Molecular mechanisms of transcriptional regulation in the Notch pathway

描述（由申请人提供）：癌症的根本原因通常与信号传导途径有关，例如Notch途径，其通过突变变得失调并异常信号传导。Notch信号异常是许多类型癌症发病机制的基础，最著名的是T细胞急性淋巴细胞白血病-一种常见的儿童白血病。此外，致癌病毒EBV和KSHV共同选择Notch信号传导以维持病毒在体内的持续存在并主要在免疫功能低下的个体中引起癌症。因此，Notch途径的药物调节对于新型抗癌化疗药物具有很大的希望。经典Notch信号传导导致基因表达的变化，其由DNA结合转录因子CSL调节。CSL通过与不同的转录辅调节因子形成复合物来调节Notch靶基因转录的抑制和激活。CSL在Notch靶基因的转录调控中的中心地位使其成为治疗干预的有吸引力的靶标。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与转录负调控因子和病毒调控因子复合的结构，使用ITC定义其热力学结合参数，并表征其在细胞测定中的功能。我们假设，通常有两种模式，转录调节Notch靶基因-一个，在蛋白质水平，其中辅助调节因子直接竞争CSL上的结合表面;和两个，在染色质水平，其中CSL辅助调节因子复合物招募组蛋白修饰机制。为了实现我们的目标和测试我们的假设，我们将追求以下三个目标：（1）表征负调节剂如何结合CSL抑制转录;（2）表征CSL如何招募染色质重塑机制;（3）表征CSL-病毒蛋白复合物的结构和功能。完成这一提案将增加我们对Notch途径中转录如何调节的理解，从而推进该领域。此外，这些研究的完成将提供新的临床干预位点，并促进靶向Notch信号传导的小分子的开发，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.