Three Prime Repair Exonuclease 1 Research Articles

Proceedings to the 7th Annual Conference of the Particle Therapy Cooperative Group North America (PTCOG-NA)

Proceedings to the 7th Annual Conference of the Particle Therapy Cooperative Group North America (PTCOG-NA)

187 Photosensitivity and cGAS-dependent type I IFN activation in lupus patients with TREX1 deficiency

The exonuclease three prime repair exonuclease 1 (TREX1) safeguards the cell against DNA accumulation in the cytosol and thereby prevents innate immune activation and autoimmunity. TREX1 mutations lead to chronic DNA damage and a cell-intrinsic type I interferon (IFN) response. Associated disease phenotypes include Aicardi-Goutières syndrome, familial chilblain lupus and systemic lupus erythematosus. Given the role of ultraviolet (UV) light in lupus pathogenesis, we assessed sensitivity to UV light in lupus patients with TREX1 mutation by phototesting which revealed an enhanced photosensitivity compared to healthy controls.

Nucleic Acid Sensing in the Tumor Vasculature.

Simple SummaryOur cells can recognize DNA or RNA from pathogens, such as viruses. The proteins that recognize these nucleic acids are known as nucleic acid sensors. Upon activation, they trigger immune responses that result in the elimination of the infected cells. Recent research has shown how we can mimic this process in cancer and recruit immune cells against the tumor. Among the different cell types within a tumor, endothelial cells that line the blood vessels play a main role as conduits for nutrients and oxygen and highways for the immune cells. In this review, we discuss two different nucleic acid sensors—the three-prime repair exonuclease 1 (TREX1) and the retinoic acid-inducible gene 1 (RIG-I)—and how they play a role in endothelial cells. We present some approaches to target these pathways within the cancer blood vessels to disrupt the blood supply and attract immune responses to cancers.Endothelial cells form a powerful interface between tissues and immune cells. In fact, one of the underappreciated roles of endothelial cells is to orchestrate immune attention to specific sites. Tumor endothelial cells have a unique ability to dampen immune responses and thereby maintain an immunosuppressive microenvironment. Recent approaches to trigger immune responses in cancers have focused on activating nucleic acid sensors, such as cGAS-STING, in combination with immunotherapies. In this review, we present a case for targeting nucleic acid-sensing pathways within the tumor vasculature to invigorate tumor-immune responses. We introduce two specific nucleic acid sensors—the DNA sensor TREX1 and the RNA sensor RIG-I—and discuss their functional roles in the vasculature. Finally, we present perspectives on how these nucleic acid sensors in the tumor endothelium can be targeted in an antiangiogenic and immune activation context. We believe understanding the role of nucleic acid-sensing in the tumor vasculature can enhance our ability to design more effective therapies targeting the tumor microenvironment by co-opting both vascular and immune cell types.

Human Three Prime Repair Exonuclease 1 Promotes HIV-1 Integration by Preferentially Degrading Unprocessed Viral DNA.

Three prime repair exonuclease 1 (TREX1) is the most abundant 3'→5' exonuclease in mammalian cells. It has been suggested that TREX1 degrades HIV-1 DNA to enable the virus to evade the innate immune system. However, the exact role of TREX1 during early steps of HIV-1 infection is not clearly understood. In this study, we report that HIV-1 infection is associated with upregulation, perinuclear accumulation, and nuclear localization of TREX1. However, TREX1 overexpression did not affect reverse transcription or nuclear entry of the virus. Surprisingly, HIV-1 DNA integration was increased in TREX1-overexpressing cells, suggesting a role of the exonuclease in the post-nuclear entry step of infection. Accordingly, preintegration complexes (PICs) extracted from TREX1-overexpressing cells retained higher levels of DNA integration activity. TREX1 depletion resulted in reduced levels of proviral integration, and PICs formed in TREX1-depleted cells retained lower DNA integration activity. Addition of purified TREX1 to PICs also enhanced DNA integration activity, suggesting that TREX1 promotes HIV-1 integration by stimulating PIC activity. To understand the mechanism, we measured TREX1 exonuclease activity on substrates containing viral DNA ends. These studies revealed that TREX1 preferentially degrades the unprocessed viral DNA, but the integration-competent 3'-processed viral DNA remains resistant to degradation. Finally, we observed that TREX1 addition stimulates the activity of HIV-1 intasomes assembled with the unprocessed viral DNA but not that of intasomes containing the 3'-processed viral DNA. These biochemical analyses provide a mechanism by which TREX1 directly promotes HIV-1 integration. Collectively, our study demonstrates that HIV-1 infection upregulates TREX1 to facilitate viral DNA integration. IMPORTANCE Productive HIV-1 infection is dependent on a number of cellular factors. Therefore, a clear understanding of how the virus exploits the cellular machinery will identify new targets for inhibiting HIV-1 infection. The three prime repair exonuclease 1 (TREX1) is the most active cellular exonuclease in mammalian cells. It has been reported that TREX1 prevents accumulation of HIV-1 DNA and enables the virus to evade the host innate immune response. Here, we show that HIV-1 infection results in the upregulation, perinuclear accumulation, and nuclear localization of TREX1. We also provide evidence that TREX1 promotes HIV-1 integration by preferentially degrading viral DNAs that are incompatible with chromosomal insertion. These observations identify a novel role of TREX1 in a post-nuclear entry step of HIV-1 infection.

Serial magnetic resonance imaging changes of pseudotumor lesions in retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations: a case report

BackgroundRetinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations (RVCL-S) is an adult-onset rare monogenic microvasculopathy. Its typical neuroimaging features are punctate white matter lesions or pseudotumor alterations. RVCL-S is often under-recognized and misdiagnosed because of its rarity and similar imaging manifestations to multiple sclerosis or brain malignant mass.Case presentationHere we report a case of a 36-year-old Chinese man who developed multiple tumefactive brain lesions spanning over two years leading to motor aphasia, cognitive decline, and limb weakness. He also presented with slight vision loss, and fundus fluorescein angiography indicated retinal vasculopathy. He underwent brain biopsies twice and showed no evidence of malignancy. Given the family history that his father died of a brain mass of unclear etiology, RVCL-S was suspected, and genetic analysis confirmed the diagnosis with a heterozygous insertion mutation in the three-prime repair exonuclease 1 gene. He was given courses of corticosteroids and cyclophosphamide but received little response.ConclusionsThe present case is one of the few published reports of RVCL-S with two-year detailed imaging data. Serial magnetic resonance images showed the progression pattern of the lesions. Our experience emphasizes that a better understanding of RVCL-S and considering it as a differential diagnosis in patients with tumefactive brain lesions may help avoid unnecessary invasive examinations and make an earlier diagnosis.

Type I interferonopathies with novel compound heterozygous TREX1 mutations in two siblings with different symptoms responded to tofacitinib

BackgroundType I interferonopathies are a group of rare autoimmune diseases characterised by excessive activation of type I interferon that leads to disturbances in immune function. Three prime repair exonuclease 1 (TREX1) is an important exonuclease and plays an important role in DNA damage repair. TREX1 mutations are associated with many type I interferonopathies. Studies have been published on the effectiveness of tofacitinib in the treatment of type I interferonopathies. The aim of this study is to identify the pathogenic variation in a Chinese family with type I interferonopathies and to observe the therapeutic effects of tofacitinib.MethodsA Chinese family with two members with type I interferonopathies was investigated. Whole exome sequencing and Sanger sequencing were applied for mutation screening using peripheral blood DNA of the patient and her family members. Sequencing results were analysed using bioinformatics software tools including VarCards and PolyPhen-2. Close clinical follow-up and observation were used to record changes in the disease before and after treatment with tofacitinib.ResultsCompound heterozygous variants of TREX1 were observed in the patient’s genome. One was a missense variant (NM_016381; c.C227T; p.Ala76Val) from the patient’s father, and the other was a frameshift variant (NM_016381; c.458dupA; p.Gln153Glnfs*3) from the patient’s mother. One of the proband’s elder brothers with similar skin lesions also carried these two variants. This brother of the proband had more serious cutaneous involvement with the comorbidity of cerebral palsy. These TREX1 variants have not been reported in previous studies and are predicted to be highly pathogenic. The proband was given tofacitinib that led to a marked improvement.ConclusionsWe identified two novel complex heterozygous variants in the TREX1 gene, which may underlie the molecular pathogenesis of the type I interferonopathies observed in members of this family. Tofacitinib could be an alternative treatment for this disease.

Carboplatin activates the cGAS-STING pathway by upregulating the TREX-1 (three prime repair exonuclease 1) expression in human melanoma

ABSTRACT Human melanoma is a highly aggressive type of cancer, causing significant mortalities despite the advances in treatment. Carboplatin is a cisplatin analog necessary for the treatment of various cancers and can also be used to treat human melanoma. We assessed the effects and mechanisms leading to inhibited proliferation and induced apoptosis of human melanoma after carboplatin therapy in vitro and in vivo. TREX1, cGAS/STING, and apoptotic protein expressions were determined through RT-qPCR and western blot assays. Cell proliferation was validated through MTT assays. The study used SK-MEL-1 and SK-HEP-1 tumor cell line inoculations along with carboplatin in nude mice to validate the results. The TREX1 levels were down-regulated in human melanoma cell lines. TREX1 overexpression-induced apoptosis and decreased proliferation in the human melanoma cell lines. TREX1 overexpression also activated the cGAS/STING pathway to induce apoptosis and decrease cell growth. Carboplatin activated TREX1, induced apoptosis, and decreased proliferation in the human melanoma cancerous cell lines. Finally, carboplatin reduced the in-vivo tumor size and weight. In conclusion, the study revealed that carboplatin activated TREX1 and cGAS/STING pathways to upregulate apoptosis. The work also provides in vitro and in vivo evidence to understand the effects of TREX overexpression on tumor suppression. Targeting of TREX1/cGAS/STING pathway could be an effective therapeutic alternative to human melanoma.

Rapidly Progressing Early Puberty in a Boy with Bilateral Basal Ganglia Germinoma and TREX1 Variant

Introduction: Organic lesions, including brain tumors, should be suspected in boys with precocious puberty. However, it is not usually suspected in children with early puberty. Case Presentation: Here we present an extremely rare case of rapidly progressing early puberty with basal ganglia germinoma coupled with three-prime repair exonuclease 1 (TREX1) variant. This was a 10-year-old-boy with borderline mental retardation and rapidly progressing puberty. Physical examination revealed 10 mL testes (Tanner stage 3 for genital development), and his bone age was that of a 12-year old boy. Laboratory findings showed abnormally elevated serum β-human chorionic gonadotropin (23.0 mIU/mL; reference, 0-10 mIU/mL), and suppressed LH level (

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.

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.

TREX1 531C>T Polymorphism is Associated with High Proviral Load Levels in HTLV-1-Infected Persons.

Human T-lymphotropic virus type 1 (HTLV-1) deregulates the immune system and cell cycle, resulting in loss of immune tolerance and disease, including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Three prime repair exonuclease 1 (TREX1) maintains innate immune tolerance of the host and host-cell permissiveness to retroviral infections. TREX1 polymorphisms may influence the course of infection and autoimmune manifestations. The influence of TREX1 531C/T polymorphism was investigated in HTLV-1 infection and development of symptoms among 151 persons infected with HTLV-1 (32 HAM/TSP, 19 rheumatologic manifestations, two dermatitis, five more than one diagnosis, two probable HAM/TSP, and 91 asymptomatic individuals) and 100 uninfected persons in the control group. Polymorphism genotyping and proviral load quantification were performed by real-time polymerase chain reaction (PCR) and antinuclear antibodies (ANAs) were screened by an indirect immunofluorescence assay. No statistically significant difference was found in polymorphism genotype and allele frequencies between the infected and control groups. HAM/TSP patients showed higher frequency of TT genotype than asymptomatic persons (p = 0.0339). Proviral load was significantly higher among individuals with CT/TT genotypes and CC genotype carriers had lower proviral load and higher levels of proinflammatory cytokines. ANAs were present only in the HAM/TSP group. TREX1 531C>T polymorphism seems to be associated with TREX-1 regulation and HTLV-1 infection.

Suppression of mosaic mutation by co-delivery of CRISPR associated protein 9 and three-prime repair exonuclease 2 into porcine zygotes via electroporation.

Gene-modified animals, including pigs, can be generated efficiently by introducing CRISPR associated protein 9 (CRISPR/Cas9) into zygotes. However, in many cases, these zygotes tend to become mosaic mutants with various different mutant cell types, making it difficult to analyze the phenotype of gene-modified founder animals. To reduce the mosaic mutations, we introduced three-prime repair exonuclease 2 (Trex2), an exonuclease that improves gene editing efficiency, into porcine zygotes along with CRISPR/Cas9 via electroporation. Although the rate of porcine blastocyst formation decreased due to electroporation (25.9 ± 4.6% vs. 41.2 ± 2.0%), co-delivery of murine Trex2 (mTrex2) mRNA with CRISPR/Cas9 did not affect it any further (25.9 ± 4.6% vs. 31.0 ± 4.6%). In addition, there was no significant difference in the diameter of blastocysts carrying CRISPR/Cas9 (164.7 ± 10.2 μm), and those with CRISPR/Cas9 + mTrex2 (151.9 ± 5.1 μm) as compared to those from the control group (178.9 ± 9.0 μm). These results revealed that mTrex2 did not affect the development of pre-implantation embryo. We also found bi-allelic, as well as mono-allelic, non-mosaic homozygous mutations in the blastocysts. Most importantly, co-delivery of mTrex2 mRNA with CRISPR/Cas9 increased non-mosaic mutant blastocysts (29.3 ± 4.5%) and reduced mosaic mutant blastocysts (70.7 ± 4.5%) as compared to CRISPR/Cas9 alone (5.6 ± 6.4% and 92.6 ± 8.6%, respectively). These data suggest that the co-delivery of CRISPR/Cas9 and mTrex2 is a useful method to suppress mosaic mutation.

TREX1 variants in Sjogren's syndrome related lymphomagenesis

Genetic variants of the three-prime repair exonuclease 1 (TREX1) -an exonuclease involved in DNA repair and degradation- have been previously found to increase susceptibility to Aicardi Goutieres syndrome, familial chilblain lupus and systemic lupus erythematosus. We aimed to explore whether TREX1 common variants could influence the risk of primary Sjogren’s syndrome (SS) and SS-related lymphoma. Three single nucleotide polymorphisms (SNPs) of the TREX1 gene (rs11797, rs3135941 and rs3135945) were evaluated in 229 SS, 89 SS-lymphoma (70 SS-MALT and 19 SS non-MALT) and 240 healthy controls by PCR-based assays. In available 52 peripheral blood and 26 minor salivary gland tissues from our SS cohort, mRNA expression of type I interferon (IFN) related genes and TREX1 was determined by real-time PCR. Significantly decreased prevalence of rs11797 A minor allele was detected in SS patients complicated by non-MALT lymphoma compared to controls (ΟR [95% CI]: 0.4 [0.2–0.9], p-value: 0.02). SS patients carrying the rs11797 AA genotype had increased type I IFN related gene mRNA expression in minor salivary gland tissues. These data support genetically related dampened type I IFN production as an additional mechanism for SS-related lymphomagenesis.

RETRACTED ARTICLE: Nucleic acid sensing activates the innate cytosolic surveillance pathway and promotes parasite survival in visceral leishmaniasis

Microbial pattern recognition critically contributes to innate response, both at extracellular and intracellular cytosolic surveillance pathway (CSP) interface. However, the role of pattern recognition by host innate receptors in CSP is poorly understood in Leishmania donovani infection. Here, we have demonstrated that cytosolic targeting of L.donovani DNA (Ld-DNA) inhibits macrophage responsiveness to IFNɣ, through decreased MHC-II expression and lowered pSTAT1 (Y701) levels, involving host three-prime repair exonuclease-1 (TREX-1). The Ld-DNA potently induced type-1 IFNs, i.e. significant over-production of IFNβ through activation of the IRF pathway. Interestingly, knockdown of TRIF or MyD88 expression in macrophages had no effect on cytosolic Ld-DNA transfection-mediated IFN-β production, indicating involvement of a TLR independent pathway. Contrastingly, Ld-DNA failed to induce IFNβ in both TBK-1 and IRF3KO knockout macrophages. Although IFNβ was not induced by Ld-DNA in STING- knockout macrophages, STING alone was not enough for the induction. Evidently, besides STING, Ld-DNA recognition for induction of IFNβ critically required cytosolic cyclic GMP-AMP synthase (cGAS). Furthermore, the cGAS dependent targeting of Ld-DNA induced IFNβ over-production that contributed to antimony resistance in L.donovani infection. We provide the first evidence that enhanced cytosolic sensing of Ld-DNA in infection by antimony resistant (SBR-LD), but not antimony sensitive L.donovani strains (SBS-LD), was critically regulated by host MDRs, multi drug resistant associated protein 1 (MRP 1) and permeability glycoprotein (P-gp) in macrophages. Collectively, our results disclose Ld-DNA as a vital pathogen associated molecular pattern (PAMP) driving host Type-I IFN responses and antimony resistance. The findings may help in future development of policies for novel anti-leishmanial therapeutics.

Three Prime Repair Exonuclease 1 (TREX1) expression correlates with cervical cancer cells growth in vitro and disease progression in vivo

Alterations in specific DNA damage repair mechanisms in the presence of human papillomavirus (HPV) infection have been described in different experimental models. However, the global effect of HPV on the expression of genes involved in these pathways has not been analyzed in detail. In the present study, we compared the expression profile of 135 genes involved in DNA damage repair among primary human keratinocytes (PHK), HPV-positive (SiHa and HeLa) and HPV-negative (C33A) cervical cancer derived cell lines. We identified 9 genes which expression pattern distinguishes HPV-positive tumor cell lines from C33A. Moreover, we observed that Three Prime Repair Exonuclease 1 (TREX1) expression is upregulated exclusively in HPV-transformed cell lines and PHK expressing HPV16 E6 and E7 oncogenes. We demonstrated that TREX1 silencing greatly affects tumor cells clonogenic and anchorage independent growth potential. We showed that this effect is associated with p53 upregulation, accumulation of subG1 cells, and requires the expression of E7 from high-risk HPV types. Finally, we observed an increase in TREX1 levels in precancerous lesions, squamous carcinomas and adenocarcinomas clinical samples. Altogether, our results indicate that TREX1 upregulation is important for cervical tumor cells growth and may contribute with tumor establishment and progression.

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.

TREX1 is expressed by microglia in normal human brain and increases in regions affected by ischemia.

BackgroundMutations in the three‐prime repair exonuclease 1 (TREX1) gene have been associated with neurological diseases, including Retinal Vasculopathy with Cerebral Leukoencephalopathy (RVCL). However, the endogenous expression of TREX1 in human brain has not been studied.MethodsWe produced a rabbit polyclonal antibody (pAb) to TREX1 to characterize TREX1 by Western blotting (WB) of cell lysates from normal controls and subjects carrying an RVCL frame‐shift mutation. Dual staining was performed to determine cell types expressing TREX1 in human brain tissue. TREX1 distribution in human brain was further evaluated by immunohistochemical analyses of formalin‐fixed, paraffin‐embedded samples from normal controls and patients with RVCL and ischemic stroke.ResultsAfter validating the specificity of our anti‐TREX1 rabbit pAb, WB analysis was utilized to detect the endogenous wild‐type and frame‐shift mutant of TREX1 in cell lysates. Dual staining in human brain tissues from patients with RVCL and normal controls localized TREX1 to a subset of microglia and macrophages. Quantification of immunohistochemical staining of the cerebral cortex revealed that TREX1+ microglia were primarily in the gray matter of normal controls (22.7 ± 5.1% and 5.5 ± 1.9% of Iba1+ microglia in gray and white matter, respectively) and commonly in association with the microvasculature. In contrast, in subjects with RVCL, the TREX1+ microglia were predominantly located in the white matter of normal appearing cerebral cortex (11.8 ± 3.1% and 38.9 ± 5.8% of Iba1+ microglia in gray and white matter, respectively). The number of TREX1+ microglia was increased in ischemic brain lesions in central nervous system of RVCL and stroke patients.Conclusions TREX1 is expressed by a subset of microglia in normal human brain, often in close proximity to the microvasculature, and increases in the setting of ischemic lesions. These findings suggest a role for TREX1+ microglia in vessel homeostasis and response to ischemic injury.

TREX1 D18N mice fail to process erythroblast DNA resulting in inflammation and dysfunctional erythropoiesis

Anaemia is commonly observed in chronic inflammatory conditions, including systemic lupus erythematosus (SLE), where ∼50% of patients display clinical signs of anaemia. Mutation at the aspartate residue 18 of the three prime repair exonuclease 1 (TREX1) gene causes a monogenic form of cutaneous lupus in humans and the genetically precise TREX1 D18N mice recapitulate a lupus-like disease. TREX1 degrades single- and double-stranded DNA (dsDNA), and the link between failed DNA degradation by nucleases, including nucleoside-diphosphate kinases (NM23H1/H2) and Deoxyribonuclease II (DNase II), and anaemia prompted our studies to investigate whether TREX1 dysfunction contributes to anaemia. Utilizing the TREX1 D18N mice we demonstrate that (1) TREX1 mutant mice develop normocytic normochromic anaemia and (2) TREX1 exonuclease participates in the degradation of DNA originating from erythroblast nuclei during definitive erythropoiesis. Gene expression, hematocrit, hemoglobin, immunohistochemistry (IHC) and flow cytometry were used to quantify dysfunctional erythropoiesis. An altered response to induced anaemia in the TREX1 D18N mice was determined through IHC, flow cytometry, and interferon-stimulated gene (ISG) expression analysis of the liver, spleen and erythroblastic islands (EBIs). IHC, flow cytometry, and ISG expression studies were performed in vitro to determine the role of TREX1 in the degradation of erythroblast DNA within EBIs. The TREX1 D18N mice exhibit altered erythropoiesis including a 20% reduction in hematocrit, 10–20 fold increased erythropoietic gene expression levels in the spleen and phenotypic signs of normocytic normochromic anaemia. Anaemia in TREX1 D18N mice is accompanied by increased erythropoietin (Epo), normal hepcidin levels and the TREX1 D18N mice display an inappropriate response to anaemic challenge. Enhanced ISG expression results from failed processing and subsequent sensing of undegraded erythroblast DNA in EBIs. TREX1 participates in the degradation of erythroblast DNA in the EBI and TREX1 D18N mice exhibit a normocytic normochromic anaemia.

Abstract 501: Anemia of Inflammation in an Exonuclease Deficient Lupus Mouse

Background: Patients with chronic inflammatory conditions, such as autoimmune diseases, infections, and cancer, often display anemia of inflammation (AI). 50% of patients with systemic lupus erythematosus display clinical signs of anemia. Mutations at aspartate residue 18 of the three prime repair exonuclease 1 ( TREX1 ) gene, which codes for the primary mammalian exonuclease, cause a monogenic form of cutaneous lupus erythematosus in humans and the genetically precise TREX1 D18N mice recapitulate a lupus-like disease. Objective: The link between failed DNA degradation during erythropoiesis and anemia prompted our studies to investigate whether TREX1 dysfunction might contribute to AI. Methods: AI in TREX1 D18N mice was determined through analysis of hematocrit, hemoglobin, gene expression analysis, immunohistochemistry (IHC) and flow cytometry. Response to phenylhydrazine (PHZ) induced anemia and activation of DNA sensing pathways were determined through IHC and interferon stimulatory gene (ISG) expression analysis of the liver, spleen and erythroblastic islands. Results: The TREX1 D18N mice exhibit altered erythropoiesis including reduced hematocrit, increased erythropoietic and hematotoxic gene expression levels in the spleen (~10-20 fold) and phenotypic signs of normocytic normochromic anemia. Anemia in TREX1 lupus mice is accompanied by increased erythropoietin (Epo) and extramedullary hematopoiesis. Furthermore, the TREX1 D18N mice display an inappropriate response to anemic challenge. Enhanced ISG expression is apparent and results from failed processing and sensing of undegraded erythroblast DNA. Conclusion: These results support a critical role for TREX1 in the degradation of erythroblast DNA and identify a distinct AI phenotype in TREX1 D18N mice.

Structural basis for overhang excision and terminal unwinding of DNA duplexes by TREX1.

Three prime repair exonuclease 1 (TREX1) is an essential exonuclease in mammalian cells, and numerous in vivo and in vitro data evidenced its participation in immunity regulation and in genotoxicity remediation. In these very complicated cellular functions, the molecular mechanisms by which duplex DNA substrates are processed are mostly elusive because of the lack of structure information. Here, we report multiple crystal structures of TREX1 complexed with various substrates to provide the structure basis for overhang excision and terminal unwinding of DNA duplexes. The substrates were designed to mimic the intermediate structural DNAs involved in various repair pathways. The results showed that the Leu24-Pro25-Ser26 cluster of TREX1 served to cap the nonscissile 5′-end of the DNA for precise removal of the short 3′-overhang in L- and Y-structural DNA or to wedge into the double-stranded region for further digestion along the duplex. Biochemical assays were also conducted to demonstrate that TREX1 can indeed degrade double-stranded DNA (dsDNA) to a full extent. Overall, this study provided unprecedented knowledge at the molecular level on the enzymatic substrate processing involved in prevention of immune activation and in responses to genotoxic stresses. For example, Arg128, whose mutation in TREX1 was linked to a disease state, were shown to exhibit consistent interaction patterns with the nonscissile strand in all of the structures we solved. Such structure basis is expected to play an indispensable role in elucidating the functional activities of TREX1 at the cellular level and in vivo.