Roles of Epstein-Barr virus nuclear antigens 2 and LP in B cell proliferation

描述(申请人提供)：Epstein Barr病毒(EBV)是非洲Burkitt淋巴瘤、霍奇金淋巴瘤、免疫抑制和HIV感染者的恶性淋巴增殖性疾病、HIV感染者的口腔上皮细胞增生性病变以及鼻咽癌和胃窦癌的主要原因。EBV通过表达潜伏期III EBV核抗原蛋白和潜伏膜蛋白1，将静止的B淋巴细胞(RBL)转化为持续增殖的淋巴母细胞(LCLS)。EBV转化为LCLS是一种相关的模型，可用于研究RBL转化为LCLS的时间过程和EBV基因依赖性。我们发现，大多数EBNA2结合位点距离最近的基因超过2kb，50个调控基因的EBNA2结合位点平均距离靶基因转录起始点330kB。许多EBNA2效应是通过长距离传递的，这需要将中间的DNA环化。由于EBNA2结合了LCL基因组中的&gt；5000个位点，受EBNA2位点影响的细胞基因仅被部分确定。此外，EBNA2用来调节环路的蛋白质还没有得到准确的鉴定。然而，我们发现，EBNA2对MYC诱导的细胞生长的影响大多是通过EBNA2增强子与MYC启动子的长距离环路介导的。AIMS1和2使用我们实验室建立的创新技术和独特的试剂来1)鉴定EBNA2增强子与细胞启动子和启动子连接处的蛋白质环复合体的相互作用。2)确定EBNA2增强子/启动子相互作用成分在LCL增殖和存活中的生物学意义。3)确定EBNALP在增强子和启动子相互作用中的作用。在AIM1中，新的基因组方法将被用来识别DNA环(CHIA-PET)带来的邻近的增强子和启动子DNA位点。CHIP-SEQ将被用来确定候选循环因子的基因组定位，并将结合与EBNA2诱导的转录效应相关联。在AIM2中，将通过下调LCLS中shRNA对EBNA2诱导c-myc和LCL生长和存活的影响来评估每个环状因子的重要性。在AIM 3中，我们将继续最近的发现，EBNA2也是由EBNALP在全基因组范围内共同激活的，EBNALP主要与启动子结合，以YY1、CTCF和ZNF143以及组蛋白H2-Az的存在为标志。在AIM3中，我们将评估EBNALP对环路的影响，以确定EBNALP介导的共激活的分子机制。我们建议使用重组病毒，针对EBNALP或环状因子的可诱导shRNA，一个显性的EBNALP突变体，以及EBNA2和EBNALP在原代B细胞中的表达，以更好地描述EBNALP在MYC、Cyclin D2和细胞生存基因调控中的作用。我们还将评估EBNALP相互作用蛋白在环化、LCL基因转录和LCL生长中的重要性。这些实验使用整合的基因组方法来阐明EBNA2和EBNALP激活LCLS转录的分子机制。由于EBNA2和EBNALP模拟Notch途径，并使用静止的B淋巴细胞基因组框架来实现它们的作用，我们的发现也将为深入了解正常和恶性B淋巴细胞基因调控的基本机制提供帮助。

DESCRIPTION (provided by applicant): Epstein Barr Virus (EBV) is a prominent cause of African Burkitt's Lymphoma, Hodgkin's Lymphomas, malignant Lymphoproliferative Diseases in immune suppressed and HIV infected people, proliferative oral epithelial cell lesions in HIV infected people, as well as Nasophyangeal Cancers and Antral Gastric Cancers. EBV converts Resting B Lymphocytes (RBLs) to continuously proliferating Lymphoblasts (LCLs) by expressing Latency III EBV nuclear antigen proteins and latent membrane protein 1. EBV conversion of RBLs to LCLs is a relevant model that can be genetically manipulated to investigate the time course and EBV gene dependence for RBL conversion to LCLs. We have shown that most EBNA2 binding sites are more than 2kB from the nearest gene and EBNA2 sites for 50 regulated genes, which are, on average, 330kB from the transcription start site of target genes. Many EBNA2 effects are mediated over long distances that require the intervening DNA to be looped out. Because EBNA2 binds >5000 sites in the LCL genome, the cell genes affected by EBNA2 sites are only partially defined. Furthermore, the proteins that EBNA2 employs to mediate looping have not been precisely identified. However, we have discovered that most of EBNA2 effects on MYC induced cell growth are mediated by long distance looping of EBNA2 enhancers to the MYC promoter. AIMS1 and 2 use innovative techniques and unique reagents established in our laboratory to 1) Identify EBNA2 enhancer interactions with cell promoters and protein looping complexes at promoter junctions. 2) Determine the biologic significance of EBNA2 enhancer/promoter interaction components in LCL proliferation and survival. and 3) Determine the role of EBNALP in enhancer and promoter interactions. In AIM1, Novel genomic approaches will be used to identify enhancer and promoter DNA sites that are brought in proximity by DNA looping (ChIA-PET). ChIP-seq will be used to determine the genomic localization of candidate looping factors and correlate binding with the transcriptional effects induced by EBNA2. In AIM2, the importance of each looping factor will be evaluated by the effect of shRNA knockdown in LCLs on EBNA2 induction of c-myc and LCL growth and survival. In AIM 3 we will pursue the more recent discovery that EBNA2 is also co-activated genome wide by EBNALP, which binds mostly to promoters, which are marked by the presence of YY1, CTCF, and ZNF143, as well as Histone H2-Az. In AIM3, we will evaluate the effect of EBNALP on looping to identify the molecular mechanisms that underlie EBNALP-mediated co-activation. We propose to use recombinant viruses, inducible shRNA targeting EBNALP or looping factors, a dominant negative EBNALP mutant, and expression of EBNA2 and EBNALP in primary B cells to better delineate the EBNALP effects in MYC, Cyclin D2, and cell survival gene regulation. We will also evaluate the importance of EBNALP interacting proteins in looping, LCL gene transcription, and LCL growth. These experiments use an integrative genomic approach to elucidate the molecular mechanism by which EBNA2 and EBNALP activate transcription in LCLs. Since EBNA2 and EBNALP mimic the Notch pathway and use the resting B-lymphocyte genome framework for their effects, our findings will also afford insight into the fundamental mechanisms of gene regulation in normal and malignant B-lymphocytes.