Modifiers of notch transcriptional activity identified by genome-wide RNAi - PubMed (original) (raw)
Modifiers of notch transcriptional activity identified by genome-wide RNAi
Philippos Mourikis et al. BMC Dev Biol. 2010.
Abstract
Background: 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.
Results: Employing 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.
Conclusions: The 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.
Figures
Figure 1
Validation of the Notch activity reporter and application for high throughput RNAi. Validation of the m3-luc reporter by RNAi targeting of known Notch pathway components. Notch induced E(spl)m3 signal normalized against A. the control viral promoter or B. uninduced E(spl)m3 promoter transcription. C. Uninduced E(spl)m3 promoter expression normalized by control promoter. For each dsRNA, 32 independent wells were measured in a 384-well plate format. Each box encloses 50% of the data with the median value displayed. The error bars mark the full range excluding the shown outliers. D. Schematic of the automated high throughput screen in 384-well plates. Drosophila Kc167 cells were incubated with a unique dsRNA per well. After a four day incubation, the cells were split into three different transfection mixes in duplicate. Firefly luciferase signals were read 24 h after transfection.
Figure 2
RNAi data analysis overview. Histograms of targeted genes binned by standard deviations from the mean (z-scores). A. Histogram of z-scores for Notch-induced E(spl)m3 reporter (NΔecn >m3-luc) normalized by the signal from the control reporter (con-luc) (Additional file 1). B. Signals of Notch-induced E(spl)m3 reporter (NΔecn >m3-luc) normalized by the signal from the non-induced E(spl)m3 reporter (Additional file 2). Cutoffs for genes selected are highlighted in grey for both A and B. C. Plot of the z-scores from histogram 2A on the x-axis and histogram 2B on the y-axis. Regions outside the red box are listed as potential hits and the overlaps between the two normalization methods are shaded in blue (area a). Area a. represents the subset of genes that either affect Notch induced transcription specifically or have opposing effects on induced and non-induced reporter transcription (e.g. Su(H)). RNAi for this overlapping set was redesigned and retested (Additional file 5). Area b. represents genes that affect both Notch induced and non-induced transcription by similar percent amounts (e.g. heph and Bap55). Area c. represents genes that primarily affect non-induced reporter transcription specifically (e.g. H, RpL19 and Bap170). Red and/or boxed genes have known genetic interactions with Notch. Blue are chromatin components, Yellow are mRNA processing factors, and Green are ribosomal components (Minute class).
Figure 3
Protein-protein interaction map of Notch transcription modifiers. The Notch interaction network was generated by connecting the Notch transcription modifiers identified in the genome-wide study with protein-protein interaction links (e.g. two-hybrid and Co-IP data from the DroID database [19]). This resulting network included 126 genes (nodes) with 237 physical interactions (edges). Genetic interactions were not used for the network and the resulting map was drawn using Cytoscape [51]. A. These physical links are shown in relation to components of the activated Notch pathway (N and Su(H)) and the Notch repressor complex (Su(H), H, CtBP and gro), shown in red. B. Expanded view of the chromatin factors identified in this study that form the central core of the interaction network (blue). C. Ttk is a known downstream target of Notch signaling. The transcriptional and physical interaction data suggests that this factor may have a positive feedback role in Notch induced transcription. D. Factors with roles in mRNA processing (yellow). The interaction network suggests that these proteins may be working though the chromatin machinery to modulate Notch transcription. E. The interaction network suggests the possibility of a similar chromatin based mechanism for the class of ribosomal proteins known as Minute. The network file is included with the supplemental data (Additional file 6) and can be viewed in detail using the open source Cytoscape viewer
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Figure 4
Analysis of retested genes. A. Retested genes (from selected set, Figure 2C area a) that show significantly reduced signaling when down regulated by RNAi. Signals are shown as (+/-) percent deviation from the control RNAi signal. Three general classes are shown. All three classes down regulate both soluble (Nicd) and membrane bound (NΔecn) Notch-induced signal, yet have different effects on the E(spl)m3 promoter in the absence of active Notch. Class I genes have positive, Class II neutral and Class III negative effect on the uninduced signal. B. Selected set of retested genes that show significantly enhanced signaling when down regulated by RNAi. Two classes of hits are noted. Class IV is only effective on the membrane bound form of Notch (NΔecn), while class V is effective on both membrane bound and soluble forms (Nicd). All deviations are calculated to be significant by two-tailed t-test with p-values < 0.05 from control RNAi treatment (Additional file 5 for full statistics).
Figure 5
Modulation of Notch transcription for subset of retested genes. A. The two constitutively active Notch constructs used to determine epistatic relationships in the pathway, NΔecn and Nicd. NΔecn is a truncated form of N missing the extracellular domain that is initially membrane bound. NΔecn undergoes constitutive cleavage to form the soluble Nicd that is transported to the nucleus to activate transcription. Su(H) is the canonical Notch pathway transcription factor that represses transcription in the absence of Nicd and is essential for the Nicd activated transcription of targets such as E(spl)m3. B. Transcriptional response to RNAi treatment of selected retested genes that promote Notch signaling. The E(spl)m3 reporter was induced with either NΔecn or Nicd or left in the uninduced repressed state. All three classes down regulate both soluble (Nicd) and membrane bound (NΔecn) Notch-induced signal, yet have different effects on the E(spl)m3 promoter in the absence of active Notch. Class I genes have positive, Class II neutral and Class III negative effect on the repressed signal. C. Transcriptional response to RNAi treatment of selected retested genes that repress Notch signaling. Two classes of hits are noted. Class IV is only effective on the membrane bound form of Notch (NΔecn), while class V is effective on both membrane bound and soluble forms (Nicd). Error bars represent the standard error of the mean (SEM). *****Significant deviation from control RNAi treatment, calculated by two-tailed t-test with a p-value < 0.05 (Additional file 5 for full statistics).
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