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

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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.