PROJECT SUMMARY/ABSTRACT The overall goal of our research program is to delineate molecular mechanisms that regulate cell proliferation and differentiation in the context of animal development. This knowledge will help to advance the understanding of normal processes in the developing multicellular organisms, explain why dysregulation of these mechanisms leads to disease, and provide strategies to ameliorate these unwanted effects. We focus on the highly conserved Retinoblastoma (RB) pathway, which is involved in plethora of biological processes but is best known for its role in cell cycle control. One of the key targets of the RB pathway is the E2F family of transcription factors that is negatively regulated by the Retinoblastoma protein (pRB). Mammalian RB research has traditionally centered around its prominent role in cancer and therefore, its function in development remains poorly understood. A major hurdle to studying the RB pathway in mammalian development is redundancy and compensation, as the large multigene Rb and E2F families make genetic analysis daunting. The Drosophila model system provides an attractive alternative because the Drosophila RB pathway is highly conserved yet it is simpler. In previous years, we have focused on identifying tissues and functions of E2F and Rbf, the fly pRB ortholog, that are essential for animal viability. During these studies, we have found that the RB pathway is particularly important in adult skeletal muscle. Surprisingly, in the muscle, both E2F and Rbf are needed in late muscle development to directly activate the expression of metabolic genes. We will use genomics approaches coupled with genetic analysis to investigate the molecular details of E2F/Rbf dependent activation. We will focus on the role of E2F/Rbf at enhancers and how the loss of E2F/Rbf binding at these regulatory regions changes chromatin state and gene expression. Previosuly, we have found that the phenotype of E2F deficient animals is a combination of both tissue intrinsic and systemic effects. Our future research will build on this discovery to determine why E2F inactivation in skeletal muscle leads to lethality. Another area of interest is focused on interaction between the RB pathway and Hippo signaling pathway. Combined inactivation of both pathways leads to photoreceptor dedifferentiation because the cells inappropriately turn on the eye progenitor transcriptional program, which is dependent on Homothorax (Hth) and Yorkie, a transcriptional effector of the Hippo pathway. Our future experiments will leverage the power of single cell genomics and single cell epigenomics to determine the upstream regulatory events and identify the role of Hth-Yorkie in transcriptional regulation in dedifferentiating photoreceptors and in normal eye progenitor cells. Collectively, the proposed research will advance our understanding of normal development and animal physiology, and how perturbations in growth pathways may lead to disease.