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 ( ; BIC). EBNA2 also activates transcription, indicating that EBV may upregulate miR-155 through direct and indirect mechanisms. The mechanism of transcriptional regulation of and by EBNA2, however, has not been defined. We demonstrate that EBNA2 can activate and 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 promoter, IRF4 can also activate via the upstream enhancer also targeted by EBNA2. Gene editing to remove the EBNA2- and IRF4-responsive enhancer located 60 kb upstream of led to reduced 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. 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 ( ) from which miR-155 is derived. We show that EBNA2 also indirectly activates expression through enhancer-mediated activation of IRF4 then activates both the promoter and the upstream enhancer, independently of EBNA2. Gene editing to remove the enhancer leads to a reduction in expression. We therefore identify enhancer-mediated activation of as a critical step in promoting B cell growth and a likely contributor to lymphoma development.