Transcriptomics of specific activation of the Integrated Stress Response compared to Arsenite and Thapsigargin treatment

The integrated stress response (ISR) is a central signaling pathway induced by a variety of insults, but how its outputs contribute to downstream physiological effects across diverse cellular contexts remains unclear. Using a synthetic tool, we specifically and tunably activated the ISR and performed multi-omics profiling to define the core modules elicited by this response in the absence of co-activation of parallel pathways commonly induced by pleiotropic stressors. We found that the ISR can elicit time- and dose-dependent gene expression changes that cluster into four modules with ATF4 driving only a small but fast and sensitive module that includes many amino acid metabolic enzymes. We showed that ATF4 was required to reroute carbon utilization towards amino acid synthesis derived both from glucose and reductive carboxylation of glutamine and away from the tricarboxylic acid cycle and fatty acid biogenesis revealing a new role for ATF4 in modulating cellular energetics. We also discovered an ATF4-independent reorganization of cellular lipids that promotes triglycerides synthesis and accumulation of lipid droplets that was essential for cell survival. Together, we demonstrate that a minimal ISR-inducing system is sufficient to trigger formation of two distinct cellular structures, stress granules and lipid droplets, and a previously unappreciated metabolic state. We generated a cell line expressing a synthetic construct that allowed us to selectively initiate the ISR in a tunable fashion. We harnessed this system to quantitatively explore the transcriptome over time and define ISR-specific and -sufficient responses. To specifically profile the cellular effects of ISR signaling, we generated a U2OS cell line stably expressing a synthetic construct, dimerizable PERK (Dmr-PERK), consisting of the eIF2ɑ kinase domain of mouse PERK fused to a chemically inducible DmrB dimerization domain. Upon addition of ligand AP20187 (dimerizer), the fusion protein dimerizes, leading to its activation and phosphorylation of eIF2ɑ. To evaluate how the transcriptional response elicited by this minimal system compared to that of commonly used pleiotropic ISR-inducing agents, we performed RNA-seq on Dmr-PERK cells treated with a time course of dimerizer (0.2nM), thapsigargin (100nM), a SERCA inhibitor that induces ER stress by depleting its Ca++ stores, or sodium arsenite (0.05mM), which generates ROS and causes oxidative stress. For each compound, we chose a dose that elicited comparable levels of phospho-eIF2ɑ. In parallel, we performed transcriptomics measurements on the parental U2OS cell line that did not express the Dmr-PERK construct to confirm that dimerizer treatment did not have any significant transcriptional effects.

整合应激反应（integrated stress response, ISR）是一类由多种损伤因素诱导的核心信号通路，但其下游输出如何在多样的细胞环境中介导生理效应仍未明确。本研究借助一种合成工具，实现了ISR的特异性与可调控激活，并在排除多效性应激原通常共激活的平行通路的前提下，通过多组学分析解析该应激反应诱导的核心模块。研究发现，ISR可引发时间和剂量依赖性的基因表达变化，这些变化可聚类为四个模块；其中激活转录因子4（ATF4）仅调控一个小型但响应快速且敏感的模块，该模块包含众多氨基酸代谢酶。我们证实，ATF4可介导碳代谢重定向：使碳流从三羧酸循环（tricarboxylic acid cycle, TCA）和脂肪酸生物合成转向源自葡萄糖及谷氨酰胺还原性羧化的氨基酸合成，这揭示了ATF4在调控细胞能量代谢中的全新功能。本研究还发现了一种不依赖ATF4的细胞脂质重编程过程，该过程可促进甘油三酯合成与脂滴积累，且对细胞存活至关重要。综上，我们证明了最小化的ISR诱导系统足以触发两种不同的细胞结构——应激颗粒与脂滴的形成，以及一种此前未被认知的代谢状态。我们构建了一株表达合成构建体的细胞系，该构建体可让我们以可调控的方式选择性启动ISR。我们利用该系统对不同时间点的转录组进行定量检测，以明确ISR特异性且足以介导的应答反应。为特异性分析ISR信号的细胞效应，我们构建了稳定表达二聚化PERK（Dmr-PERK）的U2OS细胞系，该构建体由小鼠PERK的eIF2α激酶结构域与化学诱导型DmrB二聚化结构域融合而成。当添加配体AP20187（二聚化剂）时，融合蛋白发生二聚化，进而被激活并磷酸化eIF2α。为评估该最小化系统引发的转录应答与常用多效性ISR诱导剂的应答差异，我们对经以下试剂处理的Dmr-PERK细胞进行了RNA测序：时间梯度处理的二聚化剂（0.2nM）、毒胡萝卜素（100nM，一种通过耗尽内质网钙储备诱导内质网应激的肌浆网钙ATP酶（SERCA）抑制剂），以及亚砷酸钠（0.05mM，一种产生活性氧（Reactive Oxygen Species, ROS）并引发氧化应激的试剂）。我们为每种试剂选择了可引发相当水平磷酸化eIF2α的剂量。同时，我们对未表达Dmr-PERK构建体的亲本U2OS细胞系进行了转录组学检测，以确认二聚化剂处理不会引发显著的转录效应。