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Transcriptional Repression of IFN Regulatory Factor 7 by MYC Is Critical for Type I IFN Production in Human Plasmacytoid Dendrit

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Transcriptional Repression of IFN Regulatory Factor 7 by MYC Is Critical for Type I IFN Production in Human Plasmacytoid Dendrit Transcriptional Repression of IFN Regulatory Factor 7 by MYC Is Critical for Type I IFN Production in Human Plasmacytoid Dendritic Cells This information is current as of September 28, 2021. Tae Whan Kim, Seunghee Hong, Yin Lin, Elise Murat, HyeMee Joo, Taeil Kim, Virginia Pascual and Yong-Jun Liu J Immunol 2016; 197:3348-3359; Prepublished online 14 September 2016; doi: 10.4049/jimmunol.1502385 Downloaded from http://www.jimmunol.org/content/197/8/3348 Supplementary http://www.jimmunol.org/content/suppl/2016/09/14/jimmunol.150238 http://www.jimmunol.org/ Material 5.DCSupplemental References This article cites 74 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/197/8/3348.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 28, 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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Transcriptional Repression of IFN Regulatory Factor 7 by MYC Is Critical for Type I IFN Production in Human Plasmacytoid Dendritic Cells Tae Whan Kim,*,1 Seunghee Hong,*,1 Yin Lin,* Elise Murat,* HyeMee Joo,* Taeil Kim,†,2 Virginia Pascual,* and Yong-Jun Liu*,† Type I IFNs are crucial mediators of human innate and adaptive immunity and are massively produced from plasmacytoid dendritic cells (pDCs). IFN regulatory factor (IRF)7 is a critical regulator of type I IFN production when pathogens are detected by TLR 7/9 in pDC. However, hyperactivation of pDC can cause life-threatening autoimmune diseases. To avoid the deleterious effects of aberrant pDC activation, tight regulation of IRF7 is required. Nonetheless, the detailed mechanisms of how IRF7 transcription is regulated in pDC are still elusive. MYC is a well-known highly pleiotropic transcription factor; however, the role of MYC in pDC function is not Downloaded from well defined yet. To identify the role of transcription factor MYC in human pDC, we employed a knockdown technique using human pDC cell line, GEN2.2. When we knocked down MYC in the pDC cell line, production of IFN-stimulated genes was dramatically increased and was further enhanced by the TLR9 agonist CpGB. Interestingly, MYC is shown to be recruited to the IRF7 promoter region through interaction with nuclear receptor corepressor 2/histone deacetylase 3 for its repression. In addition, activation of TLR9-mediated NF-kB and MAPK and nuclear translocation of IRF7 were greatly enhanced by MYC depletion. Pharmaceutical inhibition of MYC recovered IRF7 expression, further confirming the negative role of MYC in the antiviral response by pDC. http://www.jimmunol.org/ Therefore, our results identify the novel immunomodulatory role of MYC in human pDC and may add to our understanding of aberrant pDC function in cancer and autoimmune disease. The Journal of Immunology, 2016, 197: 3348–3359. lasmacytoid dendritic cells (pDCs) are specialized type I cells to trigger the protective defenses of the host and help eradicate IFN–producing immune cells that sense viral RNA and pathogens. However, uncontrolled type I IFN production has been im- P DNA through endosomal TLR7 and TLR9, respectively (1–3). plicated in several human autoimmune diseases, such as systemic lupus Type I IFNs, mainly IFN-a and IFN-b, are pleiotropic cytokines that erythematosus (SLE) (7, 8), rheumatoid arthritis (9, 10), inflam- have the most potent antiviral and antitumoral activity in humans (4–6). matory bowel disease (11), or psoriasis (12). To avoid the deleterious by guest on September 28, 2021 IFNs are released by host cells for communication between immune effects of aberrant pDC activation, tight regulation of type I IFN production in pDC is essential. Importantly, production of these type I IFNs in pDCs is mainly controlled by IFN regulatory factor (IRF)7 *Baylor Institute for Immunology Research, Dallas, TX 75204; and †Sanofi, Cambridge, MA 02139 (13). Regulation of IRF7 is complex and possesses many positive 1 and negative feedback mechanisms. Transcriptional, posttranscrip- T.W.K. and S.H. are cofirst authors. tional, translational, posttranslational, and epigenetic (acetylation and 2Current address: MedImmune, Gaithersburg, MD. methylation) regulation of IRF7 have been extensively studied (14– ORCID: 0000-0003-2156-334X (E.M.). 21). Nonetheless, detailed mechanisms of how IRF7 transcription Received for publication November 10, 2015. Accepted for publication August 22, 2016. and activation are directly regulated in pDC are still unclear. This work was supported by National Institutes of Health Grant R01AI097348-03 MYC is a well-known highly pleiotropic transcription factor. (to Y.-J.L.). MYC is a basic helix-loop-helix leucine zipper transcription factor T.W.K. designed research, performed experiments, analyzed data, and wrote the (22) and specifically binds to the DNA sequence 59-CACGTG-39 or paper; S.H. and Y.L. designed research, analyzed data, and wrote the paper; H.J. 9 9 and E.M. performed experiments; T.K. designed research and wrote the paper; V.P. 5 -CAGCTG-3 known as an E-box motif to activate its targets (23). and Y.-J.L. designed and supervised research. The N-terminal transactivation domain of MYC is essential for its The microarray and ChIP-sequencing datasets presented in this article have been biological function and directly interacts with components of basal submitted to the Gene Expression Omnibus under accession numbers GSE70276 transcription machinery or chromatin remodeling (the SWItch/sucrose and GSE70275, respectively. nonfermentable [SWI/SNF] chromatin-remodeling complex and his- Address correspondence and reprint requests to Dr. Yong-Jun Liu or Dr. Tae Whan Kim, Sanofi, 640 Memorial Drive, Cambridge, MA 02139 (Y.-J.L.) or tone acetyltransferase) (24–26). Additionally, several recent findings Baylor Institute for Immunology Research, 3434 Live Oak Street, Dallas, TX suggested that MYC represses its target genes by interfering with 75204 (T.W.K.). E-mail addresses: Yong-Jun.liu@sanofi.com (Y.-J.L.) and [email protected] (T.W.K.) transcriptional activators (27) or by recruiting the corepressor com- The online version of this article contains supplemental material. plexes, including histone deacetylases (HDAC) (28, 29) or DNA Abbreviations used in this article: ChIP, chromatin immunoprecipitation; co-IP, protein methyltransferase (30). Many studies have also revealed that MYC complex immunoprecipitation; DEG, differentially expressed gene; HDAC, histone induces its oncogenic activity through transcriptional repression (22, deacetylase; IP, immunoprecipitation; IRF, IFN regulatory factor; NC, nontargeting control; NCOR2, nuclear receptor corepressor 2; pDC, plasmacytoid dendritic cell; 31). Interestingly, MYC activation is reported shown to downregulate qRT-PCR, quantitative RT-PCR; siRNA, small interfering RNA; SLE, systemic lupus many genes involved in the NF-kB response and STAT1, the central erythematosus; SWI/SNF, SWItch/sucrose nonfermentable; VPA, valproic acid. player in type I and II IFN signaling via direct inhibition of tran- Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 scription in Burkitt lymphoma cells (32–34). Less, however, is known www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502385 The Journal of Immunology 3349 about how MYC regulates pDC function, and the direct target genes nmol small interfering RNA (siRNA) for each reaction of 3–5 3 106 cells. of MYC in pDC have not been identified. siMYC, siHDAC3, and siTBK1 were purchased from Dharmacon. The In this study, we describe a previously unrecognized function of program number for electroporation was A033. The NC is a nontargeting pool from Dharmacon. MYC in the TLR-mediated negative regulation of antiviral immune response of pDCs. We demonstrate that transcriptional repression of Quantitative PCR analysis IRF7 by MYC provides an additional layer of tight regulation of IRF7 For quantitative RT-PCR (qRT-PCR) experiments, total RNAwas isolated from that ensures the appropriate level of type I IFN production and prevents cells with Qiagen RNeasy mini kit and reverse transcribed into cDNA using the development of autoimmune disease. Combination studies have iScript cDNA synthesis kit (Bio-Rad). qRT-PCR for IL-6, IL-23A, TNF-a, a b identified the IRF7 gene as a critical target of MYC, although not its IL-8, IFN- 4, IFN- , CXCL10, IRF7, MYC, PLSCR1, IFIT1, TLR7, TLR9, IFNa14, CCND1, p21,TLR7, TBK1, and TLR9 was performed with specific only target. Our results characterize the novel immunomodulatory role of primers using the instructions from the manufacturer (SyBr Green mastermix; transcription factor MYC in human pDCs and may add to our under- Bio-Rad). Primer sequences are available upon request. standing of aberrant pDC function in cancer and autoimmune disease. Measurement of cytokine production from GEN2.2 cells using ELISA Materials and Methods Reagents and Abs GEN2.2 cells were stimulated with the indicated concentrations of CpGA, CpGB, or R848 for 4 h (to detect TNF-a, IL-6, and IL-8) or for 12 h (to CpGA (ODN2116), CpGB (ODN2006), and R848 were purchased from detect IFN-a and IFN-b). Concentrations of IFN-a, IFN-b, TNF-a, IL-6, Invivogen. The following Abs were used for immunoblotting: anti-MYC, and IL-8 in the culture supernatants were measured by ELISA (PBL anti-IRF7, and anti-IRAK1 (Santa Cruz Biotechnology); anti-TLR7 (Abcam); Biomedical Laboratories and R&D Systems).
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