| Background and AimsHuman hepatitis B virus(HBV),a member of Hepadnaviridae family,is the pathogen of hepatitis B.About 2 billion individuals worldwide have been infected by HBV in the past,among which 257,000,000 suffer from chronic infection,causing approximately 600,000 deaths per year.Serological evidence shows that in China,93 million people are HBsAg carriers,and 30 million of them are persistently infected.Chronic hepatitis B has become one of the major causes of hepatocellular carcinoma(HCC).As one of the top five malignancies in terms of incidence,HCC is delitescent and difficult to diagnose at the beginning,and often progresses beyond the best point of treatment when typical manifestations occur.Therefore,the control of chronic HBV infection is crucial for the prevention of HCC.Replication is one of the most important processes in the life cycle of HBV,which also tightly links the persistence of HBV infection and the pathogenesis of HCC.HBV genome is 3.2kb partial double-strand DNA with a tightly arranged and highly overlapped gene structure,composed of structural and regulatory sequences.After entering host hepatocytes mediated by sodium taurocholate cotransporting polypeptide(NTCP),the relaxed circular DNA(rcDNA)genome is released to the nucleus and further changed to covalently closed circular DNA(cccDNA).The cccDNA performs as the template for transcription,producing pregenomic RNA(pgRNA)and multiple subgenomic RNA,which could be reversely transcribed to produce new viral genomes.As the only template of viral replication,HBV cccDNA can be further bound by multiple histones and non-histones,forming a supercoiled minichromosome.HBV cccDNA is stable and hard to degrade,and can be continuously supplemented by the reverse transcription of pgRNA,thus,stabilizing the intracellular cccDNA pool and becoming the major obstacle in the clearance of persistent infection.Since HBV and HCC are strongly correlated,replication is the central process of viral life cycle,and transcriptional products are the important intermediate within it,viral replication and transcription are under complicated regulation of host transcription factors,therefore,finding the factors which suppress the transcription of HBV may provide more insights into prevention of HBV infection and related diseases.Zinc finger and homeoboxes 2(ZHX2)was first identified in mice and then cloned in human in 2003.ZHX2 can interact with nuclear factor Y(NF-Y)and inhibit its functions as a transcriptional activator.In addition,ZHX2 decreases the expression of some HCC markers,such as AFP,GPC3,and H19,and inhibits expression of cell cycle related genes as well as multi-drug resistant genes,reducing the proliferation and drug resistance of HCC cells.Therefore,ZHX2 works as a new HCC tumor suppressor.Our previous data showed that the nuclear expression levels of ZHX2 vary in patients with different levels of HBV core antigen(HBcAg),indicating ZHX2 may be related to the activities of HBV.Hiroko et al found that HBV preS2 promoter contains the CCAAT box which can be bound by HBV preS2 promoter.However,the roles of ZHX2 in the regulation of HBV remain completely unknown.In this study,we aimed to investigate the roles and mechanisms of ZHX2 in the regulation of HBV replication and transcription.In the first part,in order to explore whether ZHX2 could influence viral behaviors,we conducted a clinical study analyzing the relationship of nuclear ZHX2 expression and HBV infection in liver tissue and blood samples,and then,using in vitro and in vivo HBV models,we further confirmed what we have found by overexpressing or knocking down ZHX2.In addition,we also analyzed the domains important for the antiviral function in ZHX2.In the second part,we clarified the mechanisms of this inhibition on HBV by ZHX2.We demonstrated that ZHX2,as a transcriptional repressor,regulates the expression of HBV not only via a classical non-epigenetic manner,but also through the epigenetic regulations,which,therefore,will provide new information on the roles of ZHX2 in HBV related diseases and CHB therapy.Methods and ResultsPart 1 The roles of ZHX2 in the regulation of HBV replication and expression1.Analysis of the relationship between nuclear expression of ZHX2 and HBV activity in human liver tissues.In order to investigate the relationship between ZHX2 and HBV expression,eighty liver tissue samples of adjacent non-tumor areas from HCC surgical specimens were enrolled and stained using immunohistochemistry.The nuclear expression of ZHX2 and intracellular HBcAg were analyzed.The HBV related index in serum,such as HBV e antigen(HBeAg)and HBV DNA were collected.Then we analyzed the relationship between ZHX2 and HBV activity,and compared the difference between HBV active and inactive groups.Using Chi-square test and non-parametric test,we found that both the positive rate and mean density of ZHX2 nuclear expression staining were significant lower in HBV active group in comparison with the other group,indicating that ZHX2 might influence the HBV activity.We further analyzed the relationship between ZHX2 and HBV antigens including HBcAg and HBeAg,both of which are related to HBV replication capacity.The result showed that those with high nuclear ZHX2 expression had lower intracellular HBcAg expression,and nuclear expression of ZHX2 showed negative correlation with levels of serum HBeAg,indicating ZHX2 could affect antigen production of HBV.In order to analyze the relationship between ZHX2 and viral load,we divided the tissue samples into HBV DNA positive and negative groups,and we found that nuclear ZHX2 was significantly lower in HBV DNA positive ones compared with negative ones.This further confirmed that ZHX2 is related to HBV replication.2.ZHX2 inhibits HBV transcription,replication and virion formation in vitro.Since gene expression of HBV is under a precise regulation,and the transcription product,3.5kb pgRNA,not only performs as the messenger for translation of viral protein,but also serves as template in the synthesis of new viral genome,pgRNA is a crucial intermediate in both transcription and replication,linking transcription and replication tightly.To investigate roles of ZHX2 in this process in vitro,we did ZHX2 overexpression or knockdown using pcHBV1.1 transfection cell model,HepG2.2.15 cell line which integrated 4 copies of HBV genome,prcccDNA/Cre transfection system which takes the advantage of high cccDNA production in hepatocytes,and infection model in HCC cell line with stable expression of NTCP which can imitate the whole life course of HBV,and then,HBV related indexes were detected 48h post transfection.1)ZHX2 decreases HBV antigen production and secretion in vitroWe analyzed the levels of HBsAg and HBeAg in the culture supernatant using ELISA,the results showed that overexpression of ZHX2 significantly reduced HBsAg and HBeAg,while knockdown of ZHX2 increased both of them.Further,by using immunofluorescence,we compared the intracellular HBcAg levels between cells with and without ZHX2 overexpression.As expected,ZHX2 could also inhibit the expression of HBcAg.2)ZHX2 trancriptionally inhibits the expression of replication intermediate pgRNA and multiple HBV RNAsIn order to investigate whether ZHX2 inhibited HBV replication and antigen production through transcriptional regulation,we then extracted the cellular RNA and analyzed levels of HBV 3.5kb pgRNA in all of the cell models,and the results showed that 3.5kb RNA levels were significantly inhibited by ZHX2 overexpression and increased by ZHX2 knockdown.Northern blots further confirmed that ZHX2 transcriptionally suppressed the subgenomic mRNAs of HBV.3)ZHX2 inhibits HBV cccDNA replication and virion formationIn order to further confirm the roles of ZHX2 in the regulation of HBV replication,we detected the HBV core particle DNA in the culture supernatant after overexpressing or knocking down ZHX2.The results showed that ZHX2 overexpression significantly reduced HBV DNA level in culture supernatant,and ZHX2 knockdown increased HBV DNA.Furthermore,to observe the formation of virion directly,we conducted transmission electron microscopy in HepG2.2.15 cells or HepG2-NTCP cells with or without ZHX2 knockdown,reconfirming ZHX2 inhibited the replication of HBV,thus decreased the formation and secretion of newly synthesized virion.3.ZHX2 suppresses HBV antigen production,replication and transcription in vivo.1)ZHX2 decreases antigen production in HBV Tg miceTo further determine whether ZHX2 could also suppress HBV expression in vivo,we performed overexpression of ZHX2 using hydrodynamic injection(HDI)in BALB/c HBV Tg mice,compared the HBsAg levels pre-and post injection and calculated the inhibition rate.The result showed that the HBsAg inhibition rate was sigficantly higher in ZHX2 overexpressing mice than in control ones,indicating ZHX2 can also suppress HBV in vivo.2)ZHX2 inhibits HBV replication and expression in the acute infection modelAcute HBV infection models were constructed and overexpression of ZHX2 was performed in C57BL/6 mice by hydrodynamic injection(HDI)of prcccDNA/Cre system together with ZHX2 overexpressing plasmids,blood and liver tissues were collected 4 days post injection.We found that serum HBsAg,HBeAg,and HBV DNA,as well as intracellular 3.5kb pgRNA and HBcAg,were significantly lower in ZHX2 overexpressing mice.3)ZHX2 suppresses HBV replication and expression in the chronic infection modelpAAV-HBV1.2 plasmids and ZHX2 knockdown/NC lentivirus were co-injected into C57BL/6 mice,blood was collected 4 days and 4 weeks post injection.In the forth week,mice were sacrificed and liver tissues were collected.The results showed that knockdown of ZHX2 decelerated the clearance of HBV,reducing the inhibition rates of HBsAg and HBeAg,and increasing the intracellular HBcAg and 3.5kb pgRNA levels.All the results above support that ZHX2 can suppress HBV transcription and antigen production,decreasing HBV replication and viral formation in vivo.4.The inhibition effects of ZHX2 depend on its nuclear localization.To further examine the crucial domain which determine the inhibition effects of ZHX2,we constructed various truncated vectors containing different domains,and co-transfected them together with prcccDNA/Cre system into cells.Then changes in the capacity of antigen secretion were examined.All of the ZHX2 segments with nuclear localization signal(NLS)showed obvious inhibition on HBV expression,the functional domain located at aa242?446,which included the NLS region.And depletion of NLS impaired the suppressive effects of ZHX2,indicating that nuclear localization of ZHX2 is essential for its inhibitory effects on HBV.Part 2 ZHX2 transcriptionally suppresses HBV expression via epigenetic and non-epigenetic manners1.ZHX2 binds to HBV cccDNA minichromosomeZHX2 is considered to be a transcriptional repressor,and through interacting with the promoter regions of target genes,ZHX2 regulates the expression of multiple genes.To determine whether ZHX2 could interact with HBV cccDNA,we transfected pcZHX2-HA vector into Huh7 prcccDNA/Cre cells,performed ChIP assays using anti-HA antibody and did PCR using rcccDNA specific primers.The results showed that ZHX2 could bind to cccDNA minichromosome.2.ZHX2 inhibits the activities of HBV promotersTo investigate whether ZHX2 performs its functions by directing to HBV promoters,we constructed reporter vectors of all the four HBV promoters and transfected them into HCC cell lines.Then we focused on the three with relative high activities and did cotransfection with ZHX2 overexpression or knockdown.Dual luciferase assays were used.ZHX2 overexpression could significantly reduce the activities of core promoter(CP),preS2 promoter(SPII),and X promoter(XP),while knockdown enhanced them.3.ZHX2 regulates HBV promoters in NF-Y/CCAAT dependent or independent mannersSince ZHX2 is demonstrated to perform its functions through NF-Y which binds to the CCAAT boxes on promoters,we predicted whether there were NF-Y binding sites within HBV promoters.Software prediction results showed the existence of NF-Y binding sites CCAAT boxes in the regions of CP,SPII and XP.Then in order to investigate whether NF-Y works in the ZHX2 induced HBV inhibition,we knocked down NF-YA and observed the changes of ZHX2 effects.We found that the inhibition effects of ZHX2 were impaired when NF-YA was knocked down for SPII,while for XP,those effects were still significant,indicating the underneath mechanisms might vary in different HBV promoters.In addition,we also mutated or truncated NF-Y binding sites in relative promoters and further confirmed our findings.And when the predicted sites were mutated in CP and SPII,the inhibition of ZHX2 on HBV disappeared.However,when the XP were truncated to a segment with no NF-Y binding site,ZHX2 could still suppress its activity,indicating that ZHX2 inhibited XP in a NF-Y/CCAAT independent way.In summary,our results showed that ZHX2 inhibits multiple HBV promoters in HBV through various mechanisms,and decreases the replication and biosynthesis of HBV transcriptionally.4.ZHX2 decreases the active epigenetic modifications on cccDNAAccumulating studies have reported that apart from the classical mechanisms,transcription factors can also regulate the post-translational modifications,change the structure of chromosome,and influence the gene expression.Our data showed that ZHX2 could bind to cccDNA minichromosome,thus,we wonder if this bind also means some epigenetic regulation between ZHX2 and HBV cccDNA.To investigate whether ZHX2 changes the post-translational modifications of histones on HBV cccDNA minichromosome,we performed ChIP assays in Huh7 prcccDNA/Cre cells after overexpressing or knocking down ZHX2,and detected some active markers abundant on cccDNA,such as H3K4me3,H3K27ac,and H3K122ac.The results showed that ZHX2 significantly decreased the tri-methylation level of H3K4 as well as the acetylation levels of H3K27 and H3K122,providing another explanation of its inhibition on HBV.5.ZHX2 targets CBP,p300,and SETD1B,and participates in the epigenetic modifications.In order to further explain how ZHX2 influences HBV epigenetic regulation,we analyzed our mRNA microarray and ChIP-on-chip data,and some target genes related to epigenetic modification were found.Then qRT-PCR and western blots were performed after over-expressing or knocking down ZHX2 to further confirm the findings.The results showed that ZHX2 significantly decreased CBP,p300,and SETD1B expression,which are crucial in acetylation or methylation of histones,and these gave rise to the transcriptional inhibition on HBV.6.ZHX2 interacts with acetyl-transferases p300/CBP and decreases its binding on cccDNA,thus downregulates the transcriptional activity of HBV.To verify whether ZHX2 can interact with p300/CBP and changes the binding of it,we performed co-IP and ChIP assays.The results of co-IP showed that ZHX2 could interact with p300,and this binding did not require the presence of HBV cccDNA.And ChIP assay showed that ZHX2 could significantly reduce the cccDNA bound p300/CBP.Also,we used C646,a small molecular inhibitor of p300,to see if the inhibition of ZHX2 on HBV was,at least partially,mediated by p300/CBP.C646 was added to cells with ZHX2 knocked down,and levels of 3.5kb pgRNA were detected.The increase of HBV expression induced by knockdown of ZHX2 was impaired by C646,indicating p300/CBP is one of the main mechanism in the inhibition on HBV by ZHX2.Conclusion1.Tumor suppressor ZHX2 restricts HBV replication on transcription level.ZHX2 negatively correlates with HBV activity in patients,and suppresses the transcription and replication of HBV both in vitro and in vivo.And this transcriptional inhibition effect depends on the nuclear localization of ZHX2.2.ZHX2 suppresses HBV promoter activities through a non-epigenetic way.ZHX2 binds to the promoters of HBV cccDNA,and inhibits the activities of HBV CP,SPII,and XP,suppresses the activities of HBV CP and SPII in a NF-Y/CCAAT dependent manner and inhibits XP activity independent of the NF-Y/CCAAT element.3.ZHX2 performs in the epigenetic inhibition of HBV.ZHX2 decreases the active epigenetic modifications on cccDNA,including H3K27ac,H3K122ac,and H3K4me3 by directly targeting the expression of multiple epigenetic regulators such as p300,CBP,and SETD1B.In addition,ZHX2 interacts with p300/CBP,decreases its binding on cccDNA,and inhibits the epigenetic activation of cccDNA transcription.In summary,this study demonstrates that ZHX2 suppresses the replication,transcription,antigen secretion and virion formation of HBV in epigenetic and non-epigenetic ways,therefore,provides new information for the genetic therapy of CHB.Significance and InnovationThis study,for the first time,demonstrates that ZHX2,a transcriptional repressor and tumor suppressor abundantly expressed in hepatocytes,can regulate the transcription and replication of HBV.Our results clarify that the tumor suppressive function of ZHX2 represents not only in the direct regulation on cell cycle related genes,but also in the upstream of HCC pathogenesis,by blocking HBV replication and biosynthesis,and by playing important roles in earlier stages of 'CHB-liver cirrhosis-HCC' trilogy.In addition,this study explains the mechanisms of the HBV inhibition by ZHX2,which broadens our knowledge in the functions of ZHX2,and therefore,provides more insights into CHB and related diseases. |
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