Molecular and kinetic characterization of the Notch coactivator complex

• Applicants: Professor Dr. Christof Gebhardt; Professor Dr. Franz Oswald
• Subject Area: Biophysics
• Term: since 2026
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 578852385

The Notch signalling pathway responds to extracellular mechanical cues by activating certain genes. It plays an essential role in the regulation of tissue differentiation and homeostasis and deregulated Notch signalling is associated with serious, often malignant diseases. Ligand binding leads to cleavage and transport of the intracellular Notch domain (NICD) into the cell nucleus. There, NICD forms a DNA bound complex with the transcription factor RBPJ and various coactivators to activate Notch target genes in a time-sensitive manner. After NICD turnover, RBPJ recruits a distinct set of corepressors, thereby turning Notch signalling inactive again. The general course of the Notch signalling pathway and its main constituents have been intensively studied. However, important information on the composition and the temporal stability of the coactivator complex is missing. For example, only few coactivators are characterized yet, and even for known factors the precise interaction interfaces are mostly unclear. Moreover, the pathway of coactivator complex assembly and how the lifetime of the complex correlates with the temporal profile of gene expression are unknown. This question becomes particularly relevant for degradation-disturbed NICD variants associated with cancer. In this proposal, we aim at obtaining a detailed protein interaction network within the Notch coactivator complex and the interaction kinetics of coactivators. We will characterize the complex composition and interaction interfaces of coactivators, as well as the temporal profile of gene expression using biochemical methods. By live-cell single-molecule tracking and colocalization experiments, we will unravel the pathway of assembly and the temporal stability of the complex. Finally, we will decipher how an altered interaction network and complex stability by cancer-related NICD variants contribute to deregulating Notch signalling. Our results will point out new strategies to improve the design of pharmacological modulators of Notch signalling in cancer therapy. Moreover, our approaches will provide a blueprint for measuring biological complex assembly, kinetic stability, and lifetime in living cells.

• DFG Programme: Research Grants