Conclusion We have scientific assays identified CDT 2 and CUL 4 as novel attenua tors of LET 23 signalling during vulva development. Both of these proteins are known components of a con served CUL 4 DDB 1 CDT 2 E3 ubiquitin ligase com plex, suggesting a novel function for this complex in signalling during C. elegans development. A similar function in other organisms might have been missed because of its requirement for cell cycle progression. Studying this complex in mammalian cells using knock down conditions that bypass the cell cycle defect might reveal a conserved role in signalling. The Notch cell surface receptor is the central ele ment of one of the handful of signaling pathways that are evolutionary conserved throughout metazoans. Notch signaling directs the development of multicellular organisms through membrane anchored interactions between adjacent cells.

The response to Notch signals varies greatly and can result in diverse biological conse quences, such as cell proliferation, differentiation or apoptosis. Notch signaling is initiated by the binding of the transmembrane Notch receptor with one of its ligands, Delta or Serrate, expressed on the surface of a neighboring cell. The receptor ligand interaction induces a series of proteolytic events that releases the Notch intracellular domain, which then translocates to the nucleus and complexes with tran scription factors and co activators to regulate target gene expression. Nicd, together with Suppressor of Hair less, a DNA binding protein in the CSL Brefeldin_A Lag2 family, and mastermind proteins form the core transcriptional complex of the signaling pathway, with the Enhancer of Split locus being the most thoroughly characterized downstream tran scriptional target.

The Notch signaling pathway is modulated at many levels, Notch protein abundance, trafficking, and post translational processing, as well as the regulated formation of repressive and promoting complexes on the DNA. The final cell fate outcome is a complex interplay between all the cellular factors that regulate Notch activity. We designed a genome wide RNA interference screen using a Drosophila cell culture based system aimed to identify novel proteins that directly influence the signaling capacity of the core Notch pathway. This genome wide RNAi screen was performed on Drosophila Kc167 cell cultures that were transfected with a construct that expresses a constitutively active, membrane tethered form of the Notch receptor, Necn.

Notch pathway activity was monitored by measuring the transcriptional response of a luciferase reporter gene cassette containing the native promoter ele ment of the E m3 gene, the most Notch respon sive E target in cell culture. The results of our study reveal the identity of proteins that influence the signaling for output of the core Notch pathway.