SKIP, a CBF1-associated protein, interacts with the ankyrin repeat domain of NotchIC to facilitate NotchIC function

Notch proteins are transmembrane receptors that mediate intercell communica tion and direct individual cell fate decisions. The activated intracellular form of Notch, NotchIC, translocates to the nucleus, where it targets the DNA binding protein CBF1. CBF1 mediates transcriptional repression through the recruitment of an SMRT-histone deacetylase-containing corepressor compl ex. We have examined the mechanism whereby NotchIC overcomes CBF1-mediated transcriptional repression. We identified SKIP (Ski-interacting protein) as a CBF1 binding protein in a yeast two-hybrid screen. Both CBF1 and SKIP ar e highly conserved evolutionarily, and the SKIP-CBF1 interaction is also co nserved in assays using the Caenorhabditis elegans and Drosophila melanogas ter SKIP homologs. Protein-protein interaction assays demonstrated interact ion between SKIP and the corepressor SMRT. More surprisingly, SKIP also int eracted with NotchIC. The SMRT and NotchIC interactions were mutually exclu sive. In competition binding experiments SMRT displaced NotchIC from CBF1 a nd from SKIP. Contact with SKIP is required for biological activity of Notc hIC. A mutation in the fourth ankyrin repeat that abolished Notch signal tr ansduction did not affect interaction with CBF1 but abolished interaction w ith SKIP. Further, NotchIC was unable to block muscle cell differentiation in myoblasts expressing antisense SKIP. The results suggest a model in whic h NotchIC activates responsive promoters by competing with the SMRT-corepre ssor complex for contacts on both CBF1 and SKIP.