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-,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 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 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. © 2015 bazot et al.