Hepatitis B virus X protein induces EpCAM expression via active DNA demethylation directed by RelA in complex with EZH2 and TET2

Oncogene. 2016 Feb 11;35(6):715-26. doi: 10.1038/onc.2015.122. Epub 2015 Apr 20.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Antigens, Neoplasm / genetics*
  • Antigens, Neoplasm / metabolism
  • Base Sequence
  • Carcinoma, Hepatocellular / genetics
  • Carcinoma, Hepatocellular / metabolism
  • Carcinoma, Hepatocellular / pathology
  • Cell Adhesion Molecules / genetics*
  • Cell Adhesion Molecules / metabolism
  • Cells, Cultured
  • DNA Methylation / genetics*
  • DNA-Binding Proteins / metabolism*
  • Dioxygenases
  • Enhancer of Zeste Homolog 2 Protein
  • Epithelial Cell Adhesion Molecule
  • Gene Expression Regulation
  • Hep G2 Cells
  • Hepatitis B, Chronic / genetics
  • Hepatitis B, Chronic / metabolism
  • Hepatitis B, Chronic / pathology
  • Humans
  • Liver Neoplasms / genetics
  • Liver Neoplasms / metabolism
  • Liver Neoplasms / pathology
  • Mice
  • Molecular Sequence Data
  • Multiprotein Complexes / metabolism
  • Polycomb Repressive Complex 2 / metabolism*
  • Promoter Regions, Genetic
  • Protein Binding
  • Proto-Oncogene Proteins / metabolism*
  • Trans-Activators / physiology*
  • Transcription Factor RelA / metabolism*
  • Viral Regulatory and Accessory Proteins

Substances

  • Antigens, Neoplasm
  • Cell Adhesion Molecules
  • DNA-Binding Proteins
  • EPCAM protein, human
  • Epithelial Cell Adhesion Molecule
  • Multiprotein Complexes
  • Proto-Oncogene Proteins
  • RELA protein, human
  • Trans-Activators
  • Transcription Factor RelA
  • Viral Regulatory and Accessory Proteins
  • hepatitis B virus X protein
  • Dioxygenases
  • TET2 protein, human
  • EZH2 protein, human
  • Enhancer of Zeste Homolog 2 Protein
  • Polycomb Repressive Complex 2