Nagati Jason S, Dioum Elhadji M, Giardinetto Catarina S, Mohiuddin S M Golam, Zhang Qiuyang, Garcia Joseph A
Department of Medicine, Columbia University Medical Center, New York, New York, USA.
Department of Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
J Biol Chem. 2025 Jun 10;301(7):110355. doi: 10.1016/j.jbc.2025.110355.
Hypoxia inducible factors (HIFs) are heterodimeric, oxygen-sensitive, stress-responsive transcription factors in vertebrates composed of one of three alpha components conferring oxygen sensitivity and one of four beta components required for dimerization and DNA binding. The founding alpha member, HIF-1α, is ubiquitously expressed and regulates numerous target genes required for cellular function under physiological hypoxic states. In comparison, HIF-2α is more restricted in its expression, but nevertheless is also required for normal physiological function. The prototypical HIF-2 target gene is erythropoietin (Epo), one of the most highly hypoxia-inducible genes in mammals and in Hep3B cells, a model cell line used to study hypoxia-inducible gene regulation. However, despite cell culture and in vivo data supporting Epo as a preferential HIF-2 target, the molecular basis for selective activation of Epo by HIF-2 remains unknown. In this study, we report identification of novel evolutionary conserved cis-acting elements in the mammalian 3' Epo enhancer region that includes recognition sites for stress-responsive early growth response (Egr) transcription factors, demonstrate that select Egr2 and Egr3 isoforms augment HIF-2 activation of a reporter containing the extended 3' Epo enhancer with Egr binding sites, reveal stable Egr2/HIF-2 complex formation in hypoxic Hep3B cells, and provide conditional knockout data from mice supporting an in vivo role for Egr2 in Epo gene regulation. These results provide insights into HIF-2 selective signaling mechanisms with ramifications that extend well beyond Epo regulation.
缺氧诱导因子（HIFs）是脊椎动物中的异源二聚体、氧敏感、应激反应转录因子，由赋予氧敏感性的三种α亚基之一和二聚化及DNA结合所需的四种β亚基之一组成。最早发现的α亚基成员HIF-1α广泛表达，可调节生理缺氧状态下细胞功能所需的众多靶基因。相比之下，HIF-2α的表达更具局限性，但对正常生理功能也是必需的。典型的HIF-2靶基因是促红细胞生成素（Epo），它是哺乳动物和用于研究缺氧诱导基因调控的模型细胞系Hep3B细胞中缺氧诱导程度最高的基因之一。然而，尽管细胞培养和体内数据支持Epo是优先的HIF-2靶标，但HIF-2选择性激活Epo的分子基础仍不清楚。在本研究中，我们报告了在哺乳动物Epo基因3'增强子区域鉴定出新型进化保守的顺式作用元件，该区域包括应激反应早期生长反应（Egr）转录因子的识别位点，证明选择的Egr2和Egr3亚型可增强含有具有Egr结合位点的扩展3'Epo增强子的报告基因的HIF-2激活，揭示缺氧的Hep3B细胞中稳定的Egr2/HIF-2复合物形成，并提供来自小鼠的条件性敲除数据，支持Egr2在Epo基因调控中的体内作用。这些结果为HIF-2选择性信号传导机制提供了见解，其影响远远超出Epo调控。
