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首页> 外文学位 >Characterization of EBV nuclear protein 3 (EBNA3) genome binding and gene regulation in EBV transformed lymphoblastoid cell lines (LCLs).
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Characterization of EBV nuclear protein 3 (EBNA3) genome binding and gene regulation in EBV transformed lymphoblastoid cell lines (LCLs).

机译：EBV转化的淋巴母细胞样细胞系（LCL）中EBV核蛋白3（EBNA3）基因组结合和基因调控的表征。

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摘要

Latent infection of B lymphocytes by Epstein-Barr virus (EBV) in vitro results in their immortalization into lymphoblastoid cell lines (LCLs); this latency program is controlled by the EBNA2 viral transcriptional activator that targets promoters via RBPJ, a DNA binding protein in the Notch signaling pathway. Three other EBNA3 proteins (EBNA3A, EBNA3B, and EBNA3C) interact with RBPJ to regulate cell gene expression. The mechanism by which EBNAs regulate different genes via RBPJ remains unclear. Our ChIP-seq analysis of the EBNA3 proteins analyzed in concert with prior EBNA2 and RBPJ data demonstrate that EBNA3A, EBNA3B and EBNA3C bind to distinct, partially overlapping genomic locations. Although RBPJ interaction is critical for EBNA3A and EBNA3C growth effects, only 30-40% of EBNA3 bound sites co-localize with RBPJ. Using LCLs conditional for EBNA3A or EBNA3C activity, we demonstrate that EBNA2 binding at sites near genes regulated by EBNA3A or EBNA3C is specifically regulated by the respective EBNA3. To investigate EBNA3 binding specificity, we identified sequences and transcription factors enriched at EBNA3A, EBNA3B, and EBNA3C bound sites. This confirmed the prior observation that IRF4 is enriched at EBNA3A and EBNA3C bound sites and revealed IRF4 enrichment at EBNA3B bound sites. Using the IRF4 negative BJAB cell, we demonstrate that IRF4 is essential for EBNA3C, but not EBNA3A or EBNA3B binding to specific sites. These results support a model in which EBNA2 and EBNA3s compete for distinct subsets of RBPJ sites to regulate cell genes and where EBNA3 subset specificity is determined by interactions with other cell transcription factors. Finally, we examined changes in the H3K27me3 mark that is believed to mediate EBNA3 repression of cell genes. Surprisingly, we observed no correlation between changes in this mark with EBNA3A or EBNA3C binding upon EBNA3A or EBNA3C inactivation. Furthermore, EBNA3 bound sites were characterized by activating (H3K4me3 and H3K9ac) histone marks. This suggests that EBNA3 proteins may exert their transcriptional effects through indirect mechanisms. We performed H3K27me3 ChIP-seq analysis in the presence and absence of EBNA3C activity to determine the exact locations that undergo this mark change in response to EBNA3C activity. Our study represents the first genome-wide characterization of EBNA3A, EBNA3B and EBNA3C binding in LCLs; as well as the first genome-wide characterization of H3K27me3 pattern in response to EBNA3C activity. Moreover, our results suggest a mechanism by which EBNA3 proteins regulate distinct, but partially overlapping, sets of cell genes and provide a basis for understanding their role in lymphomagenesis.
机译：EB病毒（EBV）潜在地感染B淋巴细胞，导致它们永生成淋巴母细胞样细胞系（LCL）；该潜伏期程序由EBNA2病毒转录激活剂控制，该激活剂通过RBPJ（Notch信号传导途径中的DNA结合蛋白）靶向启动子。其他三种EBNA3蛋白（EBNA3A，EBNA3B和EBNA3C）与RBPJ相互作用以调节细胞基因表达。 EBNA通过RBPJ调控不同基因的机制仍不清楚。我们对EBNA3蛋白质的ChIP-seq分析与先前的EBNA2和RBPJ数据相一致，证明EBNA3A，EBNA3B和EBNA3C结合到不同的，部分重叠的基因组位置。尽管RBPJ相互作用对于EBNA3A和EBNA3C的生长效果至关重要，但只有30-40％的EBNA3结合位点与RBPJ共定位。使用有条件的针对EBNA3A或EBNA3C活性的LCL，我们证明了EBNA2结合在受EBNA3A或EBNA3C调控的基因附近的位点受相应的EBNA3特异性调控。为了研究EBNA3的结合特异性，我们鉴定了在EBNA3A，EBNA3B和EBNA3C结合位点富集的序列和转录因子。这证实了先前的观察结果，即IRF4在EBNA3A和EBNA3C结合位点富集，并揭示了IRF4在EBNA3B结合位点富集。使用IRF4阴性BJAB细胞，我们证明IRF4对于EBNA3C是必不可少的，但不是EBNA3A或EBNA3B与特定位点的结合。这些结果支持了一个模型，其中EBNA2和EBNA3竞争RBPJ位点的不同子集来调节细胞基因，而EBNA3子集特异性是由与其他细胞转录因子的相互作用决定的。最后，我们检查了H3K27me3标记的变化，该标记被认为可以介导EBNA3抑制细胞基因。令人惊讶地，我们观察到在EBNA3A或EBNA3C失活后，该标记的变化与EBNA3A或EBNA3C结合之间没有相关性。此外，EBNA3结合位点的特征是激活（H3K4me3和H3K9ac）组蛋白标记。这表明EBNA3蛋白可能通过间接机制发挥其转录作用。在存在和不存在EBNA3C活性的情况下，我们进行了H3K27me3 ChIP-seq分析，以确定响应EBNA3C活性而经历此标记变化的确切位置。我们的研究代表了LCL中EBNA3A，EBNA3B和EBNA3C结合的第一个全基因组表征；以及响应EBNA3C活性的H3K27me3模式的第一个全基因组范围表征。此外，我们的结果表明，EBNA3蛋白可调节不同但部分重叠的细胞基因集，并为理解其在淋巴瘤发生中的作用提供基础。

著录项

  • 作者

    Wang, Anqi.;

  • 作者单位

    The University of Wisconsin - Madison.;

  • 授予单位 The University of Wisconsin - Madison.;
  • 学科 Cellular biology.;Oncology.;Genetics.;Virology.
  • 学位 Ph.D.
  • 年度 2016
  • 页码 152 p.
  • 总页数 152
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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