pRB, p107 and p130 comprise a family of structurally and functionally related proteins. Insight into the normal functions of these proteins has been provided by the analysis of knockout mice. Mice lacking pRB die between days 13.5 and 15.5 of embryogenesis and contain many additional 5-phase cells. Although mice lacking either plO7 or p13O have no obvious abnormalities, mice deficient for both proteins die within a few hours of birth and display defects in cell cycle. Thus, these proteins normally act to restrict cell proliferation and, either directly or indirectly, promote differentiation. How do pRB, p 1O7 and p13O regulate cell proliferation? It is generally thought that their control of the E2F transcription factor is central to their biological functions. All three proteins associate with E2F in vivo in regulatory complexes that exist transiently through the cell cycle. E2F is a critical component of the cell cycle machinery. It is a highly complex activity that co-ordinates the increase in expression of a large number of genes during cell cycle progression from G1 to S phase. The elevation of E2F activity is sufficient to drive quiescent cells into S phase and E2F is required for S-phase entry pRB, p1O7 and p13O are thought to provide different aspects of E2F regulation. However, many genes have been proposed to be targets of E2F and it has been unclear how the functions of pRB, p107 and p13O are integrated to regulate the expression of any of these targets. We have begun to solve this problem by investigating how the expression of E2F target genes is changed in the absence of pRB, p107 or P130. Our initial results show that completely different sets of E2F-target genes are disregulated in primary cells lacking p1O7 and pl30 compared with cells lacking pRB. These data lead us to the hypothesis that pRB and p107 and p130 provide distinct elements of E2F regulation and that the loss of these functions disregulate cell cycle progression in different ways. In this proposal we will (l) distinguish the roles of pRB, p107 and p13O in E2F regulation by completing the analysis of E2F-target genes in primary cells prepared from single and double knockout mice. (2) investigate the functional changes in cell cycle control that occur in the absence of pRB-family proteins by comparing the ease with which these cells can be driven into the cell cycle and asking whether any of these cells are unable to respond to defined mechanisms of G I arrest. (3) identify and characterize a novel regulator E2F that is specifically upregulated in cells lacking both p1O7 and p130.
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