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Structural basis for sequestration and autoinhibition of cGAS by chromatin

Abstract

Cyclic GMP–AMP synthase (cGAS) is an innate immune sensor for cytosolic microbial DNA1. After binding DNA, cGAS synthesizes the messenger 2′3′-cyclic GMP–AMP (cGAMP)2,3,4, which triggers cell-autonomous defence and the production of type I interferons and pro-inflammatory cytokines via the activation of STING5. In addition to responding to cytosolic microbial DNA, cGAS also recognizes mislocalized cytosolic self-DNA and has been implicated in autoimmunity and sterile inflammation6,7. Specificity towards pathogen- or damage-associated DNA was thought to be caused by cytosolic confinement. However, recent findings place cGAS robustly in the nucleus8,9,10, where tight tethering of chromatin is important to prevent autoreactivity to self-DNA8. Here we show how cGAS is sequestered and inhibited by chromatin. We provide a cryo-electron microscopy structure of the cGAS catalytic domain bound to a nucleosome, which shows that cGAS does not interact with the nucleosomal DNA, but instead interacts with histone 2A–histone 2B, and is tightly anchored to the ‘acidic patch’. The interaction buries the cGAS DNA-binding site B, and blocks the formation of active cGAS dimers. The acidic patch robustly outcompetes agonistic DNA for binding to cGAS, which suggests that nucleosome sequestration can efficiently inhibit cGAS, even when accessible DNA is nearby, such as in actively transcribed genomic regions. Our results show how nuclear cGAS is sequestered by chromatin and provides a mechanism for preventing autoreactivity to nuclear self-DNA.

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Fig. 1: Nucleosomes inhibit cGAS activity in the presence of DNA.
Fig. 2: Structure of the cGAS catalytic domain–nucleosome complex.
Fig. 3: cGAS is sequestered by the acidic patch of the nucleosome.
Fig. 4: Nucleosome sequestration sterically blocks cGAS activation.

Data availability

The electron density reconstruction and final model were deposited at the Electron Microscopy Data Bank (EMDB) with accession code EMD-11601, and the Protein Data Bank (PDB) with accession code 7A08Source data are provided with this paper.

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Acknowledgements

We thank K. Schall and V. Niebauer for guidance with nucleosome preparation and A. Drescher for discussions of SPR data. C.C.O.M. is supported as a Cancer Research Institute/Eugene V. Weissman Fellow. This work was supported by the German Research Council (grants CRC/TRR237 and the Gottfried Wilhelm Leibniz-Prize to K.-P.H. and V.H.; RTG1721 to K.-P.H. and G.W.; HO2489/8 to K.-P.H.; WI3717/3-1 to G.W.; and CRC1054 to K.L.).

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Contributions

C.C.O.M. and S.M. prepared cryo-EM samples and performed biochemical analysis. C.C.O.M., S.M. and J.B. optimized and screened cryo-EM grids. K.L. collected cryo-EM data. C.C.O.M. performed structure determination and model building with assistance from J.B. and K.L. SPR experiments were performed by G.W. C.S. performed cell-based assays and V.H. supervised cell-based experiments and interpreted data. C.C.O.M. and K.-P.H. designed the overall study, analysed the results and wrote the paper with contributions from all other authors.

Corresponding author

Correspondence to Karl-Peter Hopfner.

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The authors declare no competing interests.

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Peer review information Nature thanks Yuan He and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 cGAS binds nucleosomes even in the presence of free DNA.

a, Gel-mobility shift binding analysis of purified cGAS and nucleosomes. Nucleosomes without linker DNA (0N0) were tested for binding to purified human (h) and mouse (m) full-length cGAS and cGAScat. Data are representative of two biological replicates. b, Human cGAS activity assay preincubated with dsDNA followed by titration of 0N0 nucleosomes. Data are representative of two biological replicates.

Source data

Extended Data Fig. 2 Cryo-EM data processing for cGAS–nucleosome structure.

a, Representative micrograph of the dataset used to determine the cGAS–nucleosome complex structure. b, Left, final reconstruction of the cGAS–nucleosome complex coloured by local resolution. Right, representation of angular distribution of particles contributing to the final map. c, Histogram and directional Fourier shell correlation (FSC) plot for the final 3D reconstitution of the cGAS–nucleosome complex (3.11 Å). A sphericity of 0.9 was determined indicating very isotropic angular distribution (a value of 1 stands for completely isotropic angular distribution). The global resolution was determined to 3.11 Å (0.143 criterion). Directional FSC determination was performed with the 3DFSC software. d, Flow chart for image processing using RELION and cryoSPARC.

Extended Data Fig. 3 Sample density maps for cGAS–nucleosome structure.

a, Representative examples of cryo-EM map areas of cGAS, nucleosomal DNA and histones used for model building. b, Electron density for cGAS (blue) and H2A–H2B (yellow, red) interacting residues in interface I and interface II. cGAS tethering loops 1 and 2 with key interacting residues R222 and R241 are depicted as well as DNA-binding site B residues. Dashed lines represent hydrogen bonds.

Extended Data Fig. 4 Mutational analysis of binding interface between cGAS and the nucleosome.

a, Protein–protein residue interactions across the interface of cGAS with histone H2A and cGAS with histone H2B. Interacting amino acids are joined by coloured lines, each representing a different type of interaction, as per the key below. Interaction maps for the cGAS–nucleosome complex were generated using PDBsum. b, Thermal shift assay derivative melt curve plots of human cGAScat mutants. Respective inflection temperatures are: cGAScat 61.8 °C; cGAScat(K407E/K411E) 61.3 °C; cGAScat(C396A/C397A (55.5 °C; cGAScat(R236E) 61.9 °C, cGAScat(R300E/K301E) 62.5 °C. Data are representative of two biological replicates. c, Coomassie stained SDS–PAGE gels of purified recombinant human and mouse cGAS (7 μg each) constructs used in this study. Gels are representative of one replicate. d, Representative EMSAs for mouse cGAS–nucleosome interface I and interface II mutants binding to fluorescently labelled nucleosomes. Data are representative of three biological replicates. e, EMSAs for mouse cGAS mutants in tethering loops 1 and 2 (R222E, R241E) and DNA-binding site B (R337E, R341E) binding to fluorescently labelled nucleosomal DNA. Data are representative of two biological replicates. f, EMSAs for human cGAS full-length and catalytic domain, DNA-binding site A (K407E/K411E), Zn-thumb (C396A/C397A), site B (R236E) and site C (R300E/K301E) mutants binding to fluorescently labelled nucleosomes. Data are representative of two biological replicates. g, Representative EMSAs for mouse cGAS full-length binding to fluorescently labelled acidic patch mutant nucleosomes apI (H2A(E61A/E64A/D90A)) and apII (H2A(R71A), H2B(H49A/D51A)) and apI + apII (H2A(E61A/E64A/R71A/D90A/E92A), H2B(H49A/D51A)). Data are representative of three biological replicates.

Source data

Extended Data Fig. 5 cGAS DNA-binding site B is required for cGAS tethering by the nucleosome.

a, SPR analysis of single-cycle-kinetics experiment with immobilized nucleosomes via biotinylated DNA and mouse cGAScat and cGAScat(R241E) mutant as analytes. Shown are injections of 1.1, 3.3, 9, 10, 30 and 90 nM mouse cGAScat and cGAScat(R241E). Data are representative of two biological replicates. b, SPR analysis with acidic patch mutant nucleosomes (H2A(E61A/E64A/D90A)) immobilized via biotinylated DNA and cGAScat as analyte. Shown are buffer injections, injections of 1.1, 3.3, 9, 10, 30 and 90 nM mouse cGAScat and the cGAScat background-corrected data. Mouse cGAScat has orders of magnitude lower affinity to acidic patch mutant nucleosomes than wild-type nucleosomes. Data are representative of two biological replicates. c, Human cGAS(R236E) mutant activity assay preincubated with dsDNA followed by titration of 0N0 nucleosome. Data are representative of two biological replicates. d, Human cGAS(R236E) mutant activity assay pre-incubated with 0N0 nucleosome followed by titration of dsDNA. Data are representative of two biological replicates. e, Mouse cGAS mutations tested affecting cGAS–nucleosome interactions were tested for DNA-dependent activation with plasmid DNA in the presence of 0N0 nucleosomes. cGAMP production was assayed by thin-layer chromatography. Data are representative of two biological replicates. f, Mouse cGAS mutations affecting cGAS–nucleosome interactions were tested for DNA-dependent activation with 147-bp nucleosomal DNA in the presence of 0N0 nucleosomes. Data are representative of two biological replicates. g, Mouse cGAS single and double mutations of tethering loop and DNA-binding site B were tested for cGAMP production in the presence of plasmid DNA alone or plasmid DNA and 0N0 nucleosome. Data are representative of two biological replicates. h, Mouse cGAS mutants R222E, K240E and R241E require plasmid DNA for activation. Data are representative of two biological replicates. i, Mouse cGAS mutants R222E, K240E and R241E were tested for DNA-dependent activation with plasmid DNA in the presence of 0N0 nucleosomes. Mutation of DNA-binding site A abolishes activation by plasmid DNA. Data are representative of two biological replicates. j, Human cGAS activity assay in the presence of dsDNA, followed by titration of 0N0 and 0N0 acidic patch mutant I (apI; H2A(E61A/E64A/D90A)). Data are representative of two biological replicates. k, Wild-type and R236E mutant cGAS activity assays with 40N40 nucleosomes (40-bp linker DNA on each side). Data are representative of two biological replicates. l, Fluorescence anisotropy analysis of human cGAScat human cGAScat site A mutant (K407E/K411E) binding to fluorescently labelled 20-bp dsDNA and in the presence of 0N0 nucleosomes. Data are representative of two biological replicates. m, Agarose gel of micrococcal nuclease (MNase)-digested synthetic 601 chromatin indicating a regular nucleosomal structure. Data are representative of two biological replicates.

Source data

Extended Data Fig. 6 IP-10 cytokine and ISG production upon self and non-self DNA recognition.

a, PMA-differentiated wild-type or knockout human CGAS−/− THP-1 cells were treated with doxycycline (1 μg ml−1) overnight and either left untreated or treated with HT-DNA (975 ng per 550 μl) for 8 h. Cell lysates were separated on SDS–PAGE gels, western blotted and probed with the indicated antibodies. Data are representative of three biological replicates. b, PMA-differentiated THP-1 cells were left untreated or treated with doxycycline overnight to express the indicated human cGAS mutants, followed by stimulation using HT-DNA (200 ng per well) for 8 h. Supernatant was collected and IP-10 cytokines were measured from supernatant using ELISA. Data are mean and s.e.m. of four biological replicates. ***P < 0.001, **P < 0.01, two-way ANOVA. ns, not significant. Doxycycline R236E P < 0.001; doxycycline R255E P < 0.001; doxycycline + HT-DNA R236E P = 0.002; doxycycline + HT-DNA R255E P < 0.001.

Source data

Extended Data Fig. 7 Structurally conserved loop for protein–protein interactions in MAB21 family nucleotidyltransferases.

Structures of cGAS–nucleosome (blue) and MID49–DRP1 (red, PDB code 5WP9). The structurally conserved loop is depicted in green, showing interacting residues. Dashed lines represent hydrogen bonds. Positively charged, basic amino acids on the nucleotidyltransferase loop interact with the acidic patch of the nucleosome or with the protein DRP1, respectively.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics

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Michalski, S., de Oliveira Mann, C.C., Stafford, C. et al. Structural basis for sequestration and autoinhibition of cGAS by chromatin. Nature 587, 678–682 (2020). https://doi.org/10.1038/s41586-020-2748-0

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