| Influenza virus (â…£), a member of the orthomyxoviruses family, is a negative sense single-stranded RNA virus which causes seasonal epidemics and reoccurring pandemics in birds and mammals and threats public health worldwidely. The recent epidemic of H1N1 influenza virus has resulted in 10,000 death cases globally since its prevalence in 2009.â…£can immidiately activate host immun system, and the inflammation is a early stage antivirus effects.Epigenetic has become a hot topic in gene expression regulation study. It is well accepted that epigenetic mechanisms plays important roles in many biological events. DNA methylation is the best known epigenetic marker which was closely related with gene expression. Promoter DNA hypermethylation is associated with gene silencing, while hypomethylation is related to gene expression. There have been many studies focusing on the mechanisms of promoter methylation of host gene induced by virus infection, such as HBV, EBV and baculovirus. However, whether the expression of host genes was also regulated by epigenetic events inâ…£infection was yet to be uncovered.In this study, we investigte whether epigenetic mechanisms was involved in IL-6 expression during influenza virus infection or its replicative intermediate dsRNA treatment.Firstly, upregulation of IL-6 expression was measured inâ…£infection and its replicative intermediate dsRNA treatment. The results showed that bothâ…£and dsRNA increased IL-6 promotor activity, mRNA transcription and protein secretion. Meanwhile, IL-6 promoter activity was up-regulated by dsRNA in a dose-dependent manner. Those results implied that dsRNA might be a key viral component involved inâ…£-triggered IL-6 expression during viral infection. Thus, we focused on the effect of dsRNA in the regulation of IL-6 expression in following studies.Secondly, we investigate whether epigenetic mechanisms was involved in IL-6 expression inâ…£or dsRNA. The results from siRNA and inhibitor of DNMTs and HDACs showed that DNA methylation decreased IL-6 expression, on the contrary, histone acetylation incrased its expression. Furthermore, results from in vitro methylation assays also suggested that both DNA methylation and histone acetylation might be involved in the activation of IL-6 expression in dsRNA treatment.Thirdly, we found that the IL-6 upstream regions spanning from position-1690 to-83 contained 30 CpGs and two potential CpG islands which were analyzed by bioinfromatic methods. Interestingly, region I contained many classic transcription factors binding site, such as CREB, NF-kB and SP-1. The sodium bisulfate sequencing result showed that this region was hypermethylated in IV infection or dsRNA treatment. Furthermore, we found the methylation of-162 CG was associated with CREB transcript factor binding by EMSA and ChIP.At last, the direct reason of IL-6 promoter demethylation was investigated. The results showed that DNMT1 was down-regulated in dsRNA treatment, but DNMT3A and 3B were no significant charge. Furthermore, DNMT1 which was responsible for IL-6 promoter demethylation was measured by siRNA and over-expression assays.In conclusion, this study provided evidences for the notion that the expression of IL-6 induced by influenza virus infection was regulated by epigenetic mechanism. It is reported for the first time that host gene dysregulation was modulated by epigenetic mechanism after IV infection. This result provided a new insight into further understanding the molecular mechanism under virus infection and interaction between host and virus. |
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