Cytokine Production by Suppressing Tollip Promotes TLR-Triggered

Histone Lysine Methyltransferase Ezh1 Promotes TLR-Triggered Inflammatory Cytokine Production by Suppressing Tollip

This information is current as Yiqi Liu, Qian Zhang, Yuanyuan Ding, Xia Li, Dezhi Zhao, of September 27, 2021. Kai Zhao, Zhenhong Guo and Xuetao Cao J Immunol published online 16 February 2015 http://www.jimmunol.org/content/early/2015/02/16/jimmun ol.1402087 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 © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 16, 2015, doi:10.4049/jimmunol.1402087 The Journal of Immunology

Histone Lysine Methyltransferase Ezh1 Promotes TLR-Triggered Inﬂammatory Cytokine Production by Suppressing Tollip

Yiqi Liu,*,†,1 Qian Zhang,†,1 Yuanyuan Ding,* Xia Li,* Dezhi Zhao,* Kai Zhao,† Zhenhong Guo,† and Xuetao Cao*,†,‡

Histone modifications play critical roles in the regulation of gene expression; however, their roles in the regulation of the innate response remain to be fully investigated. Using transcriptome analysis of mouse immature dendritic cells (DCs) and LPS-induced mature DCs, we identified that Ezh1 was the most upregulated histone methyltransferase during DC maturation. In this study, we investigated the role of Ezh1 in regulating the innate immune response. We found that silencing of Ezh1 significantly suppressed

TLR-triggered production of cytokines, including IL-6, TNF-a, and IFN-b, in DCs and macrophages. Accordingly, TLR-activated Downloaded from signaling pathways were impaired in Ezh1-silenced macrophages. By transcriptome analysis of Ezh1-silenced macrophages, we found that Toll-interacting protein (Tollip), one well-known negative regulator of TLR signaling, was upregulated. Silencing of Tollip rescued TLR-triggered cytokine production in Ezh1-silenced macrophages. The SET domain of Ezh1 is essential for its enhancing effect on the TLR-triggered innate immune response and downstream signaling, indicating that Ezh1 promotes a TLR- triggered innate response through its lysine methyltransferase activity. Finally, Ezh1 was found to suppress the transcription of

Tollip by directly targeting the proximal promoter of tollip and maintaining the high level of trimethylation of histone H3 lysine 27 http://www.jimmunol.org/ there. Therefore, Ezh1 promotes TLR-triggered inﬂammatory cytokine production by suppressing the TLR negative regulator Tollip, contributing to full activation of the innate immune response against invading pathogens. The Journal of Immunology, 2015, 194: 000–000.

pon recognition of invading pathogens, the innate im- inﬂammation and tissue damage, and even multiple organ failure. mune response is efﬁciently initiated against the infec- Thus, negative regulation of the innate response attracts much tion. Once the invading pathogens have been eliminated, attention, and many negative regulators of the innate response

U by guest on September 27, 2021 the immune response must be tightly regulated to be at the ap- have been reported to play important roles in maintaining the propriate level to maintain the homeostasis of the immune system. balance between the immune response and inflammation (1). If regulation of the innate immune response is out of control, It was shown that invading pathogens may develop a strategy to overproduction of inflammatory cytokines may lead to excessive use the host negative regulators to downregulate the host defense and, thereby, escape the innate response, leading to chronic infections. Suppressor of cytokine signaling 1 can be induced by *Institute of Immunology, Zhejiang University School of Medicine, Hangzhou 310058, China; †National Key Laboratory of Medical Molecular Biology and De- various pathogenic components, which restricts activation of partment of Immunology, Institute of Basic Medical Sciences, Chinese Academy of macrophage through repressing the JAK/STAT and TLR/NF-kB Medical Sciences, Beijing 100005, China; and ‡National Key Laboratory of Medical Immunology and Institute of Immunology, Second Military Medical University, pathways (2). Ebola virus VP35 protein was shown to inhibit IFN Shanghai 200433, China production in innate immune cells by promoting sumoylation of 1Y.L. and Q.Z. contributed equally to this work. IRF7 via the E3 ligase PIAS1 (3). Siglec-G, selectively upregu- Received for publication August 13, 2014. Accepted for publication January 15, lated by the RNA virus, can recruit SHP2 and the E3 ubiquitin 2015. ligase c-Cbl to bind RIG-I and then promote RIG-I degradation This work was supported by grants from the National Key Basic Research Program of via ubiquitination, attenuating the innate response to RNA virus China (2013CB530502) and the National Natural Science Foundation of China infection (4). Therefore, the expression level and function of (31390431, 81230074, 81123006, 31200654, and 31270966). negative regulators are well-tuned to maintain homeostasis of the The sequences presented in this article have been submitted to the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/genbank) under accession number immune system. A better understanding of the mechanisms for the GSE60307. negative regulation of the innate response will benefit the design Address correspondence and reprint requests to Prof. Xuetao Cao, Institute of Im- of immunotherapy for the control of inflammatory disorders or munology, Zhejiang University School of Medicine, Hangzhou 310058, China. autoimmune diseases. New negative regulators and their under- E-mail address: [email protected] lying mechanism need to be identified. Abbreviations used in this article: BMDC, bone marrow–derived DC; ChIP, chroma- TLRs are important components of the innate immune response, tin immunoprecipitation; DC, dendritic cell; EZHI vector, full-length Ezh1 vector; EZH1-DSET, SET domain–depleted Ezh1 vector; H3K27, histone H3 lysine 27; which initiates the elimination of invading micro-organisms H3K27me2, dimethylation of H3K27; H3K27me3, trimethylation of H3K27; IRAK, through the induction of inflammatory molecules, including IL-1R–associated kinase; KDM, lysine demethylase; KMT, lysine methyltransferase; ODN, oligodeoxynucleotide; PGN, peptidoglycan; Poly(I:C), polyinosinic- cytokines, chemokines, and other antimicrobial factors (5). TLRs polycytidylic acid; qPCR, quantitative PCR; RNA-seq, RNA sequencing; RPKM, activate multiple pathways rapidly upon recognizing structurally reads per kilobase per million reads; siRNA, small interfering RNA; Tollip, Toll- conserved lipid, carbohydrate, peptide, and nucleic acid molecules interacting protein; TSS, transcription start site. that are components of microbial pathogens. The transcription of Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 hundreds of genes is induced during the immune response acti-

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402087 2 EZH1 PROMOTES TLR RESPONSE VIA TARGETING TOLLIP vated by TLR signals. To determine how these genes are orga- In this study, we investigated the role of Ezh1 in the regulation of nized and transcripted, much attention has been paid to the tran- the innate immune response in DCs and macrophages and found scription factors and the modulators in the activated signaling that it promotes TLR-triggered inflammatory cytokine production pathways. by suppressing the expression of Toll-interacting protein (Tollip), In recent years, more and more evidence has shown the im- the key negative regulator of TLR signaling. portance of epigenetic regulation of the immune response. Epi- genetic changes influence gene expression without altering the Materials and Methods DNA sequence. DNA methylation, histone modification, and Mice and reagents microRNA-associated gene silencing are key epigenetic mecha- C57BL/6J mice were obtained from Joint Ventures Sipper BK Experimental nisms. Recently, the DNA-methylation adaptor Uhrf1 was shown to Animal (Shanghai, China). All animal experiments were performed in promote proliferation and functional maturation of colonic regu- accordance with the National Institutes of Health Guide for the Care and latory T cells (6). Histone deacetylase 2 was shown to regulate the Use of Laboratory Animals, with the approval of the Scientific Investiga- phenotype of myeloid-derived suppressor cells in cancer through tion Board of Second Military Medical University. LPS (0111:B4) was from Sigma-Aldrich. Polyinosinic-polycytidylic acid [Poly(I:C)] was from histone acetylation (7). Our previous study showed that death Calbiochem. Peptidoglycan (PGN; Staphylococcus aureus) was from domain–associated protein 6 selectively represses IL-6 expression Sigma-Aldrich. CpG-oligodeoxynucleotide (ODN) was synthesized and via histone acetylation at the IL-6 promoter region (8). Epigenetic repurified as described previously (22). Phosphorothioate-modified CpG- markers were recently shown to be involved in the progression of ODN was synthesized by Shenggong, and its sequence was 59-TCC ATG human inflammatory or autoimmune diseases, such as multiple ACG TTC CTG ATG CT-39. Endotoxin in these ODNs was removed using Endotoxin Removal Solution (Sigma-Aldrich), and the endotoxin level was sclerosis and asthma (9, 10). 0.015 endotoxin units/mg CpG-ODN. Anti-IRF3 (sc-9082), anti-Erk (sc- Downloaded from Histone lysine methylation is one of the epigenetic modifications 94), anti-p38 (sc-535), anti-Jnk (sc-474), anti-p65 (sc-7151), anti–b-actin that play fundamental roles in most biological processes. Meth- (sc-130656), and HRP-coupled secondary Abs (sc-2305 or sc-2302) were 202 204 ylation of lysine residues located on the tails of histone H3 and H4 from Santa Cruz. Ab to Erk phosphorylated at Thr –Tyr (E10), Ab to Jnk phosphorylated at Thr183–Tyr185 (G9), Ab to p38 phosphorylated at have been linked to the regulation of gene expression (11). Histone Thr180–Tyr182 (9211), and Ab to IRF3 phosphorylated at Ser396 (4D4G) modification on a specific site is catalyzed by enzymes, which can were from Cell Signaling Technology. Anti-H3K27me3 (ab6002), anti- be grouped into histone lysine demethylases (KDMs) and histone Ezh1 (ab13660), and the agarose used for immunoprecipitation were http://www.jimmunol.org/ lysine methyltransferases (KMTs), according to their substrates from Abcam. (12). Histone methyl modification is a balance between methyla- Cell culture tion and demethylation. Several KMTs or KDMs were shown to be involved in inflammation. The promoters of TLR-induced Thioglycollate-elicited mouse peritoneal macrophages were prepared from C57BL/6J mice (6–8 wk old) and cultured in DMEM with 10% (v/v) FCS. genes exhibit the trimethylation of histone H3 lysine 4 marker Bone marrow–derived DCs (BMDCs) were generated in RPMI 1640 me- under basal conditions by the histone methyltransferases Mll (13). dium with recombinant mouse GM-CSF (10 ng/ml) and IL-4 (1 ng/ml), as Histone methyltransferase Smyd5, which is associated with tri- described previously (23). Cells (3 3 105) were seeded into 24-well plates 6 methylation of histone H4 lysine 20, represses the basal expres- for cytokine assay, and 1 3 10 cells were seeded into 6-well plates for

immunoblot analysis. by guest on September 27, 2021 sion of inflammatory genes (14). In adaptive immunity, gene- specific histone H3 lysine 27 (H3K27) methylation markers RNA interference + were considered a signature of different CD4 Th cell subsets Thioglycollate-elicited mouse peritoneal macrophages were transfected (15). Recently, Th1 lineage-specific transcription factor T-bet was with small interfering RNA (siRNA; 20 nM) by using INTERFERin reagent shown to be under the regulation of the H3K27 methyltransferase (Polyplus Transfection), as previously indicated (22). siRNA for Ezh1 and Ezh2 (16). Ezh2 also was found to specifically enhance Th cell Tollip were designed and synthesized by GenePharma (Shanghai, China). 9 differentiation and plasticity via transcriptional regulation of The specific siRNA for Ezh1 was 5 -GGCACCUAUUUCAACAACUTT- 39, and the specific siRNA for Tollip was 59-CAGGGAAGUAAUCCG- other lineage-specifying genes (17). Ashl1, a histone H3 lysine 4 UUCUTT-39. The nonsense sequence 59-UUCUCCGAACGUGUCAC- methyltransferase, could induce A20 expression to suppress IL-6 GUTT-39 was used as negative-control siRNA. production in inflammatory autoimmune diseases (18). However, Plasmid constructs and transfection the precise roles and the underlying mechanisms of the epigenetic molecules in the regulation of the inflammatory innate response Recombinant vectors encoding Ezh1 and its SET domain mutants were remain to be fully investigated. constructed by PCR-based amplification from cDNA of mouse BMDCs, and they were subcloned into the pcDNA3.1-Myc(-)B eukaryotic expression Dendritic cells (DCs) are the most potent professional APCs vector (Invitrogen). All constructs were confirmed by DNA sequencing. that link the innate and adaptive immune responses. Using Mouse peritoneal macrophages were transfected using the mouse Macro- transcriptome analysis of mouse immature DCs and LPS-induced phage Nucleofector Transfection kit (Amaxa), according to the manu- mature DCs, we screened the LPS-upregulated histone methyl- facturer’s manual. transferases in mature DCs and initially identified histone meth- ELISA yltransferase Ezh1 as the most increased enzyme in mature DCs. 3 5 Similar to Ezh2, Ezh1 is a mammalian homolog of the Drosophila Cells (0.5 ml of 3 10 ) were seeded into each well of 24-well plates, incubated overnight, and transfected as described above. After 36 h, the protein enhancer of Zeste, a member of the PcG proteins. Poly- cells were stimulated with LPS for the indicated time periods. The con- comb group proteins are critical to maintaining gene repression by centrations of IL-6 and TNF-a in the supernatants were measured with catalyzing dimethylation of H3K27 (H3K27me2) and trimethy- ELISA kits (R&D Systems), and IFN-b was measured using an ELISA kit lation of H3K27 (H3K27me3) (19). Also, Ezh1 could partially (PBL). complement Ezh2 by sharing some target genes (20). Further- RNA sequencing detection and analysis more, Ezh1 was associated with RNA polymerase II, promoting mRNA production in skeletal muscle cells (21), suggesting Immature and mature BMDCs were sorted using MoFlo XDP (Dako a noncanonical function for Ezh1 in transcription regulation. The Cytomation), and total RNA was isolated with TRIzol reagent (TransGen). RNA was isolated using an RNA isolation kit (QIAGEN). RNA libraries transcriptional regulatory role for Ezh1 has been confirmed; were prepared for sequencing using standard Illumina protocols. At the however, there is no report about the function of Ezh1 in regu- data-processing step, Illumina CASAVA v1.6 software was used for base- lating the innate immune response and inflammation. calling. Sequenced reads were trimmed for the adaptor sequence, masked The Journal of Immunology 3 for low-complexity or low-quality sequences, and mapped to SOAPaligner/ soap2. Mismatches of no more than two bases were allowed in the alignment. The gene expression level was calculated using the reads per kilobase per million reads (RPKM) method, as described (24). A strict algorithm was de- veloped to identify differentially expressed genes, as described (25). These RNA sequencing (RNA-seq) data were deposited in the Gene Expression Omnibus public database under accession number GSE60307 (http://www. ncbi.nlm.nih.gov/genbank). RT-PCR and quantitative PCR Total RNA was extracted with TRIzol reagent (TransGen) and reverse transcribed with a Reverse Transcription System (Promega). Reverse- transcription products of different samples were amplified by a Light- Cycler System (Roche) using SYBR Green PCR MasterMix (Applied Biosystems), according to the manufacturer’s instructions, and data were normalized to the level of b-actin expression in each individual sample. The 22DDCt method was used to calculate relative expression changes. With the help of dissociation curve analysis and the sequencing of PCR products, pairs of specific primers of each cDNAs were designed and selected, without anyprimerdimersorunspecific amplification detected.

Chromatin immunoprecipitation assay Downloaded from Chromatin immunoprecipitation (ChIP) assays were done according to the protocol of the ChIP assay kit (Upstate Biology). Target-gene promoter sequences in both input DNA and recovered DNA from immune complexes were detected by quantitative PCR (qPCR), as described previously (26), or analyzed using endpoint PCR with the following parameters: predenatu- ration at 95˚C for 5 min, followed by 30 cycles of denaturation at 95˚C for 20 s, annealing at 57˚C for 30 s, and extension at 72˚C for 30 s, with a final http://www.jimmunol.org/ extension at 72˚C for 5 min. The endpoint PCR products were detected on 2% agarose gels. The following primers were used to detect the H3K27me3 level or Ezh1 binding at different loci upstream of the tollip promoter: 2500 bp to transcription start site (TSS), 59-GAAATGGGT- CTCCCGTACA-39 and 59-CAGTCTGACAGCCATTTTAAC-39; 21000 to 2500 bp, 59-CTGAAGGAGAGACAAGAGTG-39 and 59-AAGAG- AGTGCTCTGAAAAGG-39; 21500 to 21000 bp, 59-GCCCAAACTG- TCATCTCTAT-39 and 59-AGACACAAGTCACAGGTTTT-39; 21500 to 22000 bp, 59-AACTCTGGTATAGGTGGACT-39 and 59-TAGCAGCA- 9 2 2 9 A GAGCTAGTTTTC-3 ;and 2500 to 2000 bp, 5 -CCACAAGATGC- FIGURE 1. Upregulation of Ezh1 expression in DCs by TLR ligands. ( ) by guest on September 27, 2021 TACTCAACT-39 and 59-TAGATATGTGCCACTGTACC-39. In addition, Sorting strategy of mouse immature and mature DCs. BMDCs were stimulated the following primers were used: tnf promoter, 59-TTTCCGAGGGTT- or not with LPS (100 ng/ml) for 24 h after 6 d of bone marrow cell culture in 9 9 9 GAATGAGA-3 and 5 -CTGGTTGGCTGCTTGCTTTT-3 ; il6 promoter, GM-CSF and IL-4. Then immature (CD11c+,Iablo,CD86lo)andmature 9 9 9 5 -TCCCATCAAGACATGCTCA-3 and 5 -TCATGGGAAAATCCCA- (CD11c+,Iabhi,CD86hi) DCs were sorted as gated using a flow cytometer. (B) CATT-39;andifnb1 promoter, 59-GGACACTTTGGCTGTTTGAGA-39 AlloftheRNA-seqgenesexpressedinthesampleswithRPKM$ 0.1 were and 59-CATTTTATTCAAGCTCCTGGCTA-39. Data were normalized to the input DNA and compared with the control group. used for the analysis. Expression changes in KMTs (upper panel)andKDMs (lower panel) in immature and mature DCs are shown according to the Statistical analysis mRNA-seq profiling. The ratio was calculated as RPKM (mature DCs)/RPKM (immature DCs). (C) Mouse BMDCs were stimulated with LPS (100 ng/ml), The statistical significance between two groups was determined by the m m m Student t test. The p values , 0.05 were considered statistically significant. CpG-ODN (CpG; 0.3 M), Poly(I:C) (10 g/ml), PGN (10 g/ml), or PBS. Real-time qPCR analysis of Ezh1 mRNA expression was performed at the indicated times. (D) Ezh1 expression in the lysates of BMDCs was detected Results using Western blot 24 h after stimulation with TLR ligands, as described in (C). Upregulation of Ezh1 expression in DCs by TLR ligands b-actin was used as a loading control. Data in (A)and(D) are representative of Methylation of lysine residues within histones are regulated directly by three independent experiments with similar results. Data in (C) are mean 6 , KMTs and KDMs to maintain gene transcriptional activation or si- S.D. of three independent experiments. **p 0.01. lencing. To systematically analyze the roles of KMTs and KDMs in innate immune response, transcriptomes of immature DCs and mature BMDCs stimulated with various TLR ligands, including CpG- + DCs were established. Mouse BMDCs were generated, and CD11c , ODN, Poly(I:C), and PGN. The expression of Ezh1 at the lo lo + MHC II ,CD86 immature DCs were sorted at day 7. CD11c ,MHC mRNA and protein levels was confirmed to be upregulated sig- hi hi II ,CD86 mature DCs were sorted 24 h after LPS stimulation nificantly after TLR activation (Fig. 1C, 1D). (Fig. 1A). Based on this RNA-seq data, 28 KTMs and 19 KDMs were identified as being regulated in BMDCs, and most of the enzymes Silencing of Ezh1 reduces TLR-triggered inflammatory remained unchanged after LPS stimulation. Interestingly, among these cytokine production in DCs and macrophages enzymes, the amplitude of upregulation of one KMT, Ezh1, was much TLR-induced upregulation of Ezh1 expression suggests a potential more significant than that of the other enzymes (Fig. 1B). The mRNA role for it in regulating innate inflammatory responses. siRNAs level of Ezh1 was dramatically increased during LPS-induced matu- specific to mouse Ezh1 were designed to knockdown Ezh1 ex- ration of DCs. Hence, we selected Ezh1 for further investigation of its pression, and they were confirmed to be able to strongly silence the potential function in innate immunity. expression of Ezh1 in DCs and macrophages (Fig. 2A). Intrigu- To further explore whether Ezh1 was induced by other patho- ingly, Ezh1 knockdown led to significant decreases in the ex- gen-associated molecular patterns, we detected its expression in pression of LPS-induced inflammatory cytokines, including IL-6 4 EZH1 PROMOTES TLR RESPONSE VIA TARGETING TOLLIP

FIGURE 2. Decreased inﬂammatory cytokine production in Ezh1-silenced BMDCs and macrophages in response to LPS stimulation. (A)

Mouse BMDCs and peritoneal elicited macro- Downloaded from phages (MF) were transfected with control siRNA or siRNA speciﬁc for Ezh1 (siRNA Ezh1). mRNA expression of Ezh1 was detected by real-time qPCR (left panel). Protein amounts of Ezh1 were detected using Western blot (right panel). Mouse BMDCs (B) and mouse peritoneal macrophages (C) were harvested and transfected http://www.jimmunol.org/ with control siRNA or siRNA speciﬁc for Ezh1 (siRNA Ezh1). Forty-eight hours after interference, BMDCs (B) and macrophages (C) were stimulated or not with LPS (100 ng/ml), and mRNA expression of IL-6, TNF-a,andIFN-b was detected by q-PCR at the indicated times. BMDCs (D)and macrophages (E) were transfected with siRNA described in (A). Forty-eight hours later, the cells were treated with LPS (100 ng/ml), CpG-ODN by guest on September 27, 2021 (CpG; 0.3 mM), Poly(I:C) (10 mg/ml), PGN(10 mg/ml), or PBS for 12 h. IL-6, TNF-a,andIFN-b in the supernatants were detected using ELISA. Data in (A) are representative of three independent experiments with similar results. Data in (B)–(E) are mean 6 SD of three independent experiments. **p , 0.01.

and TNF-a,inBMDCs.Moreover,silencing of Ezh1 also inhibited stimulation, but this phenomenon was not observed upon Poly(I:C) IFN-b expression induced by LPS (Fig. 2B). We also found that LPS- stimulation, indicating a possible regulatory role for Ezh1 in TLR2/4/9 induced expression of IL-6, TNF-a,andIFN-b was significantly signaling. Considering that transfection of exogenous gene via decreased in Ezh1-silenced mouse peritoneal macrophages (Fig. 2C). plasmid vectors is much more efficient in primary macrophages As shown in Fig. 2D and 2E, production of inflammatory than in BMDCs, peritoneal macrophages were used in the fol- cytokines and IFN-b also was decreased in Ezh1-silenced mouse lowing experiments for the investigation of Ezh1’s role in the BMDCs and peritoneal macrophages upon CpG-ODN and PGN regulation of TLR-triggered innate inflammatory responses. The Journal of Immunology 5

Ezh1 promotes TLR-triggered inflammatory cytokine signaling molecules in TLR signaling, including NF-kB, MAPK, production through its lysine methyltransferase activity and IRF3, were analyzed in Ezh1-silenced macrophages. We k Ezh1 was shown to be related to histone dimethyl or trimethyl found that the LPS-induced phosphorylation of NF- B p65 sub- modification as an H3K27 methyltransferase (27). The SET do- unit, MAPKs ERK1/2, JNK1/2, p38, and IRF3 in Ezh1-silenced main of Ezh1 was shown to be the catalytic motif of lysine- macrophages was decreased significantly, as demonstrated by specific histone methyltransferase, and this structure is directly Western blotting (Fig. 4A). Also, LPS-induced nuclear translo- k related to H3K27 methylation, the hallmark of transcriptional cation of transcription factors, including NF- B and IRF3, was repression (19, 28). We hypothesized that Ezh1 might link histone assessed to determine whether reduced phosphorylation of tran- modification to innate immune regulation. To confirm whether scription factors was associated with their transcription when methyltransferase activity of Ezh1 is required to promote TLR- Ezh1 was silenced. As expected, less p65 and IRF3 were found triggered inflammatory cytokine production, two pcDNA3.1 vec- in the nuclear extracts of Ezh1-silenced macrophages after LPS tors were constructed encoding full-length Ezh1 (1–750; EZH1 stimulation (Fig. 4B), suggesting a weakened transcription of NF- k vector) or SET domain–depleted Ezh1 (1–615; EZH1-DSET B or IRF3 target genes due to the decrease in nuclear trans- vector) under the control of a CMV promoter. We overexpressed located p65 and IRF3. These results indicated that Ezh1 might full-length Ezh1 or SET domain–depleted Ezh1 in Ezh1-silenced promote TLR-triggered inflammatory cytokine production by macrophages and found that overexpression of full-length Ezh1, enhancing TLR-activated signaling pathways. but not SET domain–depleted Ezh1, rescued the attenuated ex- We next investigated whether Ezh1 silencing–mediated inhibi- pression of inflammatory cytokines in TLR-triggered Ezh1- tion of TLR signaling depended on Ezh1’s methyltransferase ac- silenced macrophages (Fig. 3). These data indicate that the SET tivity. We transfected Ezh1-silenced macrophages with vectors Downloaded from domain of Ezh1 is essential for its enhancing effect on the TLR- encoding the SET domain–depleted mutant or full-length Ezh1. triggered innate immune response. However, transfection of the Overexpression of full-length Ezh1 rescued the attenuated phos- EZH1 vector in control macrophages did not enhance cytokine phorylation of p65 and IRF3 in TLR-triggered Ezh1-silenced production in macrophages, indicating that external Ezh1 is re- macrophages, whereas overexpression of the SET domain–de- dundant to promote TLR activation. pleted Ezh1 mutant had no such effect (Fig. 4C), indicating that

the upregulation of TLR signaling activation was dependent on the http://www.jimmunol.org/ Silencing of Ezh1 impairs TLR-activated signaling pathways H3K27 methyltransferase activity of Ezh1. To investigate the underlying molecular mechanism by which Ezh1 Ezh1 promotes TLR-triggered inﬂammatory cytokine promotes TLR-triggered inﬂammatory cytokine production, key production by suppressing Tollip expression via its SET domain Histone H3 is the only known substrate of Ezh1 that is the core constituent of the PRC-Ezh1 complex that directly mediates gene transcription repression (29). Our hypothesis was that negative regulator(s) of the TLR signaling pathway may be suppressed

by Ezh1 via H3K27me3. According to RNA-seq data of Ezh1- by guest on September 27, 2021 silenced macrophages (data not shown), we identified Tollip as one of the potential upregulated targets of Ezh1. Tollip is reported to be a negative regulator of TLR signaling, and excessive Tollip inhibits the activity of IL-1R–associated kinase (IRAK) after TLR activation (30). Therefore, we used an siRNA strategy to test whether Tollip was responsible for the regulation of the TLR response by Ezh1. Twenty-four hours after Ezh1 knockdown in macrophages, we transfected macrophages with Tollip-specific siRNA and challenged them with LPS. Tollip was found to be involved in the Ezh1-mediated enhancement of the TLR response. Knockdown of Tollip partially rescued LPS-induced cytokine production that was inhibited initially by silencing of Ezh1 (Fig. 5A–C). Furthermore, LPS-induced phosphorylation of NF-kB and IRF3 recovered to near-normal levels, indicating that Tollip was responsible for the suppression of the TLR signaling pathway in Ezh1-silenced macrophages (Fig. 5D). To further confirm the involvement of Ezh1 in Tollip expression, we assessed Tollip expression by Western blot after silencing Ezh1; Tollip remained at elevated levels in Ezh1-silenced macrophages FIGURE 3. Ezh1 suppresses TLR-induced inflammatory cytokine pro- (Fig. 5D). Under normal conditions, Tollip expression was up- duction via its methyltransferase activity. (A) Mouse macrophages were regulated at late phase of inflammation to maintain homeostasis, transfected with Ezh1 siRNA and then transfected 24 h later with the and we found that Tollip was upregulated after 12 h of LPS D pcDNA3.1 empty vector, the EZH1 vector, or the EZH1- SET vector. stimulation in BMDCs or macrophages (Fig. 5E). To further in- Twenty-four hours later, the cells were stimulated with LPS (100 ng/ml) vestigate the relationship between Tollip and methyltransferase for the indicated times. mRNA expression of IL-6, TNF-a, and IFN-b was detected by q-PCR. (B) Macrophages were transfected as described in (A), Ezh1, we transfected Ezh1-silenced macrophages with the EZH1 D and 24 h later they were stimulated with LPS (100 ng/ml) for the indicated vector or the EZH1- SET vector and found that overexpression of times. The supernatants were collected 12 h after stimulation, and IL-6, full-length Ezh1 in Ezh1-silenced macrophages suppressed Tollip TNF-a, and IFN-b in the supernatant were detected using ELISA. Data are expression to basal levels, whereas the SET domain–depleted mean 6 SD of three independent experiments. **p , 0.01. Ezh1 mutant had no inhibitory effect (Fig. 5F). These results in- 6 EZH1 PROMOTES TLR RESPONSE VIA TARGETING TOLLIP Downloaded from

FIGURE 4. Silencing of Ezh1 impairs TLR signaling in macrophages. (A) Mouse peritoneal macrophages were transfected with control siRNA or siRNA specific for Ezh1 (siRNA Ezh1). Forty-eight hours later, the cells were stimulated with LPS (100 ng/ml) for the indicated times, and phosphorylation of p65, ERK, JNK, p38, and IRF3 in the lysates was detected using Western blot. Total protein and b-actin were used as loading controls. (B) Immunoblot of p65 and IRF3 in nuclear extractions of macrophages, as described in (A). Lamin A/C was used as a loading control. (C) Control or Ezh1-silenced mouse macrophages were transfected with the empty vector, the EZH1 vector, or the EZH1-DSET vector. Twenty-four hours later, the cells were stimulated or not http://www.jimmunol.org/ with LPS (100 ng/ml), and phosphorylation of p65, ERK, JNK, p38, and IRF3 in the lysates was measured 1 h later using Western blot. Total protein and b-actin were used as loading controls. Data are representative of three independent experiments with similar results. dicate that Ezh1 suppresses Tollip expression via its lysine Ezh1 Ab to detect whether Ezh1 could bind directly to the promoter of methyltransferase activity. TLR-induced cytokine genes. Ezh1 did not associate with the promoter of il6, tnf,orifnb1, with or without LPS stimulation (Fig. 6G–I). Ezh1 inhibits Tollip expression by inducing H3K27me3 Together, these experiments provide evidence that Ezh1 binds modification at the tollip proximal promoter the tollip promoter in macrophages and inhibits Tollip expression Next, we examined how Ezh1 suppressed Tollip expression. Be- in an H3K27me3-dependent manner. by guest on September 27, 2021 cause Ezh1 acts as a KMT that can catalyze H3K27me3, we wondered whether Ezh1 could directly target the tollip promoter Discussion and increase H3K27me3 modification there through its methyl- Ezh1 was reported to participate in some important biological transferase activity. We used ChIP experiments to determine the processes, including stem cell development and cell differentiation. level of H3K27me3 at the tollip gene locus. Primers to different Ezh1 in embryonic stem cells has been linked to the maintenance of loci upstream of the tollip TSS were designed, and we used qPCR stem cell identity and pluripotency (27). Ezh1 is involved in the to detect the locus of H3K27me3 modification in the promoter. maintenance of proliferation and survival of hematopoietic stem H3K27me3 was detected between 21500 and 2500 bp upstream cells (31) and is responsible for the development of skin stem of the tollip TSS, especially between 21000 and 2500 bp cells (28, 32). Ezh1 has been functionally linked to H3K27 (Fig. 6A). As a result, primers to this locus were used to analyze methylation-dependent gene repression, but an understanding of the H3K27me3 level of the tollip promoter. Then a ChIP assay Ezh1’s function in innate immunity and inflammation has been was performed with Ab specific to H3K27me3 or normal IgG in limited. In this study, we showed that Ezh1 expression by DCs and macrophages upon LPS stimulation (Fig. 6B), which demon- macrophages is essential for TLR-induced inflammatory cyto- strated that H3K27me3 modification was enriched at the tollip kine production, and the effect of Ezh1 is due to the Ezh1-medi- promoter. When Ezh1 was silenced, with or without LPS stimu- ated suppression of the transcription of Tollip, an inhibitor of lation, H3K27me3 levels at the tollip promoter decreased signif- TLR signaling. Ezh1 binds to the tollip promoter and induces icantly (Fig. 6C). H3K27me3 modification at the tollip promoter, which ultimately To fully investigate the role of Ezh1 as a KMT, Ezh1-silenced results in the suppression of Tollip, which inhibits TLR response macrophages were transfected with the EZH1 vector or the and leads to an increase in the TLR-triggered production of type I EZH1-DSET vector and then challenged with LPS. As shown in IFN, IL-6, and TNF-a. To our knowledge, this is the first de- Fig. 6D, ChIP-qPCR revealed that overexpression of full-length scription of the biological significance of an H3K27 trimethyl- Ezh1 restored H3K27me3 modification, which had decreased as transferase in the regulation of the innate inflammatory response. a result of knockdown of Ezh1 at the tollip promoter. Finally, we Histone lysine modification, especially H3K27 methylation, performed ChIP assay with an Ab to Ezh1, whose specificity was was shown to be involved in the transcriptional regulation of in- tested using an immunoprecipitation/immunoblotting strategy flammatory genes. H3K27 demethylase Jmjd3 was discovered to (Fig. 6E), and observed binding of Ezh1 to the tollip promoter promote TLR-induced gene expression in macrophages, and it is (Fig. 6F). This indicated that Ezh1 binds directly to the tollip essential for the alternative activation of macrophages (33, 34). The promoter and maintains a high level of H3K27me3 there. level of H3K27me3 is tightly regulated by H3K27-specific KDMs Finally, to eliminate the possibility of direct regulation of TLR- and KMTs. Ezh2, another important H3K27 methyltransferase, activated genes by Ezh1, we performed a ChIP experiment with an also was found by us to be highly expressed in DCs (data not The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 5. Ezh1 suppresses Tollip expression. (A–C) Mouse macro- by guest on September 27, 2021 phages were transfected with Ezh1 siRNA or negative control. After 24 h, the cells were transfected with siRNA specific for Tollip or negative control. After an additional 36 h, the cells were stimulated with LPS (100 FIGURE 6. Ezh1 binds the Tollip proximal promoter and increases ng/ml) for 12 h. IL-6 (A), TNF-a (B), and IFN-b (C) in the supernatants H3K27me3 level at the tollip promoter in response to LPS stimulation. were detected using ELISA. (D) Ezh1- or Tollip-silenced macrophages, as (A) Chromatin of mouse macrophages was immunoprecipitated with described in (A), were stimulated with LPS for the indicated times. H3K27me3 Ab or IgG, and primers to different loci of the tollip promoter Phosphorylation of p65 and IRF3 was detected in lysates of cells, and were used in qPCR. The value was normalized to the percentage of input. protein levels of Ezh1 and Tollip were analyzed by immunoblotting. (B) ChIP assay of H3K27me3 was performed at the tollip promoter. Mouse b-actin was used as a loading control. (E) Protein level of Tollip in BMDCs macrophages were stimulated or not with LPS (100 ng/ml) for 2 h. A ChIP and macrophages after LPS stimulation. b-actin was used as a loading assay was performed using anti-H3K27me3 Ab and anti-IgG, samples control. (F) Mouse peritoneal macrophages were transfected with control were analyzed by PCR, and the product was electrophoresed in 2% agarose siRNA or Ezh1 siRNA. Twenty-four hours later, they were transfected with gel. (C) Mouse macrophages were transfected with siRNA for Ezh1 or the empty vector, the EZH1 vector, or the EZH1-DSET vector, and the control siRNA and then stimulated with LPS (100 ng/ml) for the indi- protein level of Tollip was detected using Western blot. b-actin was used as cated time. A ChIP assay at the tollip promoter was performed using anti- a loading control. Data in (A)–(C) are shown as mean 6 SD of three in- H3K27me3 Ab and then analyzed by qPCR. The value represents the dependent experiments. Data in (D)–(F) are representative of three inde- enrichment (fold) in H3K27me3 compared with control IgG precipitation. (D) pendent experiments with similar results. **p , 0.01. Ezh1-silenced mouse macrophages were transfected with the empty vector, the EZH1 vector, or the EZH1-DSET vector. Cells were stimulated or not with LPS (100 ng/ml) for 2 h. A ChIP assay at the tollip promoter was shown). Ezh2 has stronger methyltransferase activity, and its performed using anti-H3K27me3 Ab and analyzed by qPCR. The data rep- knockdown was reported to affect global H3K27me2/3 levels (20). resent enrichment (fold) in H3K27me3 compared with control IgG precipi- E In addition, Ezh2 was shown to be responsible for the methylation tation. ( ) Ezh1 in the lysate of mouse macrophages was precipitated with F I at inflammatory gene promoters (35); however, we found that anti-Ezh1 Ab and detected by immunoblotting with the same Ab. ( – ) Mouse macrophages were stimulated or not with LPS (100 ng/ml) for 2 h. A ChIP knockdown of Ezh2 in DCs did not affect LPS-induced cytokine assayofEzh1atthetollip, tnf, il6,orifnb1 promoter was performed using production (data not shown). Furthermore, according to the RNA- anti-Ezh1 Ab and anti-IgG, samples were analyzed by PCR, and the product seq data of DCs as described in Fig. 1B, the expression of Ezh2 was electrophoresed in 2% agarose gel. Data in (B)and(E)–(I) are mean 6 SD was unaffected after LPS challenge. Although Ezh1 and Ezh2 may of three independent experiments. Data in (A), (C), and (D) are representative share some common target genes, we found that Ezh1, distinct of three independent experiments with similar results. **p , 0.01. from its homolog Ezh2, could play a unique role in the regulation of innate immunity and inflammation. specifically targets tollip. In our hypothesis, Ezh1 might bind to We performed a ChIP assay and determined that Ezh1 associated the tollip promoter via the CXC domain, which was a DNA- with the promoter of tollip; however, it is not known how Ezh1 binding domain. Epigenetic modification enzymes are often 8 EZH1 PROMOTES TLR RESPONSE VIA TARGETING TOLLIP recruited by corepressors or multiprotein transcriptional com- 3. Chang, T. H., T. Kubota, M. Matsuoka, S. Jones, S. B. Bradfute, M. Bray, and K. Ozato. 2009. Ebola Zaire virus blocks type I interferon production by plexes to gene promoters, where they regulate transcription exploiting the host SUMO modification machinery. 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Death domain-associated protein 6 (Daxx) selectively effectively without cotransfecting other proteins in the PRC2 represses IL-6 transcription through histone deacetylase 1 (HDAC1)-mediated complex. However, whether the repression role of Ezh1 in Tollip histone deacetylation in macrophages. J. Biol. Chem. 289: 9372–9379. 9. Koch, M. W., L. M. Metz, and O. Kovalchuk. 2013. Epigenetic changes in expression is assisted by other proteins in PRC2 complex needs to patients with multiple sclerosis. Nat Rev Neurol 9: 35–43. be further investigated. 10. Kabesch, M. 2014. Epigenetics in asthma and allergy. Curr. Opin. Allergy Clin. Tollip is associated with the TIR domain of TLR and IL-1R via Immunol. 14: 62–68. 11. Kouzarides, T. 2007. Chromatin modifications and their function. Cell 128: 693– its C-terminal coupling of ubiquitin to the endoplasmic reticulum 705. Downloaded from degradation domain, and it acts as a critical regulator of TLR- 12. Black, J. C., C. Van Rechem, and J. R. 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