The Retinoblastoma (RB) protein is a critical regulator of cell cycle progression, senescence, and differentiation. Until recently, the control of RB function was thought to be achieved through its phosphorylation by cyclin-dependent kinases. However, several other post-translational modifications of the RB protein have recently been uncovered, including acetylation and sumoylation. These modifications of RB may affect its functions in adult cells, ensuring homeostasis in cells of developing and aging organisms. Our long-term goal is to better understand the cellular mechanisms controlling RB function in mammalian cells, focusing on the modifications of RB and their functional interactions. Methylation of histones is a key cellular process to control chromatin structure and gene expression. Emerging evidence suggests that methylation of non-histone proteins also plays important cellular roles. Here, we propose to investigate RB methylation and its regulation in mammalian cells. We have found that RB is methylated at a specific lysine residue by the SMYD2 methyltransferase. We hypothesize that this methylation event participates in the control of RB function. Our first specific goal is to examine the regulation of this methylation event. To this end, we will study RB methylation pattern in various cellular contexts, in culture and in mice. In particular, we will investigate how the cell cycle stage, the differentiation status, senescence, and the presence of cellular stress may affect RB methylation by SMYD2. Our second specific aim is to determine the consequences of RB methylation by SMYD2. Based on previous observations regarding how methylation affects the function of p53, another cell cycle and cell death regulator, we will use a combination of biochemical, molecular, and cellular approaches to assay RB function under different conditions, including DNA damage response and cell cycle progression. Together, these experiments will provide novel insights into the mode of action of RB in cells. RB is a central regulator of multiple cellular processes. A better understanding of the mechanisms that control RB function in mammalian cells will ultimately provide novel ways to control RB function in age- related diseases such as cancer.