Polycomb chromobox Cbx2 enhances antiviral innate immunity by promoting Jmjd3-mediated demethylation of H3K27 at the Ifnb promoter
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Polycomb chromobox (CBX) proteins regulate gene transcription by maintaining chromatin states, which guide a variety of biological processes. Now, epigenetic regulation of innate immune response is an emerging field. However, the role of CBX proteins in innate immunity remains unclear. We confirmed that the expression of CBX family proteins, especially Cbx2, was decreased in macrophages upon viral infection, and then we investigated the role of Cbx2 in the antiviral immune response. Silencing or knockdown of Cbx2 in macrophages inhibited virus-induced production of IFN-β. Furthermore, heterozygous Cbx2 knockout were susceptible to VSV challenge. Mechanistically, Cbx2 binds to and recruits Jmjd3 to the Ifnb promoter, leading to demethylation of H3K27me3 and increased transcription of IFN-β. Together, our study reveals a non-traditional function of a Cbx protein and adds new insight into the epigenetic regulation of antiviral innate immunity.
KeywordsCbx2 Jmjd3 interferon beta innate immunity
The innate immune response is an important determinant in the fight against virus infection, deploying pattern-recognition receptors (PRRs), such as the RIG-I-like receptor (RLR), to sense and respond to pathogens (Cao, 2016; Kawai and Akira, 2010). Upon binding to pathogen-associated molecular patterns (PAMPs), PRRs activate a series of signaling cascades, leading to the induction of type I interferons (IFNs) by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), interferon regulatory factor (IRF) 3/7 and/or activator protein-1 (AP-1) (Iwasaki and Pillai, 2014; Wu and Chen, 2014). Subsequently, IFNs induce the expression of interferon stimulated gene (ISGs) to restrict viral replication through the activation of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway (McNab et al., 2015). Precise control of the innate immune response is critical for maintaining host immune homeostasis and ensuring proper viral clearance.
Epigenetic modifications such as DNA methylation, histone modifications, noncoding RNA regulation and chromatin remodeling have been shown to impact gene transcription. Epigenetic regulation in innate immunity has been studied extensively in recent years, yielding insights into the detailed molecular mechanisms that modulate the expression of inflammatory cytokines and type I interferons (Marcos-Villar et al., 2018; Yoshida et al., 2015; Zhang et al., 2015). For example, Tet2 recruits Hdac2 to specifically repress IL-6 by histone deacetylation, which resolves inflammation (Zhang et al., 2015). Dnmt3a promotes activation of TBK1 by maintaining high expression of the histone deacetylase HDAC9, leading to the production of type I interferons and antiviral innate immunity (Li et al., 2016). The lncRNA lnc-Lsm3b binds to RIG-I monomers and restricts RIG-I protein’s conformational shift, thereby terminating type I IFNs production (Jiang et al., 2018). However, the identification of other unknown epigenetic modifier and the underlying mechanisms for innate immunity remain to be further explored.
The Polycomb group (PcG) proteins, which are best characterized by two protein complexes (Polycomb repressive complex 1 [PRC1] and Polycomb repressive complex 2 [PRC2] have been identified as critical regulators of gene expression that maintain repressive chromatin states (Bernstein et al., 2006; Simon and Kingston, 2013). Chromobox (CBX) family proteins are canonical components of PRC1 and are divided into two groups: HP1α(Cbx5), HP1β(Cbx1), and HP1γ(Cbx3); and Cbx2, Cbx4, Cbx6, Cbx7 and Cbx8. The latter group has an N-terminal chromodomain that serves as a recognition site and binds to H3K27me3, which helps to recruit and stabilize PRC1 to specific regions of the chromatin (Aranda et al., 2015; Di Croce and Helin, 2013). Accumulating evidence has demonstrated that CBX proteins play crucial roles in a variety of biological processes, such as the differentiation of stem cells, neurite development and tumor angiogenesis (Chen et al., 2017; Santanach et al., 2017; Sparmann and van Lohuizen, 2006). Recently, Cbx7 has been shown to repress activation-induced T cell apoptosis, indicating a role in the adaptive immune response (Li et al., 2014a, b). However, the role of CBX family proteins in innate immunity remains largely unknown.
In this report, we addressed the function of Cbx2 in antiviral innate immunity. Our results demonstrate that Cbx2 enhances the production of type I interferon by binding and recruiting Jmjd3 to the promoter of IFN-β, thus activating the antivirus immune response. Therefore, our work identifies Cbx2 as a positive mediator in antiviral immunity, which provides new mechanistic insight into epigenetic modifiers in the innate immune response.
Cbx2 promotes virus-induced production of type I interferon in macrophages
In vivo knockdown of Cbx2 increases susceptibility to VSV challenge
Cbx2 promotes the transcriptional activation of Ifnb
Cbx2 enhances IFN-β activation by binding to Jmjd3
Taken together, these data demonstrated that Cbx2 promotes antiviral innate response by binding to and recruiting Jmjd3 to the Ifnb promoter, leading to demethylation of H3K27me3 and increased transcription of IFN-β.
In this study, we identified that Cbx2 enhances virus-induced production of type I interferon, and we defined a molecular mechanism for this regulation in which Cbx2 binds to and recruits Jmjd3 to the Ifnb promoter, leading to demethylation of H3K27 and promotion of Ifnb transcription. Our findings identify a new epigenetic regulator with an integral role in the antiviral innate immune response, which suggests a noncanonical function of Cbx2.
CBX family proteins play crucial roles in a variety of biological processes by maintaining repressive chromatin states of specific genes. In tumors, CBX family proteins have been shown to endow PRC1 with distinct functions, such as oncogene or tumor suppressor functions. For example, Cbx7 has been identified to act as tumor suppressor in lung carcinoma by suppressing CCNE1 expression (Forzati et al., 2012); conversely, it has been shown to act as an oncogene in gastric cancer by repressing p16 (INK4a) (Zhang et al., 2010). Cbx2 family proteins have also been shown to inhibit neurite development by directly inhibiting neuron-specific gene expression. On the other hand, CBX family proteins also have been shown to enhance gene transcription in certain cases. For example, Cbx4 enhances the expression of VEGF and promotes angiogenesis in hepatocellular carcinoma cells (Li et al., 2014a, b), which is consistent with our findings that Cbx2 enhances antiviral innate immunity by promoting the transcriptional activation of IFN-β. Therefore, it is possible that these distinct functions of Cbx2 are cell-type-specific. In tumor cells, Cbx2 is up-regulated and has been identified as a potential drug target (Chen et al., 2017; Clermont et al., 2016), whereas our results demonstrate that Cbx2 is down-regulated in macrophages infected with virus. Therefore, we speculate that the distinct functions of Cbx2 might be explained both by cell-type-dependent function and expression differences. We also observed that other Cbx family proteins exhibit decreased expression in virus-infected macrophages, which raises the possibility that other Cbx family proteins might collaborate with Cbx2 in the antiviral response.
Conventionally, PRC1 proteins bind to H3K27me3 and recruit other components of PRC1 to chromatin by protein-protein interaction to further enhance transcriptional repression of target genes (Gao et al., 2012). However, it has been shown that certain CBX proteins can associate with moderators other than the PRC1 complex, displaying a PRC1-independent role in transcriptional regulation. For instance, Cbx4 represses the expression of Runx2 and metastasis of colorectal carcinoma, which is dependent on its interaction with HDAC3 (Wang et al., 2016). Furthermore, Cbx8 associates with WDR5, thereby enhancing Notch-related gene expression and promoting breast tumorigenesis (Chung et al., 2016). Intriguingly, our study suggests that Cbx2 enhances IFN-β transcription in virus-infected macrophages by inhibiting H3K27me3 of Ifnb promoter, which is dependent on the recruitment of Jmjd3. These findings suggest that the interaction with Jmjd3 is pivotal for the function of Cbx2 in antiviral innate immunity. An important issue for the current research is the spatiotemporal dynamics between Cbx2, Jmjd3 and H3K27me3. Furthermore, it would be important to investigate whether other moderator(s) of PRC may be involved in Cbx2-mediated promotion of IFN-β transcription.
In Cbx2+/− mouse-derived macrophages, increased recruitment of H3K27me3 to the Ifnb gene promoter was also observed, suggesting that Cbx2 reduces H3K27me3 at the Ifnb gene promoter. This is in contrast to Polycomb group (PcG) protein-mediated H3K27me3 recruitment to genomic targets (Simon and Kingston, 2013). It has been shown that phosphorylation of Cbx2 is essential for its transcriptional repression of target genes, and that CK2-mediated phosphorylation inhibits Cbx2’s DNA-binding activity and transcriptional repression (Kawaguchi et al., 2017). Given that the expression of CK2 is virus-inducible in macrophages (GEO profile: GDS1271), we speculate that CK2-mediated phosphorylation inhibits recognition and binding to H3K27 in macrophages upon virus infection.
According to reported gene-profiling data, Cbx2 expression is downregulated in the brain (GEO: GSE44331) and in RAW264.7 macrophages (GEO: GSE81675), which is in agreement with our findings that Cbx2 expression decreases upon VSV infection. As a positive regulator of IFN-β transcription activation, the Cbx2 expression pattern differs from the expression pattern of other activators that are virus-inducible (Li et al., 2014a). This difference might be explained by a fine-tuning mechanism that serves to prevent superfluous IFN-β production. Proper termination of the antiviral effect is necessary to avoid excessive cytotoxicity and tissue injury. Therefore, Cbx2 down-regulation by VSV might constitute a feedback regulation mechanism in the antiviral response.
During virus infection, the host senses components derived from viruses and activates type I IFN response to eliminate viruses. On the other side, viruses have evolved kinds of ways to regulate and evade the host anti-viral immune response, such as sequestering anti-viral signaling transducers and effectors or targeting them for degradation. Furthermore, to establish virus infection-specific expression patterns of host cells for persistent infection or effective replication, virus may inhibit type I IFN response through epigenetic mechanisms (Garcia-Sastre, 2017). In this study, we observed that the expression of Cbx2, which is a coactivator of IFN-β, decreased during virus infection, although the underlying mechanism was unclear. Our study implies that virus may inhibit the host type I IFN response through blocking the positive epigenetic mechanism for transcription induction of INF-β.
In summary, we identified Cbx2 as a positive regulator for antiviral innate immunity. Our results suggest that Cbx2 promotes the production of IFN-β by associating with Jmjd3 and enhancing its recruitment to the Ifnb promoter. Our study provides mechanistic insight into epigenetic modifiers in the regulation of innate immunity.
Materials and methods
Cbx2+/− mice were obtained from Jackson Laboratory (Stock No: 003478). The first 4 exons of Cbx2 were replaced via homologous recombination with a neomycin resistance cassette in reverse orientation. The strain was maintained by heterozygous mating for more than 2 years. Male C57BL/6J mice (six to eight weeks) were from Beijing Vital River Laboratory Animal Technology Co. (Beijing, China). All animals were maintained in specific-pathogen-free conditions. All animal experiments were carried out under the supervision of the Institutional Animal Care and Use Committee (IACUC) of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China.
A recombinant expression vector encoding N-Myc-tagged Cbx2 was constructed by PCR cloning into EX-Mm01605-M43 eukaryotic expression vector (Fulengene Genecopoeia). The IL-6 and IFN-β luciferase reporter plasmids have been described previously (Li et al., 2014a). Expression vectors encoding Flag-tagged Cbx2, Myc-tagged Jmjd3 and Myc-tagged IRF3 were constructed by PCR cloning into pcDNA3.1-Flag or pcDNA3.1-Myc eukaryotic expression vectors. Flag-tagged P65 and IRF3 expression vectors were constructed by PCR cloning into pcDNA3.1 eukaryotic expression vector (Invitrogen, Carlsbad, CA). All constructs were confirmed by DNA sequencing.
Cells and cell transfection
HEK293T and RAW264.7 cell lines were from American Type Culture Collection. Thioglycolate-elicited mouse primary peritoneal macrophages were isolated and cultured, transfected with siRNA pool (10 nmol/L) through the use of Lipofectamine® RNAiMAX Reagent (Thermo Fisher Scientific). The siRNAs targeting mouse Cbx2 were as follow: CAAGUGCGGUCUCGGGCUA, GAGUCAAGUUCUCGUGUUU, UGGCAGAGCUCCAUCGUAC, CCGGGUAACUGUCUUGAAA (Designed by Dharmacon). For stable overexpression of Cbx2, we transfected cells expressing the IFN-β-Luc reporter gene (293T-IFN-β-Luc cells) or RAW264.7 cells with Myc-Cbx2 and selected the cells in G418 Sulfate (800 μg/mL; Gibco). For transient transfection of plasmids in HEK293T cells, Lipofectamine™ 3000 Reagent was used according to manufacturer’s instructions (Thermo Fisher Scientific). For transient transfection of plasmids in primary peritoneal macrophages, nuclear transfection was performed by using the AmaxaTMP3 Primary Cell 4D-nucleofectorTM X Kit (Lonza) according to the manufacturer’s instructions. All cells were cultured in endotoxin-free DMEM (Gibco), supplemented with 10% FBS (Gibco), 5 mg/mL penicillin (Gibco) and 10 mg/mL streptomycin (Gibco).
Antibodies and reagents
Antibodies against Myc-tag (2272, 2276, 2040), Flag-tag (14793), phosphorylated IRF3 (Ser396) (#4947), phosphorylated NF-κB p65 (Ser536) (#3033), and Normal Rabbit IgG (#2729) were from Cell Signaling Technology. Antibodies against Cbx2 (LS-C81651, LS-C289259) were from LifeSpan BioSciences. Antibodies against Histone H3K27me3 (39155), Histone H3K9me3 (61013), and Histone H3K4me3 (61379) were from Active Motif. Antibodies against β-actin (M177-3) and GAPDH (M171-3) were from MBL. Protein G-agarose (20397) used for immunoprecipitation was from Pierce; ChIP Grade Protein G Magnetic Beads (#9006), and Cell Lysis Buffer (#9803) were from Cell Signaling Technology. Poly (I:C) was from Invitrogen. HSV was a gift from Prof. Q. Li (Chinese Academy of Sciences, Beijing, China), VSV was a gift from Prof. W. Pan (Second Military Medical University, Shanghai, China), and Sendai virus (SeV) was a gift from Prof. B. Sun (Chinese Academy of Sciences, Shanghai, China). HJP (Jmjd3 inhibitor) was a gift from X. Bin (Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China).
For in vitro virus infection experiments, virus was added along with fresh medium to pre-treated cells for the indicated times at MOI of 10. For in vivo virus infection experiments, virus was injected via the tail vein.
Total RNA was extracted with Trizol reagent (Ambion). ReverTra Ace® qPCR RT Master Mix with gDNA Remover (Toyobo) was used to synthesize cDNA. KOD SYBR® qPCR Mix (Toyobo) was used for qRT-PCR. Sample data were normalized to GAPDH expression. Primers for qPCR are listed in Table S1.
IFN-β and IL-6 levels in supernatants or sera were measured with mouse IFN-β ELISA kits (PBL Biomedical Laboratories).
Cells and organs were lysed with RIPA Lysis Buffer (Merck Millipore), and extracted protein was homogenized by the BCA protein assay kit (Pierce). Immunoblot were performed with the indicated antibodies.
VSV plaque assay
VSV titers of supernatants and tissue homogenate samples were determined by plaque assays on Vero cells. 100 µL samples (10-fold dilution) were inoculated onto confluent Vero cell monolayers for 1 h at 37 °C, 5% CO2, and washed by DMEM. And Vero cells were overlayed with a final concentration of 1% agar containing MEM, 1% FBS, 200 μg/mL diethylaminoethyl-dextran, and 0.008% for 48 h and 72 h. 0.008% neutral red was added to the cell monolayers, which were then incubated overnight, and plaques were counted. Viral burdens are reported as the log10 plaque forming units per mL (PFU/mL).
Luciferase reporter assay
293T-IL-6-Luc cells or 293T-IFN-β-Luc cells were seeded in 96-well plates and then transfected with Cbx2 or empty plasmid and other signaling pathway-related molecular-expressing vectors as indicated, together with TK-Renilla-luciferase as a control. After 36 h of incubation, whole-cell lysates were collected for measurements of luciferase activity with the Dual-luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. The method has been described previously (Jiang et al., 2018).
Chromatin immunoprecipitation (ChIP)
For ChIP assays, we used ChIP-IT® Express Chromatin Immunoprecipitation Kits (Active Motif) according to the manufacturer’s instructions. Primers used for ChIP quantification are listed in Table S2.
Lung tissues from Cbx2+/+ mice and Cbx2+/− mice were dissected and processed as described previously (Wang et al., 2017).
Statistical significance between two groups was determined by the two tailed Student’s t-test. Differences were considered to be significant when P < 0.05. (*P < 0.05; **P < 0.01; ***P < 0.001). For mouse survival studies, Kaplan-Meier survival curves were generated and analyzed for statistical significance with GraphPad Prism 5.0.
We thank Ms. Qiaolian Wen and Mrs. Qijiang Cheng for technical assistance. This work was supported by grants from the National Natural Science Foundation of China (Grant Nos. 31770944 and 81788101); the National 135 Major Project of China (2017ZX10102032-002); and CAMS Innovation Fund for Medical Science (2016-I2M-1-003).
Cbx, chromobox 2; H3K27me, histone 3 lysine 27 methylation; IFN-β, interferon beta; IRF3, interferon regulatory factor 3; Jmjd3, Jumonji domain-containing protein D3; PcG, Polycomb group; PRC, Polycomb repressive complex.
Compliance with ethics guidelines
Donghao Sun, Xuetao Cao and Chunmei Wang declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed. This article does not contain any studies with human subjects performed by any of the authors.
C. Wang and X. Cao designed and supervised the research; D. Sun performed the experiments, analyzed data and wrote the paper with C. Wang.
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