title: Hepatitis B virus X protein induces EpCAM expression via active DNA demethylation directed by RelA in complex with EZH2 and TET2.
abstract: Chronic hepatitis B virus (HBV) infection is a major risk factor for developing hepatocellular carcinoma (HCC), and HBV X protein (HBx) acts as cofactor in hepatocarcinogenesis. In liver tumors from animals modeling HBx- and HBV-mediated hepatocarcinogenesis, downregulation of chromatin regulating proteins SUZ12 and ZNF198 induces expression of several genes, including epithelial cell adhesion molecule (EpCAM). EpCAM upregulation occurs in HBV-mediated HCCs and hepatic cancer stem cells, by a mechanism not understood. Herein we demonstrate HBx induces EpCAM expression via active DNA demethylation. In hepatocytes, EpCAM is silenced by polycomb repressive complex 2 (PRC2) and ZNF198/LSD1/Co-REST/HDAC1 chromatin-modifying complexes. Cells with stable knockdown of SUZ12, an essential PRC2 subunit, upon HBx expression demethylate a CpG dinucleotide located adjacent to NF-κB/RelA half-site. This NF-κB/RelA site is in a CpG island downstream from EpCAM transcriptional start site (TSS). Chromatin immunoprecipitation (ChIP) assays demonstrate HBx-dependent RelA occupancy of NF-κB half-site, whereas RelA knockdown suppresses CpG demethylation and EpCAM expression. Tumor necrosis factor-α activates RelA, propagating demethylation to nearby CpG sites, shown by sodium bisulfite sequencing. RelA-dependent demethylation occurring upon HBx expression requires methyltrasferase EZH2, TET2 a key factor in cytosine demethylation and inactive DNMT3L, shown by knockdown assays and sodium bisulfite sequencing. Co-immunoprecipitations and sequential ChIP assays demonstrate that RelA in the presence of HBx forms a complex with EZH2, TET2 and DNMT3L, although the role of DNMT3L remains to be understood. Interestingly, the human EpCAM gene also has a CpG island downstream from its TSS, and a NF-κB-binding site flanked by CpGs. HepG2 cells derived from human HCC exhibit demethylation of these NF-κB-flanking CpG sites, and HBV replication propagates demethylation to nearby CpG sites. DLK1, another PRC2 target gene, also upregulated in HBV-mediated HCCs, is demethylated in liver tumors at CpG dinucleotides flanking the NF-κB-binding sequence, supporting that this active DNA demethylation mechanism functions during oncogenic transformation.
authors.affiliation: Department of Basic Medical Sciences, Purdue University, West Lafayette, IN, USA.|Department of Purdue Center for Cancer Research, Purdue University, West Lafayette, IN, USA.
authors.forename: H
authors.initials: H
authors.lastname: Fan
authors.affiliation: Department of Basic Medical Sciences, Purdue University, West Lafayette, IN, USA.|Department of Purdue Center for Cancer Research, Purdue University, West Lafayette, IN, USA.
authors.forename: H
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Purdue University Libraries, Purdue University, West Lafayette, IN, USA.
authors.forename: P E
authors.initials: PE
authors.lastname: Pascuzzi
authors.affiliation: Department of Basic Medical Sciences, Purdue University, West Lafayette, IN, USA.|Department of Purdue Center for Cancer Research, Purdue University, West Lafayette, IN, USA.
authors.forename: O
authors.initials: O
authors.lastname: Andrisani
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D015815:Cell Adhesion Molecules / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D019175:DNA Methylation / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D049308:Dioxygenases; D000071221:Enhancer of Zeste Homolog 2 Protein; D000071858:Epithelial Cell Adhesion Molecule; D005786:Gene Expression Regulation; D056945:Hep G2 Cells; D019694:Hepatitis B, Chronic / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000378:metabolism; D063151:Polycomb Repressive Complex 2 / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D015534:Trans-Activators / Q000502:physiology*; D051996:Transcription Factor RelA / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D004268:DNA-Binding Proteins; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; D046912:Multiprotein Complexes; D011518:Proto-Oncogene Proteins; C498143:RELA protein, human; D015534:Trans-Activators; D051996:Transcription Factor RelA; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D049308:Dioxygenases; C541081:TET2 protein, human; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D063151:Polycomb Repressive Complex 2