cGAS surveillance of micronuclei links genome instability to innate immunity

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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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Figure 1: cGAS localizes to micronuclei resulting from endogenous or exogenous DNA damage.
Figure 2: cGAS localizes to micronuclei upon nuclear envelope rupture.
Figure 3: Continuous and chromatinized DNA activate cGAS.
Figure 4: Innate immune activation after radiation-induced DNA damage is cell-cycle dependent.
Figure 5: ISG upregulation occurs specifically in micronucleated cells following DNA damage.

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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

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.

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

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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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Extended data figures and tables

Extended Data Figure 1 Micronuclei form in RNase H2 deficiency, with cGAS localizing to these structures and inducing an ISG response.

a, Still images of live imaging in Rnaseh2b−/− MEFs, time in minutes; t = 0, prophase. Lagging DNA (blue arrowheads) and DNA bridges (orange arrowhead) at anaphase can result in interphase micronuclei (green arrowheads). b, Chromatin bridges and lagging chromosomal DNA (arrows) occur in Rnaseh2b−/− MEFs. Representative fixed cell images. c, d, Erythrocyte micronuclei assay37. c, Representative flow cytometry plot with quadrants containing reticulocytes and micronucleated normochromatic erythrocytes indicated. d, Rnaseh2bA174T/A174T mice have a significantly increased frequency of micronucleated erythrocytes. Mean ± s.e.m., n = 3 mice per group; two-tailed t-test, **P < 0.01. e, f, eGFP does not accumulate in micronuclei, whereas the majority of micronuclei show strong accumulation of GFP–cGAS. e, Representative image of micronucleus-containing Rnaseh2b−/− MEFs stably expressing eGFP. f, Quantification of GFP-positive micronuclei for GFP–cGAS-expressing and GFP-expressing Rnaseh2b−/− MEF lines. Mean ± s.e.m., n = 4 experiments (≥500 cells counted per experiment). Scale bars, 10 μm. g, Increased levels of ISG transcripts (Ifit1, Ifit3, Isg15, Cxcl10 and Oas1a) were detected in C57BL/6J (Trp53+/+) MEFs 48 h after irradiation. Transcript levels were normalized to Hprt. Mean ± s.e.m., n = 3 independent experiments. One-way ANOVA, 2 degrees of freedom, *P < 0.05. h, Endogenous cytosolic cGAS accumulates in micronuclei in U2OS cells. Representative images of cGAS distribution in cells with or without micronuclei. Images taken using different exposure times (200 vs 700 ms) to visualize weaker cytosolic cGAS signal. i, j, Verification of anti-cGAS antibody specificity in human cells. i, The percentage of cGAS-positive micronuclei, using anti-cGAS immunofluorescence, was determined microscopically after cGAS or luciferase siRNA knockdown. Mean ± s.e.m., n = 2 experiments (500 cells counted per experiment); two-tailed t-test. While several commercial cGAS antibodies were assessed, specific detection of mouse cGAS by immunofluorescence was not possible with these reagents (data not shown). j, Immunoblot after siRNA knockdown of cGAS in U2OS cells. siRNA targeting luciferase (siLUC) was used as a negative control. Probing with anti-actin antibody shows equal loading. Source data

Extended Data Figure 2 cGAS localization is associated with DNA damage in micronuclei.

γH2AX foci in micronuclei correlate with GFP–cGAS localization in Rnaseh2b−/− MEFs and endogenous cGAS localization in U2OS cells. a, Representative immunofluorescence images: γH2AX, red; cGAS, green. b, Percentage of γH2AX-stained micronuclei (γH2AX +ve), either co-stained with cGAS (cGAS +ve), or in which cGAS was not detected (cGAS −ve). Rnaseh2b−/− MEFs; ≥500 cells counted per experiment. c, Quantification for U2OS cells, ≥250 micronuclei counted per experiment. Mean ± s.e.m., n = 3 experiments; *P < 0.05, ***P < 0.001, two-tailed t-test. While our biochemical studies demonstrate that unbroken DNA and chromatin are sufficient to activate cGAS (Fig. 3, Extended Data Figs 4, 5), the increased accessibility of DNA after damage53 could further assist cGAS binding and activation. Scale bars, 10 μm.

Extended Data Figure 3 cGAS localizes to micronuclei upon nuclear envelope rupture.

a, b, cGAS localization to micronuclei in U2OS cells inversely correlates with localization of mCherry–NLS, which is present only in micronuclei with an intact nuclear envelope. a, Representative images of cells containing micronuclei with disrupted or intact nuclear envelopes. b, Percentage of intact and disrupted cGAS-positive micronuclei. Mean ± s.e.m., n = 3 independent experiments (≥250 micronuclei counted per experiment). NLS +ve and NLS −ve, mCherry–NLS present in or absent from micronuclei, respectively. cGAS +ve, GFP–cGAS present in micronuclei. ***P < 0.001, two-tailed t-test. c, Single-channel image for representative stills shown in Fig. 2d from live imaging of U2OS cells expressing mCherry–NLS and GFP–cGAS. DNA visualized with Hoechst stain. Time (min) relative to loss of mCherry–NLS from micronucleus (t = 0, micronuclear membrane rupture). Arrows indicate micronuclei undergoing rupture. Scale bars, 10 μm.

Extended Data Figure 4 cGAS is activated by circular plasmid DNA.

a, Plasmid DNA (SC, supercoiled; OC, open circle; linear and fragmented) separated by agarose gel electrophoresis. pBluescript II SK(+) supercoiled plasmid DNA was treated with Nt.BspQI nicking endonuclease to generate open circle DNA; with EcoRI to generate a single 3-kb linear fragment; or with HpaII to generate 13 fragments between 710 and 26 bp in size. b, Supercoiled, open circle, linear and fragmented pBluescript (pBS) DNA all activate recombinant cGAS to produce cGAMP. Representative images shown. Quantification of n = 3 experiments shown in Fig. 3b. c, Plasmid DNA induces cGAS-dependent CCL5 production in MEFs. Wild-type and cGAS−/− (Mb21d1−/−) MEFs were transfected with 400 ng HT-DNA or supercoiled or linearized pBluescript, and CCL5 production after 24 h measured by ELISA. Mean ± s.e.m., n = 3 independent experiments.

Extended Data Figure 5 cGAS is activated by chromatin.

a, Agarose gel of micrococcal nuclease (MNase)-digested synthetic chromatin assembled onto a 601 DNA template indicates that it has a regular nucleosomal structure. b, Chromatin and DNA bind recombinant cGAS; DNA in wells could be the result of near charge neutrality of cGAS–DNA complexes or previously reported cGAS oligomerization. Chromatin is stable under cGAS assay conditions, remaining intact during incubation in cGAS reaction buffer, as evidenced by the bandshift compared to naked DNA. c, Representative TLC image demonstrating cGAMP generation by recombinant cGAS in the presence of chromatin. d, MNase treatment confirms a nucleosomal ladder pattern for chromatin isolated from mouse NIH3T3 cells. e, cGAS binds chromatin, and cellular chromatin is stable under cGAS assay conditions. f, g, Cellular chromatin activates recombinant cGAS, but at a slower rate than the same amount of deproteinized DNA. Representative images shown. Graphs shows quantification from n = 3 independent experiments, mean ± s.d. Reduced cGAS activation in vitro by chromatin isolated from cells is expected due to the presence of linker histones in addition to the nucleosomal core histones, which has been shown to bind part of the linker DNA, reducing the available sites for cGAS binding, and the use of MNase during the isolation of cellular chromatin. Whereas MNase treatment is needed to fragment the chromatin to allow its purification, it will preferentially cleave accessible non-protein-bound portions, which will further reduce the available sites to which cGAS can bind in the final chromatin preparation. However, such nucleosome-free regions are more likely to allow efficient binding and activation of cGAS in vivo.

Extended Data Figure 6 ISG induction by ionizing radiation is abrogated in non-cycling cells.

a, Experimental setup: to arrest cells in G0, serum was withdrawn 24 h before transfection with HT-DNA, and supernatant harvested 24 h later. b, CCL5 production in response to transfected HT-DNA was equivalent in cycling and serum starved MEFs. Mean ± s.e.m., n = 2 independent experiments. c, Schematic of experimental protocol. d, Cycling and G0-arrested cells exhibit the same level of DNA damage as measured by formation of γH2AX foci. Representative images; scale bar, 10 μm. Quantifications shown in Fig. 4d. e, There is no significant increase in ISG transcripts Ifit1, Ifit3, Isg15, Cxcl10 and Oas1a for cells arrested in G0 after serum starvation (experimental setup as in c). Transcript levels were normalized to Hprt. Mean ± s.e.m. One-way ANOVA, 2 degrees of freedom, n = 3 independent experiments; NS, not significant. Compare to Extended Data Fig. 1g, showing data for matched cycling cells assessed concurrently.

Extended Data Figure 7 Micronuclear DNA is sufficient to account for the radiation-induced cytokine response.

a, b, Measurement of micronuclear DNA content. a, Representative images. DAPI-stained primary nuclei and micronuclei surrounded by dotted lines. Scale bar, 10 μm. b, Quantification of surface areas of micronuclei and primary nuclei 48 h after 1 Gy irradiation. Micronuclear surface area per cell 9.72 ± 1.46 μm2, primary nucleus surface area 303 ± 21 μm2. Horizontal line and error bars: mean ± s.e.m., n = 54 cells. Hence, micronuclear content is ~3.2% of the total MEF genome after irradiation, equating to 190 Mbp of DNA. This corresponds to a total of 8.1 ng of micronuclear DNA in 105 cells after 1 Gy irradiation (105 diploid mouse cells contain a total of 650 ng of genomic DNA, with 39% of cells containing micronuclei, Fig. 1h). c, CCL5 response of wild-type C57/BL6 MEFs to ionizing radiation plotted in pg per 105 cells. Reanalysis of this dataset (first depicted in Fig. 4b) confirms that the prior statistical analysis is robust to data normalization on the basis of cell counts at assay endpoint. 1 Gy of irradiation in cycling MEFs results in 38 ± 5 pg (mean ± s.d.) of CCL5 per 105 cells. **P < 0.01, two-tailed t-test; NS, not significant. d, Dose–response curve of secreted CCL5 in wild-type C57BL/6 (Trp53+/+) MEFs transfected with serial dilutions of transfected HT-DNA. Therefore, around 4 ng of transfected DNA resulted in a similar level of cytokine production to c. Mean and 95% confidence interval indicated by black and grey dashed lines, respectively. Given the similarity of the two estimates, within the same order of magnitude, micronuclear DNA is likely to be sufficient to account for the immune response observed. Conversely, ionizing radiation would not be expected to generate this quantity of small DNA fragments as 1 Gy irradiation generates ~40 double strand breaks (DSBs)54, and ~1,000 base lesions and single-stranded breaks. DSBs will have an average separation of 150 Mbp, and will therefore be too widely spaced to directly generate small dsDNA fragments. Repair of DNA lesions can generate small single-stranded DNA (ssDNA) fragments through endonuclease activity. The best characterized fragments are those generated by nucleotide excision repair, where endonucleolytic cleavage yields 24–32-nucleotide ssDNA fragments55. As such these are not an ideal substrate for cGAS activation, and 5 million such lesions per cell would have to be generated to produce 4 ng of cytosolic DNA in 105 cells. Hence, on the basis of our understanding of the current literature, such DNA fragments are likely to be generated at a level that is orders of magnitude lower than that of micronuclear DNA after radiation-induced damage.

Extended Data Figure 8 Induction of micronuclei originating from lagging chromosomes leads to a proinflammatory response, but not increased DNA damage in the primary nucleus.

a, Model: micronucleus formation after nocodazole treatment. b, Schematic of experimental protocol. c, d, Percentage of micronucleated cells following nocodazole (noc) treatment of Trp53−/− MEFs (c) or U2OS cells (d). Mean ± s.e.m., n = 5 experiments for Trp53−/− MEFs, n = 3 for U2OS cells. e, Percentage of U2OS cells with cGAS-positive micronuclei following nocodazole treatment. Mean ± s.e.m., n = 3 experiments. ce, ≥500 cells counted per experiment. f, CCL5 secretion following nocodazole treatment of Trp53−/− MEFs. Mean ± s.e.m. of n = 5 experiments. **P < 0.01, ***P < 0.001, two-tailed t-test. gi, Increased CCL5 production after nocodazole release is observed after 16 h and not associated with increased DNA damage in the primary nucleus. g, Experimental setup: Trp53−/− MEFs were arrested with nocodazole for 6 h and mitotic cells harvested by mitotic shake-off and re-plated in fresh medium with nocodazole omitted. Supernatants and cells were then collected at indicated time points after growth in medium. h, Increased CCL5 production was observed from 16 h after release from nocodazole block. Technical duplicate, mean ± s.d. Noc (−), asynchronously grown, plated at the same time as mitotic shake-off Noc (+) cells, arrested with nocodazole. i, No increase in the number of γH2AX foci in the primary nucleus was observed after release from nocodazole block. n ≥ 100 cells counted per condition. j, k, CCL5 response to interferon stimulatory DNA (ISD) is absent in U2OS cells (j) but present in MEFs (k). CCL5 measured by ELISA 8 h after transfection with ISD. n = 2 experiments for U2OS cells, n = 1 experiment for MEFs.

Extended Data Figure 9 Single-cell RNA sequencing quality control and microscopy images of individual LCM-captured cells.

a, Total gene feature counts (reads mapping to a protein coding gene) vs ERCC (RNA spike-in) percentage of total counts per cell. Cells with ERCC percentage counts >10% and/or with feature counts <2,000 were rejected, indicated by red shaded regions. b, Summary statistics for 21 micronucleated (MN+) cells and 14 non-micronucleated (MN−) cells that passed quality control. c, d, Microcopy images of cells captured by LCM that passed quality control after single-cell RNA sequencing. Fourteen live cells without micronuclei (c) and 21 live cells with micronuclei (d) were isolated from the same culture dish using LCM and used for single-cell mRNA sequencing. DNA was stained with picogreen dsDNA stain. Cells shown are those that passed quality control; numbers indicate the order in which cells were captured. Scale bars, 10 μm.

Extended Data Figure 10 cGAS localizes to telophase chromosomes and DNA bridges.

a, Endogenous cGAS was stained by immunofluorescence of U2OS cells in mitosis, showing a diffuse staining pattern without accumulation at the DAPI-stained condensed chromosomes at metaphase. Two representative images shown. During anaphase (and telophase), cGAS staining can be seen on DNA in some cells. Overexpressed GFP–cGAS also localizes more widely to mitotic DNA in U2OS cells and MEFs (data not shown). b, Quantification of cGAS staining during mitosis, by stage. c, Rnaseh2b−/− Trp53−/− MEFs stably expressing GFP–cGAS show localization of cGAS at DNA bridges (orange arrowheads). d, Endogenous cGAS can also be seen to localize to DNA bridges that occasionally occur in U2OS cells. cGAS also localized to micronuclei in the same cells (green arrowheads). Interphase chromatin bridges with cGAS bound in Rnaseh2b−/− Trp53−/− MEFs 0.08% of n = 1,223 cells; U2OS cells 0.06% of n = 1,632 cells. Scale bars, 10 μm.

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Reporting Summary (PDF 80 kb)

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

Micronuclei form from lagging DNA and chromatin bridges occurring during mitosis in RNaseH2 deficient cells. (AVI 149 kb)

cGAS enters micronuclei after envelope rupture

Live imaging of U2OS cells expressing mCherry-NLS and GFP-cGAS. DNA visualised with Hoechst. (AVI 125 kb)

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Mackenzie, K., Carroll, P., Martin, C. et al. cGAS surveillance of micronuclei links genome instability to innate immunity. Nature 548, 461–465 (2017) doi:10.1038/nature23449

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