Kobayashi Minoru, Morinibu Akiyo, Koyasu Sho, Goto Yoko, Hiraoka Masahiro, Harada Hiroshi
Laboratory of Cancer Cell Biology, Department of Genome Dynamics, Radiation Biology Center, Kyoto University, Japan.
Research Center for Advanced Science and Technology, The University of Tokyo, Japan.
FEBS J. 2017 Nov;284(22):3804-3816. doi: 10.1111/febs.14280. Epub 2017 Oct 16.
Hypoxia-inducible factor 1 (HIF-1) is a transcription factor functioning in cellular adaptive responses to hypoxia. Recent studies have suggested that HIF-1 activity is upregulated by one of the important circadian clock genes, period circadian clock 2 (PER2); however, its underlying mechanism remains unclear. Here, we show that PER2 functions as an effector protein for the recruitment of HIF-1α to its cognate enhancer sequence, the hypoxia-response element (HRE). We found that the forced expression of PER2 enhanced HIF-1 activity without influencing expression levels of the regulatory subunit of HIF-1, HIF-1α, at either mRNA or protein levels. A series of coimmunoprecipitation-based experiments revealed that PER2 interacted with HIF-1α and facilitated the recruitment of HIF-1α to HRE derived from vascular endothelial growth factor (VEGF) promoter. The PER2-mediated activation of HIF-1 was observed only when the asparagine residue at position 803 of HIF-1α (HIF-1α N803) was kept unhydroxylated by hypoxic stimulation, by introducing an N803A point mutation, or by an inhibitor of N803-dioxygenase, deferoxamine. However, the extent of PER-2-HIF-1α interaction was equivalent regardless of the N803 hydroxylation status. Taken together, these results suggest that, with the help of an unknown sensor molecule for the N803 hydroxylation status, PER2 functions as an effector molecule for the recruitment of HIF-1 to promoter regions of its downstream genes. Our findings reveal a novel regulatory step in the activation of HIF-1, which can be targeted to develop therapeutic strategies against HIF-1-related diseases, such as cancers.
缺氧诱导因子1（HIF-1）是一种在细胞对缺氧的适应性反应中发挥作用的转录因子。最近的研究表明，HIF-1的活性由重要的生物钟基因之一——周期生物钟2（PER2）上调；然而，其潜在机制仍不清楚。在这里，我们表明PER2作为一种效应蛋白，可将HIF-1α募集到其同源增强子序列——缺氧反应元件（HRE）。我们发现，PER2的强制表达增强了HIF-1的活性，而在mRNA或蛋白质水平上均不影响HIF-1调节亚基HIF-1α的表达水平。一系列基于免疫共沉淀的实验表明，PER2与HIF-1α相互作用，并促进HIF-1α募集到源自血管内皮生长因子（VEGF）启动子的HRE。仅当通过缺氧刺激、引入N803A点突变或使用N803-双加氧酶抑制剂去铁胺使HIF-1α第803位的天冬酰胺残基（HIF-1α N803）保持未羟基化时，才观察到PER2介导的HIF-1激活。然而，无论N803的羟基化状态如何，PER-2-HIF-1α相互作用的程度是相同的。综上所述，这些结果表明，在一种未知的N803羟基化状态传感分子的帮助下，PER2作为一种效应分子，将HIF-1募集到其下游基因的启动子区域。我们的发现揭示了HIF-1激活过程中的一个新的调节步骤，这可以作为开发针对HIF-1相关疾病（如癌症）的治疗策略的靶点。
