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An SP110–SP100 axis is a critical regulator of promyelocytic leukaemia body dynamics and mitotic fidelity

Nature Cell Biology volume 28, pages 684–695 (2026) Cite this article

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Abstract

Stimulation of the innate immune system by foreign RNA elicits a potent interferon response and can trigger cell death. The mechanisms by which cells balance a robust response with cell-intrinsic lethality are still being uncovered. Here, using genome-wide CRISPR–Cas9 genetic screens with triphosphorylated RNA stimulation, we discover that promyelocytic leukaemia (PML) nuclear body-localized speckled protein 110 (SP110) is a potent inhibitor of type 1 interferon-driven cell death. Death suppression by SP110 counteracts a toxic activity of SP100, a major constituent of PML bodies. Loss of SP110 leads to mitotic retention of SP100 and PML bodies, which associate with and perturb segregating chromosomes, leading to micronucleus formation, DNA damage and genotoxic cell death. A combination of cryo-electron microscopy, AlphaFold modelling and cellular biochemistry reveals that SP110 dissolves toxic SP100 oligomers via necessary and sufficient direct interactions between their caspase activation and recruitment domains. These data reveal the critical roles of SP100 and SP110 in governing the disassembly of PML bodies during mitosis, as well as the repercussions if this process is misregulated.

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Fig. 1: CRISPR screen reveals that SP110 protects against hypersensitivity to interferon stimulation.
The alternative text for this image may have been generated using AI.
Fig. 2: SP100 induces cellular toxicity in the absence of SP110.
The alternative text for this image may have been generated using AI.
Fig. 3: SP100 promotes genotoxicity via retention on mitotic DNA.
The alternative text for this image may have been generated using AI.
Fig. 4: SUMO-deficient SP100 drives genotoxicity.
The alternative text for this image may have been generated using AI.
Fig. 5: Physical SP110–SP100 CARD–CARD interaction prevents lethality.
The alternative text for this image may have been generated using AI.
Fig. 6: SP110 blocks toxic SP100 CARD oligomerization.
The alternative text for this image may have been generated using AI.

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Data availability

Raw sequencing data from the CRISPR screens, RNA-seq and ChIP-seq experiments are deposited in SRA BioProject PRJNA1302711. Processed data from ChIP-seq are deposited in GEO GSE304976. The SP100 structure is deposited in PDB 9TNZ and EMDB EMD-56096. Numerical source data for Supplementary Figs. 1 and 3 can be found in Supplementary Data 5. Source data are provided with this paper.

Code availability

No custom code was used in this study. All analyses were performed with publicly available tools.

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Acknowledgements

We acknowledge people at ETH Zurich facilities who aided in parts of this work: S. Kreutzer at Genome Engineering and Measurement Lab (GEML), T. Kockmann at the Functional Genomics Center Zurich (FGCZ) of University of Zurich and ETH Zurich for proteomics work, J. Hehl at the Scientific Center for Optical and Electron Microscopy (ScopeM) for imaging assistance and D. Boehringer at the Cryo-EM knowledge hub (CEMK). We thank A. Gvozdenovic for critical reading of the manuscript. E.J.A. is supported by an EMBO Postdoctoral Fellowship (ALTF 144-2021). J.E.C. is supported by the NOMIS Foundation and the Lotte und Adolf Hotz-Sprenger Stiftung. This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (855741-DDREAMM-ERC-2019-SyG to J.E.C. and S.P.J.) as well as SNSF Project Funding (310030_188858 to J.E.C. and 320030_232029 to B.G.H.). S.W.A. is supported by a Mark Foundation for Cancer Research (MFCR) ASPIRE II Award, is a recipient of the Women’s Postdoctoral Career Development Award in Science from the Weizmann Institute of Science and was a recipient of an Outstanding Postdoctoral Women Fellowship from the Israeli Council for Higher Education. Research in the S.P.J. laboratory is supported by Cancer Research UK (CRUK) Discovery Award DRCPGM\100005 and CRUK core grant SEBINT-2024/100003.

Author information

Authors and Affiliations

  1. Institute of Molecular Health Sciences, Department of Biology, ETH Zurich, Zurich, Switzerland

    Eric J. Aird, Tabea Knuesel, Boris Korablev, Lynn Scherpe & Jacob E. Corn

  2. Cryo-EM Knowledge Hub, ETH Zurich, Zurich, Switzerland

    Julius Rabl

  3. Institute of Medical Virology, University of Zurich, Zurich, Switzerland

    Kevin Groen & Benjamin G. Hale

  4. Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK

    Samah W. Awwad & Stephen P. Jackson

  5. Department of Pediatrics, College of Medicine, Kuwait University, Kuwait City, Kuwait

    Waleed Al-Herz

  6. Immunodeficiency Laboratory, Department of Biomedicine, University of Basel, Basel, Switzerland

    Robin Hupfer & Mike Recher

  7. University Center for Immunology, University Hospital, Basel, Switzerland

    Mike Recher

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  1. Eric J. Aird
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Contributions

The project was conceived by E.J.A. and J.E.C. E.J.A., K.G. and S.W.A. designed experiments. E.J.A., K.G., S.W.A., T.K., B.K. and L.S. performed experiments and analysed data. J.R. performed TEM, cryo-EM acquisition and structural refinement. W.A.H., R.H. and M.R. provided patient samples. S.P.J., B.G.H. and J.E.C. provided experimental guidance and material support. E.J.A. and J.E.C. wrote the manuscript with contributions from all other authors.

Corresponding author

Correspondence to Jacob E. Corn.

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Extended data

Extended Data Fig. 1 Validation of SP110 depletion.

a. Gene Set Enrichment Analysis (GSEA) from the genome-wide screen from either the genes depleted (negative normalized enrichment score) or enriched (positive normalized enrichment score) in the ppp-RNA treated condition classified according to the Molecular Signatures Database (MSigDB) hallmark gene lists with a nominative p-value < 0.05 (sample permutation test). b. Validation of SP110 specific knockdown by CRISPRi at the RNA level. Data points are technical replicates. Data is representative of n = 2 independent biological replicates. c. Validation of specific gene knockdown by CRISPRi from Fig. 1d. Data points are technical replicates. Data is representative of n = 2 independent biological replicates. d. Representative flow cytometry gating strategy. Live cells are gated on SSC-A versus FSC-A. Single cells are gated on FSC-H versus FSC-A. Fluorescent positive cells are gated on FSC-A versus fluorescent protein (mCherry, GFP, or BFP). e. Validation of SP110 knockdown at the protein level in the screening cells. NT = non-targeting guide RNA. f and g. Validation of SP110 specific knockout of selected clones in indicated cell backgrounds. c1 = clone 1. h. Competition assay in Jurkat SP110-KO cells ± IFNβ treatment and ± SP110 stable overexpression. Data points represent n = 3 individual biological replicates. i. Western blot assessing SP110 and SP100 expression in primary patient-derived T-lymphoblasts. WT = healthy donor. VODI = VODI patient #1. Bars in b, c and h denote mean ± s.d. Western blots in e, f, g and i were performed 2–3 independent times with similar results. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 2 Elucidating the SP110-SP100 relationship.

a. Confirmation of SP110-KO clones in the RPE1 TP53−/− CRISPRi background. b. Validation of CRISPRi from genes assayed in Fig. 2b. Data points are technical replicates. Data is representative of n = 2 independent biological replicates. c. Competition assay comparing the ratio of SP110-KO versus WT cells on day 7 with the indicated treatments. d. Competition assay in the indicated genotypes stably expressing mCherry-SP100. Bars denote mean. e. Competition assay comparing SP110-KO versus WT cells with titration of IFNβ. Line denotes sigmoidal 4 parameter logistic curve. f. SP100 versus SP110 expression across all cell lines in the Cancer Cell Line Encyclopedia (CCLE) (n = 1474 cell lines) binned by low (log2 < 1 TPM) or high (log2 > 1 TPM) expressors. r = Pearson correlation coefficient. g. Heat map of fraction of tissue origin for each category of cell lines binned in f. h. Comparison of Pearson correlation coefficients between expression of SP110 and every gene (n = 19,143) in the CCLE. i. Heat map of SP140 expression in all CCLE cell lines grouped by low (log2 < 2 TPM) or high (log2 > 2 TPM) expression levels. Bars in b and c denote mean ± s.d. Data points in c-e represent individual biological replicates (n = 2–3). Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 3 SP100 chromatin binding characteristics.

a. Heat map of endogenous SP100 ChIP-Seq peaks in and surrounding gene bodies of normalized length. Each line corresponds to a gene. The color scale indicates the relative intensity of the signal. b. Heat map of rabbit IgG isotype control ChIP-Seq peaks in and surrounding gene bodies of normalized length. Each line corresponds to a gene. The color scale indicates the relative intensity of the signal. c. SP100 peak annotations based on chromosome location. d. Comparison of SP100 peak localization relative to the transcription start site (TSS) in stimulated RPE1 cells compared to HEp-2 cells28. e. Comparison of SP100 binding in HEp-2 cells28, WT RPE1 cells and SP110-KO RPE1 cells at an example locus. The y-axis corresponds to read depth. f. SP100 peak annotations based on location within gene body in RPE1 cells. g. Western blot assessing endogenous SP100 localization in soluble or chromatin-bound fractions. The soluble fraction is marked by GAPDH and the chromatin-bound fraction by histone H2B. Experiment was performed 2 independent times with similar results. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 4 SP expression status does not broadly impact ISG expression.

a-c. MA plots of log2 fold change (FC) in gene expression versus log10 average gene expression when comparing RPE1 (a) SP110;SP100-KO versus SP110-KO, (b) SP110-KO versus WT, and (c) SP110;SP100-KO versus WT cells as measured by RNA-seq with 6 h IFNβ stimulation. Each data point represents a gene. Red and blue data points = adjusted p-value < 0.05. Experiment was performed with biological triplicates. d-f. Gene Set Enrichment Analysis (GSEA) classified according to the Molecular Signatures Database (MSigDB) hallmark gene lists with a nominative p-value < 0.05 for the RPE1 (d) SP110;SP100-KO versus SP110-KO, (e) SP110-KO versus WT, and (f) SP110;SP100-KO versus WT cells comparison under IFNβ stimulated conditions. g. Transcript count (in transcripts per million, TPM) of various ISGs. Each data point represents an individual biological replicate (n = 3). Bars denote mean ± s.d. Source numerical data are available in source data.

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Extended Data Fig. 5 SP110 loss amplifies virus-induced cell death independent of SP100.

a. Validation of SP100 and SP110 expression status in A549 WT pool or isogenic knockout clones. GAPDH served as the loading control. b-d. Time course of A549 cells infected with (b) H5N1-GFP, (c) HSV-1-GFP, or (d) VSV-GFP. Left panels: Relative cell confluence as quantified microscopically by cell area. Right panels: Normalized viral replication as quantified by GFP fluorescence measured per well area. hpi = h post-infection. Data points denote mean ± s.d. (n = 2 independent biological replicates). Source numerical data and unprocessed blots are available in source data.

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Extended Data Fig. 6 SP100 induces mitotic genotoxicity.

a,b. 53BP1 immunofluorescence in RPE1 TP53 +/+ (a) WT and (b) SP110;SP100 dual-KO cells stably expressing H2B-GFP treated with buffer, IFNβ (10 ng/mL), or etoposide (ETO; 25 µM) for 72 h. Scale bar, 20 µm. c. Western blot of the lysates from the indicated RPE1 TP53+/+ genotypes treated with buffer (mock), ETO, IFNβ, or transduced with SP100 cDNA. Note the transduction efficiency was ~30% and cells were not selected for positive integrations. Lysates were probed for phosphorylated KAP1 (S824) and cyclin B1 with GAPDH serving as the loading control. d. Representative fluorescence images of catalytically inactivate GFP-RNaseH1D210N in U2OS cells treated with the indicated siRNAs (NT = non-target). Scale bar, 50 µm. Results are quantified in Fig. 3f. e. Quantification of knockdown efficiencies of the labelled genes with the indicated siRNAs. Each data point represents a technical replicate. Results are representative from n = 2 experiments. f. RT-qPCR of CDKN1A (p21) in untreated and IFNβ treated cells of the indicated genotype. Each data point corresponds to technical replicates from a representative experiment performed n = 2 times. g. Immunofluorescence of human centromere in RPE1 TP53+/+; SP110−/− cells treated with IFNβ. Scale bar, 10 µm. h. Western blots of cellular fractions of G2/M arrested cells to identify SP100 localization. So = soluble/cytoplasmic fraction (marked by GAPDH); Nu = nuclear soluble fraction (marked by ZEB1); Ch = chromatin fraction (marked by histone H2B). Bars in e and f denote mean ± s.d. Images in a, b and g are representative from n = 3 independent biological replicates. Western blots in c and h were performed 2 independent times with similar results. Source numerical data and unprocessed blots are available in source data.

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Extended Data Fig. 7 Multiple cell types exhibit mitotic SP100 retention and genotoxicity.

a. Representative immunofluorescence images of SP100 to quantify micronuclei abundance 72 h post-treatment with buffer or IFNβ in the indicated A549 genotypes. Green arrows denote micronuclei. Scale bar, 20 µm. b. Quantification of micronuclei in A549 cells represented in a. Each data point corresponds to 51–100 cells (n = 3 biological replicates). c. Representative SP100 and PML immunofluorescence in mitotic A549 cells (WT, upper panels; SP110 KO, lower panels). Green arrows denote SP100 retained on mitotic DNA. Scale bar, 10 µm. d. Western blot validation of SP100 and SP110 expression status in U2OS WT pool or isogenic knockout clones. GAPDH served as the loading control. Experiment representative of n = 2 independent biological replicates. e. Representative DAPI-stained images of U2OS cells to quantify micronuclei abundance 72 h post-treatment with buffer or IFNβ in the indicated genotypes. Green arrows denote micronuclei. Scale bar, 20 µm. f. Quantification of micronuclei in U2OS cells represented in e. Each data point corresponds to 51–100 cells (n = 3 biological replicates). g. Representative SP100 and PML immunofluorescence in mitotic U2OS cells. Green arrows denote SP100 retained on mitotic DNA. Scale bar = 10 µm. Experiment performed n = 3 independent times with similar results. Bars in b and f denote mean ± s.d. Each data point in b and f corresponds to an individual biological replicate quantifying 51–100 cells. Statistical significance in b and f was determined with a two-tailed Welch’s t-test. Source numerical data and unprocessed blots are available in source data.

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Extended Data Fig. 8 PML bodies colocalize with SP100-induced DNA damage.

a. Immunofluorescence in RPE1 SP110-KO cells stably expressing H2B-GFP stimulated with IFNβ staining for 53BP1 and PML. Scale bar, 20 µm. b. Individual IFNβ treated SP110-KO cell stained with 53BP1 (magenta) and PML (yellow) with blue profile line drawn across. Scale bar, 10 µm. c. Quantification of intensity across the blue profile line in b. d. Individual etoposide treated SP110-KO cell stained with 53BP1 (magenta) and PML (yellow) with blue profile line drawn across. Scale bar, 10 µm. e. Quantification of intensity across the blue profile line in d. f. Pearson object correlation of colocalization between PML and 53BP1 foci. Each data point represents one cell (n = 50 for each condition). Bars denote the median. A Mann-Whitney U test was used to determine statistical significance. g. Representative SP100 and PML immunofluorescence images in RPE1 SP110 KO cells treated with the indicated conditions. sgPML = CRISPRi knockdown of PML. Green arrows denote SP100 retained on mitotic DNA. Scale bar, 20 µm. h. Competition assay comparing ratio of RPE1 SP110 KO versus WT cells on day 7 with the indicated treatments. i. Competition assay comparing the ratio of RPE1 SP110-KO CRISPRi cells expressing the indicated sgRNA versus SP110 KO CRISPRi cells treated with IFNβ (n = 3 independent biological replicates). j. Knockdown verification of both PML targeting sgRNAs. Data points are technical replicates. Data is representative of n = 2 independent biological replicates. Bars in h-j denote mean ± s.d. Immunofluorescence experiments were performed n = 3 independent times with similar results. Source numerical data are available in source data.

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Extended Data Fig. 9 PML body and SUMOylation are important in SP100-induced toxicity.

a. Representative PML immunofluorescence images used to quantify PML body size and number in Fig. 4a in RPE1 TP53−/− cells. Scale bar, 10 µm. b. Fluorescence images of stably expressed mCherry-SP100 variants. SIM = SUMO interacting motif. K297R = SUMO-deficient SP100. Scale bar, 100 µm. c. Western blot assessing SUMOylation status of SP100 when subjected to pan-SUMOylation inhibitor TAK-981 or ubiquitin inhibitor TAK-243 in RPE1 TP53−/− cells. d. Quantification of SP100 or ISG15 upregulation upon 24 h treatment with IFNβ or TAK-981 in RPE1 TP53−/− cells. Each data point represents a technical replicate. Data is representative of n = 2 independent biological replicates. Bars denote mean ± s.d. Source numerical data and unprocessed blots are available in source data.

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Extended Data Fig. 10 SP110 and SP100 localization and complementation expression.

a. Western blot of stably expressed FLAG-SP110 variants in RPE1 SP110-KO background. b. Western blot of stably expressed FLAG-SP110 CARD variants RPE1 SP110-KO background. The indicated CARD replaces the SP110 CARD in the full-length SP110. c. Fluorescence microscopy imaging of stably expressed SP110-GFP (yellow) constructs in RPE1 WT cells treated with IFNβ. Scale bar, 20 µm. d. Western blot of stably expressed mCherry-HA-SP100 variants in RPE1 SP100-KO background. e. Fluorescence microscopy imaging of stably expressed mCherry-SP100 (yellow) constructs in RPE1 WT cells treated with IFNβ. Scale bar, 20 µm. f. Western blot of stably expressed mCherry-HA-SP100 CARD variants RPE1 SP100-KO background. The indicated CARD replaces the SP100 CARD in SP100A. All experiments were performed 2–3 independent times with similar results. Unprocessed blots are available in source data.

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Aird, E.J., Rabl, J., Knuesel, T. et al. An SP110–SP100 axis is a critical regulator of promyelocytic leukaemia body dynamics and mitotic fidelity. Nat Cell Biol 28, 684–695 (2026). https://doi.org/10.1038/s41556-026-01916-w

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