BioProject

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 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 determine how the transcriptional outputs of the ISR vary with time (duration) and input strength (stress level), we performed RNA-seq on cells treated for 1, 2, 4, 8, 16, or 24 hours with one of three doses of dimerizer. We chose a low dose of dimerizer (0.01 nM) where protein synthesis inhibition would be minimal (<15%) but ATF4 protein expression was still induced, an intermediate dose (0.2 nM) that caused a partial (50%) reduction in protein synthesis, and a high dose (3 nM) that led to the strongest (75%) reduction in protein synthesis and accumulation of stress granules.