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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.

SUBMITTER: Housden BE

PROVIDER: S-EPMC3536677 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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

Housden Ben E BE Fu Audrey Q AQ Krejci Alena A Bernard Fred F Fischer Bettina B Tavaré Simon S Russell Steven S Bray Sarah J SJ

PLoS genetics 20130103 1

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 1 ...[more]

PMID: 23300480

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Project description:Cellular responses to signalling pathways are often highly dynamic, however most analyses of developmental signalling pathways focus on a single endpoint. Here we have analyzed the temporal changes in transcription following a short Notch activation treatment and have related these to the recruitment of the Notch pathway transcription factor, CSL [Suppressor of Hairless, Su(H), in Drosophila], and to the state of RNA Polymerase II (Pol II) binding. A total of 154 genes showed significant differential expression over time and their expression profiles stratified into 14 clusters based on temporal and quantitative differences in their responses. These differences were partially reflected in the profiles of Pol II and Su(H) binding. However neither could fully account for the different response profiles. Furthermore, the timing of the different responses was unaffected by more prolonged Notch activation. Instead our data suggest that regulatory relationships between genes that segregate into different response clusters can partially account for the stratification. Thus feed-forward repression, where products of early responding Enhancer of split bHLH genes (E(spl)bHLH) inhibit expression of endogenous repressors, is one mechanism that explains the profile of genes that exhibit delayed up-regulation. E(spl)bHLH genes may therefore be responsible for co-ordinating the Notch response of a wide spectrum of other targets, explaining their critical functions in many developmental and disease contexts. DmD8 cells were cultured in Schneiders medium (Invitrogen) supplemented with 10% FBS (Sigma), 5ug/ml insulin (Sigma) and 5% penicillin / streptomycin (Sigma) according to standard protocols. Notch signalling was initiated by replacing cell media with 2mM EDTA in PBS. The cells were stimulated for 5 minutes before washing out EDTA using normal culture media. The cells were then collected at 18 time points at 0M-bM-^@M-^Y, 5M-bM-^@M-^Y, 10M-bM-^@M-^Y, 15M-bM-^@M-^Y, 20M-bM-^@M-^Y, 25M-bM-^@M-^Y, 30M-bM-^@M-^Y, 35M-bM-^@M-^Y, 40M-bM-^@M-^Y, 50M-bM-^@M-^Y, 60M-bM-^@M-^Y, 70M-bM-^@M-^Y, 80M-bM-^@M-^Y, 90M-bM-^@M-^Y, 100M-bM-^@M-^Y, 110M-bM-^@M-^Y, 120M-bM-^@M-^Y and 150M-bM-^@M-^Y after stimulation in 4 independent time trials. For control samples, for each time trial, media was replaced with fresh media to mimic the addition and removal of EDTA in corresponding experimental samples and then collected at equivalent times. For each trial 50 M-BM-5g of the untreated control RNA sample of each time point was pooled to create a trial specific reference sample. Samples from trials #1 and #3 were labelled with Cy5 and trails #2 and #4 as dye-swap with Cy3.

Project description:The biosynthesis of membrane lipids is an essential pathway for virtually all bacteria. Despite its potential importance for the development of novel antibiotics, little is known about the underlying signaling mechanisms that allow bacteria to control their membrane lipid composition within narrow limits. Recent studies disclosed an elaborate feed-forward system that senses the levels of malonyl-CoA and modulates the transcription of genes that mediate fatty acid and phospholipid synthesis in many Gram-positive bacteria including several human pathogens. A key component of this network is FapR, a transcriptional regulator that binds malonyl-CoA, but whose mode of action remains enigmatic. We report here the crystal structures of FapR from Staphylococcus aureus (SaFapR) in three relevant states of its regulation cycle. The repressor-DNA complex reveals that the operator binds two SaFapR homodimers with different affinities, involving sequence-specific contacts from the helix-turn-helix motifs to the major and minor grooves of DNA. In contrast with the elongated conformation observed for the DNA-bound FapR homodimer, binding of malonyl-CoA stabilizes a different, more compact, quaternary arrangement of the repressor, in which the two DNA-binding domains are attached to either side of the central thioesterase-like domain, resulting in a non-productive overall conformation that precludes DNA binding. The structural transition between the DNA-bound and malonyl-CoA-bound states of SaFapR involves substantial changes and large (>30 Å) inter-domain movements; however, both conformational states can be populated by the ligand-free repressor species, as confirmed by the structure of SaFapR in two distinct crystal forms. Disruption of the ability of SaFapR to monitor malonyl-CoA compromises cell growth, revealing the essentiality of membrane lipid homeostasis for S. aureus survival and uncovering novel opportunities for the development of antibiotics against this major human pathogen.

Dataset Information

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

Publications

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

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