The long-term goal of this R01 is to understand how epigenetic mechanisms control Epstein-Barr Virus (EBV) latency and carcinogenesis. EBV latent infection is associated with a diverse spectrum of epithelial and lymphoid malignancies. The highly adaptive nature of EBV infection to various host cells and environments suggests that it exploits fundamental cellular processes of dynamic gene regulation. EBV is known to adapt various gene expression programs, termed latency types, in different host cell and tumor environments. These latency types and viral gene expression patterns are determined by epigenetic factors ranging from nucleosome positioning, histone modifications, CpG DNA methylation, transcription factor occupancy, and chromosome conformation. The mechanisms regulating viral and host DNA epigenetic controls are not fully understood but are critical for understanding viral latency and oncogenesis in diverse cell types. We have been investigating the process through which EBV establishes and regulates the epigenetic program of both viral and host genomes. In the previous funding cycles, we identified the viral tegument protein BNRF1 as a binding partner of DAXX-histone H3.3 complex and showed that this interaction is required for viral chromatin assembly and gene expression during the early, pre-latent phase of infection. We have identified viral and cellular transcription factor binding sites for EBNA1, EBNA2, CTCF, cohesin (RAD21), EBF1, RBP JK and chromosome conformations that change during the establishment of latency and correlate with different latency types. We have assayed chromatin accessibility and RNA expression changes during the multiple stages of EBV-induced B-cell immortalization to correlate gene expression with chromatin architecture. We have also found that viral and host DNA methylation programming depends on viral EBNA2 and vmiRNAs that coordinately regulate TET2 expression and cytosine hydroxymethylation and demethylation. We now propose to further advance these studies to better understand the role of epigenetic mechanisms in the control of EBV latency and oncogenicity. We will test the overarching hypothesis that EBV reprograms host epigenetic mechanisms to enable viral genome persistence and transcriptional plasticity that drives EBV-associated oncogenesis.