Functional Characterization Of Id1 And E2F4 On HBV Transcription And Replication

Objective:Hepatitis B virus?HBV?is a partial double-stranded DNA virus of the small viral genome.Even if benefiting from hepatitis B vaccines and antiviral drugs,Chronic hepatitis B caused by HBV is still a global public health problem.More than 200 million people still have chronic hepatitis B,and the long-term development of hepatitis B will lead to the occurrence of liver cirrhosis and hepatocellular carcinoma?HCC?,which poses a serious threat to human health.Id1 protein,a member of Inhibitor of DNA binding and/or differentiation?Id1-4?,belongs to the bHLH transcription factor family.Due to absence of basic region of binding to DNA,Id1 protein forms heterodimers by binding to other bHLH transcription factors,thus inhibiting the regulation of downstream target genes by bHLH transcription factors.The function of Id1 is mainly related to promoting cell proliferation and inhibiting differentiation.Many genes involved in the regulation of cell proliferation are also participated in the regulation of HBV expression.In addition,E2F transcription factor 4 also belongs to the Helix superfamily and have been reported to interact with Id2.However,the role of E2F4 in HBV replication regulation has not been reported.Therefore,our study focused on the regulatory role and molecular mechanism of host factor Id1 and its'interacting transcription factor E2F4in modulating HBV replication and transcription.The research results further enriched the regulatory network of host factor regulating HBV and contributed to developing new antiviral drugs based on this mechanism.Methods:1.In clinical samples of HBV-related HCC,qRT-PCR and western blot were used to analyze the HBV pgRNA levels and the expression levels of Id1 and HBV core proteins in cancer tissue and adjacent cancer tissue.2.Both at the cellular level and animal level,western blot experiments were also used to compare the expression levels of Id1 in HBV replication-positive and HBV replication-negative cells and HBV-transgenic and non-transgenic mice.3.Analysing the influence of four HBV proteins?HBc/p/s/x?on Id1and the effect of HBV on Id1 in the HepG2-NTCP infection model by western blot.4.Overexpression of Id1 was achieved in HBV replication cells?HepG2.2.15,HepAD38 and HepG2-HBV1.1?by infecting with Id1recombinant adenovirus?Id1Ad?.The levels of HBV DNA,HBeAg and HBc were detected by Southern blot,ELISA and western blot,respectively.The changes of HBV cccDNA and pgRNA in HepG2.2.15 cells were detected by qRT-PCR.5.The HBV-infected HepG2-NTCP cell model was transfected with pCDNA3.1-Id,and the changes of HBV DNA level were detected by qRT-PCR.6.The shRNA1/2 targeting-Id1 was used to reduce the Id1 level in HepG2.2.15 and HepG2-HBV1.1 cells,subsequently the HBV DNA,HBeAg and HBc levels were detected by Southern blot assay,ELISA and western blot assay,respectively.7.HBV-TgM was injected with Id1Ad by tail vein,and Id1 was overexpressed in vivo in mice.The changes of HBV DNA in liver tissues of mice were also detected by Southern blot and qRT-PCR,the changes of Id1 and HBc proteins in liver cells were detected by western blot and IHC,and the changes of HBeAg in serum of mice were detected by ELISA.8.The effects of Id1 overexpression on the classical host factors of regulating HBV were screened by qRT-PCR and western blot.9.qRT-PCR was used to screen for HLH transcription factors with altered mRNA levels in HBV-replicating cells.10.The overexpression plasmids of potential HLH factors were constructed,and their influence on HBV Cp promoter was screened.In particular,E2F4 played a significant role in promoting the activity of Cp promoter.11.Further studies compared the expression of E2F4 in HepG2.2.15,HepAD38 and HepG2-HBV1.1 cells replicating HBV and 25 pairs of clinical samples in cancer tissues and adjacent tissues.12.E2F4Ad was used to infect HepG2.2.15 and HepG2-NTCP cells to overexpress E2F4,and qRT-PCR was used to detect the changes of HBV DNA and pgRNA.13.The interaction between E2F4 and Id1 was detected by immunofluorescence assay,immunoco-precipitation assay and GST-pulldown assay.14.Cytoplasmic and nuclear protein separation were followed by western blot assay for detecting the effects of HBV replication and Id1 on E2F4 distribution in cells.15 After HepG2.2.15 infected with E2F4Ad and transfected with shRNA-1/2 targeting Id1,or infected with Id1Ad and transfected with shE2F4-1/2,Southern blot was used to detect the replication level of HBV DNA in HepG2.2.15.16.ChIP assay in HepG2.2.15 and HepAD38 were performed for observing the recruitment of E2F4 protein by Cp promoter and the influence of Id1 on the recruitment.17.Direct interaction between Cp promoter and E2F4 protein was detected by EMSA in vitro.18.According to the secondary structure and recognition site rule of E2F4,bioinformatics analysis was performed to find the potential recognition sites of E2F4 on the Cp promoter.The corresponding promoter variants were constructed,and further verified whether the loss of potential sites caused fail of E2F4 on HBV regulation by Lucifase activity experiment.19.SPR and ITC experiments detected the interaction of E2F4truncated protein and short DNA stretches of Cp promoter in vitro.20.The corresponding HBV 1.3-mer variants were constructed according to the potential sites,and the recruitment of E2F4 by mutated Cp promoter was detected by ChIP assay.21.HBV 1.3-mer variants were transfected into Huh7 cells.qRT-PCR was used to detect the changes of HBV DNA after the cells were infected with E2F4Ad and Id1Ad respectively.ELISA was used to detect the changes of HBeAg in the supernatant.22.Bioinformatics analysis was used to compare and analyze the conservation of the potential recognition sites of E2F4 in four common HBV genotypes?Gt A/B/C/D?,and then the universality of the regulatory effects of E2F4 and Id1 on four HBV replication was detected by southern blot and qRT-PCR.Results:1.In 25 pairs of HBV-related HCC tissue adjacent and carcinoma samples,the overall expression level of pgRNA in the adjacent cancer samples was about 2-folds than that of liver cancer tissues?P<0.05?,of which HBc protein levels were significantly higher in adjacent tissue than those in the carcinoma tissue from 20 pairs of the sample,and the protein expression level of Id1 in 18 pairs of samples was significantly higher in carcinoma tissues than those in adjacent cancers.2.Id1 mRNA and protein levels in HBV-stable replication cells and HBV-transiently transfected cells were significantly lower than that in control HCC cells without HBV replication.4.In HBV-infected cell model,the decrease of Id1 protein level was significantly depended on dose of HBV infection viral titer and time of infection.Overexpression of HBV-related proteins HBp and L-HBs also resulted in the decrease of Id1 protein level.Id1 protein levels in liver tissues of HBV-transgenic mice were also significantly lower than that of normal C57 mice.4.Overexpression of Id1 significantly inhibited the expression levels of HBV DNA replication intermediates,pgRNA and HBc in HBV replication cells?HepG2.2.15,HepAD38 and HEPG2-HBV1.1 cells?,and the level of HBeAg in cultural supernatant?P<0.01?.In addition,overexpression of Id1 significantly decreased the replication level of HBV cccDNA and Cp promoter activity in HepG2.2.15 cells?P<0.05?.5.Down-regulating the expression of Id1 significantly promoted the expression levels of HBV DNA replication intermediates and HBc in HepG2.2.15 and HepG2-HBV1.1 cells,and the secretion level of HBeAg in supernatant?P<0.05?.6.Id1 overexpression significantly inhibited HBV DNA replication and HBc expression levels in liver tissues of HBV-transgenic mice,and also resulted in a significant decrease in serum HBeAg secretion?P<0.05?.7.In HepG2.2.15 cells,overexpression of Id1 did not cause significant changes in classic HBV regulation factors,but HBV replication resulted in significant up-regulation of mRNA level of 4 transcription factors?E2F4,TCF3,E40 and USF1?and significant down-regulation of 2 transcription factors?Clock and HIF1?gene?.8.E2F4 overexpression can significantly promote HBV Cp promoter activity and up-regulate HBV pgRNA and HBV DNA replication.9.The level of E2F4 protein in the HBV-replicating cells was significantly higher than that in the control cells.In clinical samples of HBV-associated liver cancer,the global protein level of E2F4 was significantly higher in paracancer tissues than in cancer tissues?P<0.05?.10.The IP and GST-pulldown experiments showed that E2F4interacted with Id1,and the overexpression of Id1 hindered the enhancement of p130 and E2F4 nuclear translocation caused by HBV replication.11.Overexpression of E2F4 can further promote the up-regulation of HBV DNA by silencing Id1,while overexpression of Id1 does not significantly inhibit the down-regulation of HBV DNA by interfering with E2F4.12.ChIP experiment confirmed that E2F4 could be recruited by Cp promoter,and the overexpression of Id1 also prevented the recruitment of E2F4 by Cp promoter.13.In the EMSA experiment,with the increase of E2F4 truncated protein(E2F4_1-180),the migration rate of Cp promoter decreased significantly,and Cp cold probe could compete for binding to E2F4truncated protein.14.Bioinformatics analysis showed that E2F4 had a potential recognition site of 5'-1758TTAAAGGTC1766-3'on Cp,and the deletion of this site led to the disappearance of the promoting effect of E2F4 on HBV Cp promoter activity.In the corresponding HBV1.3 mutants?HBV1.3mut2.3?,both E2F4 and Id1 lost their regulatory effect on HBV.15.SPR and ITC experiments showed that Cp2 DNA short-chain containing 5'-1758TTAAAGGTC1766-3'could directly bind to the DNA-binding domain of E2F4.16.Further analysis found that 5'-1758TTAAAGGTC1766-3'was highly conserved in HBV Gt A/B/C/D genotypes,and E2F4 and Id1 were generally applicable to the regulation of these four genotypes of HBV.Conclusion:In this study,we elucidated the synergistic regulation of Id1 and its interacting transcription factor E2F4 in HBV replication and transcription.The results showed that the expression level of Id1 was significantly down-regulated and the expression level of E2F4 was significantly up-regulated in both cell and tissue levels under the background of HBV replication.E2F4 could promote HBV replication and corresponding transcription by promoting HBV Cp promoter activity.The upstream Id1 molecule inhibits intranuclear transfer of E2F4/p130 by interacting with E2F4,leading to inhibition of HBV replication and transcription.5'-1758TTAAAGGTC1766-3'is the direct recognition site of E2F4 on the Cp promoter,which is highly conserved among the four HBV A/B/C/D genotypes and lays foundation for the regulation of Id1 and E2F4on the four HBV genotypes.Our study further improved the regulatory network of host factors on HBV and provided new ideas for the development of new anti-HBV strategies.