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Genome-Wide CRISPR Screen Identifies Suppressors of Endoplasmic Reticulum Stress-Induced Apoptosis

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Genome-Wide CRISPR Screen Identifies Suppressors of Endoplasmic Reticulum Stress-Induced Apoptosis Genome-wide CRISPR screen identifies suppressors of endoplasmic reticulum stress-induced apoptosis Ronald A. Panganibana,1, Hae-Ryung Parka,1, Maoyun Suna, Maya Shumyatcherb, Blanca E. Himesb, and Quan Lua,c,2 aProgram in Molecular and Integrative Physiological Sciences, Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA 02115; bDepartment of Biostatistics, Epidemiology, and Informatics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; and cDepartment of Genetics & Complex Diseases, Harvard T.H. Chan School of Public Health, Boston, MA 02115 Edited by F. Ulrich Hartl, Max Planck Institute of Biochemistry, Martinsried, Germany, and approved May 24, 2019 (received for review April 12, 2019) Sensing misfolded proteins in the endoplasmic reticulum (ER), cells ER-associated protein degradation (ERAD) machinery, ER initiate the ER stress response and, when overwhelmed, undergo chaperones, and autophagy pathway genes (12–14), to allevi- apoptosis. However, little is known about how cells prevent ate ER stress. In addition, PERK-induced eIF2α phosphory- excessive ER stress response and cell death to restore homeostasis. lation attenuates global protein translation to prevent the Here, we report the identification and characterization of cellular introduction of additional misfolded proteins (8). Together, suppressors of ER stress-induced apoptosis. Using a genome-wide these responses relieve disturbances in the ER and help restore CRISPR library, we screen for genes whose inactivation further proteohomeostasis. increases ER stress-induced up-regulation of C/EBP homologous Sustained high levels of ER stress, however, trigger apoptosis — protein 10 (CHOP) the transcription factor central to ER stress- (15). The ER stress-induced apoptosis is largely mediated associated apoptosis. Among the top validated hits are two inter- through CHOP (C/EBP homologous protein, also known as acting components of the polycomb repressive complex (L3MBTL2 MGA DDIT3), the transcription factor that integrates signaling from [L(3)Mbt-Like 2] and [MAX gene associated]), and microRNA- all three branches of the ER stress pathway. XBP1s, ATF6(n), 124-3 (miR-124-3). CRISPR knockout of these genes increases CHOP and ATF4 all can bind to the CHOP promoter to increase its expression and sensitizes cells to apoptosis induced by multiple expression (16). Up-regulation of CHOP triggers apoptosis ER stressors, while overexpression confers the opposite effects. L3MBTL2 associates with the CHOP promoter in unstressed cells mainly by increasing the ratio of pro- vs. antiapoptotic proteins to repress CHOP induction but dissociates from the promoter in (16, 17). For example, CHOP increases the expression of the the presence of ER stress, whereas miR-124-3 directly targets the proapoptotic proteins BIM and PUMA, and, at the same time, IRE1 branch of the ER stress pathway. Our study reveals distinct down-regulates the expression of antiapoptotic Bcl-2 protein – mechanisms that suppress ER stress-induced apoptosis and may (18 20). These molecular events ultimately lead to the activa- lead to a better understanding of diseases whose pathogenesis tion of apoptotic caspases (e.g., caspase 3, 8, and 9) to cause cell is linked to overactive ER stress response. death (21). While the elaborate signaling pathways leading to ER stress- UPR (unfolded protein response) | proteotoxicity | environmental induced cell death have been well characterized, much less is toxicant | genetic screen known about how cells suppress excessive ER stress response to aintaining protein homeostasis is critical for the fitness and Significance Msurvival of all living cells. Newly synthesized proteins in the endoplasmic reticulum (ER) must be correctly folded before Dysregulated endoplasmic reticulum (ER) stress response con- being transported to subcellular destinations. About 30% of all tributes to the pathogenesis of myriad diseases. The molecular newly synthesized proteins are misfolded (1), and exposure of pathways leading to ER stress-induced cell death are well char- cells to environmental proteotoxicants such as arsenic (As) fur- acterized; however, much less is known about how cells suppress ther increases protein misfolding (2, 3). Misfolded proteins are excessive ER stress response to avoid apoptosis and restore ho- nonfunctional, prone to aggregation, and often toxic to cells (4). meostasis. Using a CRISPR-based loss-of-function genetic screen, As a result, high levels of misfolded proteins contribute to the our study uncovered multiple suppressors of ER stress response. pathogenesis of multiple diseases, including type 2 diabetes, These suppressors include a polycomb protein complex that di- cancer, and most neurodegenerative disorders (5). rectly inhibits the expression of the transcriptional factor central As a major site of protein synthesis, the ER is capable of sensing to ER stress-induced cell death and a microRNA that targets IRE1, and responding to the accumulation of misfolded proteins, a a canonical ER stress pathway component. Our study reveals condition widely known as ER stress. The elaborate cellular regulatory mechanisms that ameliorate potentially damaging response to ER stress, also known as unfolded protein response stress response and provides potential therapeutic targets for (UPR), is mediated by three ER-resident transmembrane pro- pathologies whose etiology is linked to overactive ER stress teins: PERK, IRE1, and ATF6 (6). In the presence of ER stress, response. misfolded proteins bind and sequester the molecular chaperone Author contributions: R.A.P., H.-R.P., and Q.L. designed research; R.A.P. and H.-R.P. per- BiP/GRP78 away from PERK, IRE1, and ATF6, leading to ac- formed research; R.A.P., H.-R.P., M. Sun, M. Shumyatcher, B.E.H., and Q.L. contributed tivation of these three molecules and their respective down- new reagents/analytic tools; R.A.P., H.-R.P., M. Sun, M. Shumyatcher, B.E.H., and Q.L. stream signaling cascades (7). Activation of PERK induces analyzed data; and R.A.P., H.-R.P., and Q.L. wrote the paper. phosphorylation of eIF2α and up-regulation of ATF4, a potent The authors declare no conflict of interest. transcription factor (8). Activation of IRE1 triggers the cleavage This article is a PNAS Direct Submission. of XBP1 mRNA into its transcriptionally active spliced form Published under the PNAS license. XBP1s (9, 10). ATF6 activation results in translocation to the 1R.A.P. and H.-R.P. contributed equally to this work. Golgi, where it is cleaved by proteases into an active form 2To whom correspondence may be addressed. Email: [email protected]. ATF6(n), another potent transcription factor (11). As tran- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. scriptional activators, ATF4, XBP1s, and ATF6(n) up-regulate 1073/pnas.1906275116/-/DCSupplemental. a myriad of UPR target genes, including antioxidant genes, Published online June 18, 2019. 13384–13393 | PNAS | July 2, 2019 | vol. 116 | no. 27 www.pnas.org/cgi/doi/10.1073/pnas.1906275116 Downloaded by guest on September 28, 2021 prevent unwanted apoptosis. In this study, using a genome-wide sociated with increased risk of developing ER stress-associated CRISPR loss-of-function screen coupled with a CHOP up- diseases (26, 27). As-treated cells were then subjected to regulation–based ER stress cell model, we identified and char- fluorescence-activated cell sorting (FACS) to isolate the bright acterized multiple genes, including members of polycomb repressive cell populations that express a high amount of mCherry (Fig. complex 1 (PRC.1), as well as microRNAs, as suppressors of ER 1A), as CRISPR knockout of genes that normally suppress stress response and associated cell death. Our study reveals CHOP up-regulation would lead to an increase in mCherry ex- distinct mechanisms that suppress ER stress response and apo- pression. To allow for identification of CRISPR cell populations ptosis, and provides insights into diseases whose pathogenesis is with increased intensity of fluorescence, we used a screen con- linked to abnormal ER stress response and cell death. dition (treatment of 5 μM As for 15 h) that initially induced a moderate increase (approximately two-fold) in the mean Results mCherry fluorescence. Sorted mCherry-bright cells were then Genome-Wide CRISPR Screen Identifies Suppressors of ER Stress allowed to repopulate once, followed by another round of ER Response. We previously developed an ER stress cell model that stress induction and cell sorting (SI Appendix, Fig. S2A). To harbors a fluorescence reporter (mCherry) under the control of minimize nonspecific bystander effect, we constructed a sub- the CHOP gene promoter (22). These CHOP-mCherry reporter CRISPR library using guides amplified from the genomic DNA cells respond in a dose-dependent manner to known ER stressors of cells isolated from the second-round sorting (SI Appendix, Fig. such as As and tunicamycin (Tm) (SI Appendix,Fig.S1A and B). S2B). Compared with vector control, the sublibrary cells We transduced the CHOP-mCherry reporter cells with a genome- exhibited greater mCherry fluorescence at basal levels. The wide lentiviral CRISPR-Cas9 knockout (KO) library that targets sublibrary cells were again subjected to As treatment and cell ∼19,000 protein-coding genes (six guide RNAs [gRNA] per gene) sorting (Fig. 1A and SI Appendix, Fig. S2C). After As treatment, and ∼1,800 miRNAs (four gRNAs per miRNA) (23). We used the there
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