Day Latasha, Chau Charles M, Nebozhyn Michael, Rennekamp Andrew J, Showe Michael, Lieberman Paul M
The Wistar Institute, 3601 Spruce St., Philadelphia, PA 19104, USA.
J Virol. 2007 Jun;81(12):6389-401. doi: 10.1128/JVI.02172-06. Epub 2007 Apr 4.
Epstein-Barr virus (EBV) escapes host immunity by the reversible and epigenetic silencing of immunogenic viral genes. We previously presented evidence that a dynamic chromatin domain, which we have referred to as the latency control region (LCR), contributes to the reversible repression of EBNA2 and LMP1 gene transcription. We now explore the protein-DNA interaction profiles for a few known regulatory factors and histone modifications that regulate LCR structure and activity. A chromatin immunoprecipitation assay combined with real-time PCR analysis was used to analyze protein-DNA interactions at approximately 500-bp intervals across the first 60,000 bp of the EBV genome. We compared the binding patterns of EBNA1 with those of the origin recognition complex protein ORC2, the chromatin boundary factor CTCF, the linker histone H1, and several histone modifications. We analyzed three EBV-positive cell lines (MutuI, Raji, and LCL3459) with distinct transcription patterns reflecting different latency types. Our findings suggest that histone modification patterns within the LCR are complex but reflect differences in each latency type. The most striking finding was the identification of CTCF sites immediately upstream of the Qp, Cp, and EBER transcription initiation regions in all three cell types. In transient assays, CTCF facilitated EBNA1-dependent transcription activation of Cp, suggesting that CTCF coordinates interactions between different chromatin domains. We also found that histone H3 methyl K4 clustered with CTCF and EBNA1 at sites of active transcription or DNA replication initiation. Our findings support a model where CTCF delineates multiple domains within the LCR and regulates interactions between these domains that correlate with changes in gene expression.
爱泼斯坦-巴尔病毒（EBV）通过免疫原性病毒基因的可逆性表观遗传沉默来逃避宿主免疫。我们之前提供的证据表明，一个动态染色质结构域，我们称之为潜伏控制区（LCR），有助于EBNA2和LMP1基因转录的可逆性抑制。我们现在探索一些已知调节因子和组蛋白修饰的蛋白质-DNA相互作用图谱，这些调节因子和组蛋白修饰调控LCR的结构和活性。采用染色质免疫沉淀试验结合实时PCR分析，以大约500 bp的间隔分析EBV基因组前60,000 bp区域的蛋白质-DNA相互作用。我们比较了EBNA1与起始识别复合物蛋白ORC2、染色质边界因子CTCF、连接组蛋白H1以及几种组蛋白修饰的结合模式。我们分析了三种EBV阳性细胞系（MutuI、Raji和LCL3459），它们具有不同的转录模式，反映了不同的潜伏类型。我们的研究结果表明，LCR内的组蛋白修饰模式很复杂，但反映了每种潜伏类型的差异。最显著的发现是在所有三种细胞类型中，均在Qp、Cp和EBER转录起始区域的紧邻上游鉴定到CTCF位点。在瞬时试验中，CTCF促进了Cp的EBNA1依赖性转录激活，这表明CTCF协调了不同染色质结构域之间的相互作用。我们还发现，组蛋白H3甲基化K4在活跃转录或DNA复制起始位点与CTCF和EBNA1聚集在一起。我们的研究结果支持一种模型，即CTCF在LCR内划定多个结构域，并调节这些结构域之间与基因表达变化相关的相互作用。
