A Single Methylated CpG In The Promoter Of Ifnb1 Mediated By UHRF1 Suppresses Antiviral Innate Immunity

Virus is one kind of the pathogens that cause body damage.The pandemic of various pathogenic viruses often causes a large number of deaths and huge economic losses.Early studies have revealed that epigenetic modification plays an important role in the attack and defense game between host cells and viruses.However,most studies rely on non-immune cells and focus on histone modification.the role of DNA methylation has not been fully elaborated.Recently,it has been reported that the incorporation of the DNA methylation inhibitor 5-AZA upregates the background level of type I interferon in tumor cells,whereas the expression of type I interferon is significantly inhibited in cell lines with naturally low levels of DNA methylation.These contradictions indicate that the regulation of type I interferon by intracellular DNA methylation is affected by the characteristics of the cell itself,and the conclusions in cancer cells cannot be generalized to primary cells directly.Innate immune cells,which are specialized cell groups responsible for recognizing viruses and initiating anti-infective immunity,are important sources of type I interferons in vivo,but the role of DNA methylation during their responses to viral infection is still under investigation.In this study,by comparing the global DNA methylation levels and the expression differences of DNA methylation-related molecules in human peripheral blood mononuclear cells infected by different strains of influenza virus,we screened out the key molecule UHRF1,which mediated DNA methylation maintenance,and constructed a gene modificated mice that specifically knocked out UHRF1 in macrophage(Uhrf1^MKO)to reveal the role of DNA methylation in the resistance of macrophages to virus infection.Our data suggested that Uhrf1^MKO mice had stronger viral resistance compared to WT mice,and that UHRF1-deficient macrophages had higher level of Ifnb expression upon stimulated by viruses or Toll-like receptor agonists.Further studies showed that the regulation of Ifnb expression by UHRF1 did not depend on the activation of classical antiviral signaling pathways,nor did it affect the entry of key transcription factors into the nucleus.The combined analysis of whole-genome bisulfit sequencing and transcriptome sequencing showed that there was a CpG site at upstream of the Ifnb1 gene transcription start site,and its methylation modification had ansignificant effect on the transcription regulation of Ifnb1 gene.This site was conserved in human cells and was also methylated.Subsequent studies showed that the methylation of the indicated CpG site had a strong inhibitory effect on the binding of IRF3,a key transcription factor that initiated the transcription of Ifnb1 gene,to the promoter region of Ifnb1 gene.Targeted erasing of methylation at this site in mouse embryonic fibroblasts significantly improved the expression level of Ifnb and its antiviral ability.The CpG mutant mice also produced higher levels of Ifnb and had stronger antiviral capacity.In conclusion,our study elucidates the mechanism by which UHRF1-mediated methylation of a single CpG site in the promoter region of Ifnb1 gene regulates the expression of type I interferon gene and its important function in antiviral innate immunity.