Robertson R W, Zhang L, Pasco D S, Fagan J B
Molecular Biology Laboratory, Maharishi International University, Fairfield, IA 52557-1078.
Nucleic Acids Res. 1994 May 11;22(9):1741-9. doi: 10.1093/nar/22.9.1741.
In vivo footprinting experiments, augmented with gel shift and transfection analyses suggest that activation of the CYP1A1 gene by aryl hydrocarbons may be a multicomponent process. During the first 30 minutes of exposure to aryl hydrocarbon carcinogens and environmental contaminants, in vivo footprints appear at nine distinct sites within a 281 bp region centered 950 bp upstream of the CYP1A1 transcription start site. Six of these sites are unrelated in sequence to the three xenobiotic response elements (XREs) within this region, at which the aryl hydrocarbon (AH) receptor is known to bind. These six display a variety of footprint patterns, are diverse in sequence and range in G-C content from 60 to 75%. This diversity suggests that multiple nuclear factors may be responsible for these six in vivo footprints. These observations are consistent with competition gel shift experiments showing that the nuclear factors binding at two of these sites are different from each other, as well as from the AH receptor. Gel shifts also indicate that the sequence-specific factors binding at these sites are expressed constitutively. This is consistent with a model in which in vivo footprints are induced at these six sites, not through direct activation or de novo synthesis of DNA-binding factors, but through a two phase mechanism in which binding of the nuclear AH receptor complex to XREs facilitates the binding of constitutive factors at these sites. This facilitation could be mediated either through specific protein-protein interactions or through alterations in chromatin structure that make these sites accessible to constitutive nuclear factors. A function for the sequences at which aryl hydrocarbons induce in vivo footprints is suggested by transfection experiments showing that one of these sequences cooperates with a weak XRE to confer on a reporter gene responsiveness to aryl hydrocarbons.
体内足迹实验，辅以凝胶迁移和转染分析表明，芳烃对CYP1A1基因的激活可能是一个多组分过程。在暴露于芳烃致癌物和环境污染物的最初30分钟内，体内足迹出现在CYP1A1转录起始位点上游950 bp处一个281 bp区域内的九个不同位点。其中六个位点的序列与该区域内已知芳烃（AH）受体结合的三个外源性反应元件（XRE）无关。这六个位点呈现出各种足迹模式，序列多样，G-C含量在60%至75%之间。这种多样性表明多种核因子可能对这六个体内足迹负责。这些观察结果与竞争凝胶迁移实验一致，该实验表明在其中两个位点结合的核因子彼此不同，也与AH受体不同。凝胶迁移还表明在这些位点结合的序列特异性因子是组成型表达的。这与一个模型一致，在该模型中，这六个位点的体内足迹不是通过DNA结合因子的直接激活或从头合成诱导的，而是通过一个两阶段机制诱导的，其中核AH受体复合物与XREs的结合促进了组成型因子在这些位点的结合。这种促进作用可以通过特定的蛋白质-蛋白质相互作用或通过染色质结构的改变来介导，使这些位点能够被组成型核因子接近。转染实验表明，这些序列之一与一个弱XRE协同作用，赋予报告基因对芳烃的反应性，这表明芳烃诱导体内足迹的序列具有功能。
