title: Epstein-Barr Virus Proteins EBNA3A and EBNA3C Together Induce Expression of the Oncogenic MicroRNA Cluster miR-221/miR-222 and Ablate Expression of Its Target p57KIP2.
abstract: We show that two host-encoded primary RNAs (pri-miRs) and the corresponding microRNA (miR) clusters--widely reported to have cell transformation-associated activity--are regulated by EBNA3A and EBNA3C. Utilising a variety of EBV-transformed lymphoblastoid cell lines (LCLs) carrying knockout-, revertant- or conditional-EBV recombinants, it was possible to demonstrate unambiguously that EBNA3A and EBNA3C are both required for transactivation of the oncogenic miR-221/miR-222 cluster that is expressed at high levels in multiple human tumours--including lymphoma/leukemia. ChIP, ChIP-seq, and chromosome conformation capture analyses indicate that this activation results from direct targeting of both EBV proteins to chromatin at the miR-221/miR-222 genomic locus and activation via a long-range interaction between enhancer elements and the transcription start site of a long non-coding pri-miR located 28 kb upstream of the miR sequences. Reduced levels of miR-221/miR-222 produced by inactivation or deletion of EBNA3A or EBNA3C resulted in increased expression of the cyclin-dependent kinase inhibitor p57KIP2, a well-established target of miR-221/miR-222. MiR blocking experiments confirmed that miR-221/miR-222 target p57KIP2 expression in LCLs. In contrast, EBNA3A and EBNA3C are necessary to silence the tumour suppressor cluster miR-143/miR-145, but here ChIP-seq suggests that repression is probably indirect. This miR cluster is frequently down-regulated or deleted in human cancer, however, the targets in B cells are unknown. Together these data indicate that EBNA3A and EBNA3C contribute to B cell transformation by inhibiting multiple tumour suppressor proteins, not only by direct repression of protein-encoding genes, but also by the manipulation of host long non-coding pri-miRs and miRs.
authors.affiliation: Molecular Virology, Department of Medicine, Imperial College London, London, United Kingdom.
authors.forename: Quentin
authors.initials: Q
authors.lastname: Bazot
authors.affiliation: Molecular Virology, Department of Medicine, Imperial College London, London, United Kingdom.
authors.forename: Kostas
authors.initials: K
authors.lastname: Paschos
authors.affiliation: Molecular Virology, Department of Medicine, Imperial College London, London, United Kingdom.
authors.forename: Lenka
authors.initials: L
authors.lastname: Skalska
authors.affiliation: Molecular Virology, Department of Medicine, Imperial College London, London, United Kingdom.
authors.forename: Jens S
authors.initials: JS
authors.lastname: Kalchschmidt
authors.affiliation: Molecular Virology, Department of Medicine, Imperial College London, London, United Kingdom.
authors.forename: Gillian A
authors.initials: GA
authors.lastname: Parker
authors.affiliation: Molecular Virology, Department of Medicine, Imperial College London, London, United Kingdom.
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Allday
mesh_terms: D001402:B-Lymphocytes / Q000821:virology; D015153:Blotting, Western; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D047369:Chromatin Immunoprecipitation; D050761:Cyclin-Dependent Kinase Inhibitor p57 / Q000096:biosynthesis; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics* / Q000378:metabolism; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D006801:Humans; D047468:Immunoprecipitation; D035683:MicroRNAs / Q000096:biosynthesis / Q000235:genetics*; D009857:Oncogenes; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D050761:Cyclin-Dependent Kinase Inhibitor p57; D019309:Epstein-Barr Virus Nuclear Antigens; D035683:MicroRNAs; C508151:EBNA-3A antigen; C498774:EBNA-3C, epstein-barr virus; C525709:MIR221, human; C525710:MIR222, human