These included several STI571 known Notch interactors, validating the robustness of the assay and our experimental approach. Molecules residing in the extracellular matrix, the plasma membrane, the cytosol, and the nucleus, as well as a large number of proteins with unknown function and localization, were also recovered. To further analyze and categorize our dataset, the Notch signaling modifiers identified in the study were combined with physical interaction data from public databases. The interaction map that was generated revealed classes of interacting Notch modifiers such as mRNA processing and ribosomal proteins. The network analysis also highlighted a central core of chromatin reg ulating genes, including chromatin modifying enzymes and remodelers that interact directly with the Su DNA binding complex.

Results and Discussion Development of a robust assay to measure changes in Notch transcriptional activity A reporter assay was developed to measure Notch activ ity in a high throughput Drosophila cell based approach. The assay consists of three components, 1 a Notch activity reporter construct with two, tandem copies of the E m3 promoter positioned upstream of the firefly luciferase gene, 2 the constitutively active, membrane tethered form of the Notch receptor with the extracellular domain removed, driven by the viral OpIE2 promoter, 3 a control construct that constitutively expresses firefly luciferase, also driven by the viral OpIE2 promoter. Con luc was used to normalize signal intensity relative to transfection effi ciency, cell density and viability, and general effects on OpIE2 mediated transcription.

To test the sensitivity and specificity of the Notch activity assay, a series of experiments were performed in cells treated with interfering RNA targeting known com ponents of the Notch signaling pathway. Cells were incubated with dsRNA against mastermind, Hairless, and the major downstream co transcription factor Suppressor of Hairless and then split and transfected for three assays. N induced luciferase expression levels were measured relative to either con luc or uninduced E m3 promoter. Uninduced promoter levels were also tested by normalizing m3 luc measure ments with corresponding con luc signals. As predicted, we found that targeting Su and mam with RNAi in cells expressing activated Notch resulted in a sharp reduction of the reporter luciferase activity.

Conversely, knock down of Su increased the basal activity of the m3 luc reporter in the absence of Necn. These opposing effects Cilengitide of Su RNAi on E m3 expression are consistent with the dual roles of Su as a transcriptional repres sor in the absence of Notch activation, as well as a tran scriptional activator when complexed with processed Nicd in the nucleus. In contrast, RNAi against Hair less resulted in a marked decrease in the ratio of induced,uninduced signal of m3 luc.