Notch signaling involves a highly conserved pathway that mediates communication between neighboring cells. Activation of Notch by its ligands, results in the release of the Notch intracellular domain (NICD), which enters the nucleus and regulates transcription. This pathway has been implicated in many developmental decisions and diseases (including cancers) over the past decades. The simplicity of the Notch pathway in Drosophila melanogaster, in combination with the availability of powerful genetics, make this an attractive model for studying fundamental principles of Notch regulation and function. In this article we present some of the established and emerging tools that are available to monitor and manipulate the Notch pathway in Drosophila and discuss their strengths and weaknesses.

The outcome of the Notch pathway on proliferation depends on cellular context, being growth promotion in some, including several cancers, and growth inhibition in others. Such disparate outcomes are evident in Drosophila wing discs, where Notch overactivation causes hyperplasia despite having localized inhibitory effects on proliferation. To understand the underlying mechanisms, we have used genomic strategies to identify the Notch-CSL target genes directly activated during wing disc hyperplasia. Among them were genes involved in both autonomous and non-autonomous regulation of proliferation, growth and cell death, providing molecular explanations for many characteristics of Notch induced wing disc hyperplasia previously reported. The Notch targets exhibit different response patterns, which are shaped by both positive and negative feed-forward regulation between the Notch targets themselves. We propose, therefore, that both the characteristics of the direct Notch targets and their cross-regulatory relationships are important in coordinating the pattern of hyperplasia.

The histone chaperone Asf1 assists in chromatin assembly and remodeling during replication, transcription activation, and gene silencing. However, it has been unclear to what extent Asf1 could be targeted to specific loci via interactions with sequence-specific DNA-binding proteins. Here, we show that Asf1 contributes to the repression of Notch target genes, as depletion of Asf1 in cells by RNAi causes derepression of the E(spl) Notch-inducible genes. Conversely, overexpression of Asf1 in vivo results in decreased expression of target genes and produces phenotypes that are strongly modified (enhanced and suppressed) by mutations affecting the Notch pathway, but not by mutations in other signaling pathways. Asf1 can be coprecipitated with the DNA-binding protein Su(H) and the corepressor Hairless and interacts directly with two components of this complex, Hairless and SKIP. Thus, in addition to playing more general roles in chromatin dynamics, Asf1 is directed via interactions with sequence-specific complexes to mediate silencing of specific target genes.

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.

Notch signaling regulates many fundamental events including lateral inhibition and boundary formation to generate very reproducible patterns in developing tissues. Its targets include genes of the bHLH hairy and Enhancer of split [E(spl)] family, which contribute to many of these developmental decisions. One member of this family in Drosophila, deadpan (dpn), was originally found to have functions independent of Notch in promoting neural development. Employing genome-wide chromatin-immunoprecipitation we have identified several Notch responsive enhancers in dpn, demonstrating its direct regulation by Notch in a range of contexts including the Drosophila wing and eye. dpn expression largely overlaps that of several E(spl) genes and the combined knock-down leads to more severe phenotypes than either alone. In addition, Dpn contributes to the establishment of Cut expression at the wing dorsal-ventral (D/V) boundary; in its absence Cut expression is delayed. Furthermore, over-expression of Dpn inhibits expression from E(spl) gene enhancers, but not vice versa, suggesting that dpn contributes to a feed-back mechanism that limits E(spl) gene expression following Notch activation. Thus the combined actions of dpn and E(spl) appear to provide a mechanism that confers an initial rapid output from Notch activity which becomes self-limited via feedback between the targets.

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 Drosophila post-embryonic neuroblasts (pNBs) are neural stem cells that persist in the larval nervous system where they proliferate to produce neurons for the adult CNS. These pNBs provide a good model to investigate mechanisms regulating the maintenance and proliferation of stem cells. The transcription factor Grainyhead (Grh), which is required for morphogenesis of epidermal and tracheal cells, is also expressed in all pNBs. Here, we show that grh is essential for pNBs to adopt the stem cell programme appropriate to their position within the CNS. In grh mutants the abdominal pNBs produced more progeny while the thoracic pNBs, in contrast, divided less and produced fewer progeny than wild type. We investigated three candidates; the Neuroblast identify gene Castor, the signalling molecule Notch and the adhesion protein E-Cadherin, to determine whether they could mediate these effects. Neither Castor nor Notch fulfilled the criteria for intermediaries, and in particular Notch activity was found to be dispensable for the normal proliferation and survival of the pNBs. In contrast E-Cadherin, which has been shown to regulate pNB proliferation, was present at greatly reduced levels in the grh mutant pNBs. Furthermore, ectopic expression of Grh was sufficient to promote ectopic E-Cadherin and two conserved Grh-binding sites were identified in the E-Cadherin/shotgun flanking sequences, arguing that this gene is a downstream target. Thus one way Grh could regulate pNBs is through expression of E-cadherin, a protein that is thought to mediate interactions with the glial niche.

In all metazoans, a small number of evolutionarily conserved signaling pathways are reiteratively used during development to orchestrate critical patterning and morphogenetic processes. Among these, Notch (N) signaling is essential for most aspects of tissue patterning where it mediates the communication between adjacent cells to control cell fate specification. In Drosophila, Notch signaling is required for several features of eye development, including the R3/R4 cell fate choice and R7 specification. Here we show that hypomorphic alleles of Notch, belonging to the Nfacet class, reveal a novel phenotype: while photoreceptor specification in the mutant ommatidia is largely normal, defects are observed in ommatidial rotation (OR), a planar cell polarity (PCP)-mediated cell motility process. We demonstrate that during OR Notch signaling is specifically required in the R4 photoreceptor to upregulate the transcription of argos (aos), an inhibitory ligand to the epidermal growth factor receptor (EGFR), to fine-tune the activity of EGFR signaling. Consistently, the loss-of-function defects of Nfacet alleles and EGFR-signaling pathway mutants are largely indistinguishable. A Notch-regulated aos enhancer confers R4 specific expression arguing that aos is directly regulated by Notch signaling in this context via Su(H)-Mam-dependent transcription.

The Notch pathway mediates cell-to-cell communication in a variety of tissues, developmental stages and organisms. Pathway activation relies on the interaction between transmembrane ligands and receptors on adjacent cells. As such, pathway activity could be influenced by the size, composition or dynamics of contacts between membranes. The initiation of Notch signalling in the Drosophila embryo occurs during cellularization, when lateral cell membranes and adherens junctions are first being deposited, allowing us to investigate the importance of membrane architecture and specific junctional domains for signalling. By measuring Notch-dependent transcription in live embryos, we established that it initiates while lateral membranes are growing and that signalling onset correlates with a specific phase in their formation. However, the length of the lateral membranes per se was not limiting. Rather, the adherens junctions, which assemble concurrently with membrane deposition, contributed to the high levels of signalling required for transcription, as indicated by the consequences of α-Catenin depletion. Together, these results demonstrate that the establishment of lateral membrane contacts can be limiting for Notch trans-activation and suggest that adherens junctions play an important role in modulating Notch activity.

Accurate Notch signalling is critical for development and homeostasis. Fine-tuning of Notch-ligand interactions has substantial impact on signalling outputs. Recent structural studies have identified a conserved N-terminal C2 domain in human Notch ligands which confers phospholipid binding in vitro. Here, we show that Drosophila ligands Delta and Serrate adopt the same C2 domain structure with analogous variations in the loop regions, including the so-called β1-2 loop that is involved in phospholipid binding. Mutations in the β1-2 loop of the Delta C2 domain retain Notch binding but have impaired ability to interact with phospholipids in vitro. To investigate its role in vivo, we deleted five residues within the β1-2 loop of endogenous Delta. Strikingly, this change compromises ligand function. The modified Delta enhances phenotypes produced by Delta loss-of-function alleles and suppresses that of Notch alleles. As the modified protein is present on the cell surface in normal amounts, these results argue that C2 domain phospholipid binding is necessary for robust signalling in vivo fine-tuning the balance of trans and cis ligand-receptor interactions.

Active Notch signalling is elicited through receptor-ligand interactions that result in release of the Notch intracellular domain (NICD), which translocates into the nucleus. NICD activates transcription at target genes, forming a complex with the DNA-binding transcription factor CSL [CBF1/Su(H)/LAG-1] and co-activator Mastermind. However, CSL lacks its own nuclear localisation sequence, and it remains unclear where the tripartite complex is formed. To probe the mechanisms involved, we designed an optogenetic approach to control NICD release (OptIC-Notch) and monitored the subsequent complex formation and target gene activation. Strikingly, we observed that, when uncleaved, OptIC-Notch sequestered CSL in the cytoplasm. Hypothesising that exposure of a juxta membrane ΦWΦP motif is key to sequestration, we masked this motif with a second light-sensitive domain (OptIC-Notch{ω}), which was sufficient to prevent CSL sequestration. Furthermore, NICD produced by light-induced cleavage of OptIC-Notch or OptIC-Notch{ω} chaperoned CSL into the nucleus and induced target gene expression, showing efficient light-controlled activation. Our results demonstrate that exposure of the ΦWΦP motif leads to CSL recruitment and suggest this can occur in the cytoplasm prior to nuclear entry.

Regulation of transcription is fundamental to development and physiology, and occurs through binding of transcription factors to specific DNA sequences in the genome. CSL (CBF1/Suppressor of Hairless/LAG-1), a core component of the Notch signaling pathway, is one such transcription factor that acts in concert with co-activators or co-repressors to control the activity of associated target genes. One fundamental question is how CSL can recognize and select among different DNA sequences available in vivo and whether variations between selected sequences can influence its function. We have therefore investigated CSL-DNA recognition using computational approaches to analyze the energetics of CSL bound to different DNAs and tested the in silico predictions with in vitro and in vivo assays. Our results reveal novel aspects of CSL binding that may help explain the range of binding observed in vivo. In addition, using molecular dynamics simulations, we show that domain-domain correlations within CSL differ significantly depending on the DNA sequence bound, suggesting that different DNA sequences may directly influence CSL function. Taken together, our results, based on computational chemistry approaches, provide valuable insights into transcription factor-DNA binding, in this particular case increasing our understanding of CSL-DNA interactions and how these may impact on its transcriptional control.

Wiring between signaling pathways differs according to context, as exemplified by interactions between Notch and epidermal growth factor receptor (EGFR) pathways, which are cooperative in some contexts but antagonistic in others. To investigate mechanisms that underlie different modes of cross talk, we have focused on argos, an EGFR pathway regulator in Drosophila melanogaster which is upregulated by Notch in adult muscle progenitors but is repressed in the wing. Results show that the alternate modes of cross talk depend on the engagement of enhancers with opposite regulatory logic, which are selected by context-determining factors. This is likely to be a general mechanism for enabling the wiring between these pathways to switch according to context.

Repressors are frequently deployed to limit the transcriptional response to signalling pathways. For example, several co-repressors interact directly with the DNA-binding protein CSL and are proposed to keep target genes silenced in the absence of Notch activity. However, the scope of their contributions remains unclear. To investigate co-repressor activity in the context of this well defined signalling pathway, we have analysed the genome-wide binding profile of the best-characterized CSL co-repressor in Drosophila, Hairless, and of a second CSL interacting repressor, SMRTER. As predicted there was significant overlap between Hairless and its CSL DNA-binding partner, both in Kc cells and in wing discs, where they were predominantly found in chromatin with active enhancer marks. However, while the Hairless complex was widely present at some Notch regulated enhancers in the wing disc, no binding was detected at others, indicating that it is not essential for silencing per se. Further analysis of target enhancers confirmed differential requirements for Hairless. SMRTER binding significantly overlapped with Hairless, rather than complementing it, and many enhancers were apparently co-bound by both factors. Our analysis indicates that the actions of Hairless and SMRTER gate enhancers to Notch activity and to Ecdysone signalling respectively, to ensure that the appropriate levels and timing of target gene expression are achieved.

During development, tissues and organs must coordinate growth and patterning so they reach the right size and shape. During larval stages, a dramatic increase in size and cell number of Drosophila wing imaginal discs is controlled by the action of several signaling pathways. Complex cross-talk between these pathways also pattern these discs to specify different regions with different fates and growth potentials. We show that the Notch signaling pathway is both required and sufficient to inhibit the activity of Yorkie (Yki), the Salvador/Warts/Hippo (SWH) pathway terminal transcription activator, but only in the central regions of the wing disc, where the TEAD factor and Yki partner Scalloped (Sd) is expressed. We show that this cross-talk between the Notch and SWH pathways is mediated, at least in part, by the Notch target and Sd partner Vestigial (Vg). We propose that, by altering the ratios between Yki, Sd and Vg, Notch pathway activation restricts the effects of Yki mediated transcription, therefore contributing to define a zone of low proliferation in the central wing discs.

Notch signalling is involved in a multitude of developmental decisions and its aberrant activation is linked to many diseases, including cancers. One example is the neural stem cell tumours that arise from constitutive Notch activity in Drosophila neuroblasts. To investigate how hyperactivation of Notch in larval neuroblasts leads to tumours, we combined results from profiling the upregulated mRNAs and mapping the regions bound by the core Notch pathway transcription factor Su(H). This identified 246 putative direct Notch targets. These genes were highly enriched for transcription factors and overlapped significantly with a previously identified regulatory programme dependent on the proneural transcription factor Asense. Included were genes associated with the neuroblast maintenance and self-renewal programme that we validated as Notch regulated in vivo. Another group were the so-called temporal transcription factors, which have been implicated in neuroblast maturation. Normally expressed in specific time windows, several temporal transcription factors were ectopically expressed in the stem cell tumours, suggesting that Notch had reprogrammed their normal temporal regulation. Indeed, the Notch-induced hyperplasia was reduced by mutations affecting two of the temporal factors, which, conversely, were sufficient to induce mild hyperplasia on their own. Altogether, the results suggest that Notch induces neuroblast tumours by directly promoting the expression of genes that contribute to stem cell identity and by reprogramming the expression of factors that could regulate maturity.

Histone chaperones are involved in a variety of chromatin transactions. By a proteomics survey, we identified the interaction networks of histone chaperones ASF1, CAF1, HIRA, and NAP1. Here, we analyzed the cooperation of H3/H4 chaperone ASF1 and H2A/H2B chaperone NAP1 with two closely related silencing complexes: LAF and RLAF. NAP1 binds RPD3 and LID-associated factors (RLAF) comprising histone deacetylase RPD3, histone H3K4 demethylase LID/KDM5, SIN3A, PF1, EMSY, and MRG15. ASF1 binds LAF, a similar complex lacking RPD3. ASF1 and NAP1 link, respectively, LAF and RLAF to the DNA-binding Su(H)/Hairless complex, which targets the E(spl) NOTCH-regulated genes. ASF1 facilitates gene-selective removal of the H3K4me3 mark by LAF but has no effect on H3 deacetylation. NAP1 directs high nucleosome density near E(spl) control elements and mediates both H3 deacetylation and H3K4me3 demethylation by RLAF. We conclude that histone chaperones ASF1 and NAP1 differentially modulate local chromatin structure during gene-selective silencing.

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.

A major challenge in cancer genetics is to determine which low-frequency somatic mutations are drivers of tumorigenesis. Here we interrogate the genomes of 7,651 diverse human cancers and find inactivating mutations in the homeodomain transcription factor gene CUX1 (cut-like homeobox 1) in ~1-5% of various tumors. Meta-analysis of CUX1 mutational status in 2,519 cases of myeloid malignancies reveals disruptive mutations associated with poor survival, highlighting the clinical significance of CUX1 loss. In parallel, we validate CUX1 as a bona fide tumor suppressor using mouse transposon-mediated insertional mutagenesis and Drosophila cancer models. We demonstrate that CUX1 deficiency activates phosphoinositide 3-kinase (PI3K) signaling through direct transcriptional downregulation of the PI3K inhibitor PIK3IP1 (phosphoinositide-3-kinase interacting protein 1), leading to increased tumor growth and susceptibility to PI3K-AKT inhibition. Thus, our complementary approaches identify CUX1 as a pan-driver of tumorigenesis and uncover a potential strategy for treating CUX1-mutant tumors.

A key feature of Notch signaling is that it directs immediate changes in transcription via the DNA-binding factor CSL, switching it from repression to activation. How Notch generates both a sensitive and accurate response-in the absence of any amplification step-remains to be elucidated. To address this question, we developed real-time analysis of CSL dynamics including single-molecule tracking in vivo. In Notch-OFF nuclei, a small proportion of CSL molecules transiently binds DNA, while in Notch-ON conditions CSL recruitment increases dramatically at target loci, where complexes have longer dwell times conferred by the Notch co-activator Mastermind. Surprisingly, recruitment of CSL-related corepressors also increases in Notch-ON conditions, revealing that Notch induces cooperative or "assisted" loading by promoting local increase in chromatin accessibility. Thus, in vivo Notch activity triggers changes in CSL dwell times and chromatin accessibility, which we propose confer sensitivity to small input changes and facilitate timely shut-down.