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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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3561-5) contains supplementary material, which is available to authorized users.

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.

The NAD؉ -dependent histone deacetylase hSirT1 regulates cell survival and stress responses by inhibiting p53-, NF-B-, 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. hSirT1, the mammalian homologue of Sir2 (silent information regulator 2), is a NAD-dependent class III deacetylase (15, 33) that regulates cell survival, stress responses, and metabolism by inhibiting p53 (3, 18, 19, 28)-, E2F1 (1, 30)-, NF-B (31)-, and Forkhead (2)-dependent transcription. The role of hSirT1 in the regulation of mammalian cell survival in response to DNA damage is supported by several observations. hSirT1-deficient mice display increased levels of radiation-induced apoptosis and p53 hyperacetylation (4). hSirT1-dependent deacetylation attenuates the ability of p53 to trans-activate the cell cycle (p21) and apoptotic (bax) target genes (19,28). The constitutive expression of the tumor suppressor hypermethylated in cancer 1 (Hic1) represses hSirT1 transcription, thereby allowing the accumulation of acetylated p53 species and the enhancement of p53-mediated growth arrest and apoptosis in response to DNA damage (3). hSirT1 deacetylase function also modulates p53-independent pathways involved in the DNA damage response. The targeted disruption of the SirT1 gene in p53-deficient cells strongly sensitizes cells to radiation-, cisplatin-, and etoposide-induced cell death (20). hSirT1 deacetylates the DNA damage repair protein Ku70, and deacetylated Ku70 prevents Bax translocation to mitochondria to initiate apoptosis (5). hSirT1 maintains the Nijmegen breakage syndrome protein (NBS1) hypoacetylated and susceptible to be phosphorylated by ATM in response to DNA damage (32). Finally, etoposide treatment results in the E2F1-dependent induction of hSirT1 expression, and the abrogation of hSirT1 expression sensitizes cells to E2F1-dependent apoptosis (30).The E2F family of transcription factors has critical roles in the control of cell proliferation and apoptosis (7). E2F transcriptional activity is tightly regulated during the cell cycle through the association with pRb or the related pocket proteins p107 and p130, leading, in quiescent cells, to the recruitment of transcriptional corepressors, including histone deacetylases (HDACs), methyltransferases, and polycomb group proteins, onto the promoters of proliferation-associated E2F target genes (7). As cells progress into the cell cycle, cyclin-dependent kinases phosphorylate pRb, releasing free E2F and allowing it to interact with transcriptional coactivators, and direc...