乙肝病毒X蛋白诱导通过在复杂的EZH2和TET2导演的RelA积极DNA去

StephenW

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Original Article

Oncogene , (20 April 2015) | doi:10.1038/onc.2015.122

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

H Fan, H Zhang, P E Pascuzzi and O Andrisani
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.

StephenW

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原来的文章

原癌基因，（2015年4月20日）| DOI：10.1038 / onc.2015.122

   
乙肝病毒X蛋白诱导通过在复杂的EZH2和TET2导演的RelA积极DNA去甲基化表达的EpCAM

ħ风扇，H张，P·E Pascuzzi和O的Andr​​isani
抽象

慢性乙型肝炎病毒（HBV）感染是一个用于开发肝细胞癌（HCC）的主要危险因素，和HBV X蛋白（HBx蛋白）作为辅助因子的肝癌。在从动物造型HBx-肝脏肿瘤和HBV介导的肝癌，染色质的下调调控蛋白SUZ12和ZNF198诱导的几个基因，包括上皮细胞粘附分子（EpCAM的）的表达。 EpCAM的上调发生在HBV介导的肝细胞癌与肝癌干细胞，通过不理解的机制。在这里我们展示的HBx通过积极DNA去甲基化引起的EpCAM表达。在肝细胞中，EpCAM的由多梳镇压复杂2（PRC2）和ZNF198 / LSD1 /联合REST / HDAC1染色质修饰复合沉默。细胞SUZ12，一个基本PRC2亚基的稳定敲低，在表达HBx的脱甲基化一的CpG二核苷酸位于邻近的NF-κB/的RelA半位点。这NF-κB/的RelA网站是在CpG岛下游EpCAM的转录起始位点（TSS）。染色质免疫沉淀（ChIP）法证明NF-κB的半位点的HBx依赖性的RelA占用，而击倒的RelA抑制的CpG去甲基化和表达的EpCAM。肿瘤坏死因子α激活的RelA，去甲基化传播到附近的CpG位点，通过亚硫酸氢钠测序所示。时的HBx表达发生的RelA依赖性去甲基化需要methyltrasferase EZH2，TET2的关键因素在胞嘧啶脱甲基化和非活动DNMT3L，由击倒测定法和亚硫酸氢钠测序所示。共免疫沉淀和连续ChIP实验证明的RelA中的HBx的存在下形成具有EZH2，TET2和DNMT3L一个复杂的，虽然DNMT3L的作用仍然被理解。有趣的是，人EpCAM基因也有一个CpG岛从其TSS下游，并通过的CpG侧翼一个的NF-κB结合位点。从这些的NF-κB侧翼CpG部位肝癌展品去甲基化，和HBV复制衍生的HepG2细胞传播去甲基化，以在附近的CpG位点。 DLK1，另一PRC2靶基因，也上调HBV介导的肝癌，是在CpG二核苷酸侧翼的NF-κB结合序列脱甲基化在肝脏肿瘤，支持了致癌性转化过程中该活性DNA去甲基化机制的功能。