Telomere-to-mitochondria signalling by ZBP1 mediates replicative crisis – Nature

Cell culture

IMR90 (CCL-186) and WI38 (AG06814-N) fibroblasts were purchased from ATCC and the Coriell Institute for Medical Research, respectively. IMR90 and WI38 fibroblasts were grown under 7.5% CO₂ and 3% O₂ in GlutaMax-DMEM (Gibco, 10569-010) supplemented with 0.1 mM non-essential amino acids (Corning, 25-025-Cl) and 15% fetal bovine serum (VWR/Seradigm, 97068-085, 311K18). Human mammary epithelial cells (HMECs; CC-2551) were purchased from Lonza and were grown under 5% CO₂ and 3% O₂ using the mammary epithelial cell medium complete kit (Lifeline Cell Technology, LL-0061). The number of PDs was calculated at each passage using the following equation: PD = log[number collected/seeded number]/log₂. Cells have been tested to be free of mycoplasma.

Plasmids

Non-coding scramble shRNA was obtained from D. Sabatini through Addgene (plasmid 1864)⁵². TRF2 shRNA in pLKO.1 was obtained from Open Biosystems. HPV16 E6E7, SV40 LT and H2B-mCherry in pLXSN3 and hTERT in pBabe were obtained from Karlseder laboratory stock². To generate shZBP1 1, oligos (forward, 5′-CCGGCCAAGTCCTCTACCGAATGAACTCGAGTTCATTCGGTAGAGGACTTGGTTTTTG-3′; and reverse, 3′-AATTCAAAAACCAAGTCCTCTACCGAATGAACTCGAGTTCATTCGGTAGAGGACTTGG-5′) were annealed by temperature ramp from 100 °C to 25 °C and cloned in a AgeI- and EcoRI-digested non-coding scramble shRNA vector using T4 DNA ligase. ZBP1 shRNA in pLKO.1 (shZBP1 2) was obtained from Sigma-Aldrich (TRCN0000123050).

To generate pooled knockout cell populations and knockout clones lentiCRISPRv2 was used. The plasmid was obtained from F. Zhang through Addgene (plasmid 52961)⁵³ and the guide target sequence was cloned as phosphorylated adapter in the Esp3I-digested lentiCRISPRv2 vector. Individual guide RNAs (gRNAs) for the targeting of corresponding genomic exons were designed using the CRISPOR web tool (http://crispor.org). The guide RNA sequences are as follows: sgZBP1 1, 5′-CACCTGGTGCCATTGAAGGG-(PAM)-3′; sgZBP1 2, 5′-GGACGATTTACCGCCCAGGT-(PAM)-3′; sgZBP1 3, 5′-TGGGACACAGCAATGAGATG-(PAM)-3′; sgGFP, 5′-GAAGTTCGAGGGCGACACCC-(PAM)-3′; sgLUC (sgrenilla_luciferase), 5′-GGTATAATACACCGCGCTAC-(PAM)-3′; sgMAVS, 5′-AAGTTACCCCATGCCTGTCC-(PAM)-3′; and sgSTING, 5′-AATATGACCATGCCAGCCCA-(PAM)-3′. ZBP1-knockout clones were generated using sgZBP1 2 5′-GGACGATTTACCGCCCAGGT-(PAM)-3′. After transduction, cells were selected with 1 μg ml⁻¹ puromycin for 1 week and individual clones were isolated using the array dilution method in 96 wells. Clones were tested by western blotting for the absence of ZBP1 protein staining.

All ZBP1 expression plasmids used here are derivates of pLenti-CMV-GFP-Hygro-(656-4) obtained from E. Campeau and P. Kaufman through Addgene (plasmid 17446)⁵⁴. The sequence of full-length human ZBP1 transcript variant 1 (GenBank: NM_030776.3) was obtained as gblock from IDT. The empty vector control pLenti-MCS-hygro was cloned in three steps: first, the pLenti-CMV-GFP-Hygro-(656-4) vector was digested with XbaI and SalI and the GFP sequence was replaced with a multiple cloning site (MCS) adapter (MCS1_fw, 5′-CTAGACCTCAGGATCCCGGGACGCGTG-3′; and MCS2_rev, 5′-TCGACACGCGTCCCGGGATCCTGAGGT-3′) to generate pLenti-CMV-MCS-Hygro. Second, the CMV promoter in the pLenti-CMV-MCS-Hygro vector was excised using ClaI and BamHI and replaced with a second adapter (MCS2_fw, 5′-CGATCCTGAGGTCTAGAGAATTCG-3′; and MCS2_rev, 5′-GATCCGAATTCTCTAGACCTCAGGAT-3′). Finally, pLenti-MCS-WPRE-Hygro was digested with SalI and XhoI to remove the WPRE sequence followed by religation to generate pLenti-MCS-Hygro. To clone N-terminally 3×Flag-tagged ZBP1 expression constructs, pLenti-CMV-3×Flag-(GS)₅-MCS-Hygro was generated first. For this, the Kozak sequence with a 3×Flag tag and a (GS)₅ linker was amplified from pLenti-FNLS-P2A-GFP-PGK-Puro (obtained from L. Dow through Addgene (plasmid 110869)⁵⁵ using primers 5′-CTAGACCTCAGGATCGCCACCATGGACTATAAGGAC-3′ and 5′-ACGCGTCCCGGGATCCAGAGCCGGACCCGCTCCCGGAGCCCTTATCGTCATCGTCTTTGTAATCAATATCATGAT-3′. The fragment was cloned into BamHI-linearized pLenti-CMV-MCS-Hygro using InFusion cloning (Takara Bio, 639650).

ZBP1 full-length (ZBP1(L)) and short isoform (ZBP1(S), also known as Delta-exon-2, Delta-Z-alpha or isoform 7) were amplified from full-length ZBP1 gblock DNA using either the primer 5′-GTCCGGCTCTGGATCCATGGCCCAGGCTCCTGCT-3′ or primer 5′-GTCCGGCTCTGGATCCATGGCCCAGGCTCCTGCTGACCCGGGCAGAGAAGCCGAGAGGCCCCAGC-3′ in combination with the primer 5′-ACGCGTCCCGGGATCCCTAAATCCCACCTCCCCACC-3′. ZBP1 fragments were cloned into BamHI-linearized pLenti-CMV-3×Flag-(GS)₅-MCS-Hygro using InFusion cloning. To clone C-terminally 3×Flag-tagged ZBP1 expression constructs, pLenti-CMV-MCS-(GS)₅-3×Flag-Hygro was generated first. For this, a (GS)₅ linker with a 3×Flag tag and STOP codon was amplified from pLenti-FNLS-P2A-GFP-PGK-Puro using the primers 5′-CGACTCTAGAGGATCCGGCTCCGGGAGCGGGTCCGGCTCTGACTATAAGGACCACGACGG-3′ and 5′-GAGGTTGATTGTCGACTCACTTATCGTCATCGTCTTTGTAATC-3′. The fragment was cloned into BamHI- and SalI-digested pLenti-CMV-GFP-Hygro-(656-4) using InFusion cloning.

ZBP1(L)-, ZBP1(S)- and ZBP1(S)-deletion mutants were PCR amplified from full-length ZBP1 gblock DNA and the pLenti-CMV-3×Flag-(GS)₅-ZBP1(S)-Hygro plasmid, respectively, with the primers listed below. Fragments were cloned into BamHI-digested pLenti-CMV-MCS-(GS)₅-3×Flag-Hygro using InFusion cloning.

The primers for the C-terminal tagged ZBP1 construct cloning were as follows: ZBP1(L) (amino acids 1–429) and ZBP1(S) (amino acids 1–11, 87–429) ZBP1, 5′-CGACTCTAGAGGATCCATGGCCCAGGCTCCTG-3′ and 5′-TCCCGGAGCCGGATCCAATCCCACCTCCCCACC-3′; ZBP1(S)-ΔZα2 (amino acids 167–429), 5′-CGACTCTAGAGGATCCATGCCAGAAGATTCTGGAAGAAGAGCAA-3′ and 5′-TCCCGGAGCCGGATCCAATCCCACCTCCCCACC-3′; ZBP1(S)-ΔZα2-ΔRHIM1-ΔRHIM2 (amino acids 278–429), 5′-CGACTCTAGAGGATCCATGCCGTCCGAGGGCCCT-3′ and 5′-TCCCGGAGCCGGATCCAATCCCACCTCCCCACC-3′; ZBP1(S)-ΔC (amino acids 1–11, 87–319), 5′-CGACTCTAGAGGATCCATGGCCCAGGCTCCTG-3′ and 5′-TCCCGGAGCCGGATCCGGCGGCTTCCCCCT-3′; ZBP1(S)-ΔRHIM1 (amino acids 1–11, 87–194, 220–429) N-terminal fragment, 5′-CGACTCTAGAGGATCCATGGCCCAGGCTCCTG-3′ and 5′-TCCCTGGAGGGTCCATTCTGGCAGATCA-3′ and C-terminal fragment 5′-TGGACCCTCCAGGGAGGACGGT-3′ and 5′-TCCCGGAGCCGGATCCAATCCCACCTCCCCACC-3′; ZBP1(S)-ΔRHIM2 (amino acids 1–11, 87–252, 278–429) N-terminal fragment, 5′-CGACTCTAGAGGATCCATGGCCCAGGCTCCTG-3′ and 5′-TCGGACGGCTGGGGCCCCCAGG-3′ and C-terminal fragment, 5′-GCCCCAGCCGTCCGAGGGCCCTGC-3′ and 5′-TCCCGGAGCCGGATCCAATCCCACCTCCCCACC-3′; and ZBP1(S)-ΔRHIM1-ΔRHIM2 (amino acids 1–11, 87–194, 278–429) N-terminal fragment, 5′-CGACTCTAGAGGATCCATGGCCCAGGCTCCTG-3′ and 5′-TCGGACGGGGGTCCATTCTGGCAGATCA-3′ and C-terminal fragment, 5′-TGGACCCCCGTCCGAGGGCC-3′ and 5′-TCCCGGAGCCGGATCCAATCCCACCTCCCCACC-3′. Zα2, RHIM1 and RHIM2 point mutations were introduced in the pLenti-CMV-ZBP1(S)-(GS)₅-3×Flag-Hygro vector by site-directed mutagenesis using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, 210518). The primers used here for site-directed mutagenesis were as follows: ZBP1(S)-mZα2 (N141A,Y145A), 5′-GGACAGCAAAAGATGTGGCCCGAGACTTGGCCAGGATGAAGAGCAGGC-3′ and 5′-GCCTGCTCTTCATCCTGGCCAAGTCTCGGGCCACATCTTTTGCTGTCC-3′; ZBP1(S)-mRHIM1 (I206A,Q207A,I208A,G209A), 5′-TGGATTTCCATTGCAAACTCCGAAGCCGCCGCGGCTGCACACGGGAACATCATTACAAGACAGAC-3′ and 5′-GTCTGTCTTGTAATGATGTTCCCGTGTGCAGCCGCGGCGGCTTCGGAGTTTGCAATGGAAATCCA-3′; and ZBP1(S)-mRHIM2 (V264A,Q265A,L266A,G267A), 5′-GCAGTCCATACTGAGACGGGCGGCGGCGGCACACAGCAATGAGATGAGGC-3′ and 5′-GCCTCATCTCATTGCTGTGTGCCGCCGCCGCCCGTCTCAGTATGGACTGC-3′.

To express untagged VP16-TRF1ΔN and TRF1ΔN from a doxycycline-inducible tight TRE promoter, VP16-TRF1ΔN and TRF1ΔN was subcloned into pLPT-Empty⁵⁶. For this, VP16-TRF1ΔN and TRF1ΔN were PCR amplified from pINDUCER20-VP16–TRF1ΔN(44–439) and pINDUCER20-TRF1ΔN(44–439), respectively (both plasmids were kind gifts from P. M. Lieberman⁵⁷), using either primer 5′-TGGAGAATTGGCTAGCATGGCCCCCCCGACCG-3′ or primer 5′-TGGAGAATTGGCTAGCATGCTTCTCGAGTGCCAGGTGC-3′ in combination with primer 5′-CCCCAACCCCGGATCCTCAGTCTTCGCTGTCTGAGGAAATCAG-3′. Fragments were cloned into the NheI and BamHI-digested pLPT-Empty vector using InFusion cloning.

To localize ZBP1(S) to the mitochondrial outer membrane, the outer mitochondrial membrane localization (OMM) signal of FIS1 was fused to the C terminus of 3×Flag-ZBP1(S). The construct was cloned in three steps: first, pLenti-CMV-MCS-3×Flag-OMM-hygro was generated by amplifying the OMM of FIS1 with the primers 5′-CGATAAGGCCATCGTGGGAGGC-3′ and 5′-GAGGTTGATTGTCGACTCAGGATTTGGACTTGGACACAG-3′ from MAVS-Mito (obtained from J. Kagan through Addgene (plasmid 44556)⁵⁸ and (GS)5-linker-3×Flag with the primers 5′-CGACTCTAGAGGATCCGGCTCCGGGAGCGGGTCCGGCTCTGACTATAAGGACCACGACGG-3′ and 5′-CACGATGGCCTTATCGTCATCGTCTTTGTAATC-3′ from pLenti-FNLS-P2A-GFP-PGK-Puro⁵. The two fragments were cloned into BamHI/SalI-digested pLenti-CMV-GFP-Hygro(656-4) (see methods of ref. ⁴) using InFusion cloning. Next, pLenti-CMV-MCS-(GS)5-3Flag-OMM-hygro was linearized with BamHI and ZBP1(S) that was amplified with the primers 5′-CGACTCTAGAGGATCCATGGCCCAGGCTCCTG-3′ and 5′-TCCCGGAGCCGGATCCAATCCCACCTCCCCACC-3′ from pLenti-CMV-3×Flag-(GS)5-ZBP1(S)-hygro was cloned into the linearized vector using InFusion cloning. Finally, pLenti-CMV-3×Flag-(GS)5-ZBP1(S)-OMM-hygro was generated by InFusion cloning using amplified fragments of pLenti-CMV-ZBP1(S)-(GS)5-3Flag-OMM with the primers 5′-GCCATCGTGGGAGGCATG-3′ and 5′-GGATCCTCTAGAGTCGGTGTCT-3′ and 3×Flag-(GS)5-ZBP1(S) with the primers 5′-GACTCTAGAGGATCCGCCACCATGGACTATAAGGACCACGAC-3′ and 5′-GCCTCCCACGATGGCAATCCCACCTCCCCACC-3′ from pLenti-CMV-3×Flag-(GS)5-ZBP1(S)-hygro.

T4 ligase (M0202S), T4 PNK (M0201S) and all restriction enzymes used, except for Esp3I (Thermo Scientific ER0452), were obtained from NEB. All primers were ordered from Eton Bioscience. PCR reactions for cloning were performed using KOD Hot Start DNA Polymerase (Novagen, 71086) according to the manufacturer’s protocol.

Cell death assays

For real-time assessment of cell death, 2,000 cells per well per condition in triplicate were seeded in 96-well plates (CytoOne, CC7682-7596) the day before the experiment. The next day, the medium was changed to medium containing 250 nM IncuCyte Cytotox green dye (Sartorius, 4633) and IncuCyte Nuclight Rapid red dye (1:100 dilution) (Sartorius, 4717). Cells were seeded in 96-well plates the day before the experiment and the medium changed to medium containing 250 nM IncuCyte Cytotox green dye on the day of the experiment. Cells were imaged with at least two fields per well every 2 h using the ×10 objective on the IncuCyte S3 or IncuCyte Zoom live-cell analysis system (Sartorius). The total number of nuclei and the Cytotox-green-dye-positive nuclei were quantified using the IncuCyte analysis software. The percentage of dead cells was calculated by dividing the Cytotox green dye positive nuclei by the total number of nuclei multiplied by 100. In Extended Data Figs. 6e and 13d, cells were transfected with siRNA 48 h before cell death measurements. Then, 24 h after transfection, the medium was changed to medium containing 250 nM IncuCyte Cytotox green dye (Sartorius, 4633) or IncuCyte Cytotox red dye (Sartorius, 4632). Then, 48 h after transfection, nuclei were stained with Hoechst (1 μg ml⁻¹), images were taken using the Revolve fluorescence microscope and analysed with CellProfiler v.4.2.1 using a customized pipeline. Total nuclei and Cytotox-dye-positive nuclei were segmented using an integrated intensity-based object detection module. The percentage of dead cells was calculated by dividing the number of Cytotox-dye-positive nuclei by the total number of nuclei multiplied by 100.

Retroviral and lentiviral transduction

Lentiviral and retroviral particles were produced by the laboratory. Production of lentivirus was performed as described previously⁵⁶. In brief, HEK293T (ATCC, CRL-11268) cells were transfected with 7 µg of DNA using Lenti-X Packaging Single-Shot system (Clontech, 631276). Then, 48 h after transfection, viral supernatant was collected, supplemented with serum and used for transduction in the presence of Lenti-Blast (Oz Biosciences, LB00500). To produce retrovirus, Phoenix cells were transfected with 20 µg of DNA using 100 µM of chloroquine. Then, 5 h after transfection, fresh medium was added. The viral supernatant was collected 24 h later and used for transduction in the presence of polybrene 4 µg ml⁻¹. Then, 48 h after infection, cells were washed and selected with 1 µg ml⁻¹ puromycin, 600 µg ml⁻¹ G418, or 90 µg ml⁻¹ hygromycin. IMR90^E6E7 and WI38^SV40LT fibroblasts were subjected to long-term culturing under antibiotic selection.

Transfections

DNA transfections were performed using the Lipofectamine 3000 kit (Thermo Fisher Scientific, 1857482) according to the manufacturer’s instructions. siRNA transfections were performed using the Lipofectamine RNAiMAX kit (Thermo Fisher Scientific, 13778030) according to the manufacturer’s instructions.

Western blotting

Western blots were performed as described previously⁵⁹. In brief, cells were lysed in NuPage LDS sample buffer (Invitrogen, NP0007) at 1 × 10⁴ cells per µl. Proteins were resolved using NuPage Bis-Tris gel electrophoresis (Invitrogen, NP0343, NP0321 or WG1402) and transferred to nitrocellulose membranes (Amersham, 10600037). Secondary antibodies were peroxidase-conjugated anti-mouse IgG (Amersham, NXA931V) or anti-rabbit IgG (Amersham, NA934V). Peroxidase activity was detected using an ECL kit (Prometheus, 20-302B) and the Syngene G-Box imager. Primary antibodies are listed below.

Metaphase spreads for quantification of chromosome fusions

Metaphase spread preparation was performed as described⁶⁰. In brief, cells were treated with 0.2 µg ml⁻¹ of Colcemid (Gibco, 15212-012) for 3 h, collected and incubated with hypotonic solution (75 mM of KCl) for 7 min at 37 °C. The cell suspension was centrifuged, washed in fixative solution (3:1 methanol:glacial acetic acid), dropped onto superfrost microscope slides (VWR, 48311-703) and air dried overnight. For telomere staining, cells were fixed in 4% formaldehyde in PBS for 10 min, dehydrated in a series of ethanol baths (70%, 90% and 100%) for 3 min each and air-dried for 20 min. Cells were then covered in 0.3 ng µl⁻¹ of the telomeric (PNA Bio, F1004) PNA probes diluted in hybridization solution (70% deionized formamide, 0.25% blocking reagent (NEN), 10 mM Tris pH 7.5). The samples were heated for 5 min at 80 °C, incubated 2 h at room temperature, washed twice in 70% formamide and 10 mM Tris-HCl pH 7.5 and three times in 50 mM Tris-HCl pH 7.5, 150 mM NaCl and 0.08% Tween-20. Slides were finally mounted in ProLong Diamond with DAPI (Invitrogen, P36971).

Immunofluorescence-FISH analysis of interphase nuclei and metaphase spreads

For interphase nuclei, cells were seeded onto glass coverslips 24 h before the experiment as described previously⁶⁰. Cells were washed in PBS, fixed in 4% formaldehyde in PBS for 10 min and permeabilized in 0.1% Triton X-100 in PBS for 10 min. For metaphase spreads, cells were treated with 20 ng ml⁻¹ of Colcemid (Gibco, 15212-012) for 1 h, collected and incubated in hypotonic solution (27 mM KCl, 6.5 mM tri-sodium citrate) for 5 min. The cell suspension was cytocentrifuged, fixed in 4% formaldehyde in PBS for 10 min and permeabilized in KCM buffer (120 mM KCl, 20 mM NaCl, 10 mM Tris pH 7.5, 0.1% Triton X-100) for 10 min. In both settings, the samples were incubated in blocking buffer (20 mM Tris pH 7.5, 2% BSA, 0.2% fish gelatin, 150 mM NaCl, 0.1% Triton X-100, 0.1% sodium azide and 100 µg ml⁻¹ RNase A) for 1 h at 37 °C. Cells were incubated with the primary antibody (γH2AX) for 2 h, washed in PBS and incubated with secondary antibody for 1 h at room temperature. The secondary antibodies used were AlexaFluor 568 anti-IgG Mouse (Thermo Fisher Scientific, A-11004) or AlexaFluor 647 anti-IgG Mouse (Thermo Fisher Scientific, A-21235). The samples were finally fixed in 4% formaldehyde in PBS before fluorescence in situ hybridization (FISH) analysis.

Immunofluorescence

Cells were seeded onto glass coverslips 24 h before the experiment. Cells were fixed in 4% paraformadehyde in PBS for 10 min, washed in PBS and incubated in blocking solution of 5% BSA in PBS at room temperature. Cells were then incubated with primary antibodies for 2 h, washed in PBS and incubated with secondary antibodies for 1 h at room temperature. The secondary antibodies used were AlexaFluor 488 goat anti-rabbit IgG (H+L) (Thermo Fisher Scientific, A-11034) and AlexaFluor 568 goat anti-Mouse IgG (H+L) (Thermo Fisher Scientific, A-11004). The samples were finally washed in PBS and mounted in ProLong Diamond with DAPI (Invitrogen, P36971). For MitoTracker staining, MitoTracker Red CMXRos (Thermo Fisher Scientific, M7512) was added to the culture medium at a final concentration of 200 nM and incubated for 20 min under tissue culture conditions. After staining, cells were washed twice in prewarmed PBS, then fixed in paraformaldehyde 4% PBS for 10 min and washed in PBS. Imaging was performed using the Zeiss LSM 880 with the Airyscan microscope. ZEN (Zeiss) and ImageJ were used for image analysis.

Crystal violet assay for determining cell viability

Cells before crisis were seeded at low density (476 cells per cm²) and kept at 37 °C for 10 days before fixing with 4% paraformaldehyde in PBS. Cells were then stained with 0.05% crystal violet in distilled water for 20 min.

fRIP

fRIP–seq was performed as previously described with minor modifications⁶¹. In brief, IMR90^E6E7 cells were cross-linked with 0.1% formaldehyde in PBS for 10 min at room temperature and unreacted formaldehyde was then neutralized with 125 mM glycine for 5 min at room temperature. Cells were washed twice with ice-cold PBS, collected by trypsinization and the cell pellets were resuspended in RIPA buffer (50 mM Tris pH 8, 150 mM KCl, 0.1% SDS, 1% Triton X-100, 5 mM EDTA, 0.5% sodium deoxycholate, 0.5 mM DTT (added fresh), protease inhibitor cocktail (Roche, 4693159001), 100 U ml⁻¹ RNasin Ribonuclease Inhibitor (Promega, N251B)), and incubated for 20 min at 4 °C under slow rotation. The cell lysates were centrifuged at maximum speed at 4 °C for 10 min, and the supernatants were collected and diluted in equal volumes of freshly made fRIP binding/wash buffer (150 mM KCl, 25 mM Tris pH 7.5, 5 mM EDTA, 0.5% NP-40, 0.5 mM DTT (added fresh), protease inhibitor cocktail (Roche, 4693159001), 100 U ml⁻¹ RNasin Ribonuclease Inhibitor (Promega, N251B)). Diluted lysates were first precleared with Dynabeads Protein G (Invitrogen, 10004D) at a concentration of 25 µl of beads per 5 million cells for 30 min at 4 °C under slow rotation, and then incubated with 10 μg anti-Flag M2 antibodies (Sigma-Aldrich, F3165) previously coupled to 40 μl protein G Dynabeads (per each 5 million cells) for 2 h at 4 °C under slow rotation. The beads were washed twice with fRIP binding/wash buffer and protein–RNA cross-links were reversed by resuspending the beads in 56 μl of RNase-free water + 33 μl of 3× reverse-cross-linking buffer (3× PBS (without Ca²⁺ and Mg²⁺), 6% N-lauroyl sarcosine, 30 mM EDTA, 15 mM DTT (added fresh), 10 μl of 20 mg ml⁻¹ proteinase K (Millipore, 70663) and 1 μl of RNasin Ribonuclease Inhibitor. Protein degradation and reverse-cross-linking was performed for 1 h at 42 °C, followed by another hour at 55 °C. RNA was recovered by resuspending the beads and reaction buffer in 1 ml TRIzol Reagent (Invitrogen, 15596018) and purified using the Direct-zol RNA Microprep kit as recommended by manufacturer (Zymo Research, R2061).

Library generation and sequencing for fRIP–seq

RNA-seq libraries were prepared with immunoprecipitated RNA using the TruSeq stranded total RNA sample preparation kit according to the manufacturer’s protocol (Illumina). RNA-seq libraries were multiplexed, normalized and pooled for sequencing. The libraries were sequenced on the MiniSeq system (Illumina) with paired-end 37 bp reads. Image analysis and base calling were performed using Illumina CASAVA v.1.8.2. on the MiniSeq system and sequenced reads were quality-tested using FASTQC. Read pairs were mapped individually to the most complete assembly available of human subtelomeres (http://www.wistar.org/lab/harold-c-riethman-phd/page/subtelomere-assemblies) using STAR v.2.5.3a allowing up to 101 mapping locations⁶². Primary alignments were assigned randomly, and each read is represented by one mapping location. Library sizes were normalized to 1 × 10⁷ for comparison and the log₂ ratio of enrichment of immunoprecipitates versus input was calculated at each base in the subtelomere using HOMER v.4.10 and the assembly available of human subtelomeres⁶³. Visualizations were performed using R v.3.6.1 (R Core Team). Graphical packages (Gviz v.1.28.3, rtracklayer v.1.44.2, gridExtra v.2.3, ggplot2 v.3.3.2) were used to visualize data.

RT–qPCR

Total RNA was isolated using TRIzol (Invitrogen, 15596018) and purified using the RNAeasy Mini Kit (Qiagen, 74106) according to the manufacturer’s instructions. Genomic DNA was eliminated by double digestion with DNase I (RNase-free DNase Set, Qiagen, 79256). For RT–qPCR, 3.5 μg of RNA was reverse-transcribed either with random hexamers for measuring ISGs or with 2 μM of telC- and GAPDH-specific RT primers⁶⁴ using the SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific, 18080-051). qPCR was performed on the CFX384 Touch Real-Time PCR Detection System (BioRad). Reactions were run in triplicates with Power SYBR Green Master Mix (Applied Biosystems, Thermo Fisher Scientific, 4367659) in a total volume of 10 μl with standard cycling conditions. Relative gene expression was normalized using GAPDH as a housekeeping gene and calculated using the Comparative CT Method (ΔΔC_T method). The primers are listed below.

RNA-dot blot

Total RNA (10 μg) was blotted onto a positively charged nitrocellulose membrane (GE, RPNBL/02/10). For fRIP, 200 ng of RNA from IP or input samples were used. For RNase controls, RNA was incubated with RNase A (Invitrogen, 12091-039) at 37 °C for 1 h. RNA was fixed by ultraviolet cross-linking (Stratalinker, 2400) and TERRA was detected by hybridizing overnight at 55 °C with a Church mix containing telomeric repeat probes generated by [CCCTAA]³-primed Klenow labelling of an 800 bp [TTAGGG]ⁿ fragment in the presence of [α³²P]dCTP. After hybridization, the membrane was washed twice in 2× SSC and 0.1% SDS for 10 min at room temperature and then once for 10 min at 50 °C. The radioactive signal was detected using the Typhoon FLA 9000 imager (GE Healthcare). After the signal acquisition, membranes were stripped and rehybridized at 50 °C overnight with ³²P-dCTP-labelled probes targeting GAPDH transcripts. Signals were measured using ImageJ.

TRF analysis

TRF analysis was performed as previously described⁶⁵. In brief, genomic DNA was isolated by phenol–chloroform extraction and digested with AluI and MboI overnight. A total of 4 µg of digested gDNA was separated on 0.7% agarose gel at 40 V and transferred to a positively charged Nylon membrane (Amersham, RPN203B). After cross-linking the DNA and prehybridization (5× SSC. 0.1% N-lauroylsarcosine sodium salt solution, 0.04% SDS) for 2 h at 65 °C, the membrane was incubated with digoxigenin-labelled TelG probe diluted in hybridization buffer (1.3 nM final concentration) overnight at 65 °C. Digoxigenin-labelled TelG probe was generated as previously described⁶⁶. Then, the membrane was washed three times with wash buffer 1 (2× SSC, 0.1% SDS), one time with wash buffer 2 (2× SSC) for 15 min each and blocked in freshly prepared blocking solution (100 mM maleic acid, 150 mM NaCl, pH 7.5, 1% (w/v) blocking reagent (Roche, 11096176001)) for 30 min. Next, the membrane was incubated for 30 min in anti-digoxigenin-AP antibodies (Roche, 11093274910) diluted in blocking solution, washed twice in wash buffer 3 (100 mM maleic acid, 150 mM NaCl, pH 7.5, 0.3% (v/v) Tween-20) for 15 min each and equilibrated in AP buffer (100 mM Tris, 100 mM NaCl, pH 9.5) for 2 min. Digoxigenin-labelled telomeric DNA was detected using CDP-star ready to use (Roche, 12041677001) solution.

Genome-wide CRISPR screen

The human Brunello CRISPR knockout pooled library was obtained from D. Root and J. Doench through Addgene (73179-LV)⁶⁷. This ready-to-use lentiviral library has 77,441 gRNAs, targeting 19,114 protein-coding genes, with approximately 4 sgRNAs per gene. For adequate representation of each sgRNA, a total of 100 million pre-crisis (PD100) IMR90^E6E7 fibroblasts were transduced with the lentiviral library at a multiplicity of infection of 0.5. Transductions were performed in six-well plates (3 million cells per well) in medium containing 4 μg ml⁻¹ polybrene while centrifuging at 1,000 rcf for 1 h at 33 °C (spinfection). The next day, cells were transferred to Cell Factory System (Thermo Fisher Scientific, 140360), and puromycin-containing medium (1 μg ml⁻¹) was added for 7 days to eliminate uninfected cells and achieve genome-edited cell pools. After selection, cells were pooled together and divided into two technical replicates of 30 million cells each corresponding to the library baseline control at day 0. Another 30 million cells were replated into one Cell Factory System and positive selection was performed by growing cells for additional 15 days, at which point crisis-associated cell death was frequent. Two technical replicates of 30 million cells each were prepared by collecting cells at day 15, and their genomic DNA was extracted using a modified version of QIAGEN’s DNeasy Blood and Tissue Kit provided by the FLI-Seq Library Prep for CRISPR kit (Eclipse Bioinnovations). Genomic DNA was also extracted from the baseline count control sample at day 0. DNA fragments containing the sgRNA sequences were first captured from sheared gDNA, amplified by PCR using the FLI-Seq Library Prep for CRISPR kit (Eclipse Bioinnovations) and processed for next-generation sequencing. CRISPR libraries were multiplexed, normalized and pooled for sequencing. To compensate for low base diversity in CRISPR libraries, high-diversity libraries or PhiX Control v3 Library were spiked in for sequencing and the libraries were sequenced on the HiSeq 2500 system (Illumina) as single reads. Image analysis and base calling were performed using Illumina CASAVA v.1.8.2 on the HiSeq 2500 system and sequenced reads were quality-tested using FASTQC. Fold changes of sgRNA read counts before (day 0) and after (day 15) enrichment were calculated with the PinAPL-Py software and hits were defined as genes with a read count ratio in the survival pool to the baseline control log₂[fold change] ≥ 2 in both replicates. GO enrichment analysis was performed using the WEB-based Gene Set Analysis Toolkit (WebGestalt). The top 20 GO terms with an FDR value of <0.05 were considered to be statistically significant and visualized using the ClueGO and ggplot2 R packages.

Whole-transcriptome analysis

Total RNA was isolated using TRIzol (Invitrogen, 15596018) and purified using the RNAeasy Mini Kit (Qiagen, 74106) according to the manufacturer’s instructions. Genomic DNA was eliminated by double digestion with DNase I (RNase-free DNase Set, Qiagen, 79256). The quality of the isolated total RNA was assessed using the Agilent TapeStation 4200 and RNA-seq libraries were prepared with 500 ng total RNA using the TruSeq stranded mRNA sample preparation kit according to the manufacturer’s protocol (Illumina). RNA-seq libraries were multiplexed, normalized and pooled for sequencing. The libraries were sequenced on the HiSeq 4000 system (Illumina) as 50 bp single reads or the NextSeq 500 system (Illumina) as 75 bp single reads. Image analysis and base calling were performed using Illumina CASAVA v.1.8.2 on the HiSeq 4000 system and sequenced reads were quality-tested using FASTQC. Sequenced reads were quality-tested using FASTQC (v.0.11.8)⁶⁸ and mapped to the hg19 human genome using the STAR aligner (v.2.5.3a)⁶² with the default parameters. Raw or transcripts per kilobase million (TPM) gene expression was quantified across all the exons of RefSeq genes with analyzeRepeats.pl in HOMER (v.4.11.1)⁶³, which used the top-expressed isoform as proxy for gene expression. Differential gene expression was performed on the raw gene counts with the R package, DESeq2 (v.1.24.0)⁶⁹, using replicates to compute within-group dispersion. Differentially expressed genes were defined as having a FDR < 0.05 and a log₂[fold change] > 0.585 (~1.5 fold) when comparing two experimental conditions. GO enrichment analysis was performed using the WEB-based Gene Set Analysis Toolkit” (WebGestalt) and the R package clusterProfiler. The top 20 GO terms with an FDR value of <0.05 were considered to be statistically significant and visualized using the ClueGO and ggplot2 R packages. Heat maps and Venn diagrams were generated using the R packages ComplexHeatmap and VennDiagram, respectively.

ELISA assay

One million cells were seeded in a 10 cm plate for 24 h. Conditioned cell culture medium was collected and centrifuged at 14,000 rpm for 10 min at 4 °C to remove cellular debris. Three technical replicates of 50 μl each were collected for the assay. For preparation of standards, GlutaMax-DMEM (Gibco, 10569-010) supplemented with 0.1 mM non-essential amino acids (Corning, 25-025-Cl) and 15% fetal bovine serum (Thermo Fisher Scientific, SH3007103) was used. IFNβ secretion in cell culture supernatant was quantified using the IFNβ enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems Human IFNβ Quantikine ELISA Kit, DIFNB0) according to the manufacturer’s instruction. Supernatants from the treated cells were collected and incubated in IFNβ ELISA kit for 2 h at room temperature and washed three times with wash buffer. The optical density at 450 nm for each sample was measured with a microplate reader and data obtained were plotted against a four-parameter logistic standard curve to determine the concentration of IFNβ.

Chemical reagents

The reagents were as follows: recombinant human IFN-beta protein: R&D Systems, 8499-IF-010/CF; poly(dA:dT): InvivoGen, tlrl-patn; poly(I:C): InvivoGen, tlrl-pic; 2′,3′-dideoxycytidine: Sigma-Aldrich, D5782; deoxyribonucleic acid sodium salt from herring testes (HT-DNA): Sigma-Aldrich, D6898; MitoTracker: Invitrogen, M22426; IncuCyte Nuclight Rapid red dye: Sartorius, 4717; IncuCyte Cytotox red dye: Sartorius, 4632; IncuCyte Cytotox green dye: Sartorius, 4633; doxycycline: Sigma-Aldrich, D9891; staurosporine: Cell Signaling Technology, 9953; recombinant human TNF-alpha protein: R&D Systems, 210-TA; BV6: Selleckchem, S7597; z-VAD-FMK: Cell Signaling Technology, 60332; LPS: Cell Signaling Technology, 14011; nigericin: Cayman Chemical, 11437; Hoechst 33342: Thermo Fisher Scientific, 62249.

Statistical analysis

Statistical analysis was performed using Prism 9. Comparisons between two groups were performed using unpaired two-tailed Student’s t-tests. Multiple comparisons were performed using one-way ANOVA followed by Tukey’s or Dunnett’s multiple-comparisons test. For all representative findings, two or three independent experiments were performed, and similar results were obtained. Significance in all figures is denoted as follows: *P <  0.05, **P < 0.01, ***P < 0.001.

Exact P values are as follows.

Figure 1c: 36 h after incubation with Cytotox. Vector versus WT (<0.0001); vector versus mRHIM2 (<0.0001).

Figure 1d: IFNB1: vector versus WT (<0.0001); vector versus mZα2 (0.9775); vector versus mRHIM1 (0.9554); vector versus mRHIM2 (<0.0001). CCL5: vector versus WT (<0.0001); vector versus mZα2 (0.101); vector versus mRHIM1 (0.9799); vector versus mRHIM2 (<0.0001). CXCL10: vector versus WT (<0.0001); vector versus mZα2 (0.8118); vector versus mRHIM1 (0.9958); vector versus mRHIM2 (<0.0001). IFI44: vector versus WT (<0.0001); vector versus mZα2 (0.9426); vector versus mRHIM1 (0.9997); vector versus mRHIM2 (<0.0001). ISG15-vector versus WT (<0.0001); vector versus mZα2 (0.1726); vector versus mRHIM1 (0.8483); vector versus mRHIM2 (<0.0001).

Figure 2b: IFNB1: shScramble versus shTRF2 (<0.0001); shTRF2-siCtrl versus shTRF2-siZBP1 1 (<0.0001); shTRF2-siCtrl versus shTRF2-siZBP1 2 (<0.0001). CCL5: shScramble versus shTRF2 (<0.0001); shTRF2-siCtrl versus shTRF2-siZBP1 1 (<0.0001); shTRF2-siCtrl versus shTRF2-siZBP1 2 (<0.0001). CXCL10: shScramble versus shTRF2 (<0.0001); shTRF2-siCtrl versus shTRF2-siZBP1 1 (<0.0001); shTRF2-siCtrl versus shTRF2-siZBP1 2 (<0.0001). IFI44: shScramble versus shTRF2 (<0.0001); shTRF2-siCtrl versus shTRF2-siZBP1 1 (<0.0001); shTRF2-siCtrl versus shTRF2-siZBP1 2 (<0.0001). ISG15-shScramble versus shTRF2 (<0.0001); shTRF2-siCtrl versus shTRF2-siZBP1 1 (<0.0001); shTRF2-siCtrl versus shTRF2-siZBP1 2 (<0.0001).

Figure 3a: 9p, growing versus crisis (<0.0001). 13q, growing versus crisis (<0.0001). 15q, growing versus crisis (<0.0001). Xp, growing versus crisis (<0.0001). Xq, growing versus crisis (<0.0001). Figure 3b: 9p, shScramble versus shTRF2 (<0.0001). 13q, shScramble versus shTRF2 (<0.0001). 15q, shScramble versus shTRF2 (<0.0001). Xp, shScramble versus shTRF2 (<0.0001). Xq, shScramble versus shTRF2 (0.0015).

Figure 3f: 28 h after incubation with Cytotox. ZBP1(S)(WT)–Flag TRF1(ΔN) versus ZBP1(S)(WT)–Flag VP16–TRF1(ΔN) (<0.0001).

Figure 4a: PD45 versus PD79 (<0.0001); PD45 versus PD99 (<0.0001); PD45 versus PD103 (<0.0001); PD45 versus PD106 (<0.0001); PD35 versus PD50 (0.0043); PD35 versus PD62 (<0.0001); PD35 versus PD71 (<0.0001); PD35 versus PD86 (<0.0001).

Figure 4c: CCL5: PD40 vector versus PD40 WT (>0.9999). PD40 vector versus PD40 mZα (>0.9999); PD40 vector versus PD40 mRHIM1 (>0.9999); PD40 vector versus PD40 mRHIM2 (>0.9999); PD95 vector versus PD95 WT (<0.0001); PD95 vector versus PD95 mZα (0.8488); PD95 vector versus PD95 mRHIM1 (0.9998); PD95 vector versus PD95 mRHIM2 (<0.0001). CXCL10: PD40 vector versus PD40 WT (>0.9999); PD40 vector versus PD40 mZα (>0.9999); PD40 vector versus PD40 mRHIM1 (>0.9999); PD40 vector versus PD40 mRHIM2 (>0.9999); PD95 vector versus PD95 WT (0.0005); PD95 vector versus PD95 mZα (0.9766); PD95 vector versus PD95 mRHIM1 (>0.9999); PD95 vector versus PD95 mRHIM2 (<0.0001). IFI44: PD40 vector versus PD40 WT (>0.9999); PD40 vector versus PD40 mZα (>0.9999); PD40 vector versus PD40 mRHIM1 (0.9965); PD40 vector versus PD40 mRHIM2 (>0.9999); PD95 vector versus PD95 WT (<0.0001); PD95 vector versus PD95 mZα (>0.9566); PD95 vector versus PD95 mRHIM1 (0.0338); PD95 vector versus PD95 mRHIM2 (<0.0001).

Figure 4e: CCL5: Flag–ZBP1(S)–FIS1 versus Flag–ZBP1(S) (<0.0001); vector versus Flag–ZBP1(S)–FIS1 (<0.0001). CXCL10: Flag–ZBP1(S)–FIS1 versus Flag-ZBP1(S) (<0.0001); vector versus Flag–ZBP1(S)–FIS1 (<0.0001). IFI44: Flag–ZBP1(S)–FIS1 versus Flag–ZBP1(S) (<0.0001); vector versus Flag–ZBP1(S)–FIS1 (<0.0001).

Figure 4g: CCL5: vector siCtrl versus vector siMAVS (0.9002); Flag–ZBP1(S) siCtrl versus Flag–ZBP1(S) siMAVS (0.3629); Flag–ZBP1(S)–FIS1 siCtrl versus Flag–ZBP1(S)–FIS1 siMAVS (<0.0001). CXCL10: vector siCtrl versus vector siMAVS (0.9537). Flag–ZBP1(S) siCtrl versus Flag–ZBP1(S) siMAVS (0.0001). Flag–ZBP1(S)–FIS1 siCtrl versus Flag–ZBP1(S)–FIS1 siMAVS (<0.0001). IFI44: vector siCtrl versus vector siMAVS (>0.9999). Flag–ZBP1(S) siCtrl versus Flag–ZBP1(S) siMAVS (0.9966). Flag–ZBP1(S)–FIS1 siCtrl versus Flag–ZBP1(S)–FIS1 siMAVS (<0.0001).

Extended Data Fig. 3d: IMR90^E6E7, 24 h after incubation with Cytotox. sgGFP versus sgZBP1 1 (<0.0001). sgGFP versus sgZBP1 2 (<0.0001); sgGFP versus sgZBP1 3 (<0.0001); sgLUC versus sgZBP1 1 (<0.0001); sgLUC versus sgZBP1 2 (<0.0001); sgLUC versus sgZBP1 3 (<0.0001). WI38^SV40LT, 24 h after incubation with Cytotox. sgGFP versus sgZBP1 1 (<0.0001); sgGFP versus sgZBP1 2 (<0.0001); sgGFP versus sgZBP1 3 (<0.0001); sgLUC versus sgZBP1 1 (<0.0001); sgLUC versus sgZBP1 2 (<0.0001); sgLUC versus sgZBP1 3 (<0.0001).

Extended Data Fig. 3e: IMR90^E6E7: sgLUC versus sgGFP (0.0668); sgLUC versus sgZBP1 1 (<0.0001); sgLUC versus sgZBP1 2 (<0.0001); sgLUC versus sgZBP1 3 (<0.0001). WI38^SV40LT: sgLUC versus sgGFP (0.1293); sgLUC versus sgZBP1 1 (<0.0001); sgLUC versus sgZBP1 2 (<0.0001); sgLUC versus sgZBP1 3 (<0.0001).

Extended Data Fig. 3f: IMR90^E6E7: sgLUC versus sgGFP (0.9995); sgLUC versus sgZBP1 1 (0.0002); sgLUC versus sgZBP1 2 (<0.0001); sgLUC versus sgZBP1 3 (0.0004). WI38^SV40LT: sgLUC versus sgGFP (0.7845); sgLUC versus sgZBP1 1 (0.0018); sgLUC versus sgZBP1 2 (<0.0001). sgLUC versus sgZBP1 3 (0.0011).

Extended Data Fig. 4b: IMR90^E6E7: growing versus crisis (<0.0001). WI38^SV40LT: growing versus crisis (<0.0001).

Extended Data Fig. 4e: experiment 1: siCtrl versus siIFNAR1 (1.39178 × 10⁻⁵); siCtrl versus siIFNAR2 (8.05855 × 10⁻⁵); siCtrl versus siCGAS (3.65474 × 10⁻⁵); siCtrl versus siAIM2 (0.57384863); siCtrl versus siTLR3 (0.028940902); siCtrl versus siTLR7 (0.130633803); siCtrl versus siTLR9 (0.481604543); siCtrl versus siRIG-I (0.067963804); siCtrl versus siMDA5 (0.078645098); siCtrl versus RIG-I + siMDA5 (0.075426998); siCtrl versus siSTING (0.000445511); siCtrl versus siPYCARD (0.272857261); siCtrl versus siMyD88 (0.16111072); siCtrl versus siTRIF (0.138938302); siCtrl versus siMAVS (3.61752 × 10⁻⁵). Experiment 2: siCtrl versus siIFNAR1 (1.48978 × 10⁻⁶); siCtrl versus siIFNAR2 (1.4765 × 10⁻⁵); siCtrl versus siCGAS (1.40243 × 10⁻⁶); siCtrl versus siAIM2 (0.081532291); siCtrl versus siTLR3 (0.244271443); siCtrl versus siTLR7 (0.413559022); siCtrl versus siTLR9 (0.1970414); siCtrl versus siRIG-I (0.015458711); siCtrl versus siMDA5 (0.027374775); siCtrl versus RIG-I + siMDA5 (0.857465154); siCtrl versus siSTING (9.9925 × 10⁻⁵); siCtrl versus siPYCARD (0.28638452); siCtrl versus siMyD88 (0.73179998); siCtrl versus siTRIF (0.0188581); siCtrl versus siMAVS (7.6601 × 10⁻⁶). Experiment 3: siCtrl versus siIFNAR1 (1.23834 × 10⁻⁵); siCtrl versus siIFNAR2 (6.7646 × 10⁻⁵); siCtrl versus siCGAS (1.04549 × 10⁻⁶); siCtrl versus siAIM2 (0.05401101); siCtrl versus siTLR3 (0.16170163); siCtrl versus siTLR7 (0.01702073); siCtrl versus siTLR9 (0.0189054); siCtrl versus siRIG-I (0.00405314); siCtrl versus siMDA5 (0.373743841); siCtrl versus RIG-I + siMDA5 (0.024471461); siCtrl versus siSTING (0.00368923); siCtrl versus siPYCARD (0.08524613); siCtrl versus siMyD88 (0.14064815); siCtrl versus siTRIF (0.12424426); siCtrl versus siMAVS (0.008386476).

Extended Data Fig. 5d: IFNB1: vector versus WT (<0.0001); vector versus ΔZα2 (0.2671); vector versus ΔZα2ΔRHIM1ΔRHIM2 (0.1795); vector versus ΔC (<0.0001); vector versus ΔRHIM1 (0.6671); vector versus ΔRHIM2 (<0.0001); vector versus ΔRHIM1ΔRHIM2 (0.0151). CCL5: vector versus WT (<0.0001); vector versus ΔZα2 (0.0367); vector versus ΔZα2ΔRHIM1ΔRHIM2 (0.978); vector versus ΔC (<0.0001); vector versus ΔRHIM1 (0.0675); vector versus ΔRHIM2 (<0.0001); vector versus ΔRHIM1ΔRHIM2 (0.0816). CXCL10: vector versus WT (<0.0001); vector versus ΔZα2 (0.1945); vector versus ΔZα2ΔRHIM1ΔRHIM2 (0.9998); vector versus ΔC (<0.0001); vector versus ΔRHIM1 (0.1365); vector versus ΔRHIM2 (<0.0001); vector versus ΔRHIM1ΔRHIM2 (0.1988). IFI44: vector versus WT (<0.0001); vector versus ΔZα2 (0.0294); vector versus ΔZα2ΔRHIM1ΔRHIM2 (0.9813); vector versus ΔC (<0.0001); vector versus ΔRHIM1 (0.0287); vector versus ΔRHIM2 (<0.0001); vector versus ΔRHIM1ΔRHIM2 (0.2269). ISG15: vector versus WT (<0.0001); vector versus ΔZα2 (0.0003); vector versus ΔZα2ΔRHIM1ΔRHIM2 (0.0242); vector versus ΔC (<0.0001); vector versus ΔRHIM1 (0.0005); vector versus ΔRHIM2 (<0.0001); vector versus ΔRHIM1ΔRHIM2 (0.0001).

Extended Data Fig. 6e: vector versus ZBP1(S)–Flag (0.0082); siCtrl versus siZBP1 (<0.0001); siCtrl versus siRIPK3 (0.9942); siCtrl versus siMLK (>0.9999); siCtrl versus siRIPK1 (>0.9999); siCtrl versus siFADD (0.9425); siCtrl versus siCASP8 (>0.9999); siCtrl versus siNLRP3 (>0.9999); siCtrl versus siPYCARD (>0.9999); siCtrl versus siCASP1 (0.9790); siCtrl versus siIFNAR1 (<0.0001); siCtrl versus siIFNAR2 (<0.0001); siCtrl versus siSTAT1 (<0.0001); siCtrl versus siSTAT2 (<0.0001); siCtrl versus siIRF9 (<0.0001); siCtrl versus siATG5 (<0.0001); siCtrl versus siATG7 (<0.0001); siCtrl versus siATG12 (<0.0001).

Extended Data Fig. 6g: mock versus shScramble day 3 (0.9934); mock versus shScramble day 6 (0.9996); mock versus shScramble day 9 (0.9994); mock versus shScramble day 12 (0.9996); mock versus shTRF2 day 3 (<0.0001); mock versus shTRF2 day 6 (<0.0001); mock versus shTRF2 day 9 (<0.0001); mock versus shTRF2 day 12 (<0.0001).

Extended Data Figure 6h: mock versus shScramble day 3 (>0.9999); mock versus shScramble day 6 (>0.9999); mock versus shScramble day 9 (>0.9999); mock versus shScramble day 12 (>0.9999); mock versus shTRF2 day 3 (<0.0001); mock versus shTRF2 day 6 (<0.0001); mock versus shTRF2 day 9 (<0.0001); mock versus shTRF2 day 12 (<0.0001).

Extended Data Figure 6j: siCtrl versus siZBP1 1 (0.9038); siCtrl versus siZBP1 2 (0.2923).

Extended Data Fig. 7c: IMR90^E6E7: PD42 versus PD45 (>0.9999); PD42 versus PD46 (0.9999); PD42 versus PD51 (0.9999); PD42 versus PD55 (0.9997); PD42 versus PD58 (>0.9999); PD42 versus PD62 (0.9998); PD42 versus PD69 (0.9996); PD42 versus PD73 (>0.9999); PD42 versus PD85 (0.9996); PD42 versus PD87 (0.9998); PD42 versus PD90 (0.9998); PD42 versus PD99 (0.9864); PD42 versus PD100 (0.0891); PD42 versus PD101 (0.0003); PD42 versus PD103 (<0.0001); PD42 versus PD105 (<0.0001); PD42 versus PD106 (<0.0001). IMR90^E6E7, hTERT: PD61 versus PD74 (0.8616); PD61 versus PD89 (0.9996); PD61 versus PD95 (0.9995); PD61 versus PD105 (>0.9999); PD61 versus PD108 (0.9882). WI38^SV40LT: PD35 versus PD38 (>0.9999); PD35 versus PD41 (>0.9999); PD35 versus PD44 (0.9997); PD35 versus PD47 (>0.9999); PD35 versus PD50 (0.9999); PD35 versus PD53 (>0.9999); PD35 versus PD58 (0.9998); PD35 versus PD62 (0.9998); PD35 versus PD67 (0.9998); PD35 versus PD69 (0.9994); PD35 versus PD71 (0.9997); PD35 versus PD79 (0.9994). PD35 versus PD82 (0.9990); PD42 versus PD86 (<0.0001); PD42 versus PD87 (<0.0001); PD42 versus PD89 (<0.0001); PD42 versus PD90 (<0.0001). WI38^SV40LT, hTERT: PD50 versus PD63 (0.9576); PD50 versus PD74 (0.9576); PD50 versus PD77 (0.9796); PD50 versus PD83 (>0.9999); PD50 versus PD92 (0.9576).

Extended Data Fig. 8a: growing versus crisis (<0.0001).

Extended Data Fig. 8b: shScramble versus shTRF2 (<0.0001).

Extended Data Fig. 9c: 9p: ZBP1(S)(WT)–Flag TRF1(ΔN) versus ZBP1(S)(WT)–Flag VP16–TRF1(ΔN) (<0.0001); ZBP1(S)(ΔZα2)–Flag TRF1(ΔN) versus ZBP1(S)(ΔZα2)–Flag VP16–TRF1(ΔN) (<0.0001). 13q: ZBP1(S)(WT)–Flag TRF1(ΔN) versus ZBP1(S)(WT)–Flag VP16–TRF1(ΔN) (0.0002); ZBP1(S)(ΔZα2)–Flag TRF1(ΔN) versus ZBP1(S)(ΔZα2)–Flag VP16–TRF1(ΔN) (0.0005). 15q: ZBP1(S)(WT)–Flag TRF1(ΔN) versus ZBP1(S)(WT)–Flag VP16–TRF1(ΔN) (<0.0001); ZBP1(S)(ΔZα2)–Flag TRF1(ΔN) versus ZBP1(S)(ΔZα2)–Flag VP16–TRF1(ΔN) (<0.0001). Xp: ZBP1(S)(WT)–Flag TRF1(ΔN) versus ZBP1(S)(WT)–Flag VP16–TRF1(ΔN) (<0.0001); ZBP1(S)(ΔZα2)–Flag TRF1(ΔN) versus ZBP1(S)(ΔZα2)–Flag VP16–TRF1(ΔN) (<0.0001). Xq: ZBP1(S)(WT)–Flag TRF1(ΔN) versus ZBP1(S)(WT)–Flag VP16–TRF1(ΔN) (0.0023); ZBP1(S)(ΔZα2)–Flag TRF1(ΔN) versus ZBP1(S)(ΔZα2)–Flag VP16–TRF1(ΔN) (0.0009).

Extended Data Figure 10b: CCL5: ZBP1(S)(WT)–Flag TRF1(ΔN) versus ZBP1(S)(WT)–Flag VP16–TRF1(ΔN) (<0.0001); ZBP1(S)(ΔZα2)–Flag TRF1(ΔN) versus ZBP1(S)(ΔZα2)–Flag VP16–TRF1(ΔN) (0.7919). CXCL10–ZBP1(S)(WT)–Flag TRF1(ΔN) versus ZBP1(S)(WT)–Flag VP16–TRF1(ΔN) (<0.0001); ZBP1(S)(ΔZα2)–Flag TRF1(ΔN) versus ZBP1(S)(ΔZα2)–Flag VP16–TRF1(ΔN) (0.9996). IFI44: ZBP1(S)(WT)–Flag TRF1(ΔN) versus ZBP1(S)(WT)–Flag VP16–TRF1(ΔN) (<0.0001); ZBP1(S)(ΔZα2)–Flag TRF1(ΔN) versus ZBP1(S)(ΔZα2)–Flag VP16–TRF1(ΔN) (>0.9999).

Extended Data Figure 10d: CCL5: ZBP1(S)(WT)–Flag TRF1(ΔN) versus ZBP1(S)(WT)–Flag VP16–TRF1(ΔN) (<0.0001); ZBP1(S)(mZα2)–Flag TRF1(ΔN) versus ZBP1(S)(mZα2)–Flag VP16–TRF1(ΔN) (0.8595). CXCL10: ZBP1(S)(WT)–Flag TRF1(ΔN) versus ZBP1(S)(WT)–Flag VP16–TRF1(ΔN) (<0.0001). ZBP1(S)mZα2–Flag TRF1(ΔN) versus ZBP1(S)(mZα2)–Flag VP16–TRF1(ΔN) (>0.9999). IFI44: ZBP1(S)(WT)–Flag TRF1(ΔN) versus ZBP1(S)(WT)–Flag VP16–TRF1(ΔN) (<0.0001). ZBP1(S)(mZα2)–Flag TRF1(ΔN) versus ZBP1(S)(mZα2)–Flag VP16–TRF1(ΔN) (0.9998).

Extended Data Figure 11c: IMR90^E6E7: TOM20–ZBP1, growing versus crisis (<0.0001); MitoTracker–ZBP1, growing versus crisis (<0.0001); TFAM–ZBP1, growing versus crisis (<0.0001). WI38^SV40LT: TOM20–ZBP1, growing versus crisis (<0.0001); MitoTracker–ZBP1, growing versus crisis (<0.0001); TFAM–ZBP1, growing versus crisis (<0.0001).

Extended Data Figure 11d: dsDNA intensity: growing versus crisis (<0.0001); dsRNA intensity: growing versus crisis (<0.0001); number of ZBP1 filaments: growing versus crisis (0.0712).

Extended Data Figure 11e: dsRNA intensity: siCtrl versus siSUV3 (<0.0001); siCtrl versus siPNPase (<0.0001); number of ZBP1 filaments: siCtrl versus siSUV3 (0.6179). siCtrl versus siPNPase (0.3240).

Extended Data Figure 12c: CCL5: ZBP1(S)(WT)–Flag TRF1(ΔN): siCtrl versus siMAVS (0.6383); siCtrl versus siMDA5 (0.9499); siCtrl versus siRIG-I (>0.9999); siCtrl versus siMDA5 + RIG-I (0.9959). ZBP1(S)(WT)–Flag VP16–TRF1(ΔN): siCtrl versus siMAVS (<0.0001), siCtrl versus siMDA5 (0.2942); siCtrl versus siRIG-I (0.9933); siCtrl versus siMDA5 + RIG-I (0.8552). CXCL10: ZBP1(S)(WT)–Flag TRF1(ΔN): siCtrl versus siMAVS (0.9997); siCtrl versus siMDA5 (0.9980); siCtrl versus siRIG-I (0.9973); siCtrl versus siMDA5 + RIG-I (>0.9999). ZBP1(S)(WT)–Flag VP16–TRF1(ΔN): siCtrl versus siMAVS (<0.0001); siCtrl versus siMDA5 (0.0645); siCtrl versus siRIG-I (0.8284); siCtrl versus siMDA5 + RIG-I (0.1366). IFI44: ZBP1(S)(WT)–Flag TRF1(ΔN): siCtrl versus siMAVS (0.9999); siCtrl versus siMDA5 (>0.9999); siCtrl versus siRIG-I (>0.9999); siCtrl versus siMDA5 + RIG-I (>0.9999). ZBP1(S)(WT)–Flag VP16–TRF1(ΔN): siCtrl versus siMAVS (<0.0001); siCtrl versus siMDA5 (<0.0001); siCtrl versus siRIG-I (<0.0001); siCtrl versus siMDA5 + RIG-I (<0.0001).

Extended Data Figure 13d: IMR90^E6E7: vector siCtrl versus siMAVS (>0.9999); vector siCtrl versus siIFNAR2 (>0.9999); Flag–ZBP1(S)–FIS1 siCtrl versus siMAVS (<0.0001); Flag–ZBP1(S)–FIS1 siCtrl versus siIFNAR2 (<0.0001). HMECs: vector siCtrl versus siMAVS (>0.9999); vector siCtrl versus siIFNAR2 (>0.9999); Flag–ZBP1(S)–FIS1 siCtrl versus siMAVS (<0.0001); Flag–ZBP1(S)–FIS1 siCtrl versus siIFNAR2 (<0.0001).

shRNAs

shRNAs were as follows: Scramble, CCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGG, pLKO.1, Addgene, 1864; TRF2, ACAGAAGCAGTGGTCGAATC, pLKO.1, Open Biosystems, TRCN0000018358; shZBP1 1, CCAAGTCCTCTACCGAATGAA, pLKO.1; shZBP1 2, GCACAATCCAATCAACATGAT, pLKO.1, Sigma-Aldrich, TRCN0000123050.

siRNAs

siRNAs were as follows: non-targeting pool (Dharmacon, D-001810-10-20): UGGUUUACAUGUCGACUAA, UGGUUUACAUGUUGUGUGA, UGGUUUACAUGUUUUCUGA, UGGUUUACAUGUUUUCCUA); non-targeting (Dharmacon, D-001810-01-05): UGGUUUACAUGUCGACUAA; ZBP1 1 (Dharmacon, J-014650-07-0005): GGACACGGGAACAUCAUUA; ZBP1 2 (Dharmacon, J-014650-08-0005): CAAAAGAUGUGAACCGAGA; ZBP1 3 (Dharmacon, J-014650-06-0005): GGAUUUCCAUUGCAAACUC; ZBP1 4 (Dharmacon, J-014650-05-0005): CAAAGUCAGCCUCAAUUAU; MB21D1 (Dharmacon, L-015607-02-0005): GAAGAAACAUGGCGGCUAU, AGGAAGCAACUACGACUAA, AGAACUAGAGUCACCCUAA, CCAAGAAGGCCUGCGCAUU; TMEM173 (Dharmacon, L-024333-02-0005): UCAUAAACUUUGGAUGCUA, CGAACUCUCUCAAUGGUAU, AGCUGGGACUGCUGUUAAA, GCAGAUGACAGCAGCUUCU; MAVS 1 (Dharmacon, J-024237-07-0005): GCAAUGUGGAUGUUGUAGA; MAVS 2 (Dharmacon, J-024237-05-0005): AAGUAUAUCUGCCGCAAUU; MAVS 3 (Dharmacon, J-024237-06-0005): CAUCCAAAGUGCCUACUAG; MAVS 4 (Dharmacon, J-024237-08-0005): CAUCCAAAUUGCCCAUCAA; PYCARD (Dharmacon, L-004378-00-0005): GGAAGGUCCUGACGGAUGA, UCACAAACGUUGAGUGGCU, GGCCUGCACUUUAUAGACC, CCACCAACCCAAGCAAGAU; MYD88 (Dharmacon, L-004769-00-0005): CGACUGAAGUUGUGUGUGU, GCUAGUGAGCUCAUCGAAA, GCAUAUGCCUGAGCGUUUC, GCACCUGUGUCUGGUCUAU; TLR7 (Dharmacon, L-004714-00-0005): CAACAACCGGCUUGAUUUA, GGAAAUUGCCCUCGUUGUU, GAAUCUAUCACAAGCAUUU, GGAAUUACUCAUAUGCUAA; TLR3 (Dharmacon, L-007745-00-0005): GAACUAAAGAUCAUCGAUU, CAGCAUCUGUCUUUAAUAA, AGACCAAUCUCUCAAAUUU, UCACGCAAUUGGAAGAUUA; TLR9 (Dharmacon, L-004066-00-0005): CAGACUGGGUGUACAACGA, GCAAUGCACUGGGCCAUAU, CGGCAACUGUUAUUACAAG, ACAAUAAGCUGGACCUCUA; TLR8 (Dharmacon, L-4715-00-0005): CAACGGAAAUCCCGGUAUA, CAGAAUAGCAGGCGUAACA, GUGCAGCAAUCGUCGACUA, CUUCCAAACUUAUCGACUA; DDX58 (Dharmacon, L-012511-00-0005): GCACAGAAGUGUAUAUUGG, CCACAACACUAGUAAACAA, CGGAUUAGCGACAAAUUUA, UCGAUGAGAUUGAGCAAGA; IFIH1 (Dharmacon, L-013041-00-0005): GAAUAACCCAUCACUAAUA, GCACGAGGAAUAAUCUUUA, UGACACAAUUCGAAUGAUA, CAAUGAGGCCCUACAAAUU; TICAM1 (Dharmacon, L-012833-00-0005): GGAGCCACAUGUCAUUUGG, CCAUAGACCACUCAGCUUU, GGACGAACACUCCCAGAUC, CCACUGGCCUCCCUGAUAC; IFNAR1 (Dharmacon, L-020209-00-0005): GCGAAAGUCUUCUUGAGAU, UGAAACCACUGACUGUAUA, GAAAAUUGGUGUCUAUAGU, GAAGAUAAGGCAAUAGUGA; IFNAR2 (Dharmacon, L-015411-00-0005): CAGAGGGAAUUTUUAAGAA, GAGUAAACCAGAAGAUUUG, CACCAGAGUUUGAGAUUGU, UCACCUAUAUCAUUGACAA; AIM2 (Dharmacon, L-011951-00-0005): GCACAGUGGUUUCUUAGAG, UCAGACGAGUUUAAUAUUG, GAAAGUUGAUAAGCAAUAC, GUUCAUAGCACCAUAAAGG; SUPV3L1 (Dharmacon, L-017841-01-0005): UGGCUAAGCUACCGAUUUA, GUAAGGAUGAUCUACGUAA, CGGUGCAGCUCAUGCGGAU, GGAAAGACUUAUCACGCAA; PNPT1 (Dharmacon, L-020112-00-0005): GACAGAAGUAGUAUUGUAA, ACAGAAAGAUUAUUGGCUA, GAAUGUAAGUUGUGAGGUA, AAUCAGAGAUACUGGUGUA; RIPK3 (Dharmacon, L-003534-00-0005): CCACAGGGUUGGUAUAAUC, AACCAGCACUCUCGUAAUG, GCUACGAUGUGGCGGUCAA, GACCGCUCGUUAACAUAUA; ATG7 (Dharmacon, L-020112-00-0005): CCAACACACUCGAGUCUUU, GAUCUAAAUCUCAAACUGA, GCCCACAGAUGGAGUAGCA, GCCAGAGGAUUCAACAUGA; TERRA 1 (Dharmacon, CTM-536949): AGGGUUAGGGUUAGGGUUAUU; TERRA 2 (Dharmacon, CTM-536950): GGGUUAGGGUUAGGGUUAGUU; MLKL (Dharmacon, L-005326-00-0005): GAGCAACGCAUGCCUGUUU, CAAACUUCCUGGUAACUCA, GAAGGAGCUCUCGCUGUUA, GGAUUUGCAUUGAUGAAAC; RIPK1 (Dharmacon, L-004445-00-0005): CCACUAGUCUGACGGAUAA, UGAAUGACGUCAACGCAAA, GCACAAAUACGAACUUCAA, GAUGAAAUCCAGUGACUUC); FADD (Dharmacon, L-003800-00-0005): CAUUUAACGUCAUAUGUGA, GGAGAAGGCUGGCUCGUCA, UGACAGAGCGUGUGCGGGA, GCAUCUACCUCCGAAGCGU); CASP8 (Dharmacon, L-003466-00-0005): GGACAAAGUUUACCAAAUG, GCCCAAACUUCACAGCAUU, GAUAAUCAACGACUAUGAA, GUCAUGCUCUAUCAGAUUU); NLRP3 (Dharmacon, L-017367-00-0005): GGAUCAAACUACUCUGUGA, UGCAAGAUCUCUCAGCAAA, GAAGUGGGGUUCAGAUAAU, GCAAGACCAAGACGUGUGA); CASP1 (Dharmacon, L-004401-00-0005): GGAAGACUCAUUGAAACAUA, GAUGGUAGAGCGCAGAUGC, CCGCAAGGUUCGAUUUUCA, GAGUGACUUUGACAAGAUG); STAT1 (Dharmacon, L-003543-00-0005): GCACGAUGGGCUCAGCUUU, CUACGAACAUGACCCUAUC, GAACCUGACUUCCAUGCGG, AGAAAGAGCUUGACAGUAA); STAT2 (Dharmacon, L-012064-00-0005): GGACUGAGUUGCCUGGUUA, GGACUGAGGAUCCAUUAUU, GAGCCCUCCCUGGCAAGUUA, GAUUUGCCCUGUGAUCUGA); IRF9 (Dharmacon, L-020858-00-0005): GCAGAGACUUGGUCAGGUA, CCACCGAAGUUCCAGGUAA, GCGUGGAGCUCUUCAGAAC, GAAAGUACCAUCAAAGCGA); ATG5 (Dharmacon, L-004374-00-0005): GGCAUUAUCCAAUUGGUUU, GCAGAACCAUACUAUUUGC, UGACAGAUUUGACCAGUUU, ACAAAGAUGUGCUUCGAGA); ATG12 (Dharmacon, L-010212-00-0005): GAACACCAAGUUUCACUGU, GCAGUAGAGCGAACACGAA, GGGAAGGACUUACGGAUGU, GGGAUGAACCACAAAGAAA).

Primers for RT–qPCR

The primers were as follows: 9p-F, GAGATTCTCCCAAGGCAAGG; 9p-R, ACATGAGGAATGTGGGTGTTAT; 13q-F, CTGCCTGCCTTTGGGATAA; 13q-R, AAACCGTTCTAACTGGTCTCTG; 15q-2-F, CAGCGAGATTCTCCCAAGCTAAG; 15q-2-R, AACCCTAACCACATGAGCAACG; Xq-F, AGCAAGCGGGTCCTGTAGTG; Xq-R, GGTGGAACTTCAGTAATCCGAAA; Xp-F, AAGAACGAAGCTTCCACAGTAT; Xp-R, GGTGGGAGCAGATTAGAGAATAAA; GAPDH-F, AGCCACATCGCTCAGACAC; GAPDH-R, GCCCAATACGACCAAATCC; GAPDH RT, GCCCAATACGACCAAATCC; TERRA RT, CCCTAACCCTAACCCTAACCCTAACCCTAA; ZBP1-F, AACATGCAGCTACAATTCCAGA; ZBP1-R, AGTCTCGGTTCACATCTTTTGC; IFNB1-F, ACGCCGCATTGACCATCTAT; IFNB1-R, GTCTCATTCCAGCCAGTGCT; ISG15-F, CGCAGATCACCCAGAAGATCG; ISG15-R, TTCGTCGCATTTGTCCACCA; IFI44-F, AGCCGTCAGGGATGTACTATAAC; IFI44-R, AGGGAATCATTTGGCTCTGTAGA; CCL5-F, CCAGCAGTCGTCTTTGTCAC; CCL5-R, CTCTGGGTTGGCACACACTT; CXCL10-F, GTGGCATTCAAGGAGTACCTC; CXCL10-R, TGATGGCCTTCGATTCTGGATT.

Antibodies

Antibodies used were as follows: TRF2 (Karlseder laboratory), ZBP1 (Novus Biologicals, NBP1-76854 and Cell Signaling Technology, 60968), LC3 (Cell Signaling Technology, 2775 and 3868), STING (Cell Signaling Technology, 13647), CGAS (Cell Signaling Technology, D1D3G), γH2AX (Millipore, 05-636-I), Flag (Sigma-Aldrich, F1804), GAPDH (Abnova, PAB17013), TBK1 (Cell Signaling Technology, 3504S), phosphorylated TBK1 (Cell Signaling Technology, 5483S), STAT1 (Cell Signaling Technology, 9172S), phosphorylated STAT1 (Cell Signaling Technology, 9167L), IRF3 (Cell Signaling Technology, 11904S), phosphorylated IRF3 (Cell Signaling Technology, D4947S), MAVS (Cell Signaling Technology, 24930S), TOMM20 (Abcam, ab56783), TFAM (Abcam, ab119684), dsDNA (Abcam, ab27156), dsRNA (Millipore, MABE1134), phosphorylated ATR (Abcam, ab223258), phosphorylated ATM (Cell Signaling Technology, 5883), the apoptosis antibody sampler kit (Cell Signaling Technology, 9915), the necroptosis antibody sampler kit (Cell Signaling Technology, 98110) and the pyroptosis antibody sampler kit (Cell Signaling Technology, 43811).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.