Nakazato M, Chung H K, Ulianich L, Grassadonia A, Suzuki K, Kohn L D
Cell Regulation Section, Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Mol Cell Biol. 2000 Nov;20(22):8499-512. doi: 10.1128/MCB.20.22.8499-8512.2000.
Follicular thyroglobulin (TG) selectively suppresses the expression of thyroid-restricted transcription factors, thereby altering the expression of thyroid-specific proteins. In this study, we investigated the molecular mechanism by which TG suppresses the prototypic thyroid-restricted transcription factor, thyroid transcription factor 1 (TTF-1), in rat FRTL-5 thyrocytes. We show that the region between bp -264 and -153 on the TTF-1 promoter contains two nuclear factor I (NFI) elements whose function is involved in TG-mediated suppression. Thus, NFI binding to these elements is critical for constitutive expression of TTF-1; TG decreases NFI binding to the NFI elements in association with TG repression. NFI is a family of transcription factors that is ubiquitously expressed and contributes to constitutive and cell-specific gene expression. In contrast to the contribution of NFI proteins to constitutive gene expression in other systems, we demonstrate that follicular TG transcriptionally represses all NFI RNAs (NFI-A, -B, -C, and -X) in association with decreased NFI binding and that the RNA levels decrease as early as 4 h after TG treatment. Although TG treatment for 48 h results in a decrease in NFI protein-DNA complexes measured in DNA mobility shift assays, NFI proteins are still detectable by Western analysis. We show, however, that the binding of all NFI proteins is redox regulated. Thus, diamide treatment of nuclear extracts strongly reduces the binding of NFI proteins, and the addition of higher concentrations of dithiothreitol to nuclear extracts from TG-treated cells restores NFI-DNA binding to levels in extracts from untreated cells. We conclude that NFI binding to two NFI elements, at bp -264 to -153, positively regulates TTF-1 expression and controls constitutive TTF-1 levels. TG mediates the repression of TTF-1 gene expression by decreasing NFI RNA and protein levels, as well as by altering the binding activity of NFI, which is redox controlled.
滤泡性甲状腺球蛋白（TG）选择性抑制甲状腺限制性转录因子的表达，从而改变甲状腺特异性蛋白的表达。在本研究中，我们调查了TG在大鼠FRTL-5甲状腺细胞中抑制典型甲状腺限制性转录因子甲状腺转录因子1（TTF-1）的分子机制。我们发现，TTF-1启动子上-264至-153碱基对之间的区域包含两个核因子I（NFI）元件，其功能与TG介导的抑制作用有关。因此，NFI与这些元件的结合对于TTF-1的组成型表达至关重要；TG与TG抑制相关，降低NFI与NFI元件的结合。NFI是一类转录因子，广泛表达并有助于组成型和细胞特异性基因表达。与NFI蛋白在其他系统中对组成型基因表达的作用不同，我们证明滤泡性TG通过降低NFI结合转录抑制所有NFI RNA（NFI-A、-B、-C和-X），并且RNA水平在TG处理后4小时就开始下降。虽然TG处理48小时导致DNA迁移率变动分析中测量的NFI蛋白-DNA复合物减少，但通过蛋白质免疫印迹分析仍可检测到NFI蛋白。然而，我们发现所有NFI蛋白的结合都受氧化还原调节。因此，用二酰胺处理核提取物会强烈降低NFI蛋白的结合，向TG处理细胞的核提取物中添加更高浓度的二硫苏糖醇可将NFI-DNA结合恢复到未处理细胞提取物中的水平。我们得出结论，NFI与-264至-153碱基对处的两个NFI元件结合，正向调节TTF-1表达并控制TTF-1的组成型水平。TG通过降低NFI RNA和蛋白水平，以及改变受氧化还原控制的NFI结合活性，介导TTF-1基因表达的抑制。
