Molecular mechanisms of transcriptional regulation in the Notch pathway

描述（由申请人提供）：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信号。异常的Notch信号是许多类型癌症的发病机制的基础，最显著的是t细胞急性淋巴细胞白血病——一种常见的儿童白血病。此外，致癌性病毒EBV和KSHV利用Notch信号维持病毒在体内的持久性，主要在免疫功能低下的个体中引起癌症。因此，Notch通路的药物调节对新型抗癌化疗药物具有很大的前景。典型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通过与不同的转录共调节因子形成复合物来调节Notch靶基因的转录抑制和激活。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一个有吸引力的靶点。我们的长期目标是在分子水平上了解Notch通路中转录是如何调节的，并确定这些过程是否可以被操纵以获得治疗益处。虽然在表征活性Notch转录复合物的结构和功能方面取得了进展，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破坏CSL功能的理解是不完整的。本提案的目的是确定CSL与阴性和病毒转录调节因子复合物的结构，使用ITC定义其热力学结合参数，并在细胞分析中表征其功能。我们假设Notch靶基因的转录调节通常有两种模式：一种是在蛋白质水平上，共调节因子直接竞争CSL上的结合表面；第二，在染色质水平，csl -共调节复合物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)描述负调控因子如何结合CSL抑制转录；(2)描述CSL如何招募染色质重塑机制；(3)鉴定csl -病毒蛋白复合物的结构和功能。这一建议的完成将增加我们对Notch通路中转录是如何调节的理解，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位点，并促进靶向Notch信号的抗癌治疗小分子的发展。

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.