Fan H, Zhang H, Pascuzzi P E, Andrisani O
Department of Basic Medical Sciences, Purdue University, West Lafayette, IN, USA.
Department of Purdue Center for Cancer Research, Purdue University, West Lafayette, IN, USA.
Oncogene. 2016 Feb 11;35(6):715-26. doi: 10.1038/onc.2015.122. Epub 2015 Apr 20.
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.
慢性乙型肝炎病毒（HBV）感染是发生肝细胞癌（HCC）的主要危险因素，而HBV X蛋白（HBx）在肝癌发生过程中作为辅助因子发挥作用。在模拟HBx和HBV介导的肝癌发生的动物肝脏肿瘤中，染色质调节蛋白SUZ12和ZNF198的下调诱导了包括上皮细胞粘附分子（EpCAM）在内的多个基因的表达。EpCAM上调发生在HBV介导的肝癌和肝癌干细胞中，其机制尚不清楚。在此我们证明HBx通过活性DNA去甲基化诱导EpCAM表达。在肝细胞中，EpCAM被多梳抑制复合物2（PRC2）和ZNF198/LSD1/Co-REST/HDAC1染色质修饰复合物沉默。在HBx表达时，稳定敲低PRC2必需亚基SUZ12的细胞会使位于NF-κB/RelA半位点附近的一个CpG二核苷酸去甲基化。这个NF-κB/RelA位点位于EpCAM转录起始位点（TSS）下游的一个CpG岛中。染色质免疫沉淀（ChIP）分析表明HBx依赖的RelA占据NF-κB半位点，而RelA敲低会抑制CpG去甲基化和EpCAM表达。肿瘤坏死因子-α激活RelA，通过亚硫酸氢盐测序显示将去甲基化传播到附近的CpG位点。HBx表达时发生的RelA依赖的去甲基化需要甲基转移酶EZH2、胞嘧啶去甲基化的关键因子TET2和无活性的DNMT3L，通过敲低实验和亚硫酸氢盐测序证明。免疫共沉淀和连续ChIP分析表明，在存在HBx的情况下，RelA与EZH2、TET2和DNMT3L形成复合物，尽管DNMT3L的作用仍有待了解。有趣的是，人类EpCAM基因在其TSS下游也有一个CpG岛，以及一个两侧有CpG的NF-κB结合位点。源自人类肝癌的HepG2细胞表现出这些NF-κB侧翼CpG位点的去甲基化，并且HBV复制将去甲基化传播到附近的CpG位点。DLK1是另一个PRC2靶基因，在HBV介导的肝癌中也上调，在肝脏肿瘤中其NF-κB结合序列侧翼的CpG二核苷酸处去甲基化，支持这种活性DNA去甲基化机制在致癌转化过程中起作用。
