C. Scisciani

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.

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.

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.

Genome-wide identification of direct HBx genomic targets

BackgroundThe Hepatitis B Virus (HBV) HBx regulatory protein is required for HBV replication and involved in HBV-related carcinogenesis. HBx interacts with chromatin modifying enzymes and transcription factors to modulate histone post-translational modifications and to regulate viral cccDNA transcription and cellular gene expression. Aiming to identify genes and non-coding RNAs (ncRNAs) directly targeted by HBx, we performed a chromatin immunoprecipitation sequencing (ChIP-Seq) to analyse HBV recruitment on host cell chromatin in cells replicating HBV.ResultsChIP-Seq high throughput sequencing of HBx-bound fragments was used to obtain a high-resolution, unbiased, mapping of HBx binding sites across the genome in HBV replicating cells. Protein-coding genes and ncRNAs involved in cell metabolism, chromatin dynamics and cancer were enriched among HBx targets together with genes/ncRNAs known to modulate HBV replication. The direct transcriptional activation of genes/miRNAs that potentiate endocytosis (Ras-related in brain (RAB) GTPase family) and autophagy (autophagy related (ATG) genes, beclin-1, miR-33a) and the transcriptional repression of microRNAs (miR-138, miR-224, miR-576, miR-596) that directly target the HBV pgRNA and would inhibit HBV replication, contribute to HBx-mediated increase of HBV replication.ConclusionsOur ChIP-Seq analysis of HBx genome wide chromatin recruitment defined the repertoire of genes and ncRNAs directly targeted by HBx and led to the identification of new mechanisms by which HBx positively regulates cccDNA transcription and HBV replication.

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.

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