CHOP induces activating transcription factor 5 (ATF5) to trigger apoptosis in response to perturbations in protein homeostasis.

Environmental stresses that disrupt protein homeostasis induce phosphorylation of eIF2, triggering repression of global protein synthesis coincident with preferential translation of ATF4, a transcriptional activator of the integrated stress response (ISR). Depending on the extent of protein disruption, ATF4 may not be able to restore proteostatic control and instead switches to a terminal outcome that features elevated expression of the transcription factor CHOP (GADD153/DDIT3). The focus of this study is to define the mechanisms by which CHOP directs gene regulatory networks that determine cell fate. We find that in response to proteasome inhibition, CHOP enhances the expression of a collection of genes encoding transcription regulators, including ATF5, which is preferentially translated during eIF2 phosphorylation. Transcriptional expression of ATF5 is directly induced by both CHOP and ATF4. Knockdown of ATF5 increases cell survival in response to proteasome inhibition, supporting the idea that both ATF5 and CHOP have proapoptotic functions. Transcriptome analysis of ATF5-dependent genes reveals targets involved in apoptosis, including NOXA, which is important for inducing cell death during proteasome inhibition. This study suggests that the ISR features a feedforward loop of stress-induced transcriptional regulators, each subject to transcriptional and translational control, which can switch cell fate toward apoptosis.

Methods for analyzing eIF2 kinases and translational control in the unfolded protein response.

Endoplasmic reticulum (ER) stress induces a program of translational and transcriptional regulation, designated the unfolded protein response (UPR), that collectively remedies stress damage and restores ER homeostasis. The protein kinase PERK facilitates the translational control arm of the UPR by phosphorylation of eIF2, a translation initiation factor that combines with GTP to escort initiator Met-tRNA(i)(Met) to the ribosomal machinery during the initiation of protein synthesis. Phosphorylation of the alpha subunit of eIF2 on serine-51 inhibits global translation initiation, which reduces the influx of nascent polypeptides into the overloaded ER. eIF2 phosphorylation also facilitates the preferential translation of stress-related mRNAs, such as ATF4 which in turn activates the transcription of UPR genes. In this chapter, we present experimental strategies and methods for establishing and characterizing global and gene-specific translation control induced by eIF2 phosphorylation (eIF2α~P) during ER stress. These methods include assays for the detection of eIF2α~P and its target genes. We also discuss strategies to address whether a given ER stress condition triggers eIF2α~P through PERK, as opposed to other stress conditions activating alternative members of the eIF2 kinase family. Additionally, experimental descriptions are provided for detecting and quantifying a repression in global translation initiation, and identifying stress-induced preferential translation, such as that described for ATF4. Together, these experimental descriptions will provide a useful molecular "toolkit" to study each feature of the translational control processes invoked during ER stress.