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.

Public Health Relevance

Epstein Barr Virus (EBV) associated malignancies are a significant human threat, causing excess lymphomas, Hodgkins Disease, and oral proliferative diseases in immune-deficient humans, especially HIV infected people, worldwide. The EBV related disease burden is highest in Africa and Asia, where HAART therapy and chemotherapy for B cell lymphomas are less available. The objectives of these studies are to further elucidate the molecular mechanisms through which EBV nuclear proteins EBNA2 and EBNALP exploit the normal B lymphocyte transcription program to achieve immortal cell growth through long range enhancer looping and short range promoter activation of cell gene expression. These studies will identify new molecular targets that mediate chromatin looping and explore the effects of knock down of these cell looping components on EBV transformed lymphoblastoid cell gene expression.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Virology - A Study Section (VIRA)
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Daschner, Phillip J
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Brigham and Women's Hospital
United States
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Jiang, Sizun; Willox, Bradford; Zhou, Hufeng et al. (2014) Epstein-Barr virus nuclear antigen 3C binds to BATF/IRF4 or SPI1/IRF4 composite sites and recruits Sin3A to repress CDKN2A. Proc Natl Acad Sci U S A 111:421-6
Zhao, Bo; Barrera, Luis A; Ersing, Ina et al. (2014) The NF-?B genomic landscape in lymphoblastoid B cells. Cell Rep 8:1595-606
Chen, Ya-Lin; Liu, Cheng-Der; Cheng, Chi-Ping et al. (2014) Nucleolin is important for Epstein-Barr virus nuclear antigen 1-mediated episome binding, maintenance, and transcription. Proc Natl Acad Sci U S A 111:243-8
Portal, Daniel; Zhou, Hufeng; Zhao, Bo et al. (2013) Epstein-Barr virus nuclear antigen leader protein localizes to promoters and enhancers with cell transcription factors and EBNA2. Proc Natl Acad Sci U S A 110:18537-42