Lee I J, Driggers P H, Medin J A, Nikodem V M, Ozato K
Laboratory of Molecular Growth Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1647-51. doi: 10.1073/pnas.91.5.1647.
The thyroid hormone and retinoid X receptors form a heterodimer with each other and mediate thyroid hormone (T3)-dependent transcription. Retinoid X receptor, in addition, forms a homodimer and mediates 9-cis-retinoic acid-dependent transcription. Here, recombinant thyroid hormone receptor and recombinant retinoid X receptor beta expressed from baculovirus vectors have been studied for ligand-mediated activation of transcription in vitro. We show that the two recombinant receptors, most likely as a heterodimer, cooperatively enhance transcription in vitro from a template containing functional T3 responsive elements. The enhancement was specific for the T3 responsive element and was greatest when T3 was added to the reaction (approximately 14-fold increase). Albeit to a lesser degree, the two receptors also directed transcription in the absence of T3. Template competition experiments suggest that the two receptors enhance formation of the preinitiation complex and that activation by T3 occurs when the ligand binds the receptor prior to (or during), but not after, the formation of the preinitiation complex. Although 9-cis-retinoic acid had no effect on the T3-dependent transcription, this ligand activated transcription in vitro directed by recombinant retinoic X receptor beta, most likely as a homodimer. This activation was observed when using nuclear extracts from embryonal carcinoma cells as a source of basal transcription factors, but not those from B lymphocytes. These results demonstrate that transcriptional activation mediated by T3 and 9-cis-retinoic acid can be reconstituted in vitro with the respective recombinant receptors.
甲状腺激素受体和视黄酸X受体相互形成异二聚体，并介导甲状腺激素（T3）依赖性转录。此外，视黄酸X受体形成同二聚体，并介导9-顺式视黄酸依赖性转录。在此，对从杆状病毒载体表达的重组甲状腺激素受体和重组视黄酸X受体β进行了体外配体介导的转录激活研究。我们表明，这两种重组受体很可能作为异二聚体，协同增强来自含有功能性T3反应元件的模板的体外转录。这种增强对T3反应元件具有特异性，当向反应中加入T3时增强作用最大（增加约14倍）。尽管程度较小，但这两种受体在没有T3的情况下也能指导转录。模板竞争实验表明，这两种受体增强起始前复合物的形成，并且当配体在起始前复合物形成之前（或期间）而非之后与受体结合时发生T3介导的激活。虽然9-顺式视黄酸对T3依赖性转录没有影响，但这种配体在体外激活由重组视黄酸X受体β指导的转录，很可能作为同二聚体。当使用胚胎癌细胞的核提取物作为基础转录因子的来源时观察到这种激活，但使用B淋巴细胞的核提取物时则未观察到。这些结果表明，由T3和9-顺式视黄酸介导的转录激活可以在体外与各自的重组受体一起重建。
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