Hepatitis B viral transactivator HBx alleviates p53-mediated repression of alpha-fetoprotein gene expression.

Chronic infection with hepatitis B virus (HBV) is associated with development of hepatocellular carcinoma (HCC). The exact mechanism by which chronic infection with HBV contributes to onset of HCC is unknown. However, previous studies have implicated the HBV transactivator protein, HBx, in progression of HCC through its ability to bind the human tumor suppressor protein, p53. In this study, we have examined the ability of HBx to modify p53 regulation of the HCC tumor marker gene, alpha-fetoprotein (AFP). By utilizing in vitro chromatin assembly of DNA templates prior to transcription analysis, we have demonstrated that HBx functionally disrupts p53-mediated repression of AFP transcription through protein-protein interaction. HBx modification of p53 gene regulation is both tissue-specific and dependent upon the p53 binding element. Our data suggest that the mechanism by which HBx alleviates p53 repression of AFP transcription is through an association with DNA-bound p53, resulting in a loss of p53 interaction with liver-specific transcriptional co-repressors.


Introduction
Chronic infection with Hepatitis B Virus (HBV) is a predominant risk factor associated with development of hepatocellular carcinoma (HCC). Multiple lines of evidence support the relationship between chronic HBV infection and HCC: geographic correlation exists between global distribution of HCC and the prevalence of HBV carrier states; a high incidence of HBV markers in blood and tissue samples is detected in HCC patients; 30% of all virally induced human tumors involve HBV infection [1,2]. Based on epidemiological studies involving chronic HBV infection, it is estimated that the relative risk of developing HCC may be between 100 to 200-fold higher for HBV carriers than for non-carriers [2,3].
The most likely scenario for HBV's role in HCC predisposition is by modification of host gene regulation [4][5][6]. Integration of viral DNA into the host genome can mediate host gene deregulation by a variety of mechanisms: integration of viral promoters can activate and/or mutate neighboring host genes [6]; integration of viral DNA encoding the HBV transactivator X protein (HBx) enhances HBx expression and subsequent interaction with cellular genes and regulatory proteins [2,7,8]. Although HBx has not been reported to bind double-stranded DNA, it can activate transcription of both viral and cellular genes through interaction with a variety of host DNA binding proteins [reviewed in 4]. HBx association with cellular transcriptional activators and general transcription and allowed to bind for five minutes at room temperature prior to addition of transcribing extract. RNA products were purified and analyzed by primer extension.
Solid-phase DNA templates for chromatin transcriptions were prepared as described [33,35]. Briefly, AFP DNA was digested with EcoRI and ClaI, then biotinylated with Biotin-21 dUTP and Biotin-14 dATP and Klenow fragment DNA polymerase (Gibco) prior to coupling to streptavidin coated, paramagnetic beads (Dynal).
In chromatin transcription reactions, p53 and HBx were added to 500 ng solid-phase DNA templates during a 20 minute pre-incubation in HepG2 or HeLa cellular extracts prior to chromatin assembly. After 1 hour chromatin assembly in fractionated Xenopus egg extract, solid-phase DNA templates were washed three times in modified nuclear dialysis buffer (mNDB) (20 mM Hepes, pH 7.9, 50 mM KCl, 0.2 mM EDTA, 10% glycerol, 1 mM DTT) plus 0.01% NP-40, then transcribed in HeLa extract and analyzed as above. p53 electromobility shift assay (EMSA). EMSA was performed using the double-stranded p53 regulatory element from the AFP distal promoter (bases -862 through -830) 5'GATCCTTAGCAAACATGTCTGGACCTCTAGAC as previously described [15], with protein-DNA binding carried out for 30 minutes at 30° C. Protein binding assays contained 7 µg of HepG2 or HeLa cell extract and approximately 1 µg purified p53 protein and 1 µg purified HBx protein, except as indicated Solid-phase DNA-protein purification. Solid-phase DNA oligomers were generated by annealing 5' biotinylated p53 regulatory element (5' Bio-GATCCTTAGCAAACATGTCTGGACCTCTAGAC) (Gibco) to complementary strand prior to coupling to streptavidin-coated paramagnetic beads (Dynal). Control reactions to assess protein binding specificity were performed in parallel with AFP site -1007 (5'Bio-GATCCAATATCCTCTTCAG) solid-phase DNA oligomers prepared in the same way.

Results
HBx disrupts p53-mediated repression of AFP transcription. In order to examine the regulatory consequences of HBx transactivator expression on a hepaticexpressed cellular gene, we performed in vitro transcription analysis of AFP DNA templates in the presence of HBx and p53 proteins. Transcription extracts isolated from the human hepatoma cell line HepG2, which actively expresses AFP but does not carry integrated HBV DNA, were used for in vitro transcription [33]. We utilized a constitutively activated p53 protein harboring a C-terminal truncation to examine the ability of p53 to regulate AFP transcription independently of post-translational modifications within the protein C-terminus, which activate p53 for DNA binding [13]. Addition of HBx without p53 resulted in modest activation (less than 2-fold) of AFP transcription ( Fig 1A, compare lanes 1 and 5), suggesting that the observed activation of AFP is dependent primarily upon HBx effects on p53, rather than HBx association with hepatoma-enriched transcriptional activators.
Effects of p53-HBx interaction are tissue-specific. AFP is expressed in the fetus by endoderm-derived cells of the yolk sac, liver and gut [39,40]. We have previously shown by cell culture transfection studies that p53 repression of AFP is tissue-  [15]. To determine if the observed effects of HBx on p53-regulated AFP expression were also tissue-specific, we performed in vitro transcription analysis in cervical cancer-derived HeLa cell extract. In contrast to the observed repression of AFP transcription following addition of p53 to HepG2 reactions, p53 introduction to HeLa transcription reactions resulted in modest activation of AFP expression (less than 2-fold) To determine if derepression of p53-regulated AFP transcription was due to a direct interaction between HBx and p53 that occurs only in a hepatoma extract, we Additionally, p53 and HBx interacted in both HeLa extract and extract buffer ( Figure 1C, lanes 6 and 8), demonstrating that p53 and HBx proteins form a stable complex in the absence of DNA or additional proteins present in hepatoma cell extracts. Taken together, these data demonstrate that the tissue-specific effects of HBx on p53-regulated AFP transcription are not due to an inability of the proteins to form a stable complex in a nonhepatic cell extract, but rather are likely due to tissue-specificity of p53 transcriptional repression.
HBx does not disrupt p53-mediated squelching of transcription. The ability of p53 to repress in vitro transcription of AFP templates could be explained by a number of mechanisms. Multiple regions of p53 protein interact with and bind a wide range of proteins mediating, in part, the pleiotropic functions of the tumor suppressor [13,14]. p53 can interact with GTF's including TFIID subunits TBP, TAF 31 and TAF70, and TFIIH subunits p62, XPB and XPD. The ability of p53 to squelch transcription through interactions with GTFs, particularly TPB, is well documented [41][42][43]. If p53-mediated AFP repression was due in part to p53 squelching of TBP in the hepatoma extract, the apparent derepression upon HBx addition could be due to HBx disruption of p53-TBP or p53-GTF interactions.
To determine if HBx could reverse p53-mediated transcriptional squelching, in vitro transcription analysis was performed using chick β-globin DNA as template. β-globin DNA has no p53 binding site and is not directly regulated by p53 (data not shown). In the presence of high levels of p53 protein, apparent transcriptional repression can be attributed, most likely, to p53-mediated squelching of basal transcription factors.
As demonstrated in Figure  Addition of HBx in the absence of p53 resulted in approximately 2-fold repression of transcription, potentially due to HBx-mediated squelching, as discussed above (Fig 2, compare lanes 1 and 7). Because HBx reactivation of p53-squelched β-globin transcription was much lower than the reactivation of p53-repressed AFP in vitro transcription, we suspected that HBx alleviation of p53-mediated AFP repression was not due simply to reversal of p53 squelching. This inability of HBx to activate transcription from a promoter lacking a p53-binding site suggested that HBx must be targeted to DNA by promoter-bound p53 in order to render its activating effects.
HBx disrupts p53 regulation of AFP chromatin transcription. In order to demonstrate conclusively that HBx-mediated derepression of AFP expression in our in vitro transcription system was not an effect on squelching, we utilized in vitro chromatin assembly of AFP DNA templates prior to transcription analysis. Solid-phase AFP DNA templates were prepared by coupling biotinylated AFP DNA to paramagnetic beads as previously described [44]. Chromatin assembly was achieved by incubating the solidphase DNA templates in fractionated Xenopus egg extracts [33]. In this system DNA templates are pre-incubated with cell extracts to allow activating and/or repressive Interestingly, addition of HBx to HeLa pre-incubation in the absence of p53 resulted in 11-fold activation over HeLa alone (compare lanes 3 and 8), suggesting that, in the absence of p53, HBx may associate with a non-hepatic derived DNA binding protein in order to activate AFP expression by a chromatin-dependent mechanism. This possibility is currently under investigation in our laboratory.
Thus far, our data demonstrated that HBx interference with p53-mediated regulation of transcription was both hepatoma-specific and required targeting by DNAbound p53. Therefore, we hypothesized that HBx might be interacting with DNA-bound p53 to potentially dislodge putative tissue specific co-repressors of transcription. An association with DNA-bound p53 in a non-hepatic derived cell extract, where p53 acts in the absence of putative co-repressors as an activator of AFP transcription, would then allow augmentation of activation.
HBx associates with DNA-bound p53. In order to assess the effect of HBx on p53 DNA binding and to determine if HBx can associate with DNA-bound p53, we performed a series of electromobility shift assays (EMSA) with both purified proteins and cellular extracts. In examining the ability of purified p53 to bind to its AFP regulatory  (Fig 6A, lanes 3-8), signaling formation of a potential p53-HBx complex, in addition to an enhancement of p53 association with the AFP regulatory element (lanes 7 and 8). HBx did not associate with the p53 regulatory element in the absence of p53 (lane 9).
To assess the possibility of a tissue-specific effect by HBx on p53 DNA binding in hepatic and non-hepatic derived cell extracts, we performed EMSA with the p53 regulatory element from the AFP distal promoter with both HepG2 and HeLa transcription extracts in the presence and absence of p53 and HBx (Fig 6B). Because it appeared that HBx-p53 interaction did not modify p53's ability to bind DNA in either the hepatic or non-hepatic cell extract, we wanted to determine if tissuespecific co-factors binding with p53 to its regulatory site could be affected by HBx addition. To this end, we performed solid-phase DNA-protein pull downs. Incubation of biotinylated, p53 regulatory element oligomers coupled to streptavidin paramagnetic beads (solid-phase DNA) with purified p53 protein in the absence (Fig 6C, lane 1) or presence of HBx (lanes 2 and 3) resulted in p53 association with the DNA template, as evidenced by silver stain (Fig 6C) and western blot (data not shown). Additionally, HBx co-purified with DNA bound p53 (lanes 2 and 3), confirming that HBx is capable of associating with DNA-bound p53. p53 DNA binding was also detected upon incubation of the solid-phase p53 binding element with HepG2 or HeLa extract in the absence (lanes 4 and 6) or presence (lanes 5 and 7) of HBx. Also, as with the purified proteins, HBx was found to co-purify with DNA bound p53 in both the HepG2 (lane 5) and HeLa (lane 7) cell extracts. Two hepatoma-specific differences were revealed in these analyses. One is that p53 incubated in HepG2 appeared to be post-translationally modified, generating a slower migrating band in lanes 4 and 5. The second difference is two proteins copurifying with p53 from the HepG2 extract were greatly reduced upon addition of HBx To serve as control, p53, HBx and extract were incubated with a non-specific oligo (-1007) lacking any defined p53 binding element. p53 and HBx association with the -1007 oligo could not be detected by silver stain (Fig 6C, lanes 8 and 9), demonstrating that p53 and HBx association with the p53 regulatory binding element is specific, and dependent upon p53 DNA binding. We have previously characterized an overlapping p53/HNF-3 binding site within the developmental repressor region that mediates opposing regulatory signals. HNF-3 is a potent activator of AFP transcription, while p53 acts to repress AFP transcription [15,33].
Aberrant activation of AFP is a hallmark of hepatocellular carcinoma, and exemplifies a loss in regulated gene expression that is common to numerous cancers.
The exact mechanism by which AFP is reactivated in the diseased liver is unknown, but likely is due to transcriptional activator and/or repressor dysfunction. One such transcription factor, p53, which is mutated or modified in over 60% of human cancers [13,54], is a target of the Hepatitis B Virus-encoded X protein. HBx binding to p53 likely evolved as a consequence of p53-mediated disruption of HBV replication in hepatic cells [62][63][64]. In conjunction with a complex of liver specific co-factors, p53 can bind and repress activation from HBV Enhancer II, the enhancer responsible for the tissue-specific replication of the virus. The p53-containing complex acts on HBV Enhancer II to block transcription from promoters under its control, resulting in loss of active HBV replication. HBx expression overcomes this hindrance by HBx binding to p53, decreasing the negative effects of the p53-containing complex on Enhancer II [65]. The exact mechanism by which HBx overcomes p53mediated repression of HBV replication has not been established, but could be due to HBx disruption of p53 association with liver-specific cofactors, as we believe to be the case with derepression of AFP transcription. losing the ability to silence AFP have a disease phenotype, it is likely that the mechanism by which AFP is re-activated is one way that HBV contributes to the development and progression of HCC. Our demonstration that HBx can derepress AFP through its interaction with p53 lends additional support to the hypothesis that HBx is the primary factor by which HBV contributes to the development of HCC, and that its interaction with p53 is an integral step in the process.
In conclusion, we have shown that HBx overcomes p53-mediated repression of a liver-specific, tumor marker gene by disrupting DNA-bound p53 interaction with potential liver-specific co-repressors. Modification of p53 interaction or communication with protein partners by HBx may be a global mechanism affecting the ability of p53 to regulate multiple genes, potentially contributing to development of HCC in infected individuals. Future studies will include examination of p53 protein partners and additional liver-specific transcriptional repressors and activators of AFP that may contribute to development of HBV-associated HCC.       70 µg, lanes 6, 7 and 9). Binding reactions were allowed to proceed for 30 minutes at 22°C . Complexes were eluted and analyzed by SDS-PAGE and silver stain. p53, HBx and putative co-factors are indicated (asterisk denotes putative p53 co-repressor).