Expanding the roles of Rb-E2F in cell cycle control and tumorigenesis

Cell division is an essential and ubiquitous process of life. In multi-cellular organisms, the need for balancing individual cell growth and proper development and homeostasis of an organism is delicately maintained through an elaborate signaling network of cell cycle control. Central to any division process is the decision to replicate the genetic content of each cell. In mammalian cells, a set of transcription factors called E2F plays pivotal roles in directing progression from G₀ to S phase and in synthesizing the components for the replication machinery. Regulation of E2F activity is mediated by the retinoblastoma protein (pRB) and related proteins, p107 and p130. RB is a classical tumor suppressor gene. Humans born with only one copy of RB will invariably develop retinoblastoma, and mice develop pituitary and thyroid tumors. In this dissertation, two studies are presented. The first study examines the contribution of pRB-E2F4 interactions to tumorigenesis while the second study utilizes a recently developed genome-wide location analysis to identify novel E2F target genes.; The E2F family consists of six members, E2F1-6. pRB has been shown to interact exclusively with E2F1-E2F4. E2F1-E2F3 behave as activators of transcription while E2F4-E2F5 proteins appear to function as repressors. E2F1-loss in RB+/− mice reduces the incidence of pituitary and thyroid tumors. Here we examine the consequences of loss of E2F4, the major repressive E2F, on tumorigenesis in RB+/− mice. We show that, contrary to the prediction that loss of E2F4 would exacerbate the tumor phenotype, E2F4-loss dramatically delays and reduces the incidence of pituitary and thyroid tumors in Rb +/−; E2f4−/− mice. Our biochemical analysis uncovers a novel “reshuffling” mechanism among the remaining E2Fs with p107 and p130 that could explain the dramatic absence of tumors in Rb+/−; E2f4−/− mice.; A major question in the area of cell cycle regulated gene expression pertains to the identification of physiological targets of relevant transcription factors such as E2F and pRB. Indeed, fewer than two dozen genes have been shown to be controlled by E2F, and each of them participates either in the G₁/S phase transition or DNA replication. In the second part of this thesis, using genome-wide location analysis we have identified 127 target genes whose promoters are bound by E2F4 in quiescent human primary fibroblasts. Fifty of these are also bound by E2F1 during G₁ phase. Clustering analysis coupled with expression profiling allowed us to discover additional novel pathways regulated by E2F beyond known roles in the G₁/S transition and DNA replication.