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. 2013 May;15(5):481-90.
doi: 10.1038/ncb2738. Epub 2013 Apr 28.

ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death

Jaeseok Han  1 , Sung Hoon Back, Junguk Hur, Yu-Hsuan Lin, Robert Gildersleeve, Jixiu Shan, Celvie L Yuan, Dawid Krokowski, Shiyu Wang, Maria Hatzoglou, Michael S Kilberg, Maureen A Sartor, Randal J Kaufman
Affiliations

ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death

Jaeseok Han et al. Nat Cell Biol. 2013 May.

Abstract

Protein misfolding in the endoplasmic reticulum (ER) leads to cell death through PERK-mediated phosphorylation of eIF2α, although the mechanism is not understood. ChIP-seq and mRNA-seq of activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), key transcription factors downstream of p-eIF2α, demonstrated that they interact to directly induce genes encoding protein synthesis and the unfolded protein response, but not apoptosis. Forced expression of ATF4 and CHOP increased protein synthesis and caused ATP depletion, oxidative stress and cell death. The increased protein synthesis and oxidative stress were necessary signals for cell death. We show that eIF2α-phosphorylation-attenuated protein synthesis, and not Atf4 mRNA translation, promotes cell survival. These results show that transcriptional induction through ATF4 and CHOP increases protein synthesis leading to oxidative stress and cell death. The findings suggest that limiting protein synthesis will be therapeutic for diseases caused by protein misfolding in the ER.

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Figures

Figure 1
Figure 1
ATF4 and CHOP bind to promoter regions of genes encoding protein synthesis and UPR functions. (a) Protein expression during ER stress-mediated cell death. Cell lysates were collected at the indicated times after Tm (2 μg ml–1) treatment for western blot analysis. (b) Effect of ATF4 and CHOP expression in WT MEFs. MEFs were infected with adenoviruses expressing CHOP and/or ATF4 at an MOI (mode of infection) of 100. At 24 h after infection, cell lysates were analysed by western blotting (upper panel). At 48 h after infection, cell viability was measured by a WST-8 assay (n = 3 independent experiments; lower panel). (c) Distribution of CHOP and ATF4 ChIP-seq peaks across the genome. The peaks were classified as: within introns (Intron), within 3′ untranslated regions (UTRs), within 5′ UTRs, or within coding sequences (Exon), <3 kb from TSSs or >3 kb from TSSs in intergenic regions. The numbers below the annotations represent the percentage of peaks across the genome. (d) Venn diagram showing overlapping and unique sets of ATF4- and CHOP-occupied genes that have peaks <3 kb from the TSS. (e) Functional enrichment analysis of ATF4 and CHOP target genes that have peaks <3 kb from the TSS. All error bars represent means±s.e.m. Uncropped images of blots are shown in Supplementary Fig. S7.
Figure 2
Figure 2
ATF4 and CHOP upregulate expression of genes encoding protein synthesis and UPR functions. (a) Clustered mRNA-seq expression data presented in a heat map with ChIP-seq peaks aligned. Tm-induced and -repressed genes in WT, Chop–/– (CK), and Atf4–/–-(AK) MEFs are shown. Genes containing ATF4- and CHOP- binding sites <3 kb from the TSSs are denoted by bars. Green vertical bar represents downregulated genes, whereas red vertical bar represents upregulated genes in WT MEFs in response to Tm (2 μg ml–1) when compared with vehicle (dimethylsulphoxide). (b) Venn diagram illustrating overlaps of gene sets from mRNA-seq and ChIP-seq for ATF4 and CHOP. A: genes bound by CHOP without expression changes in response to Tm (2 μg ml–1). B: genes bound by both ATF4 and CHOP without expression changes. C: genes bound by both ATF4 and CHOP with differential expression. Genes in C are shown in Supplementary Table S4. D: genes bound by CHOP that exhibit differential expression. E: genes bound by ATF4 that do not change expression. F: genes bound by ATF4 that exhibit differential expression. G: genes with differential expression but do not bind ATF4 or CHOP. (c) Functional enrichment analysis of gene sets in b. The EASE scores of DAVID functional enrichment of selected GO terms are represented in a heat map with –log10 (EASE score) as a colour index and number. White corresponds to an EASE score of 1.0 (no number in the block) with no statistical significance; dark red corresponds to an EASE score of 1.0×10–10 or lower.
Figure 3
Figure 3
ATF4 and CHOP interact to induce target genes involved in protein synthesis and the UPR. (a) Motif analysis of ATF4- and CHOP-binding sites. Sequences from CHOP- and ATF4-binding regions <3 kb upstream and downstream of a TSS were analysed. Significantly over-represented motifs from each gene set are shown. (b, c) Interaction of ATF4 and CHOP. (b) Flag–CHOP and ATF4 were expressed either alone or together in HEK293 cells and immunoprecipitated (IP) using anti-Flag or anti-ATF4 antibodies. Immunoprecipitated proteins were analysed by western blotting (IB) using ATF4 or Flag antibody. (c) MEFs were treated with Tm (2 mg ml–1) and immunoprecipitated using anti-ATF4, normal rabbit IgG or anti-CHOP. Immunoprecipitated proteins were analysed by western blotting using anti-CHOP antibody. (d) Co-occupancy of ATF4 and CHOP in the promoter regions of commonly targeted genes. Cells were treated with Tm for 10 h, followed by sequential ChIP assay (see Methods). The enrichment was determined for re-ChIPed chromatin by qPCR (n = 3 independent experiments). (e) Effect of ATF4 and/or CHOP overexpression on common target genes. ATF4 and CHOP were overexpressed either alone or together in WT MEFs. Gene expression was measured by qRT–PCR using β-gal as a negative control. Data are presented as means±s.e.m. (n = 3 independent experiments). (f) Expression profile of common target genes during Tm treatment. WT, Chop–/– and Atf4–/– MEFs were treated with Tm (2 μg ml–1) for 16 h and total RNAs were prepared at indicated time points for qRT–PCR (n = 3 independent experiments). All error bars represent means±s.e.m. Uncropped images of blots are shown in Supplementary Fig. S7.
Figure 4
Figure 4
ATF4 and CHOP increase protein synthesis leading to cell death. (a) Increased protein synthesis by ATF4 and CHOP. ATF4 and CHOP were expressed either alone or together in Chop+/+ or Chop–/– MEFs. After 24 h, cells were pulse-labelled with [35S]methionine/cysteine (n = 3 independent experiments; see Methods). (b, c) Ablation of translation attenuation by ATF4- and CHOP-mediated GADD34 induction. After forced expression of ATF4 and CHOP, MEFs were treated with 1 μM thapsigargin (Tg) for an hour and analysed by western blotting (b) and metabolic labelling (n = 3 independent experiments; c). (d) Effect of Gadd34 deletion on ATF4/CHOP-mediated increase in protein synthesis. ATF4 and CHOP were expressed in WT and Gadd34–/– MEFs, followed by metabolic labelling (n = 3 independent experiments). (e, f) Effect of translation attenuation on cell survival. Cells were transfected with scrambled (Scr) or Rpl24 siRNAs, followed by Ad-ATF4 and Ad-CHOP infection. Protein synthesis at 24 h (e) and cell viability at 48 h (f) were measured (n = 3 independent experiments). (g–i) Effect of unrestricted protein synthesis on survival. (g) Translational recovery after one hour Tg (1 μM) treatment in Eif2αA/A and Eif2αS / S (left panel) or Atf4+/+ and Atf4–/– MEFs (right panel; n = 3 independent experiments). (h) MEFs were treated with Tm (2 μg ml–1) for 24 h, and cell viability was measured (n = 3 independent experiments). (i) Cell lysates were collected at indicated times for western blot analysis. (j–l) Effect of GADD34 overexpression on cell survival. (j) Translational recovery after one hour Tg (1 μM) treatment in WT, Eif2αA/A and Atf4–/– MEFs after forced expression of either Gadd34ΔC or Gadd34ΔN (n = 3 independent experiments). (k) Indicated MEFs were treated with Tm (2 μg ml–1) for 24 h and viability was measured (n = 3 independent experiments). (l) Western blot analysis of cell lysates from MEFs treated with Tm (2 μg ml–1) for the indicated times. All error bars represent means ± s.e.m. Uncropped images of blots are shown in Supplementary Fig. S7.
Figure 5
Figure 5
ATF4 and CHOP increase oxidative stress and deplete ATP. (a, b) Oxidative stress induced by ATF4 and CHOP overexpression. (a) WT MEFs were mock-infected or infected with Ad-ATF4 and Ad-CHOP for 24 h and stained with CM-H2DCFDA for analysis by flow cytometry. Where indicated, 100 μM BHA was added to the medium at time of infection. Cells were treated with Tm (2 μg ml–1) at 24 h before analysis. (b) Histogram for median peaks in a (n = 3 independent experiments). (c) Effect of BHA treatment on cell viability. MEFs were treated with vehicle (Veh) or BHA (100 μM) 24 h before infection with Ad-ATF4 and Ad-CHOP for 48 h or Tm treatment (2 μg ml–1) for 24 h and cell survival was measured (n = 3 independent experiments). (d) Effect of Ero1α knockdown on cell viability. Survival was measured at 48 h after forced expression of ATF4 and CHOP or at 24 h after Tm treatment (2 μg ml–1; n = 3 independent experiments). (e) Expression profiles of anti-oxidant genes in response to ER stress. MEFs were treated with Tm (2 μg ml–1) for 10 h and total RNAs were extracted for qRT–PCR (n = 3 independent experiments). (f) ATP and ADP levels measured at the indicated times after infection with Ad-ATF4 and Ad-CHOP or Ad-GFP as control (n = 3 independent experiments). All error bars represent means±s.e.m.
Figure 6
Figure 6
ATF4 and CHOP increase protein synthesis and oxidative stress in vivo. (a) Protein synthesis was measured in the liver at 3 days after injection of Ad-β-gal (n = 6 mice) or Ad-ATF4/Ad-CHOP (n = 5 mice) applying the 2H2O tracer method (see Methods). (b) Carbonyls in the livers isolated from mice at 3 days after adenovirus infection (n = 6 mice per group). (c–e) Effect of inhibition of ATF4 function on protein synthesis and cell death. At 3 days after injection of Ad-β-gal or Ad-ATF4ΔRK, Tm was administered to each group of mice for 24 h followed by collection of liver and plasma for protein synthesis measurement (c) and staining for cleaved CASP3 (d). Scale bar, 50 μm. (e) Quantification of cleaved CASP3-positive cells was performed on cells in d (n = 3 mice per group). (f, g) Insulin and glucagon immunofluorescence staining. Insulin and glucagon staining were performed using pancreatic sections from A/A;fTg/0 (having the intact eIF2α transgene; n = 4 mice) and A/A;fTg/0;RIP-CreER/0 (having a WT Eif2α transgene deleted by tamoxifen injection; n = 4 mice) (f) and 4-month-old Atf4+/+ (n = 3 mice), and Atf4–/– mice (n = 3 mice) (g). Scale bars, 20 μm. Islet area was measured using dotSlide software. The percentage indicates the total islet area over the pancreas area in each section. (h, i) Glucose tolerance tests were performed and the area under curve from the glucose tolerance tests was quantified in A/A;fTg/0 (n = 6 mice) and A/A;fTg/0;RIP-CreER/0 (n = 4 mice) (h) mice at 12 weeks after tamoxifen injection or in Atf4+/+ (n = 5 mice) and Atf4–/– (n = 4 mice) mice at the age of 4 months (i). (j) Mechanism for ATF4- and CHOP-mediated cell death. On ER stress, eIF2α phosphorylation by PERK promotes preferential translation of ATF4 for subsequent induction of CHOP. ATF4 and CHOP act together to upregulate target genes encoding functions in protein synthesis to restore general mRNA translation. If the adaptive UPR effectively reduces the unfolded protein load, restoration of protein synthesis promotes cell survival. However, if protein synthesis increases before restoration of proteostasis, ROS are produced as a signal to promote cell death. Blue colour indicates pro-survival pathways and orange colour indicates pro-death pathways. All error bars represent means±s.e.m.
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