RESEARCH ARTICLE 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 regu-lated 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 transacti-vation 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 confor-mation 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 28kb 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 targe