SV40 Large T and Rb Effects on E2F Dependent Genes

最近的观察表明，病毒癌蛋白SV 40大T抗原（SV 40 LT）和 腺病毒E1 A在E2 F依赖基因的反式激活中发挥直接作用，以促进细胞进入周期。我们 我提出SV 40 LT转化依赖于其直接反式激活E2 F依赖性的能力， 通过结合染色质结合的Rb，p107和p130，从而募集CBP和p300激活启动子， E2 F转录。此外，我们对Rb家族如何控制E2 F依赖基因的理解， 表达方式不断演变。使用敏感的蛋白质组学方法，我们证实Rb与 SWI/SNF蛋白的BRG 1和BRM家族。然而，不知道Rb是否募集BRG 1/BRM 复合物的E2 F启动子，如果它们用于激活E2 F依赖性基因表达。解决这些 针对这些问题，我们提出以下具体目标。 具体目标1。确定SV 40 LT是否可以直接反式激活E2 F启动子。我们将执行 在选定的E2 F依赖性启动子上进行SV 40 LT抗原的染色质免疫沉淀（ChIP）。我们将 使用敲除MEF鉴定SV 40 LT与E2 F依赖性启动子结合所需的宿主细胞因子 并确定SV 40 LT的已知转化结构域是否有助于E2 F反式激活。 具体目标2。确定Rb是否招募BRG 1/BRM复合物来影响E2 F活性 静止和增殖。我们将确定Rb-BRG 1/BRM复合物特异性结合 E2 F启动子以及复合物是否有助于激活或抑制E2 F转录。 具体目标3。确定SV 40 LT是否选择性激活E2 F启动子以促进进入细胞 周期我们将使用SV 40 LT的基因组平铺阵列（ChIP-芯片）进行ChIP以鉴定启动子 SV 40 LT选择性结合的元件。将这些结果与Rb、p130、p107、 E2 F1、E2 F2、E2 F3和E2 F4。该方法将用于确定SV 40 LT是否激活所有或部分 E2 F启动子促进细胞周期进入和转化。

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.