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

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-, revert...

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Bibliographic Details
Published in:PLoS pathogens 2015-07, Vol.11 (7), p.e1005031
Main Authors: Bazot, Quentin, Paschos, Kostas, Skalska, Lenka, Kalchschmidt, Jens S, Parker, Gillian A, Allday, Martin J
Format: Article
Language:English
Subjects:
B-Lymphocytes - virology
Blotting, Western
Cell Transformation, Neoplastic - genetics
Chromatin Immunoprecipitation
Cyclin-Dependent Kinase Inhibitor p57 - biosynthesis
Epstein-Barr Virus Nuclear Antigens - genetics
Epstein-Barr Virus Nuclear Antigens - metabolism
Flow Cytometry
Gene Expression Regulation, Neoplastic - genetics
Humans
Immunoprecipitation
MicroRNAs - biosynthesis
MicroRNAs - genetics
Oncogenes
Real-Time Polymerase Chain Reaction
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Summary: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.
ISSN:1553-7374
1553-7366
1553-7374
DOI:10.1371/journal.ppat.1005031