Class-specific Regulation of Pro-inflammatory Genes byMyD88 Pathways and I�B�*□S

Received for publication, December 6, 2007, and in revised form, February 8, 2008 Published, JBC Papers in Press, March 3, 2008, DOI 10.1074/jbc.M709965200

Hisako Kayama‡§1, Vladimir R. Ramirez-Carrozzi¶1, Masahiro Yamamoto‡, Taketoshi Mizutani�, Hirotaka Kuwata§,Hideo Iba�, Makoto Matsumoto§, Kenya Honda‡, Stephen T. Smale¶2, and Kiyoshi Takeda‡§3

From the ‡Laboratory of Immune Regulation, Department of Microbiology and Immunology, Graduate School of Medicine, OsakaUniversity, Suita, Osaka 565-0871, Japan, the ¶Department of Microbiology, Immunology, and Molecular Genetics, UCLA,Los Angeles, California 90095, the §Department of Molecular Genetics, Medical Institute of Bioregulation, KyushuUniversity, Fukuoka 812-8582, Japan, and the �Department of Microbiology and Immunology, Divisionof Host-Parasite Interaction, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan

Toll-like receptors trigger the induction of primary responsegenes viaMyD88-mediated activation of NF-�B and other tran-scription factors. These factors then act in concert with primaryresponse gene products to induce secondary response genes.Although theMyD88 pathway is important for the expression ofboth primary and secondary response genes, we show that therecruitment of NF-�B, RNA polymerase, and the TATA-bind-ing protein is MyD88-dependent only at secondary responsegenes. This selective dependence correlates with the fact thatMyD88 is required for nucleosome remodeling and histoneH3K4 trimethylation at secondary response promoters, whereasrapidly induced primary response promoters are assembled intopoised MyD88-independent chromatin structures. At a subsetof secondary response promoters, I�B�was identified as a selec-tive regulator ofH3K4 trimethylation andpreinitiation complexassembly after nucleosome remodeling. These mechanistic dis-tinctions advance our understanding of the diverse molecularcascades that underlie the differential regulation of pro-inflam-matory genes.

Toll-like receptor (TLR)4-dependent recognition of micro-bial components controls immune responses through the acti-vation of innate immunity and the subsequent development of

antigen-specific adaptive immunity (1–3). Excessive activationof innate immunity has been shown to be associated with sev-eral immune disorders (4, 5). Therefore, TLR-mediated innateimmune responses are finely controlled through the regulationof signaling cascades and the modulation of gene induction(5–7). TLR-mediated signaling consists of at least two path-ways, a MyD88-dependent pathway and a TRIF-dependentpathway. In contrast to the selective role of TRIF in TLR3- andTLR4-mediated responses, MyD88 acts downstream of almostall TLRs to promote the activation of a broad range of pro-inflammatory and anti-microbial genes (8).One gene that is induced in response to TLR signaling is

Nfkbiz, which encodes a nuclear I�B family member, I�B�(9–11). Because Nfkbiz expression is induced rapidly in theabsence of new protein synthesis, it is considered to be a pri-mary response gene. Newly synthesized I�B� protein then trig-gers the induction of a subset of TLR-dependent secondarygenes through the modulation of NF-�B activity (12). Thus, inI�B�-deficient mice, rapidly induced primary response genes,includingCxcl2,Cxcl1, and Il23a,were activated normally (13).In contrast, impaired expression was observed with a subset ofsecondary response genes that, in wild-type mice, are inducedat relatively late times after TLR stimulation, including Il12b,Il6, and Lcn2 (13).Although themechanism by which I�B� regulates secondary

response genes is not known, accumulating evidence has dem-onstrated that chromatin structure plays a critical role in geneactivation and suppression in cells of the immune system (14–18). Twomainmediators, ATP-dependent nucleosome remod-eling complexes and histone-modifying enzymes, help regulatechromatin structure (19–23). ATP-dependent chromatinremodeling complexes use the energy of ATP hydrolysis to dis-rupt histone-DNA interactions, whereas histone-modifyingenzymes alter theN-terminal tails and core domains of histonesto regulate the activation and suppression of transcription.Among these histonemodifications, which include acetylation,methylation, ubiquitination, and sumoylation of lysine resi-dues, methylation of specific lysine residues of histones H3 andH4 is well associated with gene activation or suppression. Ofparticular relevance to this study, di- and tri-methylation of H3Lys-4 (H3K4) are generally found at genes that are competentfor activation, with H3K4 trimethylation often linked to activetranscription (24, 25).

* This work was supported by grants-in-aid from the Ministry of Education,Culture, Sports, Science, and Technology, the Ministry of Health, Labor, andWelfare, the Tokyo Biochemical Research Foundation, the Cell ScienceResearch Foundation, the Yakult Bio-Science Foundation, the Osaka Foun-dation for Promotion of Clinical Immunology, the Sumitomo Foundation,the Sankyo Foundation of Life Science, the Giannini Family Foundation (toV. R. R. C.), and the Howard Hughes Medical Institute (to S. T. S.). The costsof publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement” inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1–S4.

1 Both authors contributed equally to this work.2 To whom correspondence may be addressed: HHMI/UCLA 6525 MRL, 675

Charles E. Young Dr. South, Los Angeles, CA 90095-1662. Fax: 310-206-8623; E-mail: smale@mednet.ucla.edu.

3 To whom correspondence may be addressed: 2-2 Yamadaoka, Suita,Osaka 565-0871, Japan. Fax: 81-6-6879-3989; E-mail: ktakeda@ongene.med.osaka-u.ac.jp.

4 The abbreviations used are: TLR, toll-like receptor; TBP, TATA-binding pro-tein; Ab, antibody; LPS, lipopolysaccharide; pol II, polymerase II; ChIP, chro-matin immunoprecipitation; IL, interleukin; PBS, phosphate-bufferedsaline.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 18, pp. 12468 –12477, May 2, 2008© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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Initial evidence that chromatin structure may be critical forthe differential regulation of primary and secondary responsegenes following TLR stimulation was provided in an influentialstudy by Saccani et al. (26). Specifically, chromatin immuno-precipitation (ChIP) experiments revealed that NF-�B associ-ates rapidly with rapidly induced primary response genes butmuch more slowly with genes induced with delayed kinetics.Saccani and co-workers (26, 27) hypothesized that the associa-tion ofNF-�Bwas delayed because changes in chromatin struc-ture at this latter class of genes must precede NF-�B bindingand transcriptional activation. More recently, this hypothesisreceived support from studies of the SWI/SNF family of ATP-dependent nucleosome remodeling complexes (28). SWI/SNF-dependent nucleosome remodeling was found to be importantfor the activation of secondary response genes and a subset ofprimary response genes induced with delayed kinetics. How-ever, nucleosome remodeling by SWI/SNF complexes was notnecessary for induction of rapidly induced primary responsegenes. Further analysis of chromatin structure using a restric-tion enzyme accessibility assay revealed inducible nucleosomeremodeling at the promoters of secondary response and lateprimary response genes, with constitutively accessible chroma-tin observed at the promoters of early primary response genes.However, the contributions of specific signaling pathways andtranscription factors to the differential regulation of primaryand secondary response genes were not examined.The importance of TLR signaling through MyD88 for the

induction of a broad range of genes raises the intriguing ques-tion of whether the MyD88-dependent pathway makes similaror different contributions to the activation of primary and sec-ondary response genes. The selective role of the primary responsegene product I�B� in regulating a subset of secondary responsegenes is equally intriguing, as, a priori, it must carry out a functionthat is not required for the activation of primary response genes.I�B� could therefore be essential for nucleosome remodeling at asubset of secondary response genes or could catalyze anotherchromatin-relatedevent that isnotnecessary forprimaryresponsegene activation.In this study, we found that, in murine macrophages

responding to LPS through TLR4, MyD88 is required for therecruitment of NF-�B p65, RNA polymerase II (pol II), and theTATA-binding protein (TBP) to secondary response promot-ers. However, because of redundancywith the TRIF-dependentpathway, MyD88 was not required for the recruitment of thesefactors to primary response promoters, although it is essentialfor efficient induction of primary response gene transcription.At the secondary response promoters, MyD88 was also essen-tial for nucleosome remodeling and histone H3K4 trimethyla-tion, whereas primary response promoters were assembled intoconstitutively open chromatin structures in unstimulated cells,with pre-existing H3K4 trimethylation. Surprisingly, althoughthe function of I�B� was restricted to secondary responsegenes, it was not necessary for nucleosome remodeling at thesegenes, but rather was important for H3K4 trimethylation andpreinitiation complex assembly downstream of the remodelingevent.

EXPERIMENTAL PROCEDURES

Antibodies and Mice—Antibodies against NF-�B p65 (C-20)(sc-372), pol II (H-224) (sc-9001), and TFIID (TBP) (Sl-1) (sc-273) were purchased from Santa Cruz Biotechnology. Antibod-ies to trimethyl histone H3 (Lys-4) (07-473) and SNF2�/BRG1(07-478) were purchased from Upstate Biotechnology, Inc.Polyclonal anti-I�B� Ab was obtained by immunizing rabbitwith a recombinant protein containing the N-terminal regionof murine I�B� (1–380 amino acids).Myd88�/�, Trif�/�, and Nfkbiz�/� mice were generated as

described previously (13, 29). All animal experimentswere con-ducted in accordance with the guidelines of the Animal Careand Use Committee of Kyushu University and OsakaUniversity.Stable Cell Lines—RAW264.7 cells were transfected with

pcDNA 3.1 (�)-FLAG-I�B�. The cells resistant to G418 wereselected in the presence of 0.4mg/ml G418 and cloned. Expres-sion of I�B� mRNA was determined by real time reverse tran-scription-PCR, and expression of FLAG-I�B�proteinwasmon-itored by Western blotting using anti-M2 monoclonal Ab(Sigma).Cell Culture—For isolation peritoneal macrophages, mice

were intraperitoneally injected with 2 ml of 4% thioglycollatemedium (Sigma). Peritoneal exudate cells were isolated fromthe peritoneal cavity 3 days post-injection. Cells were incubatedovernight andwashedwith PBS. Remaining adherent cells wereused as peritoneal macrophages for the experiments. To pre-pare bone marrow-derived macrophages, bone marrow cellswere prepared from femora and passed through nylon mesh.Then cells were cultured in RPMI 1640 medium supplementedwith 10% fetal calf serum, 100 �M 2-mercaptoethanol, and 30%supernatants of culturedL cells. After 6 days, the cellswere usedas macrophages for experiments. Macrophage cell lineRAW264.7 cells and J774 cells were maintained in RPMI 1640medium containing 10% fetal calf serum, 100 �M 2-mercapto-ethanol. Peritoneal macrophages, bone marrow-derived mac-rophages, and RAW264.7 cells were stimulated with Esche-richia coliO55:B5 LPS (Sigma).Quantitative Real Time Reverse Transcription-PCR—Total

RNAwas isolated with TRIzol reagent (Invitrogen), and 1–2�gof RNA was reverse-transcribed using Moloney murine leuke-mia virus reverse transcriptase (Promega) and random primers(Toyobo) after treatment with RQ1 DNase I (Promega). Quan-titative real time PCR was performed on an ABI 7000 (AppliedBiosystems) using TaqMan Universal PCR Master Mix(Applied Biosystems). All data were normalized to the corre-sponding gene Eef1a1 encoding elongation factor-1� or 18 SrRNA expression, and the fold difference relative to the elon-gation factor-1� or 18 S rRNA level was shown. Amplificationconditions were 50 °C (2min), 95 °C (10min), 40 cycles of 95 °C(15 s), and 60 °C (60 s). Primers of 18 S ribosomal RNA, Cxcl2,Il23a, Tnf, Lcn2, and Nfkbiz were purchased from Assay onDemand (Applied Biosystems). Sequence for Eef1a1, Il12b, Il6,and Cxcl1 are follows: Eef1a1probe, 5�-gcacctgagcagtgaagc-cagctgct-3�, forward primer 5�-gcaaaaacgacccaccaatg-3� andreverse primer 5�-ggcctggatggttcaggata-3�; Il12b probe, 5�-ctg-cagggaacacatgcccacttg-3�, forward primer 5�-gctcaggatcgctat-

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tacaat-3� and reverse primer 5�-tcttccttaatgtcttccact3�; Il6probe, 5�-ccttcttgggactgatgctggtgaca-3�, forwardprimer5�-ctg-caagagacttccatccagtt-3� and reverse primer 5�-aagtagggaaggc-cgtggtt-3�; Cxcl1 probe, 5�-ttgccctcagggccccactg-3�, forwardprimer 5�-caagaacatccagagcttgaaggt-3� and reverse primer5�-gtg gctatgacttcggtttgg-3�.Chromatin Immunoprecipitation Assay—Peritoneal macro-

phages, bone marrow-derived macrophages, or RAW264.7cells were stimulatedwith 100 ng/ml LPS for the indicated peri-ods. Chromatin was cross-linked by 1% formaldehyde at roomtemperature for 10 min. The cells were scraped after washingwith PBS and centrifuging at 3000 rpm, and then the pellet wasresuspended in SDS buffer (50 mM Tris-HCl (pH 8.0), 10 mM

EDTA, 0.5% SDS). Chromatin was sonicated eight times with30-s pulses, centrifuged at 14,000 rpm to remove debris, diluted5-fold with ChIP dilution buffer (16.7 mM Tris-HCl, 167 mM

NaCl, 1.2 mM EDTA, 1.1% X-100) supplemented with proteaseinhibitor, and precleared with salmon sperm DNA/proteinA-agarose (Upstate). Diluted chromatin was immunoprecipi-tated at 4 °C overnight, and immune complexes were absorbedwith salmon spermDNA/protein A-agarose beads, and washedone timewith low salt buffer (20mMTris-HCl (pH 8.1), 150mM

NaCl, 0.1% SDS, 1%TritonX-100, 2mMEDTA), high salt buffer(20 mM Tris-HCl (pH 8.1), 500 mM NaCl, 0.1% SDS, 1% TritonX-100, 2mMEDTA), LiCl buffer (10mMTris-HCl (pH8.1), 0.25M LiCl, 1 mM EDTA, 1% deoxycholic acid, 1% Nonidet P-40),and two times with TE buffer (10 mM Tris-HCl (pH 8.1), 1 mM

EDTA). Immune complexes extracted in elution buffer (1%SDS, 100mMNaHCO3)were incubated for 4 h at 65 °C to revertDNA-protein cross-links. Then the DNA was extracted byincubation in proteinase K (final 50 �g/ml) buffer for 1 h at45 °C.The purifiedDNAwas used in PCR to assess the presenceof target sequences. Promoter-specific primer was designed toincludeNF-�B-binding site. Sequence of primers are as follows:5�-caacagtgtacttacgcagacg-3� and 5�-ctagctgcctgcctcattctac-3�in the Cxcl2 promoter; 5�-ctgagcactggagactctgaag-3� and5�-gctgggatcatggtgctgtgtt-3� in the Cxcl1 promoter; 5�-gccact-tcctccaagaac-3� and 5�-tttggaaagttggggacacc-3� in the Tnf pro-moter; 5�-atccaaagccctgggaatgtc-3� and 5�-gggtagtccatcctttac-caa-3� in the Lcn2 promoter; 5�-agtatctctgcctccttcctt-3� and5�-gcaacactgaaaactagtgtc-3� in the Il12b promoter; 5�-agaa-gagtgctcatgcttc-3� and 5�-agctacagacatccccagtctc-3� in the Il6promoter; and 5�-gagatggccttgcatgaggat-3� and 5�-gccagagtct-cagtcttcaac-3� in the iNOS promoter. Chromatin immunopre-cipitation using J774 cells with reduced expression of BRG1/BRMwas performed essentially as described (28). In brief, J774cells (7.5 � 105/well) were seeded in 6-well plates and weretransduced with either empty vector or BRG1/BRM short hair-pin RNA vector. The BRG1/BRM short hairpin RNA targets aconserved region between BRG1 and BRM mRNAs(TGGAGAAGCAGCAGAAGAT). The cells were infected viaspin infections on consecutive days at 2500 rpm for 1.5 h and at30 °C. After the second spin infection, puromycin (3 �g/ml)selection was started. The enrichment of transduced cells wasfollowed by flow cytometry, and RNA interference-mediateddepletion was monitored by Western blot. For chromatinimmunoprecipitation experiments, BRG1/BRM RNA interfer-

ence-depleted cells and control cells were stimulated and cross-linked 5 days after the first spin infection.Nuclei Preparation—Peritoneal macrophages or bone mar-

row-derived macrophages were stimulated with 10 �g/ml LPSfor the indicated periods. Cells were scraped and pelleted at1500 rpm. Cells were washed once with PBS. The cell pellet wasresuspended in Nonidet P-40 lysis buffer (10 mM Tris-HCl (pH7.4), 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40, 0.15 mMspermine, and 0.5 mM spermidine) and incubated on ice for 5min. Nuclei were pelleted at 1000 rpm, followed by washingwith RE buffer (10 mM Tris-HCl (pH 7.4), 50 mM NaCl, 10 mMMgCl2, 0.2 mM EDTA, 0.2 mM EGTA, 1 mM �-mercaptoetha-nol, 0.15 mM spermine, 0.5 mM spermidine).Restriction Enzyme Accessibility Assay—Restriction enzyme

accessibility assay was performed essentially as described (30,31). Isolated cell nuclei and restriction enzyme (100 units)(Il12b promoter and enhancer, SpeI; Il6 promoter, AfIII) wereincubated for 15 min at 37 °C. Reactions were stopped by add-ing proteinase K buffer (100 mM Tris-HCl (pH 8.5), 200 mMNaCl, 5 mM EDTA, 0.2% SDS, 100 ng/ml proteinase K), incu-bated overnight at 56 °C, followed by genomic DNA isolation.Purified DNA (10–15 �g) was digested to completion to gen-erate reference cleavage products using the following restric-tion enzymes: KpnI and SphI for the Il12b promoter andenhancer and XbaI and SpeI for Il6. Samples were analyzed bySouthern blotting with 32P-labeled gene-specific probesdesigned at the following regions, Il12b promoter (�64 to�437), Il12b enhancer (�8711 to �9113), and Il6 promoter(�544 to �1043).

RESULTS

Different Roles ofMyD88 at Primary and Secondary ResponseGenes—Tounderstand how theMyD88 pathway contributes tothe regulation of primary and secondary response genes, wecompared LPS-stimulated wild-type and Myd88�/� macro-phages. In the mutant cells, the expression of a large number ofprimary and secondary response genes is known to be severelyreduced, despite an intact TRIF-dependent pathway (1). Forthis study, the Cxcl2, Cxcl1, and Tnf genes (encoding MIP2,GRO1, and tumor necrosis factor-�, respectively) were moni-tored as examples of primary response genes, which areinduced rapidly (supplemental Fig. S1A) in the absence of arequirement for new protein synthesis (based on resistance tocycloheximide) (28). The Lcn2, Il12b, and Il6 genes (encodinglipocalin 2, IL-12 p40, and IL-6, respectively) were used asexamples of secondary response genes, which are induced withdelayed kinetics (supplemental Fig. S1B) in a cycloheximide-sensitive manner (28). Although expression of all seven geneswas greatly reduced in Myd88�/� macrophages, residualinduction was observed with the four primary response genes.This induction was largely eliminated in Myd88�/�Trif�/�

macrophages (supplemental Fig. S1C).To determine how the absence ofMyD88 signaling alters the

cascade of events leading to transcription initiation, ChIPexperiments were performed. At the Lcn2 secondary responsepromoter, the recruitment of theNF-�Bp65 subunit, pol II, andTBP was greatly reduced in LPS-stimulated peritoneal macro-phages from Myd88�/� mice, when compared with macro-

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phages from wild-type mice (Fig. 1A). Remarkably, these samefactors were recruited normally to the Cxcl2 primary responsepromoter inMyd88�/� macrophages, with only amodest delayin p65 recruitment and perhaps more transient association ofpol II (Fig. 1B). Consistent with previous findings (26), factorrecruitment was observed at earlier time points at the primaryresponse promoter than at the secondary response promoter.Similar results were obtained using bone marrow-derivedmacrophages (data not shown). Importantly, recruitment ofp65, pol II, and TBP to the Cxcl2 promoter was eliminated inMyd88�/�Trif�/� macrophages (Fig. 1C), consistent withprevious evidence that NF-�B activation by LPS is only mod-estly delayed in Myd88�/� macrophages but eliminated inMyD88�/�Trif�/� macrophages (29).

These findings suggest a hypothesis in which MyD88 isessential for a change in chromatin structure at secondaryresponse promoters that must precede the binding of NF-�Band the assembly of a transcription preinitiation complex.However, at primary response promoters, preinitiation complexassembly is relatively unperturbed because of the following: 1)these genes possess a poised MyD88-independent chromatinstructure in unstimulated cells, and 2) the TRIF-dependentpathway can support NF-�B activation in the absence ofMyD88. It is important to emphasize thatCxcl2 transcription isseverely reduced in Myd88�/� macrophages (supplementalFig. 1A), despite the efficient recruitment of p65, pol II, andTBPto the Cxcl2 promoter. Possible reasons primary responsegenes require the MyD88 pathway for efficient induction areconsidered below (see under “Discussion”).

MyD88-dependent H3K4 Trimethylation and NucleosomeRemodeling at Secondary Response Promoters—To test theabove hypothesis, we evaluated the importance of MyD88 forthe chromatin changes that accompany gene activation in LPS-stimulated macrophages. Trimethylation of histone H3K4 wasfirst examined because of its close association with transcrip-tionally active genes (23, 32, 33). At the promoters of two rep-resentative primary response genes, Cxcl2 and Cxcl1, ChIPexperiments revealed constitutively high H3K4 trimethylationin unstimulated wild-type macrophages, with no significantchange following LPS stimulation (Fig. 2A). These results areconsistent with previous evidence that early primary responsepromoters possess constitutively acetylated histones and con-stitutively open chromatin structures (28). Importantly, similarH3K4 trimethylation levels were observed at these promotersinMyd88�/� macrophages (Fig. 2A).In contrast to the constitutiveH3K4 trimethylation observed

at the primary response promoters, this modification wasstrongly induced in LPS-stimulated wild-type macrophages at

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FIGURE 1. MyD88-dependent regulation of primary and secondaryresponse genes. Peritoneal macrophages from wild-type, Myd88�/�, andMyd88�/�Trif�/� mice were stimulated with 100 ng/ml LPS for the indicatedperiods, and ChIP assay was performed with antibodies to NF-�Bp65, pol II, orTBP. The immunoprecipitated Lcn2 promoter (A) or Cxcl2 promoter (B and C)was analyzed by PCR with promoter-specific primers. PCR amplification of thetotal input DNA in each sample is shown (Input). This is representative of fiveindependent experiments. The same result was obtained when bone mar-row-derived macrophages were used.

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FIGURE 2. MyD88-dependent nucleosome remodeling of secondaryresponse promoters. Peritoneal macrophages from wild-type andMyd88�/� mice were stimulated with 100 ng/ml LPS for the indicated peri-ods, and chromatin immunoprecipitation (ChIP) assay was performed withanti-trimethyl histone H3 (Lys-4) Ab (Me3-H3K4). Precipitated DNA for theCxcl2 promoter, Cxcl1 promoter (A), Lcn2 promoter, or Il12b promoter (B) wasanalyzed by PCR. This is representative of two independent experiments.C, bone marrow macrophages from wild-type and Myd88�/� mice were stim-ulated with 10 �g/ml LPS for the indicated periods. Restriction enzyme acces-sibility assay at Il12b promoter region used nuclei from bone marrowmacrophages.

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the promoters for two representative secondary responsegenes, Lcn2 and Il12b (Fig. 2B). Significantly, the LPS-inducedH3K4 trimethylation observed at these promoters wasMyD88-dependent. We further examined nucleosome remodeling atthe Il12b promoter using a Southern blot-based restrictionenzyme accessibility assay (28, 31). As demonstrated previ-ously, strong increases in restriction enzyme cleavage efficiencywere observed following LPS stimulation in wild-type macro-phages (Fig. 2C) (28). However, restriction enzyme cleavagewas greatly reduced in Myd88�/� macrophages (Fig. 2C).Taken together, the results in Figs. 1 and 2 strongly suggest thatNF-�B, TBP, and pol II cannot associate with the promoters ofsecondary response genes in LPS-stimulated macrophagesfromMyd88�/� mice because the MyD88 pathway is requiredfor LPS-induced H3K4 trimethylation and nucleosome remod-eling at these promoters. In contrast, the recruitment ofNF-�B,TBP, and pol II to primary response promoters does not dependon MyD88 because these promoters are assembled into poisedchromatin structures in unstimulated cells.I�B� Mediates the Activation of a Subset of Secondary

Response Genes—The above results suggest that, although theTRIF pathway can support NF-�B activation in LPS-stimulatedmacrophages from Myd88�/� mice, TRIF cannot support theactivation of one or more factors that act prior to nucleosomeremodeling and H3K4 trimethylation at MyD88-dependentsecondary response promoters. Thus, an MyD88-specific tar-get is essential for nucleosome remodeling andH3K4 trimethy-lation at these promoters. It is noteworthy that LPS-inducedremodeling at a collection of secondary response promoterswas previously found to require new protein synthesis (28).This previous finding suggests that theMyD88 target of interestmay be a primary response gene product, as opposed to a tran-scription factorwhose activity is induced post-translationally inresponse to TLR4 signaling.One primary response gene product that is an attractive can-

didate for contributing to the activation of a subset of secondaryresponse genes is the nuclear I�B protein, I�B�, encoded by theNfkbiz gene. As described previously, expression of a subset ofsecondary response genes is impaired in Nfkbiz�/� macro-phages, whereas primary response genes are expressed nor-mally (supplemental Fig. S2, A and B) (13).

To gain further insight into theimportance of I�B� for the expres-sion of secondary response genes,I�B� was constitutively overex-pressed in the RAW264.7 macro-phage line. When I�B� was presentat the time of LPS stimulation,three I�B�-dependent secondaryresponse genes, Lcn2, Il12b, and Il6,were induced more rapidly than incontrol RAW264.7 cells (supple-mental Fig. S2C). Consistent withthe more rapid induction in thepresence of constitutively expressedI�B�, ChIP assays revealed that theassociation of p65, pol II, and TBPreached a detectable level at the

Lcn2 promoter more rapidly than in control cells (Fig. 3A). Incontrast, the kinetics of factor recruitment to theCxcl2 primaryresponse promoter was unchanged (Fig. 3B). Importantly, his-tone H3K4 trimethylation was also inducedmore rapidly at theLcn2 promoter following LPS stimulation of the I�B�-express-ing cells (Fig. 3C), whereas the constitutive H3K4 trimethyla-tion observed at the Cxcl2 promoter remained unchanged (Fig.3D). These findings are consistent with a model in which I�B�plays amajor role in the changes in chromatin structure that areassociated with the induction of I�B�-dependent secondaryresponse genes.I�B�-dependentH3K4Trimethylation at Secondary Response

Promoters—To complement the I�B� gain-of-function experi-ments, loss of function experiments were performed with bonemarrow-derived macrophages from Nfkbiz�/� mice. Strik-ingly, although the constitutive H3K4 trimethylation at threerepresentative primary response promoters was comparable inwild-type and Nfkbiz�/� macrophages (Cxcl2, Cxcl1, Fig. 4A;Tnf, data not shown), the inducible H3K4 trimethylationobserved in wild-type macrophages at the promoters of threeI�B�-dependent secondary response promoters was greatlyreduced in Nfkbiz�/� cells (Lcn2, Il12b; Fig. 4B, Il6; data notshown). Furthermore, the recruitment ofNF-�Bp65, pol II, andTBP was greatly diminished at secondary response promotersin Nfkbiz�/� cells (Lcn2, Il12b; Fig. 4D, Il6; supplemental Fig.S3), whereas the recruitment of these proteins to promoters ofprimary response or I�B�-independent secondary responsegenes was unaffected (Cxcl2, Tnf, or iNOS Fig. 4C and supple-mental Fig. S4). These results are consistent with the gain-of-function results and support the view that I�B� is a selectivemajor regulator of chromatin structure and preinitiation com-plex assembly at I�B�-dependent secondary response genes.To determine whether I�B� directly regulates I�B�-depend-

ent genes, ChIP experiments were performed. The resultsrevealed that I�B� associates with the Il12b and Il6 promotersin LPS-stimulatedmacrophages (Fig. 5). The kinetics of bindingwas similar to that observed with two other factors previouslyshown to associate with these control regions, BRG1 andC/EBP� (28). It is important to note that, in addition to itsassociation with the promoters of I�B�-dependent genes,inducible I�B� association was observed in our hands at a

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recently described enhancer for the Il12b gene (Fig. 5), aswell asat the promoters for a number of primary response genes thatdo not require I�B� for expression (data not shown). Thus,although the analysis of Nfkbiz�/� macrophages providesstrong evidence that I�B� is selectively required for H3K4 tri-methylation and for the recruitment of p65, TBP, and pol II to asubset of I�B�-dependent secondary response genes, I�B� asso-ciates with other LPS-induced genes with no apparent func-tional consequences.

I�B�-independent NucleosomeRemodeling at Secondary ResponsePromoters—As shown previously(28) and in Fig. 2C, nucleosomeremodeling by ATP-dependentremodeling complexes is generallyrequired for the activation of sec-ondary response genes followingLPS stimulation. To determinewhether I�B� is required for nucleo-some remodeling at I�B�-dependent genes, we first used aChIP assay to monitor recruitmentof the BRG1 catalytic subunit of theSWI/SNF remodeling complexes.Following LPS stimulation, BRG1was found to associate with repre-sentative I�B�-dependent genes (Il6and Lcn2 in Fig. 6A and Il6 and Il12bin Fig. 6B). Interestingly, this induc-ible association was eliminated inMyd88�/� macrophages but wasretained in Nfkbiz�/� macrophages(Fig. 6). These results suggest thatI�B� acts downstream of theremodeling event, with anotherMyD88 target required for remodel-ing. In contrast to the resultsobtained with secondary responsegenes, BRG1 associated constitu-tively with the Cxcl2 primaryresponse promoter (Fig. 6), as previ-ously described, even though BRG1is not important for the induction ofthis and other primary responsegenes (28). This constitutive asso-ciation was retained in bothMyd88�/� and Nfkbiz�/� cells(Fig. 6).To further evaluate the role of

I�B� in nucleosome remodeling,restriction enzyme accessibilityexperiments were performed.Consistent with the BRG1 ChIPdata, the LPS-induced increases inrestriction enzyme cleavageobserved at the Il12b enhancer,Il12b promoter, and Il6 promoterwere comparable in wild-type and

Nfkbiz�/� macrophages stimulated with LPS (Fig. 7A). Finally,to determine whether I�B� association with secondaryresponse genes requires nucleosome remodeling, BRG1 andthe closely related BRM catalytic subunits of the SWI/SNFremodeling complexes were depleted from J774 macrophagesusing a retrovirus that expresses a short interfering RNA tar-geted to a conserved region of BRG1 andBRM(28). In cells withreduced BRG1/BRM expression, LPS-induced recruitment ofI�B� to the Il6 and Lcn2 promoters and the Il12b enhancer was

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bone marrow-derived macrophages treated with 100 ng/ml LPS for the indicated periods was immunopre-cipitated with antibody against trimethyl histone H3 (Lys-4). Precipitated DNA was analyzed by PCR withpromoter-specific primers for Cxcl2, Cxcl1 (A), Lcn2, and Il12b (B). This is representative of three independentexperiments. C and D, peritoneal macrophages from wild-type and Nfkbiz�/� mice were stimulated with 100ng/ml LPS for the indicated periods and then chromatins were prepared and immunoprecipitated with anti-body against NF-�Bp65, pol II, or TBP. Precipitated DNA was analyzed by PCR with promoter-specific primersfor Cxcl2, Tnf (C), Lcn2, and Il12b (D). The results are representative of three independent experiments.

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reduced compared with controlcells, whereas recruitment to theconstitutively open Cxcl2 and Tnfpromoters was unchanged (Fig. 7B).These findings indicate that nucleo-some remodeling is required for theefficient recruitment of I�B� to thetranscriptional control regions ofsecondary response genes.

DISCUSSION

The results described in thisstudy highlight the diverse mecha-nisms by which chromatin struc-ture, signal transduction pathways,and transcription factors can con-trol the activation of a large panel ofinducible pro-inflammatory genesexpressed by macrophages follow-

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FIGURE 7. Nucleosome remodeling at secondary response gene regulatory regions in the absence of I�B�. A, bone marrow-derived macrophages fromwild-type and Nfkbiz�/� mice were stimulated with 10 �g/ml LPS for the indicated periods. Restriction enzyme accessibility assay at the Il12b enhancer (upper),Il12b promoter (middle), and Il6 promoter (bottom) regions was performed. B, ChIP assay was performed with chromatin prepared from J774 cells infected withthe empty RNA interference vector (white bars) and from BRG1/BRM short interfering RNA-depleted cells (black bars) treated with LPS for 0 and 240 min.Antibodies against I�B� and BRG1 were used. Precipitated DNA was quantified by real time PCR using primers specific for the indicated control regions. Datawere plotted relative to input DNA (% Input).

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ing TLR4 stimulation. At one key class of genes that is inducedrapidly in the absence of new protein synthesis, the early pri-mary response class, the promoters appear to be assembled intochromatin structures that are poised for activation. In unstimu-lated cells, these chromatin structures consist of high histoneacetylation andH3K4 trimethylation levels and high accessibil-ity to nuclease cleavage (26, 28). After macrophage activation,the chromatin structure remains largely unchanged, butNF-�Brapidly associates with the promoters and presumably contrib-utes to the rapid assembly of a preinitiation complex containingTBP and pol II. Preinitiation complex assembly in response toTLR4 signaling does not specifically require MyD88 because ofits redundancy with TRIF. However, MyD88 is critical for theefficient induction of primary response gene transcription. It ispossible that reduced expression of primary response genes inMyd88�/� cells is because of earlier shutdown of transcription.But there might be another unknown mechanism, because theMyD88 effect is just as strong at the 1-h time point.Although secondary response genes can be induced quite

rapidly after TLR4 stimulation, theirmechanismof activation is

dramatically different. In unstimulated macrophages, second-ary response promoters are usually assembled into chromatinstructures that are inaccessible to nuclease cleavage and exhibitlow levels of histone acetylation and H3K4 trimethylation (26,28). Substantial changes in chromatin structure are thereforerequired for transcriptional activation (27). One critical eventappears to be the remodeling of nucleosomes by ATP-depend-ent nucleosome remodeling complexes of the SWI/SNF family.A previous study showed that nucleosome remodeling at thepromoters of secondary response genes requires new proteinsynthesis (28). The results of this study show thatMyD88 path-ways are also required for nucleosome remodeling. AlthoughI�B� was an attractive candidate for anMyD88-dependent pri-mary response gene product that might drive nucleosomeremodeling at a subset of secondary response genes, our resultsstrongly suggest that other MyD88-dependent primaryresponse gene products carry out this critical function. I�B�instead plays an important role downstream of the nucleosomeremodeling step but prior to the binding of NF-�B p65, TBP,and pol II, and prior to histone H3K4 trimethylation (Fig. 8).

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FIGURE 8. Schematic model of primary and secondary response gene activation pathway. Early primary response gene promoters have open nucleosomestructures and are activated immediately after LPS stimulation. In contrast, secondary response gene promoters have closed nucleosome structures and are remod-eled through an unknown primary response gene product (X)-dependent recruitment of the SWI/SNF complexes, including BRG1. Then another primary responsegene product, I�B�, mediates preinitiation complex assembly and histone H3K4 trimethylation, resulting in activation of the secondary response genes.

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The precise physiological reasons for the evolution of thesediverse pro-inflammatory gene activation pathways are notknown.However, one important consequence is the capacity toregulate different subsets of genes with greater selectivity.Indeed, many of the primary response gene productsmay func-tion properly only if activated rapidly and if produced at anappropriate level. For example, tumor necrosis factor-� andIL-1� are cytokines that trigger a series of inflammatory andhost defense responses, and when produced in excess, theyinduce serious multiple organ failure (34). Other primaryresponse gene products, the MIP-2 and GRO1 chemokines,mediate the recruitment of neutrophils during an acute phaseof inflammation (35).The rapid induction of many early primary response genes

may therefore be of considerable benefit during the initiation ofan immune response. At the same time, the need to overcome anucleosome barrier may provide important benefits for sec-ondary response genes, by allowing them to be activated with ahigher degree of selectivity by different stimuli and in differentbiological scenarios. Consistent with this hypothesis, only asubset of secondary response genes require I�B� for activation,whereas other transcription factors presumably carry out thesame functions at other subsets of secondary response genes. Infact, our results provide evidence that the nucleosome barriercan confer a requirement for at least two transcription factorsthat are not generally required by early primary response genes.Therefore, at least oneMyD88 target genemust be required forinducible nucleosome remodeling by SWI/SNF complexes,with I�B� or its equivalent functioning at a later stage of thegene activation cascade to promote preinitiation complexassembly and histone H3K4 trimethylation. In this context, it isinteresting to note that I�B�-dependent secondary responsegene products include cytokines and chemokines that areinvolved in the regulation of T cell-mediated immune respons-es; IL-12 p40 is a key subunit of the IL-12 and IL-23 het-erodimeric cytokines, which are critical to Th1 andTh17 devel-opment, respectively (36), and IL-6 has recently been shown tobe essential for initiation of Th17 cell development (37). OtherI�B�-dependent secondary response gene products, such asEbi3, IL-18, and TARC (13), also regulate Th1/Th2/Th17 cell-mediated immune responses (38–42). Therefore, the nucleo-some barrier for secondary response genesmay have evolved toensure tight regulation of adaptive immunity during TLRsignaling.Although the results of this analysis provide a framework

toward understanding the differential regulation of pro-inflam-matory genes, a number of important mechanistic questionsremain to be answered. First, why is MyD88 required for pri-mary response gene activation, despite the efficient recruit-ment of p65, TBP, and pol II to primary response promoters inMyd88�/� macrophages? A likely explanation is that thesegenes require a direct target of the MyD88 signaling pathwaythat remains to be characterized.However, an alternative is thatthe moderately delayed induction of NF-�B somehow disruptstranscriptional activation.A second unanswered question is, what primary response

gene products and MyD88 target genes are responsible forinducible remodeling at I�B�-dependent secondary response

genes, as well as other subsets of secondary response genes?This question has been especially difficult to answer. I�B�appeared to be an ideal candidate because its regulatory func-tions are restricted to secondary response genes. However, wewere unable to find evidence implicating I�B� in the regulationof nucleosome remodeling. Analyses of several other primaryresponse gene products for a possible role in the regulation ofnucleosome remodeling at secondary response genes have alsoyielded negative results.5 As an alternative to the candidate-gene approach, it may be possible to identify DNA sequenceelements in secondary response promoters that are required forinducible nucleosome remodeling, which may lead to the crit-ical transcription factors. However, it will first be necessary todevelop an assay in which secondary response promotersassemble into a native chromatin structure that depends on anucleosome remodeling event for transcriptional activation.A third unanswered question is how I�B� contributes to the

recruitment of NF-�B p65 complexes, TBP, and pol II, and howit facilitates histone H3K4 trimethylation. A previous studyprovided evidence that I�B� is recruited to target promoters byNF-�Bp50homodimers (13).One possibility is that the bindingof p50 homodimers recruits I�B� through a direct interaction,which then recruits a p65-containing dimer to a differentNF-�B site, or to the same site through dimer exchange. Thep65 dimer could then act in concert with other transcriptionfactors bound to the promoter to recruit TBP and pol II andfacilitate H3K4 trimethylation and transcription initiation.Other possible mechanisms of I�B� function must also be con-sidered, such as a direct role in the recruitment of an H3K4methyltransferase.The importance of chromatin for the differential regulation

of TLR-dependent genes was recently highlighted in an elegantanalysis of the negative regulation of the inflammatoryresponse (18). Further dissection of the diverse mechanismsunderlying these key regulatory events will help elucidate themolecular basis of immune disorders caused by abnormal acti-vation of innate immunity.

Acknowledgments—We thank Y. Yamada and K. Takeda for techni-cal assistance, T. Nakayama for helpful discussion, and M. Kurataand M. Yasuda for secretarial assistance.

REFERENCES1. Akira, S., and Takeda, K. (2004) Nat. Rev. Immunol. 4, 499 –5112. Iwasaki, A., and Medzhitov, R. (2004) Nat. Immunol. 5, 987–9953. Beutler, B., Jiang, Z., Georgel, P., Crozat, K., Croker, B., Rutschmann, S.,

Du, X., and Hoebe, K. (2006) Annu. Rev. Immunol. 24, 353–3894. Marshak-Rothstein, A. (2006) Nat. Rev. Immunol. 6, 823– 8355. Liew, F. Y., Xu, D., Brint, E. K., and O’Neill, L. A. (2005) Nat. Rev. Immunol.

5, 446 – 4586. Negishi, H., Ohba, Y., Yanai, H., Takaoka, A., Honma, K., Yui, K., Mat-

suyama, T., Taniguchi, T., and Honda, K. (2005) Proc. Natl. Acad. Sci.U. S. A. 102, 15989 –15994

7. Kuwata, H., Matsumoto, M., Atarashi, K., Morishita, H., Hirotani, T.,Koga, R., and Takeda, K. (2006) Immunity 24, 41–51

8. Akira, S., Uematsu, S., and Takeuchi, O. (2006) Cell 124, 783– 8019. Yamazaki, S., Muta, T., and Takeshige, K. (2001) J. Biol. Chem. 276,

5 V. Ramirez-Carrozzi and S. T. Smale, unpublished data.

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by guest on Novem

ber 28, 2020http://w

ww

.jbc.org/D

ownloaded from

27657–2766210. Kitamura, H., Kanehira, K., Okita, K., Morimatsu, M., and Saito, M. (2000)

FEBS Lett. 485, 53–5611. Haruta, H., Kato, A., and Todokoro, K. (2001) J. Biol. Chem. 276,

12485–1248812. Motoyama, M., Yamazaki, S., Eto-Kimura, A., Takeshige, K., and Muta, T.

(2005) J. Biol. Chem. 280, 7444 –745113. Yamamoto, M., Yamazaki, S., Uematsu, S., Sato, S., Hemmi, H., Hoshino,

K., Kaisho, T., Kuwata, H., Takeuchi, O., Takeshige, K., Saitoh, T.,Yamaoka, S., Yamamoto, N., Yamamoto, S., Muta, T., Takeda, K., andAkira, S. (2004) Nature 430, 218 –222

14. Smale, S. T., and Fisher, A. G. (2002) Annu. Rev. Immunol. 20, 427– 46215. Ansel, K. M., Lee, D. U., and Rao, A. (2003) Nat. Immunol. 4, 616 – 62316. Mostoslavsky, R., Alt, F. W., and Bassing, C. H. (2003) Nat. Immunol. 4,

603– 60617. Natoli, G., Saccani, S., Bosisio, D., and Marazzi, I. (2005) Nat. Immunol. 6,

439 – 44518. Foster, S. L., Hargreaves, D. C., and Medzhitov, R. (2007) Nature 447,

972–97819. Roth, S. Y., Denu, J. M., and Allis, C. D. (2001) Annu. Rev. Biochem. 70,

81–12020. Lusser, A., and Kadonaga, J. T. (2003) BioEssays 25, 1192–120021. Cairns, B. R. (2005) Curr. Opin. Genet. Dev. 15, 185–19022. de la Serna, I. L., Ohkawa, Y., and Imbalzano, A. N. (2006) Nat. Rev. Genet.

7, 461– 47323. Li, B., Carey, M., and Workman, J. L. (2007) Cell 128, 707–71924. Bernstein, B. E., Humphrey, E. L., Erlich, R. L., Schneider, R., Bouman, P.,

Liu, J. S., Kouzarides, T., and Schreiber, S. L. (2002) Proc. Natl. Acad. Sci.U. S. A. 99, 8695– 8700

25. Martin, C., and Zhang, Y. (2005) Nat. Rev. Mol. Cell Biol. 6, 838 – 84926. Saccani, S., Pantano, S., and Natoli, G. (2001) J. Exp. Med. 193, 1351–135927. Natoli, G. (2006) FEBS Lett. 580, 2843–284928. Ramirez-Carrozzi, V. R., Nazarian, A. A., Li, C. C., Gore, S. L., Sridharan,

R., Imbalzano, A. N., and Smale, S. T. (2006) Genes Dev. 20, 282–29629. Yamamoto, M., Sato, S., Hemmi, H., Hoshino, K., Kaisho, T., Sanjo, H.,

Takeuchi, O., Sugiyama, M., Okabe, M., Takeda, K., and Akira, S. (2003)Science 301, 640 – 643

30. Weinmann, A. S., Plevy, S. E., and Smale, S. T. (1999) Immunity 11,665– 675

31. Zhou, L., Nazarian, A. A., and Smale, S. T. (2004) Mol. Cell. Biol. 24,2385–2396

32. Santos-Rosa, H., Schneider, R., Bannister, A. J., Sherriff, J., Bernstein, B. E.,Emre, N. C., Schreiber, S. L., Mellor, J., and Kouzarides, T. (2002) Nature419, 407– 411

33. Bernstein, B. E., Kamal, M., Lindblad-Toh, K., Bekiranov, S., Bailey, D. K.,Huebert, D. J., McMahon, S., Karlsson, E. K., Kulbokas, E. J., III, Gingeras,T. R., Schreiber, S. L., and Lander, E. S. (2005) Cell 120, 169 –181

34. Dinarello, C. A. (1991) J. Infect. Dis. 163, 1177–118435. Zhang, X. W., Liu, Q., Wang, Y., and Thorlacius, H. (2001) Br. J. Pharma-

col. 133, 413– 42136. Trinchieri, G., Pflanz, S., and Kastelein, R. A. (2003) Immunity 19,

641– 64437. Bettelli, E., Carrier, Y., Gao, W., Korn, T., Strom, T. B., Oukka, M., Weiner,

H. L., and Kuchroo, V. K. (2006) Nature 441, 235–23838. Hunter, C. A. (2005) Nat. Rev. Immunol. 5, 521–53139. Batten, M., Li, J., Yi, S., Kljavin, N. M., Danilenko, D. M., Lucas, S., Lee, J.,

de Sauvage, F. J., and Ghilardi, N. (2006) Nat. Immunol. 7, 929 –93640. Stumhofer, J. S., Laurence, A., Wilson, E. H., Huang, E., Tato, C. M., John-

son, L. M., Villarino, A. V., Huang, Q., Yoshimura, A., Sehy, D., Saris, C. J.,O’Shea, J. J., Hennighausen, L., Ernst, M., and Hunter, C. A. (2006) Nat.Immunol. 7, 937–945

41. Nakanishi, K., Yoshimoto, T., Tsutsui, H., and Okamura, H. (2001) Annu.Rev. Immunol. 19, 423– 474

42. Imai, T., Nagira, M., Takagi, S., Kakizaki, M., Nishimura, M., Wang, J.,Gray, P. W., Matsushima, K., and Yoshie, O. (1999) Int. Immunol. 11,81– 88

I�B�-mediated Activation of TLR-dependent Genes

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Smale and Kiyoshi TakedaMizutani, Hirotaka Kuwata, Hideo Iba, Makoto Matsumoto, Kenya Honda, Stephen T.

Hisako Kayama, Vladimir R. Ramirez-Carrozzi, Masahiro Yamamoto, Taketoshiζ

BκClass-specific Regulation of Pro-inflammatory Genes by MyD88 Pathways and I

doi: 10.1074/jbc.M709965200 originally published online March 3, 20082008, 283:12468-12477.J. Biol. Chem. 

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VOLUME 283 (2008) PAGES 12468 –12477DOI 10.1074/jbc.A114.709965

Class-specific regulation of pro-inflammatory genes byMyD88 pathways and I�B�.Hisako Kayama, Vladimir R. Ramirez-Carrozzi, Masahiro Yamamoto, TaketoshiMizutani, Hirotaka Kuwata, Hideo Iba, Makoto Matsumoto, Kenya Honda,Stephen T. Smale, and Kiyoshi Takeda

PAGE 12471:

The data shown for TBP binding to the Lcn2 promoter in Fig. 1A arenot correct. The correct data are shown in the version of the article thatwas published as a JBC Paper in Press on March 3, 2008, but a duplicateof the pol II panel was incorrectly substituted for the TBP panel in theredacted version that was published on May 2, 2008.

56p

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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 8, p. 4815, February 20, 2015© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

FEBRUARY 20, 2015 • VOLUME 290 • NUMBER 8 JOURNAL OF BIOLOGICAL CHEMISTRY 4815

ADDITIONS AND CORRECTIONS

Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice ofthese corrections as prominently as they carried the original abstracts.

VOLUME 283 (2008) PAGES 12468 –12477DOI 10.1074/jbc.A115.709965

Class-specific regulation of pro-inflammatory genes byMyD88 pathways and I�B�.Hisako Kayama, Vladimir R. Ramirez-Carrozzi, Masahiro Yamamoto,Taketoshi Mizutani, Hirotaka Kuwata, Hideo Iba, Makoto Matsumoto,Kenya Honda, Stephen T. Smale, and Kiyoshi Takeda

PAGE 12473:

The following sentence should be added to the legend for Fig. 5. “TheIl12� profiles for BRG1 and C/EBP� are reproduced with permissionfrom Ramirez-Carrozzi et al. (28), to allow comparison with the IkB�

profile for Il12� and the three profiles for Il6.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 37, p. 22446, September 11, 2015© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

22446 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 37 • SEPTEMBER 11, 2015

ADDITIONS AND CORRECTIONS

Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice ofthese corrections as prominently as they carried the original abstracts.