Identification of a Functional Antioxidant Response Element within the Eighth Intron of the Human ABCC3 Gene
Identification of a Functional Antioxidant Response Element within the Eighth Intron of the Human ABCC3 Gene
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DOI：
10.1124/dmd.114.060103
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发表时间：
2015-01-01
影响因子：
3.9
通讯作者：
Cherrington, Nathan J.
中科院分区：
文献类型：
--
作者：
Canet, Mark J.;Merrell, Matthew D.;Cherrington, Nathan J.
ATP结合盒（ABC）转运蛋白家族，包括ABCC 3，是一个大家族的外排泵，在消除体内外源性物质中起着关键作用。据报道，ABCC 3在肝脏应激条件下以及通过某些形式的癌症的进展而被诱导。有几条证据表明转录因子核因子（红细胞衍生2）样2（Nrf 2）参与了这种诱导。然而，尽管啮齿动物模型已被调查，在人类ABCC 3基因的功能性抗氧化反应元件（ARE）尚未确定。本研究的目的是鉴定和表征负责介导Nrf 2依赖性诱导人类ABCC 3基因的ARE。在A549细胞中进行的高通量染色质免疫沉淀测序分析揭示了Nrf 2和人类ABCC 3基因的第八内含子之间的特异性相互作用，而不是该基因的更典型的侧翼区域。随后的计算机分析的内含子确定了两个推定的ARE元件，包含核心共识ARE序列常见于几个Nrf 2响应基因。这两个战神的功能特性，使用内切酶-报告构建体与ARE突变体构建体揭示，这些推定的战神之一是功能活性。最后，DNA下拉试验证实了这些内含子战神在体外与Nrf 2的特异性结合。我们的研究结果确定了ABCC 3基因的第八内含子内的功能性Nrf 2反应元件，这可能会提供抗氧化反应刺激过程中ABCC 3的诱导机制的见解。
The ATP-binding cassette (ABC) family of transporters, including ABCC3, is a large family of efflux pumps that plays a pivotal role in the elimination of xenobiotics from the body. ABCC3 has been reported to be induced during hepatic stress conditions and through the progression of some forms of cancer. Several lines of evidence have implicated the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in this induction. However, although rodent models have been investigated, a functional antioxidant response element (ARE) in the human ABCC3 gene has not been identified. The purpose of this study was to identify and characterize the ARE(s) responsible for mediating the Nrf2-dependent induction of the human ABCC3 gene. A high-throughput chromatin immunoprecipitation-sequencing analysis performed in A549 cells revealed a specific interaction between Nrf2 and the eighth intron of the human ABCC3 gene rather than the more prototypical flanking region of the gene. Subsequent in silico analysis of the intron identified two putative ARE elements that contained the core consensus ARE sequence commonly found in several Nrf2-responsive genes. Functional characterization of these two AREs using luciferase-reporter constructs with ARE mutant constructs revealed that one of these putative AREs is functionally active. Finally, DNA pull-down assays confirmed specific binding of these intronic AREs by Nrf2 in vitro. Our findings identify a functional Nrf2 response element within the eighth intron of the ABCC3 gene, which may provide mechanistic insight into the induction of ABCC3 during antioxidant response stimuli.