Suen C S, Yen P M, Chin W W
Department of Medicine, Brigham and Women's Hospital, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115.
J Biol Chem. 1994 Jan 14;269(2):1314-22.
Thyroid hormone receptors (TRs) are ligand-dependent nuclear transcription factors that are encoded by two different genes, TR alpha and TR beta, and bind to thyroid hormone response elements (TREs) in the promoters of thyroid hormone (T3)-regulated genes. Retinoid X receptors (RXRs), major members of the thyroid hormone receptor auxiliary proteins, have recently been shown to enhance the binding of TRs to TREs. We previously showed that TRs extracted from rat pituitary GH3 cells retain ligand (T3) and DNA binding specificity and stimulate rat growth hormone (rGH) promoter activity in a cell-free in vitro transcription system. In this report, we have studied further how T3 activates endogenous TRs and stimulates transcription from different TRE-containing promoters. We found that T3 (10(-8) M) selectively stimulates transcription from rGH-TRE- and TREpal-, but not ME-TRE- and F2-TRE-, containing templates in which these TREs are linked in front of the rGH minimal promoter containing only the TATA box binding protein, but not any other proximal binding protein, sequence. In contrast, only the TREpal/AdML template, in which TREpal oligonucleotide was linked in front of the adenovirus major late gene (AdML) minimal promoter, was stimulated by T3. Electrophoretic mobility shift assay (EMSA) demonstrates that endogenous TR complexes specifically bind to either natural or idealized TRE (rGH-TRE, TREpal, ME-TRE, and F2-TRE) oligonucleotides. To further understand these receptor-DNA complexes formed on various TREs, isoform-specific anti-receptor antisera (TR alpha, TR beta 1, TR beta 2, and RXR beta) were added in the EMSA. These antisera differentially supershifted TR.DNA complexes formed on the TREs. These data suggest either that endogenous TR isoforms and RXR beta may form different complexes on the various TREs or that TR.RXR complexes have distinct conformations when bound to the various TREs. Taken together, these data suggest that particular TREs in which specific TR.RXR complexes are formed and different minimal promoters may provide specificity in T3-mediated transcriptional stimulation of gene expression.
甲状腺激素受体（TRs）是依赖配体的核转录因子，由两个不同的基因TRα和TRβ编码，并与甲状腺激素（T3）调节基因启动子中的甲状腺激素反应元件（TREs）结合。视黄酸X受体（RXRs）是甲状腺激素受体辅助蛋白的主要成员，最近已被证明可增强TRs与TREs的结合。我们之前表明，从大鼠垂体GH3细胞中提取的TRs保留配体（T3）和DNA结合特异性，并在无细胞体外转录系统中刺激大鼠生长激素（rGH）启动子活性。在本报告中，我们进一步研究了T3如何激活内源性TRs并刺激来自不同含TRE启动子的转录。我们发现，T3（10^(-8) M）选择性地刺激来自含rGH-TRE和TREpal-的模板的转录，但不刺激含ME-TRE和F2-TRE-的模板的转录，在这些模板中，这些TREs连接在仅含TATA盒结合蛋白而不含任何其他近端结合蛋白序列的rGH最小启动子之前。相比之下，只有TREpal/AdML模板（其中TREpal寡核苷酸连接在腺病毒主要晚期基因（AdML）最小启动子之前）受到T3的刺激。电泳迁移率变动分析（EMSA）表明，内源性TR复合物特异性结合天然或理想化的TRE（rGH-TRE、TREpal、ME-TRE和F2-TRE）寡核苷酸。为了进一步了解在各种TREs上形成的这些受体-DNA复合物，在EMSA中加入了亚型特异性抗受体抗血清（TRα、TRβ1、TRβ2和RXRβ）。这些抗血清使在TREs上形成的TR.DNA复合物发生不同程度的超迁移。这些数据表明，内源性TR亚型和RXRβ可能在各种TREs上形成不同的复合物，或者TR.RXR复合物在与各种TREs结合时具有不同的构象。综上所述，这些数据表明，形成特定TR.RXR复合物的特定TREs和不同的最小启动子可能在T3介导的基因表达转录刺激中提供特异性。
