Transcriptional Dynamics Elicited by a Short Pulse of Notch Activation Involves Feed-Forward Regulation by E(spl)/Hes Genes

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.

Mapping signaling pathway cross-talk in Drosophila cells

“…Indeed, at least one incoherent feedforward or feedback loop was present among the targets of each pathway, with the exception of Notch and Wnt. We note, however, that incoherent feedforward loops have previously been identified downstream of Notch in another cell type (13,14).…”

“…1C). In addition, these time points are likely optimal for detecting the expression of primary target genes (12,13,16,27).…”

“…Specifically, we examined changes in gene expression in homogeneous cultured cells at a time scale allowing us to detect primary transcriptional targets (12)(13)(14)(15)(16). We selected Drosophila cell lines for these studies, because the signaling systems are simpler than in mammals.…”

A cautionary note on the impact of protocol changes for genome-wide association SNP × SNP interaction studies: an example on ankylosing spondylitis

“…terms highlighted in bold in Table 1 and Table S6 the correct development and growth of nerve tissue (Housden et al 2013). It was shown by (Gao et al 2013) that the…”

Notch Signaling in Cell–Cell Communication Pathways

“…Furthermore, the different signaling pathways are not only in an ON/OFF state, but there are also oscillatory feedback loops and intensity effects that determine their final outcome. Genomic studies in Drosophila have shown that Hes repressors are at the base of the oscillatory feedback loops observed in the Notch pathway [67], an observation that has been confirmed in several mammalian systems. In the embryonic aorta, when HSCs are formed, Notch signaling leads to the transcriptional activation of both Hes1 and the hematopoietic gene Gata2.…”