Live imaging of NICD occupancy on a single locus

Notch signaling is a highly conserved pathway that plays a crucial role during normal development and disease. Targets of Notch signaling are activated by the cleavage of the Notch intracellular domain (NICD), which then translocates to the nucleus creating an activating complex with RBPj, its DNA binding co-factor and Mastermind. Interestingly, different Notch signaling targets are activated in different cells and to different levels. Despite our detailed knowledge of the activation mechanism of Notch we still do not understand the factors that differentially regulate the activation of Notch targets in the nucleus. Here, we report the development of a method to quantitatively measure the dynamics of NICD occupancy on a target promoter. We use live imaging of Halo-tagged NICD in cells that contain synthetic promoters with different number and orientation of RBPj binding sites. For large enough number of binding sites, we can observe a bright puncta corresponding to accumulation of NICD at the promoter. The ultimate goal of this project is to obtain a quantitative understanding of the relation between NICD occupancy and transcriptional response. These studies will improve our understanding and provide insights on how Notch signaling operates reliably in different contexts, both during normal development, and in disease states.