Brd3 In The Regulation Of Virus Or LPS Triggered Innate Immune Response And The Underlying Mechanism

Innate immune response is the first defense line in hosts to fight against pathogens.It can be initiated by the pattern recognition receptors(PRRs)and sensors in immune cells for recognition of bacteria or viruses to produce inflammatory cytokines and the type I IFN,IFN-β.Upon virus infection,IFN-β is the mostly produced cytokine which is very powerful and has important consequences in anti-virus response.Multiple molecules are involved in the regulation of this process in order to fine tune the prod uction of IFN-β.However,the components of the pathways,especially the epigenetic regulators involved have not been fully elucidated.Bromodamain protein 3(Brd3)is a family member of bromodomain and extraterminal motif proteins(BET)which can functio n as protein scaffolds,mitotic bookmarks,cell cycle regulators and transcriptional regulators.O ne of the family members,Brd4 has been found acting as a co-activator for the transcriptional activation of NF-κB,suggesting that BET might participate in innate immune response.Our previous genome-wide screening data in macrophages have shown that the mRNA level of Brd3 was decreased by nearly two folds after VSV infection,suggesting that Brd3 might be involved in virus triggered innate immune response.He nce,in this study we focused our attention on the regulation of Brd3 in innate immune response and explored the underlying mechanism.In the first research part,whether Brd3 was involved in virus or LPS triggered innate immune response was investigated.Using Q-PCR experiments,we found that Brd3 was ubiquitously expressed in mouse immune organs and immune cells.Then we detected the expression pattern of Brd3 in macrophages upon virus infection or LPS challenge.Q-PCR and WB results showed that both the mRN A and protein level of Brd3 was significantly down regulated.To further investigate the role of Brd3 in cytokine production,we screened an RAW264.7 cell line having Brd3 knocked out by CRISPR-CAS9 technology.ELISA results showed that knock out of Brd3 in RAW264.7 cells significantly inhibited virus or LPS induced production of IFN-β,while the production of inflammatory cytokine IL-6 decreased slightly and TNF-α showed no difference.So in this part,we confirmed the participation of Brd3 in virus or LPS triggered innate immune response,and found that Brd3 promotes the production of IFN-β.In the second research part,we explored the underlying mechanism by which Brd3 promotes the production of IFN-β after virus infection.We firstly screened the signal pathways which regulate IFN-β production.WB results showed that knock out of Brd3 barely affected the nuclear translocation of transcription factors IRF3 and p65.Then immunoprecipitation assays showed that Brd3 can associate with the transcription factor IRF3,co-activator p300 protein and enhance IRF3/p300 complex formation upon virus infection,these three proteins may function as a complex to regulate gene transcription;we also found that Brd3 could promote p300 mediated acetylation of IRF3.Furthermore,ChIP assays showed that the recruitment of IRF3 and p300 to Ifnb gene promoter was significantly down regulated in Brd3 knocked out cells,and the acetylation level of histone3/histone4 also decreased.So in this part,we found that Brd3 could enha nce the association between transcription factor IRF3 and co-activator p300,Brd3 could also promotes IRF3/p300 recruitment to Ifnb gene promoter,thus promotes the transcription and production of IFN-β.Collectively,we explored the role of Brd3 in virus or LPS induced innate immune response,and investigated the underlying mechanism by which Brd3 promotes the production of IFN-β.Our work revealed the important function of Brd3 in innate immune response,and will facilitate to understand the regulatory mechanism of virus-induced immune response better.