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Defective removal of ribonucleotides from DNA promotes systemic autoimmunity - PubMed

. 2015 Jan;125(1):413-24.

doi: 10.1172/JCI78001. Epub 2014 Dec 15.

Defective removal of ribonucleotides from DNA promotes systemic autoimmunity

Claudia Günther et al. J Clin Invest. 2015 Jan.

Abstract

Genome integrity is continuously challenged by the DNA damage that arises during normal cell metabolism. Biallelic mutations in the genes encoding the genome surveillance enzyme ribonuclease H2 (RNase H2) cause Aicardi-Goutières syndrome (AGS), a pediatric disorder that shares features with the autoimmune disease systemic lupus erythematosus (SLE). Here we determined that heterozygous parents of AGS patients exhibit an intermediate autoimmune phenotype and demonstrated a genetic association between rare RNASEH2 sequence variants and SLE. Evaluation of patient cells revealed that SLE- and AGS-associated mutations impair RNase H2 function and result in accumulation of ribonucleotides in genomic DNA. The ensuing chronic low level of DNA damage triggered a DNA damage response characterized by constitutive p53 phosphorylation and senescence. Patient fibroblasts exhibited constitutive upregulation of IFN-stimulated genes and an enhanced type I IFN response to the immunostimulatory nucleic acid polyinosinic:polycytidylic acid and UV light irradiation, linking RNase H2 deficiency to potentiation of innate immune signaling. Moreover, UV-induced cyclobutane pyrimidine dimer formation was markedly enhanced in ribonucleotide-containing DNA, providing a mechanism for photosensitivity in RNase H2-associated SLE. Collectively, our findings implicate RNase H2 in the pathogenesis of SLE and suggest a role of DNA damage-associated pathways in the initiation of autoimmunity.

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Figures

Figure 6
Figure 6. Increased sensitivity to photodamage in ribonucleotide-containing DNA.

(A) Increased CPD formation in RNase H2–deficient fibroblasts (SLE1, SLE2, AGS1, and AGS2) compared with WT in response to 20 J/m2 UVC, measured immediately or 7 and 24 hours after irradiation. Box plots depict interquartile range (box), mean (square), median (line) and SD (whisker) of ≥3 independent experiments per patient and for 5 WT cell lines. *P < 0.05, **P < 0.01, ***P < 0.001 versus WT, t test. (B and C) Dihedral angles between covalent C5-C6 bonds (B) and pyrimidine ring planes (C) of 2 consecutive thymidines of the unmodified (1NAJ) and the ribonucleotide-containing (2L7D) Dickerson dodecamer. Ribonucleotide substitution increased colinearity between C5-C6 covalent bonds and coplanarity between pyrimidine rings, enhancing the probability of a photoreactive dimerization. For each dodecamer, 5 structures deposited in PDB were compared. Mean and SD shown. ***P < 0.001 versus 1NAJ, t test. (D) CPD formation in genomic DNA from fibroblasts of individual RNase H2–deficient versus WT patients and from Rnaseh2b–/– versus WT MEFs (n = 2 per group). Representative dot blot and mean intensities with SEM of 3 independent experiments are shown. *P < 0.05, **P < 0.01, ***P < 0.001 versus WT, t test. (E) CPD formation in double-stranded concatamers of chimeric DNA oligonucleotides containing 3 Dickerson dodecamer sequences in tandem. Replacement of a deoxyriboguanosine at position 4 of the dodecamer by a riboguanosine (R) is indicated.

Figure 5
Figure 5. Type I IFN production by patient cells is enhanced in response to nucleic acids and UV light.

(A) Increased type I IFN activation in fibroblasts of patients with SLE and AGS compared with WT controls after treatment with poly(I:C) and/or UVC irradiation (30 J/m2 UVC for 3 seconds). Samples were processed after subsequent culture for 4, 8, and 24 hours. IFNB mRNA was normalized to GAPDH mRNA. Mean and SEM of ≥3 independent experiments per group (SLE and AGS, n = 2; WT, n = 5). *P < 0.05, **P < 0.01, ***P < 0.001 versus poly(I:C) at the same time point, Mann-Whitney U test. (B) Lesional skin from lupus patients carrying the indicated mutations in RNASEH2B and RNASEH2C showed high expression of type I IFN–inducible MxA (red) and CXCL10 (green). Scale bars: 100 μm.

Figure 4
Figure 4. Reduced proliferation and increased DNA damage response activation in SLE and AGS patient fibroblasts.

(A) Reduced proliferation rate of fibroblasts from patients with SLE (SLE1 and SLE2) and AGS (AGS1 and AGS2) compared with the mean of 5 WT control fibroblast lines (2 children, 3 adults). P < 0.05 (3 and 5 hours), P < 0.001 (7 hours), AGS1 vs. WT, t test. (B) Cell cycle analysis of propidium iodide–stained fibroblasts after synchronization in G1 by 24 hours of serum starvation. Representative flow cytometry images depict cell cycle delay in RNase H2–deficient fibroblasts. (C) Activation of a p53-dependent DNA damage response in patient fibroblasts in the absence of exogenous genotoxic stress, as shown by increased levels of phosphorylated p53 (Ser15). The same immunoblot probed for β-actin shows equal loading. (D) Increased number of p16-positive cells in RNase H2–deficient fibroblasts, measured by flow cytometry. (E) Increased senescence in patient fibroblasts, measured by β-galactosidase staining. Scale bar: 100 μm. (F) Increased dsDNA damage in RNase H2–deficient human fibroblasts. dsDNA breaks were visualized by immunostaining of γH2AX (red) and 53BP1 (green). The number of nuclei with 0–3 foci stained for both (yellow) among 50 randomly selected cells was quantified. Original magnification, ×400. Data are mean and SEM (D and E) or mean and SD (A, B, and F) of 3 independent experiments per patient and 5 independent WT control cell lines (A and B) or of 4 experiments per patient and 5 WT controls (D–F). (B–F) *P < 0.05, **P < 0.01, #P < 0.001 versus WT, t test.

Figure 3
Figure 3. Increased levels of misincorporated ribonucleotides in genomic DNA in cells from patients with AGS and SLE.

(A) Representative images of comets formed by human fibroblasts after single-cell electrophoresis at alkaline and neutral pH without or with RNase H2 pretreatment. Scale bar: 50 μm. (B and C) Quantification of Olive tail moment (see Supplemental Methods) after alkaline comet assay, indicative of single-strand DNA breaks, in (B) fibroblasts from SLE patients (SLE1 and SLE2), AGS patients (AGS1 and AGS2), and WT controls (WT1 and WT2) and in (C) Rnaseh2b-deficient or WT MEFs. (D and E) Quantification of Olive tail moment in comet assays of human fibroblasts and MEFs (as in B and C, respectively) under neutral condition, without and with RNase H2 pretreatment, showed increased Olive tail moments for RNase H2–deficient cells, consistent with elevated levels of embedded ribonucleotides in their genomic DNA. (B–E) Mean and SEM of 30–70 cells from 3 independent experiments per cell line. **P < 0.01, ***P < 0.001 versus WT2 (B and D) or WT (C and E), t test.

Figure 2
Figure 2. Rare sequence variants impair RNase H2 function in SLE and AGS.

RNASEH2 variants identified by resequencing caused reduced enzyme activity or complex stability. (A) Effects of RNASEH2 variants on recombinant enzyme activity on dsDNA substrate with an embedded ribonucleotide. Cleavage of an RNA/DNA substrate was similarly affected (data not shown). Mean (n = 3) and SD shown. (B) Thermal stability (expressed as thermal shift; ΔTm) of recombinant mutant RNase H2 complexes relative to WT; negative values denote less stable complexes. Mean (n ≥ 9) and SEM shown. (A and B) Unless specifically indicated as P = NS, activity was significantly different compared with WT (P < 0.01, t test). (C) Burden analysis demonstrated that increased risk of SLE correlated with functional severity of the RNASEH2 variants. Variant frequencies for neutral, mild, and severe functional consequences (see Supplemental Table 3) were calculated using number of occurrences as a percentage of total SLE cases (n = 600) or controls (n = 1,056) respectively. Fold change relative to control is also denoted.

Figure 1
Figure 1. Heterozygosity for RNASEH2 mutations promotes systemic autoimmunity.

(A) Prevalence of ANAs in heterozygous parents of AGS patients. Shown is the percentage of ANA-positive parents of AGS patients (with mutations in TREX1, RNASEH2A–RNASEH2C, or SAMHD1; n = 28; P < 0.001) and in parents carrying RNASEH2 mutations (n = 16; P < 0.01, Fisher’s exact test) compared with data from a control population (n = 1,000) measured in the same laboratory. (B) RNASEH2B, RNASEH2C, and RNASEH2A gene structures. Variants identified in individuals with SLE (n = 600) are shown in bold above; variants in controls (n = 1,056) are shown below (boxes indicate exons). Colors correspond to those used for each subunit in the crystal structure in C: blue, RNASEH2A; green, RNASEH2B; red/pink, RNASEH2C. Except for variants L202S and D205E in RNASEH2A, all variants occurred with allele frequencies of <0.002 (Table 1). Variant A156A in RNASEH2C affected splicing (Table 1). (C) RNase H2 residues (yellow sticks) affected by missense mutations identified in SLE patients, mapped on the structure of the human enzyme (PDB ID, 3PUF; ref. 63).

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