Henry E C, Kent T A, Gasiewicz T A
Department of Environmental Medicine, University of Rochester School of Medicine, Rochester, New York, 14642, USA.
Arch Biochem Biophys. 1997 Mar 15;339(2):305-14. doi: 10.1006/abbi.1996.9873.
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcriptional enhancer which mediates the biochemical and toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and structurally related chemicals and which may have a role in the normal development of some tissues. We have previously reported the purification of the transformed TCDD.receptor complex from rat liver cytosol based on binding to its dioxin-responsive enhancer sequence (DRE) and that it comprises the AhR ligand-binding monomer and its dimerization partner, ARNT. The present studies were designed to compare the DRE-binding characteristics of the purified receptor with the cruder preparations that are commonly used and ultimately to determine whether the purified receptor complex itself (in the absence of additional cytosolic or nuclear factors) is capable of enhancing transcription in an in vitro system. The purified AhR retained in vitro DRE binding activity in the presence of carrier protein and dithiothreitol, and its affinity for the DRE oligonucleotide was equivalent to that of the other receptor preparations (crude and partially purified cytosolic and crude nuclear). When the ligand.receptor complex was bound to a DRE oligonucleotide containing BrdU and then UV-irradiated, two proteins in each of the receptor preparations were found to crosslink to BrdU-DRE, and we concluded that they are the AhR monomer and ARNT protein. All receptor preparations also gave a similar footprint of interaction with G-residues within the DRE consensus sequence, as assessed by methylation interference. Furthermore, purified and partially purified receptors were able to stimulate transcription from a DRE-containing template in a cell-free system in the presence of HeLa cell nuclear extract. Transcriptional enhancement was receptor dose-dependent, TCDD-dependent, and specific for the DRE sequence upstream of the promotor in our template construct. These data document for the first time that a purified TCDD.Ah receptor complex retains both specific DNA binding and transcriptional activities. This observation constitutes an important step toward understanding the mechanism of gene regulation by TCDD since it implies that the transformed receptor.ligand complex itself is competent as a transcriptional enhancer without a requirement for other factors.
芳烃受体（AhR）是一种配体激活的转录增强子，它介导2,3,7,8-四氯二苯并对二恶英（TCDD）及结构相关化学物质的生化和毒性作用，并且可能在某些组织的正常发育中发挥作用。我们之前报道过基于与二恶英反应增强子序列（DRE）的结合，从大鼠肝脏胞质溶胶中纯化转化的TCDD-受体复合物，该复合物由AhR配体结合单体及其二聚化伴侣ARNT组成。本研究旨在比较纯化受体与常用的粗制制剂的DRE结合特性，并最终确定纯化的受体复合物本身（在没有其他胞质或核因子的情况下）是否能够在体外系统中增强转录。在存在载体蛋白和二硫苏糖醇的情况下，纯化的AhR保留了体外DRE结合活性，并且其对DRE寡核苷酸的亲和力与其他受体制剂（粗制和部分纯化的胞质溶胶及粗制核提取物）相当。当配体-受体复合物与含有BrdU的DRE寡核苷酸结合然后进行紫外线照射时，在每种受体制剂中发现有两种蛋白质与BrdU-DRE发生交联，我们得出结论，它们是AhR单体和ARNT蛋白。通过甲基化干扰评估，所有受体制剂与DRE共有序列内的G残基的相互作用足迹也相似。此外，在存在HeLa细胞核提取物的情况下，纯化和部分纯化的受体能够在无细胞系统中刺激来自含DRE模板的转录。转录增强是受体剂量依赖性、TCDD依赖性的，并且对我们模板构建体中启动子上游的DRE序列具有特异性。这些数据首次证明纯化的TCDD-Ah受体复合物保留了特异性DNA结合和转录活性。这一观察结果是理解TCDD基因调控机制的重要一步，因为这意味着转化的受体-配体复合物本身作为转录增强子具有活性，而无需其他因子。
