Saatcioglu F, Perry D J, Pasco D S, Fagan J B
Molecular Biology Laboratory, Maharishi International University, Fairfield, Iowa 52556.
Mol Cell Biol. 1990 Dec;10(12):6408-16. doi: 10.1128/mcb.10.12.6408-6416.1990.
Three nuclear factors, the Ah receptor, XF1, and XF2, bind sequence specifically to the Ah response elements or xenobiotic response elements (XREs) of the cytochrome P450IA1 (P450c) gene. The interactions of these factors with the Ah response element XRE1 were compared by three independent methods, methylation interference footprinting, orthophenanthroline-Cu+ footprinting, and mobility shift competition experiments, using a series of synthetic oligonucleotides with systematic alterations in the XRE core sequence. These studies established the following (i) all three factors interact sequence specifically with the core sequence of XRE1; (ii) the pattern of contacts made with this sequence by the Ah receptor are different from those made by XF1 and XF2; and (iii) although XF1 and XF2 can be distinguished by the mobility shift assay, the sequence specificities of their interactions with XRE1 are indistinguishable. Further characterization revealed the following additional differences among these three factors: (i) XF1 and XF2 could be extracted from nuclei under conditions quite different from those required for extraction of the Ah receptor; (ii) XF1 and XF2 were present in the nuclei of untreated cells and did not respond to polycyclic compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-napthoflavone, while nuclear Ah receptor was undetectable in untreated cells and rapidly increased in response to TCDD; (iii) inhibition of protein synthesis did not affect the TCDD-induced appearance of the Ah receptor but substantially decreased the constitutive activities of XF1 and XF2, suggesting that the Ah receptor must be present in untreated cells in an inactive form that can be rapidly activated by polycyclic compounds, while the constitutive expression of XF1 and XF2 depends on the continued synthesis of a relatively unstable protein; (iv) the receptor-deficient and nuclear translocation-defective mutants of the hepatoma cell line Hepa1, which are known to lack nuclear Ah receptor, expressed normal levels of XF1 and XF2, suggesting that the former factor is genetically distinct from the latter two; and (v) a divalent metal ion, probably Zn2+, is known to be an essential cofactor for the Ah receptor but was not required for the DNA-binding activities of XF1 and XF2. Together, these findings indicate that the Ah receptor is distinct from XF1 and XF2, while the latter two activities may be related. Because the DNA-binding domains of these three factors overlap substantially, their binding to XREs is probably mutually exclusive, which suggests that the interplay of these factors at Ah response elements may be important to the regulation of CYP1A1 gene transcription. The results of preliminary transfection experiments with constructs harboring XREs upstream of the chloramphenicol acetyltransferase gene driven by a minimal simian virus 40 promoter are presented that are consistent with this hypothesis.
三种核因子，即芳烃受体、XF1和XF2，能特异性地与细胞色素P450IA1（P450c）基因的芳烃反应元件或外源性物质反应元件（XREs）结合。利用一系列XRE核心序列有系统改变的合成寡核苷酸，通过三种独立方法，即甲基化干扰足迹法、邻菲罗啉 - Cu⁺足迹法和迁移率变动竞争实验，比较了这些因子与芳烃反应元件XRE1的相互作用。这些研究得出以下结果：（i）所有这三种因子都能与XRE1的核心序列特异性地相互作用；（ii）芳烃受体与该序列的接触模式不同于XF1和XF2；（iii）虽然通过迁移率变动分析可以区分XF1和XF2，但它们与XRE1相互作用的序列特异性难以区分。进一步的特性分析揭示了这三种因子之间的其他差异：（i）XF1和XF2可以在与提取芳烃受体所需条件截然不同的情况下从细胞核中提取出来；（ii）XF1和XF2存在于未处理细胞的细胞核中，对多环化合物如2,3,7,8 - 四氯二苯并 - p - 二恶英（TCDD）和β - 萘黄酮没有反应，而在未处理细胞中未检测到细胞核芳烃受体，且其在TCDD刺激下迅速增加；（iii）蛋白质合成的抑制并不影响TCDD诱导的芳烃受体的出现，但显著降低了XF1和XF2的组成型活性，这表明芳烃受体在未处理细胞中必定以一种无活性形式存在，可被多环化合物迅速激活，而XF1和XF2的组成型表达依赖于一种相对不稳定蛋白质的持续合成；（iv）肝癌细胞系Hepa1的受体缺陷型和核转位缺陷型突变体，已知缺乏细胞核芳烃受体，却表达正常水平的XF1和XF2，这表明前一种因子在基因上与后两种不同；（v）已知一种二价金属离子，可能是Zn²⁺，是芳烃受体的必需辅因子，但XF1和XF2的DNA结合活性并不需要它。总之，这些发现表明芳烃受体与XF1和XF2不同，而后两者的活性可能相关。由于这三种因子的DNA结合结构域有很大重叠，它们与XREs的结合可能是相互排斥的，这表明这些因子在芳烃反应元件处相互作用对于CYP1A1基因转录的调控可能很重要。展示了用含有由最小猿猴病毒40启动子驱动的氯霉素乙酰转移酶基因上游XREs的构建体进行的初步转染实验结果，这些结果与该假设一致。
