DNA Methyltransferase Dnmt3a Promotes Typs â…  Interferon Production Though HDAC9-mediated TBK1 Deacetylation

Part Ⅰ Dnmt3a up-regulates TLR and RIG-Ⅰ -induced type Ⅰ interferon production by affecting phosphorylation of IRF3 in macrophagesMacrophage, as one of the major innate immune cells, is the first line of host innate defense against virus infection. Dnmt3a, a de nove DNA methyltransferase, is responsible for the establishment of DNA methylation patterns during development. In the preliminary studies, we found the higher level of Dnmt3a expression in terminally differentiated cells-mouse peritoneal macrophages, compared to other detected tissues and immune cells. However, the function of Dnmt3a remains unknown in macrophage and innate immune response. To examine the function of Dnmt3a in macrophage, we measured the gene expression profile in Dnmt3a-knockdown macrophages by gene-chip and screened the cytokine production in TLR-triggered macrophages. Here we found Dnmt3a could positively regulate TLR-induced IFN-β production in macrophages. Dnmt3a knockdown inhibits TLR and RIG-Ⅰ-induced interferon-beta production in macrophages. We generated the mice that lacked Dnmt3a by breeding Dnmt3aflox/flox mice with transgenic mice (lyz2Cre) expressing Cre recombinase under the control of lyz2 promoter (collectively termed Dnmt3afl/fl lyz2Cre+) facilitating deletion of Dnmt3a in macrophages. Compared with control mice, Dnmt3afl/fl lyz2Cre+ mice displayed normal macrophage populations with almost same cell number and phenotype. However, gene expression and secretion of IFN-β, but not TNF-a and IL-6, was found to be decreased in Dnmt3afl/fl lyz2Cre+ peritoneal macrophages as compared with that in littermate control cells. We challenged Dnmt3a conditional knockout mice with RNA viruses VSV. The mortality to VSV infection in Dnmt3a conditional knockout mice was higher than the littermate control mice in overall survival assays. Dnmt3a conditional knockout mice produced much lower levels of IFN-β than littermate control mice in response to VSV infection. VSV titer and replication in organs from Dnmt3a conditional knockout mice were significantly enhanced compared to the littermate control mice. More infiltration of inflammatory cells was observed in the lungs of Dnmt3a conditional knockout mice after infection with VSV. Dnmt3a knockdown specifically suppressed LPS and VSV-induced IRF3 activation in macrophages. So, Dnmt3a can selectively up-regulate TLR and RIG-Ⅰ-induced type Ⅰ interferon production by affecting phosphorylation of IRF3 in macrophage.Part II Dnmt3a promotes type I interferon production though HDAC9-mediated TBK1 deacetylationReversible acetylation of lysine which ranks similar to the important master switch phosphorylation crucially modulates protein function such as signal transduction, DNA binding activity, protein stability, subcellular localization. Acetylation modification controls many cellular processes, and increasing evidence has implicated protein acetylation in immunological pathways. Importantly, lysine residues can be post-translationally modified in numerous ways, and crosstalk between different modification systems can also have an impact on inflammatory and immune responses. HDAC9 is a class Ⅱa histone deacetylases (HDACs) which can shuttle between the nucleus and cytoplasm. To elucidate the underlying mechanism by which Dnmt3a enhances TBK1 mediated IRF3 activation, we measured the gene expression profile in Dnmt3a-knockdown macrophage by gene-chip, we found Dnmt3a knockdown decreased HDAC9 mRNA level. We detecded the subcellular location of HDAC9 in mouse peritoneal macrophages by confocal and indicated HDAC9 mostly located in cytoplasm. We synthesized interfering RNA targeting mouse HDAC9, and found HDAC9 siRNA significantly inhibited TLR and RIG-I-induced IFN-β mRNA level in mouse peritoneal macrophage. And we found HDAC9 knockdown specifically suppressed phosphorylation of IRF3 (Ser396) in macrophages. Coimmunoprecipitation revealed that the C-terminal part of the deacetylation domain of HDAC9 was necessary for the interaction of HDAC9 and TBK1. We analyzed the acetylation of TBK1 immunoprecipitated from whole-cell extracts of mouse peritoneal macrophages, and found TBK1 acetylation inhibited TBK1 activation in macrophages. In vitro kinase assay results suggested that HDAC9 directly binds and deacetylates TBK1, which leads to the increased activation of TBK1. IP-MS analysis the acetylation of TBK1 immunoprecipitated from whole-cell extracts of mouse peritoneal macrophages treated with TSA. So, DNMT3a promotes type I interferon production though HDAC9-mediated TBK1 deacetylation.Part Ⅲ Dnmt3a maintains high expression of HDAC9 by antagonizing H3k27me3 enrichment at the distal promoter of HDAC9Currently, there are the two opponent perspectives on relationship between DNA methylation and gene expression. The traditional view is that DNA methylation in most of the proximal promoter in CpG rich regions (CpG islands) associates with gene silencing. However, recent reports suggest that nonpromoter DNA methylation by Dnmt3a maintains active chromatin states at some gene loci by functionally antagonizing Polycomb repression. To investigate the mechanisms for promoting HDAC9 expression by Dnmt3a, firstly, we analysed the promoter region of HDAC9, and found that Dnmt3a bound to the HDAC9 promoter. We analyzed the DNA methylation state of the HDAC9 distal promoter by MeDIP, methylation state of the CpG sites decreased in Dnmt3afl/fl lyz2Cre+ peritoneal macrophages compared with the littermate control cells. And then, Dnmt3a knockdown increased H3K27me3 level in the promoter region of HDAC9 in mouse peritoneal macrophages. Similarly we also found the higher level of H3K27me3 binding in the HDAC9 distal promoter region in Dnmt3afl/fl lyz2Cre+ peritoneal macrophages compared with littermate control cells by ChIP assay. Therefore, Dnmt3a-dependent DNA methylation promotes HDAC9 expression by antagonizing H3k27me3 binding to the distal promoter of HDAC9 in naive macrophages.