Mycobacterium Avium Macrophages with Receptor 2 Promoter During

Rapid Chromatin Remodeling of Toll-Like Receptor 2 Promoter During Infection of Macrophages with Mycobacterium avium

This information is current as Tianyi Wang, William P. Lafuse, Kiyoshi Takeda, Shizuo of September 26, 2021. Akira and Bruce S. Zwilling J Immunol 2002; 169:795-801; ; doi: 10.4049/jimmunol.169.2.795 http://www.jimmunol.org/content/169/2/795 Downloaded from

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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 © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Rapid Chromatin Remodeling of Toll-Like Receptor 2 Promoter During Infection of Macrophages with Mycobacterium avium1

Tianyi Wang,* William P. Lafuse,† Kiyoshi Takeda,‡ Shizuo Akira,‡ and Bruce S. Zwilling2*†

We have previously reported that NF-␬B and stimulating factor 1 elements within the proximal mouse Toll-like receptor 2 (TLR2) promoter region are required for the transcriptional activation of TLR2 expression following infection with Mycobacterium avium. In the present study, we found that a rapid increase in both DNase I sensitivity and restriction enzyme accessibility at the TLR2 promoter region occurred following infection with M. avium. Increase in restriction enzyme accessibility at the TLR2 promoter region covering the NF-␬B and stimulating factor 1 elements was associated with the induction of TLR2 expression at the mRNA

level. Furthermore, the increase in restriction enzyme accessibility at the TLR2 promoter region did not appear to result from Downloaded from binding of NF-␬B, but rather depended on a TLR2-myeloid differentiation factor 88 signaling pathway. Together our results indicate that chromatin remodeling occurs at TLR2 promoter region following infection with M. avium, allowing the access of transcription factors to initiate the transcription of TLR2. The Journal of Immunology, 2002, 169: 795–801.

oll is a type I transmembrane receptor in Drosophila that two NF-␬B and stimulating factor 1 (Sp1) sites are required for the is involved in dorsal-ventral patterning in larvae and in

full induction of TLR2 transcription following infection with M. http://www.jimmunol.org/ T the induction of an antifungal response in adult flies (1Ð avium. 3). Activation of the Toll receptor by its ligand Spatzle results in Genes in eukaryotic cells are packed into chromatin. The pri- the interaction and stimulation of several signaling molecules that mary repeating units of chromatin are nucleosomes (17). Forma- are homologous to proteins involved in NF-␬B activation by the tion of nucleosomes and other higher order chromatin structures IL-1R in mammalian cells. Medzhitov et al. (4) cloned a human can render the DNA inaccessible to transcription factors and RNA receptor, homologous to Toll, which participates in activation of polymerases. The repressive chromatin structure must be remod- cells during both innate and adaptive immune responses. Ten eled to allow transcription to occur. Studies at the Saccharomyces 3 members of the human Toll-like receptor (TLR) family have been cerevisiae Pho5, HIV, and murine mammary tumor virus (MMTV) identified (5). At least some of these are involved in mediating promoters have indicated that nucleosomes contribute important by guest on September 26, 2021 NF-␬B activation following the interaction of macrophages with regulatory functions for genes that are rapidly induced in response different bacterial pathogens or bacterial cell wall components (6Ð to extracellular signals. The Pho5 promoter is contained within 8). TLR4 has been shown to mediate the response of macrophages positioned nucleosomes. Induction with low phosphate results in to LPS (9, 10). The hyporesponsiveness of C3H/HeJ mice (Lpsd) dephosphorylation of activator Pho4, which binds a DNase I-hy- to LPS is the result of a missense mutation in TLR4 (11, 12). TLR2 persensitive region flanked by the promoter, ultimately allowing has been implicated in the activation of NF-␬B following interaction of macrophages with LPS, lipoarabinomannan from mycobac- DNA binding and the selective remodeling of four nucleosomes in terial species, and lipoteichoic acids from the cell walls of Gram- the vicinity of the promoter (18, 19). Nucleosomes are positioned positive bacteria (13Ð16). We have previously reported that the downstream of the start site of the nucleosome-free HIV-1 pro- transcription of TLR2 promoter was induced following infection moter (20). During T cell activation, transcription factors bind the of macrophages with Mycobacterium avium. We also found that promoter, leading to the remodeling of the downstream nucleosome and the initiation of the transcription. Finally, the glucocorticoid-inducible MMTV promoter is contained within a nucleo-

*Department of Microbiology, College of Biological Sciences, and †Department of some array, with one nucleosome spanning the glucocorticoid Molecular Virology, Immunology and Medical Genetics, College of Medicine and receptor binding sites (21, 22). Addition of ligand selectively re- Public Health, Ohio State University, Columbus, OH 43210; and ‡Department of Host models this nucleosome via the interaction with a remodeling pro- Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan tein, BRG1, facilitating the binding of additional factors required Received for publication November 16, 2001. Accepted for publication May 10, 2002. for transcription (23). The costs of publication of this article were defrayed in part by the payment of page A recent report by Weinmann et al. (24) further supports the charges. This article must therefore be hereby marked advertisement in accordance hypothesis that nucleosome remodeling is important for inducible with 18 U.S.C. Section 1734 solely to indicate this fact. transcription in mammalian cells. High resolution micrococcal nu- 1 This work was supported by National Institutes of Health Grants AI42901, clease analyses revealed that a positioned nucleosome, nucleosome HL59795, and DK57667. 1, spanned the IL-12 promoter, while three additional positioned 2 Address correspondence and reprint requests to Dr. Bruce S. Zwilling, Department of Microbiology, 484 W. 12th Avenue, Ohio State University, Columbus, OH 43210. nucleosomes were found further upstream. Upon activation, nu- E-mail address: [email protected] cleosome 1 was rapidly and selectively remodeled in a protein 3 Abbreviations used in this paper: TLR, Toll-like receptor; MMTV, murine mam- synthesis-dependent manner. These results suggested that remod- mary tumor virus; MNase, micrococcal nuclease; MyD88, myeloid differentiation factor 88; PDTC, pyrrolidine dithiocarbamate; RPA, RNase protection assay; Sp1, eling complexes are selectively targeted to a single, promoter-en- stimulating factor 1. compassing nucleosome.

To investigate whether the induction of TLR2 transcription also Southern blot correlates with chromatin remodeling, we determined whether in- Purified genomic DNA (15Ð25 ␮g) was incubated overnight at 37¡C with fection with M. avium resulted in an alteration in DNase 1 sensi- an excess of resriction enzyme SSP1. The DNA was then cleaned by phe- tivity or restriction enzyme accessibility. We found that infection nol-chloroform extraction and analyzed on a 1% agarose gel, followed by with M. avium resulted in an increase in restriction enzyme acces- transfer and hybridization with a [32P]dCTP-radiolabeled probe specific for sibility and in sensitivity to digestion with DNase 1 within the the TLR2 promoter. The prehybridization and hybridization process was done within 3 h using the Stratagene (La Jolla, CA) QuickHyb hybridiza- TLR2 promoter. The chromatin change required TLR2 and my- tion buffer, according to the manufacturer’s instruction. Films were ex- eloid differentiation factor 88 (MyD88), but not new protein syn- posed for 2Ð18 h before development. thesis. We interpret our findings as indicating that chromatin re- Nuclear extracts and EMSA modeling has occurred at the TLR2 promoter following infection with M. avium. Nuclear extracts were prepared as described by Hussain et al. (26). Protein concentration was determined by the bicinchoninic acid protein assay (Pierce, Rockford, IL). Nuclear extracts were either assayed immediately Materials and Methods or stored at Ϫ70¡C until further use. The oligonucleotide used in the EMSA was: NF-␬B5Ј-ACACCTGGGGAATTCCCACACG-3Ј. Oligonu- Cell lines and reagents cleotide was end labeled with [␥-32P]ATP using T4 polynucleotide kinase. The murine alveolar macrophage cell line AMJ2-C8 (ATCC CRL-2455) The binding reactions were conducted using the gel shift assay core system ␮ was a generous gift from M. J. Fenton (Boston University, Boston, MA). from Promega (Madison, WI). Binding reactions contained 2Ð5 g nuclear Another murine macrophage cell line P388D1 (CCL-46) was obtained extract and 20,000 cpm radiolabeled oligonucleotide in 10 mM Tris-HCl, ␮ from American Type Culture Collection (Manassas, VA). Cells were cul- pH 7.5, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 1 g Downloaded from tured in IMDM (Life Technologies, Grand Island, NY) supplemented with poly(dI:dC), and 4% glycerol. Binding reactions were electrophoresed at ϫ 10% FBS (HyClone Laboratories, Logan, UT), 1% penicillin, and strep- 4¡C in 5% native polyacrylamide gels using 0.5 Tris-borate buffer, fol- tomycin. The expression and regulation of TLR2 by AMJ2-C8 cells were lowed by autoradiography. the same as those previously described by us for J774.1 macrophage cell RNase protection assay line (25). Total RNA was extracted using the RNAqueous kit (Ambion, Austin, TX). RNase protection assays (RPA) were performed using reagents from BD

Primary macrophages http://www.jimmunol.org/ PharMingen (San Diego, CA), as previously described (27). Probe tem- C57BL/6 mice (Charles River Breeding Laboratories, Wilmington, MA) plates for mouse TLR2 and GAPDH were made in this laboratory and have and TLR2Ϫ/Ϫ and MyD88Ϫ/Ϫ (generous gifts from S. Akira, Osaka Uni- been previously described (27). A total of 10 ␮g RNA was used for each versity, Osaka, Japan) mice were injected i.p. with thioglycolate. Three RPA. Reactions were run on 6% polyacrylamide/8 M urea sequencing gels, days later, the peritoneal exudate cells were isolated and allowed to adhere followed by autoradiography. to the tissue culture plates. After overnight incubation, the cells were washed with PBS before treatment. Monolayers were infected with M. Transient transfection avium (ATCC 35712) at an 8:1 bacteria-macrophage ratio for 30 min or A total of 1 ␮g plasmid expressing NF-␬B p65 was transfected into C8 speciﬁed times. Before use, the M. avium was grown in Middlebrook 7H9 cells with 4 ␮l lipofectamine plus 6 ␮l lipofectamine. After 24 h, the cell broth cultures, aliquoted in 1-ml amounts, and stored frozen at Ϫ70¡C. nuclei were isolated for restriction enzyme accessibility determination or ϫ 8 Each vial contained 2.5 10 CFU, as determined by plate count on 7H11 RNA was isolated for RPA. The transfection has been proved effective by guest on September 26, 2021 agar plates. when a luciferase reporter gene was used (25). To measure luciferase ac- tivity, 15 ng pCMV-NF-␬B p65 or pCMV alone was cotransfected into C8 Preparation of nuclei cells with 0.2 ␮g 326-bp TLR2 promoter-driven luciferase reporter. Forty- eight hours after transfection, the cells were lysed and luciferase was mea- Cells grown to conﬂuence were removed by scraping and centrifuged at sured, as described before (25). 1500 rpm in a microcentrifuge. Cells were washed once with ice-cold PBS and centrifuged. The cell pellet was resuspended in ice-cold Nonidet P-40 Results lysis buffer (10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% Increase in DNase 1 sensitivity and restriction enzyme Nonidet P-40, 0.15 mM spermine, and 0.5 mM spermidine) (17) and incubated on ice for 5 min. Nuclei were centrifuged at 1000 rpm, followed accessibility at the murine TLR2 promoter following infection with M. avium by washing in the respective digestion buffer (without CaCl2). To investigate the active element in TLR2 promoter region, DNase Nuclease digestions and isolation of genomic DNA 1 hypersensitivity of the chromosomal region covering TLR2 gene For DNase 1 hypersensitivity assay, cell nuclei were resuspended in buffer

A (100 mM NaCl, 50 mM Tris, pH 8.0, 3 mM MgCl2, 0.15 mM spermine, and 0.5 mM spermidine) (17) supplemented with CaCl2 (1 mM). DNase 1 (at specified concentration) was added, and the reaction (total volume 100 ␮l) was incubated at 37¡C for 2 min. Micrococcal nuclease (MNase) digestion proceeded as described by Carey and Smale (17) with minor modifications. Briefly cell nuclei were resuspended in MNase digestion buffer (10 mM Tris, pH 7.4, 15 mM NaCl, 60 mM KCl, 0.15 mM spermine, and 0.5 mM spermidine) containing ϭ ␮ CaCl2 (1 mM). MNase (stock solution 25 U/ l) was added, and the reaction mixture was incubated at room temperature for 2, 5, and 10 min. The reaction was terminated by addition of 80 ␮l MNase digestion buffer and 20 ␮l stop buffer (100 mM EDTA and 10 mM EGTA). FIGURE 1. Infection of M. avium resulted in the appearance of a DNase For restriction enzyme digestion, cell nuclei were washed in restriction I-hypersensitive band at TLR2 promoter. Nuclei from infected or unin- enzyme buffer (10 mM Tris, pH 7.4, 50 mM NaCl, 10 mM MgCl2, 0.2 mM fected C8 cells were digested with 25 U DNase I for specified time. EDTA, 0.2 mM EGTA, 1 mM 2-ME, 0.15 mM spermine, and 0.5 mM Genomic DNA was isolated and digested overnight with SSP1, followed spermidine) (24). Nuclei were resuspended in the specific buffer for re- by separation on a 1% agarose gel. Southern blot was conducted using the striction enzyme digestion (50 ␮l). Digestion reactions were initiated with 2 ␮l restriction enzyme and incubated at 37¡C for 10 min. probe derived from PstI-SSP1 region of mouse TLR2 gene. The arrow After digestion with nucleases, the total volume of reaction was brought indicates the band that appeared following infection, labeled as “H.” In- ␮ cubation of the nuclei from infected cells with of DNase 1 for 8 min to 200 l by adding ddH2O. The reaction was stopped, and genomic DNA was isolated using the DNeasy Tissue kit from Qiagen (Valencia, CA), completely digested the DNA. The results are representative of three in- according to the manufacturer’s instructions. dependent experiments. The Journal of Immunology 797

FIGURE 2. Restriction enzyme accessibility increases at TLR2 promoter region following infection with M. avium. Nuclei from infected or uninfected C8 cells were digested with speciﬁed restriction enzymes (20 U) for 10 min at 37¡C. Genomic DNA was isolated and then completely digested with SSP1 overnight. Southern blot was performed using the probe derived from PstI- SSP1 region of mouse TLR2 gene. The upper band represents the fragment derived from in vitro digestion by SSP1, while the lower band represents the fragment derived from nuclear digestion by EcoRI or HaeII or EcoI 109. The in- tensity of the band representing nuclear cleavage or in vitro cleavage product was determined by scanning using software Sigma Pro (SPSS, Chi- cago, IL). The ratio of nuclear cleavage vs in vitro cleavage is provided in which a band representing nuclear cleavage can be seen. The exact position of each nucleosome is purely hypothetical and re-

mains to be deﬁned. The results are representative Downloaded from of three independent experiments.

was measured. The results in Fig. 1 show that a band appeared high level of TLR2, while TLR2 mRNA was not detectable in when cells were infected with M. avium. The size of this band is P388D1 cells under the same conditions (Fig. 4A). The chromatin

consistent with it being the part of the TLR2 promoter region that from P388D1 cells was much more resistant to digestion with http://www.jimmunol.org/ includes the NF-␬B and Sp1 elements. The DNA was completely EcoRI and HaeII than the chromatin from C8 cells (Fig. 4B). Fur- digested by DNase 1 within 8 min in the M. avium-infected cells. thermore, infection with M. avium induced TLR2 at mRNA level DNase 1 hypersensitivity is not precise, and it is difficult to in C8 cells, but failed to do so in P388D1 cells (Fig. 4A). Infection determine the exact region in which DNase 1 digestion occurs. with M. avium led to an increase in restriction enzyme sensitivity Therefore, we measured restriction enzyme accessibility to more of the chromatin from C8 cells, but not P388D1 cells (Fig. 4B). precisely map the chromosomal site. C8 cell nuclei were treated with a limiting concentration of an appropriate restriction enzyme Increased restriction enzyme accessibility is independent of ␬ (EcoRI, HaeII, and EcoI 109). The cleaved genomic DNA was NF- B binding then purified and digested to completion in vitro with a different The EcoRI cuts the NF-␬B binding site, which is necessary for the by guest on September 26, 2021 restriction enzyme (SSP1) that recognizes sites on either side of induction of TLR2 transcription by LPS stimulation or infection the EcoRI, HaeII, and EcoI 109 restriction sites (Fig. 2). To nor- with M. avium (25, 28). To investigate the role of NF-␬Binin- malize the reaction, the results are also presented as the ratio of creasing the restriction enzyme accessibility of TLR2 promoter nuclear cleavage to in vitro cleavage. An increase in the ratio upon region covering NF-␬B site and Sp1 sites, two approaches were cell activation would suggest that a nucleosome becomes more used. First, we tested whether the overexpression of NF-␬B p65 accessible to nuclear cleavage and thus remolded. C8 cell activa- can result in the increased restriction enzyme accessibility at TLR2 tion led to a strong and reproducible increase in nuclear cleavage promoter. Overexpression of NF-␬B p65 induced the 326-bp by EcoRI, HaeII, but not EcoI 109 (Fig. 2). Nuclear cleavage at the TLR2 promoter-driven luciferase expression (Fig. 5A), but failed EcoRI site increased by 5-fold, while the nuclear cleavage at the HaeII site increased 3-fold after infection. The nuclear cleavage at the EcoI 109 site remained unchanged following infection. The exact position of the nucleosome, as presented in Fig. 2, is hypothetical and remains to be defined. To demonstrate that the TLR2 promoter region may be assembled into nucleosomes, the isolated nuclei were digested with MNase. MNase digests genomic DNA at the internucleosomal region, resulting in a DNA ladder when separated on an agarose gel, which may be detected when using a specific probe for TLR2. The results in Fig. 3 indicate that upon digestion with MNase, a DNA FIGURE 3. The TLR2 promoter region is incorporated into nucleo- ladder appeared, while MNase digested naked DNA much more somes. Nuclei from uninfected C8 cells were digested with 50 U MNase efficiently and the DNA ladder was not observed. These data sug- for the specified time. Genomic DNA was isolated and separated on an gest that TLR2 promoter region is incorporated into nucleosomes. agarose gel. Southern blot was then conducted using the same probe described in Fig. 2. A nucleosome array was observed, and the first three Increased restriction enzyme accessibility correlates with the bands were indicated. Lane 1, Genomic DNA from nuclei without MNase induction of TLR2 expression digestion; lane 2, naked genomic DNA digested with MNase (50 U) for 5 min; lane 3, naked genomic DNA digested with MNase (50 U) for 10 min; Increased restriction enzyme accessibility occurs before activation lane 4, nuclei digested with MNase (50 U) for 2 min; lane 5, nuclei di- of some genes. To test whether an increase in restriction enzyme gested with MNase (50 U) for 5 min; lane 6, nuclei digested with MNase accessibility correlates with the activation of TLR2, we compared (50 U) for 10 min. The time of the digestion was chosen based on the the expression of TLR2 at the mRNA level in two different mac- appearance of a DNA ladder on the agarose gel. Ten-minute digestion of rophage cell lines, P388D1 and C8. C8 cells express a relatively nuclei by MNase (50 U) yielded a clear DNA ladder (not shown). 798 CHROMATIN REMODELING OF THE TLR2 PROMOTER Downloaded from

FIGURE 4. Increased restriction enzyme accessibility correlates with http://www.jimmunol.org/ the expression of TLR2 mRNA. A, Expression of TLR2 mRNA was analyzed by RPA using C8 or P388D1 cells. Cells were infected with M. avium for 3 h before mRNA extraction. The TLR2 mRNA in P388D1 cells was detected using RT-PCR (unpublished results). B, Restriction enzyme accessibility was determined using nuclei from C8 or P388D1 cell. Infec- tion with M. avium for 30 min increased the restriction enzyme accessibility in C8 cells. A probe derived from the TLR2 promoter region covering NF-␬B and Sp1 sites (Ϫ294 to ϩ32 relative to ﬁrst transcription start site) was used for Southern blot. Two bands can be visualized after nuclear ␬ cleavage. Lanes 1, 3, 5, and 7, Digested by HaeII; lanes 2, 4, 6, and 8, FIGURE 5. Overexpression of NF- B failed to increase restriction en- by guest on September 26, 2021 digested by EcoRI. Lanes 1, 2, 5, and 6, Uninfected; lanes 3, 4, 7, and 8, zyme accessibility or to induce TLR2 expression. A, pCMV-NF-␬B p65 infected with M. avium for 30 min before digestion. Again the ratio of plasmid (15 ng/well) or pCMV alone was cotransfected into C8 cells with nuclear cleavage vs in vitro cleavage is provided only where a band rep- 326-bp TLR2 promoter-driven luciferase reporter gene. Twenty-four hours resenting nuclear cleavage can be seen. The results are representative of after transfection, cells were lysed and luciferase was measured. Lane 1, three independent experiments. No transfection; lane 2, luciferase reporter alone; lane 3, luciferase reporter ϩ pCMV-NF-␬B; lane 4, luciferase reporter ϩ pCMV. B, C8 cells were transiently transfected with a pCMV-NF-␬B p65 plasmid (1 ␮g/well). to increase the sensitivity to EcoRI and HaeII of the endogenous Twenty-four hours later, the nuclei were collected and restriction enzyme TLR2 promoter (Fig. 5B). Additionally, overexpression of NF-␬B accessibility was determined. The ratio of nuclear cleavage vs in vitro p65 did not lead to an induction of endogenous TLR2 expression cleavage was provided as the number below the blot. B, Transfection was done similarly as in A, and RNA was isolated 24 h after transfection. The (Fig. 5C). Another approach was to determine whether blocking of ␬ expression of TLR2 was determined by RPA. The TLR2-GAPDH ratio NF- B binding to TLR2 promoter can abolish the increased re- was indicated as the number below the blot. The results are representatives striction enzyme accessibility following infection with M. avium. of three independent experiments. Pyrrolidine dithiocarbamate (PDTC), an antioxidant, has been reported to block the binding of NF-␬B to numerous genes (29). The results in Fig. 6A show that PDTC blocked the binding of NF-␬B to the TLR2 promoter. PDTC also suppressed the induction of determine whether the TLR2-MyD88 signaling is essential for the TLR2 following infection. However, it failed to block the increase increase in the restriction enzyme accessibility within the TLR2 in restriction enzyme accessibility following infection with M. promoter in response to M. avium infection, peritoneal macro- avium (Fig. 6B). phages from TLR2Ϫ/Ϫ mice and MyD88Ϫ/Ϫ mice and the control To further characterize the increased restriction enzyme acces- C57BL/6 mice were analyzed. As a control, we also analyzed the sibility, its kinetics and requirement for de novo protein synthesis response of macrophages from TLR2Ϫ/Ϫ mice following stimula- were evaluated. The results in Fig. 7 show that substantial restriction with LPS. The results in Fig. 8 show that restriction enzyme tion enzyme accessibility occurred 30 min after infection and was accessibility did not increase in macrophages from TLR2Ϫ/Ϫ mice not affected by protein synthesis inhibitor, cycloheximide. and MyD88Ϫ/Ϫ mice following infection with M. avium. In a par- allel experiment, restriction enzyme accessibility increased in mac- Increased restriction enzyme accessibility of the TLR2 promoter rophages from the wild-type C57BL/6 mice following infection is TLR2 and MyD88 dependent with M. avium. Restriction enzyme accessibility also increased in Activation of macrophages upon infection with M. avium requires macrophages from TLR2Ϫ/Ϫ treated with LPS. These results sug- the activation of TLR2 signaling pathways. This signaling pathway gest that the increase in restriction enzyme accessibility of the also requires the participation of an adapter protein, MyD88. To TLR2 promoter following M. avium infection appears to require The Journal of Immunology 799

FIGURE 6. Binding of NF-␬B is not required for an increase in restriction enzyme accessibility to occur at the TLR2 promoter region. A, PDTC at speciﬁed

concentration was added to medium 1 h before infection. Nuclear protein was then extracted. EMSA was carried using a probe derived from the NF-␬B site of Downloaded from TLR2 promoter. Increasing concentrations of PDTC inhibited binding of NF-␬B. B, RPA was conducted to analyze the induction of TLR2 mRNA following infection. PDTC (200 ␮M) suppressed the induction of TLR2 mRNA following infection with M. avium. C, PDTC (200 ␮M)-pretreated cells were infected with M. avium for 30 min. Restriction enzyme accessibility assay was performed, as described above. The numbers below the blot indicated the ratio of nuclear cleavage vs in vitro cleavage. PDTC did not inhibit the increase in restriction enzyme accessibility. The results are representatives of three independent experiments.

the TLR2-MyD88 signaling pathway. The ability of LPS stimula- rules out the possibility that the failure of M. avium infection to http://www.jimmunol.org/ tion to induce the restriction enzyme accessibility at TLR2 pro- increase the restriction enzyme accessibility at TLR2 promoter moter region in cells from TLR2Ϫ/Ϫ mice (Fig. 8B, lanes 3 and 4) was due to the changes of TLR2 gene locus in TLR2Ϫ/Ϫ mice. The promoter region of the TLR2Ϫ/Ϫ mice is intact (30).

Discussion Previously, we have reported that the expression of TLR2 was induced following infection of macrophages with M. avium.We also analyzed the promoter region of mouse TLR2 and found that by guest on September 26, 2021 both NF-␬B and Sp1 elements were required for the induction of

FIGURE 8. Increase in restriction enzyme accessibility of TLR2 promoter following infection with M. avium is TLR2-MyD88 dependent. A, Thioglycolate-elicited cells from C57BL/6, TLR2Ϫ/Ϫ, and MyD88Ϫ/Ϫ mice were infected with M. avium for 30 min. Restriction enzyme accessibility was assessed, as described in Materials and Methods. Lane 1, HaeII; lane 2, EcoRI; lane 3, HaeII; lane 4, EcoRI. Lanes 1 and 2, Unin- FIGURE 7. Characterization of restriction enzyme accessibility follow- fected samples; lanes 3 and 4, infected samples. The promoter region of ing infection with M. avium at TLR2 promoter. A, C8 cells were infected TLR2 knockout mice is intact, although the coding region of TLR2 in these with M. avium for speciﬁed times, and the nuclei were isolated for diges- mice was knocked out. Again the numbers below the blot indicated the tion with restriction enzymes. Southern blot was then conducted as de- ratio of nuclear cleavage vs in vitro cleavage. B, The thioglycolate-elicited scribed. The numbers below the blot indicated the ratio of nuclear cleavage cells from the TLR2Ϫ/Ϫ mice were stimulated with 1 ␮g/ml LPS (Esche- vs in vitro cleavage. B, Protein synthesis inhibitor cycloheximide (10 ␮g/ richia coli 055.B5) for 30 min. Lanes 1, 3, and 5, Digested with EcoRI; ml) was added into medium 1 h before infection with M. avium. Southern lanes 2, 4, and 6, digested with HaeII. Lanes 1 and 2, M. avium infected; blot was conducted following restriction enzyme digestion. The results are lanes 3 and 4, LPS stimulated; lanes 5 and 6, infected samples. The results representatives of three independent experiments. are representative of three independent experiments. 800 CHROMATIN REMODELING OF THE TLR2 PROMOTER

TLR2 transcription following infection (25). In this study, we restriction enzyme accessibility. Thus, activation of NF-␬B alone found an inducible DNase 1-hypersensitive site around the NF-␬B is not sufficient to lead to the chromatin remodeling within the site of TLR2 promoter following infection with M. avium, indi- TLR2 promoter region. In contrast, we have previously reported cating that this region may undergo chromatin remodeling. By that overexpression of NF-␬B p65 alone was sufficient to activate determining the restriction enzyme accessibility, we were able to a TLR2 promoter-driven reporter construct transiently transfected map the site that undergoes chromatin remodeling to the elements into macrophage cell lines (25). Transiently transfected plasmids in which NF-␬B and Sp1 bind. This observation supports our pre- are not assembled into chromatin; thus, the activation of transcrip- vious results using a transient transfection system: both NF-␬B and tion of the reporter construct is not dependent upon chromatin SP1 elements are required for the full induction of the TLR2 pro- remodeling. In the present study, exogenous NF-␬B failed to in- moter. Although the restriction enzyme accessibility increased at crease the restriction enzyme accessibility of the endogenous NF-␬B and Sp1 site after infection, the inefficient cleavage at EcoI TLR2 promoter; neither did it induce the expression of 109 site may indicate that this region is not undergoing a chroma- endogenous TLR2. tin remodeling following infection. NF-␬B did not appear to be necessary for the chromatin remod- Not all promoters are assembled into nucleosomes and remodeling. PDTC inhibited the binding of NF-␬B to TLR2 promoter eled during transcriptional activation. Only the first nucleosome and also suppressed the induction of TLR2 at the mRNA level was remodeled during activation of both the IL-12 and the MMTV following infection, while it did not have any effect on the chro- promoters (24). In contrast, analysis of Pho5 and HIV-1 promoters matin remodeling. Taken together, these results suggest that the showed that at least one binding site for an inducible activator was chromatin remodeling within TLR2 promoter following infection in a nucleosome-free region (18, 20). To determine whether the with M. avium is NF-␬B independent. Alternatively, chromatin Downloaded from TLR2 promoter region is incorporated into nucleosomes before remodeling may occur before the binding of NF-␬B to TLR2 pro- activation, the sensitivity of chromatin, including TLR2 promoter moter. Similarly, chromatin remodeling at IL-12 p40 promoter, region, to MNase was determined. The appearance of a DNA lad- following LPS stimulation, did not require NF-␬B (34). der after digestion with MNase indicates that the TLR2 promoter The activation of TLR2 signaling involves TLR2-MyD88-NF- is assembled into a nucleosome array. ␬B-dependent pathways (30, 35). Recently, other pathways were

The regulation of gene expression in mammalian cells is not reported to become activated following TLR2 activation (36). The http://www.jimmunol.org/ only restricted to the control of transcriptional, posttranscriptional, increase in restriction enzyme accessibility within the TLR2 pro- translational, or posttranslational events. Chromatin remodeling is moter following infection was TLR2-MyD88 dependent and did another target for controlling the gene expression. Chromatin re- not require new protein synthesis. Because NF-␬B is neither suf- modeling has been shown to be important for IL-4 gene activation ﬁcient nor necessary for the chromatin remodeling of TLR2 pro- (31). We observed that an increase in restriction enzyme accessi- moter, different signals may mediate the chromatin remodeling of bility following infection with M. avium, within the TLR2 pro- TLR2 promoter following infection with M. avium. moter, correlated with the induction of TLR2 expression. P388D1 Our results demonstrate that the TLR2 promoter region becomes cells express almost no detectable levels of TLR2 mRNA. The more susceptible for DNase I or restriction enzyme digestion fol- chromatin from these cells was more resistant to restriction en- lowing infection with M. avium. The increase in sensitivity to by guest on September 26, 2021 zyme digestion. In contrast, C8 cells expressed a relatively high DNase I or restriction enzymes digestion correlated with induction level of TLR2, and its chromatin was easily digested by the re- of TLR2 expression. Both M. avium (TLR2 agonist) and LPS striction enzymes. The restriction enzyme accessibility assays (TLR4 agonist) that induce TLR2 expression resulted in an in- shown in Figs. 4b,6c, 7, and 8 appear to indicate that there may be crease in restriction enzyme accessibility. The increased restriction additional cleavage sites for HaeII and EcoRI. However, the probe enzyme accessibility induced following infection with M. avium used for these studies encompassed nucleotides Ϫ292 to ϩ32, i.e., was TLR2-MyD88 dependent, but not NF-␬B dependent. Thus, we the region containing the restriction sites for both EcoRI and HaeII propose that chromatin remodeling occurs within TLR2 promoter (see Fig. 2). In contrast, the probe used to detect the increase in following infection with M. avium. The chromatin remodeling may restriction enzyme accessibility for the experiments shown in Figs. occur before binding of NF-␬B to TLR promoter through TLR2- 2 and 5 was remote from this site and between restriction sites PstI MyD88 pathway, thus allowing transcription factor NF-␬Btoin- and SSP1, as shown in Fig. 2. Therefore, only one nuclear cleav- teract with TLR2 promoter to initiate transcription. age fragment was apparent following restriction enzyme digestion. 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