Recent observations have suggested that the viral oncoproteins SV40 Large T antigen (SV40LT) and Adenovirus E1A play a direct role in transactivation of E2F-dependent genes to promote cell cycle entry. We propose that SV40LT transformation is dependent on its ability to directly transactivate E2F-dependent promoters by binding to chromatin-bound Rb, p107 and p130, thereby recruiting CBP and p300 to activate E2F transcription. In addition, our understanding of how the Rb family controls E2F-dependent gene expression has continued to evolve. Using a sensitive proteomic approach, we confirmed that Rb binds to the BRG1 and BRM family of SWI/SNF proteins. However, it is not known if Rb recruits the BRG1/BRM complex to E2F promoters and if they serve to activate E2F dependent gene expression. To address these questions, we propose the following specific aims.
Specific Aim 1. Determine if SV40LT can directly transactivate E2F promoters. We will perform Chromatin immunoprecipitation (ChIP) for SV40LT antigen on selected E2F dependent promoters. We will identify the host cell factors required for SV40LT binding to E2F-dependent promoters using knockout MEFs and determine if the known transforming domains of SV40LT contribute to E2F transactivation.
Specific Aim 2. Determine if Rb recruits the BRG1/BRM complex to affect E2F activity during quiescence and proliferation. We will determine the ability of the Rb-BRG1/BRM complex to bind specifically to E2F promoters and whether the complex contributes to activation or repression of E2F transcription.
Specific Aim 3. Determine if SV40LT selectively activates E2F promoters to promote entry into the cell cycle. We will perform ChIP using genomic tiling arrays (ChlP-chip) for SV40LT to identify promoter elements that SV40LT selectively binds. These results will be compared with ChlP-chip for Rb, p130, p107, E2F1, E2F2, E2F3 and E2F4. This approach will be used to determine if SV40LT activates all or a subset of E2F promoters to promote cell cycle entry and transformation.
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