Drosophila EYA regulates the immune response against DNA through an evolutionarily conserved threonine phosphatase motif Xi Liu, Teruyuki Sano, Yongsheng Guan, Shigekazu Nagata, Jules Hoffmann, Hidehiro Fukuyama To cite this version: Xi Liu, Teruyuki Sano, Yongsheng Guan, Shigekazu Nagata, Jules Hoffmann, et al.. Drosophila EYA regulates the immune response against DNA through an evolutionarily conserved threonine phosphatase motif. PLoS ONE, Public Library of Science, 2012, 7 (8), 10.1371/journal.pone.0042725. hal-02346287 HAL Id: hal-02346287 https://hal.archives-ouvertes.fr/hal-02346287 Submitted on 4 Nov 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Drosophila EYA Regulates the Immune Response against DNA through an Evolutionarily Conserved Threonine Phosphatase Motif Xi Liu1,2., Teruyuki Sano4,5.¤, Yongsheng Guan1., Shigekazu Nagata4,5*, Jules A. Hoffmann2,3, Hidehiro Fukuyama1* 1 INSERM Equipe Avenir, CNRS UPR9022, Institut de Biologie Mole´culaire et Cellulaire, Strasbourg, France, 2 University of Strasbourg, Strasbourg, France, 3 CNRS UPR9022, Institut de Biologie Mole´culaire et Cellulaire, Strasbourg, France, 4 Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto, Japan, 5 Solution Oriented Research for Science and Technology, and Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Kyoto, Japan Abstract Innate immune responses against DNA are essential to counter both pathogen infections and tissue damages. Mammalian EYAs were recently shown to play a role in regulating the innate immune responses against DNA. Here, we demonstrate that the unique Drosophila eya gene is also involved in the response specific to DNA. Haploinsufficiency of eya in mutants deficient for lysosomal DNase activity (DNaseII) reduces antimicrobial peptide gene expression, a hallmark for immune responses in flies. Like the mammalian orthologues, Drosophila EYA features a N-terminal threonine and C-terminal tyrosine phosphatase domain. Through the generation of a series of mutant EYA fly strains, we show that the threonine phosphatase domain, but not the tyrosine phosphatase domain, is responsible for the innate immune response against DNA. A similar role for the threonine phosphatase domain in mammalian EYA4 had been surmised on the basis of in vitro studies. Furthermore EYA associates with IKKb and full-length RELISH, and the induction of the IMD pathway-dependent antimicrobial peptide gene is independent of SO. Our data provide the first in vivo demonstration for the immune function of EYA and point to their conserved immune function in response to endogenous DNA, throughout evolution. Citation: Liu X, Sano T, Guan Y, Nagata S, Hoffmann JA, et al. (2012) Drosophila EYA Regulates the Immune Response against DNA through an Evolutionarily Conserved Threonine Phosphatase Motif. PLoS ONE 7(8): e42725. doi:10.1371/journal.pone.0042725 Editor: Justin Kumar, Indiana University, United States of America Received April 2, 2012; Accepted July 11, 2012; Published August 15, 2012 Copyright: ß 2012 Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding provided by Avenir, Institut National de la Sante´ et de la Recherche Me´dicale, France (HF), Chinese Scholarship Council, China (XL), Solution Oriented Research for Science and Technology, Japan Science and Technology Agency, Japan (SN) and Centre National de la Recherche Scientifique, France (HF and JH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (HF); [email protected] (SN) . These authors equally contributed equally to this work. ¤ Current address: Molecular Pathology, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, New York, United States of America Introduction deficient animals or cells to further our understanding on the DNA sensing mechanisms [13] and on the downstream signaling events. In humans, innate inflammation is frequently linked to various DNaseII is an evolutionarily conserved acid DNase localized in types of diseases, namely to autoimmune diseases [1]. However, it lysosomes [14]. Macrophages in the fetal liver and thymus of is still unclear in many instances how inflammation is initiated and DNaseII2/2 mice cannot digest DNA of engulfed dead cells, or chronically maintained. Pathogen invasions and/or subsequent DNA of nuclei expelled from erythroid precursors [15,16]. This changes of cellular integrity can cause inflammation and DNA has results in their production of pro-inflammatory cytokines such as been reported to be a strong immune stimulator under such interferon-b (IFN-b) and tumor necrosis factor-a (TNF-a) [17,18]. conditions [2,3]. How cells recognize, and respond to, DNA are As a result of excessive cytokine production, DNaseII2/2 mice among the challenging questions in the field of inflammation, suffer from anemia during embryonic stages and later, as adults, particularly in humans. from polyarthritis. Crosses of DNaseII2/2 mice with mice deficient Several DNA recognition molecules have been recently for various immune response-related signaling molecules conclu- identified. Among these are TLR-9 [4], Absent in Melanoma 2 sively showed that (1) the DNA-dependent IFN-b gene expression (AIM2) [5,6], Interferon gamma-inducible protein 16 (IFI16) [7], is TLR-independent and IRF-3/7-dependent and that (2) the DNA-dependent activator of IRFs (DAI) [8], High-mobility group DNA-dependent TNF-a gene expression is both independent of box protein 1 (HMGBP1) [9], and Leucine-rich repeat flightless- TLR- and IRF-3/7- signaling [19,20]. interacting protein 1 (LRRFIP1) [10]. These identifications were To elucidate the molecular mechanisms of DNA sensing in routinely based on the analysis of synthetic DNA-mediated DNaseII2/2 mice, Okabe et al. recently performed expression immune reactions [11,12]. Recent studies have addressed DNaseII cloning and reported that Eyes absent 4 (EYA4) enhances the PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e42725 Drosophila EYA Immune Signaling innate immune responses against DNA by activating IRF3 and (eyacli-IID) that causes truncation of the EYA protein at amino acid NF-kB [21]. EYA was originally identified in Drosophila as a 335 [33]. Eya1 or eya2 flies develop to adults deprived of eyes. Eyacli-IID transcription factor that is essential for eye development [22]. flies are lethal at the embryonic stage. The DNaseIIlo allele has a Mammals have four EYA paralogs, EYA1-4. Mutations in human missense G-to-A mutation at nucleotide 668, converting a Serine EYA1 cause branchio-oto-renal (BOR) syndrome, an autosomal residue to an Aspargine residue at amino acid position 223, which dominant genetic disorder, affecting necks, ears, and kidneys results in a reduction of the acid DNase activity [33]. DNaseII [23,24,25]. EYA12/2 mice also show renal abnormalities and a mutants develop normally to the adult stage. Interestingly, these flies conductive hearing loss similar to BOR syndrome [26]. EYA32/2 constitutively express the antimicrobial peptide Attacin A [31]. We mice have a minor defect in locomotion with some deficits of generated eya1;DNaseIIlo, eya2;DNaseIIlo,andeyacli-IID;DNaseIIlo double respiratory, muscle and heart functions [27], and EYA42/2 mice mutant flies and monitored Attacin A expression. We noted that flies show a defect in eustachian tube and middle ear [28]. EYA22/2 carrying homozygous eya and DNaseIIlo double mutations died at the mice have not yet been generated. Several groups recently showed pupal stage and we therefore used adult eya/+;DNaseIIlo flies to that EYA proteins carry C-terminally an evolutionarily conserved evaluate the effect of the eya gene in this context. As shown in Fig. 1A, domain with tyrosine phosphatase activity [29,30]. The detailed DNaseII mutant flies constitutively expressed Attacin A, as previously biochemical analysis by Okabe et al. [21]. Further identified a N- reported [31]. In contrast, a heterozygous eya1 mutation combined terminal threonine phosphatase domain in mouse EYA4, and with DNaseII deficiency significantly reduced Attacin A mRNA levels. showed that this domain is responsible for the innate immune Similar results were obtained with the eya2 and eyacli-IID alleles responses observed against DNA. (Fig. 1A). To exclude possible effects of genetic background and/or Drosophila has a single orthologue of mammalian DNaseII. aberrant development due to the gene deficiency/insufficiency, we Interestingly, Mukae et al. found that flies carrying a hypomorphic generated an inducible eya/DNaseII knockdown system by RNA mutation in the DNaseII gene (DNaseIIlo)