Inappropriate stimulation or defective negative regulation of the type I interferon response can lead to autoinflammation. In genetically uncharacterized cases of the type I interferonopathy Aicardi–Goutières syndrome, we identified biallelic mutations in LSM11 and RNU7-1, which encode components of the replication-dependent histone pre-mRNA–processing complex. Mutations were associated with the misprocessing of canonical histone transcripts and a disturbance of linker histone stoichiometry. Additionally, we observed an altered distribution of nuclear cyclic guanosine monophosphate–adenosine monophosphate synthase (cGAS) and enhanced interferon signaling mediated by the cGAS–stimulator of interferon genes (STING) pathway in patient-derived fibroblasts. Finally, we established that chromatin without linker histone stimulates cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) production in vitro more efficiently. We conclude that nuclear histones, as key constituents of chromatin, are essential in suppressing the immunogenicity of self-DNA.
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The RNA-seq data have been deposited at the National Center for Biotechnology Information Gene Expression Omnibus (accession no. GSE153079). The exome and genome sequencing data are not publicly available due to the possibility of compromising privacy. Human fibroblasts are primary cells and therefore a limited resource. Availability is through the corresponding author subject to technical constraints and completion of a material transfer agreement required to ensure patient privacy.
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We thank W. Bickmore, F. Taglini, P. Heyn, T. Chandra, R. O’Keefe, Z. Dominski, W. Marzluff, L. Murphy, L. Sanchez-Pulido and C. Ponting for help and advice. We acknowledge the help of the Genetics Core, Edinburgh Clinical Research Facility, University of Edinburgh. Y.J.C. acknowledges the European Research Council (grant nos. GA309449 and 786142-E-T1IFNs), a state subsidy managed by the National Research Agency (France) under the ‘Investments for the Future’ program bearing (no. ANR-10-IAHU-01) and the National Institute for Health Research UK Rare Genetic Disease Research Consortium. The project was supported by MSDAVENIR (Devo-Decode Project) and the Fondation Maladies Rare (GenOmics of rare diseases 2016-1). N.G., D.S., J.L.G.-P., A.P.J. and M.A.M.R. are supported by a UK Medical Research Council (MRC) Human Genetics Unit core grant (no. U127580972). D.S. is a Cancer Research UK Career Development fellow (no. C47648/A20837). This work was supported by a Chancellor’s fellowship of the University of Edinburgh and an Institutional Strategic Research Fund (ISSF3) to A. Dhir. J.R. is supported by the MRC (core funding of the MRC Human Immunology Unit). J.H. was supported by the European Commission under the Horizon 2020 program. We acknowledge the Edinburgh Super-Resolution Imaging Consortium, supported by the Wellcome Trust (grant no. 208345/Z/17/Z).
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, CLUSTAL Omega alignment of RNU7-1 sequences. Single nucleotide substitutions identified in probands with Aicardi-Goutières syndrome shaded individually for ease of visualization. Human NR_023317.1 ENSG00000238923; Orangutan U7 ENSPPYG00000029105; Horse U7 ENSECAG00000027042; Chicken U7 ENSGALG00000025670; Anole U7 ENSACAG00000019795; Zebrafish CR388102.4 ENSDARG00000082384; Tetraodon U7 ENSTNIG00000020484; Xenopus U7-201 ENSXETT00000102447.1; Drosophila snRNA:U7 FBgn0053504. b, Structure of pedigrees in which individuals with an Aicardi-Goutières syndrome phenotype were recorded to harbor biallelic rare (allelic frequency ≤ 0.005 on gnomAD) variants in RNU7-1 encoding the snRNA U7. nt, not tested. Variants in grey (n.59C>T and n.62C>G) have a frequency < 0.005 but are considered likely non-pathogenic on the basis of familial segregation.
a,b, The effect of knockdown of LSM11 in HCT116 (a) and U2OS (b) cells on the processing of replication-dependent histone mRNAs. Primers surrounding the cleavage site were used to assess the presence of polyadenylated RDH transcripts after 24 h knockdown of LSM11 by siRNA. Error bars indicate standard deviation from the mean. Two-tailed Mann-Whitney U test was used to compare differences between two groups. Experiments were performed with a minimum of 3 technical replicates. c, Misprocessing of RDH mRNAs in fibroblasts. Bar charts showing the expression of polyadenylated (Poly(A)+) and non-polyadenylated (Poly(A)-) replication dependent linker (H1) or core (H2A, H2B, H3, H4) histone transcripts in control (n = 3) and AGS8/9 patient-derived (n = 3) fibroblasts.
Relative quantification (RQ) values of a panel of six interferon-stimulated genes (ISGs) measured in whole blood in 12 AGS families (AGS114 mutated in LSM11; all others mutated in RNU7-1), compared to the combined results of 29 healthy controls. Numbers in brackets refer to decimalized age at sampling, followed by an interferon score calculated from the median fold change in relative quantification value for the panel of six ISGs. Shades denote individuals, with repeat samples indicated by different bars of the same shade.
Extended Data Fig. 4 ISGs expression and treatment with BX795 and ruxolitinib in patient-derived fibroblasts.
a, Gene set enrichment analysis (GSEA) of genome-wide RNA-seq data derived from control (n = 3) and patient-derived (n = 3) fibroblasts. P value is estimated using GSEA empirical phenotype-based permutation test. b, Treatment of patient fibroblasts with BX795 and ruxolitinib. Assessment of IFIT1 expression following treatment of patient fibroblasts with the TBK1 chemical inhibitor BX795, and the JAK1/2 inhibitor ruxolitinib. Kruskal-Wallis H test, followed by Dunn’s multiple comparison test. Experiments were performed with a minimum of 3 technical replicates. Box plots show the minimum and maximum scores (bottom and top whisker respectively) outside the interquartile range, the lower quartile, the median, and the upper quartile. Data points in the box plots represent the average value per cell line for each experiment.
a, Expression of interferon beta and selected interferon stimulated genes (ISGs) following 4 to 5 days LSM11 lentivirus shRNA transduction of THP-1 cells. b,c, Expression of interferon beta and selected ISGs (b) and of ISG15 protein (c) following LSM11 lentivirus shRNA transduction of THP-1 cells wild-type or null for cGAS, STING or MAVS. Cofilin was used as a housekeeping protein. The white asterisk indicates a non-specific band. Box plots show the minimum and maximum scores (bottom and top whisker, respectively) outside the interquartile range, the lower quartile, the median, and the upper quartile. Data points in the box plots represent the value for each experiment. Differences in expression within groups were assessed by Kruskal-Wallis H test, followed by Dunn’s multiple comparison test. Error bars indicate standard deviation. Experiments were performed with a minimum of 3 technical replicates.
a, Full blot corresponding to Fig. 5e. b, Expression of phospho-STING (Ser366) following knockdown of LSM1 in THP-1 cells wild-type or null for cGAS, STING or MAVS. Vinculin was used as a housekeeping protein. The black arrows indicate the band corresponding to phospho-STING. Experiments were performed with a minimum of 3 biological and/or technical replicates.
a, Knockdown efficiency of MYD88, and expression of ISG15 following MYD88 downregulation. b, Knockdown efficiency of STING, cGAS, MYD88 or MAVS corresponding to Fig. 5f. Two-tailed Mann-Whitney U test was used to compare differences between two groups.
Extended Data Fig. 8 ISG expression in fibroblasts following knockdown of nucleic acid sensing pathway molecules in two different patient lines.
a, AGS8. b, AGS9 (full blot corresponding to Fig. 5f). Experiments were performed with a minimum of 3 biological and/or technical replicates. Vertical white dotted line in a and b (left panel) indicates where gel was cropped.
a, Coomassie blue gel of fibroblast nuclear extract in control (C) and patient (AGS) fibroblasts, and in HEK293 cells. Bands corresponding to linker histones (H1) and core histones (H2A, H2B, H3 and H4) are indicated. The bottom panel shows the same samples run with higher amount of nuclear extract. n = 2 biologically independent samples of control and/or patient fibroblasts. b, Western blot of chromatin-bound histones using antibodies specific to H1.2 and H1.4. Full blot corresponding to Fig. 6b showing that samples were loaded on the same gel.
Extended Data Fig. 10 Proposed model explaining the induction of interferon signaling secondary to mutations in LSM11 or RNU7-1 (AGS8/9).
The U7 snRNP complex is essential for the processing of RDH pre-mRNAs in histone locus bodies (HLB). Mutations in LSM11 and RNU7-1 result in disturbed histone stoichiometry (perhaps particularly relating to linker histones), leading to increased binding of cGAS (green dots) to nuclear DNA and/or enhanced cGAS activation, and the subsequent induction of interferon signaling.
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Uggenti, C., Lepelley, A., Depp, M. et al. cGAS-mediated induction of type I interferon due to inborn errors of histone pre-mRNA processing. Nat Genet 52, 1364–1372 (2020). https://doi.org/10.1038/s41588-020-00737-3
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