Zhang Leying, Zheng Wenchao, Jefcoate Colin R
Department of Pharmacology, Medical Science Center, University of Wisconsin, 1300 University Avenue, Madison, WI 53706, USA.
Toxicol Appl Pharmacol. 2003 Oct 15;192(2):174-90. doi: 10.1016/s0041-008x(03)00276-x.
Cyp1B1 is expressed constitutively in many extrahepatic cells and is induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). An enhancer region (AhER-810 to -1075 of the mouse Cyp1B1 promoter), which mediates aryl hydrocarbon receptor (AhR) regulation of transcription, contains three consensus XRE sequences (designated XRE1, XRE4, and XRE5) and a central Ebox. XRE5 is essential for both basal and induced activity in C3H10T1/2 cells. AhR/ARNT binding to XRE1, XRE4, and the Ebox complex function in combination to support the AhR/ARNT complex at XRE5. The identical 12 base cores of XRE1 and XRE4 differ from the core of XRE5 by two bases outside of the consensus XRE. These sites bind a constitutive complex slightly smaller than AhR/ARNT (anomalous complex; anC), which is not formed at XRE5 or six Cyp1A1 XREs. Exchange of these bases (m3 mutations) restores selective AhR/ARNT binding at XRE1/XRE4 and introduces anC binding at XRE5. The activities of multimeric XRE1 and XRE5 luciferase reporters responded in parallel to the extent of AhR/ARNT binding. The consensus anC binding sequence ((C/T)GCG(C/T)GCGC(C/A)GC) overlaps the XRE1/XRE4 AhR/ARNT element. Gel mobility analyses show that anC binds to XRE1/XRE4 under basal conditions, while AhR/ARNT partially displaces anC following TCDD induction. Selective depletion of anC with biotin-oligonucleotides increases AhR/ARNT binding. M3-mutations at, respectively, XRE1 and XRE4 of the AhER sequence, had opposite effects on luciferase reporters. Activities increased for the XRE1 mutation and decreased for the XRE4 mutation, but also depended on the level of AhR transfected into AhR -/- fibroblasts. AnC compete with AhR at XRE1 while playing an activating role at XRE4. This positive effect of constitutive anC binding at XRE4 may contribute to the characteristic basal Cyp1B1 expression in embryo fibroblasts, which is mediated by low constitutive activities of AhR.
Cyp1B1在许多肝外细胞中组成性表达，并由2,3,7,8-四氯二苯并对二恶英（TCDD）诱导。一个增强子区域（小鼠Cyp1B1启动子的AhER - 810至 - 1075）介导芳烃受体（AhR）对转录的调控，它包含三个共有XRE序列（分别命名为XRE1、XRE4和XRE5）以及一个中心E盒。XRE5对于C3H10T1/2细胞中的基础活性和诱导活性均至关重要。AhR/ARNT与XRE1、XRE4以及E盒复合物结合，共同发挥作用以支持XRE5处的AhR/ARNT复合物。XRE1和XRE4相同的12个碱基核心与XRE5的核心在共有XRE之外有两个碱基不同。这些位点结合一种组成性复合物，其大小略小于AhR/ARNT（异常复合物；anC），在XRE5或六个Cyp1A1 XRE处不会形成这种复合物。交换这些碱基（m3突变）可恢复XRE1/XRE4处AhR/ARNT的选择性结合，并在XRE5处引入anC结合。多聚体XRE1和XRE5荧光素酶报告基因的活性与AhR/ARNT结合程度呈平行响应。共有anC结合序列（(C/T)GCG(C/T)GCGC(C/A)GC）与XRE1/XRE4的AhR/ARNT元件重叠。凝胶迁移分析表明，在基础条件下anC与XRE1/XRE4结合，而在TCDD诱导后AhR/ARNT会部分取代anC。用生物素寡核苷酸选择性耗尽anC会增加AhR/ARNT的结合。AhER序列的XRE1和XRE4处的M3突变对荧光素酶报告基因有相反的影响。XRE1突变时活性增加，XRE4突变时活性降低，但这也取决于转染到AhR -/- 成纤维细胞中的AhR水平。在XRE1处anC与AhR竞争，而在XRE4处发挥激活作用。XRE4处组成性anC结合的这种正向作用可能有助于胚胎成纤维细胞中Cyp1B1的特征性基础表达，这是由AhR的低组成性活性介导的。
