Analysis of the transcriptional regulation and molecular function of the aryl hydrocarbon receptor repressor in human cell lines
Analysis of the transcriptional regulation and molecular function of the aryl hydrocarbon receptor repressor in human cell lines
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DOI：
10.1124/dmd.107.016253
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发表时间：
2007-12-01
影响因子：
3.9
通讯作者：
Fritsche, Ellen
中科院分区：
文献类型：
--
作者：
Haarmann-Stemmann, Thomas;Bothe, Hanno;Fritsche, Ellen
芳烃受体阻遏物（AhRR）是芳烃受体（AhR）信号级联的成员，其介导二恶英毒性并参与调节细胞生长和分化。AhRR被描述为反馈调节剂，其抵消AhR依赖性基因表达。我们通过克隆位于AhRR内含子I的调控DNA区域，研究了人类AhRR转录调控的分子机制。通过报告基因分析和产生的缺失变体，我们确定了一个功能，3-甲基胆蒽敏感的异生素反应元件（XRE）网站。染色质免疫沉淀分析表明，AhRR结合到这个XRE，显示AhRR表达的自动调节环。此外，我们表明，一个相邻的GC盒AhRR转录的功能相关性，因为这个GC盒的阻断导致组成型和诱导型AhRR基因活性的降低。在HepG2、原代成纤维细胞和HeLa细胞中观察到的组成型AhRR mRNA水平的差异与CYP1A1诱导直接相关。我们发现，高AhRR表达细胞对AhR激动剂的无反应性与CYP1A1的AhRR与XRE位点的组成性结合有关。组蛋白去乙酰化酶抑制剂丁酸钠的治疗恢复了CYP1A1在这些细胞系中的反应性，由于AhRR从XRE的解离。此外，在未经处理的HeLa细胞中，瞬时AhRR mRNA沉默伴随着基础CYP1A1表达的增加，这表明AhRR在CYP1A1调节中的组成性作用。高AhRR表达的原代成纤维细胞的AhRR的功能相关性进行了讨论。
The aryl hydrocarbon receptor repressor (AhRR) is a member of the aryl hydrocarbon receptor (AhR) signaling cascade, which mediates dioxin toxicity and is involved in regulation of cell growth and differentiation. The AhRR was described as a feedback modulator, which counteracts AhR- dependent gene expression. We investigated the molecular mechanisms of transcriptional regulation of the human AhRR by cloning its regulatory DNA region located in intron I of the AhRR. By means of reporter gene analyses and generation of deletion variants, we identified a functional, 3- methylcholanthrene-sensitive xenobiotic response element (XRE) site. Chromatin immunoprecipitation analyses revealed that the AhRR binds to this XRE, displaying an autoregulatory loop of AhRR expression. In addition we show that an adjacent GC-box is of functional relevance for AhRR transcription, since blocking of this GC-box resulted in a decrease of constitutive and inducible AhRR gene activity. The differences in constitutive AhRR mRNA level observed in HepG2, primary fibroblast, and HeLa cells are directly correlated with CYP1A1 inducibility. We show that the nonresponsiveness of high AhRR-expressing cells toward AhR-agonists is associated with a constitutive binding of the AhRR to XRE sites of CYP1A1. Treatment with the histone deacetylase inhibitor sodium butyrate restored the responsiveness of CYP1A1 in these cell lines, due to the dissociation of AhRR from the XREs. Furthermore, transient AhRR mRNA silencing in untreated HeLa cells was accompanied by an increase of basal CYP1A1 expression, pointing to a constitutive role of the AhRR in regulation of CYP1A1. The functional relevance of the AhRR in high AhRR-expressing primary fibroblasts is discussed.