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. Overall design: 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 eIF2? 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 eIF2?. 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-eIF2?. 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.