Roles of the ankyrin repeats and C-terminal region of the mouse Notch1 intracellular region

The Notch intracellular region (RAMIC) interacts with a DNA binding protein RBP-J to activate transcription of genes that inhibit cell differentiation . The RAM domain and ankyrin (ANK) repeats of mouse Notch1 RAMIC were shown to be responsible for RBP-J binding and necessary for transactivation. The C-terminal portion of Notch1 RAMIC has also been suggested to be important for transactivation. Using GAL4 fusion constructs, we identified a novel t ransactivation domain (TAD) between the ANK repeats and the PEST sequence o f mouse Notch1. The C-terminal half of mouse Notch2 RAMIC also exhibited TA D activity. Unexpectedly, the RBP-J chimeric protein with the Notch1 TAD fa iled to activate transcription but the activity was recovered by addition o f either the RAM domain or ANK repeats. The results suggest that the activi ty of Notch1 TAD is repressed by fusion with RBP-J because of the presence of a RBP-J-associated co-repressor(s), which could be displaced by either t he RAM domain or ANK repeats. Taken together, mouse Notch1 RAMIC can experi mentally be separated into three functional domains: the RAM domain and ANK repeats for RBP-J binding and co-repressor displacement and the C-terminal TAD.