Francesca Guerrieri

IFN-α inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome

HBV infection remains a leading cause of death worldwide. IFN-α inhibits viral replication in vitro and in vivo, and pegylated IFN-α is a commonly administered treatment for individuals infected with HBV. The HBV genome contains a typical IFN-stimulated response element (ISRE), but the molecular mechanisms by which IFN-α suppresses HBV replication have not been established in relevant experimental systems. Here, we show that IFN-α inhibits HBV replication by decreasing the transcription of pregenomic RNA (pgRNA) and subgenomic RNA from the HBV covalently closed circular DNA (cccDNA) minichromosome, both in cultured cells in which HBV is replicating and in mice whose livers have been repopulated with human hepatocytes and infected with HBV. Administration of IFN-α resulted in cccDNA-bound histone hypoacetylation as well as active recruitment to the cccDNA of transcriptional corepressors. IFN-α treatment also reduced binding of the STAT1 and STAT2 transcription factors to active cccDNA. The inhibitory activity of IFN-α was linked to the IRSE, as IRSE-mutant HBV transcribed less pgRNA and could not be repressed by IFN-α treatment. Our results identify a molecular mechanism whereby IFN-α mediates epigenetic repression of HBV cccDNA transcriptional activity, which may assist in the development of novel effective therapeutics.

Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function

HBV cccDNA, the template for transcription of all viral mRNAs, accumulates in the nucleus of infected cells as a stable episome organized into minichromosomes by histones and non-histone viral and cellular proteins. Using a cccDNA-specific chromatin immunoprecipitation (ChIP)-based quantitative assay, we have previously shown that transcription of the HBV minichromosome is regulated by epigenetic changes of cccDNA-bound histones and that modulation of the acetylation status of cccDNA-bound H3/H4 histones impacts on HBV replication. We now show that the cellular histone acetyltransferases CBP, p300, and PCAF/GCN5, and the histone deacetylases HDAC1 and hSirt1 are all recruited in vivo onto the cccDNA. We also found that the HBx regulatory protein produced in HBV replicating cells is recruited onto the cccDNA minichromosome, and the kinetics of HBx recruitment on the cccDNA parallels the HBV replication. As expected, an HBV mutant that does not express HBx is impaired in its replication, and exogenously expressed HBx transcomplements the replication defects. p300 recruitment is severely impaired, and cccDNA-bound histones are rapidly hypoacetylated in cells replicating the HBx mutant, whereas the recruitment of the histone deacetylases hSirt1 and HDAC1 is increased and occurs at earlier times. Finally, HBx mutant cccDNA transcribes significantly less pgRNA. Altogether our results further support the existence of a complex network of epigenetic events that influence cccDNA function and HBV replication and identify an epigenetic mechanism (i.e., to prevent cccDNA deacetylation) by which HBx controls HBV replication.

Transcriptional regulation of miR-224 upregulated in human HCCs by NFκB inflammatory pathways

BACKGROUND & AIMS miR-224 is up-regulated in human HCCs as compared to both paired peri-tumoral cirrhotic tissues and cirrhotic livers without HCC. Here, we have cloned the miR-224 regulatory region and characterized its transcriptional regulation by the NFκB-dependent inflammatory pathways. METHODS Mature miRNA expression was evaluated by a 2 step stem-loop real-time RT-PCR. The recruitment of polymerase II and transcription factors on the pre-miR-224 promoter has been assessed by ChIPSeq and ChIP. RESULTS We found miR-224 levels strongly up-regulated in both peri-tumoral cirrhotic livers and HCC samples as compared to normal livers. In silico analysis of the putative miR-224 promoter revealed multiple NFκB sites. We showed that LTα and TNFα activate transcription from the miR-224 promoter and of endogenous miR-224 expression in HCC cell lines, whereas the expression of miR-224 target API5 was reduced. Exogenously expressed p65/RelA activates the miR-224 promoter and a dominant negative form of IκBα (IκBSR) represses it. ChIP analysis showed that p65/NFκB is recruited on the miR-224 promoter and that its binding sharply increases after exposure to LPS, TNFα, and LTα. Altogether these findings link the inflammatory signals to NFκB-mediated activation of miR-224 expression. An antago-miR specific for miR-224 blocked LPS and LTα stimulated HCC cells migration and invasion. Conversely, the IKK inhibitor BMS-345541 blocks pre-miR-224-induced cellular migration and invasion. CONCLUSIONS Our results identify p65/NFκB as a direct transcriptional regulator of miR-224 expression and link miR-224 up-regulation with the activation of the LPS, LTα, and TNFα inflammatory pathways and cell migration/invasion in HCC.

Molecular Mechanisms of HBV-Associated Hepatocarcinogenesis

Hepatitis B virus (HBV) contributes to hepatocellular carcinoma (HCC) development through direct and indirect mechanisms. HBV-DNA integration into the host genome occurs at early steps of clonal tumor expansion and induces both genomic instability and direct insertional mutagenesis of diverse cancer-related genes. Prolonged expression of the viral regulatory protein HBx and the large envelope protein deregulate the cellular transcription program and proliferation control and sensitize liver cells to carcinogenic factors. Epigenetic changes targeting the expression of tumor suppressor genes occur early in the development of HCC. A major role is played by HBx that is recruited on cellular chromatin and modulates chromatin dynamics at specific gene loci. Compared with tumors associated with other risk factors, HBV-related tumors have a higher rate of chromosomal alterations and p53 inactivation by mutations, overexpress fetal liver/hepatic progenitor cells genes, and show a specific activation of the AKT pathway. The wnt/β-catenin pathway is also often activated, but HBV-related tumors display a low rate of activating β-catenin mutations. All available evidence strongly supports the notion that chronic HBV infection triggers both common and etiology-specific oncogenic pathways, thus playing a direct role beyond stimulation of host immune responses and chronic necroinflammatory liver disease.

hSirT1-dependent regulation of the PCAF-E2F1-p73 apoptotic pathway in response to DNA damage.

ABSTRACT The NAD+-dependent histone deacetylase hSirT1 regulates cell survival and stress responses by inhibiting p53-, NF-κB-, and E2F1-dependent transcription. Here we show that the hSirT1/PCAF interaction controls the E2F1/p73 apoptotic pathway. hSirT1 represses E2F1-dependent P1p73 promoter activity in untreated cells and inhibits its activation in response to DNA damage. hSirT1, PCAF, and E2F1 are corecruited in vivo on theP1p73 promoter. hSirT1 deacetylates PCAF in vitro and modulates PCAF acetylation in vivo. In cells exposed to apoptotic DNA damage, nuclear NAD+ levels decrease and inactivate hSirT1 without altering the hSirT1 interaction with PCAF and hSirT1 binding to the P1p73 promoter. The reactivation of hSirT1 by pyruvate that increases the [NAD+]/[NADH] ratio completely abolished the DNA damage-induced activation of TAp73 expression, thus linking the modulation of chromatin-bound hSirT1 deacetylase activity by the intracellular redox state with P1p73 promoter activity. The release of PCAF from hSirT1 repression favors the assembly of transcriptionally active PCAF/E2F1 complexes onto the P1p73 promoter and p53-independent apoptosis. Our results identify hSirT1 and PCAF as potential targets to modulate tumor cell survival and chemoresistance irrespective of p53 status.

Targeting mitochondrial dysfunction can restore antiviral activity of exhausted HBV-specific CD8 T cells in chronic hepatitis B

Hepatitis B virus (HBV)-specific CD8 T cells are functionally exhausted in chronic hepatitis B infection, and this condition can be corrected only partially through the modulation of inhibitory pathways, which suggests that a more complex molecular interplay underlies T cell exhaustion. To gain broader insight into this process and identify additional targets for the restoration of T cell function, we compared the transcriptome profiles of HBV-specific CD8 T cells from patients with acute and chronic disease with those of HBV-specific CD8 T cells from patients able to resolve HBV infection spontaneously and influenza (FLU)-specific CD8 T cells from healthy participants. The results indicate that exhausted HBV-specific CD8 T cells are markedly impaired at multiple levels and show substantial downregulation of various cellular processes centered on extensive mitochondrial alterations. A notable improvement of mitochondrial and antiviral CD8 functions was elicited by mitochondrion-targeted antioxidants, which suggests a central role for reactive oxygen species (ROS) in T cell exhaustion. Thus, mitochondria represent promising targets for novel reconstitution therapies to treat chronic hepatitis B infection.

Chromatin Dynamics of Gene Activation and Repression in Response to Interferon α (IFNα) Reveal New Roles for Phosphorylated and Unphosphorylated Forms of the Transcription Factor STAT2

Signal transducer and activator of transcription 2 (STAT2), the critical component of type I interferons signaling, is a prototype latent cytoplasmic signal-dependent transcription factor. Activated tyrosine-phosphorylated STAT2 associates with STAT1 and IRF9 to bind the ISRE elements in the promoters of a subset of IFN-inducible genes (ISGs). In addition to activate hundreds of ISGs, IFNα also represses numerous target genes but the mechanistic basis for this dual effect and transcriptional repression is largely unknown. We investigated by ChIP-chip the binding dynamics of STAT2 and “active” phospho(P)-STAT2 on 113 putative IFNα direct target promoters before and after IFNα induction in Huh7 cells and primary human hepatocytes (PHH). STAT2 is already bound to 62% of our target promoters, including most “classical” ISGs, before IFNα treatment. 31% of STAT2 basally bound promoters also show P-STAT2 positivity. By correlating in vivo promoter occupancy with gene expression and changes in histone methylation marks we found that: 1) STAT2 plays a role in regulating ISGs expression, independently from its phosphorylation; 2) P-STAT2 is involved in ISGs repression; 3) “activated” ISGs are marked by H3K4me1 and H3K4me3 before IFNα; 4) “repressed” genes are marked by H3K27me3 and histone methylation plays a dominant role in driving IFNα-mediated ISGs repression.

IL6 Inhibits HBV Transcription by Targeting the Epigenetic Control of the Nuclear cccDNA Minichromosome

The HBV covalently closed circular DNA (cccDNA) is organized as a mini-chromosome in the nuclei of infected hepatocytes by histone and non-histone proteins. Transcription from the cccDNA of the RNA replicative intermediate termed pre-genome (pgRNA), is the critical step for genome amplification and ultimately determines the rate of HBV replication. Multiple evidences suggest that cccDNA epigenetic modifications, such as histone modifications and DNA methylation, participate in regulating the transcriptional activity of the HBV cccDNA. Inflammatory cytokines (TNFα, LTβ) and the pleiotropic cytokine interleukin-6 (IL6) inhibit hepatitis B virus (HBV) replication and transcription. Here we show, in HepG2 cells transfected with linear HBV monomers and HBV-infected NTCP-HepG2 cells, that IL6 treatment leads to a reduction of cccDNA-bound histone acetylation paralleled by a rapid decrease in 3.5kb/pgRNA and subgenomic HBV RNAs transcription without affecting cccDNA chromatinization or cccDNA levels. IL6 repressive effect on HBV replication is mediated by a loss of HNF1α and HNF4α binding to the cccDNA and a redistribution of STAT3 binding from the cccDNA to IL6 cellular target genes.

TP63 and TP73 in cancer, an unresolved “family” puzzle of complexity, redundancy and hierarchy

TP53 belongs to a small gene family that includes, in mammals, two additional paralogs, TP63 and TP73. The p63 and p73 proteins are structurally and functionally similar to p53 and their activity as transcription factors is regulated by a wide repertoire of shared and unique post‐translational modifications and interactions with regulatory cofactors. p63 and p73 have important functions in embryonic development and differentiation but are also involved in tumor suppression. The biology of p63 and p73 is complex since both TP63 and TP73 genes are transcribed into a variety of different isoforms that give rise to proteins with antagonistic properties, the TA‐isoforms that act as tumor‐suppressors and DN‐isoforms that behave as proto‐oncogenes. The p53 family as a whole behaves as a signaling “network” that integrates developmental, metabolic and stress signals to control cell metabolism, differentiation, longevity, proliferation and death. Despite the progress of our knowledge, the unresolved puzzle of complexity, redundancy and hierarchy in the p53 family continues to represent a formidable challenge.

p53-paralog DNp73 oncogene is repressed by IFNα/STAT2 through the recruitment of the Ezh2 polycomb group transcriptional repressor.

The DNp73 proteins act as trans-repressors of p53 and p73-dependent transcription and exert both anti-apoptotic activity and pro-proliferative activity. DNp73s are frequently up-regulated in a variety of human cancers, including human hepatocellular carcinomas (HCCs). Increased levels of DNp73 proteins confer to HCC cells resistance to apoptosis and, irrespective to p53 status, a chemoresistant phenotype. Here, we show that interferon (IFN)α down-regulates DNp73 expression in primary human hepatocytes (PHHs) and HCC cell lines. IFNα has been used as pro-apoptotic agent in the treatment of malignancies and there is increasing evidence of IFNα effectiveness in HCC treatment and prevention of recurrence. The precise mechanisms by which class I IFNs exert their anti-proliferative and anti-tumor activity remain unclear. IFNα binding to its receptor activates multiple intracellular signaling cascades regulating the transcription of numerous direct target genes through the recruitment of a complex comprising of STAT1, STAT2 and IFN regulatory factor (IRF)9 to their promoters. We found that, in response to IFNα, the P2p73 promoter undergoes substantial chromatin remodeling. Histone deacetylases (HDACs) replace histone acetyl transferases. STAT2 is recruited onto the endogenous P2p73 promoter together with the polycomb group protein Ezh2, leading to increased H3K27 methylation and transcriptional repression. The reduction of DNp73 levels by IFNα is paralleled by an increased susceptibility to IFNα-triggered apoptosis of Huh7 hepatoma cells. Our results show, for the first time, that IFN-stimulated gene factor 3 recruitment may serve both in activating and repressing gene expression and identify the down-regulation of DNp73 as an additional mechanism to counteract the chemoresistance of liver cancer cells.