Intestinal epithelial cells (IECs) exist in a metabolic state of low oxygen tension termed “physiologic hypoxia.” An important factor in maintaining intestinal homeostasis is the transcription factor hypoxia-inducible factor (HIF), which is stabilized under hypoxic conditions and mediates IEC homeostatic responses to low oxygen tension. To identify HIF transcriptional targets in IEC, chromatin immunoprecipitation (ChIP) was performed in Caco-2 IECs using HIF-1α or HIF-2α specific antibodies. ChIP-enriched DNA was hybridized to a custom promoter microarray (termed ChIP-chip). This unbiased approach identified autophagy as a major HIF-1 targeted pathway in IEC. Binding of HIF-1 to the ATG9A promoter, the only transmembrane component within the autophagy pathway, was particularly enriched by exposure of IEC to hypoxia. Validation of this ChIP-chip revealed prominent induction of ATG9A, and luciferase promoter assays identified a functional hypoxia response element upstream of the TSS. Hypoxia-mediated induction of ATG9A was lost in cells lacking HIF-1. Strikingly, we found that lentiviral-mediated knockdown of ATG9A in IECs prevents epithelial barrier formation by >95% and results in significant mislocalization of multiple tight junction (TJ) proteins. Extensions of these findings showed that ATG9A knockdown cells have intrinsic abnormalities in the actin cytoskeleton, including mislocalization of the TJ binding protein vasodilator-stimulated phosphoprotein (VASP). These results implicate ATG9A as essential for multiple steps of epithelial TJ biogenesis and actin cytoskeletal regulation. Our findings have novel applicability for disorders that involve a compromised epithelial barrier and suggest that targeting ATG9A may be a rational strategy for future therapeutic intervention.

肠上皮细胞（IEC）处于低氧张力的代谢状态，称为“生理性缺氧”。维持肠道稳态的一个重要因素是转录因子缺氧诱导因子（HIF），它在缺氧条件下稳定并介导IEC对低氧张力的稳态反应。为了识别 IEC 中的 HIF 转录靶标，使用 HIF-1α 或 HIF-2α 特异性抗体在 Caco-2 IEC 中进行染色质免疫沉淀 (ChIP)。富含 ChIP 的 DNA 与定制启动子微阵列（称为 ChIP 芯片）杂交。这种公正的方法将自噬确定为 IEC 中主要的 HIF-1 靶向途径。IEC 暴露于缺氧条件下，HIF-1 与 ATG9A 启动子（自噬途径中唯一的跨膜成分）的结合特别丰富。该 ChIP 芯片的验证揭示了 ATG9A 的显着诱导，并且荧光素酶启动子测定鉴定了 TSS 上游的功能性缺氧反应元件。缺氧介导的 ATG9A 诱导在缺乏 HIF-1 的细胞中消失。引人注目的是，我们发现慢病毒介导的 IEC 中 ATG9A 的敲低可阻止超过 95% 的上皮屏障形成，并导致多个紧密连接 (TJ) 蛋白的显着错误定位。这些发现的扩展表明，ATG9A 敲低细胞的肌动蛋白细胞骨架具有内在异常，包括 TJ 结合蛋白血管舒张刺激磷蛋白 (VASP) 的错误定位。这些结果表明 ATG9A 对于上皮 TJ 生物发生和肌动蛋白细胞骨架调节的多个步骤至关重要。我们的研究结果对于涉及上皮屏障受损的疾病具有新的适用性，并表明针对 ATG9A 可能是未来治疗干预的合理策略。