67
IRUS Total
Downloads

Genetic analysis of the role of RBPJ (CBF1) in Epstein-Barr Virus nuclear antigen 3C-mediated gene regulation

File Description SizeFormat 
Gillman-A-2019-PhD-Thesis.pdf13.87 MBAdobe PDFView/Open
Title: Genetic analysis of the role of RBPJ (CBF1) in Epstein-Barr Virus nuclear antigen 3C-mediated gene regulation
Authors: Gillman, Adam
Item Type: Thesis or dissertation
Abstract: Epstein-Barr nuclear antigen 3C (EBNA3C) is an oncoprotein essential to Epstein-Barr virus (EBV) mediated transformation of B cells. Exon-microarrays and ChIP-seq studies have shown EBNA3C directly regulates hundreds of genes yet the mechanisms behind how regulation is brought about are largely unknown. EBNA3C cannot bind DNA directly but instead must be tethered to chromatin through association with DNA binding transcription factors, the best described being the downstream effector molecular of the Notch signalling pathway, RBPJ (RBPJK/CBF1). Two RBPJ interaction motifs have been ascribed to EBNA3C, the 209TFGC212 and 226VWTP229 motif, but their functional significance had not been assessed in the context of B cell infection. To investigate the role of the EBNA3C:RBPJ interaction in B cell transformation, novel recombinant EBVs were generated carrying EBNA3C with either RBPJ interaction motif mutated, HDmut-EBNA3C or W227S-EBNA3C, or both motifs mutated creating a Double mutant-EBNA3C. Infection of resting B cells consistently lead to establishment of HDmut and W227S lymphoblastoid cell lines (LCLs) but HDmut-EBNA3C infected cells proliferated more slowly than W227S-EBNA3C which resembled wild-type EBNA3C in its proliferation rate. Infection with Double mutant-EBNA3C virus established LCLs only in a few cases with a proliferation rate less than that of HDmut-EBNA3C. Gene expression analysis revealed HDmut-EBNA3C and Double mutant-EBNA3C were defective in regulating certain genes whereas no impairment in regulation was apparent in W227S LCLs. Focusing on the well characterised EBNA3C repressed genes COBLL1, ADAM28 and ADAMDEC1, chromatin immunoprecipitation (ChIP) revealed both HDmut-EBNA3C and W227S-EBNA3C were equally able to bind to chromatin and recruit RBPJ. Double mutant-EBNA3C failed to bind chromatin, showing that the impaired regulatory function of HDmut-EBNA3C is distinct from that of Double mutant-EBNA3C. Therefore, HDmut-EBNA3C allowed for investigation of the regulatory mechanism behind EBNA3C repression decoupled from the role of RBPJ. Despite failing to effectively represses these genes, HDmut-EBNA3C recruited polycomb group proteins BMI1 and SUZ12, resulting in deposition of the repression-associated mark H3K27me3. However, HDmut-EBNA3C failed to effectively remove the activation-associated marks H3K4me3 and H3K9Ac from these genes. EBNA3C was found to interact with the H3K4me3 demethylase KDM2B and this interaction was disrupted in HDmut-EBNA3C. Lentiviral knockdown of KDM2B in the conditional EBNA3C system (EBNA3C-HT) prevented EBNA3C directed removal of H3K4me3 from the transcription start sites of COBLL1, ADAM28 and ADAMDEC1 and prevented repression. Taken together, these data provide strong support for the proposed two-step repression model of EBNA3 regulation and implicate KDM2B as an important factor in EBNA3C mediated repression.
Content Version: Open Access
Issue Date: Dec-2018
Date Awarded: Jul-2019
URI: http://hdl.handle.net/10044/1/71438
DOI: https://doi.org/10.25560/71438
Copyright Statement: Creative Commons Attribution-NonCommercial 4.0 International Licence (CC BY-NC).
Supervisor: Allday, Martin
White, Rob
Farrell, Paul
Sponsor/Funder: Wellcome Trust
Funder's Grant Number: WT099273/Z/12/Z
Department: Department of Medicine
Publisher: Imperial College London
Qualification Level: Doctoral
Qualification Name: Doctor of Philosophy (PhD)
Appears in Collections:Medicine PhD theses