GoKawiilCell lines and culture
HAP1 cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM) (Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal calf serum (FCS) (BioSell) and 1% penicillin–streptomycin–glutamine (PSG) solution (Thermo Fisher Scientific). 293T (HEK293T), A549, HeLa, U2OS, BJEH and HCT116 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific) supplemented with 10% heat-inactivated FCS and 1% PSG. SH-SY5Y cells were maintained in DMEM supplemented with 15% FCS, 1% PSG and 1 mM sodium pyruvate. K562 cells were maintained in Roswell Park Memorial Institute 1640 (RPMI) medium (Thermo Fisher) supplemented with 10% heat-inactivated FCS and 1% PSG. K562 differentiation was induced as previously described52. In brief, cells were pretreated with 200 nM imatinib (MedChemExpress, HY-15463) in IMDM at a density of 200,000 cells per ml for 24 h followed by treatment with 1 µM decatibine (MedChemExpress, HY-A0004R) in HEMA medium (IMDM, 20% FCS, 1% PSG and 2% BSA (Sigma Aldrich, A7030-50G)), 0.5 mg ml–1 holo-transferrin (Merck-Sigma, T0665), 2 U ml–1 erythropoietin (MedChemExpress, HY-P7164) and 20 ng ml–1 insulin (MedChemExpress, HY-P0035) for 3 days. HAP1 CHOPNeon WT and ∆HRI cells and clonal 293T ∆OMA1, ∆DELE1, ∆HRI and DELE1HA cells have been previously described21. All cell lines were tested initially for mycoplasma contamination.
Gene editing
To mutate EIF2S1 (which encodes eIF2α) endogenously into a non-phosphorylatable variant (EIF2S1S49A,S52A), 293T cells were transfected with a pX330 CRISPR plasmid (Addgene, 42230) containing a sgRNA targeting EIF2S1 exon 2, a donor vector encoding the serine 49 and 52 to alanine mutation and around 700 bp homology arms upstream and downstream of the sgRNA target site, and a puromycin-resistance vector. After 24 h of transfection, cells were selected with puromycin (1 μg ml–1), and single cell clones were derived and analysed for gene editing by PCR and Sanger sequencing.
Clonal and polyclonal knockout cell lines were also generated using the CRISPR–Cas9 system. Specifically, transient transfection of a pX330 containing the sgRNA of interest together with a puromycin-resistance or blasticidin-resistance vector was used to generate clonal 293T cells lacking OMA1, DELE1, HRI, HMOX1 and/or HMOX2. Lentiviral transduction of a pLentiCRISPR v.2 variant (derived from Addgene, 52961) containing the sgRNA of interest was used to delete OMA1, DELE1, HRI or NIX in A549, HeLa, U2OS, BJEH, HCT116, SH-SY5Y or K562 cells. After 24 h of transduction or transfection, cells were selected using puromycin (1 μg ml–1) or blasticidin (10 μg ml–1), and knockout efficiency was assessed by immunoblotting. Where indicated, clonal progeny of the polyclonal knockout populations were generated by single-cell cloning and verified by PCR, Sanger sequencing and immunoblotting. sgRNA sequences and primers for genotyping PCR used in this study are listed in Supplementary Table 1.
Haploid genetic screen for identification of CHOP regulators
Genome-wide mutagenesis of haploid HAP1 cells was carried out as previously described21. In brief, gene-trap virus particles were produced in 293T cells, concentrated by ultracentrifugation at 22,800 rpm for 2 h at 4 °C and stored at 4 °C overnight. To generate random genomic mutations via insertional mutagenesis by gene trapping, 1.5 × 107 haploid HAP1 CHOPNeon cells were transduced with concentrated retroviral particles 24 h after plating, followed by two additional transductions. The resulting library of mutants was expanded, plated at 20% confluence in a total of 20 T175 flasks (Sarstedt) and treated for 9 h with 5 μM DHA 48 h after plating. Cells were collected using trypsin–EDTA (0.25%, Gibco), passed through a 40 μm cell strainer (Greiner, 542040) and fixed with one volume of BD fix buffer I (BD Biosciences) for 10 min at 37 °C. The fixation was stopped with PBS (Gibco) containing 1% FCS, cells were passed through a 40 μm cell strainer, and approximately 1.5 × 109 cells were permeabilized with 1 pellet volume of cold BD Perm Buffer III (BD Biosciences) for 30 min on ice. Permeabilization was stopped with PBS containing 1% FCS, and cells were blocked in PBS with 1% FCS and 3% bovine serum albumin (BSA) for 30 min at room temperature. To increase fluorescence intensity of the CHOP(Neon) protein, cells were stained with anti-mNeonGreen antibodies (ProteinTech, 32F6) diluted 1:2,500 in PBS with 1% FCS and 1% BSA for 2.5 h on a rotor wheel at room temperature, followed by three 15-min washing steps with PBS and 1% FCS at room temperature. Primary antibodies were detected using AlexaFluor 488-conjugated secondary antibodies (anti-mouse-AF488, Life Technologies) diluted 1:500 in PBS supplemented with 1% FCS and 1% BSA for 1 h at room temperature on a rotor wheel protected from light. DAPI (Sigma-Aldrich, D9542) was added to the secondary antibody dilution at a final concentration of 2.5 μg ml–1 for a DNA counterstain. After three 15-min washing steps, cells were resuspended in PBS and 1% FCS, stored at 4 °C until sorting on a BD Fusion cell sorter (BD Biosciences, via FACSDiva v.8.0.2) using a 70 μm nozzle. Staining specificity was determined using a secondary antibody-only control. Haploid cells were identified on the basis of DNA content in the DAPI channel and of those, approximately 107 cells of the bottom 4% CHOP(Neon)-low and top 4% CHOP(Neon)-high cells were sorted into PBS and 10% FCS for isolation of gDNA.
Insertion site mapping and analysis
To extract gDNA from the sorted cell populations de-crosslinking was performed at 56 °C overnight followed by DNA isolation using a QIAamp DNA Mini kit (Qiagen, 51306) according to the manufacturer’s instructions. Gene-trap insertion sites of CHOP(Neon)-high and CHOP(Neon)-low populations were recovered as previously described21. The amplified libraries were sequenced on a NextSeq1000 (Illumina) with a read length of 60 nucleotides. Demultiplexing of indexed sequencing reactions was performed, allowing one mismatch. Reads were aligned to the human reference genome (hg19) and analysed as previously described21. Bowtie53 (v.1.0.1) was used to align reads to the human genome, allowing one mismatch, followed by mapping to the coordinates of RefSeq protein-coding genes with intersectBED54 (v.2.26.0). Only integrations in the sense orientation were considered disruptive and used for downstream analyses. To identify CHOP(Neon) regulators in DHA-treated cells per gene, the number of unique gene-trap insertion sites in the query gene versus the whole sample was compared between the CHOP(Neon)-high and CHOP(Neon)-low cell populations using a two-sided Fisher’s exact test and Benjamini–Hochberg FDR correction. Data were plotted as the combined number of unique mutations identified in the CHOP(Neon)-high and CHOP(Neon)-low population (x axis) versus their mutation ratio (high versus low) normalized to the respective sizes of the datasets (y axis). Fishtail plots were created using GraphPad Prism 10.
Treatments, transfections and transductions
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