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B P50 and Represses Gene Expression* S Supplemental Material can be found at: http://www.jbc.org/content/suppl/2012/07/16/M112.365601.DC1.html THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 37, pp. 31207–31217, September 7, 2012 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. EHMT1 Protein Binds to Nuclear Factor-␬B p50 and Represses Gene Expression*□S Received for publication, March 22, 2012, and in revised form, July 14, 2012 Published, JBC Papers in Press, July 16, 2012, DOI 10.1074/jbc.M112.365601 Chee-Kwee Ea‡, ShengLi Hao§, Kok Siong Yeo‡, and David Baltimore§1 From the ‡Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia and the §Division of Biology, California Institute of Technology, Pasadena, California 91125 Background: Methylation of histone H3 lysine 9 (H3K9) is commonly correlated with gene repression. Results: p50 recruits EHMT1 to repress gene expression. Conclusion: EHMT1 is a negative regulator of NF-␬B and type I interferon induction pathways. Downloaded from Significance: p50-dependent changes in the chromatin structure importantly contribute to the repression of the expression genes in resting cells. Transcriptional homeostasis relies on the balance between most studied H3K9-HMTs are SUV39H1 and SUV39H2, which positive and negative regulation of gene transcription. Methyla- promote the formation of pericentric heterochromatin (6, 7). www.jbc.org tion of histone H3 lysine 9 (H3K9) is commonly correlated with Methylation of H3K9 by SUV39H1 creates a docking site for gene repression. Here, we report that a euchromatic H3K9 subsequent binding of the heterochromatin protein HP1, pro- methyltransferase, EHMT1, functions as a negative regulator in viding compelling evidence linking H3K9 methylation to het- ␬ both the NF- B- and type I interferon-mediated gene induction erochromatin formation and transcriptional repression (8–10). at CALIFORNIA INSTITUTE OF TECHNOLOGY, on October 25, 2012 pathways. EHMT1 catalyzes H3K9 methylation at promoters of In contrast, we know that EHMT1 and EHMT2 are the major ␬ NF- B target genes. Moreover, EHMT1 interacts with p50, and, euchromatic H3K9-HMTs because cells lacking either EHMT1 surprisingly, p50 appears to repress the expression of type I or EHMT2 show decreased H3K9me2 in euchromatin, whereas interferon genes and genes activated by type I interferons by the level of H3K9me2 remains unchanged in heterochromatin recruiting EHMT1 to catalyze H3K9 methylation at their pro- (11, 12). EHMT1 is ubiquitously expressed. Knocking out the moter regions. Silencing the expression of EHMT1 by RNA EHMT1 gene in mice leads to embryonic lethality (12). The interference enhances expression of a subset NF-␬B-regulated global levels of H3K9me1 and H3K9me2, but not H3K9me3, genes, augments interferon production, and augments antiviral are lower in EHMT1-deficient ES cells than in wild-type ES immunity. cells (12). However, it has been shown that EHMT1 is capable of catalyzing formation of H3K9me1, H3K9me2, and H3K9me3 in an in vitro methyltransferase assay (13). Covalent modifications of chromatin have emerged as key NF-␬B is a transcription factor that plays a pivotal role in determinants of the transcriptional competence of the genome. regulating multiple biological functions including inflamma- Indeed, a strong correlation exists between the expression sta- tion, immunity, cell proliferation, and apoptosis. In resting tus of a gene and specific histone modification (1). Although cells, NF-␬〉 is sequestered in the cytoplasm by interaction with histone methylation was discovered 4 decades ago (2), its phys- ␬ ␣ ␬ ␣ iological role was unclear until recently. Site-specific methyla- I B . Upon activation, I B is rapidly degraded by the ubiqui- ␬ tions of specific histone residues correlate with either activa- tin-proteasome pathway. Free NF- B is then translocated into tion or repression of transcription. For example, H3K42 the nucleus where it turns on expression of a large array of methylation is a mark of active genes, whereas H3K9 methyla- target genes (14, 15). Although inducible nuclear translocation ␬ tion has been correlated with gene repression (3–5). is critical for NF- B activation, many post-translational modi- There are seven known H3K9 histone methyltransferases fications have been shown to regulate the nuclear function of (H3K9-HMTs) in the human genome: SUV39H1, SUV39H2, NF-␬B, including phosphorylation, ubiquitination, nitrosyla- ESET, RIZ, EHMT1 (GLP), EHMT2 (G9a), and SETDB2. The tion, acetylation, and methylation (16, 17). We previously demonstrated that NF-␬B is positively regu- lated by methylation of the p65 subunit (17). In this report, we * This work was supported, in whole or in part, by National Institutes of Health show that NF-␬B action as well as that of type I interferon are Grant 2R01GM039458. This work was also supported by the University of Malaya HIR Grant UM.C/625/1/HIR/MOHE/CHAN-02; H-50001-A000022. negatively regulated by EHMT1. We show that the expression □S This article contains supplemental Figs. S1–S5 and Tables S1 and S2. of 44% of TNF-induced genes is enhanced in cells lacking The data reported in this paper have been deposited in the Gene Expression EHMT1. Using IL8 as a model, we further show that EHMT1 is Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE39744). required for establishing H3K9 methylation at the IL8 pro- 1 To whom correspondence should be addressed. Tel.: 626-395-8541; Fax: moter. Furthermore, EHMT1 interacts with the p50 subunit of 626-585-9495; E-mail: [email protected]. NF-␬B. Surprisingly, p50 recruits EHMT1 to the promoters of 2 The abbreviations used are: H3K4, histone H3 lysine 4; H3K9, histone H3 lysine 9; BMM, bone marrow-derived macrophage; HMT, histone methyl- genes that respond to type I interferon and represses the transferase; MEF, mouse embryonic fibroblast. expression of these genes. Silencing the expression of either p50 SEPTEMBER 7, 2012•VOLUME 287•NUMBER 37 JOURNAL OF BIOLOGICAL CHEMISTRY 31207 Mechanism of EHMT1-mediated Gene Repression or EHMT1 augments interferon production and strengthens washing buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 1% Triton the interferon-mediated inhibition of virus replication. X-100, 2 mM EDTA, 0.5% Nonidet P-40) followed by two times with a high salt washing buffer (50 mM HEPES, pH 7.9, 500 mM EXPERIMENTAL PROCEDURES NaCl, 1% Triton X-100, 0.1% SDS, 0.1% 2,5-dimethoxy-4- Cell Culture—HeLa, Human embryonic kidney (HEK) 293, chloramphetamine) and one time with TE buffer. The beads HEK293-PB1, A549-PB1, wild-type, and p50-deficient mouse were then resuspended with 100 ␮l of 10% Chelex 100 beads, embryonic fibroblast (MEF) cells were cultured in DMEM sup- and cross-linking was reversed by boiling the beads for 10 min. plemented with 10% fetal calf serum (FCS), penicillin G (100 The beads were then treated with 1 ␮l of proteinase K and ␮g/ml), and streptomycin (100 ␮g/ml). Bone marrow-derived incubated at 56 °C for 30 min. The proteinase K was inactivated macrophages (BMMs) were cultured as described earlier (18). by boiling for 10 min. Supernatants containing DNA were col- Antibodies—Antibodies against p65 (F6, C20), I␬B␣ (C21), ␮ lected, and the beads were washed with 120 lofdH2O. Super- p50 (H119, C19), JNK (C17), p38 (H147), HDAC1 (H-51), and natants were collected and analyzed by quantitative PCR. The pol II (N20) were purchased from Santa Cruz Biotechnology. sequences of the primers are listed in supplemental Table S2. Downloaded from Antibodies against FLAG (M2, Sigma), HA (Covance), p-JNK Immunoprecipitation—For immunoprecipitation of EHMT1, (Cell Signaling), p-p38 (Cell Signaling), histone H3 (Abcam, HEK293 cells were transfected with FLAG-tagged p65 or p50 ab1791), H3K9me1 (Abcam, ab8896), H3K9me2 (Abcam, along with HA-EHMT1. Twenty-four hours later, the cells were ab1220), H3K9me3 (Abcam, ab8898), and EHMT1 (R&D Sys- lysed in radioimmune precipitation assay buffer (20 mM Tris, pH tems, Bethyl Laboratories) were purchased from the respective 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% 2,5-dimethoxy-4-chlor- commercial sources. www.jbc.org amphetamine, 0.1% SDS, 0.5 mM DTT, 0.5 mM PMSF, and 1ϫ Flow Cytometry—A549-PB1 and 293T-PB1 cells were protease inhibitor mixture (Roche Applied Science)). Two hun- infected with influenza virus for 12 h. Cells were washed and dred micrograms of lysates was incubated with 8 ␮lof fixed with paraformaldehyde (1% final). Fixed cells were FLAG-m2 beads (Sigma) or HA beads (F7, Santa Cruz Biotech- at CALIFORNIA INSTITUTE OF TECHNOLOGY, on October 25, 2012 assayed using a BD FACScan flow cytometer (BD Biosciences) nology) at 4 °C for 1 h. The beads were washed three times with and further analyzed with FlowJo software. radioimmune precipitation assay buffer. Bound proteins were RT-PCR—Cells treated with TNF␣ for 2 h were lysed with examined by immunoblotting with FLAG or HA antibodies. TRIzol LS reagent (Invitrogen) to isolate total RNAs. cDNAs For immunoprecipitation of endogenous p65, p50, or were synthesized with the SuperScript௡ III Reverse Transcrip- EHMT1, HEK293 cells were treated with TNF␣ (10 ng/ml) and tase kit (Invitrogen). Quantitative PCR was performed with the harvested at different time points. The cytoplasmic extracts 2ϫ SYBR Green PCR Master Mix (Applied Biosystems) and run were prepared using a hypotonic lysis buffer containing 0.2% on the Applied Biosystems ABI 7300 Real-time PCR System. All Nonidet P-40 (10 mM Tris, pH 7.5, 1.5 mM MgCl ,10mM KCl, data were normalized to L32. The sequences of the primers are 2 0.5 mM DTT, 0.5 mM PMSF, and 1ϫ protease inhibitor mix- listed in supplemental Table S2. ture). The nuclear extracts were prepare by resuspending nuclei RNA Interference—All siRNAs were purchased from Santa in micrococcus nuclease digestion buffer (50 mM Tris, pH 7.4, 1 Cruz Biotechnology: control (sc-37007), EHMT1a (sc-62261), mM CaCl ,25mM KCl, 4 mM MgCl , 12.5% glycerol, 0.5 mM EHMT1b (Invitrogen HSS129760), ESET (sc-45659), RIZ (sc- 2 2 ϫ 106513), SUV39H1 (sc-38463), SUV39H2 (sc-97240), SETDB2 DTT, 0.5 mM PMSF, and 1 protease inhibitor mixture).
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