Notch signalling in context.

The highly conserved Notch signalling pathway functions in many different developmental and homeostatic processes, which raises the question of how this pathway can achieve such diverse outcomes. With a direct route from the membrane to the nucleus, the Notch pathway has fewer opportunities for regulation than do many other signalling pathways, yet it generates exquisitely patterned structures, including sensory hair cells and branched arterial networks. More confusingly, its activity promotes tissue growth and cancers in some circumstances but cell death and tumour suppression in others. Many different regulatory mechanisms help to shape the activity of the Notch pathway, generating functional outputs that are appropriate for each context. These mechanisms include the receptor-ligand landscape, the tissue topology, the nuclear environment and the connectivity of the regulatory networks

SWI/SNF chromatin remodeling controls Notch-responsive enhancer accessibility.

Notch signaling plays a key role in many cell fate decisions during development by directing different gene expression programs via the transcription factor CSL, known as Su(H) in Drosophila Which target genes are responsive to Notch signaling is influenced by the chromatin state of enhancers, yet how this is regulated is not fully known. Detecting a specific increase in the histone variant H3.3 in response to Notch signaling, we tested which chromatin remodelers or histone chaperones are required for the changes in enhancer accessibility to Su(H) binding. We show a crucial role for the Brahma SWI/SNF chromatin remodeling complex, including the actin-related BAP55 subunit, in conferring enhancer accessibility and enabling the transcriptional response to Notch activity. The Notch-responsive regions have high levels of nucleosome turnover which depend on the Brahma complex, increase in magnitude with Notch signaling, and primarily involve histone H3.3. Together these results highlight the importance of SWI/SNF-mediated nucleosome turnover in rendering enhancers responsive to Notch

Notch after cleavage.

The discovery that Notch activation involves a proteolytic cleavage to release the intracellular domain (NICD) revolutionized the field of Notch signaling. It resulted in a simple model whereby the cleaved NICD enters the nucleus and activates expression of genes by forming a DNA bound complex with CSL. However, is it really this simple? The realization that the outcome from activating Notch varies greatly from cell to cell raised many questions about what governs the target gene selections in different cell types. Insights have come from recent genome-wide studies, which highlight the importance of tissue-specific transcription factors and epigenetics. Co-factors also have been identified that participate in the regulation of enhancers. Finally, it is generally assumed that once cleaved, NICD goes on to do its job, but with a burgeoning number of post-translations, it may not be that simple

Drosophila Reporter Vectors Compatible with ΦC31 Integrase Transgenesis Techniques and Their Use to Generate New Notch Reporter Fly Lines

Complex spatial and temporal regulation of gene activity is fundamental to development and homeostasis. The ability to decipher the DNA sequences that accurately coordinate gene expression is, therefore, of primary importance. One way to assess the functions of DNA elements entails their fusion to fluorescent reporter genes. This powerful approach makes it possible to visualize their regulatory capabilities when reintroduced into the developing animal. Transgenic studies in Drosophila have recently advanced with the introduction of site-specific, ΦC31 integrase–mediated approaches. However, most existing Drosophila reporter vectors are not compatible with this new approach and have become obsolete. Here we describe a new series of fluorescent reporter vectors optimized for use with ΦC31 transgenesis. By using these vectors to generate a set of Notch reporter fly lines, we demonstrate their efficacy in reporting the function of gene regulatory elements

Rme-8 depletion perturbs Notch recycling and predisposes to pathogenic signaling.

Notch signaling is a major regulator of cell fate, proliferation, and differentiation. Like other signaling pathways, its activity is strongly influenced by intracellular trafficking. Besides contributing to signal activation and down-regulation, differential fluxes between trafficking routes can cause aberrant Notch pathway activation. Investigating the function of the retromer-associated DNAJ protein Rme-8 in vivo, we demonstrate a critical role in regulating Notch receptor recycling. In the absence of Rme-8, Notch accumulated in enlarged tubulated Rab4-positive endosomes, and as a consequence, signaling was compromised. Strikingly, when the retromer component Vps26 was depleted at the same time, Notch no longer accumulated and instead was ectopically activated. Likewise, depletion of ESCRT-0 components Hrs or Stam in combination with Rme-8 also led to high levels of ectopic Notch activity. Together, these results highlight the importance of Rme-8 in coordinating normal endocytic recycling route and reveal that its absence predisposes toward conditions in which pathological Notch signaling can occur.This work was funded by an MRC programme grant [G0800034] to SJB. LAS was the recipient of a BBSRC PhD studentship. ES and TK were funded by the DFG [Sachbeihilfe KL 1028/5-­1].This is the author accepted manuscript. The final version is available from Rockefeller University Press via http://dx.doi.org/10.1083/jcb.20141100

Enhancer Priming Enables Fast and Sustained Transcriptional Responses to Notch Signaling.

Information from developmental signaling pathways must be accurately decoded to generate transcriptional outcomes. In the case of Notch, the intracellular domain (NICD) transduces the signal directly to the nucleus. How enhancers decipher NICD in the real time of developmental decisions is not known. Using the MS2-MCP system to visualize nascent transcripts in single cells in Drosophila embryos, we reveal how two target enhancers read Notch activity to produce synchronized and sustained profiles of transcription. By manipulating the levels of NICD and altering specific motifs within the enhancers, we uncover two key principles. First, increased NICD levels alter transcription by increasing duration rather than frequency of transcriptional bursts. Second, priming of enhancers by tissue-specific transcription factors is required for NICD to confer synchronized and sustained activity; in their absence, transcription is stochastic and bursty. The dynamic response of an individual enhancer to NICD thus differs depending on the cellular context.Wellcome Trus

The atypical mammalian ligand Delta-like homologue 1 (Dlk1) can regulate Notch signalling in Drosophila

<p>Abstract</p> <p>Background</p> <p>Mammalian <it>Delta-like 1 </it>(<it>Dlk-1</it>) protein shares homology with Notch ligands but lacks a critical receptor-binding domain. Thus it is unclear whether it is able to interact with Notch <it>in vivo</it>. Unlike mammals, <it>Drosophila </it>have a single Notch receptor allowing a simple <it>in vivo </it>assay for mammalian <it>Dlk1 </it>function.</p> <p>Results</p> <p>Here we show that membrane-bound DLK1 can regulate Notch leading to altered cellular distribution of Notch itself and inhibiting expression of Notch target genes. The resulting adult phenotypes are indicative of reduced Notch function and are enhanced by <it>Notch </it>mutations, confirming that DLK1 action is antagonistic. In addition, cells expressing an alternative <it>Dlk1 </it>isoform exhibit alterations in cell size, functions previously not attributed to Notch suggesting that DLK1 might also act via an alternative target.</p> <p>Conclusion</p> <p>Our results demonstrate that DLK1 can regulate the Notch receptor despite its atypical structure.</p

Notch stimulates growth by direct regulation of genes involved in the control of glycolysis and the tricarboxylic acid cycle.

Glycolytic shift is a characteristic feature of rapidly proliferating cells, such as cells during development and during immune response or cancer cells, as well as of stem cells. It results in increased glycolysis uncoupled from mitochondrial respiration, also known as the Warburg effect. Notch signalling is active in contexts where cells undergo glycolytic shift. We decided to test whether metabolic genes are direct transcriptional targets of Notch signalling and whether upregulation of metabolic genes can help Notch to induce tissue growth under physiological conditions and in conditions of Notch-induced hyperplasia. We show that genes mediating cellular metabolic changes towards the Warburg effect are direct transcriptional targets of Notch signalling. They include genes encoding proteins involved in glucose uptake, glycolysis, lactate to pyruvate conversion and repression of the tricarboxylic acid cycle. The direct transcriptional upregulation of metabolic genes is PI3K/Akt independent and occurs not only in cells with overactivated Notch but also in cells with endogenous levels of Notch signalling and in vivo. Even a short pulse of Notch activity is able to elicit long-lasting metabolic changes resembling the Warburg effect. Loss of Notch signalling in Drosophila wing discs as well as in human microvascular cells leads to downregulation of glycolytic genes. Notch-driven tissue overgrowth can be rescued by downregulation of genes for glucose metabolism. Notch activity is able to support growth of wing during nutrient-deprivation conditions, independent of the growth of the rest of the body. Notch is active in situations that involve metabolic reprogramming, and the direct regulation of metabolic genes may be a common mechanism that helps Notch to exert its effects in target tissues

The SLC36 transporter Pathetic is required for neural stem cell proliferation and for brain growth under nutrition restriction

Abstract: Background: Drosophila neuroblasts (NBs) are neural stem cells whose maintenance relies on Notch activity. NBs proliferate throughout larval stages to generate a large number of adult neurons. Their proliferation is protected under conditions of nutrition restriction but the mechanisms responsible are not fully understood. As amino acid transporters (Solute Carrier transporters, SLCs), such as SLC36, have important roles in coupling nutrition inputs to growth pathways, they may have a role in this process. For example, an SLC36 family transporter Pathetic (Path) that supports body size and neural dendrite growth in Drosophila, was identified as a putative Notch target in genome-wide studies. However, its role in sustaining stem cell proliferation and maintenance has not been investigated. This study aimed to investigate the function of Path in the larval NBs and to determine whether it is involved in protecting them from nutrient deprivation. Methods: The expression and regulation of Path in the Drosophila larval brain was analysed using a GFP knock-in allele and reporter genes containing putative Notch regulated enhancers. Path function in NB proliferation and overall brain growth was investigated under different nutrition conditions by depleting it from specific cell types in the CNS, using mitotic recombination to generate mutant clones or by directed RNA-interference. Results: Path is expressed in both NBs and glial cells in the Drosophila CNS. In NBs, path is directly targeted by Notch signalling via Su(H) binding at an intronic enhancer, PathNRE. This enhancer is responsive to Notch regulation both in cell lines and in vivo. Loss of path in neural stem cells delayed proliferation, consistent with it having a role in NB maintenance. Expression from pathNRE was compromised in conditions of amino acid deprivation although other Notch regulated enhancers are unaffected. However, NB-expressed Path was not required for brain sparing under amino acid deprivation. Instead, it appears that Path is important in glial cells to help protect brain growth under conditions of nutrient restriction. Conclusions: We identify a novel Notch target gene path that is required in NBs for neural stem cell proliferation, while in glia it protects brain growth under nutrition restriction