M. Levrero

Hepatitis B virus X gene product acts as a transactivator in vivo

It has previously been shown that the hepatitis B virus X gene product, pX, transactivates homologous and heterologous transcriptional regulatory sequences of viruses and various cellular genes in vitro. However, there is no evidence about the reproducibility and the relevance of this phenomenon in vivo. In this study we crossbred transgenic mice expressing the X gene under the control of the human antithrombin III (ATIII) gene regulatory sequences with transgenics carrying either the chloramphenicol acetyl-transferase or the LacZ bacterial reporter genes driven by the HIV1-LTR, which is known to be activated in trans by pX. Expression of pX in the liver stimulates the HIV1-LTR driven expression of both chloramphenicol acetyl-transferase and beta-galactosidase reporter genes in double transgenic mice. No detectable increase in chloramphenicol acetyl-transferase expression was observed in tissues, such as the spleen, brain and heart, that do not express pX. Our results confirm the transactivating properties of pX in vivo for the first time and support the hypothesis that pX might indeed modify gene expression in HBV-infected hepatocytes and influence viral pathogenesis.

Characterization of the hepatitis B virus preS/S region encoded transcriptional transactivator

A transactivating function generated by carboxy-terminal truncation of the HBV envelope proteins has been recently described. To characterize the preS/S protein domains responsible for transactivation, preS1/S2/S and preS2/S 3 deletion mutants under the control of the adenoviral major late promoter were tested for their transactivating potential in cotransfection experiments using the c-myc and c-fos regulatory sequences as targets. Deletion of the carboxyterminal hydrophobic domain of the S protein and the presence of the endoplasmic reticulum insertion signal I (ER signal I) are required for the generation of the preS/S transactivating function. Multiple transcription factors binding sites (i.e., TRE, SRE, and NFkB sites) mediated the truncated preS/S-induced activation of the target regulatory sequences. The transactivation phenomenon is linked, at least in part, to the protein kinase C signaling pathway.

The hepatitis B virus X protein transactivation of c-fos and c-myc proto-oncogenes is mediated by multiple transcription factors.

We have constructed two expression vectors in order to study the action of the HBV 17 Kd X protein on the c-fos and c-myc promoters. The results show that the promoters contain multiple elements that respond to X protein, suggesting involvement of multiple transcription factors. The exact mechanism of the interaction remains elusive, but our data allow speculation about the factors that may be influenced.

The hepatitis B virus X gene product transactivates the HIV-LTR in vivo

It has previously been shown that the hepatitis B virus (HBV) X gene product, HBx, transactivates homologous and heterologous transcriptional regulatory sequences of viruses, including the human immunodeficiency virus type 1 (HIV1) long terminal repeat (LTR), and various cellular genes in vitro. To evaluate the transactivating function of HBx in vivo, we generated transgenic mice carrying the X open reading frame under the control of the human antithrombin III (ATIII) gene regulatory sequences. These mice express the 16 Kd HBx protein in the liver, as demonstrated by immunoprecipitation studies. Crossbreeding of HBx mice with transgenics carrying either the chloramphenicol acetyl transferase (CAT) bacterial or the lacZ reporter gene driven by the HIV1-LTR allowed us to demonstrate, for the first time, the in vivo transactivating function of HBx protein.

1121 MIR-224 IS A DIRECT TARGET OF HBX AND MODULATES HBV REPLICATION

Background: miR224 is frequently up-regulated in human HCCs, peri-tumoral cirrhotic tissues and cirrhotic livers without HCCs. We have recently shown that miR224 expression in the liver is induced by the NFkB-dependent inflammatory pathways (i.e. LTa, and TNFa) that are activated in a large proportion of chronic viral hepatitis, cirrhosis and HCC patients. Differently from other HCCrelated miRNAs that are over-expressed in several other cancers and are considered as bona fide onco-miRs (i.e. miR-21, miR-221, miR-222), miR-224 is apparently more HCC specific, suggesting a possible role of miR224 in liver physiopathology and/or chronic hepatitis infection before HCC development. Aim: To characterize the transcriptional regulation of miR224 by HBx and its effects on HBV replication. Methods: In vivo recruitment of HBx, transcription factors and chromatin remodeling enzymes was analyzed in ChIP-Seq and conventional ChIP experiments. miR224(pr) luciferase reporter constructs were generated and used to assess the ability of HBx to modulate miR224 expression. Endogenous miR224 expression and the impact of miR224 on HBV replication were assessed in HBV replicating HepG2 cells. Results: In a genome wide search of HBx cellular targets by ChIP-Seq we found that HBx protein binds in vivo to the miR224 regulatory region. HBx binding is accompanied by the co-recruitment of p65/NFkB, a loss of Pol II occupancy, the recruitment of the DNMT3a methyltransferase and reduced H4 histone acetylation. Accordingly, miR224 levels were reduced in HBV replicating cells and HBx repressed the miR224(pr) in luciferase reporter assays. pre-miR224 overexpression resulted in reduced HBV pgRNA levels and a 50% reduction in HBV replication. In silico analysis revealed the presence of several miR224 seed sequences on the HBV genome that were conserved across HBV genotypes, suggesting a direct effect of miR224 on the HBV pgRNA. Conclusions: Our results identify a functional regulatory loop between HBx, miR224 and HBV replication where HBx repression of miR224 expression relieves the negative effects of miR224 on HBV replication. These results are compatible with the low HBV replication observed in HCC tissues and support the hypothesis that the loss of the described regulatory loop might occur at the time of transformation or HCC progression.

Truncated pre-S/S proteins transactivate multiple target sequences

In order to investigate the transactivational function of HBV truncated preS/S proteins we have constructed two sets of plasmids and have tested their transactivational potential on the c-myc regulatory sequences and the TPA-responsive element. We found that preS/S proteins only become transactivationally active when truncated at the carboxy terminal end. Furthermore, using immunofluorescence microscopy we determined that the proteins are located exclusively in the cytoplasm, apparently ruling out DNA binding and activation of factors in the nucleus.

545 THE P53-PARALOG DNP73 ONCOGENE IS REPRESSED BY α-IFN/STAT2 THROUGH THE RECRUITMENT OF YY1 AND HDAC1 TRANSCRIPTIONAL REPRESSORS

ackground. Expression of the p53-paralog DNp73 oncoene increases progressively in chronic hepatitis, cirrhosis nd HCC and greatly contributes to the chemoresistant henotype of HCC cells. DNp73 isoforms do not activate ranscription and do not induce apoptosis but act as domiant negative inhibitors of both p53 and TAp73. DNp73s are xpressed from the intragenic P2p73 promoter and we have ecently shown that its activity is positively regulated in heptocytes by beta-catenin and p65/NFkB. In silico analisys of he P2p73 promoter indicates that it contains two conserved SRE elements. im. To characterize the effects of -interferon on DNp73 xpression and to evaluate the functional consequences on ell proliferation and viability. ethods. Chromatin was immunoprecipatated from ntreated and IFN treated Huh7 cells with anti-AcH3, nti-STAT2, anti-P-STAT2, anti-HDAC1, anti-p300, antiY1 and anti-Pol II antibodies. DNp73 mRNA levels were uantitated real-time PCR. esults. ChIP analysis showed that both ISRE sites are ound in vivo by Stat2 and by its phosphorilated form after h of IFN treatment. Endogenous DNp73 mRNA levels re downregulated by IFN . Immunoblot analysis showed a trong DNp73 protein downregulation after IFN-treatment. hIP analysis showed that, before the IFN treatment, H3 ysines are acetylated and p300 and Pol II are bound to the 2p73 promoter, mirroring active transcription, whereas after h treatment (when the ISGF3 complex is recruited), Pol II nd p300 binding is lost whereas HDAC1, a well-known trancription cofactor related to transcriptional repression, and he YY1 co-repressor are actively recruited. These results ndicate that the recruitment of the ISGF3 complex to the 2p73promoter has a repressive activity and suggest that this h o a a isease 41 (2009) A1–A19