Abstract
Activation of cyclic GMP-AMP synthase (cGAS) through sensing cytosolic double stranded DNA (dsDNA) plays a pivotal role in innate immunity against exogenous infection as well as cellular regulation under stress. Aberrant activation of cGAS induced by self-DNA is related to autoimmune diseases. cGAS accumulates at chromosomes during mitosis or spontaneously in the nucleus. Binding of cGAS to the nucleosome competitively attenuates the dsDNA-mediated cGAS activation, but the molecular mechanism of the attenuation is still poorly understood. Here, we report two cryo-electron microscopy structures of cGAS–nucleosome complexes. The structures reveal that cGAS interacts with the nucleosome as a monomer, forming 1:1 and 2:2 complexes, respectively. cGAS contacts the nucleosomal acidic patch formed by the H2A–H2B heterodimer through the dsDNA-binding site B in both complexes, and could interact with the DNA from the other symmetrically placed nucleosome via the dsDNA-binding site C in the 2:2 complex. The bound nucleosome inhibits the activation of cGAS through blocking the interaction of cGAS with ligand dsDNA and disrupting cGAS dimerization. R236A or R255A mutation of cGAS impairs the binding between cGAS and the nucleosome, and largely relieves the nucleosome-mediated inhibition of cGAS activity. Our study provides structural insights into the inhibition of cGAS activity by the nucleosome, and advances the understanding of the mechanism by which hosts avoid the autoimmune attack caused by cGAS.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
Data availability
The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession numbers: EMDB-30339 (monomer-overall and monomer-subtract), EMDB-30340 (dimer-overall and dimer-subtract). The atomic coordinates have been deposited in the Protein Data Bank (PDB) under accession codes: 7CCQ (monomer) and 7CCR (dimer).
References
Wu, J. & Chen, Z. J. Innate immune sensing and signaling of cytosolic nucleic acids. Annu. Rev. Immunol. 32, 461–488 (2014).
Sun, L., Wu, J., Du, F., Chen, X. & Chen, Z. J. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339, 786–791 (2013).
Li, X. D. et al. Pivotal roles of cGAS-cGAMP signaling in antiviral defense and immune adjuvant effects. Science 341, 1390–1394 (2013).
Wu, J. et al. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 339, 826–830 (2013).
Cai, X., Chiu, Y. H. & Chen, Z. J. The cGAS-cGAMP-STING pathway of cytosolic DNA sensing and signaling. Mol. Cell 54, 289–296 (2014).
Ablasser, A. et al. cGAS produces a 2’-5’-linked cyclic dinucleotide second messenger that activates STING. Nature 498, 380–384 (2013).
Gao, P. 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).
Ishikawa, H. & Barber, G. N. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature 455, 674–678 (2008).
Gao, P. et al. Structure-function analysis of STING activation by c[G(2’,5’)pA(3’,5’)p] and targeting by antiviral DMXAA. Cell 154, 748–762 (2013).
Tanaka, Y. & Chen, Z. J. STING specifies IRF3 phosphorylation by TBK1 in the cytosolic DNA signaling pathway. Sci. Signal. 5, ra20 (2012).
Fitzgerald, K. A. et al. IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway. Nat. Immunol. 4, 491–496 (2003).
Crow, Y. J. & Manel, N. Aicardi-Goutières syndrome and the type I interferonopathies. Nat. Rev. Immunol. 15, 429–440 (2015).
Gao, D. et al. Activation of cyclic GMP-AMP synthase by self-DNA causes autoimmune diseases. Proc. Natl. Acad. Sci. USA 112, E5699–E5705 (2015).
Gray, E. E., Treuting, P. M., Woodward, J. J. & Stetson, D. B. Cutting edge: cGAS is required for lethal autoimmune disease in the Trex1-deficient mouse model of Aicardi-Goutières syndrome. J. Immunol. 195, 1939–1943 (2015).
Xiao, N. et al. cGAS activation causes lupus-like autoimmune disorders in a TREX1 mutant mouse model. J. Autoimmun. 100, 84–94 (2019).
Civril, F. et al. Structural mechanism of cytosolic DNA sensing by cGAS. Nature 498, 332–337 (2013).
Li, X. et al. Cyclic GMP-AMP synthase is activated by double-stranded DNA-induced oligomerization. Immunity 39, 1019–1031 (2013).
Xie, W. et al. Human cGAS catalytic domain has an additional DNA-binding interface that enhances enzymatic activity and liquid-phase condensation. Proc. Natl. Acad. Sci. USA 116, 11946–11955 (2019).
Yang, H., Wang, H., Ren, J., Chen, Q. & Chen, Z. J. cGAS is essential for cellular senescence. Proc. Natl. Acad. Sci. USA 114, E4612–E4620 (2017).
Harding, S. M. et al. Mitotic progression following DNA damage enables pattern recognition within micronuclei. Nature 548, 466–470 (2017).
Gentili, M. et al. The N-terminal domain of cGAS determines preferential association with centromeric DNA and innate immune activation in the nucleus. Cell Rep. 26, 2377–2393.e13 (2019).
Volkman, H. E., Cambier, S., Gray, E. E. & Stetson, D. B. Tight nuclear tethering of cGAS is essential for preventing autoreactivity. Elife 8, e47491 (2019).
Jiang, H., Xue, X. & Panda, S. Chromatin-bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death. EMBO J. 38, e102718 (2019).
Liu, H. et al. Nuclear cGAS suppresses DNA repair and promotes tumorigenesis. Nature 563, 131–136 (2018).
Zierhut, C. et al. The cytoplasmic DNA sensor cGAS promotes mitotic cell death. Cell 178, 302–315.e23 (2019).
Kato, K. et al. Structural and functional analyses of DNA-sensing and immune activation by human cGAS. PLoS One 8, e76983 (2013).
Kranzusch, P. J., Lee, A. S., Berger, J. M. & Doudna, J. A. Structure of human cGAS reveals a conserved family of second-messenger enzymes in innate immunity. Cell Rep. 3, 1362–1368 (2013).
Zhang, X. et al. The cytosolic DNA sensor cGAS forms an oligomeric complex with DNA and undergoes switch-like conformational changes in the activation loop. Cell Rep. 6, 421–430 (2014).
Zhou, W. et al. Structure of the human cGAS-DNA complex reveals enhanced control of immune surveillance. Cell 174, 300–311.e11 (2018).
Andreeva, L. et al. cGAS senses long and HMGB/TFAM-bound U-turn DNA by forming protein-DNA ladders. Nature 549, 394–398 (2017).
Du, M. & Chen, Z. J. DNA-induced liquid phase condensation of cGAS activates innate immune signaling. Science 361, 704–709 (2018).
Tao, J., Zhang, X. W. & Jin, J. Nonspecific DNA binding of cGAS N terminus promotes cGAS activation. J. Immunol. 198, 3627–3636 (2017).
Lama, L., Adura, C. & Xie, W. Development of human cGAS-specific small-molecule inhibitors for repression of dsDNA-triggered interferon expression. Nat. Commun. 10, 2261 (2019).
Fang, Q. et al. Human cytomegalovirus IE1 protein alters the higher-order chromatin structure by targeting the acidic patch of the nucleosome. Elife 5, e11911 (2016).
Yang, D. et al. Nα-acetylated Sir3 stabilizes the conformation of a nucleosome-binding loop in the BAH domain. Nat. Struct. Mol. Biol. 20, 1116–1118 (2013).
Barbera, A. J. et al. The nucleosomal surface as a docking station for Kaposi’s sarcoma herpesvirus LANA. Science 311, 856–861 (2006).
McGinty, R. K., Henrici, R. C. & Tan, S. Crystal structure of the PRC1 ubiquitylation module bound to the nucleosome. Nature 514, 591–596 (2014).
Makde, R. D., England, J. R., Yennawar, H. P. & Tan, S. Structure of RCC1 chromatin factor bound to the nucleosome core particle. Nature 467, 562–566 (2010).
Kato, H. et al. A conserved mechanism for centromeric nucleosome recognition by centromere protein CENP-C. Science 340, 1110–1113 (2013).
Mackenzie, K. J. et al. cGAS surveillance of micronuclei links genome instability to innate immunity. Nature 548, 461–465 (2017).
Pathare, G. R. & Decout, A. Structural mechanism of cGAS inhibition by the nucleosome. Nature https://doi.org/10.1038/s41586-020-2750-6 (2020).
Zhao, B. & Xu, P. The molecular basis of tight nuclear tethering and inactivation of cGAS. Nature https://doi.org/10.1038/s41586-020-2749-z (2020).
Michalski, S., de Oliveira Mann, C. C., Stafford, C. & Witte, G. Structural basis for sequestration and autoinhibition of cGAS by chromatin. Nature https://doi.org/10.1038/s41586-020-2748-0 (2020).
Kujirai, T. & Zierhut, C. Structural basis for the inhibition of cGAS by nucleosomes. Science https://doi.org/10.1126/science.abd0237 (2020).
Boyer, J. A. & Spangler, C. J. Structural basis of nucleosome-dependent cGAS inhibition. Science https://doi.org/10.1126/science.abd0609 (2020).
Liu, Z. S. et al. G3BP1 promotes DNA binding and activation of cGAS. Nat. Immunol. 20, 18–28 (2019).
Hu, S. & Sun, H. PKR-dependent cytosolic cGAS foci are necessary for intracellular DNA sensing. Sci. Signal. 12, eaav7934 (2019).
Dyer, P. N. et al. Reconstitution of nucleosome core particles from recombinant histones and DNA. Methods Enzymol. 375, 23–44 (2004).
Lowary, P. T. & Widom, J. New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning. J. Mol. Biol. 276, 19–42 (1998).
Wu, C., Huang, X., Cheng, J., Zhu, D. & Zhang, X. High-quality, high-throughput cryo-electron microscopy data collection via beam tilt and astigmatism-free beam-image shift. J. Struct. Biol. 208, 107396 (2019).
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J Struct. Biol. 192, 216–221 (2015).
Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J Struct. Biol. 180, 519–530 (2012).
Wakamori, M. et al. Intra- and inter-nucleosomal interactions of the histone H4 tail revealed with a human nucleosome core particle with genetically-incorporated H4 tetra-acetylation. Sci. Rep. 5, 17204 (2015).
Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).
Pettersen, E. F. et al. UCSF Chimera-a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).
Acknowledgements
We thank X. Huang, B. Zhu, X. Li, L. Chen and other staff members at the Center for Biological Imaging (CBI), Core Facilities for protein Science at the Institute of Biophysics, Chinese Academy of Science (IBP, CAS) for the support in cryo-EM data collection; L. Kong for cryo-EM data storage and backup. The project was funded by the National Key R&D Program of China (2017YFA0504700, 2019YFA0508900), the Ministry of Science and Technology of China, the National Natural Science Foundation of China (31930069, 31521002 and 31991162), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB37040101, XDB37010101). X.Z. received scholarships from the ‘National Thousand (Young) Talents Program’ from the Office of Global Experts Recruitment in China. D.C. is sponsored by the Youth Innovation Promotion Association at the Chinese Academy of Sciences (2018124).
Author information
Authors and Affiliations
Contributions
X.Z. and R.-M.X. conceived the project; X.F. expressed and purified the cGAS proteins. X.H. purified the nucleosomes, prepared the samples of the cGAS-nucleosome complexes and carried out the EMSA experiments. D.C. and X.F. performed the cryo-EM sample preparation, sample screening, data collection and data processing. D.C. carried out the pull-down assays, the activity assays, model building and structure refinement. X.Z., R.-M.X. and D.C. analyzed the structures and wrote the manuscript with the help of all the other authors.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Rights and permissions
About this article
Cite this article
Cao, D., Han, X., Fan, X. et al. Structural basis for nucleosome-mediated inhibition of cGAS activity. Cell Res 30, 1088–1097 (2020). https://doi.org/10.1038/s41422-020-00422-4
Received:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/s41422-020-00422-4
This article is cited by
-
Emerging role of the cGAS-STING pathway in cardiovascular diseases: biologic function, mechanisms and targeted therapy
Molecular Medicine (2025)
-
Regulation of cGAS–STING signalling and its diversity of cellular outcomes
Nature Reviews Immunology (2025)
-
Cyclic GMP-AMP synthase recognizes the physical features of DNA
Acta Pharmacologica Sinica (2025)
-
Emerging regulatory mechanisms and functions of biomolecular condensates: implications for therapeutic targets
Signal Transduction and Targeted Therapy (2025)
-
The cGAS-STING axis: a comprehensive review from immune defense to disease pathogenesis
Immunologic Research (2025)
