormone biosynthesis. To obtain new insights in the regulation of Tg gene expression, we investigated the interaction of the human Tg promoter with the thyroid-specific transcription factors TTF-1 and Pax8. A reporter gene, containing a 202 bp fragment from the human Tg 5'-flanking region including the promoter sequence and the transcriptional start site, and expression vectors containing the cDNAs for human TTF-1 and Pax8 were used in cotransfection experiments, in the non-thyroidal cell lines COS-7 and HeLa. Pax8 increased the specific transcriptional activity of the Tg promoter about threefold, whereas cotransfection with the homeodomain-containing protein TTF-1 stimulated promoter activity from six- to tenfold. The simultaneous expression of both factors stimulated the Tg promoter activity in a multiplicative manner up to 25-fold. TTF-1 binding sites could be localized precisely by lectron mobility shift assay. The two binding elements corresponded to sites A and C in the rat Tg promoter. Site-directed mutagenesis of three nucleotides in each binding element inhibited binding of TTF-1 to the two oligonucleotides. In cotransfection experiments, the mutant site C decreased TTF-1 transactivation to 26% of the wild-type, whereas an additional mutation in the site A reduced this value to almost zero, thus proving the physiological relevance of these sites. The present results demonstrate that the activity of the human Tg promoter is closely dependent on the function of TTF-1 and Pax8, opening the field for further investigations of pathological alterations of Tg gene expression.
甲状腺球蛋白（Tg）是一种重要的甲状腺特异性蛋白，它作为甲状腺激素生物合成的基质。为了深入了解Tg基因表达的调控机制，我们研究了人类Tg启动子与甲状腺特异性转录因子TTF-1和Pax8之间的相互作用。在非甲状腺细胞系COS-7和HeLa中进行的共转染实验中，使用了一个报告基因，该基因包含来自人类Tg 5'侧翼区域的202 bp片段，包括启动子序列和转录起始位点，以及包含人类TTF-1和Pax8 cDNA的表达载体。Pax8使Tg启动子的特异性转录活性增加了约三倍，而与含同源结构域的蛋白TTF-1共转染则刺激启动子活性提高了6至10倍。两种因子的同时表达以倍增方式刺激Tg启动子活性，最高可达25倍。通过电泳迁移率变动分析可以精确地定位TTF-1结合位点。这两个结合元件对应于大鼠Tg启动子中的A位点和C位点。对每个结合元件中的三个核苷酸进行定点突变可抑制TTF-1与这两个寡核苷酸的结合。在共转染实验中，突变的C位点使TTF-1的反式激活作用降至野生型的26%，而A位点的额外突变则使该值几乎降至零，从而证明了这些位点的生理相关性。目前的结果表明，人类Tg启动子的活性紧密依赖于TTF-1和Pax8的功能，这为进一步研究Tg基因表达的病理改变开辟了领域。
