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

Oncogene volume 35, pages 715–726 (2016)Cite this article

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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.

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Acknowledgements

This work was supported by NIH grant DK044533 to OA. Shared Resources (flow cytometry and DNA sequencing) are supported by NIH grant P30CA023168 to Purdue Cancer Research Center. The authors thank Dr R Hullinger for critical review of the manuscript.

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Authors and Affiliations

  1. Department of Basic Medical Sciences, Purdue University, West Lafayette, IN, USA,

    H Fan, H Zhang & O Andrisani

  2. Department of Purdue Center for Cancer Research, Purdue University, West Lafayette, IN, USA,

    H Fan, H Zhang & O Andrisani

  3. Purdue University Libraries, Purdue University, West Lafayette,, IN, USA

    P E Pascuzzi

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Fan, H., Zhang, H., Pascuzzi, P. et al. Hepatitis B virus X protein induces EpCAM expression via active DNA demethylation directed by RelA in complex with EZH2 and TET2. Oncogene 35, 715–726 (2016). https://doi.org/10.1038/onc.2015.122

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