The latent infection of Epstein-Barr Virus (EBV) is associated with human lymphoid and epithelial cell malignancies. Reversible epigenetic silencing of EBV-encoded oncogenes is known to provide the virus with a survival strategy that includes controlling B-cell maturation and evading immune system detection. The cellular signals and mechanisms regulating the epigenetic silencing have not been fully elucidated. We have investigated the chromatin organization and epigenetic modifications in the control regions of the major latency transcripts of EBV in different latency types. Our studies suggest that epigenetic modifications play a key role in regulating the different gene expression programs of EBV latency. We focus on the control of the major latency promoters Cp and Qp, and how they are regulated by the tumor suppressor protein Rb (aim1), the viral- encoded origin binding protein EBNA1 (aim 2), and the chromatin boundary factor CTCF (aim 3). Rb and Rb-associated factors bind to Cp and regulate a cell cycle-dependent change in chromatin modifications. We propose to explore the role of Rb and Rb-associated factors in the epigenetic silencing that occurs at Cp in type I latency, and cell cycle regulation in type III. For aim 2, we focus on the role of EBNA1 in the transcription enhancement of Cp and how this is regulated in different cell types. We propose to identify a B-cell specific mediated of EBNA1 enhancer function and whether enhancer-promoter activation correlates with changes in viral chromosome conformation. In aim 3, we focus on the role of CTCF in regulating the interactions between enhancers and promoters, and in insulating active promoters from silent genes during latency. The long-term goal of this project is to understand the epigenetic mechanisms regulating viral gene expression during latency, and how these mechanisms contribute to viral pathogenesis.