Probing complex assembly and dynamics of the transcription factor RBPJ by live cell single-molecule microscopy

Notch途径将细胞间的交流转化为特定的转录程序，从而在调节组织分化和动态平衡方面发挥重要作用。Notch信号的失调通常会导致癌症。当Notch激活时，Notch胞内域(NICD)移位到细胞核内，在转录因子RBPJ周围招募多蛋白复合体，激活Notch靶基因。在没有Notch激活的情况下，RBPJ通过招募不同的辅因子来抑制Notch靶基因。虽然Notch信号通路的整个激活和抑制周期得到了很好的研究，但关于复合体形成和靶基因激活的重要动力学信息仍然缺失。例如，目前还不清楚从抑制物到激活复合体的转换是否涉及NICD和辅助抑制物结合之间的竞争，以及RBPJ不同的基因调控活性是否反映在抑制物和激活复合体的不同DNA结合特性中。在这个建议中，我们旨在了解Notch信号调节的动力学基础，特别是抑制物和激活物复合体的形成以及Notch靶基因激活的时间分布。我们将对Notch信号通路的组成部分进行定量的活细胞单分子荧光跟踪实验，以获得动力学信息，并利用最先进的生化和光遗传学方法激活Notch信号并表征其时间分布。此外，我们还想分析肿瘤相关的Notch突变对这些动力学参数的影响。

The Notch pathway translates cell-to-cell communication into a specific transcription program and thereby plays an important role in regulating tissue differentiation and homeostasis. Dysregulation of Notch signalling often results in cancer. Upon Notch activation, the Notch intracellular domain (NICD) translocates into the nucleus, recruits a multi-protein complex around the transcription factor RBPJ and activates Notch target genes. In the absence of Notch activation, RBPJ represses Notch target genes by recruiting different cofactors. While the overall activating and repression cycle of the Notch signalling pathway is well investigated, important kinetic information on complex formation and target gene activation is missing. For example, it is unclear whether the switch from a repressing to an activating complex involves competition between NICD and co-repressor binding and whether the differential gene regulatory activity of RBPJ is reflected in different DNA-binding properties of the repressing and activating complexes.In this proposal, we aim at understanding the kinetic underpinnings of Notch signalling regulation, in particular those of repressor and activator complex formation and the temporal profile of Notch target gene activation. We will perform quantitative live cell single molecule fluorescence tracking experiments of components of the Notch signalling pathway to obtain kinetic information and utilize state of the art biochemical and optogenetic methods to activate Notch signalling and characterize its temporal profile. In addition, we want to analyse the impact of tumor associated Notch mutations on these kinetic parameters.