The novel methyltransferase SETD4 regulates TLR agonist-induced expression of cytokines through methylation of lysine 4 at histone 3 in macrophages
Introduction
The host response to infection involves the detection of pathogen-associated molecular patterns (PAMPs) by innate immune cells via pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) (Vijay, 2018). TLRs recognize PAMPs such as lipopolysaccharide (LPS), bacterial lipoprotein (BLP) and polyinosinic:polycytidylic acid (poly (I:C), trigger multiple signalling pathways including the MAPK and NF-κB pathways, and induce the expression of inflammatory cytokines (Kaukonen et al., 2015). Uncontrolled and excessive expression of inflammatory cytokines leads to severe tissue injury and a variety of life-threatening pathological conditions including septic shock and multiple organ dysfunction syndrome (MODS) (Lakshmikanth et al., 2016; Liu and Yao, 2010). Therefore, the tight control and modulation of transcriptional expression of inflammatory cytokines during the pathological process are an important area of research.
Gene transcription is regulated epigenetically through alterations in the patterns of DNA methylation and covalent histone modifications (Berger, 2007; Nishioka et al., 2002). Histone methylation, one of the most characterized posttranslational modifications, regulates the transcription and replication of genes by providing binding platforms for diverse transcription factors that specify whether the promoter of an associated gene is in an active or repressed chromatin state, thereby controlling specific biological outcomes such as the inflammatory response (Falnes et al., 2016). Histone methylation can be associated with either transcriptional activation or transcriptional repression depending on its position and methylation state. For example, tri- or dimethylation at residue lysine 4 of histone H3 (H3K4) correlates with the activation of gene expression, whereas H3K36 dimethylation and H3K27 trimethylation is commonly linked to gene silencing (Kubicek and Jenuwein, 2004; Wei et al., 2009). Histone lysine methylation, which is tightly regulated by specific lysine methyltransferases and lysine demethylases, plays a critical role in the transcriptional expression of inflammatory cytokines (Medzhitov and Horng, 2009). For example, Smyd2, a SET (Su[var]3-9, E[z] and trithorax) domain family protein, can specifically facilitate H3K36 dimethylation at the Tnf and Il6 promoters, which suppresses the production of proinflammatory cytokines induced by macrophage activation (Xu et al., 2015). Ezh1 promotes TLR-triggered production of inflammatory cytokines, including IL-6, TNF-α and IFN-β, by suppressing the TLR negative regulator Tollip through its H3K27me3 methyltransferase activity (Liu et al., 2015). However, the detailed roles of histone methyltransferases, especially in TLR-mediated innate immune responses, remain poorly understood. A better understanding of the function of these enzymes in innate immunity and the pathophysiology of relevant diseases, as well as the underlying mechanisms, will provide new insight into the epigenetic regulation of immune responses.
SETD4 is a member of the SET family, which contains a highly conserved SET domain structure. To date, only a few studies have reported that SETD4 may play a role in the development of breast cancer (Faria et al., 2013), hepatocellular carcinoma (Li et al., 2014) and cell quiescence during diapause formation in Artemia (Dai et al., 2017). However, no report has investigated the role of SETD4 in innate immunity-associated inflammatory responses. Here, we report that SETD4 is a positive regulator of the expression of inflammatory cytokines including IL-6 and TNF-α in TLR agonist-triggered macrophages and SETD4 might exert its effect by enhancing H3K4 methylation directly. We propose that this newly identified SETD4 protein may play an important role in TLR agonist-induced inflammatory cytokine expression.
Section snippets
Reagents and antibodies
LPS (E. coli, serotype O111:B4) and Poly(I:C) were purchased from Sigma-Aldrich (St. Louis, MO, USA). BLP (Pam3CysSKKKK) was purchased from EMC Microcollections (Tubingen, Germany). TRIzol RNA was purchased from Invitrogen (Thermo Fisher Scientific, USA). SYBR® Green Real-time PCR and ReverTra Ace®qPCR RT Kit were obtained from TOYOBO (Osaka, Japan). Small interfering RNAs (siRNA) including specifically targeting SETD4 and its scrambled siRNA were obtained from Gene Pharma (Shanghai, China). A
SETD4 is involved in the inflammatory response
TLR-induced proinflammatory cytokines such as IL-6 and TNF-α are critical mediators in the immune response and inflammation against pathogens. To investigate whether SETD4 is involved in the regulation of inflammation, down-regulation of SETD4 by small RNA interference was used to assess the role of SETD4 in regulating inflammatory cytokines. After determining the efficiency of SETD4 RNA interference (Fig. 1A), IL-6 and TNF-α production upon LPS treatment were examined in SETD4 down-regulated
Discussion
Epigenetic modifications such as histone methylation are involved in the transcriptional regulation of multiple TLR agonist-mediated genes. For example, SMYD4, an SET domain family protein, is a negative regulator of inflammatory response genes. SMYD4 can trimethylate H4K20 as a repression checkpoint that restricts the expression of TLR4 signal pathway-mediated target genes in macrophages through its association with nuclear receptor co-repressor (NCoR) complexes (Stender et al., 2012). Ash1l,
Conclusions
Taken together, SETD4 positively regulates LPS-stimulated and other TLR agonist-stimulated a wild range of inflammatory mediators including both pro- and anti-inflammatory cytokines as well as chemokines, indicating that by up-regulating inflammatory cytokine expression and production, SETD4 plays an important role in host innate immune responses against microbial infection. Results from the present study indicate that SETD4 is essential for initiating an efficient inflammatory response via its
Funding
This work was supported by the National Natural Science Foundation of China (grant numbers 81471901, 81272149, 81873620), the Guangdong Provincial Key Laboratory Construction Project of China (grant number 2014B030301044), the Guangdong Provincial Natural Science Foundation (grant number 2015A030311031), and the South Wisdom Valley Innovative Research Team Program (grant number CXTD-001).
Declaration of Competing Interest
None.
Acknowledgements
We thank the Wellcome Trust Sanger Institute Mouse Genetics Project (Sanger MGP) and its funders for providing the mutant mouse line (Allele: SETD4), and the distribution centre from which we received the mouse line, the KOMP Repository at the University of California, Davis (www.komp.org). Funding and associated primary phenotypic information may be found at www.sanger.ac.uk/mouseportal.
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