Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013;9(1):e1003162.
doi: 10.1371/journal.pgen.1003162. Epub 2013 Jan 3.

Transcriptional dynamics elicited by a short pulse of notch activation involves feed-forward regulation by E(spl)/Hes genes

Ben E Housden  1 , Audrey Q Fu, Alena Krejci, Fred Bernard, Bettina Fischer, Simon Tavaré, Steven Russell, Sarah J Bray
Affiliations

Transcriptional dynamics elicited by a short pulse of notch activation involves feed-forward regulation by E(spl)/Hes genes

Ben E Housden et al. PLoS Genet. 2013.

Abstract

Dynamic activity of signaling pathways, such as Notch, is vital to achieve correct development and homeostasis. However, most studies assess output many hours or days after initiation of signaling, once the outcome has been consolidated. Here we analyze genome-wide changes in transcript levels, binding of the Notch pathway transcription factor, CSL [Suppressor of Hairless, Su(H), in Drosophila], and RNA Polymerase II (Pol II) immediately following a short pulse of Notch stimulation. A total of 154 genes showed significant differential expression (DE) over time, and their expression profiles stratified into 14 clusters based on the timing, magnitude, and direction of DE. E(spl) genes were the most rapidly upregulated, with Su(H), Pol II, and transcript levels increasing within 5-10 minutes. Other genes had a more delayed response, the timing of which was largely unaffected by more prolonged Notch activation. Neither Su(H) binding nor poised Pol II could fully explain the differences between profiles. Instead, our data indicate that regulatory interactions, driven by the early-responding E(spl)bHLH genes, are required. Proposed cross-regulatory relationships were validated in vivo and in cell culture, supporting the view that feed-forward repression by E(spl)bHLH/Hes shapes the response of late-responding genes. Based on these data, we propose a model in which Hes genes are responsible for co-ordinating the Notch response of a wide spectrum of other targets, explaining the critical functions these key regulators play in many developmental and disease contexts.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Transient activation of Notch and classification of transcripts according to Pol II class.
A: Schematic outlining the experimental strategy. Arrows indicate the time-points at which data were collected. B: Levels of Nicd that co-immunoprecipitated with Su(H) after a 5 min pulse of Notch activation (Nact) using EDTA treatment. C: Graph shows a quantification of Nicd levels relative to Su(H) from B, normalised to 0 min. D: Representative genomic regions, gene models are indicated in black. Red graphs represent enrichment with anti-pSer2pSer5-Pol II relative to total input (0–0.47 fold enrichment on a log2 scale). Pol II binding classes, AP (active poised), P (poised), UB (unbound) and AU (active uniform) are illustrated. A ratio of log2(max)/log2(median)≥2 cut-off was used to distinguish AP from AU (see Text S1). E: Relationship between log2 absolute expression levels at 0 min and Pol II class at 0 min. RNA expression levels were approximated, up to a constant, by the spot intensity levels (logC0). The four Pol II classes have significantly different mean logC0 (ANOVA p value<2.2e-16). All pairs of classes, except for AU and AP, have significantly different means (pair-wise one-sided two-sample t test p values<3e-15).
Figure 2
Figure 2. Different temporal profiles in response to Notch activation.
Examples of 6 clusters of genes that exhibit different temporal expression profiles in Notch activated cells. Left graphs: profiles for all the genes in the cluster, coloured line represents the mean profile. Right graphs: profiles for a single gene from each cluster as indicated; Error bars indicate standard error of the mean from four replicates. Vertical axes for all graphs indicate median M values. Cluster types are indicated to the right of the graphs.
Figure 3
Figure 3. Rapid and transient recruitment of Su(H) and Pol II to genes in the E(spl) complex.
A: Enrichment for Su(H) (blue) and Pol II (red) across the E(spl) complex at different time points (min) after Notch activation (Su(H) 0.5–4.5, Pol II 0–4.7 fold enrichment on a log2 scale). Cluster 1 genes: mβ (orange) m3 (brown) and m7 (yellow) have poised Pol II at 0 min, m6 (light blue), mα (dark blue) and m2 (mid blue) have no Pol II present at 0 min. Cluster 2 genes: mδ (purple) and mγ(pink) have no Pol II present at 0 mins. Pol II is recruited at all expressed genes by 10 mins. Su(H) occupancy increases after Notch activation at all loci. B: Log2 fold changes in mRNA levels for the indicated genes at different times (min) after Notch activation.
Figure 4
Figure 4. Patterns of Su(H) and Pol II recruitment.
A: Table showing, for each cluster, the proportion of genes with Su(H) binding within 10 kb [ # Su(H)] and with each Pol II state (Pol II: AU, AP, P and UB as described in main text). In cases where individual transcripts of a gene had different Pol II states, the gene was assigned a state as follows: AU>AP>P>UB. Conditions where >30% of genes are ascribed to a particular class are indicated in bold. B–D: Enrichment for Su(H) (blue) and Pol II (red) across the W/hid (B), CG4398 (C) and hairy (D) genes at different time points (min) after Notch activation (Su(H) 0.5–4.5, Pol II 0–4.7 fold enrichment on a log2 scale).
Figure 5
Figure 5. Relationship between Su(H) binding and gene responses.
A: Enrichment for Su(H) at the indicated times, calculated from the area under the peak of ChIP enrichment (Blue: genes from cluster 1; Green: genes from cluster 3). B: Log2 fold changes in mRNA levels for the corresponding genes. C: Diagram illustrating whether Su(H) binding, Pol II class and initial absolute expression level (log C0) have statistically significant positive (red) or negative (black) effects on the log odds of differential expression (DE). Effects were calculated using logistic regression models (see Text S1). Solid lines indicate statistically significant effects (p<0.005 after adjusting for multiple testing); the numbers indicate the estimated mean effect on the log odds (see Text S1). For example, in considering all expressed genes these effects equate to: (i) Su(H) bound within 10 kb at 0 min increases odds of DE by e2.78 = 16.12. (ii) P class increases the odds of DE by e0.77 = 2.16, (iii) AU class decreases odds of DE by e−0.49 = 0.61.
Figure 6
Figure 6. Evidence for cross-regulatory relationships between genes in different clusters.
A: mRNA expression levels of the indicated genes in Notch activated cells relative to controls (log2) in untreated (grey) and cycloheximide treated (black) cells at the times indicated. Cells were exposed to cycloheximide for 60 min prior to Notch activation at 0 min as well as during the timecourse. B,C: Expression of Hairy in the wing imaginal disc pouch from control (B, ptc-Gal4 ; UAS-lacZ) and HLHmβ-VP16 (C, ptc-Gal4 ; UAS-HLHmβ-VP16), arrows indicate the stripe of HLHmβ-VP16 expression where Hairy is induced. D,E: Expression of Hairy in muscle progenitors (brackets) from control wing imaginal discs (D, 1151-Gal4; UAS-lacZ) and from those expressing HLHmβ (E, 1151-Gal4; UAS-HLHmβ). Hairy is reduced in the muscle progenitors (brackets) but not in neighbouring epithelial cells (asterisks). F,G: Quantification of expression levels of Hairy (F) and edl-GFP (G) in muscle progenitors expressing β-galactosidase (con), HLHmβ (mβ) or HLHmβ-VP16 (mβ-VP16). Average pixel intensities from a defined region within the expression domain were measured using ImageJ and normalized relative to background levels from a comparable region in the same discs, >5 discs per genotype. Error bars indicate standard error of the mean. Asterisks indicate that results are significantly different from control (p≤0.05; using an unpaired, 2-tailed student T-test). H: Fold change of the indicated mRNAs in cells treated with dsRNA against hairy, edl or btn in comparison to controls (no RNAi). RNA levels were reduced by 65%, 71% and 61% for hairy, edl and btn respectively. These experiments were performed in the basal state (no Notch activation). Bars represent the average of three biological replicates and error bars indicate standard error of the mean. I: Log2 fold changes in mRNA levels of hairy (h, brown) hibris (hbs, green) and HLHmβ (mβ, blue) from the microarray study. Scale for hbs and h is indicated by left axis and for mβ, which had larger fold changes, by right axis. J: Summary model of the feed-forward regulatory relationships, dotted line indicates that the direct regulation of hbs (cluster 3) by Notch signaling has not been directly tested here, although hbs and other genes in cluster 3 exhibit Su(H) binding, which implies that at least some undergo direct Notch regulation.
Figure 7
Figure 7. Differential sensitivities to the dose of Notch activation.
A: Comparison of fold changes in mRNA expression levels (log2) from 5 and 30 min Notch activation regimes. Symbols indicate fold-change at 30 min time-point after commencing activation (EDTA) treatment, where colours represent cluster assignments according to the legend in B. Dashed line represents the expected trend if each treatment produced the same response; solid line indicates the line of best fit from the data (regression coefficient = 0.84, r2 = 0.71). B: Ratio of the fold change in mRNA levels at 30 min, with a 30 versus 5 min treatment for the indicated genes. Higher bars indicate greater sensitivity to the differences in the activation regime. Colours indicate cluster assignments as in the legend. C: Levels of Nicd that co-immunoprecipitate with Su(H) under continuous treatment for the times indicated (compare with Figure 1A). D: Fold changes for the indicated mRNAs at the times indicated (red lines: 5 min activation, blue lines: continuous activation). T = 0 corresponds to the time at which the activation regime commenced. Error bars indicate standard error of the mean from four biological replicates.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Akimov V, Rigbolt KT, Nielsen MM, Blagoev B (2011) Characterization of ubiquitination dependent dynamics in growth factor receptor signaling by quantitative proteomics. Molecular BioSystems 7: 3223–3233. - PubMed
    1. Ilagan MX, Lim S, Fulbright M, Piwnica-Worms D, Kopan R (2011) Real-time imaging of notch activation with a luciferase complementation-based reporter. Science Signaling 4: rs7. - PMC - PubMed
    1. Kawahashi K, Hayashi S (2010) Dynamic intracellular distribution of Notch during activation and asymmetric cell division revealed by functional fluorescent fusion proteins. Genes to Cells : Devoted to Molecular & Cellular Mechanisms 15: 749–759. - PubMed
    1. Krouk G, Mirowski P, LeCun Y, Shasha DE, Coruzzi GM (2010) Predictive network modeling of the high-resolution dynamic plant transcriptome in response to nitrate. Genome Biology 11: R123. - PMC - PubMed
    1. Andersson ER, Sandberg R, Lendahl U (2011) Notch signaling: simplicity in design, versatility in function. Development 138: 3593–3612. - PubMed

Publication types

MeSH terms