A detailed functional and structural analysis of a major thyroid hormone inhibitory element in the human thyrotropin beta-subunit gene.
A detailed functional and structural analysis of a major thyroid hormone inhibitory element in the human thyrotropin beta-subunit gene.
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对人促甲状腺素β亚基基因中主要甲状腺激素抑制元件的详细功能和结构分析。
DOI：
10.1016/s0021-9258(18)54688-3
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
F. Wondisford
中科院分区：
文献类型：
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作者：
D. L. Bodenner;Melisa A. Mroczynski;Bruce D. Weintraub;Sally Radovick;F. Wondisford
我们的实验室已经证明人类促甲状腺素β（hTSH β）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。为了充分表征该元件，我们对该元件进行了详细的功能和结构扫描突变分析。将含有第一外显子连续5个脱氧胸苷取代突变的hTSH β基因的各种-1192至+37（碱基对）bp片段插入荧光素酶报告质粒中，并瞬时转染至人胚胎细胞（293）和稳定转染至大鼠垂体细胞（GH 3）。通过扫描对甲状腺激素抑制元件的功能至关重要的突变鉴定了两个结构域（结构域1和2）：+3至+13 bp和+28至+37 bp。含有-12至+43 bp hTSH β基因和相同扫描突变的生物素化DNA片段表明，体外合成的c-erbA-β结合被-3至+17 bp突变破坏高达95%，而被+23至+27 bp和+33至+43 bp突变破坏程度较小（20-30%）。在抗生物素蛋白-生物素复合物DNA结合和凝胶迁移率测定中，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的c-erbA-β，我们无法证明一个以上的蛋白质-DNA复合物在凝胶迁移率测定。然而，使用抗生物素蛋白-生物素复合物DNA结合试验和交联剂，1，6-bismaleimidohexane，我们能够证明甲状腺激素受体二聚体形成域1，但没有任何显着的程度上域2。总之，功能和DNA结合的研究表明，甲状腺激素受体结合到两个不同的区域在第一个外显子的hTSH β基因。上游位点（结构域1）以较高的亲和力结合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c-erbA-β的单个分子。这些结果表明，甲状腺激素受体，结合到第一个外显子中的至少两个位点，共同介导T3抑制hTSH β表达。
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.