PROJECT SUMMARY Cell growth triggers human cell division at the G1/S transition before DNA is replicated. This process is important because it determines the size of proliferating cells, which is important for their physiological functions. Larger cells, including macrophages and hepatocytes, often have additional copies of their genome in proportion to their increased cell size. Typically, such large cells maintain their DNA-to-cytoplasm ratio by triggering DNA synthesis, but not division, at cell sizes in proportion to their ploidy. However, while cell size and ploidy are frequently correlated, the function of maintaining the DNA-to-cytoplasm ratio is unclear. Moreover, we do not know the molecular mechanisms controlling the DNA-to-cytoplasm ratio despite having identified many key cell cycle regulatory proteins. Here, we propose to determine both the function of the DNA-to-cytoplasm ratio and the regulatory mechanisms linking cell growth to DNA replication in vivo by examining mouse hepatocytes in developmental and regenerative contexts. The scientific premise of this work is a recent breakthrough that my laboratory made in understanding how cell growth triggers division. Contrary to expectations that growth would increase Cyclin D-Cdk4,6 activity, we found instead that cell growth dilutes the cell cycle inhibitor Rb to trigger division in cultured cells. Our discovery of the Rb dilution mechanism in vitro raises the question if this mechanism operates in vivo. Here, we propose to definitively test the Rb dilution, and an alternative model in which small cells activate p38 to inhibit cell division in mouse hepatocytes. We will measure changes in hepatocyte cell size and how cell growth is coupled to cell cycle progression in a series of mouse lines in which Rb1 has been conditionally deleted, knocked down or over-expressed. Preliminary data indicate that for hepatocytes of the same ploidy, the DNA-to-cytoplasm ratio is inversely correlated with Rb1 gene dosage, consistent with the Rb- dilution model. We will use these genetic models that change the DNA-to-cytoplasm ratio to test its function in the liver. More specifically, we will use inducible knockdown and over-expression alleles to generate hepatocytes that are larger and smaller than wild type and have aberrant DNA-to-cytoplasm ratios. We will then perform a panel of liver function and regeneration tests. This is important because aberrant DNA-to-cytoplasm ratio is associated with various pathological states, but its function in vivo is still unclear in any cell type. Taken together, the proposed work is important because determining how cell growth triggers cell division is a fundamental question in cell and developmental biology. Its understanding will also provide insight into cancers, where this process is misregulated.