Lymphomagenesis in the presence of deregulated MYC requires suppression of MYC-driven apoptosis, often through downregulation of the pro-apoptotic BCL2L11 gene (Bim). Transcription factors (EBNAs) encoded by the lymphoma-associated Epstein-Barr virus (EBV) activate MYC and silence BCL2L11. We show that the EBNA2 transactivator activates multiple MYC enhancers and reconfigures the MYC locus to increase upstream and decrease downstream enhancer-promoter interactions. EBNA2 recruits the BRG1 ATPase of the SWI/SNF remodeller to MYC enhancers and BRG1 is required for enhancer-promoter interactions in EBV-infected cells. At BCL2L11, we identify a haematopoietic enhancer hub that is inactivated by the EBV repressors EBNA3A and EBNA3C through recruitment of the H3K27 methyltransferase EZH2. Reversal of enhancer inactivation using an EZH2 inhibitor upregulates BCL2L11 and induces apoptosis. EBV therefore drives lymphomagenesis by hijacking long-range enhancer hubs and specific cellular co-factors. EBV-driven MYC enhancer activation may contribute to the genesis and localisation of MYC-Immunoglobulin translocation breakpoints in Burkitt's lymphoma.DOI: http://dx.doi.org/10.7554/eLife.18270.001

In B cells infected by the cancer-associated Epstein-Barr virus (EBV), RUNX3 and RUNX1 transcription is manipulated to control cell growth. The EBV-encoded EBNA2 transcription factor (TF) activates RUNX3 transcription leading to RUNX3-mediated repression of the RUNX1 promoter and the relief of RUNX1-directed growth repression. We show that EBNA2 activates RUNX3 through a specific element within a −97 kb super-enhancer in a manner dependent on the expression of the Notch DNA-binding partner RBP-J. We also reveal that the EBV TFs EBNA3B and EBNA3C contribute to RUNX3 activation in EBV-infected cells by targeting the same element. Uncovering a counter-regulatory feed-forward step, we demonstrate EBNA2 activation of a RUNX1 super-enhancer (−139 to −250 kb) that results in low-level RUNX1 expression in cells refractory to RUNX1-mediated growth inhibition. EBNA2 activation of the RUNX1 super-enhancer is also dependent on RBP-J. Consistent with the context-dependent roles of EBNA3B and EBNA3C as activators or repressors, we find that these proteins negatively regulate the RUNX1 super-enhancer, curbing EBNA2 activation. Taken together our results reveal cell-type-specific exploitation of RUNX gene super-enhancers by multiple EBV TFs via the Notch pathway to fine tune RUNX3 and RUNX1 expression and manipulate B-cell growth.

Epstein-Barr virus (EBV) is implicated in the pathogenesis of multiple human tumours of lymphoid and epithelial origin. The virus infects and immortalizes B cells establishing a persistent latent infection characterized by varying patterns of EBV latent gene expression (latency 0, I, II and III). The CDK1 activator, Response Gene to Complement-32 (RGC-32, C13ORF15), is overexpressed in colon, breast and ovarian cancer tissues and we have detected selective high-level RGC-32 protein expression in EBV-immortalized latency III cells. Significantly, we show that overexpression of RGC-32 in B cells is sufficient to disrupt G2 cell-cycle arrest consistent with activation of CDK1, implicating RGC-32 in the EBV transformation process. Surprisingly, RGC-32 mRNA is expressed at high levels in latency I Burkitt's lymphoma (BL) cells and in some EBV-negative BL cell-lines, although RGC-32 protein expression is not detectable. We show that RGC-32 mRNA expression is elevated in latency I cells due to transcriptional activation by high levels of the differentially expressed RUNX1c transcription factor. We found that proteosomal degradation or blocked cytoplasmic export of the RGC-32 message were not responsible for the lack of RGC-32 protein expression in latency I cells. Significantly, analysis of the ribosomal association of the RGC-32 mRNA in latency I and latency III cells revealed that RGC-32 transcripts were associated with multiple ribosomes in both cell-types implicating post-initiation translational repression mechanisms in the block to RGC-32 protein production in latency I cells. In summary, our results are the first to demonstrate RGC-32 protein upregulation in cells transformed by a human tumour virus and to identify post-initiation translational mechanisms as an expression control point for this key cell-cycle regulator.

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.

The EBNA 2 (Epstein-Barr nuclear antigen 2) transcription factor is essential for B-cell transformation by the cancer-associated EBV (Epstein-Barr virus) and for the continuous proliferation of infected cells. EBNA 2 activates transcription from the viral Cp (C promoter) during infection to generate the 120 kb transcript that encodes all nuclear antigens required for immortalization by EBV. EBNA 2 contains an acidic activation domain and can interact with a number of general transcription factors and co-activators. It is now becoming clear, however, that the regulation of transcription elongation in addition to initiation by EBNA 2, at least in part through CDK9 (cyclin-dependent kinase 9)-dependent phosphorylation of the RNA polymerase C-terminal domain, is likely to play a crucial role in the mechanism of action of this key viral protein.

Page 5, Fig. 2D: Due to errors in final figure assembly, the actin loading control blots were placed in the incorrect positions under the left and right sections. The right-hand blot should be on the left, and the left-hand blot should be on the right.Figure 2D should appear as shown below.

No abstract

16The oncogenic microRNA-155 (miR-155) is the most frequently upregulated miRNA in 17 Epstein-Barr virus (EBV)-positive B cell malignancies and is upregulated in other non-18 viral lymphomas. Both the EBV nuclear antigen 2 (EBNA2), and B cell transcription 19 factor, interferon regulatory factor 4 (IRF4) are known to activate transcription of the host 20 cell gene from which miR-155 is processed (miR-155HG, BIC). EBNA2 also activates 21IRF4 transcription indicating that EBV may upregulate miR-155 through direct and 22 indirect mechanisms. The mechanism of transcriptional regulation of IRF4 and miR-23 155HG by EBNA2 however has not been defined. We demonstrate that EBNA2 can 24 activate IRF4 and miR-155HG expression through specific upstream enhancers that are 25 dependent on the Notch signaling transcription factor RBPJ, a known binding partner of 26 EBNA2. We demonstrate that in addition to activation of the miR-155HG promoter, IRF4 27 can also activate miR-155HG via the upstream enhancer also targeted by EBNA2. Gene 28 editing to remove the EBNA2-and IRF4-responsive miR-155HG enhancer located 60 kb 29 upstream of miR-155HG led to reduced miR155HG expression in EBV-infected cells. Our 30 data therefore demonstrate that specific RBPJ-dependent enhancers regulate the IRF4-31 miR-155 expression network and play a key role in the maintenance of miR-155 expression 32 in EBV-infected B cells. These findings provide important insights that will improve our 33 understanding of miR-155 control in B cell malignancies. 34 35 IMPORTANCE 36MicroRNA-155 (miR-155) is expressed at high level in many human cancers particularly 37 lymphomas. Epstein-Barr virus (EBV) infects human B cells and drives the development 38. CC-BY 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/311886 doi: bioRxiv preprint first posted online May. 1, 2018; 3 of numerous lymphomas. Two EBV-encoded genes (LMP1 and EBNA2) upregulate miR-39 155 expression and miR-155 expression is required for the growth of EBV-infected B cells. 40We show that the EBV transcription factor EBNA2 upregulates miR-155 expression by 41 activating an enhancer upstream from the miR-155 host gene (miR-155HG) from which 42 miR-155 is derived. We show that EBNA2 also indirectly activates miR-155 expression 43 through enhancer-mediated activation of IRF4. IRF4 then activates both the miR-155HG 44 promoter and the upstream enhancer, independently of EBNA2. Gene editing to remove 45 the miR-155HG enhancer leads to a reduction in miR-155HG expression. We therefore 46 identify enhancer-mediated activation of miR-155HG as a critical step in promoting B cell 47 growth and a likely driver of lymphoma development. 48 49. CC-BY 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in pe...