Mechanisms of notch signaling diagram.

Upon ligand binding, Notch receptors undergo ectodomain shedding followed by γ-secretase-mediated release of the Notch intracellular domain (NICD), which translocates to the nucleus and associates with the DNA-binding protein CSL (CBF1/RBPjk/Su(H)/Lag1). NICD does not bind DNA, acting at low protein concentration to modulate expression of select target genes. To monitor activation of specific Notch paralogs in live cells and in real time, we developed luciferase complementation imaging (LCI) reporters for NICD/CSL association and validated them as a specific, robust and sensitive assay system that enables structure-function and pharmacodynamic analyses. Notch-LCI represents a powerful approach for characterizing modulators that target Notch signaling and for studying pathway dynamics in normal and disease contexts.

To understand how the NICD protein selects targets, we launched a method development effort that is near completion. We created a tool that permits us to label DNA bound by NICD and any protein partner in a very small number of cells. we are attempting to bring this analysis to a single cell level. We are recruiting talented postdocs with interest in protein DNA interactions for this project.