High-throughput evaluation of aryl hydrocarbon receptor-binding sites selected via chromatin immunoprecipitation-based screening in Hepa-1c1c7 cells stimulated with 2,3,7,8-tetrachlorodibenzo-p-dioxin
High-throughput evaluation of aryl hydrocarbon receptor-binding sites selected via chromatin immunoprecipitation-based screening in Hepa-1c1c7 cells stimulated with 2,3,7,8-tetrachlorodibenzo-p-dioxin
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DOI：
10.1266/ggs.83.455
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发表时间：
2008-12-01
影响因子：
1.1
通讯作者：
Ashida, Hitoshi
中科院分区：
文献类型：
--
作者：
Kinehara, Masaki;Fukuda, Itsuko;Ashida, Hitoshi
在与配体如2，3，7，8-四氯二苯并-对-二恶英（TCDD）结合后，芳烃受体（AhR）被激活以与芳烃受体核转位器（Arnt）形成异二聚体并与DNA结合。研究表明，AhR与DNA的结合依赖于二恶英反应元件（DRE），并控制异源反应基因。AhR-结合DNA片段从小鼠肝癌Hepa-1c 1c 7细胞刺激TCDD的染色质免疫沉淀（ChIP）DNA文库中富集，并通过高通量西南化学为基础的酶联免疫吸附试验（SW-ELISA）筛选。在筛选了1，700个片段后，ChIP-SW-ELISA筛选策略使我们能够在TCDD存在的情况下分离出77个与AhR紧密相互作用的片段。77个片段中只有39个似乎含有典型的DRE，表明在某些情况下DRE对于AhR结合是不稳定的，而75个片段位于启动子远端区域内。77个片段的基因组定位使我们能够估计121个潜在的AhR靶点，包括已知的靶点，如Cyp 1A 1和Cyp 1B 1，但只有有限的数量表现出依赖于TCDD的表达改变。这项研究揭示了这样一个事实，即TCDD激活的AhR经常结合到启动子远端区域，即使没有DRE，并不总是参与转录调控，这表明在基因组内，AhR的DNA结合可能发生在许多区域，没有顺式调控元件，可能不是建立其调控功能的关键决定因素。
Upon binding to ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an aryl hydrocarbon receptor (AhR) is activated to form a heterodimer with an aryl hydrocarbon receptor nuclear translocator (Arnt) and binds to DNA. It has been shown that the binding of AhR to DNA depends on the dioxin response element (DRE) and controls xenobiotic-response genes. AhR-binding DNA fragments from mouse hepatoma Hepa-1c1c7 cells stimulated with TCDD were once enriched in a chromatin immunoprecipitation (ChIP) DNA library and screened through a high-throughput southwestern chemistry-based enzyme-linked immunosorbent assay (SW-ELISA). After screening 1,700 fragments, the ChIP-SW-ELISA screening strategy allowed us to isolate 77 fragments tightly interacting with AhR in the presence of TCDD. Only 39 of the 77 fragments appeared to contain a typical DRE, indicating that in some cases the DRE was dispensable for AhR-binding, while 75 fragments were located within promoter-distal regions. Genomic mapping of the 77 fragments enabled us to estimate 121 potential AhR targets including known targets such as Cyp1A1 and Cyp1B1, but only a limited number exhibited an altered expression dependent on TCDD. This study revealed the fact that TCDD-activated AhR frequently binds to promoter-distal regions even without a DRE and is not always involved in transcriptional regulation, suggesting that within the genome DNA-binding of AhR could take place often in many regions without cis-regulatory elements and might not be a key determinant to establish its regulatory function.