Transcriptional Regulation of the Human TLR9 Gene Fumihiko Takeshita, Koichi Suzuki, Shin Sasaki, Norihisa Ishii, Dennis M. Klinman and Ken J. Ishii This information is current as of October 2, 2021. J Immunol 2004; 173:2552-2561; ; doi: 10.4049/jimmunol.173.4.2552 http://www.jimmunol.org/content/173/4/2552 Downloaded from References This article cites 49 articles, 31 of which you can access for free at: http://www.jimmunol.org/content/173/4/2552.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 2, 2021 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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Transcriptional Regulation of the Human TLR9 Gene1 Fumihiko Takeshita,2* Koichi Suzuki,† Shin Sasaki,‡ Norihisa Ishii,‡ Dennis M. Klinman,* and Ken J. Ishii3* To clarify the molecular basis of human TLR9 (hTLR9) gene expression, the activity of the hTLR9 gene promoter was charac- terized using the human myeloma cell line RPMI 8226. Reporter gene analysis and EMSA demonstrated that hTLR9 gene transcription was regulated via four cis-acting elements, cAMP response element, 5؅-PU box, 3؅-PU box, and a C/EBP site, that interacted with the CREB1, Ets2, Elf1, Elk1, and C/EBP␣ transcription factors. Other members of the C/EBP family, such as C/EBP␤, C/EBP␦, and C/EBP⑀, were also important for TLR9 gene transcription. CpG DNA-mediated suppression of TLR9 gene transcription led to decreased binding of the trans-acting factors to their corresponding cis-acting elements. It appeared that suppression was mediated via c-Jun and NF-␬B p65 and that cooperation among CREB1, Ets2, Elf1, Elk1, and C/EBP␣ culmi- nated in maximal transcription of the TLR9 gene. These findings will help to elucidate the mechanism of TLR9 gene regulation and to provide insight into the process by which TLR9 evolved in the mammalian immune system. The Journal of Immunology, 2004, Downloaded from 173: 2552–2561. he mammalian innate immune system has evolved to ex- flammatory cytokines (IL-1, IL-6, IL-12, IL-18, and TNF-␣), and press TLRs able to sense pathogen-associated molecular chemokines (MIP-1␣, MIP-1␤, MIP-2, RANTES, JE/MCP-1, and T patterns, enabling early responses against pathogens. Of IP-10) (9–12). the 10 TLRs so far discovered, TLR9 is thought to recognize the The signal transduction pathway mediated by the interaction specific conformation of CpG DNA, thereby mediating signaling between CpG DNA and TLR9 is shared with the other functionally http://www.jimmunol.org/ pathways that direct innate immune activation (1, 2). CpG motifs, characterized members of the TLR family and involves the my- consisting of unmethylated CpG dinucleotides flanked by two 5Ј eloid differentiation marker 88, IL-1R-associated kinase, TNFR- purines and two 3Ј pyrimidines, are more common in bacterial associated factor 6, TGF␤-activated kinase1, and I␬B kinases, DNA than in vertebrate DNA, which suggests that TLR9 plays an I␬B, and NF-␬B (13). Recent studies have demonstrated that the important role in discriminating between bacterial and self DNA formation and maturation of CpG DNA-containing endosomes are (3). CpG oligodeoxynucleotides (ODNs)4 have been demonstrated regulated by PI3Ks and the ras-associated GTP-binding protein to have clinically promising properties in a number of areas, in- Rab5 and are essential for the initiation of TLR9-mediated signal- cluding stimulation of innate immunity to combat pathogens and ing (14, 15). Suppressive ODNs compete with CpG DNA for bind- by guest on October 2, 2021 cancer cells, enhanced vaccine antigenicity via acquired immunity, ing to TLR9, which results in blocking of this signaling pathway and immune modulation toward Th1-dominant responses that can (7). Chloroquine (CQ) also blocks CpG DNA-mediated signaling suppress allergic responses (4–8). Recent studies have shown that by down-regulating the maturation of CpG DNA-containing a subset of CpG ODN, termed D-type CpG ODN (or CpG-A) can endosomes (8). directly activate plasmacytoid dendritic cells (PDCs) and NK cells Mouse TLR9 (mTLR9) mRNA is abundantly expressed in the to produce IFN-␣␤ and IFN-␥, respectively. Another CpG ODN spleen. Of the various cell types, PDCs show the strongest TLR9 subset, termed K-type CpG ODN (or CpG-B), preferentially stim- gene expression (16, 17). Although expression is weak in B cells ulates B cells, monocytes, and macrophages to produce Ig, proin- and monocytes, augmented TLR9 expression in response to anti-Ig cross-linking or IFN-␥ stimulation correlates with increased re- sponsiveness to CpG DNA (18, 19). Of the commercially available *Section of Retroviral Immunology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892; †Laboratory of Molecular En- cell lines, the human myeloma cell line RPMI 8226 expresses suf- docrinology, Medstar Research Institute, Washington Hospital Center, Washington, ficient TLR9 to respond to CpG DNA. K-type CpG ODN, but not DC 20010; and ‡Department of Bioregulation, National Institute of Infectious Dis- eases, Tokyo, Japan D-type CpG ODN, activates RPMI 8226 to produce IL-6, IL-12 p40, MIP-1␣, MIP-1␤, and RANTES (11, 20, 21). To examine the Received for publication December 29, 2003. Accepted for publication June 1, 2004. cis-acting elements essential for constitutive activation of the hu- The costs of publication of this article were defrayed in part by the payment of page Ј charges. This article must therefore be hereby marked advertisement in accordance man TLR9 (hTLR9) gene promoter in RPMI 8226, the 5 -flanking with 18 U.S.C. Section 1734 solely to indicate this fact. region of the hTLR9 gene was cloned and characterized. Our re- 1 This work was supported in part by the Yokohama Medical Foundation. sults identified four cis-acting elements and demonstrated the bind- 2 Address correspondence and reprint requests to Dr. Fumihiko Takeshita at the cur- ing of various CREB, Ets, and C/EBP transcription factors. rent address: Department of Molecular Biodefense Research, Yokohama City Uni- versity School of Medicine, 3-9 Fukuura, Kanazawaku, Yokohama 236-0004, Japan. E-mail address: [email protected] 3 Current address: Exploratory Research for Advanced Technology (Japan), Japan Materials and Methods Science and Technology Agency, Department of Host Defense, Research Institute for Reagents Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan. ODNs were synthesized (Center for Biologics, Evaluation, and Research 4 Abbreviations used in this paper: ODN, oligodeoxynucleotide; PDC, plasmacytoid core facility, Bethesda, MD) according to the following sequences: K3, dendritic cell; CQ, chloroquine; m, mouse; h, human; ATF, activating transcription ATC GAC TCT CGA GCG TTC TC; K3 flip, ATG CAC TCT GCA GG factor; CRE, cAMP response element; MDC, myeloid dendritic cell; IRF, IFN reg- C TTC TC (2); D19, GGt gca tcg atg caG GGG G (9); H154, CCT CAA ulatory factor. GCT TGA GGG G (22); A151, TTA GGG TTA GGG TTA GGG TTA Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 2553 GGG (23); and 1471, TCA AGC TTG A (uppercase letters indicate phos- The sequence of each PCR product was confirmed using an ABI PRISM phorothioates and lowercase letters indicate phosphodiesters). LPS, IFN-␥, Genetic Analyzer (PE Applied Biosystems, Foster City, CA). and CQ were purchased from Sigma-Aldrich (St. Louis, MO). Anti- CREB1, -activating transcription factor 2 (ATF2), -Ets1/2, -Ets2, -Elf1, Construction of transcription factor expression vectors ␣ ␤ ␥ ␦ ⑀ -Elk1, -Spi1 (PU.1), -C/EBP , -C/EBP , -C/EBP , -C/EBP , -C/EBP , Human cDNAs encoding Ets1, Ets2, Elf1, Elk1, Spi1 (PU.1), SpiB, SpiC, and -Sp1 Abs were from Santa Cruz Biotechnology (Santa Cruz, CA). CREB1, ATF2, C/EBP␣, C/EBP␤, C/EBP␥, C/EBP␦, and C/EBP⑀ were amplified by PCR using a Marathon-Ready cDNA library derived from Cell lines and cell cultures human spleen (BD Clontech, Palo Alto, CA) and primers as shown in ␬ RPMI 8226 and THP-1 cells were purchased from American Type Culture Table I. Mouse cDNA encoding c-Fos, c-Jun, or NF- B p65 were also PCR Collection (Manassas, VA). Cells were grown in RPMI 1640 (Sigma-Aldrich) amplified from the mouse spleen cDNA library using primers shown in supplemented with 10% FBS, 50 ␮g/ml penicillin/streptomycin, and 0.1 mM Table I. All PCR products were gel-purified and cloned into the mamma- nonessential amino acids. Cells were maintained at 37°Cin5%CO. lian expression vector pCIneo (Promega). Sequences of the PCR products 2 were confirmed using an ABI PRISM Genetic Analyzer (PE Applied RT-PCR Biosystems). Semiquantitative RT-PCR was performed as previously described (21). 5Ј-RACE Briefly, total RNA was extracted with TRIzol reagent (Invitrogen, Carls- 5Ј-RACE was performed on hTLR9 cDNA using a SMART RACE cDNA bad, CA) according to the manufacturer’s protocol, and 5 ␮ g of total RNA Amplification Kit (BD Clontech). A 36-cycle touch-down PCR, in which was reverse transcribed in first strand buffer (50 mM Tris-HCl (pH 7.5), 75 the annealing temperature was reduced 2°C every 12 cycles from 70°C, mM KCl, and 2.5 mM MgCl ) containing 25 ng/␮l oligo(dT) , 200 U 2 12–18 was performed using the AP-1 primer (BD Clontech), the ϩ365/TLR9 AS of Moloney leukemia virus reverse transcriptase, 2 mM dNTP, and 10 mM primer, 5Ј-ACA GCC AAG AAG GTG CTG GGC TCG ATGG-3Ј, and the DTT.