This map does not include the known genetic interactions identifi

This map does not include the known genetic interactions identified between the candi date genes and caution should be noted as to the presence of possible false positives in the protein inter action data. The importance of chromatin in Notch regulation has recently become apparent and this transcription based screen was suited to uncover this class of regulators. On average, chromatin modifying genes scored relatively high in the data analysis. The interac tion map reveals a central core of chromatin modifying components that have multiple physical connections to the nuclear elements of the Notch pathway such as Su and H. Many of these chromatin com ponents are known to interact genetically and physically with the Notch pathway.

The protein interaction network also shows a number of protein classes that have no known mechanistic link to Notch transcriptional regulation. For these classes of mole cules, the network suggests that they may be affecting Notch signaling through direct interactions with these core chromatin components. Epistatic analysis of candidate genes The subset of candidate Notch modifiers that over lapped between the two normalization methods was retested with redesigned dsRNAs. Luciferase reporter activity was assessed in cells in which Notch had been activated by either the mem brane tethered Necn or the downstream intracellular Nicd, aiming to discriminate between factors that regu late Notch processing at the plasma membrane versus factors that affect Notch signaling downstream in the nucleus.

Of the re designed dsRNA, 79% retested by either normalization method, 67% re tested the m3 luc normalized signal and 64% the con luc normalized signal. Three genes were identified that exclusively promote the activity of the membrane bound Notch and may function to inhibit the intramembrane proteolysis of the receptor. This class includes Patj and two genes of unknown function, CG7099 and CG17189. The solu ble protein Patj has not been shown to modulate Notch activity directly, but is known to associate with the transmembrane protein Crumbs that, in turn, is known to repress Notch activity. Crumbs is a central regu lator of epithelial apical basal polarity in Drosophila and has been shown to down regulate g secretase activity and the membrane proteolysis of Notch.

Our obser vation in Kc167 cell culture, a non polarized cell line, suggests that Patj may be modifying Notch signaling not via influencing the localization of the receptor, but instead Batimastat by acting in the Crumbs based complex to down regulate membrane proteolysis of Notch. In contrast, RNAi against nuclear factors such as Su, His3. 3A B, Nipped A, ttk and Sin3A, had similar effects on Necn and Nicd induced transcription, indi cating interactions with Notch downstream of the pro teolytic processing events.

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