Targeting Bcl6 in the TREX1 D18N murine model ameliorates autoimmunity by modulating T-follicular helper cells and germinal center B cells.

The Three Prime Repair EXonuclease I (TREX1) is critical for degrading post-apoptosis DNA. Mice expressing catalytically inactive TREX1 (TREX1 D18N) develop lupus-like autoimmunity due to chronic sensing of undegraded TREX1 DNA substrates, production of the inflammatory cytokines, and the inappropriate activation of innate and adaptive immunity. This study aimed to investigate Thelper (Th) dysregulation in the TREX1 D18N model system as a potential mechanism for lupus-like autoimmunity. Comparison of immune cells in secondary lymphoid organs, spleen and peripheral lymph nodes (LNs) between TREX1 D18N mice and the TREX1 null mice revealed that the TREX1 D18N mice exhibit a Th1 bias. Additionally, the T-follicular helper cells (Tfh) and the germinal celter (GC) B cells were also elevated in the TREX1 D18N mice. Targeting Bcl6, a lineage-defining transcription factor for Tfh and GC B cells, with a commercially available Bcl6 inhibitor, FX1, attenuated Tfh, GC, and Th1 responses, and rescued TREX1 D18N mice from autoimmunity. The study presents Tfh and GC B-cell responses as potential targets in autoimmunity and that Bcl6 inhibitors may offer therapeutic approach in TREX1-associated or other lupus-like diseases.

T Cells Produce IFN-α in the TREX1 D18N Model of Lupus-like Autoimmunity.

Autoimmunity can result when cells fail to properly dispose of DNA. Mutations in the three-prime repair exonuclease 1 (TREX1) cause a spectrum of human autoimmune diseases resembling systemic lupus erythematosus. The cytosolic dsDNA sensor, cyclic GMP-AMP synthase (cGAS), and the stimulator of IFN genes (STING) are required for pathogenesis, but specific cells in which DNA sensing and subsequent type I IFN (IFN-I) production occur remain elusive. In this study, we demonstrate that TREX1 D18N catalytic deficiency causes dysregulated IFN-I signaling and autoimmunity in mice. Moreover, we show that bone marrow-derived cells drive this process. We identify both innate immune and, surprisingly, activated T cells as sources of pathological IFN-α production. These findings demonstrate that TREX1 enzymatic activity is crucial to prevent inappropriate DNA sensing and IFN-I production in immune cells, including normally low-level IFN-α-producing cells. These results expand our understanding of DNA sensing and innate immunity in T cells and may have relevance to the pathogenesis of human disease caused by TREX1 mutation.

Identification of Inhibitors of the dNTP Triphosphohydrolase SAMHD1 Using a Novel and Direct High-Throughput Assay.

The dNTP triphosphohydrolase SAMHD1 is a regulator of cellular dNTP pools. Given its central role in nucleotide metabolism, SAMHD1 performs important functions in cellular homeostasis, cell cycle regulation, and innate immunity. It therefore represents a high-profile target for small molecule drug design. SAMHD1 has a complex mechanism of catalytic activation that makes the design of an activating compound challenging. However, an inhibitor of SAMHD1 could serve multiple therapeutic roles, including the potentiation of antiviral and anticancer drug regimens. The lack of high-throughput screens that directly measure SAMHD1 catalytic activity has impeded efforts to identify inhibitors of SAMHD1. Here we describe a novel high-throughput screen that directly measures SAMHD1 catalytic activity. This assay results in a colorimetric end point that can be read spectrophotometrically and utilizes bis(4-nitrophenyl) phosphate as the substrate and Mn2+ as the activating cation that facilitates catalysis. When used to screen a library of Food and Drug Administration-approved drugs, this HTS identified multiple novel compounds that inhibited SAMHD1 dNTPase activity at micromolar concentrations.

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.

Solid-State Nanopore Analysis of Diverse DNA Base Modifications Using a Modular Enzymatic Labeling Process.

Many regulated epigenetic elements and base lesions found in genomic DNA can both directly impact gene expression and play a role in disease processes. However, due to their noncanonical nature, they are challenging to assess with conventional technologies. Here, we present a new approach for the targeted detection of diverse modified bases in DNA. We first use enzymatic components of the DNA base excision repair pathway to install an individual affinity label at each location of a selected modified base with high yield. We then probe the resulting material with a solid-state nanopore assay capable of discriminating labeled DNA from unlabeled DNA. The technique features exceptional modularity via selection of targeting enzymes, which we establish through the detection of four DNA base elements: uracil, 8-oxoguanine, T:G mismatch, and the methyladenine analog 1,N6-ethenoadenine. Our results demonstrate the potential for a quantitative nanopore assessment of a broad range of base modifications.

DNase-active TREX1 frame-shift mutants induce serologic autoimmunity in mice.

TREX1/DNASE III, the most abundant 3'-5' DNA exonuclease in mammalian cells, is tail-anchored on the endoplasmic reticulum (ER). Mutations at the N-terminus affecting TREX1 DNase activity are associated with autoimmune and inflammatory conditions such as Aicardi-Goutières syndrome (AGS). Mutations in the C-terminus of TREX1 cause loss of localization to the ER and dysregulation of oligosaccharyltransferase (OST) activity, and are associated with retinal vasculopathy with cerebral leukodystrophy (RVCL) and in some cases with systemic lupus erythematosus (SLE). Here we investigate mice with conditional expression of the most common RVCL mutation, V235fs, and another mouse expressing a conditional C-terminal mutation, D272fs, associated with a case of human SLE. Mice homozygous for either mutant allele express the encoded human TREX1 truncations without endogenous mouse TREX1, and both remain DNase active in tissues. The two mouse strains are similar phenotypically without major signs of retinal, cerebral or renal disease but exhibit striking elevations of autoantibodies in the serum. The broad range of autoantibodies is primarily against non-nuclear antigens, in sharp contrast to the predominantly DNA-related autoantibodies produced by a TREX1-D18N mouse that specifically lacks DNase activity. We also found that treatment with an OST inhibitor, aclacinomycin, rapidly suppressed autoantibody production in the TREX1 frame-shift mutant mice. Together, our study presents two new mouse models based on TREX1 frame-shift mutations with a unique set of serologic autoimmune-like phenotypes.

Mutations in the gene encoding the 3'-5' DNA exonuclease TREX1 are associated with systemic lupus erythematosus.

TREX1 acts in concert with the SET complex in granzyme A-mediated apoptosis, and mutations in TREX1 cause Aicardi-Goutières syndrome and familial chilblain lupus. Here, we report monoallelic frameshift or missense mutations and one 3' UTR variant of TREX1 present in 9/417 individuals with systemic lupus erythematosus but absent in 1,712 controls (P = 4.1 x 10(-7)). We demonstrate that two mutant TREX1 alleles alter subcellular targeting. Our findings implicate TREX1 in the pathogenesis of SLE.

The Arg-62 residues of the TREX1 exonuclease act across the dimer interface contributing to catalysis in the opposing protomers.

TREX1 is a 3'-deoxyribonuclease that degrades single- and double-stranded DNA (ssDNA and dsDNA) to prevent inappropriate nucleic acid-mediated immune activation. More than 40 different disease-causing TREX1 mutations have been identified exhibiting dominant and recessive genetic phenotypes in a spectrum of autoimmune disorders. Mutations in TREX1 at positions Asp-18 and Asp-200 to His and Asn exhibit dominant autoimmune phenotypes associated with the clinical disorders familial chilblain lupus and Aicardi-Goutières syndrome. Our previous biochemical studies showed that the TREX1 dominant autoimmune disease phenotype depends upon an intact DNA-binding process coupled with dysfunctional active site chemistry. Studies here show that the TREX1 Arg-62 residues extend across the dimer interface into the active site of the opposing protomer to coordinate substrate DNA and to affect catalysis in the opposing protomer. The TREX1(R62A/R62A) homodimer exhibits ∼50-fold reduced ssDNA and dsDNA degradation activities relative to TREX1(WT). The TREX1 D18H, D18N, D200H, and D200N dominant mutant enzymes were prepared as compound heterodimers with the TREX1 R62A substitution in the opposing protomer. The TREX1(D18H/R62A), TREX1(D18N/R62A), TREX1(D200H/R62A), and TREX1(D200N/R62A) compound heterodimers exhibit higher levels of ss- and dsDNA degradation activities than the homodimers demonstrating the requirement for TREX1 Arg-62 residues to provide necessary structural elements for full catalytic activity in the opposing TREX1 protomer. This concept is further supported by the loss of dominant negative effects in the TREX1 D18H, D18N, D200H, and D200N compound heterodimers. These data provide compelling evidence for the required TREX1 dimeric structure for full catalytic function.

The TREX1 C-terminal region controls cellular localization through ubiquitination.

TREX1 is an autonomous 3'-exonuclease that degrades DNA to prevent inappropriate immune activation. The TREX1 protein is composed of 314 amino acids; the N-terminal 242 amino acids contain the catalytic domain, and the C-terminal region (CTR) localizes TREX1 to the cytosolic compartment. In this study, we show that TREX1 modification by ubiquitination is controlled by a highly conserved sequence in the CTR to affect cellular localization. Transfection of TREX1 deletion constructs into human cells demonstrated that this sequence is required for ubiquitination at multiple lysine residues through a "non-canonical" ubiquitin linkage. A proteomic approach identified ubiquilin 1 as a TREX1 CTR-interacting protein, and this interaction was verified in vitro and in vivo. Cotransfection studies indicated that ubiquilin 1 localizes TREX1 to cytosolic punctate structures dependent upon the TREX1 CTR and lysines within the TREX1 catalytic core. Several TREX1 mutants linked to the autoimmune diseases Aicardi-Goutières syndrome and systemic lupus erythematosus that exhibit full catalytic function were tested for altered ubiquitin modification and cellular localization. Our data show that these catalytically competent disease-causing TREX1 mutants exhibit differential levels of ubiquitination relative to WT TREX1, suggesting a novel mechanism of dysfunction. Furthermore, these differentially ubiquitinated disease-causing mutants also exhibit altered ubiquilin 1 co-localization. Thus, TREX1 post-translational modification indicates an additional mechanism by which mutations disrupt TREX1 biology, leading to human autoimmune disease.

Synonymous mutations in RNASEH2A create cryptic splice sites impairing RNase H2 enzyme function in Aicardi-Goutières syndrome.

Aicardi-Goutières syndrome is an inflammatory disorder resulting from mutations in TREX1, RNASEH2A/2B/2C, SAMHD1, or ADAR1. Here, we provide molecular, biochemical, and cellular evidence for the pathogenicity of two synonymous variants in RNASEH2A. Firstly, the c.69G>A (p.Val23Val) mutation causes the formation of a splice donor site within exon 1, resulting in an out of frame deletion at the end of exon 1, leading to reduced RNase H2 protein levels. The second mutation, c.75C>T (p.Arg25Arg), also introduces a splice donor site within exon 1, and the internal deletion of 18 amino acids. The truncated protein still forms a heterotrimeric RNase H2 complex, but lacks catalytic activity. However, as a likely result of leaky splicing, a small amount of full-length active protein is apparently produced in an individual homozygous for this mutation. Recognition of the disease causing status of these variants allows for diagnostic testing in relevant families.

Functional consequences of the RNase H2A subunit mutations that cause Aicardi-Goutieres syndrome.

Mutations in the three genes encoding the heterotrimeric RNase H2 complex cause Aicardi-Goutières Syndrome (AGS). Our mouse RNase H2 structure revealed that the catalytic RNase H2A subunit interfaces mostly with the RNase H2C subunit that is intricately interwoven with the RNase H2B subunit. We mapped the positions of AGS-causing RNase H2A mutations using the mouse RNase H2 structure and proposed that these mutations cause varied effects on catalytic potential. To determine the functional consequences of these mutations, heterotrimeric human RNase H2 complexes containing the RNase H2A subunit mutations were prepared, and catalytic efficiencies and nucleic acid binding properties were compared with the wild-type (WT) complex. These analyses reveal a dramatic range of effects with mutations at conserved positions G37S, R186W, and R235Q, reducing enzymatic activities and substrate binding affinities by as much as a 1000-fold, whereas mutations at non-conserved positions R108W, N212I, F230L, T240M, and R291H reduced activities and binding modestly or not at all. All mutants purify as three-subunit complexes, further supporting the required heterotrimeric structure in eukaryotic RNase H2. These kinetic properties reveal varied functional consequences of AGS-causing mutations in the catalytic RNase H2A subunit and reflect the complex mechanisms of nuclease dysfunction that include catalytic deficiencies and altered protein-nucleic acid interactions relevant in AGS.

Aicardi-Goutieres syndrome gene and HIV-1 restriction factor SAMHD1 is a dGTP-regulated deoxynucleotide triphosphohydrolase.

The SAMHD1 protein is an HIV-1 restriction factor that is targeted by the HIV-2 accessory protein Vpx in myeloid lineage cells. Mutations in the SAMHD1 gene cause Aicardi-Goutières syndrome, a genetic disease that mimics congenital viral infection. To determine the physiological function of the SAMHD1 protein, the SAMHD1 gene was cloned, recombinant protein was produced, and the catalytic activity of the purified enzyme was identified. We show that SAMHD1 contains a dGTP-regulated deoxynucleotide triphosphohydrolase. We propose that Vpx targets SAMHD1 for degradation in a viral strategy to control cellular deoxynucleotide levels for efficient replication.

The TREX1 exonuclease R114H mutation in Aicardi-Goutières syndrome and lupus reveals dimeric structure requirements for DNA degradation activity.

Mutations in the TREX1 gene cause Aicardi-Goutières syndrome (AGS) and are linked to the autoimmune disease systemic lupus erythematosus. The TREX1 protein is a dimeric 3' DNA exonuclease that degrades DNA to prevent inappropriate immune activation. One of the most common TREX1 mutations, R114H, causes AGS as a homozygous and compound heterozygous mutation and is found as a heterozygous mutation in systemic lupus erythematosus. The TREX1 proteins containing R114H and the insertion mutations aspartate at position 201 (D201ins) and alanine at position 124 (A124ins), found in compound heterozygous AGS with R114H, were prepared and the DNA degradation activities were tested. The homodimer TREX1(R114H/R114H) exhibits a 23-fold reduced single-stranded DNA (ssDNA) exonuclease activity relative to TREX1(WT). The TREX1(D201ins/D201ins) and TREX1(A124ins/A124ins) exhibit more than 10,000-fold reduced ssDNA degradation activities. However, the TREX1(R114H/D201ins) and TREX1(R114H/A124ins) compound heterodimers exhibit activities 10-fold greater than the TREX1(R114H/R114H) homodimer during ssDNA and double-stranded DNA (dsDNA) degradation. These higher levels of activities measured in the TREX1(R114H/D201ins) and TREX1(R114H/A124ins) compound heterodimers are attributed to Arg-114 residues of TREX1(D201ins) and TREX1(A124ins) positioned at the dimer interface contributing to the active sites of the opposing TREX1(R114H) protomer. This interpretation is further supported by exonuclease activities measured for TREX1 enzymes containing R114A and R114K mutations. These biochemical data provide direct evidence for TREX1 residues in one protomer contributing to DNA degradation catalyzed in the opposing protomer and help to explain the dimeric TREX1 structure required for full catalytic competency.

Dominant mutation of the TREX1 exonuclease gene in lupus and Aicardi-Goutieres syndrome.

TREX1 is a potent 3' → 5' exonuclease that degrades single- and double-stranded DNA (ssDNA and dsDNA). TREX1 mutations at amino acid positions Asp-18 and Asp-200 in familial chilblain lupus and Aicardi-Goutières syndrome elicit dominant immune dysfunction phenotypes. Failure to appropriately disassemble genomic DNA during normal cell death processes could lead to persistent DNA signals that trigger the innate immune response and autoimmunity. We tested this concept using dsDNA plasmid and chromatin and show that the TREX1 exonuclease locates 3' termini generated by endonucleases and degrades the nicked DNA polynucleotide. A competition assay was designed using TREX1 dominant mutants and variants to demonstrate that an intact DNA binding process, coupled with dysfunctional chemistry in the active sites, explains the dominant phenotypes in TREX1 D18N, D200N, and D200H alleles. The TREX1 residues Arg-174 and Lys-175 positioned adjacent to the active sites act with the Arg-128 residues positioned in the catalytic cores to facilitate melting of dsDNA and generate ssDNA for entry into the active sites. Metal-dependent ssDNA binding in the active sites of the catalytically inactive dominant TREX1 mutants contributes to DNA retention and precludes access to DNA 3' termini by active TREX1 enzyme. Thus, the dominant disease genetics exhibited by the TREX1 D18N, D200N, and D200H alleles parallel precisely the biochemical properties of these TREX1 dimers during dsDNA degradation of plasmid and chromatin DNA in vitro. These results support the concept that failure to degrade genomic dsDNA is a principal pathway of immune activation in TREX1-mediated autoimmune disease.

The structure of the mammalian RNase H2 complex provides insight into RNA.NA hybrid processing to prevent immune dysfunction.

The mammalian RNase H2 ribonuclease complex has a critical function in nucleic acid metabolism to prevent immune activation with likely roles in processing of RNA primers in Okazaki fragments during DNA replication, in removing ribonucleotides misinserted by DNA polymerases, and in eliminating RNA.DNA hybrids during cell death. Mammalian RNase H2 is a heterotrimeric complex of the RNase H2A, RNase H2B, and RNase H2C proteins that are all required for proper function and activity. Mutations in the human RNase H2 genes cause Aicardi-Goutières syndrome. We have determined the crystal structure of the three-protein mouse RNase H2 enzyme complex to better understand the molecular basis of RNase H2 dysfunction in human autoimmunity. The structure reveals the intimately interwoven architecture of RNase H2B and RNase H2C that interface with RNase H2A in a complex ideally suited for nucleic acid binding and hydrolysis coupled to protein-protein interaction motifs that could allow for efficient participation in multiple cellular functions. We have identified four conserved acidic residues in the active site that are necessary for activity and suggest a two-metal ion mechanism of catalysis for RNase H2. An Okazaki fragment has been modeled into the RNase H2 nucleic acid binding site providing insight into the recognition of RNA.DNA junctions by the RNase H2. Further structural and biochemical analyses show that some RNase H2 disease-causing mutations likely result in aberrant protein-protein interactions while the RNase H2A subunit-G37S mutation appears to distort the active site accounting for the demonstrated substrate specificity modification.

DNA binding induces active site conformational change in the human TREX2 3'-exonuclease.

The TREX enzymes process DNA as the major 3'-->5' exonuclease activity in mammalian cells. TREX2 and TREX1 are members of the DnaQ family of exonucleases and utilize a two metal ion catalytic mechanism of hydrolysis. The structure of the dimeric TREX2 enzyme in complex with single-stranded DNA has revealed binding properties that are distinct from the TREX1 protein. The TREX2 protein undergoes a conformational change in the active site upon DNA binding including ordering of active site residues and a shift of an active site helix. Surprisingly, even when a single monomer binds DNA, both monomers in the dimer undergo the structural rearrangement. From this we have proposed a model for DNA binding and 3' hydrolysis for the TREX2 dimer. The structure also shows how TREX proteins potentially interact with double-stranded DNA and suggest features that might be involved in strand denaturation to provide a single-stranded substrate for the active site.

TREX1 as a Novel Immunotherapeutic Target.

Mutations in the TREX1 3' → 5' exonuclease are associated with a spectrum of autoimmune disease phenotypes in humans and mice. Failure to degrade DNA activates the cGAS-STING DNA-sensing pathway signaling a type-I interferon (IFN) response that ultimately drives immune system activation. TREX1 and the cGAS-STING DNA-sensing pathway have also been implicated in the tumor microenvironment, where TREX1 is proposed to degrade tumor-derived DNA that would otherwise activate cGAS-STING. If tumor-derived DNA were not degraded, the cGAS-STING pathway would be activated to promote IFN-dependent antitumor immunity. Thus, we hypothesize TREX1 exonuclease inhibition as a novel immunotherapeutic strategy. We present data demonstrating antitumor immunity in the TREX1 D18N mouse model and discuss theory surrounding the best strategy for TREX1 inhibition. Potential complications of TREX1 inhibition as a therapeutic strategy are also discussed.

RNaseH2 mutants that cause Aicardi-Goutieres syndrome are active nucleases.

Mutations in the genes encoding the RNaseH2 and TREX1 nucleases have been identified in patients with Aicardi-Goutieres syndrome (AGS). To determine if the AGS RNaseH2 mutations result in the loss of nuclease activity, the human wild-type RNaseH2 and four mutant complexes that constitute the majority of mutations identified in AGS patients have been prepared and tested for ribonuclease H activity. The heterotrimeric structures of the mutant RNaseH2 complexes are intact. Furthermore, the ribonuclease H activities of the mutant complexes are indistinguishable from the wild-type enzyme with the exception of the RNaseH2 subunit A (Gly37Ser) mutant, which exhibits some evidence of altered nuclease specificity. These data indicate that the mechanism of RNaseH2 dysfunction in AGS cannot be simply explained by loss of ribonuclease H activity and points to a more complex mechanism perhaps mediated through altered interactions with as yet identified nucleic acids or protein partners.

TREX1 - Apex predator of cytosolic DNA metabolism.

The cytosolic Three prime Repair EXonuclease 1 (TREX1) is a powerful DNA-degrading enzyme required for clearing cytosolic DNA to prevent aberrant inflammation and autoimmunity. In the absence of TREX1 activity, cytosolic DNA pattern recognition receptors of the innate immune system are constitutively activated by undegraded TREX1 substrates. This triggers a chronic inflammatory response in humans expressing mutant TREX1 alleles, eliciting a spectrum of rare autoimmune diseases dependent on the nature of the mutation. The precise origins of cytosolic DNA targeted by TREX1 continue to emerge, but DNA emerging from the nucleus or taken up by the cell could represent potential sources. In this Review, we explore the biochemical and immunological data supporting the role of TREX1 in suppressing cytosolic DNA sensing, and discuss the possibility that TREX1 may contribute to maintenance of genome integrity.

Measuring TREX1 and TREX2 exonuclease activities.

Three-prime Repair Exonuclease (TREX1) degrades ssDNA and dsDNA. TREX1 localizes to the perinuclear space in cells and degrades cytosolic DNA to prevent aberrant nucleic acid sensing and immune activation in humans and mice. Mutations in the TREX1 gene cause a spectrum of human autoimmune diseases including Aicardi-Goutières syndrome, familial chilblain lupus, retinal vasculopathy with cerebral leukodystrophy, and are associated with systemic lupus erythematosus. More than 60 disease-causing TREX1 variants have been identified including dominant and recessive, missense, and frameshift mutations that map to the catalytic core region and to the C-terminal cell localization region. The TREX1-disease causing mutations affect exonuclease activity at varied levels. In this chapter, we describe methods to purify variant recombinant TREX1 enzymes and measure the exonuclease activity using ssDNA and dsDNA substrates. The relationships between TREX1 activities, types of TREX1 mutations, and TREX1-associated autoimmune diseases are considered.