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Innate immune sensing of cytosolic chromatin fragments through cGAS promotes senescence

Abstract

Cellular senescence is triggered by various distinct stresses and characterized by a permanent cell cycle arrest. Senescent cells secrete a variety of inflammatory factors, collectively referred to as the senescence-associated secretory phenotype (SASP). The mechanism(s) underlying the regulation of the SASP remains incompletely understood. Here we define a role for innate DNA sensing in the regulation of senescence and the SASP. We find that cyclic GMP-AMP synthase (cGAS) recognizes cytosolic chromatin fragments in senescent cells. The activation of cGAS, in turn, triggers the production of SASP factors via stimulator of interferon genes (STING), thereby promoting paracrine senescence. We demonstrate that diverse stimuli of cellular senescence engage the cGAS–STING pathway in vitro and we show cGAS-dependent regulation of senescence following irradiation and oncogene activation in vivo. Our findings provide insights into the mechanisms underlying cellular senescence by establishing the cGAS–STING pathway as a crucial regulator of senescence and the SASP.

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Figure 1: Absence of cGAS attenuates the senescence response.
Figure 2: cGAS facilitates oxidative-stress-induced senescence.
Figure 3: cGAS regulates the senescence-associated secretory phenotype.
Figure 4: cGAS interacts with chromatin fragments in senescent cells.
Figure 5: Engagement of cGAS is a common feature of multiple senescence triggers.
Figure 6: cGAS contributes to cellular senescence in vivo.

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Acknowledgements

We thank N. Jordan for the preparation of viral particles, and B. Mangeat from the Gene Expression Core Facility and E. Cabello from Bioinformatics and Biostatistics Core Facility the for assistance in the RNA sequencing study. This research was supported by grants from the SNF (BSSGI0-155984, 31003A_159836), the Gebert Rüf Foundation (GRS-059_14) and the Else Kröner-Fresenius Stiftung (2014_A250) to A.A. In addition, this work was supported by the German Research Foundation (DFG; grants FOR2314 (L.Z.) and SFB685 (L.Z.)), the Gottfried Wilhelm Leibniz Program (L.Z.), the European Research Council (projects ‘CholangioConcept’ (L.Z.), ‘Heptromic’ (L.Z.)), the German Ministry for Education and Research (BMBF) (eMed (Multiscale HCC)) (L.Z.), the German Universities Excellence Initiative (third funding line: ‘future concept’) (L.Z.), the German Center for Translational Cancer Research (DKTK) (L.Z.), and the German-Israeli Cooperation in Cancer Research (DKFZ-MOST) (L.Z.). B.G. is supported by a long-term EMBO fellowship (ALTF 203-2016). J.R. is supported by grants from the UK Medical Research Council [MRC core funding of the MRC Human Immunology Unit] and the Wellcome Trust (grant number 100954).

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S.G., B.G., M.F.G., K.W., T.-W.K., L.Z. and A.A. designed experiments and analysed the data. S.G., B.G., M.F.G., K.W., T.-W.K. and A.A. performed experiments. N.A.S. assisted in the establishment of methods. A.B. and J.R. provided reagents, cells and mice. S.G., B.G. and A.A. wrote the manuscript with help from all authors. A.A. supervised the project.

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Correspondence to Andrea Ablasser.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Absence of cGAS attenuates the establishment of senescence.

(a) Heat maps of RNA-seq analysis of WT MEFs and cGAS KO MEFs (Day 33, 36, 42). For each genotype one sample per day; MEFs collected at Day 33, 36, 42. Genes in cGAS KO MEFs exhibiting statistically significant (n = 3 biological replicates; P < 0.05; student t-test) twofold or greater increases relative to WT MEFs are shown. E2F target genes are highlighted in green1. (b) Proliferation curves of primary WT MEFs, cGAS KO MEFs and STING KO MEFs cultured under 20% O2. (c) WT MEFs, cGAS KO MEFs and STING KO MEFs were cultured under 20% O2 for 27 days and expression of p16Ink4a was determined by immunoblotting. (d) WT MEFs, cGAS KO MEFs or STING KO MEFs were harvested after 3 weeks of culture and expression of depicted genes was measured via RT-qPCR. One representative experiment out of 2 independent experiments (b,c) independent experiments is shown. Data shown in (d) are from n = 2 independent experiments with the column representing the mean. Source data are available in Supplementary Table 1. Unprocessed original blots are shown in Supplementary Fig. 7.

Supplementary Figure 2 cGAS-dependent regulation of cytokines.

(a,b) Conditioned medium (CM) was collected from WT MEFs or cGAS KO MEFs exposed to 40% O2 for 7 days. Percentages of proliferating cGAS KO MEFs were assessed by BrdU incorporation assay (a) or induction of SA-β-Gal activity was determined by microscopy (b). (c) Cytokine profile of the CM from WI-38 cells exposed to 40% O2 for 7 days and treated with a control siRNA (si Control) or a cGAS-targeting siRNA (si cGAS #1) on day 3. Numbers indicate specific cytokines or chemokines and are highlighted in red rectangles. (d) WT MEFs, cGAS KO MEFs or STING KO MEFs were incubated in 40% O2 for 7 days and Ifi44 expression was quantified by RT-qPCR. (e) WI-38 cells were exposed to 40% O2 treatment for 9 days. On day 7 cells were transfected with a non-targeting control siRNA (si Control) or with siRNAs against cGAS (si cGAS #1, si cGAS #2). Expression of IFI44 was determined by RT-qPCR. (f) WT MEFs and cGAS KO MEFs were treated with recombinant IFN-β as indicated every day and after 14 days expression levels of p16Ink4a were assessed by immunoblotting. (g) cGAS KO MEFs were treated with recombinant IFN-β as indicated and after 14 days expression levels of depicted genes was assessed by RT-qPCR. (h) WT MEFs and IFNAR KO MEFs were exposed to 40% O2 for 7 days. Expression of mRNA levels of depicted genes was assessed by RT-qPCR. One representative experiment out of 2 independent experiments (c,f), mean of n = 2 (a) or mean and s.d. of n = 3 (e) independent experiments are shown. Data shown in (b,d,g,h) are from n = 2 independent experiments with the column representing the mean. Source data are available in Supplementary Table 1. Unprocessed original blots are shown in Supplementary Fig. 7.

Supplementary Figure 3 Endogenous cGAS binds to chromatin at the nuclear-cytosolic border.

WI-38 cells were cultured under 40% O2 for 9 days and stained for cGAS (green) and DAPI (grey). Images are representative from 3 independent experiments. Scale bar: 20 μm.

Supplementary Figure 4 cGAS is a common regulator of senescence.

(a) MEFs with the indicated genotypes were exposed to 12 Gy ionizing irradiation or stimulated with Palbociclib. After 7 days cells were stained for SA-β-Gal activity. (b,c) WI-38 cells (left panel) were exposed to 12 Gy ionizing irradiation or WI-38 ER:RAS cells (right panel) were treated with 4-OHT (500 nM) for 7 days. At day 3 cells were transfected with a non-targeting control siRNA (si Control) or with siRNAs against cGAS (si cGAS #1, si cGAS #2). At day 7 SA-β-Gal activity was determined by FACS (b) or expression of CDKN2A was quantified by RT-qPCR (c). (d) WI-38 cells (left panel) were exposed to 12 Gy ionizing irradiation or WI-38 ER:RAS cells (right panel) were treated with 4-OHT (500 nM) for 7 days. Cells were stained for cGAS (green) and DAPI (grey) and analysed by fluorescence microscopy. One representative experiment out of two (b,d) independent experiments is shown. Data shown in (a) and (c) are from n = 2 independent experiments with the column representing the mean. Scale bar, 20 μm. Source data are available in Supplementary Table 1.

Supplementary Figure 5 Irradiation-induced activation of cGAS and STING in vivo.

(a) Depicted mRNA levels of lungs from WT, cGAS KO and STING KO mice 24 h after irradiation are shown. Non-irradiated mice served as controls. (b) Quantification of Nras-positive cells 12 days after intrahepatic delivery of NrasG12V or NrasG12V/D38A into WT or cGAS KO mice are shown (n = 8–10 per group). Mean and s.d. of n = 3 (a) or n = 8 (WT G12V/D38A; cGAS KO G12V/D38A) or n = 10 (WT G12V; cGAS KO G12V) (b) mice are shown. P values were calculated by two-way ANOVA (P < 0.01, P < 0.001, ns = not significant). Source data are available in Supplementary Table 1.

Supplementary Figure 6 Model of cGAS-mediated propagation of senescence.

Within senescent cells cGAS senses herniated chromatin, which triggers the production of SASP factors. Cytokine signalling in turn induces the expression of cell-cycle inhibitors, including p16Ink4a, which enforces cell-cycle arrest and senescence in an autocrine and paracrine manner.

Supplementary Figure 7 Uncropped images from western-blots.

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Glück, S., Guey, B., Gulen, M. et al. Innate immune sensing of cytosolic chromatin fragments through cGAS promotes senescence. Nat Cell Biol 19, 1061–1070 (2017). https://doi.org/10.1038/ncb3586

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