Bodenner D L, Mroczynski M A, Weintraub B D, Radovick S, Wondisford F E
Molecular, Cellular, and Nutritional Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
J Biol Chem. 1991 Nov 15;266(32):21666-73.
The first exon of the human thyrotropin-beta (hTSH beta) gene has been demonstrated in our laboratory to contain a major thyroid hormone inhibitory element. In order to characterize fully this element, we have performed a detailed functional and structural scanning mutational analysis of this element. Various -1192 to +37 (base pairs) bp fragments of the hTSH beta gene containing consecutive five deoxythymidine substitution mutations of the first exon were inserted into a luciferase reporter plasmid and transiently transfected into human embryonal cells (293) and stably transfected into rat pituitary cells (GH3). Two domains (domain 1 and 2) were identified by scanning mutations that were essential for function of the thyroid hormone inhibitory element: +3 to +13 bp and +28 to +37 bp. Biotinylated DNA fragments containing -12 to +43 bp of the hTSH beta gene and the identical scanning mutations demonstrate that in vitro synthesized c-erbA-beta binding is disrupted as much as 95% by mutations from -3 to +17 bp and to a lesser extent (20-30%) by mutations from +23 to +27 bp and from +33 to +43 bp. Domain 1 displayed a higher affinity for c-erbA-beta than domain 2 in avidin-biotin complex DNA-binding and gel-mobility assays. Using increasing amounts of in vitro synthesized c-erbA-beta, we were unable to demonstrate more than one protein-DNA complex in gel-mobility assays. However, using the avidin-biotin complex DNA-binding assay and the cross-linking reagent, 1,6-bismaleimidohexane, we were able to demonstrate thyroid hormone receptor dimer formation on domain 1 but not to any significant extent on domain 2. In conclusion, functional and DNA-binding studies suggest that the thyroid hormone receptor binds to two distinct regions in the first exon of the hTSH beta gene. The upstream site (domain 1) binds c-erbA-beta with higher affinity and is capable of binding c-erbA-beta as a dimer under some conditions, while the downstream site (domain 2) appears to bind a single molecule of c-erbA-beta with lower affinity. These results suggest that thyroid hormone receptor, binding to at least two sites in the first exon, act in conjunction to mediate T3 inhibition of hTSH beta expression.
在我们实验室已证实，人类促甲状腺激素β（hTSHβ）基因的第一个外显子含有一个主要的甲状腺激素抑制元件。为了全面表征该元件，我们对其进行了详细的功能和结构扫描突变分析。将包含hTSHβ基因第一个外显子连续五个脱氧胸苷替代突变的各种-1192至+37（碱基对）碱基片段插入荧光素酶报告质粒，并瞬时转染到人胚胎细胞（293）中，并稳定转染到大鼠垂体细胞（GH3）中。通过扫描突变鉴定出两个对甲状腺激素抑制元件功能至关重要的结构域（结构域1和结构域2）：+3至+13碱基对和+28至+37碱基对。含有hTSHβ基因-12至+43碱基对以及相同扫描突变的生物素化DNA片段表明，体外合成的c-erbA-β结合在-3至+17碱基对的突变中被破坏多达95%，而在+23至+27碱基对以及+33至+43碱基对的突变中被破坏程度较小（20%-30%）。在抗生物素蛋白-生物素复合物DNA结合和凝胶迁移分析中，结构域1对c-erbA-β的亲和力高于结构域2。使用增加量的体外合成c-erbA-β，我们在凝胶迁移分析中无法证明存在多个蛋白质-DNA复合物。然而，使用抗生物素蛋白-生物素复合物DNA结合分析和交联剂1,6-双马来酰亚胺己烷，我们能够证明甲状腺激素受体在结构域1上形成二聚体，但在结构域2上没有明显形成。总之，功能和DNA结合研究表明，甲状腺激素受体与hTSHβ基因第一个外显子中的两个不同区域结合。上游位点（结构域1）以更高的亲和力结合c-erbA-β，并且在某些条件下能够以二聚体形式结合c-erbA-β，而下游位点（结构域2）似乎以较低的亲和力结合单个c-erbA-β分子。这些结果表明，甲状腺激素受体与第一个外显子中的至少两个位点结合，共同作用以介导T3对hTSHβ表达的抑制。
