ABSTRACTThe 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 activatesIRF4transcription, indicating that EBV may upregulate miR-155 through direct and indirect mechanisms. The mechanism of transcriptional regulation ofIRF4andmiR-155HGby EBNA2, however, has not been defined. We demonstrate that EBNA2 can activateIRF4andmiR-155HGexpression 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 themiR-155HGpromoter, IRF4 can also activatemiR-155HGvia the upstream enhancer also targeted by EBNA2. Gene editing to remove the EBNA2- and IRF4-responsivemiR-155HGenhancer located 60 kb upstream ofmiR-155HGled to reducedmiR-155HGexpression 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.IMPORTANCEMicroRNA 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 activatesmiR-155expression through enhancer-mediated activation ofIRF4. IRF4 then activates both themiR-155HGpromoter and the upstream enhancer, independently of EBNA2. Gene editing to remove themiR-155HGenhancer leads to a reduction inmiR-155HGexpression. We therefore identify enhancer-mediated activation ofmiR-155HGas a critical step in promoting B cell growth and a likely contributor to lymphoma development.
The Nuclear Chaperone Nucleophosmin Escorts an Epstein-Barr Virus Nuclear Antigen to Establish Transcriptional Cascades for Latent Infection in Human B Cells
