Exonuclease TREX1 degrades double-stranded DNA to prevent spontaneous lupus-like inflammatory disease

The TREX1 gene encodes a potent DNA exonuclease, and mutations in TREX1 cause a spectrum of lupus-like autoimmune diseases. Most lupus patients develop autoantibodies to double-stranded DNA (dsDNA), but the source of DNA antigen is unknown. The TREX1 D18N mutation causes a monogenic, cutaneous form of lupus called familial chilblain lupus, and the TREX1 D18N enzyme exhibits dysfunctional dsDNA-degrading activity, providing a link between dsDNA degradation and nucleic acid-mediated autoimmune disease. We determined the structure of the TREX1 D18N protein in complex with dsDNA, revealing how this exonuclease uses a novel DNA-unwinding mechanism to separate the polynucleotide strands for single-stranded DNA (ssDNA) loading into the active site. The TREX1 D18N dsDNA interactions coupled with catalytic deficiency explain how this mutant nuclease prevents dsDNA degradation. We tested the effects of TREX1 D18N in vivo by replacing the TREX1 WT gene in mice with the TREX1 D18N allele. The TREX1 D18N mice exhibit systemic inflammation, lymphoid hyperplasia, vasculitis, and kidney disease. The observed lupus-like inflammatory disease is associated with immune activation, production of autoantibodies to dsDNA, and deposition of immune complexes in the kidney. Thus, dysfunctional dsDNA degradation by TREX1 D18N induces disease in mice that recapitulates many characteristics of human lupus. Failure to clear DNA has long been linked to lupus in humans, and these data point to dsDNA as a key substrate for TREX1 and a major antigen source in mice with dysfunctional TREX1 enzyme.The TREX1 gene encodes a powerful DNA exonuclease (1–7). The amino terminal domain of the TREX1 enzyme contains all of the structural elements for full exonuclease activity, and the carboxy terminal region controls cellular trafficking to the perinuclear space (8–10). Mutations in TREX1 cause a spectrum of autoimmune disorders, including Aicardi–Goutieres syndrome, familial chilblain lupus, and retinal vasculopathy with cerebral leukodystrophy and are associated with systemic lupus erythematosus (9, 11–19). The TREX1 disease-causing alleles locate to positions throughout the gene, exhibit dominant and recessive genetics, include inherited and de novo mutations, and cause varied effects on catalytic function and cellular localization. These genetic discoveries have established a causal relationship between TREX1 mutation and nucleic acid-mediated immune activation disease. The spectrum of TREX1-associated disease parallels the diverse effects on enzyme function and localization, indicating multiple mechanisms of TREX1 dysfunction might explain the overlapping clinical symptoms related to failed DNA degradation and immune activation.The TREX1 enzyme structure reveals the uniquely stable dimer that is relevant to its function and to disease mechanisms. The backbone contacts between the protomer β3-strands generate a stable, central antiparallel β-sheet that stretches the length of the dimer and an extensive hydrogen-bonding network of sidechain–sidechain, sidechain–backbone, and water-bridged contacts that coordinate residues across the TREX1 dimer interface (8, 20). TREX1 catalytic function depends on the dimeric structure, with residues from one protomer contributing to DNA binding and degradation in the opposing protomer (21, 22). Some TREX1 disease-causing mutants exhibit complete loss of catalytic function, whereas others exhibit altered cellular localization (8, 10). A subset of TREX1 catalytic mutants at amino acid positions Asp-18 and Asp-200 exhibit selectively dysfunctional activities on dsDNA. These mutations cause autosomal-dominant disease by retaining DNA-binding proficiency and blocking access to DNA 3′ termini for degradation by TREX1 WT enzyme (21, 23, 24). The TREX1 catalytic sites accommodate four nucleotides of ssDNA, and additional structural elements are positioned adjacent to the active sites for potential DNA polynucleotide interactions.The connection between failure to degrade DNA by TREX1 and immune activation was first made in the TREX1 null mouse that showed a dramatically reduced survival associated with inflammatory myocarditis (25). However, the origin and nature of the disease-driving DNA polynucleotides resulting from TREX1 deficiency have not been clearly established. One model posits that TREX1 acts in the SET complex to degrade genomic dsDNA during granzyme A-mediated cell death by rapidly degrading DNA from the 3′ ends generated by the NM23-H1 endonuclease (26). Two additional models propose that TREX1 prevents immune activation by degrading ssDNA, but these models differ on the possible source of offending DNA polynucleotide. In TREX1-deficient cells there is an accumulation of ssDNA fragments within the cytoplasm proposed, in one model, to be generated from failed processing of aberrant replication intermediates that result in chronic activation of the DNA damage response pathway (27, 28). Another model proposes the source of accumulating ssDNA in TREX1-deficient cells to be derived from unrestrained endogenous retroelement replication, leading to activation of the cytosolic DNA-sensing cGAS–STING pathway (29–33). This concept is also supported by the participation of TREX1 in degradation of HIV-derived cytosolic DNA (34). Thus, disparate concepts on the DNA polynucleotide-driving immune activation in TREX1 deficiency have been proposed, and it is possible that the robust TREX1 exonuclease participates in multiple DNA degradation pathways. We present here structural and in vivo data supporting the concept that TREX1 degradation of dsDNA is critical to prevent immune activation.