Hapgood J, Cuthill S, Denis M, Poellinger L, Gustafsson J A
Department of Medical Nutrition, Karolinska Institute, Huddinge University Hospital, Sweden.
Proc Natl Acad Sci U S A. 1989 Jan;86(1):60-4. doi: 10.1073/pnas.86.1.60.
Upon binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (called dioxin or TCDD), the dioxin receptor exhibits increased affinity for the cell nucleus in vivo and for DNA in vitro. To define the recognition sequence of the dioxin receptor and its relationship with that of the glucocorticoid receptor, oligonucleotides derived from dioxin-responsive elements of the rat cytochrome P-450c gene were tested for their ability to form specific protein-DNA complexes in a gel retardation assay. We found that a previously defined sequence motif that is similar to the glucocorticoid-responsive element and exhibits strong enhancer activity in response to dioxin receptor ligands bound a dioxin-inducible factor with high specificity but was not recognized by the DNA-binding domain of the glucocorticoid receptor. Binding to this element was only observed in nuclear extracts of wild-type mouse hepatoma cells in a time- and dose-dependent manner and not in nuclear extracts from a nonresponsive mutant cell line deficient in DNA binding of the dioxin receptor. The specific DNA-binding activity in wild-type nuclear extracts comigrated in a Superose size-exclusion column and cosedimented on sucrose gradients with the in vivo labeled dioxin receptor. These experiments strongly suggest that the dioxin receptor is a sequence-specific DNA-binding protein and is not only biochemically but also functionally similar to the steroid receptor family.
在2,3,7,8-四氯二苯并对二恶英（称为二恶英或TCDD）结合后，二恶英受体在体内对细胞核以及在体外对DNA的亲和力会增加。为了确定二恶英受体的识别序列及其与糖皮质激素受体识别序列的关系，在凝胶阻滞试验中测试了源自大鼠细胞色素P-450c基因二恶英反应元件的寡核苷酸形成特异性蛋白质-DNA复合物的能力。我们发现，一个先前定义的与糖皮质激素反应元件相似的序列基序，在对二恶英受体配体的反应中表现出强大的增强子活性，它能以高特异性结合一种二恶英诱导因子，但不被糖皮质激素受体的DNA结合结构域识别。只有在野生型小鼠肝癌细胞核提取物中才能观察到与该元件的结合，且呈时间和剂量依赖性，而在缺乏二恶英受体DNA结合能力的无反应突变细胞系的核提取物中则未观察到。野生型核提取物中的特异性DNA结合活性在Superose尺寸排阻柱中迁移，并在蔗糖梯度上与体内标记的二恶英受体共沉降。这些实验有力地表明，二恶英受体是一种序列特异性DNA结合蛋白，不仅在生化方面，而且在功能上与类固醇受体家族相似。
