Mandl Markus, Depping Reinhard
Institute of Physiology, Center for Structural and Cell Biology in Medicine, University of Luebeck, Ratzeburger Allee 160, 23562 Lübeck, Germany.
Division of Cell Metabolism and Differentiation Research, Institute for Biomedical Aging Research, University of Innsbruck, Rennweg 10, 6020 Innsbruck, Austria.
Cancer Cell Int. 2017 Aug 24;17:77. doi: 10.1186/s12935-017-0446-2. eCollection 2017.
The transcription factor aryl hydrocarbon receptor nuclear translocator (ARNT) participates in the hypoxia-inducible factor (HIF) pathway which senses a decline in cellular oxygen tension. In hypoxia, HIF-1α and ARNT form the transcriptional active complex HIF-1 followed by the expression of target genes. ARNT is considered as constitutively expressed and unaffected by hypoxia. However, certain tumour cell lines derived from different entities are capable to elevate ARNT expression under hypoxic conditions which implies a survival benefit. It was demonstrated that high ARNT protein levels mediate radioresistance in tumour cells. Furthermore, a HIF-1α-driven feed-forward loop leading to augmented HIF signalling was discovered in Hep3B cells. Herein HIF-1α elevates the mRNA and protein expression of its binding partner ARNT in hypoxia. However, the detailed mechanism remained unclear. The objective of this study was to test whether HIF-1α might directly regulate ARNT expression by recruitment to the promoter.
Chromatin immunoprecipitation (ChIP), CRISPR/Cas9 genome editing, Western blotting, quantitative RT-PCR and reporter gene assays were applied. The unpaired test was used for statistical analysis.
ChIP assays revealed the binding of both HIF-1α and ARNT to the promoter in hypoxia. The relevance of this particular region for hypoxic ARNT induction was confirmed by CRISPR/Cas9 genome editing. ARNT normoxic basal expression and hypoxic inducibility was reduced in genome-edited Hep3B cells. This phenotype was accompanied with impaired HIF signalling and was rescued by ARNT overexpression.
The results indicate to be a putative HIF-1 target gene and a limiting factor in this model.
转录因子芳烃受体核转运蛋白（ARNT）参与感知细胞氧张力下降的缺氧诱导因子（HIF）途径。在缺氧条件下，HIF-1α与ARNT形成转录活性复合物HIF-1，随后靶基因表达。ARNT被认为是组成性表达且不受缺氧影响。然而，某些源自不同实体的肿瘤细胞系在缺氧条件下能够提高ARNT表达，这意味着具有生存优势。已证明高ARNT蛋白水平介导肿瘤细胞的放射抗性。此外，在Hep3B细胞中发现了一个由HIF-1α驱动的前馈环，导致HIF信号增强。在此，HIF-1α在缺氧条件下提高其结合伴侣ARNT的mRNA和蛋白表达。然而，具体机制仍不清楚。本研究的目的是测试HIF-1α是否可能通过募集到启动子直接调节ARNT表达。
应用染色质免疫沉淀（ChIP）、CRISPR/Cas9基因组编辑、蛋白质免疫印迹、定量逆转录-聚合酶链反应（RT-PCR）和报告基因检测。采用非配对检验进行统计分析。
ChIP检测显示在缺氧条件下HIF-1α和ARNT均与启动子结合。CRISPR/Cas9基因组编辑证实了该特定区域与缺氧诱导ARNT的相关性。在基因组编辑的Hep3B细胞中，ARNT常氧基础表达和缺氧诱导性降低。该表型伴有HIF信号受损，并通过ARNT过表达得以挽救。
结果表明是一个假定的HIF-1靶基因，并且是该模型中的一个限制因素。
