Animals
Table of Contents
All experiments were performed in accordance with UK Home Office regulations and the UK Animals (Scientific Procedures) Act of 2013 under UK Home Office licences. These licences were approved by the Wellcome Sanger Institute (WSI) Animal Welfare and Ethical Review Board. Mice were maintained in a specific-pathogen-free unit under a 12 h light and 12 h dark cycle with lights off at 19:30 and no twilight period. The ambient temperature was 21 ± 2 °C, and the humidity was 55 ± 10%. Mice were housed at 3–5 mice per cage (overall dimensions of caging: 365 mm × 207 mm × 140 mm (length × width × height), floor area, 530 cm2) in individually ventilated caging (Tecniplast, Sealsafe 1284L) receiving 60 air changes per hour. In addition to Aspen bedding substrate, standard environmental enrichment of Nestlets, a cardboard tube/tunnel and wooden chew blocks were provided. Mice were given water and diet ad libitum.
Mouse generation
A complete list of the mouse lines used in this study is provided in the Source Data. Most mouse mutants were generated using the well-validated ‘KO-first allele’. This strategy relies on the identification of an exon common to all transcript variants, upstream of which a LacZ cassette is inserted to make a constitutive KO/gene-trap known as a tm1a allele. In contrast to the tm1a allele, tm1b creates a frameshift mutation after Cre-mediated deletion of the loxP-flanked exon. Other allele types are also possible and have been described previously60. Mouse production was performed as described previously61. We maintained most mutant lines (73% of the mice tested in this study) on a pure inbred C57BL/6N background, with the other lines on mixed C57BL/6 backgrounds (for example, C57BL/6N;C57BL/6BrdTyrc-Brd). For the C57BL/6N background, a core colony was established using mice from Taconic Biosciences, which was refreshed at set generational points (typically ten generations) and cryopreserved at regular intervals to avoid genetic drift. The sex and age for all mice analysed is available in the Source Data. For tumour-watch studies, mice were aged for spontaneous tumour formation until they became moribund in keeping with the above-mentioned Home Office Guidelines. To ensure compliance, mice were examined twice daily for symptoms including weight loss, poor coat condition and hunched back. Tumour histology was analysed by a consultant pathologist to confirm cancer diagnoses. Mice were assigned randomly to groups on the basis of Mendelian inheritance.
In vivo MN screen
The in vivo MN screen was performed according to a previously described protocol20. The samples were analysed on the LSRFortessa or Cytomics FC500 (Becton Dickinson) system with a minimum of 100,000 events collected per sample. The gating strategy used is shown in Supplementary Fig. 1. For the analysis of MN screening data, a mixed linear effect beta regression model exploring the effect of genotype on the percentage of MN, was used. This was implemented within R (glmmTMB, v.1.0.1). In detail, a regression model was fitted using flow.cytometer as a fixed effect to account for any differences arising from the instrumentation, while assay.date was fitted as a random effect to account for the variance introduced by batch (Y ~ genotype + flow.cytometer + (1|batch). The genotype effect and associated error were estimated as a marginal mean using the emmeans package (R; v.1.4.4). The significance of the genotype effect was assessed using a likelihood ratio test. Analysis code is available at GitHub.
High-throughput phenotypic screen
The high-throughput phenotyping pipeline used was a series of standardized tests conducted in accordance with standard operating procedures (available at IMPReSS (https://www.mousephenotype.org/impress/index) and were performed by the Mouse Genetics Project (MGP) at the Wellcome Sanger Institute (WSI). Tests covered a broad range of biological areas, including metabolism, cardiovascular, neurological and behavioural, bone, sensory and haematological systems, and plasma chemistry. Factors predicted to affect the variables were standardized where possible. If this was not possible, measures were taken to reduce potential biases, for example, the impact of different people performing the test (known as the minimized operator), and the time of day of the test, as defined by the Mouse Experimental Design Ontology (MEDO)62. The data captured with the MEDO ontology can be found at http://www.mousephenotype.org/about-impc/arrive-guidelines. Moreover, pre-established reasons were defined for quality-control failures (for example, insufficient sample, error with equipment during test) and detailed using IMPRESS, and the data inclusion/exclusion criteria were therefore standardized. All discarded data were retained and tracked in a database to enable quality-control-failed data to be audited. Phenotyping data were collected at regular intervals on age-, sex- and strain-matched WT (control) mice. On average, at least seven homozygote mice of each sex per KO line were generated for phenotyping. If no homozygotes were obtained from ≥28 offspring from heterozygote intercrosses at postnatal day 14 (P14), the line was declared homozygous lethal. Similarly, if less than 13% of the pups resulting from heterozygote intercrosses were homozygous at P14, the line was judged as being homozygous subviable. In this event, heterozygote mice were examined in the phenotyping screen. The random allocation of mice to experimental group (WT versus KO) was driven by Mendelian inheritance. Owing to the high-throughput nature of the phenotyping screen, blinding the operators to the identity of KO lines during phenotyping was not used as the cage cards used to identify the mice included genotype information. However, in a high-throughput environment without a defined hypothesis, the potential bias is minimized. In all cases, the individual mouse was considered to be the experimental unit. Further experimental design strategies (for example, exact definition of a control animal) are defined using a standardized ontology as described previously62 and are available from the IMPC portal (http://www.mousephenotype.org/about-impc/arrive-guidelines). For a few lines, phenotyping data were also generated on a mutant of the same gene at another IMPC phenotyping centre and used to augment/enrich phenotypes from WSI. In figures that show phenotyping data, if the same phenotype was assessed by multiple assays, the most statistically robust result is shown.
Characterization of MN gene candidates in human datasets
MN gene candidates were mapped to orthologous genes in the human genome using ENSEMBL and integrated with GWAS data on mosaic LOY26. This was performed using PAR-LOYq calls from 205,011 male participants from the UK Biobank study27. An enrichment analysis was performed across the whole dataset to test for the over-representation of MN genes at LOY GWAS loci. To do this, we first performed MAGMA analyses (v.1.08)28 using all genomic variants within each MN gene extracting gene-level associations to the LOY phenotype. Genes were annotated on the basis of their proximity to genome-wide significant loci (P < 5 × 10−8) associated with LOY, specifically if they were 500 kb up- or downstream of the LOY gene start or end position. Second, further MAGMA analyses were performed using only those variants that were predicted to have deleterious effects (for example, non-synonymous and loss of function). Genes exhibiting an FDR-corrected MAGMA P < 0.05 were considered to be significant. Finally, for genomic loci reaching at least a suggestive level of significance in the GWAS (P < 5 × 10−5), we performed SMR and HEIDI tests (v.1.02)63 using blood gene expression level data from the eQTLGen study64 and blood protein level data from the Fenland study65. For both datasets, we considered expression of a gene to be influenced by the same genomic variation as that seen in the LOY GWAS if the FDR-corrected P value for the SMR test was P < 0.05 and the P value for the HEIDI test was P > 0.01. Human genomic variation within or around the DSCC1 gene was further studied by querying associations towards the human-equivalent phenotypic traits to those observed in Dscc1-mutant mice. Specifically, GWAS on BMD66, body mass index, number of children ever born67 and LOY26 were used to ascertain gene-level associations using all available variants within the DSCC1 gene and to perform SMR and HEIDI tests against the eQTLGen data, as described above (Supplementary Table 4). For the same four traits, exome gene-burden tests were performed using phenotypic and genetic data from the UK Biobank study. Rare exome variants (minor allele frequency < 0.1%) were identified on the basis of their predicted consequence on protein function and, using VEP68 and LOFTEE69, high-confidence protein truncation variants within DSCC1 were collapsed and tested for associations towards the four traits using BOLT-LMM70,71 (Supplementary Table 4). Finally, a phenome-wide association study for common variants within DSCC1 was performed using the Open Targets Genetics Portal72 (Supplementary Table 4).
HREM analysis
For analysis with HREM, embryos were collected at E14.5 and fixed in Bouin’s solution overnight. After washing in PBS, the embryos were dehydrated in a graded series of methanol. They were then infiltrated and embedded in methacrylate resin (JB4, Polysciences Europe) and stained with eosin B and acridine orange, according to previously published protocols73. The polymerized resin blocks were analysed using HREM resulting in volume datasets with isotropic voxel sizes of 2.55–3 µm. Visualization and further analysis of the HREM data were performed using Amira v.6.7.0 (Thermo Fisher Scientific) and OsiriX (v.5.6, 64 bit, Pixmeo). The embryos were staged and systematically screened for abnormalities according to a standardized protocol74,75.
Cell lines
MEFs were prepared from E13.5 embryos, after timed matings between Dscc1+/− mice. In brief, embryos were dissected from the decidua, mechanically disrupted and cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 1.0 mM l-glutamine, 0.1 mM minimal essential medium, non-essential amino acids and penicillin–streptomycin. The initial plating was defined as passage zero, and cells were subsequently maintained on a standard protocol76. SIRT1-KO HEK293 cells were obtained from Kerafast (ENH131‐FP). Cells were grown in DMEM supplemented with 10% FBS, penicillin–streptomycin and 1% GlutaMAX. RPE-1 DSCC1Δ/flox cells were obtained from Jallepalli Laboratory43, and RPE-1 TP53-KO and TP53/DSCC1-double-KO cells were obtained from the de Lange laboratory51; these cell lines were grown in DMEM supplemented with 10% FBS and 1% GlutaMAX. iPS cells were grown in Tesr-E8 supplemented with 10 μM Y-27632 ROCK inhibitor (Stem Cell). HAP1 cells77 were cultured in Iscove’s modified Dulbecco’s medium (Invitrogen), supplemented with 10% FCS (Clontech), 1% UltraGlutamin (Lonza) and 1% penicillin–streptomycin (Invitrogen). ∆PDS5A and ∆WAPL HAP1 cells were generated using CRISPR–Cas9 as described previously78,79. The CHP-212 neuroblastoma cell line (CRL-2273) was grown in RPMI with 10% FBS.
Modification of human iPS cells was performed according to established protocols80. In brief, the Gene Editing facility at WSI generated the DSCC1-KD BOB/iPS lines. We believe these cells to be null with just 2–3% of protein expression retained (Extended Data Fig. 7) but, nonetheless, designate this a KD allele. An asymmetrical exon within the target gene was replaced with a puromycin cassette, and a frameshift indel was introduced into the other allele. A template vector containing an EF1a-puromycin cassette was constructed for each gene, incorporating two 1.5 kb homology arms designed to align with the sequence surrounding the targeted exon. Two guide RNAs (gRNAs) were designed for each exon (Extended Data Fig. 7). The template vector (2 μg), both gRNA vectors (3 μg) and hSpCas9 (4 μg) were transfected into 2 × 106 cells using the Human Stem Cell Nucleofector Kit 2 (VPH-5022, Lonza). Subsequently, cells were seeded in 10 cm2 dishes and, after 72 h, they underwent selection with 3 μg ml−1 puromycin. Single cells were then expanded and subjected to genotyping for the verification of a frameshift indel using Sanger sequencing. The resulting KO lines were cultured in the presence of 1 μg ml−1 puromycin (ant-pr-1, InvivoGen). All of the cell lines in the research laboratories that participated in this study are routinely tested for mycoplasma and STR profiled and/or validated on the basis of the presence of unique engineered alleles as described in the Reporting Summary.
Chromosome preparation and FISH
Metaphase preparations were performed using a standard protocol81. For M-FISH analysis, mouse-chromosome-specific DNA libraries were provided by the Flow Cytometry Core Facility of Wellcome Sanger Institute82. To make 10 tests of M-FISH probe, 500 μl of sonicated DNA was precipitated with 100 μl mouse Cot-1 DNA (Invitrogen) and resuspended in 120 μl hybridization buffer (50% formamide, 2× saline-sodium citrate (SSC), 10% dextran sulfate, 0.5 M phosphate buffer, 1× Denhardt’s solution, pH 7.4). Metaphase preparations were dropped onto precleaned microscopy slides, and then fixed in acetone (Sigma-Aldrich) for 10 min followed by dehydration through an ethanol series (70%, 90% and 100%). Metaphase spreads on slides were denatured by immersion in an alkaline denaturation solution (0.5 M NaOH, 1.0 M NaCl) for approximately 40 s, followed by rinsing in 1 M Tris-HCl (pH 7.4) solution for 3 min, 1× PBS for 3 min and dehydration through a 70%, 90% and 100% ethanol series. The M-FISH probe (10 μl for each 22 × 22 mm hybridization area) was denatured at 65 °C for 10 min before being applied onto the denatured slides. The hybridization area was sealed with a 22 × 22 mm2 coverslip and rubber cement. Hybridization was performed in a 37 °C incubator for 40–44 h. The post-hybridization washes included a 5 min stringent wash in 0.5× SSC at 75 °C, followed by a 5 min rinse in 2× SSC containing 0.05% Tween-20 (VWR) and a 2 min rinse in 1× PBS, both at room temperature. Finally, the slides were mounted with SlowFade Diamond Antifade Mountant containing 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen). Images were visualized on the Zeiss AxioImager D1 fluorescence microscope equipped with narrow band-pass filters for DAPI, DEAC, FITC, CY3, TEXAS RED and CY5 fluorescence and an ORCA-EA CCD camera (Hamamatsu). M-FISH digital images were captured using the SmartCapture software (Digital Scientific UK) and processed using the SmartType Karyotyper software (Digital Scientific). At least 20 metaphases from each sample were fully karyotyped on the basis of M-FISH and enhanced DAPI banding.
CRISPR–Cas9 screen and sequencing
WT and DSCC1-KD iPS cells (1 × 108) were independently infected with a human genome-wide guide RNA (gRNA) lentiviral library83 that had been recloned to swap the puromycin-resistance cassette with a neomycin-resistance cassette. Both lines were infected at a multiplicity of infection of 0.1–0.2 and a library coverage of 500× in three independent replicates, which were kept independent throughout the screen. Three days after infection, 1 mg ml−1 G418 was added to the medium and cells were cultured for an additional 10 days. When cells required passaging, a minimum of 5 × 107 cells per technical replicate was maintained at a library coverage of 500×. From each replicate, PCR was performed to amplify the gRNA region, and gRNAs were sequenced as described previously83. Single-end Illumina sequencing reads of 19 nucleotides were counted for each gRNA. To identify depleted and enriched genes in the DSCC1-KD iPS cells the software package MAGeCK84 v.0.5.6 was used. Extensive quality control of the screen was performed, and this analysis is available at the GitHub for this project (https://github.com/team113sanger/Large-scale-analysis-of-genes-that-regulate-micronucleus-formation/tree/main/CRISPR_screen_QC).
Mini-arrayed CRISPR analyses
The CHP-212 cell line was transduced with the lentiviral Cas9 plasmid (Addgene, 52962) and selected with 5 µg ml−1 blasticidin (Thermo Fisher Scientific, 61120) for 5 days. To test the expression and cutting efficiency of Cas9, we took transformed and untransformed cells and further transduced them with a lentiviral BFP-GFP reporter virus (Addgene 67980). After 4 days, the cells were analysed using flow cytometry (CytoFLEX, Beckman Coulter) and the cutting and transduction efficiency were determined on the basis of the ratio of BFP- and GFP-positive cells as previously described85. Notably, we confirmed that cells continued to cycle and grow throughout the experiment.
The sgRNA-BFP plasmids were from the arrayed Sanger Institute CRISPR library (Sigma-Aldrich, HSANGERV; the sequences are provided in Supplementary Table 6). Bacteria were grown in 5 ml of LB medium overnight and DNA was extracted using a DNA purification kit (Amresco) and AcroPrep Advance 96-well filter plates (Cytavia, 8032). DNA concentrations were measured using the Quant-iT PicoGreen dsDNA Reagent (10535213). For each gene, two DNA vectors containing unique sgRNAs were mixed at equal amounts and then diluted to the same concentrations and blinded. Virus was produced by transfection of the mix of sgRNAs and the packaging plasmids psPAX (Addgene, 12260) and pMD2.G (Addgene, 12259) into HEK293FT cells. Virus was collected 3 days after transfection, and the viral titre was determined by measuring BFP expression using flow cytometry (CytoFLEX, Beckman Coulter). For the arrayed targeting screen, cells were seeded into PhenoPlate 96-well plates (Perkin Elmer, 6055302), leaving the outer wells blank. After the cells had adhered, they were transduced with lentivirus at a multiplicity of infection of >80% (each gene is targeted by two distinct gRNAs to increase the KO efficiency to >80% in our hands). Cells were allowed to recover before the addition of 2 µg ml−1 of puromycin (Santa Cruz Biotechnology, sc-108071) for 48 h. After recovery, CHP-212 cells were treated with 12.5 µM of hydroxyurea (Merck, H8627) for three cell doublings and hTERT RPE-1 cells with 50 µM for 72 h (Supplementary Table 7 (HU titration)). Next, cells were fixed with 4% PFA (Alfa Aesar, 43368) and stained with TOPRO3 (Thermo Fisher Scientific, T3605). Cells were imaged using the Operetta CLS system (Perkin-Elmer) and analysed using the Harmony software (Imaging facility, CRUK Cambridge). A prescan (×5 air objective) of each well was performed to determine 180 fields of view of each well with the ideal seeding density. These fields of view were then reimaged using a ×40 water objective. For the analysis, z planes were transformed into a maximum projection, a sliding paraboloid filter was used, and the find nuclei and find cytoplasm functions were optimized to detect our cell lines in culture. Furthermore, the find spots function was used to find MN located in the cytoplasm. Other particles were excluded on the basis of staining intensity, roundness and size. Deblinding was performed after statistical analyses.
SIRT1 KO rescue cell viability experiment
HEK293 cells were grown in antibiotic free medium for two passages before seeding at a density of 1 × 104 cells per well in quintuplicate into 96-well plates. Then, 24 h later, ON-TARGETplus Human DSCC1 (79095) siRNA-SMARTpool (L-014300-00-0005) at 25 nM final concentration was added to the cells, along with 0.2 ml per well of Dharmafect 2 (Dharmacon) transfection reagent in serum-free medium. The next day, complete antibiotic-free medium was added. Then, 48 h after transfection, the medium was refreshed on all wells with complete antibiotic-free medium. Three days after transfection, the cell viability was determined using the Promega Cell Titer Glo 2.0 cell viability assay. Medium was aspirated from wells and 175 μl of medium along with 25 μl Cell-Titer Glo reagent were added to each well and left to incubate for 10 min at room temperature. Medium and cell viability reagent mixture (150 μl) was transferred to black-walled, clear and flat-bottom 96-well plates for reading. Luminescence was read on the CLARIOstar microplate reader (BMG LABTECH). Cell viability was calculated by normalizing to untransfected control wells.
DSCC1 transcript analyses
RNA extraction was performed using the Monarch total RNA miniprep kit (New England BioLabs). RNA was converted to cDNA using the High-Capacity RNA-to-cDNA kit (Thermo Fisher Scientific) using 500 ng of total RNA. Gene expression was measured on the QuantStudio 5 qPCR System (Thermo Fisher Scientific) using TaqMan gene expression assays for human DSCC1 (Hs00900361_m1) or mouse Dscc1 (Mm01195386_m1). TaqMan Universal Master Mix II with UNG-1 was used (Thermo Fisher Scientific; 4440038). Amplification parameters were as follows: 50 °C for 2 min; 95 °C for 10 min; followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Relative gene expression was determined on the basis of the ΔCt values between the gene of interest and housekeeping genes GAPDH (Hs02786624_g1) and 18S rRNA (Hs03003631_g1) using the Design & Analysis v.2.6.0 software from Applied Biosystems (Thermo Fisher Scientific).
Antibodies
The following antibodies were used: anti-CD71-FITC (SouthernBiotech, 1720-02, 0.5 mg ml−1, 1:500)20, anti-SIRT1 (rabbit, Cell Signalling, 2496S, 1:1,000), anti-centromere (Antibodies, 15-234-0001, 1:1,000), anti-rabbit Alexa 488 (Thermo Fisher Scientific, A11034, 1:2,000), goat anti-human Alexa 647 (Thermo Fisher Scientific, A21445, 1:2,000), anti-DSCC1 (H0079075-B01P, Novus Biologicals, 1:1,000), anti-HSP90 (F-8, Santa Cruz, 1:10,000), anti-HP1γ (05-690, Millipore, 1:1,000), goat-anti-mouse-PO (DAKO, P044701, 1:2,000), anti-SMC3 (Abcam, AB 9263, 1:250), anti-SMC3 (Thermo Fisher Scientific, A300-060A, 1:1,000), anti-acetyl SMC3 mouse (Sigma-Aldrich, MABE1073, 21A7, Lys105/106, 385016, 1:1,000), anti-p53 (Cell Signaling Technology, 1C12, 2524S), anti-acetyl p53 (p53-K382Ac, Abcam, ab75754, EPR358(2) to p53 acetyl K382, 1:1,000), anti-phosphorylated-histone H2A.X (Ser139) (JBW301, Sigma-Aldrich, 05-636-I, 1:1,000), anti-β-actin (Merck, A5441, 1:10,000, 5% milk), anti-GAPDH (6C5, Abcam, ab8245, 1:1,000), anti-p21 (Abcam, ab109520, 1:1,000). Uncropped western blots are provided in the Supplementary Information.
Immunoprecipitation
Flash-frozen cell pellets were thawed on ice and resuspended in 1 ml cell lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol) freshly supplemented with 1:100 Pierce Universal Nuclease (Thermo Fisher Scientific, 88702), 1 mM DTT (Thermo Fisher Scientific, A39255) and Halt protease (Thermo Fisher Scientific, 1860932) and phosphatase inhibitor (Roche, PhosSTOP, REF: 04906845001) and incubated on ice for 30 min. The lysis buffer was also used as wash buffer. Protein was collected by centrifugation (15,000 rcf, 10 min at 4 °C), the supernatant was transferred to a fresh tube and the pellet was discarded. The protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23225) according to the manufacturer’s protocol. To start the immunoprecipitation, beads (Thermo Fisher Scientific, Immunoprecipitation Kit Dynabeads Protein A, 10006D) were conjugated to the antibody according to the manufacturers protocol. After optimization, 50 µl of beads were used to conjugate 2 µg of total antibody. The protein sample was diluted (using the lysis buffer) to 1 mg ml−1 for immunoprecipitation and 1 ml of this sample was added to 50 μl of antibody conjugated beads. The protein–bead–antibody mixture was incubated on a rotator overnight at 4 °C. The sample was placed onto a magnet and the supernatant was transferred to a new tube (this was the flow-through that was retained to assess the antibody–bead uptake). The sample was washed on a rotator three times for 10 min in 1 ml lysis buffer at room temperature. In between the second and third wash, the sample was moved to a new Eppendorf tube to eliminate any proteins stuck to the tube. A single PBS wash was performed to the sample for 5 min on a rotator at room temperature, then the sample was placed onto the magnet for the supernatant to be removed. The result was assessed using western blotting. To prepare the reagents for this, 50 μl of 2× SDS loading buffer and 5 μl of 10× reducing buffer were added to the beads. The input and the flow-through were prepared by adding the correct amount of protein, 4× SDS loading buffer, 10× reducing buffer and lysis buffer to volume. These samples were boiled at 95 °C for 5 min then loaded onto the gel (Bio-Rad, 4–12% gel) and run at 180 V for 45 min.
Full proteome analysis
The samples were lysed in RIPA buffer plus HaltTM protease and phosphatase inhibitor cocktail (final concentration 2×, ThermoFisher Scientific) with probe sonication and heating. Samples were then centrifuged at 13,000 rpm for 15 min to remove the pellet. Protein concentrations were measured using a Pierce BCA protein assay (Thermo Fisher Scientific). A total of 100 µg of protein per sample was taken. Proteins were reduced by addition of TCEP (Tris(2-carboxyethyl) phosphine, Sigma-Aldrich), alkylated by iodoacetamide (Sigma-Aldrich) and then purified by trichloroacetic acid precipitation. Purified proteins were digested in 100 mM TEAB by trypsin (Thermo Fisher Scientific) at 1:25 (by weight) at 37 °C for 18 h. A total of 40 or 50 µg of peptides were labelled using 0.4 mg TMT10plex (Thermo Fisher Scientific) according to the manufacturer’s instructions. The samples were mixed, dried in a SpeedVac and then fractionated on the XBridge BEH C18 column (2.1 mm inner diameter (i.d.) × 150 mm, Waters) with a gradient of 5% acetonitrile/0.1% NH4OH (pH 10) to 35% CH3CN/0.1% NH4OH in 30 min (total cycle 60 min). The flow rate was at 200 µl min−1. The peptides were reconstituted in 0.1% formic acid/H2O and analysed on the Orbitrap Fusion hybrid mass spectrometer coupled with the Ultimate 3000 RSLCnano system (both from Thermo Fisher Scientific). The samples were first loaded and desalted onto a PepMap C18 nano trap (100 µm i.d. × 20 mm, 100 Å, 5 µm; Thermo Fisher Scientific), then peptides were separated on the PepMap C18 column (75 µm i.d. × 500 mm, 2 µm; Thermo Fisher Scientific) over a linear gradient of 4–33.6% CH3CN/0.1% formic acid in 180 min, with a cycle time of 210 min and a flow rate at 300 nl min−1. The MS acquisition used MS3-level quantification with Synchronous Precursor Selection (SPS) with the top speed 3 s cycle time. In brief, the Orbitrap full MS survey scan was m/z 380–1,500 with a resolution of 120,000 at m/z 200, with AGC set at 4 × 105 and 50 ms maximum injection time. Multiply charged ions (z = 2–6) with an intensity threshold at 5,000 were fragmented in an ion trap at 35% collision energy, with AGC set at 1 × 104 and 50 ms maximum injection time, and isolation width of 0.7 Da in quadrupole. The top ten MS2 fragment ions were SPS selected with an isolation width of 0.7 Da, and fragmented in higher-energy collisionally activated dissociation (HCD) at 60% normalized collision energy (NCE), and detected in the Orbitrap to obtain reporter ion intensities at a better accuracy. The resolution was set at 60,000, and the AGC set at 6 × 104 with maximum injection time at 105 ms. The dynamic exclusion was set 60 s with a ±7 ppm exclusion window. The raw files were processed using Proteome Discoverer v.2.4 (Thermo Fisher Scientific) using the Sequest HT search engine. Spectra were searched against fasta files of reviewed UniProt Homo sapiens entries (December 2021) and an in-house contamination database. The search parameters were as follows: trypsin with 2 maximum miss-cleavage sites; mass tolerances at 30 ppm for precursor and 0.6 Da for fragment ions; dynamic modifications of deamidated (N, Q) and oxidation (M); static modifications of carbamidomethyl (C) and TMT6plex (peptide N-terminus and K). Peptides were validated by Percolator with the q value set at 0.01 (strict) and 0.05 (relaxed). The TMT10plex reporter ion quantifier included 20 ppm integration tolerance on the most confident centroid peak at the MS3 level. Only unique peptides were used for quantification. The co-isolation threshold was set at 100%. Peptides with average reported S/N > 3 were used for protein quantification, and the SPS mass matches threshold was set at 50%.
Chromatin enrichment and MS analysis
Flash-frozen cell pellets were thawed on ice and resuspended in nuclear-extraction buffer (15 mM Tris-HCl pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 250 mM sucrose, 0.3% NP-40, freshly supplemented with 1 mM DTT and Halt protease and phosphatase inhibitor (Thermo Fisher Scientific)) and incubated on ice for 5 min. Nuclei were collected by centrifugation (600 rcf, 5 min at 4 °C), washed once with nuclear-extraction buffer without NP-40, pelleted again, then resuspended in prechilled hypotonic buffer (3 mM EDTA, 0.2 mM EGTA and freshly supplemented with 1 mM DTT and Halt protease and phosphatase inhibitor) and incubated on ice for 30 min to release chromatin. Chromatin was pelleted for 5 min at 1,700 rcf at 4 °C in a cooled centrifuge and subsequently washed twice with hypotonic buffer. Chromatin pellets were solubilized using probe sonication in lysis buffer 100 mM triethylammonium bicarbonate (TEAB), 1% sodium deoxycholate (SDC), 10% isopropanol, 50 mM NaCl, 1:1,000 Pierce Universal Nuclease (Thermo Fisher Scientific) supplemented with Halt protease and phosphatase inhibitor. The protein concentration was measured using the Quick Start Bradford protein assay (BioRad) according to the manufacturer’s protocol. A total of 5 mg of protein with an equal contribution from each individual sample was reduced with 5 mM Tris-2-carboxyethyl phosphine (TCEP) for 1 h, followed by alkylation with 10 mM iodoacetamide for 30 min, then digested by adding trypsin (Pierce) at final concentration 75 ng μl−1 to each sample followed by incubation for 18 h at room temperature. For chromatin proteomics, 15 μg of protein digest was taken from each sample and labelled with TMTpro multiplexing reagents (Thermo Fisher Scientific), according to the manufacturer’s protocol. SDC was precipitated with formic acid at a final concentration of 2% (v/v) and centrifuged for 5 min at 10,0000 rpm. Supernatant containing TMTpro-labelled peptides were dried with a centrifugal vacuum concentrator. The remaining peptides were cleaned up using Pierce Peptide Desalting Spin Columns (Thermo Fisher Scientific), and then dried using a speed vacuum. Acetylated peptides were enriched with the PTMScan HS Acetyl-Lysine Motif (Ac-K) Kit (Cell Signalling Technologies, 46784) according to the manufacturer’s instructions, dried using a speed vacuum, resuspended in 100 mM TEAB and labelled with TMTpro according to the manufacturer’s protocol. Acetyl-enriched peptides were fractionated using the Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher Scientific, 84868) according to the manufacturer’s protocol, dried using a speed vacuum and resuspended in 0.1% trifluoroacetic acid (TFA). Before MS analysis of the chromatin proteome, TMTpro-labelled peptides were fractionated with high-pH reversed-phase (RP) chromatography using the Waters XBridge C18 column (2.1 mm × 150 mm, 3.5 μm) on the Dionex UltiMate 3000 high-performance liquid chromatography (HPLC) system. Mobile phase A was 0.1% ammonium hydroxide (v/v) and mobile phase B was 100% acetonitrile and 0.1% ammonium hydroxide (v/v). Peptide separation was performed with a gradient elution of 200 μl min−1 with the following steps: isocratic for 5 min at 5% phase B, gradient for 40 min to 35% phase B, gradient to 80% phase B in 5 min, isocratic for 5 min, and re-equilibrated to 5% phase B. The fractions were collected in a 96-well plate every 42 s to a total of 65 fractions, then concatenated into 12 fractions, dried and reconstituted in 0.1% TFA. The samples were analysed using a Real Time Search-SPS-MS3 method on the Orbitrap Ascend mass spectrometer coupled to a Dionex UltiMate 3000 system. From each fraction, an estimated amount of 3 μg of peptides per fraction was injected onto a C18 trapping column (Acclaim PepMap 100, 100 μm × 2 cm, 5 μm, 100 Å) at a flow rate of 10 μl min−1. The samples were processed via a 120 min low-pH gradient elution on a nanocapillary reversed-phase column (Acclaim PepMap C18, 75 μm × 50 cm, 2 μm, 100 Å) at 50 °C. MS1 scans were collected from the 400–1,600 m/z range in the Orbitrap with the following settings: resolution, 120,000; AGC, standard; injection time, auto; and including 2–6 precursor charge states. Dynamic exclusion was set to 45 s, repeat count of 1, mass tolerance of 10 ppm and the exclude isotope option was enabled. MS2 spectra were acquired in the ion trap at Turbo scan rate, HCD collision energy was set to 32% and 35 ms maximum-injection time was allowed. MS2 scans were searched against the human canonical and isoforms database (UniProt, 16 December 2022) using the Comet search engine in real time with the following filters: tryptic peptides with maximum of 1 missed cleavages, static modifications included Cys carbamidomethylation (+57.0215) and N-terminal/Lys TMTpro (+304.2071), variable modifications Asn/Gln deamidation (+0.984) and Met oxidation (+15.9949), with maximum of variable modifications set to 2; close-out was enabled with a maximum of 4 peptides per protein. Precursors matching these criteria were selected for SPS10-MS3 scans performed at an orbitrap resolution of 45,000 with the normalized HCD collision energy set to 55%, AGC set at 200% and 200 ms maximum injection time. Acetyl-enriched peptides were analysed using an MS2-HCD method with collision energy set to 35%, AGC set at 1 × 105 and 105 ms maximum injection time. The SequestHT and Comet search engines were used to analyse the acquired spectra in Proteome Discoverer v.3.0 (Thermo Fisher Scientific) for protein identification and quantification. For analysis of the chromatin proteome, the precursor mass was set to 20 ppm and fragment mass tolerance was 0.5 Da. Spectra were searched for fully tryptic peptides with a maximum of two missed cleavages. N-terminal/Lys TMTpro and carbamidomethyl at Cys were defined as static modifications. Dynamic modifications included oxidation of Met and deamidation of Asn/Gln. For peptides enriched for acetylated lysine, the precursor mass was set to 10 ppm and the fragment mass tolerance was set to 0.02 Da. Spectra were searched for fully tryptic peptides with a maximum of three missed cleavages. N-terminal TMTpro and carbamidomethyl at Cys were defined as static modifications, while dynamic modifications included oxidation of Met, deamidation of Asn/Gln, and TMTpro or acetyl at Lys. Peptide confidence was estimated using the Percolator node. Peptide FDR was set at 1% and validation was based on q value and a target–decoy database search. Spectra were searched against reviewed UniProt human protein entries. The reporter ion quantifier node included a TMTpro quantification method with an integration window tolerance of 15 ppm and an integration method based on the most confident centroid peak at the MS3 or MS2 level. Only unique peptides were used for quantification, considering protein groups for peptide uniqueness. Peptides with an average reporter signal-to-noise ratio of >3 were used for quantification. For the chromatin proteome, the data were normalized to total loading at the proteome level, whereas, for the respective acetylome, the data were corrected for loading for acetylated peptides only. Relative abundances were calculated by dividing normalized protein/peptide abundances by the average abundance of all TMTpro channels per biological replicate.
Immunoblotting and immunofluorescence
Cells were scraped from dishes in 2× SDS buffer (120 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol). After total protein quantification, equal protein amounts were run on 4–12% Bis-Tris NuPAGE precast gels, transferred to nitrocellulose membrane (GE Healthcare) and immunoblotted with the indicated antibodies. For chromatin fractionation, cells were washed with cold PBS and resuspended in CSK buffer (10 mM PIPES pH 7.0, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, protein inhibitor cocktail (Roche, EDTA-free, 1 tablet per 10 ml), EGTA-free phosphatase inhibitors (1 mM NaF, 0.7 mM β-glycerol phosphate, 0.2 mM Na3VO4, 8.4 mM Na4P2O7), 0.7% Triton X-100), incubated on ice for 30 min and centrifuged at 20,000g for 10 min at 4 °C. The supernatant (soluble fraction) was collected and maintained on ice. The pellet was washed twice with cold PBS and sonicated (four pulses of 10 s at 30% amplitude with 10 s resting on ice between cycles) in CSK buffer. The protein concentration of soluble and chromatin fractions was determined using the Bradford assay and Laemmli buffer was added to the samples. Finally, the samples were boiled, centrifuged at 16,000g for 1 min and equal amounts were loaded onto SDS–PAGE gels. For immunofluorescence studies, cells on coverslips were fixed in a formaldehyde lysis solution (4% formaldehyde, 0.5% Triton X-100, 1× PBS), washed with 1× PBS and permeabilized in 0.5% Triton X-100, 1× PBS. Blocking was performed in 1× PBS, 0.1% Triton X-100, 10% FBS for 1 h, followed by incubation with primary or secondary antibodies in the same solution. Washes were performed using 1× PBST (1× PBS, 0.1% Triton X-100). Coverslips were mounted in Vectashield Mounting Medium with DAPI (Vector Laboratories, H1200-10). Images were collected on the Leica SP8 with ×63/1.4 NA oil objectives, using the Leica Application Suite X software (LAS-X). Images were deconvolved using Huygens Professional v.19.04 software (Scientific Volume Imaging); processing and analysis were performed using ImageJ v.1.53a and Adobe Illustrator 2021. All of the images shown are the projections of z optical sections.
SIRT1 inhibition assays
Cells were preincubated with EX 527 (selisistat; SIRT1i; Selleckchem) resuspended in DMSO or with DMSO alone for 3 days and then seeded at a density of 2.5 × 105 cells per 10 cm dish, maintaining either SIRT1i or DMSO in the culture medium. The next day, cells were treated with tamoxifen or mock treated for 24 h. The number of living cells at each timepoint was determined after trypsinization using the Countess II machine (Life Technologies). To determine the dose of tamoxifen that resulted in full depletion of DSCC1 in the hTERT RPE-1 DSCC1Δ/floxcretam cell line, cells were grown in the presence of different concentrations of the compound. After 3 days of tamoxifen treatment (Sigma-Aldrich), cell survival was observed by staining with crystal violet (Sigma-Aldrich; 1% aqueous solution). The dose that killed all DSCC1Δ/floxcretam cells, but not WT hTERT RPE-1 control cells (100 nM), was used for subsequent experiments (Extended Data Fig. 7). To determine the dose of SIRT1i that fully inhibits SIRT1 activity in cultured hTERT RPE-1 cells, the acetylation of p53 at Lys382, a bona fide SIRT1 substrate86, was examined. Cells were grown in the presence of different concentrations of SIRT1i for 3 days (Extended Data Fig. 9). To avoid interference from other histone deacetylases 5 µM vorinostat (Sigma-Aldrich) was added to the cells 2 h before gamma irradiation (5 Gy). Then, 3 h later, the samples were collected and acetylation of p53 at Lys382 was examined using western blotting.
SIRT1 in vitro deacetylation assay
These experiments were performed in HDAC8-KO HAP1 cells (Horizon Discovery) and also hTERT RPE-1 cells (Extended Data Fig. 9). p53 (a known SIRT1 target) was purified 5 h after gamma irradiation (10 Gy) of hTERT RPE-1 cells that were previously treated with 10 µM selisistat and 5 µM vorinostat (SAHA; Sigma-Merk, SML0061). SMC3 was purified from exponentially growing HDAC8-KO HAP1 cells. Both proteins (p53 and SMC3) were purified by immunoprecipitation as follows: cell pellets were resuspended in lysis buffer (50 mM Tris-Cl pH 8, 150 mM NaCl, 1 mM EDTA, 0.5% igepal, complete EDTA-free protein inhibitor cocktail from Roche and phosphatase inhibitor cocktails 2 and 3 from Sigma-Aldrich) and quantified. For p53, 2 mg of protein from hTERT-RPE-1 cell lysates was incubated with 20 μl of Dynabeads (protein G) and 6 μl of anti-p53 antibodies. For SMC3, 10 mg of HAP1 cell lysate was incubated with 40 μl of Dynabeads (protein A) and 12 μg of anti-SMC3 antibodies. Both incubations were performed overnight at 4 °C. The next morning, the beads were washed four times with cold lysis buffer and twice with reaction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl2) and resuspended in 50 μl of reaction buffer. For the deacetylation reaction, 10 μl of beads was incubated with 1 μl of human recombinant SIRT1 (Sigma-Aldrich) in a total volume of 30 μl of reaction buffer supplemented with 1.5 μM NAD+. The reactions were incubated for 3 h at 30 °C with shaking. Finally, the reactions were stopped by the addition of 10 μl of 4× Laemmli sample buffer and incubation at 95 °C for 5 min. The samples were then immunoblotted with the respective antibodies.
siRNA experiments in RPE-1 cells
A total of 200,000 RPE-1 DSCC1Δ/floxcretam cells were seeded per well of a six-well plate and allowed to attach overnight. The cells were then transfected with either non-targeting (referred to as SCR control) or targeting siRNA at 25 nM using 5 µl DharmaFECT 1 transfection reagent (Horizon Discovery T-2001-02) according to the manufacturer’s instructions. After 24 h, the medium was replaced in all wells and cells were treated with or without 100 nM 4-OHT. Cells were incubated for a further 5 days before collecting and cell counting by trypan blue exclusion. A list of all siRNAs used is provided in Supplementary Table 6.
MN counting in HAP1 cells
HAP1 cells were seeded at an equal density, grown on coverslips and transfected with siRNAs targeting luciferase or DSCC1. All siRNAs were ON-TARGETplus SMARTpools manufactured by Dharmacon and used at a final concentration of 20 μM per siRNA. Transfections were performed using Invitrogen RNAiMAX (Life Technologies) according to the manufacturer’s instructions. Transfections were repeated after 48 h. After an additional 24 h, the coverslips were fixed with freshly prepared 3.7% paraformaldehyde in PBS for 7 min at room temperature. Cells were permeabilized and stained for 10 min with 0.1% Triton X-100 in PBS, supplemented with 1 μg ml−1 DAPI at room temperature. The coverslips were washed once with PBS, and mounted onto glass slides using Prolong Gold (Invitrogen). The slides were imaged and deconvolved on the THUNDER Imager (Leica Microsystems) and analysed using ImageJ (v.2.1.0/1.53k). A cell was scored as harbouring MN when the nucleus had one or more MN in its proximity. At least 400 cells were scored per condition.
Analysis of cohesion defects in RPE-1 TP53-KO and RPE-1 TP53/
DSCC1-double-KO cells
RPE-1 TP53/DSCC1-double-KO cells were generated as previously reported51 and were cultured in DMEM + 8% FCS. For analysis of cohesion defects, cells were incubated for 20 min with 200 ng ml−1 demecolcine (Sigma-Aldrich), collected, incubated for 20 min in 0.075 M KCl and fixed in 3:1 methanol:acetic acid. Cells were washed in fixative three times, dropped onto microscopy slides and stained with 5% Giemsa (Merck). For each condition, cohesion defects were counted in 50 metaphases on two coded slides.
Statistics and reproducibility
Statistical analyses were performed using Prism (v.9.1.0/v.10.1, GraphPad) or R (v.3/v.4.3.1). All statistical details are provided in the figure legends. All experiments were performed independently at least three times, and were replicated by independent researchers using multiple models and using blinding where possible. T-tests were unpaired.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.