Lu Fang, Wiedmer Andreas, Martin Kayla A, Wickramasinghe Priyankara J M S, Kossenkov Andrew V, Lieberman Paul M
The Wistar Institute, Philadelphia, Pennsylvania, USA.
The Wistar Institute, Philadelphia, Pennsylvania, USA
J Virol. 2017 Sep 27;91(20). doi: 10.1128/JVI.00804-17. Print 2017 Oct 15.
Epstein-Barr virus (EBV) latency and its associated carcinogenesis are regulated by dynamic changes in DNA methylation of both virus and host genomes. We show here that the ten-eleven translocation 2 (TET2) gene, implicated in hydroxymethylation and active DNA demethylation, is a key regulator of EBV latency type DNA methylation patterning. EBV latency types are defined by DNA methylation patterns that restrict expression of viral latency genes. We show that TET2 mRNA and protein expression correlate with the highly demethylated EBV type III latency program permissive for expression of EBNA2, EBNA3s, and LMP transcripts. We show that short hairpin RNA (shRNA) depletion of TET2 results in a decrease in latency gene expression but can also trigger a switch to lytic gene expression. TET2 depletion results in the loss of hydroxymethylated cytosine and a corresponding increase in cytosine methylation at key regulatory regions on the viral and host genomes. This also corresponded to a loss of RBP-jκ binding and decreased histone H3K4 trimethylation at these sites. Furthermore, we show that the TET2 gene itself is regulated in a fashion similar to that of the EBV genome. Chromatin immunoprecipitation high-throughput sequencing (ChIP-seq) revealed that the TET2 gene contains EBNA2-dependent RBP-jκ and EBF1 binding sites and is subject to DNA methylation-associated transcriptional silencing similar to what is seen in EBV latency type III genomes. Finally, we provide evidence that TET2 colocalizes with EBNA2-EBF1-RBP-jκ binding sites and can interact with EBNA2 by coimmunoprecipitation. Taken together, these findings indicate that TET2 gene transcripts are regulated similarly to EBV type III latency genes and that TET2 protein is a cofactor of EBNA2 and coregulator of the EBV type III latency program and DNA methylation state. Epstein-Barr virus (EBV) latency and carcinogenesis involve the selective epigenetic modification of viral and cellular genes. Here, we show that TET2, a cellular tumor suppressor involved in active DNA demethylation, plays a central role in regulating the DNA methylation state during EBV latency. TET2 is coordinately regulated and functionally interacts with the viral oncogene EBNA2. TET2 and EBNA2 function cooperatively to demethylate genes important for EBV-driven B-cell growth transformation.
爱泼斯坦-巴尔病毒（EBV）的潜伏状态及其相关的致癌作用受病毒和宿主基因组DNA甲基化动态变化的调控。我们在此表明，与羟甲基化和活性DNA去甲基化有关的10-11易位蛋白2（TET2）基因是EBV潜伏型DNA甲基化模式的关键调节因子。EBV潜伏型由限制病毒潜伏基因表达的DNA甲基化模式定义。我们表明，TET2 mRNA和蛋白表达与对EBNA2、EBNA3s和LMP转录本表达具有许可性的高度去甲基化的EBV III型潜伏程序相关。我们表明，TET2的短发夹RNA（shRNA）缺失导致潜伏基因表达下降，但也可触发向裂解基因表达的转变。TET2缺失导致羟甲基化胞嘧啶的丢失以及病毒和宿主基因组关键调控区域胞嘧啶甲基化相应增加。这也对应于这些位点RBP-jκ结合的丧失和组蛋白H3K4三甲基化的减少。此外，我们表明TET2基因本身的调控方式与EBV基因组类似。染色质免疫沉淀高通量测序（ChIP-seq）显示，TET2基因含有EBNA2依赖的RBP-jκ和EBF1结合位点，并且与EBV III型基因组中所见情况类似，受到DNA甲基化相关的转录沉默作用。最后，我们提供证据表明TET2与EBNA2-EBF1-RBP-jκ结合位点共定位，并且可通过免疫共沉淀与EBNA2相互作用。综上所述，这些发现表明TET2基因转录本的调控方式与EBV III型潜伏基因类似，并且TET2蛋白是EBNA2的辅助因子以及EBV III型潜伏程序和DNA甲基化状态的共调节因子。爱泼斯坦-巴尔病毒（EBV）的潜伏状态和致癌作用涉及病毒和细胞基因的选择性表观遗传修饰。在此，我们表明，参与活性DNA去甲基化的细胞肿瘤抑制因子TET2在调控EBV潜伏期间的DNA甲基化状态中起核心作用。TET2与病毒癌基因EBNA2协同调控并在功能上相互作用。TET2和EBNA2共同发挥作用，使对EBV驱动的B细胞生长转化重要的基因去甲基化。
