| Summary: | Epstein-Barr virus (EBV) is a widespread human B cell virus that is linked to many malignancies. EBV modulates the transcriptome of B lymphocytes to drive immortalisation and viral persistence. This is primarily coordinated by the EBV nuclear antigens (EBNA) 2 and the EBNA 3 family (3A, 3B and 3C), which regulate overlapping sets of cellular genes. Using Chromatin immunoprecipitation (ChIP) coupled to next generation sequencing we found >21000 EBNA 2 and >7000 EBNA 3 binding sites in the human genome, providing the first evidence of EBNA 3 association with the human genome in vivo. Binding sites were predominantly distal to transcription start sites (TSS) indicating a key role in long-range gene control. This was especially pronounced for EBNA 3 proteins (84% of sites over 4kb from any TSS). 56% of genes previously reported to be regulated by these EBNA proteins in micro array experiments were bound by an EBNA. Using ChIP-QPCR we confirmed EBNA 3C bound to and promoted epigenetic silencing of a subset of integrin receptor signalling genes (ITGA4, ITGB1, ADAM28, ADAMDEC1). Indirect silencing of CXCL10 and CXCL11 chemokines by EBNA 3C was also demonstrated. 75% of sites bound by EBNA 3 were also bound by EBNA 2 implicating extensive interplay between EBNA proteins in gene regulation. By examining novel (WEE1, CTBP2) and known (BCL2L11, ITGAL) targets of EBNA 3 proteins bound at promoter-proximal or distal binding sites, we found both cell-type and locus-specific binding and transcriptional regulation. Importantly, genes differentially regulated by a subset EBNA 3 proteins were bound by the same subset, providing a mechanism for selective regulation of host genes by EBNA 3 proteins. In summary, this research demonstrates that EBNA proteins primarily act through long-range enhancer elements and regulate gene expression in a locus and gene-specific manner through differential binding.
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