The protein product of the retinoblastoma susceptibility gene (Rb) is a tumor suppressor whose inactivation is associated with the etiology of a subset of human tumors. Although the exact mechanism(s) through which Rb exerts its negative effects on cell growth is unknown, it is thought the ability of Rb to regulate transcription is important for tumor suppression. Rb modulates transcription through the regulation of the activity of specific transcription factors. Rb binds directly to certain transcription factors such as E2F, MyoD, Elf-1, and AFT-2, resulting in either stimulation and/or inhibition of transcription depending upon the cell type. In addition, the activity of certain transcription factors such as Sp1 is regulated by Rb, but no association with Rb has been detected. The PI has demonstrated that Rb stimulation of Sp1-mediated transcription and repression of E2F-mediated is conferred in part through a TAFII250-dependent pathway. TAFII250 is a TBP- associated factor, identified as a cell cycle regulator, that is important for mediating activation by transcription factors such as Sp1. Recently the investigator has demonstrated that Rb and p107 can bind to TAFII250 in vitro and in vivo. Since temperature-sensitive mutations in TAFII250 can lead to a block in the cell cycle at the nonpermissive temperature, it is likely that the interaction between Rb and TAFII250 is important in mediating transcriptional regulation, cell cycle control, and possibly tumor suppression. In addition, the PI has shown that G1 cyclin D1 (PRAD1), identified as a proto-oncogene, also can bind to and confer phosphorylation of TAFII250 in vivo. Thus one focus of this proposal is to examine the role of the Rb/TAFII250 and D1/TAFII250 interactions in modulating transcription and cell cycle control. He also has demonstrated that Rb can negatively regulate transcription when brought to the DNA through the use of the GAL4 DNA binding domain. This observation suggests that Rb can regulate transcription by an active mechanism independent of simply binding to and blocking the transactivation domain of certain transcription factors such as E2F. A second focus of this proposal is to examine the ability of GAL-Rb to negatively regulate transcription when bound to the DNA. Particular emphasis will be placed on determining if an interaction with TAFII250 is required for the observed negative regulation of transcription by GAL4-Rb.
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