Transport, processing and regulation of the Notch receptor

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The Notch receptor is involved in important developmental and differentiation decisions. After activation of Notch the intracellular domain (NICD) is cleaved by gamma-secretase and transported into the nucleus. There it activates gene transcription, together with the co-activator RBP-J. Hyperactive Notch can cause cancer, for example T-ALL leukemia. In the first funding period using image-based high throughput screens we screened a compound library as well as a genome-wide siRNA library for factors involved in transport and processing of Notch. We identified novel gamma-secretase inhibitors and a secretion inhibitor with a novel mode of action. In the next funding period we want to focus on three hits from the siRNA screen, Nup214, a nuclear pore protein, exportin 4 (XPO4), a nuclear export factor and a phosphatase, MTMR14. Our previous work showed that Nup214 negatively regulates Notch signaling by exporting RBP-J out of the nucleus. We now want to address the following questions. What is the molecular role of Nup214 in Notch signaling? Is Nup214 directly interacting with RBP-J, or with the transport protein RITA? Is the nuclear export of RBP-J regulated, for example upon Notch activation, in certain differentiation stages, under stress?Preliminary data show that a T-ALL causing Set-Nup fusion aggregates in the nucleus and sequesters other nucleoporins. This might activate Notch signaling and contribute to tumorgenesis. We want to study the effects of several T-ALL causing Nup214 fusions on Notch signaling.We also want to analyze the function of XPO4 in Notch signaling. To this end we want to study its role in nuclear import/export of NICD in cell culture and in vitro.In zebrafish we want to determine the spatial and temporal expression pattern of Nup214 and XPO4 and want to study the phenotypes of specific Morpholinos against both mRNAs. To study the pathologic functions of Nup214 fusion proteins, we also want to establish a zebrafish model for T-ALL-causing fusions.The phosphatase MTMR14 seems to play a role in transport of Notch. Using our established assays for ER-export and transport in the secretory pathway we plan to elucidate the function of MTMR14 in Notch signaling.Finally we want to reassess the images from the siRNA screen by improved segmentation and classification approaches, to find additional phenotypes.All planned experiments make use of established methods like immuno-precipitation, GST-pull down, fluorescence microscopy, siRNA technology, in vitro assays, morpholino injections and in situ hybridization.