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At least 433 records · Page 24Linked to original sources
PubMed2005

Target selectivity of vertebrate notch proteins. Collaboration between discrete domains and CSL-binding site architecture determines activation probability.

All four mammalian Notch proteins interact with a single DNA-binding protein (RBP-jkappa), yet they are not equivalent in activating target genes. Parallel assays of three Notch-responsive promoters in several cell lines revealed that relative activation strength is dependent on protein module and promoter context more than the cellular context. Each Notch protein reads binding site orientation and distribution on the promoter differently; Notch1 performs extremely well on paired sites, and Notch3 prefers single sites in conjunction with a proximal zinc finger transcription factor. Although head-head sites can elicit a Notch response on their own, use of CBS (CSL binding site) in tail-tail orientation is context-dependent. Bias for specific DNA elements is achieved by interplay between the N-terminal RAM (RBP-jkappa-associated molecule/ankyrin region), which interprets CBS proximity and orientation, and the C-terminal transactivation domain that interacts specifically with the transcription machinery or nearby factors. To confirm the prediction that modular design underscores the evolution of functional divergence between Notch proteins, we generated a synthetic Notch protein (Notch1 ankyrin with Notch3 transactivation domain) that displayed superior signaling strength on the hes5 promoter. Consistent with the prediction that "preferred" targets (Hes1) should respond faster and at lower Notch concentration than other targets, we showed that Hes5-GFP was extinguished fast and recovered slowly, whereas Hes1-GFP was inhibited late and recovered quickly after a pulse of DAPT in metanephroi cultures.

Animals↗
PubMed2006

Notch signaling suppresses p38 MAPK activity via induction of MKP-1 in myogenesis.

Cross-talks among intracellular signaling pathways are important for the regulation of cell fate decisions and cellular responses to extracellular signals. Both the Notch pathway and the MAPK pathways play important roles in many biological processes, and the Notch pathway has been shown to interact with the ERK-type MAPK pathway. However, its interaction with the other MAPK pathways is unknown. Here we show that Notch signaling activation in C2C12 cells suppresses the activity of p38 MAPK to inhibit myogenesis. Our results show that Notch specifically induces expression of MKP-1, a member of the dual-specificity MAPK phosphatase, which directly inactivates p38 to negatively regulate C2C12 myogenesis. The Notch-induced expression of MKP-1 is shown to depend on RBP-J. Moreover, inhibition of MKP-1 expression by short interfering RNA suppresses p38 inactivation and partially rescues the negative regulation of myogenesis. These results reveal a novel cross-talk between the Notch pathway and the p38 MAPK pathway that is mediated by Notch induction of MKP-1.

Animals↗
PubMed2000

Analysis of notch lacking the carboxyl terminus identified in Drosophila embryos.

The cell surface receptor Notch is required during development of Drosophila melanogaster for differentiation of numerous tissues. Notch is often required for specification of precursor cells by lateral inhibition and subsequently for differentiation of tissues from these precursor cells. We report here that certain embryonic cells and tissues that develop after lateral inhibition, like the connectives and commissures of the central nervous system, are enriched for a form of Notch not recognized by antibodies made against the intracellular region carboxy-terminal of the CDC10/Ankyrin repeats. Western blotting and immunoprecipitation analyses show that Notch molecules lacking this region are produced during embryogenesis and form protein complexes with the ligand Delta. Experiments with cultured cells indicate that Delta promotes accumulation of a Notch intracellular fragment lacking the carboxyl terminus. Furthermore, Notch lacking the carboxyl terminus functions as a receptor for Delta. These results suggest that Notch activities during development include generation and activity of a truncated receptor we designate NDeltaCterm.

Animals↗
PubMed2004

Loss of negative regulation by Numb over Notch is relevant to human breast carcinogenesis.

The biological antagonism between Notch and Numb controls the proliferative/differentiative balance in development and homeostasis. Although altered Notch signaling has been linked to human diseases, including cancer, evidence for a substantial involvement of Notch in human tumors has remained elusive. Here, we show that Numb-mediated control on Notch signaling is lost in approximately 50% of human mammary carcinomas, due to specific Numb ubiquitination and proteasomal degradation. Mechanistically, Numb operates as an oncosuppressor, as its ectopic expression in Numb-negative, but not in Numb-positive, tumor cells inhibits proliferation. Increased Notch signaling is observed in Numb-negative tumors, but reverts to basal levels after enforced expression of Numb. Conversely, Numb silencing increases Notch signaling in normal breast cells and in Numb-positive breast tumors. Finally, growth suppression of Numb-negative, but not Numb-positive, breast tumors can be achieved by pharmacological inhibition of Notch. Thus, the Numb/Notch biological antagonism is relevant to the homeostasis of the normal mammary parenchyma and its subversion contributes to human mammary carcinogenesis.

Breast Neoplasms↗
PubMed2006

Drosophila melanogaster auxilin regulates the internalization of Delta to control activity of the Notch signaling pathway.

We have isolated mutations in the Drosophila melanogaster homologue of auxilin, a J-domain-containing protein known to cooperate with Hsc70 in the disassembly of clathrin coats from clathrin-coated vesicles in vitro. Consistent with this biochemical role, animals with reduced auxilin function exhibit genetic interactions with Hsc70 and clathrin. Interestingly, the auxilin mutations interact specifically with Notch and disrupt several Notch-mediated processes. Genetic evidence places auxilin function in the signal-sending cells, upstream of Notch receptor activation, suggesting that the relevant cargo for this auxilin-mediated endocytosis is the Notch ligand Delta. Indeed, the localization of Delta protein is disrupted in auxilin mutant tissues. Thus, our data suggest that auxilin is an integral component of the Notch signaling pathway, participating in the ubiquitin-dependent endocytosis of Delta. Furthermore, the fact that auxilin is required for Notch signaling suggests that ligand endocytosis in the signal-sending cells needs to proceed past coat disassembly to activate Notch.

Animals↗
PubMed2006

E proteins and Notch signaling cooperate to promote T cell lineage specification and commitment.

The helix-loop-helix protein, E47, is essential for both B- and T-lineage development. Here we demonstrate that in vitro E47 and Notch signaling act in concert to promote T cell development from fetal hematopoietic progenitors and to restrain development into the natural killer and myeloid cell lineages. The expression of an ensemble of genes associated with Notch signaling is activated by E47, and additionally, Notch signaling and E47 act in parallel pathways to induce a T lineage-specific program of gene expression. Enforced expression of the intracellular domain of Notch rescues the developmental arrest at the T cell commitment stage in E2A-deficient fetal thymocytes. Finally, we demonstrate that regulation of Hes1 expression by Notch signaling and E47 is strikingly similar to that observed during Drosophila melanogaster sensory development. Based on these observations, we propose that in developing fetal thymocytes E47 acts to induce the expression of an ensemble of genes involved in Notch signaling, and that subsequently E47 acts in parallel with Notch signaling to promote T-lineage maturation.

Animals↗
PubMed1997

A functional analysis of Notch mutations in Drosophila.

The Notch gene encodes a receptor protein that is involved in many processes during development. Its best understood role is during neurogenesis in a process called "lateral inhibition." However, it has been proposed that Notch also has a role in defining the proneural clusters in the first place. This raises the possibility that the Notch protein is acting as a multifunctional receptor. To test this hypothesis, we have carried out a genetic analysis of molecularly characterized Notch alleles to identify alleles that affect only one of the two proposed functions. Here we present evidence that Notch alleles can be identified that appear to affect the function of Notch during either lateral inhibition or the definition of proneural clusters. In addition our results indicate that there may be discrete regions of the Notch protein required for each function.

Alleles↗
PubMed2003

Enhanced gene activation by Notch and BMP signaling cross-talk.

The signaling systems of Notch and bone morphogenetic protein (BMP) are highly conserved from flies to mammals and have been shown to be important in the development of multiple organs. For instance, in the fate determination of mouse neuroepithelial cells, Notch signaling plays a role in keeping the progenitors from differentiating into neurons. BMP is also known to inhibit neuronal differentiation. In this paper, we show that BMP2 enhances Notch-induced transcriptional activation of Hes-5 and Hesr-1 in mouse neuroepithelial cells. BMP2 stimulation, in addition to the introduction of the intracellular domain of Notch (NIC), resulted in enhanced activation of the Hes-5 gene promoter. RBP-Jkappa binding to its target sequence is important not only for Notch signaling, but also for BMP2 signaling, to activate the Hes-5 gene promoter. Smad1, a Smad species that is activated by BMP2, barely interacted with NIC, but did form a complex with NIC in the simultaneous presence of the coactivators P/CAF and p300. Recruitment of p300 to the NIC-containing complex was facilitated by activated Smad1, which is suggested to contribute to BMP2-mediated enhancement of Notch-induced Hes-5 expression. These data suggest a novel functional cooperation between Notch signaling and BMP signaling.

Acetyltransferases↗
PubMed2001

Requirement of Notch in adulthood for neurological function and longevity.

Although Notch proteins rely upon presenilins for activation and can modulate neuritic architecture, their role in aging adults and Alzheimer's disease is unknown. Here we examine Drosophila in which Notch function was selectively diminished in adulthood. An outcrossing strategy was employed to reduce the effect of recessive modifiers of lifespan, and a temperature-sensitive allele or inducible dominant-negative Notch transgenes were used to reduce Notch function. A progressive neurological syndrome with loss of flight and shortened lifespan was observed in adults with compromised Notch function. Notch protein persists in aging adult Drosophila brains. However, no evidence of neurodegeneration in the central nervous system was detected. We conclude that Notch activity is constitutively required in the adult fly for neurological function.

Alleles↗
PubMed2003

Notch promotes epithelial-mesenchymal transition during cardiac development and oncogenic transformation.

Epithelial-to-mesenchymal transition (EMT) is fundamental to both embryogenesis and tumor metastasis. The Notch intercellular signaling pathway regulates cell fate determination throughout metazoan evolution, and overexpression of activating alleles is oncogenic in mammals. Here we demonstrate that Notch activity promotes EMT during both cardiac development and oncogenic transformation via transcriptional induction of the Snail repressor, a potent and evolutionarily conserved mediator of EMT in many tissues and tumor types. In the embryonic heart, Notch functions via lateral induction to promote a selective transforming growth factor-beta (TGFbeta)-mediated EMT that leads to cellularization of developing cardiac valvular primordia. Embryos that lack Notch signaling elements exhibit severely attenuated cardiac snail expression, abnormal maintenance of intercellular endocardial adhesion complexes, and abortive endocardial EMT in vivo and in vitro. Accordingly, transient ectopic expression of activated Notch1 (N1IC) in zebrafish embryos leads to hypercellular cardiac valves, whereas Notch inhibition prevents valve development. Overexpression of N1IC in immortalized endothelial cells in vitro induces EMT accompanied by oncogenic transformation, with corresponding induction of snail and repression of VE-cadherin expression. Notch is expressed in embryonic regions where EMT occurs, suggesting an intimate and fundamental role for Notch, which may be reactivated during tumor metastasis.

Animals↗
PubMed2005

Chaperone activity of protein O-fucosyltransferase 1 promotes notch receptor folding.

Notch proteins are receptors for a conserved signaling pathway that affects numerous cell fate decisions. We found that in Drosophila, Protein O-fucosyltransferase 1 (OFUT1), an enzyme that glycosylates epidermal growth factor-like domains of Notch, also has a distinct Notch chaperone activity. OFUT1 is an endoplasmic reticulum protein, and its localization was essential for function in vivo. OFUT1 could bind to Notch, was required for the trafficking of wild-type Notch out of the endoplasmic reticulum, and could partially rescue defects in secretion and ligand binding associated with Notch point mutations. This ability of OFUT1 to facilitate folding of Notch did not require its fucosyltransferase activity. Thus, a glycosyltransferase can bind its substrate in the endoplasmic reticulum to facilitate normal folding.

Amino Acid Motifs↗
PubMed2006

Notch signaling pathway.

Notch is a receptor that mediates intercellular signaling through a pathway conserved across the metazoa. It is involved in cell fate assignation and pattern formation during development. The receptor acts as a membrane-tethered transcription factor and is activated by members of the Delta, Serrate, Lag-2 family of Notch ligands, which trigger two successive proteolytic cleavages of the receptor. The second cleavage releases the intracellular domain of Notch, which translocates to the nucleus, where it interacts with the CSL family of transcriptional regulators and forms part of a Notch target gene-activating complex. In the absence of signaling, CSL [CBF1, Su(H), Lag-1] regulators repress Notch target genes through interactions with several transcriptional co-repressors that recruit histone deacetylases and other chromatin-modifying enzymes. After forming, the transcription-activating binary Notch intracellular domain-CSL complex recruits several proteins that facilitate transcription, among them the coactivator MAM and histone acetylases. Transcription of target genes is terminated when the Notch intracellular domain is degraded in a proteasome-dependent manner.

Animals↗
PubMed1997

Synergy between suppressor of Hairless and Notch in regulation of Enhancer of split m gamma and m delta expression.

The Notch signaling pathway is known to regulate cell fate decisions in a variety of organisms from worms to humans. Although several components of the pathway have been characterized, the actual mechanism and molecular results of signaling remain elusive. We have examined the role of the Notch signaling pathway in the transcriptional regulation of two Drosophila Enhancer of split [E(spl)] genes, whose gene products have been shown to be downstream players in the pathway. Using a reporter assay system in Drosophila tissue culture cells, we have observed a significant induction of E(spl) m gamma and m delta expression after cotransfection with activated Notch. Characterization of the 5' regulatory regions of these two genes led to the identification of a number of target sites for the Suppressor of Hairless [Su(H)] protein, a transcription factor activated by Notch signaling. We show that Notch-inducible expression of E(spl) m gamma and m delta both in cultured cells and in vivo is dependent on functional Su(H). Although overexpression of Su(H) augments the level of induction of the reporter genes by activated Notch, Su(H) alone is insufficient to produce high levels of transcriptional activation. Despite the synergy observed between activated Notch and Su(H), the former affects neither the nuclear localization nor the DNA binding activity of the latter.

Animals↗
PubMed2003

Growth suppression of pre-T acute lymphoblastic leukemia cells by inhibition of notch signaling.

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-amino-acid 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.

Alleles↗
PubMed2005

RBP-Jkappa/SHARP recruits CtIP/CtBP corepressors to silence Notch target genes.

Notch is a transmembrane receptor that determines cell fates and pattern formation in all animal species. After ligand binding, proteolytic cleavage steps occur and the intracellular part of Notch translocates to the nucleus, where it targets the DNA-binding protein RBP-Jkappa/CBF1. In the absence of Notch, RBP-Jkappa represses Notch target genes through the recruitment of a corepressor complex. We and others have identified SHARP as a component of this complex. Here, we functionally demonstrate that the SHARP repression domain is necessary and sufficient to repress transcription and that the absence of this domain causes a dominant negative Notch-like phenotype. We identify the CtIP and CtBP corepressors as novel components of the human RBP-Jkappa/SHARP-corepressor complex and show that CtIP binds directly to the SHARP repression domain. Functionally, CtIP and CtBP augment SHARP-mediated repression. Transcriptional repression of the Notch target gene Hey1 is abolished in CtBP-deficient cells or after the functional knockout of CtBP. Furthermore, the endogenous Hey1 promoter is derepressed in CtBP-deficient cells. We propose that a corepressor complex containing CtIP/CtBP facilitates RBP-Jkappa/SHARP-mediated repression of Notch target genes.

Alcohol Oxidoreductases↗
PubMed1999

Regulation of T cell fate by Notch.

The transmembrane receptor Notch participates in diverse cell fate decisions throughout embryonic development. Notch receptors and their ligands are expressed in the mammalian thymus, raising the possibility that Notch could regulate T cell fate decisions. Expression of a constitutively activated form of Notch in developing thymocytes causes thymocytes normally destined for the CD4 lineage to adopt the CD8 lineage instead. This suggests that Notch activity normally acts to direct CD4+CD8+ precursors to the CD8 lineage. The choice between CD4 and CD8 T cell fates is also controlled by MHC recognition during positive selection, implying that recognition of class I or II MHC might regulate Notch signaling. Possible models for the regulation of Notch by MHC recognition during CD4 versus CD8 lineage determination are discussed.

Animals↗
PubMed2006

Aberrant activation of notch signaling in human breast cancer.

A role for Notch signaling in human breast cancer has been suggested by both the development of adenocarcinomas in the murine mammary gland following pathway activation and the loss of Numb expression, a negative regulator of the Notch pathway, in a large proportion of breast carcinomas. However, it is not clear currently whether Notch signaling is frequently activated in breast tumors, and how it causes cellular transformation. Here, we show accumulation of the intracellular domain of Notch1 and hence increased Notch signaling in a wide variety of human breast carcinomas. In addition, we show that increased RBP-Jkappa-dependent Notch signaling is sufficient to transform normal breast epithelial cells and that the mechanism of transformation is most likely through the suppression of apoptosis. More significantly, we show that attenuation of Notch signaling reverts the transformed phenotype of human breast cancer cell lines, suggesting that inhibition of Notch signaling may be a therapeutic strategy for this disease.

Apoptosis↗
PubMed2006

Loss of notch activity in the developing central nervous system leads to increased cell death.

Many cells in the mammalian brain undergo apoptosis as a normal and critical part of development but the signals that regulate the survival and death of neural progenitor cells and the neurons they produce are not well understood. The Notch signaling pathway is involved in multiple decision points during development and has been proposed to regulate the survival and apoptosis of neural progenitor cells in the developing brain; however, previous experiments have not resolved whether Notch activity is pro- or anti-apoptotic. To elucidate the function of Notch signaling in the survival and death of cells in the nervous system, we have produced single and compound Notch conditional mutants in which Notch1 and Notch3 are removed at different times during brain development and in different populations of cells. We show here that a large number of neural progenitor cells, as well as differentiating neurons, undergo apoptosis in the absence of Notch1 and Notch3, suggesting that Notch activity promotes the survival of both progenitors and newly differentiating cells in the developing nervous system. Finally, we show that postmitotic neurons do not require Notch activity indefinitely to regulate their survival since elevated levels of cell death are observed only during embryogenesis in the Notch mutants and are not detected in neonates.

Animals↗