p300 Acts as a Transcriptional Coactivator for Mammalian Notch-1

Abstract

Notch-1 belongs to a family of transmembrane receptor proteins that direct the decisions as to various cell fates. After ligand binding, a proteolytic cleavage step occurs and the intracellular part of Notch-1, Notch-1-IC, translocates into the nucleus, where it targets the DNA binding protein RBP-Jκ/CBF1. RBP-Jκ mediates repression through recruitment of a histone deacetylase-containing complex. The Notch-1-IC/RBP-Jκ complex overcomes repression and activates the transcription of Notch target genes. We have identified a novel domain in Notch-1-IC, the EP domain, which is indispensable for full transcriptional activation. This transactivation domain is localized adjacent to the ankyrin repeats of Notch-1-IC. In cotransfection experiments, Notch-1-IC-mediated transcriptional activation was inhibited by E1A12S and p53, two proteins, which interfere with the function of the common coactivator p300. Protein-protein interaction assays demonstrated the association of Notch-1-IC and the CH3 region of p300. In addition, the interaction of mammalian Notch-1-IC with p300 was destabilized after deletion of the EP domain of Notch-1-IC. Based on physical interaction with Notch-1-IC and coactivator functions of p300, we propose a model for Notch-1-mediated gene regulation via p300.

ACKNOWLEDGMENTS

We thank U. Wegenka, G. Schneider, U. Zechner, and H. Häcker for critically reading the manuscript. We also thank T. Honjo for providing the RBPJκ-specific antibody, K0043, and N. Perkins for providing the p53-expressing plasmids. For excellent technical assistance we thank J. Koehler, R. Rittelmann, and C. Heber.

This study was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 322,C4, to S.L. and R.M.S.