Physical interaction between a novel domain of the receptor Notch and the transcription factor RBP-Jκ/Su(H)

Abstract Background: The mammalian transcription factor RBP-Jκ binds to the DNA sequence motif CGTGGGAA and is involved in the regulation of gene expression; for example, it plays a part in the transactivation of viral and cellular genes by Epstein–Barr virus nuclear antigen-2. The Drosophila homologue of RBP-Jκ is the product of the Suppressor of Hairless ( Su(H) ) gene. Su(H) is a neurogenic gene that acts downstream of Notch , which encodes a cell-surface receptor. Furthermore, in the mouse, the phenotypes of homozygous mutant Notch 1 embryos are very similar to those of homozygous mutant RBP-J κ embryos. Recent studies, using the yeast two-hybrid system, have led to the suggestion that the CDC10/ankyrin-like repeats of the Drosophila Notch protein interact with the Su(H) protein. Results We searched for proteins that interact with mouse RBP-Jκ using the yeast two-hybrid system, and in this way identified a short intracellular region (mRAM23) of the mouse Notch1 protein that lacks any known sequence motif. In vitro interaction studies, using proteins fused to glutathione-S-transferase, showed that RBP-Jκ and Su(H) bind directly to the RAM23 regions of mouse Notch1 and Drosophila Notch, respectively. Immunoprecipitation analysis showed that RBP-Jκ and the mRAM23 region of mouse Notch1 also interact in vivo . Further studies, including site-directed mutagenesis experiments, narrowed down the region of mouse Notch1 that interacts with RBP-Jκ. The results indicate that this region is less than 50 amino-acid residues in length, and lies immediately downstream of the transmembrane region. Conclusion We show that the transcription factor RBP-Jκ/Su(H) interacts directly with a novel intracellular domain of the cell-surface receptor Notch. RBP-Jκ/Su(H) does not appear to interact with Notch via the CDC10/ankyrin repeats implicated in previous studies.