Epigenetic Repression of p16INK4A by Latent Epstein-Barr Virus Requires the Interaction of EBNA3A and EBNA3C with CtBP

• Published in: PLoS pathogens 2010-06, Vol.6 (6), p.e1000951
• Main Authors: Skalska, Lenka, White, Robert E., Franz, Melanie, Ruhmann, Michaela, Allday, Martin J.
• Format: Article
• Language: English
• Subjects: Cell Biology/Cell Growth and Division; Cell Biology/Cellular Death and Stress Responses; Cyclin-dependent kinases; Deoxyribonucleic acid; DNA; DNA methylation; Epigenetics; Epstein-Barr virus; Gene expression; Genomes; Infectious Diseases/Viral Infections; Kinases; Molecular Biology/Histone Modification; Molecular Biology/Transcription Initiation and Activation; Oncology/Hematological Malignancies; Oncology/Myelomas and Lymphoproliferative Diseases; Proteins; Virology; Virology/Effects of Virus Infection on Host Gene Expression; Virology/Persistence and Latency; Virology/Viruses and Cancer; Viruses
• ISSN: 1553-7374; 1553-7366; 1553-7374
• DOI: 10.1371/journal.ppat.1000951

As an inhibitor of cyclin-dependent kinases, p16INK4A is an important tumour suppressor and inducer of cellular senescence that is often inactivated during the development of cancer by promoter DNA methylation. Using newly established lymphoblastoid cell lines (LCLs) expressing a conditional EBNA3C from recombinant EBV, we demonstrate that EBNA3C inactivation initiates chromatin remodelling that resets the epigenetic status of p16INK4A to permit transcriptional activation: the polycomb-associated repressive H3K27me3 histone modification is substantially reduced, while the activation-related mark H3K4me3 is modestly increased. Activation of EBNA3C reverses the distribution of these epigenetic marks, represses p16INK4A transcription and allows proliferation. LCLs lacking EBNA3A express relatively high levels of p16INK4A and have a similar pattern of histone modifications on p16INK4A as produced by the inactivation of EBNA3C. Since binding to the co-repressor of transcription CtBP has been linked to the oncogenic activity of EBNA3A and EBNA3C, we established LCLs with recombinant viruses encoding EBNA3A- and/or EBNA3C-mutants that no longer bind CtBP. These novel LCLs have revealed that the chromatin remodelling and epigenetic repression of p16INK4A requires the interaction of both EBNA3A and EBNA3C with CtBP. The repression of p16INK4A by latent EBV will not only overcome senescence in infected B cells, but may also pave the way for p16INK4A DNA methylation during B cell lymphomagenesis. We previously showed that two Epstein-Barr virus latency-associated proteins-EBNA3A and EBNA3C-contribute to enhanced B cell survival by inhibiting the expression of the death-inducing protein BIM. This repression involves remodelling of the BIM gene promoter by polycomb proteins and DNA methylation within an unusually large CpG-island that flanks the transcription initiation site. Here we show that the same two proteins, EBNA3A and EBNA3C, functionally cooperate in the polycomb-mediated chromatin remodelling of another tumour suppressor gene, p16INK4A, that encodes a cyclin-dependent kinase inhibitor capable of blocking cell proliferation. Both EBV proteins can bind the highly conserved co-repressor of transcription CtBP, and these interactions appear to be required for the efficient repression of p16INK4A. Thus by utilising the polycomb system to induce the heritable repression of two major tumour suppressor genes-one that induces cell death (BIM) and one that induces grow