Watson A J, Weir-Brown K I, Bannister R M, Chu F F, Reisz-Porszasz S, Fujii-Kuriyama Y, Sogawa K, Hankinson O
Laboratory of Biomedical and Environmental Sciences, School of Public Health, University of California, Los Angeles 90024-1786.
Mol Cell Biol. 1992 May;12(5):2115-23. doi: 10.1128/mcb.12.5.2115-2123.1992.
A dominant mutant of Hepa-1 cells, c31, expresses a repressor that prevents 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-dependent stimulation of Cyp1a1 transcription. The repressor acts via the xenobiotic-responsive elements (XREs), which are the DNA-binding sites for the aryl hydrocarbon (Ah) receptor-TCDD complex during transcriptional activation of the gene. High-salt nuclear extracts prepared from c31 cells grown with TCDD contained normal levels of the Ah receptor which bound the XRE with normal affinity, as judged by in vitro gel mobility shift assays. Furthermore, extracts prepared from these cells, grown either with or without TCDD, contained no novel XRE-binding proteins compared with extracts from wild-type Hepa-1 cells. However, in vivo genomic footprinting demonstrated that TCDD treatment leads to binding of the Ah receptor to the XREs in Hepa-1 but not mutant cells. This finding suggests that the repressor associates with the Ah receptor to prevent its binding to the XREs and that high-salt treatment either causes dissociation of the receptor/repressor complex or fails to extract the repressor from nuclei. The results underscore the importance of using both in vivo and in vitro assays for analyzing DNA-protein interactions.
Hepa-1细胞的一个显性突变体c31表达一种阻遏蛋白，该蛋白可阻止2,3,7,8-四氯二苯并对二恶英（TCDD）依赖性的Cyp1a1转录激活。该阻遏蛋白通过外源性反应元件（XREs）发挥作用，在该基因的转录激活过程中，XREs是芳烃（Ah）受体-TCDD复合物的DNA结合位点。用TCDD培养的c31细胞制备的高盐核提取物中，Ah受体水平正常，通过体外凝胶迁移率变动分析判断，其与XREs的结合亲和力也正常。此外，与野生型Hepa-1细胞提取物相比，无论是否用TCDD培养，这些细胞制备的提取物中均未发现新的XRE结合蛋白。然而，体内基因组足迹分析表明，TCDD处理可导致Ah受体与Hepa-1细胞而非突变细胞中的XREs结合。这一发现表明，阻遏蛋白与Ah受体结合，阻止其与XREs结合，高盐处理要么导致受体/阻遏蛋白复合物解离，要么无法从细胞核中提取阻遏蛋白。这些结果强调了同时使用体内和体外分析方法来分析DNA-蛋白质相互作用的重要性。
