Search

Notch signaling relies on ligand-induced proteolysis of the transmembrane receptor Notch to liberate a nuclear effector that drives cell fate decisions. Upon ligand binding, sequential cleavage of Notch by the transmembrane protease ADAM10 and the intracellular protease γ-secretase releases the Notch intracellular domain (NICD), which translocates to the nucleus and forms a complex that induces target gene transcription. To map the location and timing of the individual steps required for the proteolysis and movement of Notch from the plasma membrane to the nucleus, we used proximity labeling with quantitative, multiplexed mass spectrometry to monitor the interaction partners of endogenous NOTCH2 after ligand stimulation in the presence of a γ-secretase inhibitor and as a function of time after inhibitor removal. Our studies showed that γ-secretase–mediated cleavage of NOTCH2 occurred in an intracellular compartment and that formation of nuclear complexes and recruitment of chromatin-modifying enzymes occurred within 45 min of inhibitor washout. These findings provide a detailed spatiotemporal map tracking the path of Notch from the plasma membrane to the nucleus and identify signaling events that are potential targets for modulating Notch activity. Editor's summary: Ligand binding to the transmembrane receptor Notch induces two proteolytic cleavage events that liberate its intracellular domain (NICD), which translocates to the nucleus to modulate gene expression. Martin et al. generated a time-resolved map of the intracellular proteins encountered by endogenous NOTCH2/NICD2 as it transited to the nucleus in stimulated human cells. The second cleavage event triggering NICD2 release occurred at an intracellular compartment, and NICD2 then transiently interacted with proteins that link membranes to the actin cytoskeleton. Such time-resolved proximity mapping may be used to track the paths of other signaling proteins in cells. —Annalisa M. VanHook [ABSTRACT FROM AUTHOR]

Mastermind proteins are required for transcription of Notch target genes, yet the molecular basis for mastermind function remains incompletely understood. Previous work has shown that Notch can induce transcriptional responses by binding to promoters, but more often by binding to enhancers, with HES4 and DTX1 as representative mammalian examples of promoter and enhancer responsiveness, respectively. Here, we show that mastermind dependence of the Notch response at these loci is differentially encoded in Jurkat T-cell acute lymphoblastic leukemia (T-ALL) cells. Knockout of Mastermind-like1 (MAML1) eliminates Notch responsive activation of both these genes, and reduced target gene expression is accompanied by a decrease in H3K27 acetylation, consistent with the importance of MAML1 for p300 activity. Add-back of MAML1 variants in knockout cells identifies residues 151-350 of MAML1 as essential for expression of either Notch-responsive gene. Fusion of the Notch-binding region of MAML1 to the histone acetyltransferase (HAT) domain of p300 rescues expression of HES4 but not DTX1, suggesting that an additional activity of MAML1 is needed for gene induction at a distance. Together, these studies establish the functional importance of the MAML1 151-350 region for Notch-dependent transcriptional induction, and reveal differential requirements for MAML1- dependent recruitment activities at different Notch responsive loci, highlighting the molecular complexity of Notch-stimulated transcription. [ABSTRACT FROM AUTHOR]

Notch is a major oncogenic driver in T cell acute lymphoblastic leukemia (T-ALL), in part because it binds to an enhancer that increases expression of MYC. Here, we exploit the capacity of activated NOTCH1 and NOTCH3 to induce T-ALL, despite substantial divergence in their intracellular regions, as a means to elucidate a broad, common Notch-dependent oncogenomic program through systematic comparison of the transcriptomes and Notch-bound genomic regulatory elements of NOTCH1- and NOTCH3-dependent T-ALL cells. ChIP-seq studies show a high concordance of functional NOTCH1 and NOTCH3 genomic binding sites that are enriched in binding motifs for RBPJ, the transcription factor that recruits activated Notch to DNA. The interchangeability of NOTCH1 and NOTCH3 was confirmed by rescue of NOTCH1-dependent T-ALL cells with activated NOTCH3 and vice versa. Despite remarkable overall similarity, there are nuanced differences in chromatin landscapes near critical common Notch target genes, most notably at a Notch-dependent enhancer that regulates MYC, which correlates with responsiveness to Notch pathway inhibitors. Overall, a common oncogenomic program driven by binding of either Notch is sufficient to maintain T-ALL cell growth, whereas cell-context specific differences appear to influence the response of T-ALL cells to Notch inhibition. [ABSTRACT FROM AUTHOR]

Ligand-induced proteolysis of Notch produces an intracellular effector domain that transduces essential signals by regulating the transcription of target genes. This function relies on the formation of transcriptional activation complexes that include intracellular Notch, a Mastermind co-activator and the transcription factor CSL bound to cognate DNA. These complexes form higher-order assemblies on paired, head-to-head CSL recognition sites. Here we report the X-ray structure of a dimeric human Notch1 transcription complex loaded on the paired site from the human HES1 promoter. The small interface between the Notch ankyrin domains could accommodate DNA bending and untwisting to allow a range of spacer lengths between the two sites. Cooperative dimerization occurred on the human and mouse Hes5 promoters at a sequence that diverged from the CSL-binding consensus at one of the sites. These studies reveal how promoter organizational features control cooperativity and, thus, the responsiveness of different promoters to Notch signaling. [ABSTRACT FROM AUTHOR]

Aberrant activation of Notch receptors has been implicated in breast cancer; however, the mechanisms contributing to Notch-dependent transformation remain elusive because Notch displays dichotomous functional activities, promoting both proliferation and growth arrest. We investigated the cellular basis for the heterogeneous responses to Notch pathway activation in 3D cultures of MCF-10A mammary epithelial cells. Expression of a constitutively active Notch-1 intracellular domain (NICD) was found to induce two distinct types of 3D structures: large, hyperproliferative structures and small, growth-arrested structures with reduced cell-to-matrix adhesion. Interestingly, we found that these heterogeneous phenotypes reflect differences in Notch pathway activation levels; high Notch activity caused down-regulation of multiple matrix-adhesion genes and inhibition of proliferation, whereas low Notch activity maintained matrix adhesion and provoked a strong hyperproliferative response. Moreover, micro-array analyses implicated NICD-induced p63 down-regulation in loss of matrix adhesion. In addition, a reverse-phase protein array-based analysis and subsequent loss-of-function studies identified STAT3 as a dominant downstream mediator of the NICD-induced outgrowth. These results indicate that the phenotypic responses to Notch are determined by the dose of pathway activation; and this dose affects the balance between growth-stimulative and growth-suppressive effects. This unique feature of Notch signaling provides insights into mechanisms that contribute to the dichotomous effects of Notch during development and tumorigenesis. [ABSTRACT FROM AUTHOR]

Gain-of-function experiments have demonstrated the potential of Notch signals to expand primitive hematopoietic progenitors, but whether Notch physiologically regulates hematopoietic stem cell (HSC) homeostasis in vivo is unclear. To answer this question, we evaluated the effect of global deficiencies of canonical Notch signaling in rigorous HSC assays. Hematopoietic progenitors expressing dominant- negative Mastermind-likel (DNMAML), a potent inhibitor of Notch-mediated transcriptional activation, achieved stable long-term reconstitution of irradiated hosts and showed a normal frequency of progenitor fractions enriched for long-term HSCs. Similar results were observed with cells lacking CSLI RBPJ, a DNA-binding factor that is required for canonical Notch signaling. Notch-deprived progenitors provided normal long-term reconstitution after secondary competitive transplantation. Furthermore, Notch target genes were expressed at low levels in primitive hematopoietic progenitors. Taken together, these results rule out an essential physiological role for cell-autonomous canonical Notch signals in HSC maintenance. [ABSTRACT FROM AUTHOR]

The NOTCH1 signaling pathway directly links extracellular signals with transcriptional responses in the cell nucleus and plays a critical role during T cell development and in the pathogenesis over 50% of human T cell lymphoblastic leukemia (T-ALL) cases. However, little is known about the transcriptional programs activated by NOTCH1. Using an integrative systems biology approach we show that NOTCH1 controls a feed-forward-loop transcriptional network that promotes cell growth. Inhibition of NOTCH1 signaling in T-ALL cells led to a reduction in cell size and elicited a gene expression signature dominated by down-regulated biosynthetic pathway genes. By integrating gene expression array and ChiP-on-chip data, we show that NOTCH1 directly activates multiple biosynthetic routes and induces c-MYC gene expression. Reverse engineering of regulatory networks from expression profiles showed that NOTCH1 and c-MYC govern two directly interconnected transcriptional programs containing common target genes that together regulate the growth of primary T-ALL cells. These results identify c-MYC as an essential mediator of NOTCH1 signaling and integrate NOTCH1 activation with oncogenic signaling pathways upstream of c-MYC. [ABSTRACT FROM AUTHOR]

In Drosophila, developmental signaling via the transmembrane Notch receptor modulates branching morphogenesis and neuronal differentiation. To determine whether the notch gene family can regulate mammalian organogenesis, including neuroendocrine cell differentiation, we evaluated developing murine lung. After demonstrating gene expression for notch-1, notch-2, notch-3, and the Notch ligands jagged-1 and jagged-2 in embryonic mouse lung, we tested whether altering expression of these genes can modulate branching morphogenesis. Branching of embryonic day (E) 11.5 lung buds increased when they were treated with notch-1 antisense oligodeoxynucleotides in culture compared with the corresponding sense controls, whereas notch-2, notch-3, jagged-1, or jagged-2 antisense oligos had no significant effect. To assess cell differentiation, we immunostained lung bud cultures for the neural/neuroendocrine marker PGP9.5. Antisense to notch-1 or jagged-1 markedly increased numbers of PGP9.5-positive neuroendocrine cells alone without affecting neural tissue, whereas only neural tissue was promoted by notch-3 antisense in culture. There was no significant effect on cell proliferation or apoptosis in these antisense experiments. Cumulatively, these observations suggest that interactions between distinct Notch family members can have diverse tissue-specific regulatory functions during development, arguing against simple functional redundancy. [ABSTRACT FROM AUTHOR]

Constitutive NOTCH signaling in lymphoid progenitors promotes the development of immature T-cell lymphoblastic neoplasms (T-ALLs). Although it is clear that Notch signaling can initiate leukemogenesis, it has not previously been established whether continued NOTCH signaling is required to maintain T-ALL growth. We demonstrate here that the blockade of Notch signaling at two independent steps suppresses the growth and survival of NOTCH1-transformed T-ALL cells. First, inhibitors of presenilin specifically induce growth suppression and apoptosis of a murine T-ALL cell line that requires presenilin-dependent proteolysis of the Notch receptor in order for its intracellular domain to translocate to the nucleus. Second, a 62-aminoacid peptide derived from a NOTCH coactivator, Mastermind-like-1 (MAML1), forms a transcriptionally inert nuclear complex with NOTCH1 and CSL and specifically inhibits the growth of both murine and human NOTCH1-transformed T-ALLs. These studies show that continued growth and survival of NOTCH1-transformed lymphoid cell lines require nuclear access and transcriptional coactivator recruitment by NOTCH1 and identify at least two steps in the Notch signaling pathway as potential targets for chemotherapeutic intervention. [ABSTRACT FROM AUTHOR]

Focuses on the role of Notch signaling in the early events of hematopoiesis. Structure of the Notch protein; Mechanism of Notch signaling; Regulators of Notch signaling.

Truncated Notch receptors have transforming activity in vitro and in vivo. However, the role of wild-type Notch signaling in neoplastic transformation remains unclear. Ras signaling is deregulated in a large fraction of human malignancies and is a major target for the development of novel cancer treatments. We show that oncogenic Ras activates Notch signaling and that wild-type Notch-1 is necessary to maintain the neoplastic phenotype in Ras-transformed human cells in vitro and in vivo. Oncogenic Ras increases levels and activity of the intracellular form of wild-type Notch-1, and upregulates Notch ligand Delta-1 and also presenilin-1, a protein involved in Notch processing, through a p38-mediated pathway. These observations place Notch signaling among key downstream effectors of oncogenic Ras and suggest that it might be a novel therapeutic target. [ABSTRACT FROM AUTHOR]

Notch1 signaling drives T cell development at the expense of B cell development from a common precursor, an effect that is dependent on a C-terminal Notch1 transcriptional activation domain. The function of Deltex1, initially identified as a positive modulator of Notch function in a genetic screen in Drosophila, is poorly understood. We now demonstrate that, in contrast to Notch1, enforced expression of Deltex1 in hematopoietic progenitors results in B cell development at the expense of T cell development in fetal thymic organ culture and in vivo. Consistent with these effects, Deltex1 antagonizes Notch1 signaling in transcriptional reporter assays by inhibiting coactivator recruitment. These data suggest that a balance of inductive Notch1 signals and inhibitory signals mediated through Deltex1 and other modulators regulate T-B lineage commitment. [Copyright &y& Elsevier]

Notch receptors are involved in cell-fate determination in organisms as diverse as flies, frogs and humans[SUP1]. In Drosophila melanogaster, loss-of-function mutations of Notch produce a 'neu-rogenic' phenotype in which cells destined to become epidermis switch fate and differentiate to neural cells. Upon ligand activation, the intracellular domain of Notch (ICN) translocates to the nucleus[SUP2], and interacts directly with the DNA-binding protein Suppressor of hairless (Su(H)) in flies, or recombination signal binding protein Jκ (RBP-Jκ) in mammals[SUP3], to activate gene transcription[SUP4]. But the precise mechanisms of Notch-induced gene expression are not completely understood. The gene mastermind has been identified in multiple genetic screens for modifiers of Notch mutations in Drosophila[SUP5-8]. Here we clone MAML1, a human homologue of the Drosophila gene Mastermind, and show that it encodes a protein of 130 kD localizing to nuclear bodies. MAML1 binds to the ankyrin repeat domain of all four mammalian NOTCH receptors, forms a DNA-binding complex with ICN and RBP-Jκ, and amplifies NOTCH-induced transcription of HES1. These studies provide a molecular mechanism to explain the genetic links between mastermind and Notch in Drosophila and indicate that MAML1 functions as a transcriptional co-activator for NOTCH signalling. [ABSTRACT FROM AUTHOR]

Mammalian Notch signaling occurs when the binding of Delta or Jagged to Notch stimulates the proteolytic release of the Notch intracellular domain (NICD), which enters the nucleus to control target gene expression. To determine the temporal dynamics of events associated with Notch signaling under native conditions, we fluorescently tagged Notch and Delta at their endogenous genomic loci and visualized them upon pairing of receiver (Notch) and sender (Delta) cells as a function of time after cell contact. At contact sites, Notch and Delta immediately accumulated at 1:1 stoichiometry in synapses, which resolved by 15–20 min after contact. Synapse formation preceded the entrance of the Notch extracellular domain into the sender cell and accumulation of NICD in the nucleus of the receiver cell, which approached a maximum after ∼45 min and was prevented by chemical and genetic inhibitors of signaling. These findings directly link Notch-Delta synapse dynamics to NICD production with spatiotemporal precision. [Display omitted] • Notch and Delta form synapses with 1:1 stoichiometry at sites of cell-cell contact • Synapses are transient, resolving after ∼20 min of cell contact • Synapse formation precedes entry of the Notch ectodomain into the Delta cell • Synapse formation precedes nuclear accumulation of the Notch intracellular domain Tveriakhina, Scanavachi, et al. employed live-cell microscopy to visualize the stoichiometry and dynamics of Notch and Delta at natural protein abundance in sender and receiver cells after contact. This work directly connects Notch-Delta synapse dynamics to Notch ectodomain transendocytosis and Notch intracellular domain nuclear accumulation with spatiotemporal precision. [ABSTRACT FROM AUTHOR]

Notch signaling regulates squamous cell proliferation and differentiation and is frequently disrupted in squamous cell carcinomas, in which Notch is tumor suppressive. Here, we show that conditional activation of Notch in squamous cells activates a context-specific gene expression program through lineage-specific regulatory elements. Among direct Notch target genes are multiple DNA damage response genes, including IER5, which we show is required for Notch-induced differentiation of squamous carcinoma cells and TERT-immortalized keratinocytes. IER5 is epistatic to PPP2R2A, a gene that encodes the PP2A B55a subunit, which we show interacts with IER5 in cells and in purified systems. Thus, Notch and DNA-damage response pathways converge in squamous cells on common genes that promote differentiation, which may serve to eliminate damaged cells from the proliferative pool. We further propose that crosstalk involving Notch and PP2A enables tuning and integration of Notch signaling with other pathways that regulate squamous differentiation. [ABSTRACT FROM AUTHOR]

Notch signaling makes critical contributions to cell fate determination in all metazoan organisms, yet remarkably little is known about the binding affinity of the four mammalian Notch receptors for their three Delta-like and two Jagged family ligands. Here, we utilized signaling assays and biochemical studies of purified recombinant ligand and receptor molecules to investigate the differences in signaling behavior and intrinsic affinity between Notch1-Dll1 and Notch1-Dll4 complexes. Systematic deletion mutagenesis of the human Notch1 ectodomain revealed that epidermal growth factor (EGF) repeats 6-15 are sufficient to maintain signaling in a reporter assay at levels comparable with the full-length receptor, and identified important contributions from EGF repeats 8-10 in conveying an activating signal in response to either Dll1 or Dll4. Truncation studies of the Dll1 and Dll4 ectodomains showed that the MNNL-EGF3 region was both necessary and sufficient for full activation. Plate-based and cell binding assays revealed a specific, calcium-dependent interaction between cell-surface and recombinant Notch receptors and ligand molecules. Finally, direct measurement of the binding affinity of Notch1 EGF repeats 6-15 for Dll1 and Dll4 revealed that Dll4 binds with at least an order of magnitude higher affinity than Dll1. Together, these studies give new insights into the features of ligand recognition by Notch1, and highlight how intrinsic differences in the biochemical behavior of receptor-ligand complexes can influence receptor mediated responses of developmental signaling pathways. [ABSTRACT FROM AUTHOR]

Summary: Notch1 is a rational therapeutic target in several human cancers, but as a transcriptional regulator, it poses a drug discovery challenge. To identify Notch1 modulators, we performed two cell-based, high-throughput screens for small-molecule inhibitors and cDNA enhancers of a NOTCH1 allele bearing a leukemia-associated mutation. Sarco/endoplasmic reticulum calcium ATPase (SERCA) channels emerged at the intersection of these complementary screens. SERCA inhibition preferentially impairs the maturation and activity of mutated Notch1 receptors and induces a G0/G1 arrest in NOTCH1-mutated human leukemia cells. A small-molecule SERCA inhibitor has on-target activity in two mouse models of human leukemia and interferes with Notch signaling in Drosophila. These studies “credential” SERCA as a therapeutic target in cancers associated with NOTCH1 mutations. [Copyright &y& Elsevier]

The leukemia stem cell (LSC) hypothesis proposes that a subset of cells in the bulk leukemia population propagates the leukemia. We tested the LSC hypothesis in a mouse model of Notch-induced T-cell acute lymphoblastic leukemia (T-ALL) in which the tumor cells were largely CD4+CD8+ T cells. LSC activity was enriched but rare in the CD8+CD4-HSAhi immature single-positive T-cell subset. Although our murine T-ALL model relies on transduction of HSCs, we were unable to isolate Notch-activated HSCs to test for LSC activity. Further analysis showed that Notch activation in HSCs caused an initial expansion of hematopoietic and T-cell progenitors and loss of stem cell quiescence, which was followed by progressive loss of long-term HSCs and T-cell production over several weeks. Similar results were obtained in a conditional transgenic model in which Notch activation is induced in HSCs by Cre recombinase. We conclude that although supraphysiologic Notch signaling in HSCs promotes LSC activity in T-cell progenitors, it extinguishes self-renewal of LT-HSCs. These results provide further evidence for therapeutically targeting T-cell progenitors in T-ALL while also underscoring the need to tightly regulate Notch signaling to expand normal HSC populations for clinical applications. [ABSTRACT FROM AUTHOR]

Notch signaling regulates myriad cellular functions by activating transcription, yet how Notch selectively activates different transcriptional targets is poorly understood. The core Notch transcriptional activation complex can bind DNA as a monomer, but it can also dimerize on DNA-binding sites that are properly oriented and spaced. However, the significance of Notch dimerization is unknown. Here, we show that dimeric Notch transcriptional complexes are required for T-cell maturation and leukemic transformation but are dispensable for T-cell fate specification from a multipotential precursor. The varying requirements for Notch dimerization result from the differential sensitivity of specific Notch target genes. In particular, c-Myc and pre-T-cell antigen receptor a (Ptcra) are dimerization-dependent targets, whereas Hey1 and CD25 are not. These findings identify functionally important differences in the responsiveness among Notch target genes attributable to the formation of higherorder complexes. Consequently, it may be possible to develop a new class of Notch inhibitors that selectively block outcomes that depend on Notch dimerization (e.g., leukemogenesis). [ABSTRACT FROM AUTHOR]

Summary: Notch receptors transduce essential developmental signals between neighboring cells by forming a complex that leads to transcription of target genes upon activation. We report here the crystal structure of a Notch transcriptional activation complex containing the ankyrin domain of human Notch1 (ANK), the transcription factor CSL on cognate DNA, and a polypeptide from the coactivator Mastermind-like-1 (MAML-1). Together, CSL and ANK create a groove to bind the MAML-1 polypeptide as a kinked, 70 Å helix. The composite binding surface likely restricts the recruitment of MAML proteins to promoters on which Notch:CSL complexes have been preassembled, ensuring tight transcriptional control of Notch target genes. [Copyright &y& Elsevier]

Notch1 is a member of a conserved family of large modular heterodimeric type 1 transmembrane receptors that control differentiation in multicellular animals. Receptor maturation is accompanied by a furin-dependent cleavage that converts the Notch1 precursor polypeptide into a heterodimer consisting of an extracellular ligand-binding subunit (NEC) and a transmembrane signaling subunit (NTM). Binding of a physiologic ligand to NEC induces signaling by triggering additional proteolytic cleavages in NTM, which allow its intracellular region to translocate to the nucleus where it participates in a transcriptional activation complex. In the absence of ligand, the three conserved LNR modules of the NEC subunit participate in maintaining the receptor in its resting conformation. Here, we report the solution structure of the first LNR module (LNR_A) of human Notch1, and identify residues of LNR_A perturbed by the presence of the adjacent module LNR_B. LNR_A is held together by a unique arrangement of three disulfide bonds and a single bound Ca[sup 2+] ion, and adopts a novel fold that falls in the general class of irregular disulfide-bonded structures. Residues perturbed by the presence of the adjacent LNR_B module are predominantly hydrophobic, and lie on one face of the module. These studies represent an initial step toward understanding the structural interrelationships among the three contiguous LNR modules required for proper regulation of Notch signaling. [ABSTRACT FROM AUTHOR]

Focuses on the significance of notch signaling on lymphopoiesis. Regulation of cell life by notch signals in metazoans; Influence of notch genes on morphogenesis; Facilitation of renewal and differentiation during hematopoiesis.