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.

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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...