Project 3: Defining and targeting mechanisms of E2F transcription factor regulation

项目摘要 E2F转录因子的激活是视网膜母细胞瘤蛋白（Rb）肿瘤中最下游的事件 抑制途径，其控制进入细胞周期和增殖。E2F刺激表达 S期DNA合成和进一步进入有丝分裂所需的基因。Rb失活 E2F控制的转录程序的异常激活是癌症的标志。当Rb E2F的抑制作用已被广泛表征，但关于E2F如何激活转录却知之甚少 以及其他调节机制如何控制E2F功能。在这里，我们探索的结构机制， E2F的功能和翻译后调节。 我们的第一个目标是确定E2F如何与染色质结合并影响染色质结构， 诱导基因表达。我们将使用结构、生化和细胞分析来研究E2F如何结合 核小体以及这些相互作用如何在细胞激活核小体时对调节染色质结构至关重要。 转录程序用于S期进入。我们的第二个目标是鉴定新的E2F翻译后修饰 以及包括磷酸化和乙酰化在内的修饰如何调节E2F与染色质的结合， 辅激活蛋白。我们的第三个目标是揭示E3连接酶接头如何识别E2F蛋白 蛋白质细胞周期蛋白F（CycF）用于泛素化和降解。一个结构和细胞生物学的方法将是 应用于定义CycF结合基序，我们将测试的假设，化学抑制CycF可以 解除模型癌细胞中细胞周期的调控。 通过与其他项目和核心的合作，我们的研究将提供前所未有的分子水平。 分析E2F的功能和调节。我们的实验结果将提供基础知识， 将被应用于设计针对不同癌症中E2F活性的新治疗策略。

PROJECT SUMMARY Activation of E2F transcription factors is the most downstream event in the retinoblastoma protein (Rb) tumor suppressor pathway, which controls entry into the cell cycle and proliferation. E2F stimulates expression of genes needed for DNA synthesis during S phase and for further progression into mitosis. Inactivation of Rb and aberrant activation of the E2F-controlled transcription program is a hallmark of cancer. While Rb repression of E2F has been extensively characterized, little is known regarding how E2F activates transcription and how additional regulatory mechanisms control E2F function. Here we explore the structural mechanisms underlying the function and posttranslational regulation of E2F. Our first aim is to determine how E2F associates with chromatin and influences chromatin architecture to induce gene expression. We will use structural, biochemical, and cellular assays to investigate how E2F binds nucleosomes and how those interactions are critical for modulating chromatin architecture as cells activate the transcription program for S phase entry. Our second aim is to identify novel E2F posttranslational modifications and how modifications including phosphorylation and acetylation modulate E2F association with chromatin and co-activator proteins. Our third aim is to reveal how E2F proteins are recognized by the E3 ligase adaptor protein Cyclin F (CycF) for ubiquitylation and degradation. A structural and cell biology approach will be applied to define the CycF-binding motif, and we will test the hypothesis that chemical inhibition of CycF can deregulate the cell cycle in model cancer cells. In collaboration with the other program projects and cores, our study will provide unprecedented molecular analysis of E2F function and regulation. Our experimental results will provide foundational knowledge that can be applied to design new therapeutic strategies that target E2F activity in diverse cancers.