PROJECT SUMMARY/ABSTRACT Retinoblastoma is a rare pediatric cancer that is caused by loss of both copies of the retinoblastoma (Rb) tumor suppressor gene. Rb is a highly conserved and critical transcription factor for the proper regulation of gene expression in eukaryotes. Mutations in Rb have also been observed in many human cancers including breast and lung cancer. In humans, the Rb family comprises Rb, p107, and p130, which exhibit both overlapping and non-redundant roles in gene regulation. Similarly, the Drosophila lineage experienced a gene duplication event leading to the expression of the Rbf1 and Rbf2 paralogs. The significance of the multiplicity of the Rb family and their division of labor in regulating gene expression in different contexts is not clear. Their activity has been studied in the context of specific promoters; however, we lack deeper mechanistic understanding of these factors in the context of normal tissues, in development. This proposal will investigate the tissue-specific gene regulatory activities of retinoblastoma family proteins using the fly model system. We hypothesize that the Rb family has diversified to have gene-specific activities through differential targeting to promoters and through different repression activities. In Aim 1, we will investigate how Rb paralogs exhibit mechanistically different transcriptional control while in Aim 2 we will determine how regulation of the Rbf1 protein itself controls promoter-specific repression. We will use our newly developed method for highly precise targeting of Rb paralogs to diverse gene promoters through Rb fusions to a nuclease dead Cas9. This powerful tool allows for direct comparison of the Rb family members in a developmental system to study their gene- and tissue-specific activities. Using this tool, we will determine the differences between Rbf1 and Rbf2 impact on gene expression, and differences in modulation of the chromatin environment. This project will be carried out at Michigan State University in the lab of Dr. David Arnosti as part of a comprehensive academic and professional training plan to prepare the applicant for a career as an independent research scientist investigating gene regulation in the context of disease. With the completion of the proposed work, the results will uncover the mechanisms of gene regulation by Rb family proteins, which will improve our understanding of why certain tissues are especially sensitive to inactivation of Rb. Understanding the mechanisms of Rb-mediated gene regulation and its activity as a transcriptional repressor will allow for the development of gene therapies to treat Retinoblastoma and other cancer types.