Notch signaling is an evolutionarily conserved pathway used by metazoans to direct cell fate decisions, proliferation or apoptosis at all stages of development, including self renewing adult tissues. Aberrant Notch signaling has been directly linked to multiple human disorders from developmental syndromes to adult onset diseases and cancer, leading to the emergence of the Notch pathway as an important therapeutic target. Most if not all Notch-mediated cellular decisions depend on proteolytic cleavage of Notch. Ligand binding leads to ectodomain shedding of the Notch extracellular domain followed by ?-secretase-mediated release of the Notch intracellular domain (NICD), which translocates to the nucleus and associates with the DNA-binding protein RBPj? to regulate transcription. The dependence of Notch activity on proteolysis has permitted pharmacological modulation primarily through ?-secretase inhibitors (GSI). However, because Notch can act as both an oncogene and a tumor suppressor and different Notch paralogs can have opposing functions in the same tissue, the usefulness of GSIs as a therapeutic approach can be compromised by off-target activities and lack of receptor specificity. Therefore, we are proposing to miniaturize our existing high throughput screening method to permit screening of compound libraries with the aim of identifying novel pharmacological agents that can modulate individual receptors in a context-dependent manner. Our proven reporter system, which is based on the luciferase complementation imaging technology, allows visualization of the interactions between a specific NICD and the common nuclear cofactor RBPj?. Biologically active molecules identified in the proposed screening project will be useful for manipulating Notch signaling in cancer, inherited diseases, stem cell differentiation and tissue engineering for both research and potential clinical applications. [unreadable] [unreadable] [unreadable]