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

J Virol. 2018 Sep 12;92(19):e00716-18. doi: 10.1128/JVI.00716-18. Print 2018 Oct 1.

Abstract

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.

Keywords: EBNA2; Epstein-Barr virus; enhancer; miR-155; transcriptional regulation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • B-Lymphocytes / metabolism*
  • B-Lymphocytes / pathology
  • B-Lymphocytes / virology
  • Cells, Cultured
  • Enhancer Elements, Genetic*
  • Epstein-Barr Virus Infections / metabolism*
  • Epstein-Barr Virus Infections / pathology
  • Epstein-Barr Virus Infections / virology
  • Epstein-Barr Virus Nuclear Antigens / genetics
  • Epstein-Barr Virus Nuclear Antigens / metabolism
  • Gene Expression Regulation*
  • Herpesvirus 4, Human / physiology*
  • Humans
  • Interferon Regulatory Factors / genetics
  • Interferon Regulatory Factors / metabolism
  • MicroRNAs / genetics*
  • Promoter Regions, Genetic
  • Viral Proteins / genetics
  • Viral Proteins / metabolism

Substances

  • EBNA-2 protein, Human herpesvirus 4
  • Epstein-Barr Virus Nuclear Antigens
  • Interferon Regulatory Factors
  • MIRN155 microRNA, human
  • MicroRNAs
  • Viral Proteins
  • interferon regulatory factor-4