Institute of Physiology and Zürich Center for Integrative Human Physiology (ZIHP), University of Zürich, 8057 Zürich, Switzerland.
Nucleic Acids Res. 2012 Mar;40(5):1928-43. doi: 10.1093/nar/gkr978. Epub 2011 Nov 10.
The human prolyl-4-hydroxylase domain (PHD) proteins 1-3 are known as cellular oxygen sensors, acting via the degradation of hypoxia-inducible factor (HIF) α-subunits. PHD2 and PHD3 genes are inducible by HIFs themselves, suggesting a negative feedback loop that involves PHD abundance. To identify novel regulators of the PHD2 gene, an expression array of 704 transcription factors was screened by a method that allows distinguishing between HIF-dependent and HIF-independent promoter regulation. Among others, the E-twenty six transcription factor ETS translocation variant 4 (ETV4) was found to contribute to PHD2 gene expression particularly under hypoxic conditions. Mechanistically, complex formation between ETV4 and HIF-1/2α was observed by mammalian two-hybrid and fluorescence resonance energy transfer analysis. HIF-1α domain mapping, CITED2 overexpression and factor inhibiting HIF depletion experiments provided evidence for cooperation between HIF-1α and p300/CBP in ETV4 binding. Chromatin immunoprecipitation confirmed ETV4 and HIF-1α corecruitment to the PHD2 promoter. Of 608 hypoxically induced transcripts found by genome-wide expression profiling, 7.7% required ETV4 for efficient hypoxic induction, suggesting a broad role of ETV4 in hypoxic gene regulation. Endogenous ETV4 highly correlated with PHD2, HIF-1/2α and several established markers of tissue hypoxia in 282 human breast cancer tissue samples, corroborating a functional interplay between the ETV4 and HIF pathways.
人脯氨酰-4-羟化酶结构域（PHD）蛋白 1-3 被认为是细胞氧传感器，通过降解低氧诱导因子（HIF）α亚基发挥作用。PHD2 和 PHD3 基因可被 HIF 自身诱导，表明存在涉及 PHD 丰度的负反馈环。为了鉴定 PHD2 基因的新调控因子，通过一种能够区分 HIF 依赖性和 HIF 非依赖性启动子调控的方法，筛选了 704 个转录因子的表达谱。其中，E 区二十六个转录因子 ETS 易位变体 4（ETV4）被发现特别在低氧条件下有助于 PHD2 基因表达。从机制上讲，通过哺乳动物双杂交和荧光共振能量转移分析观察到 ETV4 与 HIF-1/2α 之间形成复合物。HIF-1α 结构域作图、CITED2 过表达和因子抑制 HIF 耗竭实验为 HIF-1α 和 p300/CBP 在 ETV4 结合中的合作提供了证据。染色质免疫沉淀证实了 ETV4 和 HIF-1α 共同募集到 PHD2 启动子。通过全基因组表达谱分析发现的 608 个低氧诱导转录物中，有 7.7%需要 ETV4 才能有效地进行低氧诱导，这表明 ETV4 在低氧基因调控中具有广泛的作用。内源性 ETV4 与 PHD2、HIF-1/2α 和 282 个人乳腺癌组织样本中的几个已建立的组织缺氧标志物高度相关，证实了 ETV4 和 HIF 通路之间的功能相互作用。
