Molecular mechanisms of transcriptional regulation in the Notch pathway

描述(申请人提供)：癌症的潜在原因通常与信号通路有关，例如Notch通路，它通过突变变得不受控制并发出异常的信号。异常的Notch信号是许多类型癌症发病的基础，最著名的是T细胞急性淋巴细胞性白血病--一种常见的儿童白血病。此外，致癌病毒EBV和KSHV共同选择Notch信号来维持病毒在体内的持久性，并主要在免疫受损的个体中导致癌症。因此，对Notch通路的药物调控为新的抗癌化疗药物带来了巨大的希望。规范的Notch信号导致基因表达的变化，这是由DNA结合转录因子CSL调节的。CSL通过与不同的转录辅助调节因子形成复合体来调节Notch靶基因转录的抑制和激活。CSL在Notch靶基因转录调控中的中心地位使其成为治疗干预的靶点。我们的长期目标是在分子水平上了解转录是如何在Notch途径中调节的，并确定这些过程是否可以被操纵以获得治疗效益。虽然在鉴定活性Notch转录复合体的结构和功能方面已经取得了进展，但我们在分子水平上对CSL如何作为转录抑制因子以及病毒蛋白如何颠覆CSL功能的了解还不完整。这项建议的目的是确定CSL与负转录调控因子和病毒转录调控因子的复合体的结构，使用ITC定义它们的热力学结合参数，并表征它们在细胞检测中的功能。我们假设，通常有两种模式在Notch靶基因上调控转录-一种在蛋白质水平上，协同调节器直接竞争CSL上的结合表面；另两种，在染色质水平上，CSL-协同调节复合体招募组蛋白修饰机制。为了实现我们的目标并验证我们的假设，我们将追求以下三个目标：(1)表征负调控因子如何结合CSL来抑制转录；(2)表征CSL如何招募染色质重塑机制；以及(3)表征CSL-病毒蛋白复合体的结构和功能。这一提议的完成将增加我们对转录如何在Notch途径中调控的理解，从而推动该领域的发展。此外，这些研究的完成将为临床干预提供新的场所，并促进针对Notch信号的小分子的开发，用于抗癌治疗。

DESCRIPTION (provided by applicant): The underlying causes of cancer are often linked to signaling pathways, such as the Notch pathway, which through mutation become deregulated and signal abnormally. Aberrant Notch signaling underlies the pathogenesis of many types of cancer, most notably T-cell acute lymphoblastic leukemia - a common childhood leukemia. Additionally, the oncogenic viruses EBV and KSHV co-opt Notch signaling to maintain viral persistence in vivo and cause cancer primarily in immunocompromised individuals. Therefore, medicinal modulation of the Notch pathway holds great promise for novel anti-cancer chemotherapeutics. Canonical Notch signaling results in changes in gene expression, which is regulated by the DNA binding transcription factor CSL. CSL regulates both repression and activation of transcription from Notch target genes by forming complexes with different transcriptional coregulators. The centrality of CSL in the transcriptional regulation of Notch target genes makes it an attractive target for therapeutic intervention. Our long-term goal is to understand at the molecular level how transcription is regulated in the Notch pathway and to determine whether these processes can be manipulated for therapeutic benefit. While progress has been made in characterizing the structure and function of active Notch transcription complexes, our understanding at the molecular level for how CSL functions as transcriptional repressor and how viral proteins subvert CSL function is incomplete. The objective of this proposal is to determine structures of CSL in complex with negative and viral regulators of transcription, define their thermodynamic binding parameters using ITC, and characterize their function in cellular assays. We hypothesize that there are generally two modes by which transcription is regulated at Notch target genes - one, at the protein level, whereby coregulators directly compete for binding surfaces on CSL; and two, at the chromatin level, whereby CSL-coregulator complexes recruit the histone modification machinery. To achieve our objective and test our hypothesis we will pursue the following three aims: (1) Characterize how negative regulators bind CSL to repress transcription; (2) Characterize how CSL recruits the chromatin remodeling machinery; and (3) Characterize the structure and function of CSL-viral protein complexes. Completion of this proposal will increase our understanding of how transcription is regulated in the Notch pathway, thereby advancing the field. Additionally, completion of these studies will provide new sites of clinical intervention and facilitate the development of small molecules that target Notch signaling for anti-cancer therapeutics.