A Unique ISR Program Determines Cellular Responses to Chronic Stress Bo-Jhih Guan, Vincent van Hoef, Raul Jobava, Orna Elroy-Stein, Leos S. Valasek, Marie Cargnello, Xing-Huang Gao, Dawid Krokowski, William C. Merrick, Scot R. Kimball, Anton A. Komar, Antonis E. Koromilas, Anthony Wynshaw-Boris, Ivan Topisirovic, Ola Larsson, Maria Hatzoglou  Molecular Cell  Volume 68, Issue 5, Pages 885-900.e6 (December 2017) DOI: 10.1016/j.molcel.2017.11.007 Copyright © 2017 Elsevier Inc. Terms and Conditions

Molecular Cell 2017 68, 885-900.e6DOI: (10.1016/j.molcel.2017.11.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Translational Recovery during Chronic ER Stress Occurs without Recovery of eIF2B Activity (A–C) Western blot analysis (A), protein synthesis (B), and eIF2B GEF activity (C) measured in WT and eIF2αS51A/S51A MEFs treated with Tg as indicated. (D) Distribution of ATF4 and GAPDH mRNAs on polyribosomes isolated from MEFs treated with Tg and PERKi for the indicated times. (E and F) Western blot analysis (E) and protein synthesis (F) in MEFs expressing control or eIF2Bε shRNAs and treated with Tg as indicated. The mean ± SEM of triplicate determinations is shown. ∗p < 0.01. (G–I) eIF2B GEF activity (G), protein synthesis (H), and western blot analysis (I) for the indicated treatments. ∗p < 0.01; n.s., not significant. Molecular Cell 2017 68, 885-900.e6DOI: (10.1016/j.molcel.2017.11.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 The Unique ISR Program during Chronic ER Stress Promotes Adaptation (A and B) Western blot analysis (A) and protein synthesis (B) in MEFs treated with CPA for the indicated times. (C–F) Cell viability (C), western blot analysis (D), protein synthesis (E), and eIF2B GEF activity (F) in MEFs treated with CPA for the indicated times, or treated with CPA for 12 hr followed by washout or no wash for the indicated times. In (C), cell viability of CPA for 12 hr is set as 100%. The mean ± SEM of triplicate determinations is shown. ∗p < 0.01; n.s., not significant. Molecular Cell 2017 68, 885-900.e6DOI: (10.1016/j.molcel.2017.11.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Remodeling of Translation Initiation during Chronic ER Stress Is eIF4F Independent (A) Protein synthesis in WT and eIF2αS51A/S51A MEFs treated with Tg and 4EGI-1 or its vehicle (DMSO) as indicated. Note that the concentrations of 4EGI-1 used to treat WT and eIF2αS51A/S51A MEFs are 100 and 200 μM, respectively, to reduce protein synthesis to a similar level (by 50%). The mean ± SEM of triplicate determinations is shown. (B) Western blot analysis of the indicated proteins isolated via a cap (m7GTP) pull-down assay from cell extracts (input) of MEFs treated with Tg for the indicated times. Torin 1 treatment for 1 hr was used as a control. (C–E) Western blot analysis (C), protein synthesis (D), and eIF2B GEF activity (E) measured in MEFs expressing control or eIF4E shRNAs and treated with Tg as indicated. ∗p < 0.01; n.s., not significant. Molecular Cell 2017 68, 885-900.e6DOI: (10.1016/j.molcel.2017.11.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Reprogramming of Translation Initiation during Chronic ER Stress Is eIF3 Dependent (A) Overview of the UV crosslinking and oligo (dT) capture assay for mRNA-binding proteins. (B and C) Silver staining (B) and western blot analysis (C) of SDS-PAGE as described in (A). See STAR Methods for details. Input represents loading of equal amount of proteins. Oligo (dT) represents loading of equal optical density (OD) (260 nm) of eluted poly(A+) RNA. (D–F) Protein synthesis (D) and western blot analysis (E and F) in MEFs expressing the indicated shRNAs (control, eIF3a, eIF3c, eIF3g, eIF3d, and eIF3l) and treated with Tg as indicated. (G) Protein synthesis and eIF2B GEF activity in MEFs expressing eIF3d shRNAs and treated as indicated. The mean ± SEM of triplicate determinations is shown. ∗p < 0.01; n.s., not significant. (H) qRT-PCR analysis of the indicated RNAs isolated from cytosolic extracts (top) or anti-eIF3d immunoprecipitates (bottom) from MEFs treated as indicated. Top: signals were normalized to β-actin mRNA first and set as value 1 in unstressed samples subsequently. Bottom: the eIF3d-IP RNA signals were first normalized to respective cytosolic RNA levels (from top) and set as value 1 in unstressed samples subsequently. In both panels, data are presented as fold change. (I) Model of the eIF3-dependent translation initiation during chronic ER stress. Molecular Cell 2017 68, 885-900.e6DOI: (10.1016/j.molcel.2017.11.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Regulation of PIC Assembly during Chronic ER Stress MEFs were treated as indicated and formaldehyde crosslinked cytosolic extracts were analyzed on sucrose gradients followed by western blot analysis (STAR Methods). Equal amount of protein (input) was analyzed in parallel. Fractions containing the PIC were marked (40S-48S). Molecular Cell 2017 68, 885-900.e6DOI: (10.1016/j.molcel.2017.11.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 Chronic ER Stress Is Associated with Modulation of mRNA Levels Whose Translation Is Insulated from Mechanisms Controlling Translational Efficiency during the Acute Phase (A) Overview of experimental setup. (B) Scatterplots of fold changes in Tg:1h versus Tg:0h (acute phase) and Tg:16h versus Tg:1h (chronic phase) using data from cytosolic or polysome-associated RNA. Transcripts showing differential translation or congruent changes are color coded and summarized in pie charts (colors correspond to those of regulatory patterns in scatterplot; white indicates the proportion of the chronic phase response that was not observed during acute phase). (C) Boxplots of residuals from a linear regression of polysome-associated mRNA on cytosolic mRNA (Tg:1h versus Tg:0h, left) (Tg:16h versus Tg:1h, right) (scatterplots shown in Figure S5E). D, downregulated; N, non-regulated; U, upregulated (color scheme as in B). p values assess differences to the non-regulated group. ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005. (D) 5′ UTR characteristics of differentially translated mRNAs during acute stress (top row) or genes regulated congruently during chronic stress (bottom row). Color scheme described as in (B). D, N, U, and statistics defined as in (C). (E) Scatterplot of log2 fold changes comparing last 4 hr of PERKi treatment during chronic stress to chronic stress (Tg:16h+PERKi versus Tg:16h) using data as described and pie chart summarized in (B). (F) Cumulative distributions for log2 fold changes of selected gene signatures compared to all genes for the acute phase, chronic phase, and Tg:16h+PERKi versus Tg:16h using cytosolic (top row) and polysome-associated (bottom row) data. Mean log2 fold change as compared to background and associated p values are indicated for each signature. (G) Number of genes overlapping between those congruently regulated during chronic phase, congruently regulated following PERKi, and transcriptional ATF4/CHOP targets (Han et al., 2013). Molecular Cell 2017 68, 885-900.e6DOI: (10.1016/j.molcel.2017.11.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 Recovery of eIF2B Activity during Chronic ER Stress Causes ER Dysfunction and Induces a “Foamy Cell” Phenotype (A) Pathway analysis of the “congruent up” genes from polysome/RNA-seq of Tg:16h versus Tg:1h (Figure 6B). x axis shows p value for the top five most significant pathways identified. (B) Cell viability, caspase 3 activity, and proteasome activity in MEFs treated as indicated. (C) Representative phase-contrast microscopic images of MEFs treated with Tg for 12 hr (first panel) or for 24 hr with addition of PERKi after the first 12 hr of Tg (panels 2–5). (D) Quantification of foamy cells in the indicated MEFs treated with Tg:24h alone or with PERKi and cycloheximide (CHX), hippuristanol (Hipp.), or harringtonine (Harri.) for last 12 hr as indicated. (E and F) Protein synthesis and eIF2B GEF activity in eIF2αS51A/S51A MEFs (E) and human iPSC-derived NPCs (F) treated as indicated. The mean ± SEM of triplicate determinations is shown. ∗p < 0.01; n.s., not significant. (G) Schematic representation of ISR programs that determine cellular responses to ER stress. In acute/transient ER stress, protein homeostasis is achieved via GADD34-dependent feedback on de-phosphorylating eIF2α-P(S51) and restoring the eIF2B GEF activity. In chronic ER stress, a unique program, defined here as chronic ISR, is achieved via an eIF3-dependent translation initiation reprogramming. Chronic ISR sustains ER function and protects cells from a foamy-type cell death. Molecular Cell 2017 68, 885-900.e6DOI: (10.1016/j.molcel.2017.11.007) Copyright © 2017 Elsevier Inc. Terms and Conditions