The Retinoblastoma protein (RB) is a well known tumor suppressor that controls cell cycle and developmentally regulated gene expression. Germ-line mutation of RB is closely linked with retinoblastoma in early childhood and osteosarcoma in adolescence, and is mutated in about half of all human cancers. During normal growth, interactions between E2Fs and RB family proteins, including the Drosophila RB homolog, Rbf1 are regulated by phosphorylation by cyclins and cyclin-dependent kinases (CDKs) and proteolytic destruction by the proteasome. To better understand the mechanism for RB family protein instability, we characterized Rbf1 turnover in Drosophila, and the protein motifs required for its destabilization. We show that specific point mutations in a C-terminal instability element (IE) stabilized Rbf1, but sacrifice repression activity. Rbf1 is destabilized especially in actively proliferating tissues of the larva, indicating that controlled degradation of Rbf1 is linked to developmental signals. The positive linkage between Rbf1 activity and its destruction indicates that the instability and activity relation is similar to that observed in the case of transcriptional activators such as VP16 and Myc. Physical and functional targets of RB and its paralogs p107/p130 have been studied largely in cultured cells, but the full biological context of this family of proteins' activities will likely be revealed only in whole organismal studies. To identify direct targets of the major Drosophila RB counterpart in a developmental context, we carried out ChIP-Seq analysis of Rbf1 in the embryo. The association of the protein with promoters is developmentally controlled; early promoter access is globally inhibited, while later in development Rbf1 was found to associate with promoter-proximal regions of approximately 2,000 genes. In addition to the conserved cell cycle-related genes, a wholly unexpected finding was that Rbf1 targets many components of the insulin, Hippo, JAK/STAT, Notch and other conserved signaling pathways. Rbf1 may thus directly affect output of these essential growth-control and differentiation pathways by regulation of receptor, kinase and downstream effector expression. Rbf1 was also found to target multiple levels of its own regulatory hierarchy. Bioinformatic analysis indicates that different classes of bound genes exhibit distinct promoter motifs, suggesting that the context of Rbf1 recruitment involves diverse transcription factors, which may allow for independent regulation of Rbf1 bound genes. Many of these targeted genes are bound by Rbf1 homologs in human cells, indicating that a conserved role of retinoblastoma proteins may be to adjust the set point of interlinked signaling networks essential for growth and development.
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