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Epigenetic and Transcriptional Regulation of IRAK-M Expression in Macrophages Konstantina Lyroni, Andreas Patsalos, Maria G

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Epigenetic and Transcriptional Regulation of IRAK-M Expression in Macrophages Konstantina Lyroni, Andreas Patsalos, Maria G Epigenetic and Transcriptional Regulation of IRAK-M Expression in Macrophages Konstantina Lyroni, Andreas Patsalos, Maria G. Daskalaki, Christina Doxaki, Birte Soennichsen, Mike Helms, Ioannis This information is current as Liapis, Vassiliki Zacharioudaki, Sotirios C. Kampranis and of September 25, 2021. Christos Tsatsanis J Immunol 2017; 198:1297-1307; Prepublished online 23 December 2016; doi: 10.4049/jimmunol.1600009 Downloaded from http://www.jimmunol.org/content/198/3/1297 Supplementary http://www.jimmunol.org/content/suppl/2016/12/23/jimmunol.160000 Material 9.DCSupplemental http://www.jimmunol.org/ References This article cites 56 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/198/3/1297.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 25, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Epigenetic and Transcriptional Regulation of IRAK-M Expression in Macrophages Konstantina Lyroni,* Andreas Patsalos,*,1 Maria G. Daskalaki,† Christina Doxaki,* Birte Soennichsen,‡,2 Mike Helms,‡,3 Ioannis Liapis,* Vassiliki Zacharioudaki,* Sotirios C. Kampranis,† and Christos Tsatsanis* During macrophage activation, expression of IL-1R–associated kinase (IRAK)-M is induced to suppress TLR-mediated responses and is a hallmark of endotoxin tolerance. Endotoxin tolerance requires tight regulation of genes occurring at the transcriptional and epigenetic levels. To identify novel regulators of IRAK-M, we used RAW 264.7 macrophages and performed a targeted RNA interference screen of genes encoding chromatin-modifying enzymes, signaling molecules, and transcription factors involved in macrophage activation. Among these, the transcription factor CCAAT/enhancer binding protein (C/EBP)b, known to be involved in macrophage inactivation, was necessary for the induction of IRAK-M expression. Chromatin immunoprecipitation showed that Downloaded from C/EBPb was recruited to the IRAK-M promoter following LPS stimulation and was indispensable for IRAK-M transcriptional activation. Among histone 3–modifying enzymes, our screen showed that knockdown of the histone 3 lysine 27 (H3K27) meth- yltransferase and part of the polycomb recessive complex 2, enhancer of Zeste 2, resulted in IRAK-M overexpression. In contrast, knockdown of the H3K27 demethylase ubiquitously transcribed tetratricopeptide repeat X chromosome suppressed the induction of IRAK-M in response to LPS stimulation. Accordingly, we demonstrated that H3K27 on the IRAK-M promoter is trimethylated in unstimulated cells and that this silencing epigenetic mark is removed upon LPS stimulation. Our data propose a mechanism for http://www.jimmunol.org/ IRAK-M transcriptional regulation according to which, in the naive state, polycomb recessive complex 2 repressed the IRAK-M promoter, allowing low levels of expression; following LPS stimulation, the IRAK-M promoter is derepressed, and transcription is induced to allow its expression. The Journal of Immunology, 2017, 198: 1297–1307. ffective recognition and elimination of pathogenic mi- associated molecular patterns on microorganisms and initiate croorganisms is vital in mammals, and tight control of proinflammatory responses (1). Bacterial LPS is sensed by TLR4 these mechanisms is essential for homeostasis. Hence, (2) on the surface of phagocytes to initiate the signaling pathway E by guest on September 25, 2021 innate immune responses have evolved to protect organisms from that leads to the activation of MyD88/PI3K/AKT and NF-kB (3). bacteria, fungi, and viruses using TLRs that recognize pathogen- TLR4 signal transduction is mediated by a family of serine/ threonine kinases called IL-1R–associated kinases (IRAKs) (4). IRAK-3, also known as IRAK-M, is the only member of the *Laboratory of Clinical Chemistry, Medical School, University of Crete, Heraklion 71003, Crete, Greece; †Laboratory of Biochemistry, Medical School, University of Crete, Heraklion IRAKs that lacks kinase activity; hence, it acts as a suppressor of 71003, Crete, Greece; and ‡Cenix Bioscience GmbH, D01307 Dresden, Germany the TLR4 signaling pathway (5). This renders IRAK-M important 1Current address: University of Debrecen Medical and Health Science Center, Debre- for dampening the inflammatory response after pathogen elimi- cen, Hungary. nation and is indispensable for the establishment for endotoxin 2Current address: Life Science Inkubator Sachsen GmbH & Co. KG, Dresden, Germany. tolerance (5). The role of IRAK-M in human disease also was 3Current address: Sanofi-Aventis Deutschland GmbH, Frankfurt am Main, Germany. established. The importance of IRAK-M in disease development 2/2 ORCIDs: 0000-0001-7446-0999 (K.L.); 0000-0002-9022-4985 (A.P.); 0000-0001- and regulation was highlighted in studies using IRAK-M mice. 6208-1684 (S.C.K.); 0000-0003-1214-4151 (C.T.). In addition to its contribution to endotoxin tolerance, IRAK-M is a Received for publication January 4, 2016. Accepted for publication November 21, key player in different host defense mechanisms, including viral 2016. and bacterial pneumonia (6–8). In humans, IRAK-M was shown to This work was supported by Research Projects for Excellence IKY/Siemens Contract be a key player in autoimmune diseases, such as systemic lupus 23416, Greek national and European Union funds under Aristeia Grant 2071, and by 2/2 the European Union under FP7 Industry-Academia Partnerships and Pathways erythematosus (9). IRAK-M NOD mice are more prone to the (GA-2008-230725-TACIT). development of type I diabetes and the related glucose intolerance Address correspondence and reprint requests to Prof. Christos Tsatsanis or Prof. as a result of the altered function of dendritic cells (10). IRAK-M Sotirios C. Kampranis, Laboratory of Clinical Chemistry, School of Medicine, Uni- is also involved in obesity-induced atherogenesis (11), obesity- versity of Crete, P.O. Box 2203, Heraklion 71003, Crete, Greece (C.T.) or Laboratory of Biochemistry, School of Medicine, University of Crete, P.O. Box 2203, Heraklion induced metabolic inflammation (12), and adiponectin-induced 71003, Crete, Greece (S.C.K.). E-mail addresses: [email protected] (C.T.) or regulation of macrophage sensitivity to LPS (13), suggesting that [email protected] (S.C.K.) it can be activated under different conditions and diverse down- The online version of this article contains supplemental material. stream signaling cascades. Abbreviations used in this article: C/EBP, CCAAT/enhancer binding protein; ChIP, Despite its importance and the relatively good understanding of chromatin immunoprecipitation; Ezh2, enhancer of Zeste 2; H3, histone 3; H3K27, histone 3 lysine 27; H3K27me3, trimethylation of lysine 27 on histone 3; IP, immu- IRAK-M function, little is known about the transcriptional regulation noprecipitation; IRAK, IL-1R–associated kinase; PRC2, polycomb recessive com- of the IRAK-M gene. The IRAK-M promoter is not fully charac- plex 2; RT, room temperature; siRNA, small interfering RNA; UTX, ubiquitously terized, and the only firm association between specific transcription transcribed tetratricopeptide repeat X chromosome. factors and IRAK-M expression was shown in promoter-cloning as- Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 says (14). According to that report, the AP1 complex and NF-kB, www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600009 1298 IRAK-M TRANSCRIPTIONAL AND EPIGENETIC REGULATION both central transcriptional complexes induced upon TLR4 activation, ABI PRISM 7900HT Fast Real-Time PCR System with 384-Well Block Module were shown to bind to and activate the IRAK-M promoter. for a maximum of 45 cycles, as follows: start steps of 30 min at 50˚C and 15 min Transcriptional regulation of genes involves transcription factors at 95˚C and repeat steps of 15 s at 95˚C, 30 s at 60˚C, and 30 s at 72˚C. The primers used were EZH2 forward: 59-GTGCAGTTATTCCTTCCATGC-39 (activators and repressors) (15), as well as chromatin-modifying en- and reverse: 59-ACGCTCAGCAGTAAGAGCAG-39;UTXforward:59- zymes (histone methyltransferases, histone acetyl transferases, histone GCACCACCTCCAGTAGAACAA-39 and reverse: 59-GTCTCATTTGGT- deacetylases, and histone demethylases) that introduce epigenetic GTTGCTGCAT-39;C/EBPbforward:59-GACAAGCTGAGCGACGAGTA- marks on chromatin to activate or suppress transcription (16). One 39 and reverse: 59-GCTTGAACAAGTTCCGCAGG-39; actin-b forward: 59- GTCATCACTATTGGCAACGAGC-39 and reverse: 59-GCACTGTGTTGGCA- major silencing epigenetic mark is the trimethylation of lysine 27 on TAGAGGTC-39; and IRAK-M forward: 59-TTCCTGGCACGTTCGAATCA-39 histone 3 (H3K27me3), which is catalyzed by the enhancer of Zeste 2 and reverse: 59-CGCTGCAGCAAAATCCGTTA-39. (Ezh2) methyl transferase, part of the
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