Regulation of B cell receptor signalling by Epstein-Barr virus nuclear antigens.

The cancer-associated Epstein-Barr virus (EBV) latently infects and immortalises B lymphocytes. EBV latent membrane protein 2A and EBV-encoded microRNAs are known to manipulate B cell receptor signalling to control cell growth and survival and suppress lytic replication. Here, we show that the EBV transcription factors EBNA2, 3A, 3B and 3C bind to genomic sites around multiple B cell receptor (BCR) pathway genes, regulate their expression and affect BCR signalling. EBNA2 regulates the majority of BCR pathway genes associated with binding sites, where EBNA3 proteins regulate only 42% of targets predicted by binding. Both EBNA2 and 3 proteins predominantly repress BCR pathway gene expression and target some common genes. EBNA2 and at least one EBNA3 protein repress the central BCR components CD79A and CD79B and the downstream genes BLNK, CD22, CD72, NFATC1, PIK3CG and RASGRP3. Studying repression of CD79B, we show that EBNA2 decreases transcription by disrupting binding of Early B cell Factor-1 to the CD79B promoter. Consistent with repression of BCR signalling, we demonstrate that EBNA2 and EBNA3 proteins suppress the basal or active BCR signalling that culminates in NFAT activation. Additionally, we show that EBNA2, EBNA3A and EBNA3C expression can result in reductions in the active serine 473 phosphorylated form of Akt in certain cell contexts, consistent with transcriptional repression of the PI3K-Akt BCR signalling arm. Overall, we identify EBNA2, EBNA3A and EBNA3C-mediated transcription control of BCR signalling as an additional strategy through which EBV may control the growth and survival of infected B cells and maintain viral latency.

Enhancer Control of MicroRNA miR-155 Expression in Epstein-Barr Virus-Infected B Cells.

The oncogenic microRNA (miRNA) miR-155 is the most frequently upregulated miRNA in Epstein-Barr virus (EBV)-positive B cell malignancies and is upregulated in other nonviral lymphomas. Both EBV nuclear antigen 2 (EBNA2) and the B cell transcription factor interferon regulatory factor 4 (IRF4) are known to activate transcription of the host cell gene from which miR-155 is processed (miR-155HG; BIC). EBNA2 also activates IRF4 transcription, indicating that EBV may upregulate miR-155 through direct and indirect mechanisms. The mechanism of transcriptional regulation of IRF4 and miR-155HG by EBNA2, however, has not been defined. We demonstrate that EBNA2 can activate IRF4 and miR-155HG expression through specific upstream enhancers that are dependent on the Notch signaling transcription factor RBPJ, a known binding partner of EBNA2. We demonstrate that in addition to the activation of the miR-155HG promoter, IRF4 can also activate miR-155HG via the upstream enhancer also targeted by EBNA2. Gene editing to remove the EBNA2- and IRF4-responsive miR-155HG enhancer located 60 kb upstream of miR-155HG led to reduced miR-155HG expression in EBV-infected cells. Our data therefore demonstrate that specific RBPJ-dependent enhancers regulate the IRF4-miR-155 expression network and play a key role in the maintenance of miR-155 expression in EBV-infected B cells. These findings provide important insights that will improve our understanding of miR-155 control in B cell malignancies.IMPORTANCE MicroRNA miR-155 is expressed at high levels in many human cancers, particularly lymphomas. Epstein-Barr virus (EBV) infects human B cells and drives the development of numerous lymphomas. Two genes carried by EBV (LMP1 and EBNA2) upregulate miR-155 expression, and miR-155 expression is required for the growth of EBV-infected B cells. We show that the EBV transcription factor EBNA2 upregulates miR-155 expression by activating an enhancer upstream from the miR-155 host gene (miR-155HG) from which miR-155 is derived. We show that EBNA2 also indirectly activates miR-155 expression through enhancer-mediated activation of IRF4 IRF4 then activates both the miR-155HG promoter and the upstream enhancer, independently of EBNA2. Gene editing to remove the miR-155HG enhancer leads to a reduction in miR-155HG expression. We therefore identify enhancer-mediated activation of miR-155HG as a critical step in promoting B cell growth and a likely contributor to lymphoma development.

Modulation of enhancer looping and differential gene targeting by Epstein-Barr virus transcription factors directs cellular reprogramming.

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.