Transcriptional Regulation of the Human TLR9 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

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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) , 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) (9Ð12). 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 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 (4Ð8). 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 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 12Ð18 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. Reactions were incubated at 42¡C for 1 h, after which 1 ␮ l of aliquots Marathon-Ready cDNA library derived from human spleen (BD Clontech). A of the cDNA synthesis mixture was subjected to standard PCR for 27 or 35 35-cycle nested PCR was then performed using the AP2 primer (BD Clon- Downloaded from cycles using primers shown in Table I. PCR products were separated on tech), HindIII/Ϫ1/TLR9 AS primer, and 5 ␮l of aliquots of first-round PCR 1.5% agarose gels and visualized under UV light after ethidium bromide products. Resultant PCR products were cloned into the pGEM-T vector (Pro- staining. mega) and inserts of 10 independent clones were sequenced. Cloning of the hTLR9 gene 5Ј-flanking region and construction Transient transfection and luciferase assay of hTLR9 gene promoter-dependent luciferase expression Transient transfections were conducted using FuGene 6 (Roche Diagnos- vectors tics, Indianapolis, IN) according to the manufacturer’s protocol. RPMI http://www.jimmunol.org/ ϫ 5 The 5Ј-flanking region of the hTLR9 gene (from the translation start site to 8226 cells (1 10 ) were cotransfected with Firefly luciferase vector, 3227 bp upstream) was PCR amplified from human genomic DNA using the control Renilla luciferase vector (pTK-RL), and/or expression vectors for primers MluI/Ϫ3227/TLR9 S primer (5Ј-CGA CGC GTC CGG GGA GCT each transcription factor to give a constant 550 ng of DNA/reaction. Eight ␮ GAG ACT AGG GTC CCA GCA CAG-3Ј) and HindIII/-1/TLR9 AS primer hours after transfection, cells were treated with medium alone, 1 g/ml ␥ ␮ (5Ј-CCC AAG CTT GCT GGG GGG CAG GGG CTT CTC CAG AGG LPS, 100 U/ml IFN- ,or1 M ODN in the presence or absence of CQ for ␮ ϫ GTC-3Ј). Truncated mutants of the 5Ј-flanking region were also PCR ampli- an additional 40 h. Cells were then lysed in 200 lof1 Passive Lysis ␮ fied. PCR fragments were cloned into the MluI-HindIII site of the pGL3 basic Buffer (Promega), and luciferase activity in 10 l of aliquots of the cell vector (Promega, Madison, WI). Two-step PCR mutagenesis was performed to lysates was measured using the Dual Luciferase Reporter Assay System obtain site-directed mutants. Mutated sequences included the cAMP response (Promega) according to the manufacturer’s protocols. Firefly luciferase ac- element (CRE) (R1: Ϫ688AGTGAϪ684 was mutated to mR1: tivity of individual cell lysates was normalized against Renilla luciferase Ϫ688GTACCϪ684), 5Ј-PU box (R2: Ϫ251AGGAAϪ247 was mutated to mR2: activity or protein concentration as measured using the BCA protein assay by guest on October 2, 2021 Ϫ251CTAGCϪ247), 3Ј-PU box (R3: Ϫ122TCCTCϪ118 was mutated to mR3: kit (Bio-Rad, Hercules, CA). Ϫ122 Ϫ118 Ϫ91 Ϫ86 CTAGT ), and C/EBP site (R4: TGGGAA was mutated to EMSA mR4: Ϫ91GGTACCϪ86). To obtain pRL mR1R2R3R4, with the expression of Renilla luciferase under the control of the Ϫ700 to Ϫ68 bp promoter region Nuclear extracts were prepared as previously described (20). All buffers

carrying mR1, mR2, mR3, and mR4, the corresponding region was cut from used contained 1 mM Na3VO4 and Complete Mini protease inhibitor mix- pGL mR1R2R3R4 and was introduced into the pRL null vector (Promega). ture (Roche Diagnostics). DNA probes were end-labeled with [␥-33P]ATP

Table I. Primers used for RT-PCR and construction of mammalian expression vectors

Target cDNA Forward Primer Reverse Primer

TLR1 5Ј-CAT AAC TCT GCT GAT CGT CAC C-3Ј 5Ј-TGC TAG GAA TGG AGT ACT GCG-3Ј TLR2 5Ј-GAT GAC TCT ACC AGA TGC CTC C-3Ј 5Ј-CAG AAG AAT GAG AAT GGC AGC-3Ј TLR3 5Ј-CTC AGA AGA TTA CCA GCC GC-3Ј 5Ј-TTC TAG TTG TGG AAG CCA AGC-3Ј TLR4 5Ј-TTA CCT GTG TGA CTC TCC ATC C-3Ј 5Ј-CAG AAG AAT GAG AAT GGC AGC-3Ј TLR5 5Ј-CCT TGA CTA TTG ACA AGG AGG C-3Ј 5Ј-TTG TAG GCA AGG TTC AGA ACC-3Ј TLR6 5Ј-TCT CAT GAC GAA GGA TAT GCC-3Ј 5Ј-CGA TCA GCA GAG TTA TGT TGC-3Ј TLR7 5Ј-ACG AAC ACC ACG AAC CTC AC-3Ј 5Ј-GGC ACA TGC TGA AGA GAG TTAC-3Ј TLR8 5Ј-TGG CTT GAA TAT CAC AGA CGG-3Ј 5Ј-ACC AGG CAG CAT TAA TCT TCC-3Ј TLR9 5Ј-CAA CAA CCT CAC TGT GGT GC-3Ј 5Ј-GAG TGA GCG GAA GAA GAT GC-3Ј TLR10 5Ј-GAA CTG ATG ACC AAC TGC TCC-3Ј 5Ј-GAA GTC TTG ATT CCA TCA CGC-3Ј RP105 5Ј-CTG CTT CTT TTG GGT GGT GCT-3Ј 5Ј-CTC GAG ATT GGA TAT TCC CGT-3Ј GAPDH 5Ј-ACC ACC ATG GAG AAG GCT GG-3Ј 5Ј-CTC AGT GTA GCC CAG GAT GC-3Ј Ets1 5Ј-CGA CGC GTC CCC CAC TCC TGG CAC CAT GAA-3Ј 5Ј-GAC TAG TCA CTC GTC GGC ATC TGG CTT GAC-3Ј Ets2 5Ј-GGA ATT CAG GAT GAA TGA TTT CGG AAT CAA-3Ј 5Ј-GCT CTA GAC CTC AGT CCT CCG TGT CGG GCT-3Ј Elf1 5Ј-CGA CGC GTA TTA TGG CTG CTG TTG TCC AAC-3Ј 5Ј-GCT CTA GAC TAA AAA GAG TTG GGT TCC AGC-3Ј Elk1 5Ј-CGA CGC GTG AGC ACT CCC CCA GCG ATG GAC-3Ј 5Ј-GCT CTA GAA GGG GTG GTG GTG GTG GTG GTA-3Ј Spi1 (PU.1) 5Ј-GGA ATT CAA AAT GGA AGG GTT TCC CCT CGT-3Ј 5Ј-GCT CTA GAT CAG TGG GGC GGG TGG CGC CGC-3Ј SpiB 5Ј-GGA ATT CAC CAT GCT CGC CCT GGA GGC TGC-3Ј 5Ј-GCT CTA GAG CCT CGG GTG TGC TCA GGC CCG-3Ј SpiC 5Ј-GGA ATT CAA TAT GAC GTG TGT TGA ACA AGA-3Ј 5Ј-GCT CTA GAA AGT ATA TTT AGC AAT CAT GGT-3Ј CREB1 5Ј-GGA ATT CGA AAT GAC CAT GGA ATC TGG AGC-3Ј 5Ј-GCT CTA GAT TAA TCT GAT TTG TGG CAG TAA-3Ј ATF2 5Ј-CGA CGC GTT ATT CAA GTT ATG AAA TTC AAG TTA CAT G-3Ј 5Ј-TTG GCT AGC AGG TTT TTA ATC AAC TTC CTG AGG GC-3Ј C/EBP␣ 5Ј-GGA ATT CCG CCA TGG AGT CGG CCG ACT TCT-3Ј 5Ј-GCT CTA GAG GCT GGC CCA GGG CGG TCC CAC-3Ј C/EBP␤ 5Ј-GGA ATT CCC CGG GCA CCC GCG GTC ATG CAA-3Ј 5Ј-GCT CTA GAC GCG CGC GGG GGC CGC GCT AGC-3Ј C/EBP␥ 5Ј-GGA ATT CGA AAT GAG CAA GAT ATC GCA GCA-3Ј 5Ј-GCT CTA GAA AGG GTG AGG TCT ACT GTC CTG-3Ј C/EBP␦ 5Ј-GGA ATT CGC TTG GAC GCA GAG CCC GGC CCG-3Ј 5Ј-GCT CTA GAG TCT CTC CCG CCC CGG CCG CGC-3Ј C/EBP␧ 5Ј-GGA ATT CCG GCC ATG TCC CAC GGG ACC TAC-3Ј 5Ј-CAT CTA GAC TCA GCT GCA ACC CCC CAC GCC-3Ј c-Fos 5Ј-CCC CAA GCT TCG ACC ATG ATG TTC TC-3Ј 5Ј-CGT CTG GAT CCC GGC TGC CTT GCC-3Ј c-Jun 5Ј-CCC AAG CTT GCT ATG ACT GCA AAG ATG GAA-3Ј 5Ј-CGG GAT CCT CAA AAC GTT TGC AAC TGC TGC-3Ј NF-␬B p65 5Ј-GGG AAG CTT ACC ATG GAC GAT CTG TTT CC-3Ј 5Ј-GGT GCC TCG AGC AGG GTC GCT GTC AGC-3Ј 2554 CREB, Ets, AND C/EBP REGULATE THE hTLR9 GENE PROMOTER

GAPDH was performed. As shown in Fig. 1, RPMI 8226 cells constitutively expressed mRNA for TLR1, 4, 6, 7, 9, and 10 and RP105, whereas THP-1 cells expressed all TLR family members except TLR9. The TLR expression profile in RPMI 8226 cells was identical with that of primary B cells, as previously reported by Hornung et al. (18). It has been demonstrated previously that CpG DNA reduces TLR9 mRNA expression levels in both macrophages and B cells. To confirm this finding in RPMI 8226 cells, TLR9 mRNA levels were monitored by semiquantitative RT-PCR after incubation of cells with or without 1 ␮M CpG DNA (K3), 1 ␮g/ml LPS, or 100 U/ml IFN-␥ (Fig. 2). TLR9 mRNA levels significantly decreased 16 h after K3 treatment (to ϳ27% of original level), whereas LPS treatment decreased expression levels ϳ69% (Fig. 2, a and c). In contrast, IFN-␥ treatment did not alter hTLR9 expression levels.

Determination of transcription start sites of the hTLR9 gene To examine the molecular mechanisms underlying transcriptional regulation of the hTLR9 gene, the 5Ј-flanking region, from the Downloaded from translation start site to ϳ3.2 kb upstream, was cloned and 5Ј- RACE was performed to determine the transcription start site. Nine of 10 clones showed identical cDNA ends (referred to as the major transcription start site), and one cloned defined a shorter cDNA end (referred to as the minor transcription start site). The major and minor transcription start sites were mapped to Ϫ91 and FIGURE 1. Profile of TLR family gene expression in RPMI 8226 and Ϫ79 bp from the translation start site, respectively (Fig. 3, large http://www.jimmunol.org/ THP-1 cell lines. Total RNA was extracted from RPMI 8226 and THP-1 arrow and small arrow, respectively). cells. After RT reaction, PCRs targeting TLR1Ð10, RP105, and GAPDH were performed. PCR products were separated on 1.5% agarose gels, and Identification of cis-acting elements within the hTLR9 gene DNA fragments of expected length were visualized under UV light after promoter ethidium bromide staining. Similar results were obtained from two inde- pendent experiments. As shown in Fig. 3, a number of putative binding sites for tran- scription factors were found within an 800-bp upstream region. To using T4 polynucleotide kinase. Sequences of R1, mutR1, R2, mutR2, R3, examine TLR9 gene promoter activity, a reporter gene assay was mutR3, R4, and mutR4 probes are shown in Fig. 6. Binding buffer for the

performed using truncated and mutagenized forms of the TLR9 by guest on October 2, 2021 R1 probe contained 1 ␮g of poly(dI-dC), 12 mM HEPES (pH 7.9), 60 mM gene promoter within 5Ј-flanking region-luciferase gene hybrid KCl, 1 mM ZnSO4, 1 mM EDTA, 6% glycerol, and 1 mM DTT. Binding buffer for the R2, R3, mR3, and R4 probes contained 1 ␮g of poly(dI-dC),

12 mM HEPES (pH 7.9), 60 mM KCl, 4 mM MgCl2, 1 mM EDTA, 6% glycerol, and 1 mM DTT. Binding reactions were performed in 10 ␮lof buffer containing 10 fmol of labeled probe and 5Ð20 ␮g of nuclear extract at 25¡C for 30 min. For supershift assays, nuclear extracts were preincu- bated with each Ab at 25¡C for 30 min before the addition of labeled probe. For competition assays, nuclear extracts were preincubated with a 5-, 10-, 25-, or 100-fold molar excess of unlabeled probe at 25¡C for 30 min before the addition of labeled probe. Samples were loaded onto 6% native poly- acrylamide gels and electorphoresed at 160 V for 2 h. After the electro- phoresis, the gels were dried and analyzed by autoradiography using BAS 2000 (Fuji Film, Tokyo, Japan). Western blot analysis RPMI 8226 cells (1 ϫ 105) were transiently transfected with pFLAG- CMV-4 vector expressing Ets1, Ets2, Elf1, Elk1, Spi1 (PU.1), SpiB, SpiC, CREB1, ATF2, C/EBP␣, C/EBP␤, C/EBP␥, C/EBP␦, or C/EBP⑀. Thirty- six hours after transfection, cells were lysed with SDS sample buffer (60 mM Tris-HCl, 2% SDS, 12% glycerol, and 1 mM DTT). After boiling, 20 ␮g of sample was analyzed by SDS-PAGE on a 10% gel and then was transferred to an Immobilon-P polyvinylidene difluoride membrane (Mil- lipore, Vienna, VA). After blocking with 5% skim milk in PBS containing 0.05% Tween 20 (Sigma-Aldrich), the membrane was incubated with anti- FIGURE 2. Suppression of TLR9 mRNA expression by LPS and CpG FLAG Ab M2 (Sigma-Aldrich), washed three times, and incubated with ODN. RPMI 8226 cells were treated with 1 ␮g/ml LPS, 100 U/ml IFN-␥, peroxidase-conjugated goat anti-mouse IgG (Amersham, Arlington ␮ Heights, IN). After further washing, the blots were incubated with ECL or 1 M CpG ODN K3 for the times shown. Total RNA was extracted and plus substrate (Amersham) and then were analyzed by autoradiography. RT-PCR targeting TLR9 and GAPDH was performed. DNA fragments of expected length were visualized after 1.5% agarose gel electrophoresis and Results ethidium bromide staining. A representative result of three independent Characterization of TLR family gene expression in RPMI 8226 experiments is shown. The density of each DNA fragment was calculated cells using Fuji Image Gauge software. Relative band density was defined as the density of the TLR9 DNA fragment normalized to that of GAPDH. Graph To characterize the gene expression profile of TLR family in shows the mean Ϯ SD of three independent experiments. a and b, p Ͻ 0.05; RPMI 8226 and THP-1, RT-PCR targeting TLR1Ð10, RP105, and c, p Ͻ 0.0001. Statistical analysis was performed by t test. The Journal of Immunology 2555 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 3. between promoter regions from human (upper sequence) and mouse (lower sequence) TLR9 genes and determination of transcription start sites. The 5Ј-flanking regions of human and mouse TLR9 genes from the translation start site (asterisk) to Ϫ800 bp were compared. Putative transcription factor binding sites are boxed. The transcription start sites were determined by 5Ј-RACE as described in Materials and Methods. The large and small arrows indicate the major (9 of 10 clones) and minor (1 of 10 clones) transcription start sites, respectively. constructs, as shown in Fig. 4. RPMI 8226 cells transfected with creased activity to ϳ53% (Fig. 4Ab). Deletion between Ϫ160 and pGLϪ3227/Ϫ1, which contained the region from Ϫ3227 to Ϫ1bp Ϫ130 bp also resulted in an ϳ40% decrease in activity (Fig. 4Ac). relative to the translation start site, showed Ͼ15.2-fold more lu- Deletion of the 3Ј-end of the flanking region from Ϫ1toϪ68 bp ciferase activity compared with cells transfected with pGL basic. did not affect luciferase activity, whereas further deletion from This suggested that the promoter was constitutively active in Ϫ68 to Ϫ120 bp resulted in an ϳ80% decrease in activity (Fig. RPMI 8226 cells. To identify essential cis-acting elements impor- 4Ad). These results suggested that there were at least four regions tant for promoter activity, 5Ј- and/or 3Ј-truncated forms of the that critically regulated promoter activity (between Ϫ700 and construct were tested. Although deletion of the 5Ј-end of the flank- Ϫ640 bp, between Ϫ290 and Ϫ240 bp, between Ϫ160 and Ϫ130 ing region from Ϫ3227 to Ϫ700 bp did not alter promoter activity, bp, and between Ϫ120 and Ϫ68 bp). A typical TATA box was further deletion of the 5Ј-end from Ϫ700 to Ϫ640 bp resulted in an found in the Ϫ160 to Ϫ130 bp region, which suggested that this ϳ62% reduction in luciferase activity (Fig. 4Aa). Further deletion region was required for basic transcriptional initiation. Consensus of the 5Ј-end from Ϫ640 to Ϫ290 bp did not affect luciferase motifs recognized by known transcription factors were identified activity, but deletion between Ϫ290 to Ϫ240 bp resulted in de- within the four regions, including CRE (Ϫ686 to Ϫ679 bp, termed 2556 CREB, Ets, AND C/EBP REGULATE THE hTLR9 GENE PROMOTER Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 4. Deletion analysis (A) and site-directed mutation analysis (B)oftheTLR9 gene promoter. Truncated and/or mutated promoter fragments were introduced into the Firefly luciferase reporter plasmid pGL3 basic. RPMI 8226 cells (1 ϫ 105) were transfected with 500 ng each of pGL-TLR9 promoter construct plus 50 ng of pTK-RL. Firefly luciferase activity was assayed 48 h after transfection and normalized to Renilla luciferase activity. Values represent the mean Ϯ SD of four independent experiments. A model showing the putative transcription factor binding sites in the 5Ј-flanking region of the TLR9 gene is provided at the left side of each figure. The numbers shown to the right of each fragment indicate the distance from the translation start site. Aa and Ac, p Ͻ 0.05; Ab and Ad, p Ͻ 0.0001; Bc and Bs, p Ͻ 0.05; Bk and Bl, p Ͻ 0.01; Bg, Bi, and Bj, p Ͻ 0.005; Bo and Br, p Ͻ 0.001; Bb, Bh, Bm, Bw, Bx, Bz, and BB, p Ͻ 0.0005; Ba, BdÐBf, Bn, Bp, Bq, BtÐBv, By, BA, and BC, p Ͻ 0.0001. ϫ, Targets of site-directed mutagenesis. Statistical analysis was performed by t test.

R1), 5Ј-PU box (Ϫ252 to Ϫ247 bp, termed R2), 3Ј-PU box (Ϫ123 transfected with pGL mR1R2R3 (4.2 Ϯ 0.1-fold) was significantly to Ϫ118 bp, termed R3), and C/EBP (Ϫ93 to Ϫ85 bp, termed R4). lower than in cells transfected with pGL mR1R2 (17.6 Ϯ 0.4-fold; To confirm the role of individual and cooperative contributions Fig. 4Bn), pGL mR1R3 (6.0 Ϯ 0.2-fold, Fig. 4Bo), or pGL mR2R3 to promoter activation by R1, R2, R3, and R4, single and com- (8.1 Ϯ 0.2-fold; Fig. 4Bp). These results suggested that R1, R2, bined mutations were introduced by site-directed mutagenesis and and R3 synergistically enhanced promoter activity. Similar syner- resultant promoter activity was examined (Fig. 4B). Cells trans- gistic enhancements by any triple combination of individual cis- fected with pGL Naive, which carried the intact promoter (the acting elements were observed (Fig. 4, BqÐBy). Luciferase activity region between Ϫ700 and Ϫ68 bp), showed 22.2-fold more lucif- in cells transfected with pGL mR1R2R3R4 (2.8 Ϯ 0.1-fold) was erase activity than did cells transfected with pGL basic. Of con- significantly lower than in cells transfected with triple mutation structs carrying a single mutation, pGL mR3 showed a 54.5% re- construct (vs pGL mR1R2R3, 4.2 Ϯ 0.1-fold, Fig. 4BA;vspGL duction in luciferase activity compared with pGL Naive (Fig. mR1R2R4, 8.3 Ϯ 0.3-fold, Fig. 4BB; vs pGL mR1R3R4, 4.3 Ϯ 4Ba), whereas single mutations at R1, R2, or R4 had no effect on 0.2-fold, Fig. 4BC; vs pGL mR2R3R4, 5.5 Ϯ 0.1-fold, Fig. 4BD). luciferase activity. Luciferase activity in cells transfected with This suggested that all four cis-acting elements synergistically pGL mR1R2 (17.6 Ϯ 0.4-fold) was significantly lower than in contributed to promoter activation (Fig. 4B). cells transfected with pGL mR1 (23.8 Ϯ 0.7-fold; Fig. 4Bb)or with pGL mR2 (21.2 Ϯ 1.8-fold; Fig. 4Bc). This suggested that R1 Suppression of the hTLR9 gene promoter by CpG ODN and R2 acted cooperatively and synergistically in promoter regu- To investigate the molecular mechanisms underlying CpG ODN- lation. Similar synergistic effects were observed between R1 and mediated decrease in TLR9 mRNA, as shown in Fig. 2, the effects R4, between R2 and R4, and between R3 and other individual of CpG ODN and other compounds on TLR9 gene promoter ac- cis-acting elements (Fig. 4, BdÐBm). Luciferase activity in cells tivity were examined using RPMI 8226 cells transfected with pGL The Journal of Immunology 2557

nuclear extracts from cells preincubated with anti-CREB1, but were present after treatment with anti-ATF2 Ab. This suggested that CREB1 constitutively bound R1, which dissociated after CpG ODN stimulation (Fig. 6, A and B). EMSA also demonstrated two specific complexes for the R2 probe that contained the 5Ј-PU box site (Ϫ252 to Ϫ247 bp), after incubation with nuclear extracts from control cells (Fig. 6, D and F). Interestingly, the upper com- plex was not observed in nuclear extracts from cells treated with K3, but was present after LPS treatment. Preincubation of the nu- clear extracts with Ab against various PU box binding , such as Ets2, Elf1, or Elk1, resulted in decreased formation of both complexes, which suggested that Ets2, Elf1, and Elk1 bound to R2. Preincubation of nuclear extracts with anti-Spi1 (PU.1) Ab did not affect complex formation, which suggested that Spi1 (PU.1) was not involved in the complexes (Fig. 6E). A single specific complex was detected using a probe for R3, which contained the 3Ј-PU box (Ϫ123 to Ϫ118 bp) (Fig. 6G). This complex was not affected by preincubation with Ab against PU box binding proteins, such as Ets, Elf1, Elk1, or Spi1 (PU.1) (Fig. 6H). Moreover, the R3 com- plex was not competed with a 5- or 25-fold excess of mutated R3 Downloaded from probe (mutR3) or R2 probe, which suggested that an unknown protein bound to R3 (Fig. 6I). For the R4 probe, which contained the C/EBP site (Ϫ93 to Ϫ85 bp), two specific complexes were detected (Fig. 6, J and L). The amount of the upper complex de- creased and the lower complex increased in nuclear extracts from

FIGURE 5. Suppression of the TLR9 gene promoter by LPS and CpG http://www.jimmunol.org/ ϫ 5 cells treated with K3, but not with LPS. Of the anti-C/EBP family ODN. RPMI 8226 cells (1 10 ) were transfected with 500 ng of pGL ␣ Naive plus 50 ng of pRL mR1R2R3R4. Fourteen hours after transfection, Abs tested, preincubation of nuclear extracts with anti-C/EBP Ab cells were treated with medium alone, 1 ␮g/ml LPS, 100 U/ml IFN-␥,1 resulted in decreased intensity of both complexes, which suggested ␮M ODN (K-type CpG ODN (K3), D-type CpG ODN (D19), suppressive that C/EBP␣ was present in the R4 complexes (Fig. 6K). ODN (H154 or A151), or control ODN (1471)), or 10 ␮g/ml CQ. In some cases, cells were treated with LPS or K3 in the presence of CQ, H154, A151, or 1471. Firefly luciferase activity was assayed 48 h after transfec- Characterization of trans-activity of the hTLR9 gene promoter tion and normalized to Renilla luciferase activity. Values represent the by known transcription factors Ϯ Ͻ Ͻ mean SD of four independent experiments. c and d, p 0.01; e, p As shown above, several transcription factors appeared to be in- Ͻ by guest on October 2, 2021 0.0005; a and b, p 0.0001. Statistical analysis was performed by t test. volved in the regulation of the hTLR9 gene promoter. We next tried to characterize the effect of these transcription factors. An overexpression study was performed using expression vectors for Naive (Fig. 5). When cells were treated with LPS or K3, luciferase CRE-associating proteins (CREB1 and ATF2), PU box-binding activity levels were significantly decreased, as expected (LPS, proteins (Ets1, Ets2, Elf1, Elk1, Spi1 (PU.1), SpiB, and SpiC), 24 Ϯ 1%, Fig. 5a; K3, 46 Ϯ 3%, Fig. 5b). Treatment of cells with C/EBPs (C/EBP␣, C/EBP␤, C/EBP␥, C/EBP␦, and C/EBP⑀), and IFN-␥, CQ, D-type CpG ODN (D19), control ODNs (K3-flip and proteins up-regulated by CpG DNA or LPS signaling (NF-␬B p65, 1471), or suppressive ODNs (H154 and A151) did not affect lu- c-Fos, and c-Jun). Cotransfection of cells with pGL Naive plus a ciferase activity levels. TLR9 gene promoter suppression by K3 vector expressing CREB1, C/EBP␣, C/EBP␤, C/EBP␦, or C/EBP⑀ was reversed by simultaneous treatment with H154, A151, or CQ, alone resulted in significantly increased luciferase activity com- whereas the effect of K3 was not reversed by treatment with 1471 pared with cells cotransfected with pGL Naive plus an empty vec- (Fig. 5, cÐe). CQ did not affect LPS-mediated suppression of pro- tor (Fig. 7, AbÐAf). In contrast, cotransfection with pGL Naive plus moter activity (Fig. 5). These results suggested that both LPS/ a vector expressing Spi1 (PU.1), c-Jun, or NF-␬B p65 alone re- TLR4/RP105- and CpG DNA/TLR9-mediated signaling pathways sulted in decreased luciferase activity (Fig. 7, Aa, Ag, and Ah). The were involved in the suppression of TLR9 gene expression in expression levels of these transcription factors were confirmed by RPMI 8226 cells. Western blot analysis (Fig. 7A). The cooperative effects of com- binations of CREB1, Ets2, Elf1, Elk1, and C/EBP␣ on trans-ac- Identification of transcription factors interacting with essential tivation of the TLR9 gene promoter were also examined. Cotrans- cis-acting elements fection with vectors expressing CREB1 and Ets2 increased the To identify the transcription factors that interact with the cis-acting luciferase activity compared with cotransfection with vectors ex- promoter elements, EMSA targeting R1, R2, R3, or R4 was per- pressing CREB1 alone (Fig. 7Ba) or Ets2 alone (Fig. 7Bd), which formed (Fig. 6). When the R1 probe, which spanned the CREB site suggested that CREB1 and Ets2 synergistically trans-activated the (Ϫ686 to Ϫ679 bp), was incubated with nuclear extracts obtained promoter. from RPMI 8226 cells, two complexes were detected (Fig. 6A). Similar synergistic enhancements were observed between These complexes were specific for the R1 probe as the band in- CREB1 and Elf1 (Fig 7, Bb and Be), CREB1 and C/EBP␣ (Fig. 7, tensity in the nuclear extracts decreased after preincubation with a Bc and Bf), Ets2 and Elf1 (Fig. 7, Bg and Bh), Elf1 and C/EBP␣ 10- or 100-fold excess of unlabeled R1 probe, but not with unla- (Fig. 7, Bk and Bl), and Elk1 and C/EBP␣ (Fig. 7, Bm and Bn). beled mutated R1 probe (mutR1; Fig. 6C). Amounts of both com- Addition of a vector expressing CREB1 to each double combina- plexes were decreased in nuclear extracts from cells treated with tion of transfectant vectors expressing Ets2, Elf1, Elk1, and K3, but not with LPS. These complexes were not observed in C/EBP␣ resulted in increased luciferase activity (Fig. 7, BoÐBt). 2558 CREB, Ets, AND C/EBP REGULATE THE hTLR9 GENE PROMOTER Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 6. EMSA targeting of CRE (R1), 5Ј-PU box (R2), 3Ј-PU box (R3), and C/EBP site (R4). RPMI 8226 cells were treated with medium alone, 1 ␮g/ml LPS, or 1 ␮M K3. Nuclear extracts were incubated with probes for R1 (AÐC), R2 (DÐF), R3 (GÐI), or R4 (JÐL). Specificity was shown by addition of 10- or 100-molar excess of unlabeled probe. Supershift experiments were performed by preincubating nuclear extracts with anti-CREB1, -ATF2, -Ets1/2, -Ets2, -Elf1, -Elk1, -Spi1 (PU.1), -C/EBP␣, -C/EBP␤, -C/EBP␥, -C/EBP␦, -C/EBP⑀, or -Sp1 Abs. Arrowheads indicate specific complexes. A represen- tative result of three independent experiments is shown. A model showing each cis-acting element in the 5Ј-flanking region of the TLR9 gene and sequences for each probe are shown at the top of the figure. Boxed letters indicate transcription factor binding sites. Lowercase letters indicate the mutagenized sequences in the mutR1, mutR2, mutR3, and mutR4 probes.

Similar synergistic trans-activation was observed among Ets2, Discussion Elf1, and Elk1 (Fig 7, Bu, Bx, and Bw), among Ets2, Elf1, and This study demonstrated that interactions between CREB1 and the ␣ C/EBP (Fig 7, Bv, By, and Bz), and among Elf1, Elk1, and CRE, the Ets2-Elf1-Elk1 complex and the 5Ј-PU box, and C/EBP␣ C/EBP␣ (Fig 7, BAÐBC). However, addition of Ets2 to a combi- and the C/EBP site critically regulated the hTLR9 gene promoter. nation of Elk1 and C/EBP␣ did not change promoter activity. Overexpression of known transcription factors suggested that: 1) Trans-activation by quadruple combinations of the transcription CREB1 and C/EBP␣ potently trans-activated the TLR9 gene pro- factors was also examined. Elf1 enhanced the activity induced by moter, 2) although Ets2, Elf1, and Elk1 did not trans-activate the Ets2, Elk1, and C/EBP␣ (Fig. 7BH), whereas CREB1 markedly enhanced the promoter activity induced by any triple combination promoter themselves, they enhanced the trans-activity of factors act- of Ets2, Elf1, Elk1, and C/EBP␣ (Fig 7, BDÐBG). Finally, the ing on other elements, 3) Spi1 (PU.1) acted, directly or indirectly, as removal of CREB1, Ets2, Elf1, Elk1, or C/EBP␣ resulted in a a suppressor on the TLR9 gene promoter, 4) factors up-regulated by ␬ significantly decreased promoter activation compared with cells CpG DNA/TLR9 or LPS/TLR4/RP105 signaling (NF- B and c-Jun, expressing all five factors (Fig. 7, BIÐBM). These results suggested a component of AP-1) also acted as suppressors, and 5) other mem- that the participation of the transcription factors CREB1, Ets2, bers of the C/EBP family, including C/EBP␤, C/EBP␦, and C/EBP⑀, Elf1, Elk1, and C/EBP␣ culminated in the maximal transcription may also be involved in the up-regulation of TLR9 gene expression in of the TLR9 gene. other cell types or via other stimuli. The Journal of Immunology 2559 Downloaded from http://www.jimmunol.org/

FIGURE 7. Characterization of the trans-activity of known transcription factors in the TLR9 gene promoter. A, RPMI 8226 cells (1 ϫ 105) were transfected with 50 ng of pGL Naive plus 500 ng of expression vector for Ets1, Ets2, Elf1, Elk1, Spi1 (PU.1), SpiB, SpiC, CREB1, ATF2, C/EBP␣, C/EBP␤, C/EBP␥, C/EBP␦, C/EBP⑀, c-Fos, c-Jun, or NF-␬B p65. Arrowhead shows the expression levels of transcription factors analyzed by Western blotting, as described in Materials and Methods. B, RPMI 8226 cells (1 ϫ 105) were transfected with 50 ng of pGL Naive plus various combinations of 100-ng expression vectors for Ets1, Ets2, Elf1, Elk1, Spi1 (PU.1), SpiB, SpiC, CREB1, ATF2, C/EBP␣, C/EBP␤, C/EBP␥, C/EBP␦, C/EBP⑀, c-Fos, c-Jun, or NF-␬B p65, supplemented with an empty vector (pCIneo) to allow a constant 550 ng of DNA per reaction. Firefly luciferase activity was assayed 48 h after transfection and was normalized to the protein concentration of each lysate. Values represent the mean Ϯ SD of four independent experiments. Ad, Af, and Ah, p Ͻ 0.05; Aa, Ac, Ae, and Ag, p Ͻ 0.01; Ab, p Ͻ 0.005; BaÐBc, BfÐBh, Bj, Bk, Bn, BD, BIÐBL, and BM, p Ͻ 0.05; Be, Bl, Bo, BrÐBt, Bz, BB, by guest on October 2, 2021 BE, BG, and BM, p Ͻ 0.01; Bd, Bm, Bv, By, BC, and BF, p Ͻ 0.005; Bp, Bu, Bw, and Bx, p Ͻ 0.001; Bq and BA, p Ͻ 0.0005; BI, p Ͻ 0.0001. Statistical analysis was performed by t test.

CREB1 is expressed in a wide variety of cell types. It has been of tissues. C/EBP␣ expression levels tend to be prominent in im- established that cAMP induces phosphorylation of CREB1, which mature myeloid cells and can vary during differentiation. C/EBP␣ then activates cAMP-responsive genes, leading to increased cell is essential for granulocyte development and blocks Spi1 (PU.1)- proliferation, differentiation, or modulation of various cell func- induced dendritic cell development from CD34ϩ human cord tions (24). In B cells, CREB1 phosphorylation is enhanced by cell blood cells (35). C/EBP␣ has been shown to cooperate with other surface Ig cross-linking (25). transcription factors to enhance or suppress promoter trans-activ- The Ets family of transcription factors is a diverse group of ϳ30 ity. The in the DNA binding domain of C/EBP␣ proteins that share a conserved DNA binding domain. Most Ets physically binds to Ets transcription factors, such as Spi1 (PU.1), family members bind to PU box/Ets sites within gene promoters. Fli1, Ets1, and Elk1 (36). Due to the close proximity of the C/EBP Ets2 is expressed in a variety of tissues, and transgenic animal site to the TATA box within the TLR9 gene promoter (Fig. 4), it is studies have demonstrated that Ets2 plays a critical role in thymo- possible that C/EBP␣ may interact with the basal transcription- cyte and macrophage development (26). Elf1 is a lymphoid-spe- initiation complex. Indeed, CBP/p300, which forms the basal tran- cific Ets transcription factor that regulates the germline Ig ␣ gene scription apparatus by interacting with the TATA box-binding pro- promoter in B cells and IL-3 and IL-2R␣ gene expression in T cells tein TFIIB and RNA polymerase II, forms a binding domain for (27Ð29). Elf1 has been shown to cooperate with C/EBP␣ to up- C/EBP␣␤, CREB, and Ets family transcription factors (24). Thus, regulate promoter activity of the IgA FcR (CD89) gene in the CBP/p300 may play a central role in the transcriptional regulation monocytic cell line U937 (30). Another family member, Elk1, is a of the TLR9 gene by controlling the interaction between various direct target of MAPKs, such as ERK1/2. Once phosphorylated, it trans-acting factors. Taken together, our findings suggest that translocates to the nucleus and serves as a transcription factor (31). CREB1, Ets2, Elf1, Elk1, and C/EBP␣ might physically and func- Physical interaction between Elk1 and C/EBP␣␤ confers insulin tionally interact with each other, leading to maximal transcription sensitivity in Chinese hamster ovary cells and synergistic trans- of the TLR9 gene. activation of the c-fos gene (32Ð34). Constitutive and sustained Overexpression of Spi1 (PU.1) resulted in suppression of the Elk1 phosphorylation after cell surface Ig cross-linking has been TLR9 gene promoter (Fig. 7A). Although functional redundancy reported in mature B cells (25). has been demonstrated between Spi1 (PU.1) and SpiB, other mem- C/EBPs belong to the basic region/leucine zipper class of tran- bers of the Ets family, such as Ets1 or Elf1, are not functionally scription factor and are involved in differentiation in a broad range similar to Spi1 (PU.1) (37). Thus, it is possible that overexpressed 2560 CREB, Ets, AND C/EBP REGULATE THE hTLR9 GENE PROMOTER

PU.1 might compete with endogenous Ets2, Elf1, and Elk1 for PU which then reduced promoter activity and resultant TLR9 gene box binding rather than serving as a trans-activator for the TLR9 mRNA levels. The CpG ODN-mediated suppression of TLR9 gene gene promoter, or alternatively, PU.1 might directly interact with promoter was further confirmed by the use of CpG-specific inhib- endogenous transcription factors, blocking their trans-activity. In a itors, such as suppressive ODNs and CQ (22, 47). This effect was case of the I-A␤ gene expression, Spi1 (PU.1) competes with other also observed after overexpression of the transcription factors c- transcription factors for DNA binding, which results in transcrip- Jun or NF-␬B p65, which mediate CpG DNA-induced cytokine/ tional suppression (38). It has also been demonstrated that the de- chemokine gene expression (20, 47). This indicated that these fac- velopment of dendritic cells from cord blood stem cells transduced tors and/or gene products up-regulated by CpG DNA were, with Spi1 (PU.1) was blocked by C/EBP␣ through a physical in- directly or indirectly, involved in the mechanism underlying the teraction (35). Thus, it is likely that Spi1 (PU.1) suppressed TLR9 gene suppression. However, this finding contradicts studies C/EBP␣-mediated activation of the TLR9 gene promoter via a pro- independently reported by Bourke et al. (48)and Bernasconi et al. tein-protein interaction. (49), in which activation by surface Ig cross-linking, Staphylococ- Tissue- and developmental stage-specific expression of a given cus aureus Cowan I bacteria, or B-type CpG ODN 2006 increased gene is not achieved through the action of a single transcription TLR9 expression in primary human B cells. However, both groups factor. Rather, unique combinations of cell type-specific and more used tonsillar resting or naive B cells to study TLR9 gene expres- generally expressed nuclear factors account for the enormous spec- sion. These immature B cells express lower levels of TLR9 than do ificity and diversity in gene expression profiles (24). For instance, activated mature B cells, such as germinal center or memory B expression of the hTLR2 gene in monocytic cells is regulated by cells. Also, the responsiveness to B cell Ag- complex or Sp1, Sp3, and Spi1 (PU.1), whereas hTLR4 gene expression is polyclonal B cell activators, such as LPS or CpG DNA, is depen- regulated by IFN consensus sequence binding protein and Spi1 dent on differentiation stage and can vary from donor to donor. Downloaded from (PU.1) (39, 40). Interestingly, hemopoietic progenitor cells from Indeed, we tested several human B cell lines at different stages of Spi1 (PU.1) gene-knockout mice are unable to generate myeloid differentiation and found that only RPMI 8226 cells expressed the dendritic cells (MDCs) in vitro, which express high levels of TLR2 TLR9 gene after treatment with CpG ODN or LPS (data not and TLR4, but not TLR9 (41). With regard to its role in hTLR9 shown). More precise studies will be needed to clarify the - gene expression, Spi1 (PU.1) might play a key role in regulating tionship between modulation of TLR9 gene expression by CpG the cell type-specific profile of TLR family gene expression. DNA and B cell differentiation stage. http://www.jimmunol.org/ Species-specific regulation of TLR family gene expression has In this study, we identify four cis-acting elements, CRE, 5Ј-PU recently been demonstrated. Murine TLR2 gene expression is up- box, 3Ј-PU box, and C/EBP site, that synergistically regulate the regulated by LPS, mycobacterial products, and NF-␬B activation, hTLR9 gene promoter. CREB1, Ets2, Elf1, Elk1, and C/EBP␣ whereas hTLR2 gene expression is not induced by these stimuli or trans-activate the promoter by interacting with these cis-acting el- by NF-␬B activation. Indeed, the mTLR2 gene promoter contains ements. Synergism between the cis- and trans-factors appear to an NF-␬B motif, which functions as a positive cis-acting element account for the maximal hTLR9 gene transcription. Our findings (42). Basal and IFN-␤-induced activation of the TLR3 gene pro- will extend our understanding of cell type-specific TLR9 gene ex- moter from mice and humans involves similar IFN regulatory fac- pression and will assist in the development of therapeutic strate- by guest on October 2, 2021 tor (IRF) elements that constitutively bind IRF-2 and recruit IRF-1 gies able to modulate responsiveness to CpG ODN for clinical after stimulation. However, in mouse macrophages, LPS up-regu- purposes. lates TLR3 gene expression through IFN-␤ in an autocrine manner, whereas in human MDCs, LPS blocks the IFN-␤-induced up-reg- Ј References ulation of TLR3 (43). Comparison of the 5 -flanking regions be- 1. Hemmi, H., O. Takeuchi, T. Kawai, T. Kaisho, S. Sato, H. Sanjo, M. Matsumoto, tween mTLR9 and hTLR9 genes showed 56% homology and both K. Hoshino, H. Wagner, K. Takeda, and S. Akira. 2000. A Toll-like receptor of them contained the CRE, the PU boxes, and the C/EBP site recognizes bacterial DNA. 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