Investigating the mechanism of cellular gene activation and repression by the EBV transcription factor EBNA 2

Ojeniyi, Opeoluwa O. (2017) Investigating the mechanism of cellular gene activation and repression by the EBV transcription factor EBNA 2. Masters thesis (MPhil), University of Sussex.

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Epstein-Barr virus (EBV) is a widespread human tropic B cell virus that is linked to several malignancies. EBV modulates the transcriptome of B lymphocytes to drive immortalisation and viral persistence. EBV nuclear antigens (EBNA) 2,3A, 3B and 3C are transcriptional regulators of both viral and cellular genes and are the primary drivers of the immortalisation and the continued proliferation of infected B-cells. EBNA 2 activates all EBV gene promoters and cellular growth control genes while EBNA3A, 3B and 3C activates or represses transcription. EBNA2 and 3 proteins do not bind directly to DNA. They bind through cellular DNA-binding proteins like RBP-Jκ and PU.1. The focus of this research was to investigate how EBNA 2 promotes immortalisation through the epigenetic reprogramming of cellular genes and how EBNA 3A, 3B and 3C antagonise or cooperate with EBNA 2 in gene regulation. Previous ChIP-seq results in our lab identified significant binding sites for EBNA 2 and EBNA 3s. I targeted three important novel shared EBNA 2 and EBNA 3s binding sites; the integrin ITGAL, cell cycle kinase WEE1 and transcription repressor CTBP2 genes. I investigated if these shared sites are functional as EBNA 2 response elements in reporter assay by transiently transfecting the endogenous promoter and any associated long range enhancer region of genes and performing luciferase assays. EBNA 2 activates the ITGAL promoter and EBNA 3s inhibits the activation while WEE1 and CTBP2 does not respond in reporter assay. I also performed site-directed mutagenesis to determine which cellular transcription factor was important for the activation of EBNA 2 at the ITGAL promoter. RBP-Jk site mutation disrupted the EBNA 2 activation. Another research focus was EBNA 2 association with gene activation and repression. BCR components CD79A and CD79B are involved in signal transduction and the regulation of B-cell growth and survival and transcription factor EBF1 plays an important role in B cell differentiation. I investigated the association of EBNA 2 with these repressed gene targets and if EBF1 plays a role in the mechanism of repression using reporter assay. CD79A and CD79B activates EBNA 2 and EBF1 does not significantly repress the activation in luciferase reporter assay. EBNA 2 have been mapped binding to enhancers at a new target gene interferon response factor 4 IRF4 and microarray data implicates EBNA 2 in its activation. When IRF4 expression is reduced in EBV transformed cells, cell proliferation rate is decreased and apoptosis enhanced so this activation may be important for B-cell transformation by EBV. I carried out reporter assays to determine if the site is EBNA 2 responsive and whether it interacts with the IRF4 promoter and enhancers. EBNA 2 slightly activates the promoter and enhancers.

Item Type: Thesis (Masters)
Schools and Departments: School of Life Sciences > Biochemistry
Subjects: Q Science > QD Chemistry > QD0241 Organic chemistry > QD0415 Biochemistry
R Medicine > RC Internal medicine > RC0109 Infectious and parasitic diseases > RC0141.5 Epstein-Barr virus disease
Depositing User: Library Cataloguing
Date Deposited: 07 Jun 2017 09:49
Last Modified: 07 Jun 2017 09:49

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