Epigenetic Mechanisms And Methods Of Hepatitis B Virus Regulating Both Its And Host Cells' Chromatin

Hepatocellular carcinoma(HCC)is one of the most common malignant tumors all over the world.The number of new cases and deaths of HCC in China ranks first of the world.In China,hepatitis B virus(HBV)infection is the primary cause.At present,the pathogenesis mechanism of HBV is not completely clear.Chronic HBV infection and HCC caused by HBV are still challenges of public health in China.On the one hand,HBV specifically infects hepatocytes and replicates itself in hepatocytes.The covalent closed circle DNA(ccc DNA)of HBV virus is entangled with histone to form the microchromatin in host cells.ccc DNA microchromatin is a template for the transcription and replication of HBV virus.However,anti-HBV drugs such as nucleoside analogue(NA)and interferon-alpha(IFN-?)are difficult to clear or effectively inhibit the transcription of ccc DNA microchromatin.At present,we have limited understanding of the molecular mechanisms involved in the formation,maintenance and transcriptional regulation of ccc DNA microchromatin.It is necessary and urgent to further study the regulatory mechanism of HBV ccc DNA microchromatin.On the other hand,the HBV genome will integrate into the host hepatocyte genome,which will induce disorders of transcriptional and epigenetic regulation of hepatocytes through various mechanisms,thus promoting the occurrence and development of HCC.Epigenetic disorders are widespread in tumors and play an important role.However,the epigenetic mechanism of HCC caused by HBV is not thorough enough.Epigenetics refers to the inheritable regulation mechanism of gene expression without changing DNA sequence.It mainly includes DNA methylation,histone modification,nucleosome position and spatial conformation of chromatin.Its core function is to control the open or closed status of chromatin,thus translating the DNA code in time and space order according to external signal instructions.Among many epigenetic mechanisms,posttranslational modification of histone has attracted extensive research due to its importance and diversity.Histone H3 4 lysine trimethylation(H3K4me3)is a key marker of gene activation and expression,which is mainly enriched in promoters and enhancers of genes.In the past,the main method to detect H3K4me3 modification was chromatin immunoprecipitation(Ch IP),but this technology requires a large initial cell number.For some rare clinical samples,such as liver perforator specimens,it is difficult to carry out.A new technique for the detection from low starting cell number needs to be developed.Our previous studies have found that HBV can up-regulate the H3K4me3 modification of host cells at the genome-wide level,thus promoting expression of corresponding genes.Similar studies have found that H3K4me3 modification also enriches on HBV ccc DNA microchromatin,which is essential for its self transcription and replication of HBV virus.These studies suggest that H3K4me3 epigenetic modification may play an important role in both HBV life-cycle and pathogenesis of liver,but the specific mechanism needs to be further studied.WD repeat domain 5(WDR5)is a core protein in the complex of histone methylation enzymes.It has been reported to be highly expressed in many tumors and plays an oncogenic role.However,the expression level of WDR5 in HCC caused by HBV is not clear.Therefore,in the first section of this project,we focused on epigenetic histone modification of H3K4me3,studied the detailed molecular mechanism of H3K4me3 upregulation by HBV,elaborated the importance of H3K4me3 modification to host cell's carcinogenesis and HBV ccc DNA transcription,and found potential therapeutic targets and clinical applications.Meanwhile,in the second section,we focused on the methodology of single-cell epigenetics,constructed the single-cell chromatin immunocleavage sequencing(sc Ch IC-seq)technology to detect histone modification in samples with low starting cell number and the single-cell Micrococcal nuclease sequencing(scMNase-seq)technology to detect the nucleosome position and chromatin accessibility simultaneously from single cells.The research contents and results are as follows: Section 1.Upregulation of WDR5 by HBx underlies pathology of Chronic HBV infection and HCC1.WDR5 is elevated in HCC caused by chronic hepatitis B and associated withdisease prognosis.(1)The expression level of WDR5 protein in HBV infected HCC was significantly higher than that in adjacent and normal tissues.(2)The up-regulation of WDR5 was associated with HCC prognosis.(3)The expression level of wdr5 was positively correlated with that of HBx in 95 HCC species.2.WDR5 promotes the proliferation and migration of HCC.(1)WDR5 promoted the proliferation of HCC in vitro.(2)WDR5 promoted the migration of HCC in vitro.(3)WDR5 promoted tumor formation,tumor growth rate and tumor volume in nude mice.3.WDR5 promotes the transcription of HBV ccc DNA.(1)WDR5 promoted the transcription of ccc DNA in HBV-infection cell models.(2)WDR5 promoted the replication of HBV in HBV-infection cell models.4.HBV inhibits ubiquitination of WDR5 protein and stabilize WDR5 protein.(1)HBV upregulated the level of WDR5 protein rather than its m RNA.(2)HBV prolonged the half-life of WDR5 protein in HCC and HBV-infection cell models.(3)The ubiquitination level of WDR5 protein was decreased after HBV infection.5.HBx plays a direct role in ubiquitination and stabilization of WDR5 protein.(1)HBx prolonged the half-life of WDR5 protein.(2)HBx directly regulated the ubiquitination of WDR5 protein.(3)HBx upregulated the level of WDR5 protein in vivo and in vitro.6.HBx prevents WDR5 degradation by compromising the DNA damage-bindingprotein 1(DDB1)-WDR5 interaction through competitive binding of HBx withDDB1.(1)DDB1 is the catalytic enzyme for ubiquitination of WDR5 protein.(2)The binding of HBx to DDB1 is essential for the stability of WDR5 protein.(3)The binding of HBx and DDB1 upregulated the level of WDR5 protein.(4)The binding of HBx and DDB1 inhibited the ubiquitination of WDR5 protein by DDB1.(5)The binding affinity of HBx to DDB1 was stronger than that of DDB1 to WDR5.7.HBx promotes H3K4me3 modification by WDR5.(1)HBV promoted genome H3K4me3 modification.(2)WDR5 promoted genome H3K4me3 modification.(3)HBx/WDR5 promoted H3K4me3 modification of target genes promoter.(4)WDR5 promoted H3K4me3 modification of ccc DNA.8.HBx recruits WDR5 to promote H3K4me3 modification.(1)HBx and WDR5 co-localized on the promoter region of target gene.(2)HBx colocalized with WDR5 on chromatin genome-wide.(3)HBx/WDR5 co-binding peaks enriched in oncogenes and cancer signaling pathways.(4)HBx bound to wdr5 directly.(5)HBx "?-helix" motif was responsible for its interaction with WDR5.(6)HBx "?-helix" motif played critical roles for HBx to recognize the chromatin.(7)HBx "?-helix" motif played critical roles for HBx to exert its carcinogenesis effect.9.WDR5 inhibitor has therapeutic effect on HCC and ccc DNA transcription.(1)WDR5-0103,as a functional inhibitor of WDR5 protein,significantly inhibited the proliferation,migration and tumor formation of HCC cells,suggesting potential clinical application value.(2)WDR5-0103 inhibited transcription of ccc DNA.Section 2.Development of new epigenomics methods from low-cell input or single cell.1.scCh IC-seq detects histone modification from single cells.(1)We developed single-cell chromatin immunocleavage sequencing method(sc Ch ICseq)which measures the epigenetic H3K4me3 profiles from low cell inputor at a single-cell level.(2)scCh IC-seq detected the apparent heterogeneity of histone modification in PBMC.2.scMNase-seq detects nucleosome position and chromatin accessibility simultaneously from single cells.(1)To understand the principles that underline chromatin heterogeneity as related to nucleosome positioning and chromatin accessibility,we developed the scMNase-seq technique to simultaneously measure nucleosome positioning and chromatin accessibility in single cells.(2)scMNase-seq detected the heterogeneity of nucleosome position.(3)scMNase-seq discovered new principle of nucleosome positioning and spacing.(4)scMNase-seq discovered heterogeneity of nucleosome space at DHS sites.(5)scMNase-seq predicted the direction of cell differentiation.The main conclusions and significance of this study are as follows:1.HBV virus up-regulates the level of WDR5 protein,a key subunit of histone methylation enzyme of host cells,through its encoded HBx protein,and then regulates the epigenetic H3K4me3 modification of host and its own ccc DNA genome.On the one hand,the abnormally high expression of H3K4me3 resulted in the disorder of the host chromatin and the activation of oncogene expression;on the other hand,HBV maintained the open state of its ccc DNA chromatin through H3K4me3,which promoted the transcription and replication of the virus itself.2.The mechanism of HBx-WDR5-H3K4me3 axis: HBx hijacked host ubiquitinase DDB1,and through competitive binding with DDB1,antagonized the ubiquitination modification of WDR5 by DDB1,thereby preventing WDR5 from being degraded;Meanwhile,HBx interacts with WDR5 directly by its "?-helix" motif,recruits WDR5 to chromatin for the H3K4me3 modification.3.sc Ch IC-seq and scMNase-seq were developed to detect histone modification and nucleosome position from single-cells or samples with low starting cell number.sc Ch IC-seq and scMNase-seq revealed the epigenetic heterogeneity between cells,discovered new principle of epigenetic regulation and predicited cell destiny.This study reported that WDR5 is a key epigenetic determinant of HBV-induced tumorigenesis and that the HBx-WDR5-H3K4me3 axis may be a potential therapeutic target in HBV-induced liver pathogenesis.Besides,new methods detecting H3K4me3 modification from low cell input(sc Ch IC-seq)and detecting nucleosome position and chromatin accessibility from single cells(scMNase-seq)were developed.Our study emphasized the HBx-WDR5-H3K4me3 axis and provided potential targets,methods and mechanisms for clinical treatment.