Abstract

DNA is strictly compartmentalized within the nucleus to prevent autoimmunity1; despite this, cyclic GMP–AMP synthase (cGAS), a cytosolic sensor of double-stranded DNA, is activated in autoinflammatory disorders and by DNA damage2,3,4,5,6. Precisely how cellular DNA gains access to the cytoplasm remains to be determined. Here, we report that cGAS localizes to micronuclei arising from genome instability in a mouse model of monogenic autoinflammation, after exogenous DNA damage and spontaneously in human cancer cells. Such micronuclei occur after mis-segregation of DNA during cell division and consist of chromatin surrounded by its own nuclear membrane. Breakdown of the micronuclear envelope, a process associated with chromothripsis7, leads to rapid accumulation of cGAS, providing a mechanism by which self-DNA becomes exposed to the cytosol. cGAS is activated by chromatin, and consistent with a mitotic origin, micronuclei formation and the proinflammatory response following DNA damage are cell-cycle dependent. By combining live-cell laser microdissection with single cell transcriptomics, we establish that interferon-stimulated gene expression is induced in micronucleated cells. We therefore conclude that micronuclei represent an important source of immunostimulatory DNA. As micronuclei formed from lagging chromosomes also activate this pathway, recognition of micronuclei by cGAS may act as a cell-intrinsic immune surveillance mechanism that detects a range of neoplasia-inducing processes.

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References

  1. 1.

    , & Recognition of endogenous nucleic acids by the innate immune system. Immunity 44, 739–754 (2016)

  2. 2.

    et al. Inflammation-driven carcinogenesis is mediated through STING. Nat. Commun. 5, 5166 (2014)

  3. 3.

    et al. Activation of cyclic GMP-AMP synthase by self-DNA causes autoimmune diseases. Proc. Natl Acad. Sci. USA 112, E5699–E5705 (2015)

  4. 4.

    et al. DNA damage primes the type I interferon system via the cytosolic DNA sensor STING to promote anti-microbial innate immunity. Immunity 42, 332–343 (2015)

  5. 5.

    et al. Ribonuclease H2 mutations induce a cGAS/STING-dependent innate immune response. EMBO J. 35, 831–844 (2016)

  6. 6.

    et al. RNase H2 catalytic core Aicardi-Goutières syndrome-related mutant invokes cGAS–STING innate immune-sensing pathway in mice. J. Exp. Med. 213, 329–336 (2016)

  7. 7.

    et al. Chromothripsis from DNA damage in micronuclei. Nature 522, 179–184 (2015)

  8. 8.

    Activation and regulation of DNA-driven immune responses. Microbiol. Mol. Biol. Rev. 79, 225–241 (2015)

  9. 9.

    et al. Cyclic [G(2′,5′)pA(3′,5′)p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase. Cell 153, 1094–1107 (2013)

  10. 10.

    , , , & Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339, 786–791 (2013)

  11. 11.

    et al. cGAS produces a 2′-5′-linked cyclic dinucleotide second messenger that activates STING. Nature 498, 380–384 (2013)

  12. 12.

    et al. STING contributes to antiglioma immunity via triggering type I IFN signals in the tumor microenvironment. Cancer Immunol. Res. 2, 1199–1208 (2014)

  13. 13.

    et al. Genome-derived cytosolic DNA mediates type I interferon-dependent rejection of B cell lymphoma cells. Cell Reports 11, 460–473 (2015)

  14. 14.

    STING: infection, inflammation and cancer. Nat. Rev. Immunol. 15, 760–770 (2015)

  15. 15.

    , & Regulation and function of the cGAS–STING pathway of cytosolic DNA sensing. Nat. Immunol. 17, 1142–1149 (2016)

  16. 16.

    et al. Enzymatic removal of ribonucleotides from DNA is essential for mammalian genome integrity and development. Cell 149, 1008–1022 (2012)

  17. 17.

    et al. DNA breaks and chromosome pulverization from errors in mitosis. Nature 482, 53–58 (2012)

  18. 18.

    , , & Catastrophic nuclear envelope collapse in cancer cell micronuclei. Cell 154, 47–60 (2013)

  19. 19.

    et al. The cytosolic DNA sensor cGAS forms an oligomeric complex with DNA and undergoes switch-like conformational changes in the activation loop. Cell Reports 6, 421–430 (2014)

  20. 20.

    et al. Cyclic GMP-AMP synthase is activated by double-stranded DNA-induced oligomerization. Immunity 39, 1019–1031 (2013)

  21. 21.

    , , , & Dnase2a deficiency uncovers lysosomal clearance of damaged nuclear DNA via autophagy. Cell Reports 9, 180–192 (2014)

  22. 22.

    et al. Nuclear envelope rupture and repair during cancer cell migration. Science 352, 353–358 (2016)

  23. 23.

    et al. ESCRT III repairs nuclear envelope ruptures during cell migration to limit DNA damage and cell death. Science 352, 359–362 (2016)

  24. 24.

    & Breaching the nuclear envelope in development and disease. J. Cell Biol. 205, 133–141 (2014)

  25. 25.

    , , , & Chromothripsis and kataegis induced by telomere crisis. Cell 163, 1641–1654 (2015)

  26. 26.

    et al. Abnormal nuclear shape in solid tumors reflects mitotic instability. Am. J. Pathol. 158, 199–206 (2001)

  27. 27.

    & Genome instability: a mechanistic view of its causes and consequences. Nat. Rev. Genet. 9, 204–217 (2008)

  28. 28.

    & Chromosomal instability and cancer: a complex relationship with therapeutic potential. J. Clin. Invest. 122, 1138–1143 (2012)

  29. 29.

    & The role of cGAS in innate immunity and beyond. J. Mol. Med. (Berl.) 94, 1085–1093 (2016)

  30. 30.

    et al. STING-dependent cytosolic DNA sensing mediates innate immune recognition of immunogenic tumors. Immunity 41, 830–842 (2014)

  31. 31.

    , , & Deregulation of STING signaling in colorectal carcinoma constrains DNA damage responses and correlates with tumorigenesis. Cell Reports 14, 282–297 (2016)

  32. 32.

    , , & DNA tumor virus oncogenes antagonize the cGAS-STING DNA-sensing pathway. Science 350, 568–571 (2015)

  33. 33.

    et al. Tumor spectrum analysis in p53-mutant mice. Curr. Biol. 4, 1–7 (1994)

  34. 34.

    et al. Viruses transfer the antiviral second messenger cGAMP between cells. Science 349, 1228–1232 (2015)

  35. 35.

    ., ., & in Current Protocols in Immunology Chapter 10, Unit 10 17C (Wiley, 2001)

  36. 36.

    et al. Analysis of the cellular uptake and nuclear delivery of insulin-like growth factor binding protein-3 in human osteosarcoma cells. Int. J. Cancer 130, 1544–1557 (2012)

  37. 37.

    et al. A high-throughput in vivo micronucleus assay for genome instability screening in mice. Nat. Protoc. 10, 205–215 (2015)

  38. 38.

    et al. Chromatin architecture of the human genome: gene-rich domains are enriched in open chromatin fibers. Cell 118, 555–566 (2004)

  39. 39.

    , , & Analysis of active and inactive X chromosome architecture reveals the independent organization of 30 nm and large-scale chromatin structures. Mol. Cell 40, 397–409 (2010)

  40. 40.

    et al. DNA methylation affects nuclear organization, histone modifications, and linker histone binding but not chromatin compaction. J. Cell Biol. 177, 401–411 (2007)

  41. 41.

    , & A method for the in vitro reconstitution of a defined “30 nm” chromatin fibre containing stoichiometric amounts of the linker histone. J. Mol. Biol. 345, 957–968 (2005)

  42. 42.

    et al. Assembly of nucleosomal arrays from recombinant core histones and nucleosome positioning DNA. J. Vis. Exp. 79, 50354 (2013)

  43. 43.

    et al. Full-length RNA-seq from single cells using Smart-seq2. Nat. Protoc. 9, 171–181 (2014)

  44. 44.

    et al. Proliferation drives aging-related functional decline in a subpopulation of the hematopoietic stem cell compartment. Cell Reports 19, 1503–1511 (2017)

  45. 45.

    et al. The External RNA Controls Consortium: a progress report. Nat. Methods 2, 731–734 (2005)

  46. 46.

    . et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013)

  47. 47.

    , & The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Res. 41, e108 (2013)

  48. 48.

    , , & Scater: pre-processing, quality control, normalization and visualization of single-cell RNA-seq data in R. Bioinformatics 33, 1179–1186 (2017)

  49. 49.

    , & Bayesian approach to single-cell differential expression analysis. Nat. Methods 11, 740–742 (2014)

  50. 50.

    et al. Central role of ULK1 in type I interferon signaling. Cell Reports 11, 605–617 (2015)

  51. 51.

    & Matrix2png: a utility for visualizing matrix data. Bioinformatics 19, 295–296 (2003)

  52. 52.

    et al. Pan-viral specificity of IFN-induced genes reveals new roles for cGAS in innate immunity. Nature 505, 691–695 (2014)

  53. 53.

    & When repair meets chromatin. First in series on chromatin dynamics. EMBO Rep. 3, 28–33 (2002)

  54. 54.

    & The DNA damage response: making it safe to play with knives. Mol. Cell 40, 179–204 (2010)

  55. 55.

    et al. Coordination of dual incision and repair synthesis in human nucleotide excision repair. EMBO J. 28, 1111–1120 (2009)

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Acknowledgements

We thank J. Rehwinkel, N. Hastie, I. Adams, D. Papadopoulos, C. Ponting and W. Bickmore for discussions and comments on the manuscript; A. Wood, G. Taylor, D. Jamieson, H. Kato, P. Gao and B. Ramsahoye for technical advice and assistance; K. S. Mackenzie, P. Vagnarelli, H. Kato and T. Fujita for sharing reagents; R. Greenberg for discussion of unpublished data; the IGMM Transgenic, Sequencing, Imaging and Flow Cytometry facilities; and C. Nicol and A. Colley for graphics assistance. This work was funded by the Medical Research Council HGU core grant (MRC, U127580972) (A.P.J., N.G.), Newlife the Charity for Disabled Children (K.J.M.), the Wellcome Trust–University of Edinburgh Institutional Strategic Support Fund 2 (K.J.M.), MRC Discovery Award (MC_PC_15075, T.C.), an International Early Career Scientist grant from the Howard Hughes Medical Institute (M.N.), an EMBO Long-Term Fellowship (ALTF 7-2015), the European Commission FP7 (Marie Curie Actions, LTFCOFUND2013, GA-2013-609409) and the Swiss National Science Foundation (P2ZHP3_158709) (O.M.).

Author information

Author notes

    • Karen J. Mackenzie
    •  & Paula Carroll

    These authors contributed equally to this work.

Affiliations

  1. MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh, UK

    • Karen J. Mackenzie
    • , Paula Carroll
    • , Carol-Anne Martin
    • , Olga Murina
    • , Adeline Fluteau
    • , Daniel J. Simpson
    • , Nelly Olova
    • , Hannah Sutcliffe
    • , Jacqueline K. Rainger
    • , Andrea Leitch
    • , Ruby T. Osborn
    • , Ann P. Wheeler
    • , Nick Gilbert
    • , Tamir Chandra
    • , Martin A. M. Reijns
    •  & Andrew P. Jackson
  2. Laboratory of Protein Structure, International Institute of Molecular and Cell Biology, Warsaw, Poland

    • Marcin Nowotny

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Contributions

K.J.M., P.C., M.A.M.R., C.-A.M., O.M., A.F., D.J.S., N.O., H.S., J.K.R., A.L., R.T.O., A.P.W., M.N. and N.G. performed experiments and analysed data. K.J.M., N.G., T.C., M.A.M.R. and A.P.J. planned the project and supervised experiments. M.A.M.R., K.J.M. and A.P.J. wrote the manuscript.

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The authors declare no competing financial interests.

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Correspondence to Martin A. M. Reijns or Andrew P. Jackson.

Reviewer Information Nature thanks N. Gekara and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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    This file contains Supplementary Text, additional references, Supplementary table 1 and Supplementary Figure 1.

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Videos

  1. 1.

    Live imaging of Rnaseh2b^-/- MEFs transiently expressing RFP-H2B

    Micronuclei form from lagging DNA and chromatin bridges occurring during mitosis in RNaseH2 deficient cells.

  2. 2.

    cGAS enters micronuclei after envelope rupture

    Live imaging of U2OS cells expressing mCherry-NLS and GFP-cGAS. DNA visualised with Hoechst.

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https://doi.org/10.1038/nature23449

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