Abstract
BackgroundThe Notch signaling pathway regulates a diverse array of developmental processes, and aberrant Notch signaling can lead to diseases, including cancer. To obtain a more comprehensive understanding of the genetic network that integrates into Notch signaling, we performed a genome-wide RNAi screen in Drosophila cell culture to identify genes that modify Notch-dependent transcription.ResultsEmploying complementary data analyses, we found 399 putative modifiers: 189 promoting and 210 antagonizing Notch activated transcription. These modifiers included several known Notch interactors, validating the robustness of the assay. Many novel modifiers were also identified, covering a range of cellular localizations from the extracellular matrix to the nucleus, as well as a large number of proteins with unknown function. Chromatin-modifying proteins represent a major class of genes identified, including histone deacetylase and demethylase complex components and other chromatin modifying, remodeling and replacement factors. A protein-protein interaction map of the Notch-dependent transcription modifiers revealed that a large number of the identified proteins interact physically with these core chromatin components.ConclusionsThe genome-wide RNAi screen identified many genes that can modulate Notch transcriptional output. A protein interaction map of the identified genes highlighted a network of chromatin-modifying enzymes and remodelers that regulate Notch transcription. Our results open new avenues to explore the mechanisms of Notch signal regulation and the integration of this pathway into diverse cellular processes.
Highlights
The Notch signaling pathway regulates a diverse array of developmental processes, and aberrant Notch signaling can lead to diseases, including cancer
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 components of the Notch signaling pathway
We found that targeting Su(H) and mam with RNA interference (RNAi) in cells expressing activated Notch resulted in a sharp reduction of the reporter luciferase activity (Figure 1A and 1B)
Summary
The Notch signaling pathway regulates a diverse array of developmental processes, and aberrant Notch signaling can lead to diseases, including cancer. To obtain a more comprehensive understanding of the genetic network that integrates into Notch signaling, we performed a genome-wide RNAi screen in Drosophila cell culture to identify genes that modify Notch-dependent transcription. We designed a genome-wide RNA interference (RNAi) 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, NΔecn [2]. Notch pathway activity was monitored by measuring the transcriptional response of a luciferase-reporter gene cassette (m3-luc) containing the native promoter element of the E(spl)m3 gene [3], the most Notch responsive E(spl) target in cell culture [4]
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