Glass C K, Franco R, Weinberger C, Albert V R, Evans R M, Rosenfeld M G
Centre for Molecular Genetics, University of California, San Diego, La Jolla 92093.
Nature. 1987;329(6141):738-41. doi: 10.1038/329738a0.
The substance 3,5,3-triiodothyronine (T3) stimulates growth hormone gene transcription in rat pituitary tumour cells. This stimulation is thought to be mediated by the binding of nuclear T3 receptors to regulatory elements 5' to the transcriptional start site. Understanding of the mechanism by which thyroid hormone activates gene transcription has been limited by failure to purify nuclear T3 receptors because of their low abundance, and by the absence of defined T3 receptor-DNA binding sites affecting T3 regulation. Recently, human and avian c-erb-A gene products have been shown to bind thyroid hormone with high affinity and to have a molecular weight and nuclear association characteristic of the thyroid hormone receptor. In the present report, we describe the development of an avidin-biotin complex DNA-binding assay which can detect specific, high-affinity binding of rat pituitary cell T3 receptors to the sequence 5'CAGGGACGTGACCGCA3', located 164 base pairs 5' to the transcriptional start site of the rat growth hormone gene. An oligonucleotide containing this sequence transferred T3 regulation to the herpes simplex virus thymidine kinase promoter in transfected rat pituitary GC2 cells, and specifically bound an in vitro translation product of the human placental c-erb-A gene. The data provide supporting evidence that the human c-erb-A gene product mediates the transcriptional effects of T3 and also that GC2 cell nuclear extracts contain additional factors that modify the binding of pituitary T3 receptors to the rat growth hormone gene T3 response element.
物质3,5,3-三碘甲状腺原氨酸（T3）可刺激大鼠垂体肿瘤细胞中生长激素基因的转录。这种刺激作用被认为是由核T3受体与转录起始位点上游5'端的调控元件结合介导的。由于核T3受体丰度低，未能纯化，以及缺乏影响T3调控的明确的T3受体-DNA结合位点，甲状腺激素激活基因转录的机制一直未明。最近，已证明人和禽类的c-erb-A基因产物能与甲状腺激素高亲和力结合，且具有甲状腺激素受体的分子量和核定位特征。在本报告中，我们描述了一种抗生物素蛋白-生物素复合物DNA结合测定法的建立，该方法可检测大鼠垂体细胞T3受体与位于大鼠生长激素基因转录起始位点上游164个碱基对处的序列5'CAGGGACGTGACCGCA3'的特异性、高亲和力结合。含有该序列的寡核苷酸将T3调控作用转移至转染的大鼠垂体GC2细胞中的单纯疱疹病毒胸苷激酶启动子，并特异性结合人胎盘c-erb-A基因的体外翻译产物。这些数据提供了支持性证据，表明人c-erb-A基因产物介导了T3的转录效应，并且GC2细胞核提取物含有其他可改变垂体T3受体与大鼠生长激素基因T3反应元件结合的因子。
