Abstract
Epstein-Barr virus (EBV) epigenetically reprogrammes B-lymphocytes to drive immortalization and facilitate viral persistence. Host-cell transcription is perturbed principally through the actions of EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated by specific combinations of these EBNAs through unknown mechanisms. Comparing human genome binding by these viral transcription factors, we discovered that 25% of binding sites were shared by EBNA 2 and the EBNA 3s and were located predominantly in enhancers. Moreover, 80% of potential EBNA 3A, 3B or 3C target genes were also targeted by EBNA 2, implicating extensive interplay between EBNA 2 and 3 proteins in cellular reprogramming. Investigating shared enhancer sites neighbouring two new targets (WEE1 and CTBP2) we discovered that EBNA 3 proteins repress transcription by modulating enhancer-promoter loop formation to establish repressive chromatin hubs or prevent assembly of active hubs. Re-ChIP analysis revealed that EBNA 2 and 3 proteins do not bind simultaneously at shared sites but compete for binding thereby modulating enhancer-promoter interactions. At an EBNA 3-only intergenic enhancer site between ADAM28 and ADAMDEC1 EBNA 3C was also able to independently direct epigenetic repression of both genes through enhancer-promoter looping. Significantly, studying shared or unique EBNA 3 binding sites at WEE1, CTBP2, ITGAL (LFA-1 alpha chain), BCL2L11 (Bim) and the ADAMs, we also discovered that different sets of EBNA 3 proteins bind regulatory elements in a gene and cell-type specific manner. Binding profiles correlated with the effects of individual EBNA 3 proteins on the expression of these genes, providing a molecular basis for the targeting of different sets of cellular genes by the EBNA 3s. Our results therefore highlight the influence of the genomic and cellular context in determining the specificity of gene deregulation by EBV and provide a paradigm for host-cell reprogramming through modulation of enhancer-promoter interactions by viral transcription factors.
Highlights
Epstein-Barr virus (EBV) was discovered in cells cultured from a Burkitt’s lymphoma (BL) biopsy in 1964 [1] and has since been associated with the development of numerous cancers including Hodgkin’s disease, post-transplant lymphoma, certain natural killer and T-cell lymphomas and the epithelial cell tumour nasopharyngeal carcinoma
Epstein-Barr nuclear antigens (EBNAs) 2 and 3 proteins target common sites and genes To examine the mechanism of interplay in cellular gene reprogramming by EBNA 2, 3A, 3B and 3C proteins, we performed comparative analysis of the human genome regulatory elements targeted by these factors in a BL cell line expressing all EBV latent proteins (Mutu III) ([60] and Figure S1)
ChIP-seq was carried out using an EBNA 2-specific monoclonal antibody and compared to ChIP-seq data generated in our laboratory using an anti-EBNA 3C polyclonal antibody that we found independently precipitates EBNA 3A and 3B, albeit at a lower efficiency (Figure S2) [37] (ChIP-seq data are available through GEO Series accession number GSE47629)
Summary
Epstein-Barr virus (EBV) was discovered in cells cultured from a Burkitt’s lymphoma (BL) biopsy in 1964 [1] and has since been associated with the development of numerous cancers including Hodgkin’s disease, post-transplant lymphoma, certain natural killer and T-cell lymphomas and the epithelial cell tumour nasopharyngeal carcinoma. EBV immortalizes host B lymphocytes generating latently infected lymphoblastoid cell lines (LCLs) that proliferate indefinitely in culture. LCLs express 9 viral latent proteins: six Epstein-Barr nuclear antigens (EBNAs) and three latent membrane proteins (LMPs). EBNA 2 functions as the master controller of latent viral gene transcription and activates the EBV C promoter that drives production of the long pre-mRNA encoding all EBNAs, in addition to the promoters of the LMP genes [4,5,6,7]. EBNA 2 activates transcription of numerous cellular genes involved in growth control and B-cell activation including MYC, RUNX3, CD23, CD21 and FGR [8,9,10,11]. EBNA 2 cannot bind DNA directly and interacts with target genes via cellular transcription factors that include RBP-Jk (C promoter binding factor 1, CBF1) and Author Summary
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