The many facets of Notch ligands.

The Notch signaling pathway regulates a diverse array of cell types and cellular processes and is tightly regulated by ligand binding. Both canonical and noncanonical Notch ligands have been identified that may account for some of the pleiotropic nature associated with Notch signaling. This review focuses on the molecular mechanisms by which Notch ligands function as signaling agonists and antagonists, and discusses different modes of activating ligands as well as findings that support intrinsic ligand signaling activity independent of Notch. Post-translational modification, proteolytic processing, endocytosis and membrane trafficking, as well as interactions with the actin cytoskeleton may contribute to the recently appreciated multifunctionality of Notch ligands. The regulation of Notch ligand expression by other signaling pathways provides a mechanism to coordinate Notch signaling with multiple cellular and developmental cues. The association of Notch ligands with inherited human disorders and cancer highlights the importance of understanding the molecular nature and activities intrinsic to Notch ligands. Oncogene (2008) 27, 5148-5167; doi:10.1038/onc.2008.229.

Functional analysis of a recurrent missense mutation in Notch3 in CADASIL.

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited vascular dementia characterised by recurrent ischemic strokes in the deep white matter. Mutations in the gene encoding the cell surface receptor, Notch3, have been identified in CADASIL patients, and accumulation of the extracellular domain of Notch3 has been demonstrated in affected vessels. Almost all CADASIL mutations alter the number of cysteine residues in the epidermal growth factor (EGF)-like repeats in the extracellular domain of the protein. OBJECTIVES: To understand the functional consequences of a recurrent CADASIL mutation on furin processing, cell surface expression, ligand binding, and activation of a downstream effector CBF1 by the Notch3 receptor. METHODS: We expressed wild type and mutant Notch3 receptors in cultured cells and examined cell surface expression of the proteins. We also applied a new flow cytometry based approach to semi-quantitatively measure binding to three Notch ligands. Additionally, we used a well characterised co-culture system to examine ligand dependent activation of transcription from a CBF1-luciferase reporter construct. RESULTS: These studies revealed subtle abnormalities in furin processing of the mutant receptor, although both heterodimeric and full length receptors are present on the cell surface, are capable of interacting with soluble forms of three ligands, Delta1, Delta4, and Jagged1, and retain the ability to activate CBF1 in a ligand dependent manner. CONCLUSIONS: By comparison with other mutant forms of Notch3, these data indicate that individual CADASIL mutations can have disparate effects on Notch3 expression and function.

Transcription factor expression and Notch-dependent regulation of neural progenitors in the adult rat spinal cord.

Recent studies have demonstrated that neural stem cells and other progenitors are present in the adult CNS. Details of their properties, however, remain poorly understood. Here we examined the properties and control mechanisms of neural progenitors in the adult rat spinal cord at the molecular level. Adult and embryonic progenitors commonly expressed various homeodomain-type (Pax6, Pax7, Nkx2.2, and Prox1) and basic helix-loop-helix (bHLH)-type (Ngn2, Mash1, NeuroD1, and Olig2) transcriptional regulatory factors in vitro. Unlike their embryonic counterparts, however, adult progenitors could not generate specific neurons that expressed markers appropriate for spinal motoneurons or interneurons, including Islet1, Lim1, Lim3, and HB9. Cells expressing the homeodomain factors Pax6, Pax7, and Nkx2.2 also emerged in vivo in response to injury and were distributed in unique patterns in the lesioned spinal cord. However, neither the expression of the neurogenic bHLH factors including Ngn2, Mash1, and NeuroD1 nor subsequent generation of new neurons could be detected in injured tissue. Our results suggest that signaling through the cell-surface receptor Notch is involved in this restriction. The expression of Notch1 in vivo was enhanced in response to injury. Furthermore, activation of Notch signaling in vitro inhibited differentiation of adult progenitors, whereas attenuation of Notch signals and forced expression of Ngn2 significantly enhanced neurogenesis. These results suggest that both the intrinsic properties of adult progenitors and local environmental signals, including Notch signaling, account for the limited regenerative potential of the adult spinal cord.

The notch receptor and its ligands are selectively expressed during hematopoietic development in the mouse.

Members of the Notch family of transmembrane receptors are found on primitive hematopoietic precursors, and Notch ligand expression has been demonstrated on the surface of stromal cells, suggesting a role for Notch signaling in mammalian blood cell development. The current report examines the expression of Notch receptors and their ligands in murine hematopoietic tissues to determine: A) which blood cell lineages in the adult are influenced by Notch activity, and B) whether fetal hematopoiesis in the embryo involves the Notch pathway. In the adult mouse, a combination of flow cytometry, immunohistochemistry and Northern analysis was used to examine Notch receptor or ligand expression in bone marrow and spleen. In the embryo, Northern analysis and in situ hybridization were used to characterize Notch receptor and ligand expression in fetal liver on embryonic day 12 (E12) through E17, an active period encompassing both erythropoiesis and granulopoeisis. Flow cytometry demonstrated the presence of Notch1 and Notch2 receptors on bone marrow-derived myeloid cells but not on erythroid cells positive for the marker, Ter-119. In situ hybridization of E12 through E17 fetal liver demonstrated widespread expression of Jagged1 and Delta1 in a pattern similar to but less abundant than that of the erythropoietin receptor. Taken together with earlier functional results, the current expression data suggest a role for Notch activity in establishing definitive hematopoiesis in fetal liver, as well as a selective use of Notch signaling in adult erythropoiesis and granulopoiesis. Notch receptors in the adult are most likely utilized by early erythroid precursors and intermediate-stage granulocytes, but not by terminally differentiating cells of either subset.

Vascular expression of Notch pathway receptors and ligands is restricted to arterial vessels.

Mice with targeted mutations in genes required for Notch signal transduction die during embryogenesis, displaying overt signs of hemorrhage due to defects in their vascular development. Surprisingly, directed expression of a constitutively active form of Notch4 within mouse endothelial cells produces a similar vascular embryonic lethality. Moreover, patients with mutations in Notch3 exhibit the cerebral vascular disorder, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). These findings underscore the importance of Notch signaling in vascular development; however, they do not identify the specific functional defect. Here, we report that Notch1, Notch3, Notch4, Delta4, Jagged1 and Jagged2 are all expressed in arteries, but are not expressed by veins. These findings identify an aspect of Notch signaling that could contribute to the mechanism by which this pathway modulates vascular morphogenesis.

Role of Deltex-1 as a transcriptional regulator downstream of the Notch receptor.

Intercellular signaling through the cell-surface receptor Notch plays important roles in a variety of developmental processes as well as in pathogenesis of some human cancers and genetic disorders. However, the mechanisms by which Notch signals are transduced into cells still remain elusive. Here we investigated the signaling mechanisms for Notch in the cell fate control of neural progenitor cells. We show that Deltex-1 (DTX1), a mammalian homolog of Drosophila Deltex, mediates a Notch signal to block differentiation of neural progenitor cells. We found that a significant fraction of DTX1 proteins were localized in the nucleus and physically interacted with the transcriptional coactivator p300. Through its binding to p300, DTX1 inhibited transcriptional activation by the neural-specific helix-loop-helix-type transcription factor MASH1, and this mechanism is likely responsible for the differentiation inhibition of neural progenitor cells. Our results further suggest that DTX1 regulates transcription independently of the previously characterized Notch signaling pathway involving RBP-J and HES1/HES5. Thus, DTX1 serves as an important signaling component downstream of Notch that regulates transcription in the nucleus.

Expression patterns of Notch1, Notch2, and Notch3 suggest multiple functional roles for the Notch-DSL signaling system during brain development.

The Notch-DSL signaling system consists of multiple receptors and ligands, and plays many roles in development. The function of Notch receptors and ligands in mammalian brain, however, is poorly understood. In the current study, we examined the expression patterns for three receptors of this system, Notch1, 2, and 3, in late embryonic and postnatal rat brain by in situ hybridization. The three receptors have overlapping but different patterns of expression. Messenger RNA for all three proteins is found in postnatal central nervous system (CNS) germinal zones and, in early postnatal life, within numerous cells throughout the CNS. Within zones of cellular proliferation of the postnatal brain, Notch1 mRNA is found in both the subventricular and the ventricular germinal zones, whereas Notch2 and Notch3 mRNAs are more highly localized to the ventricular zones. Both Notch1 and Notch3 mRNAs are expressed along the inner aspect of the dentate gyrus, a site of adult neurogenesis. Notch2 mRNA is expressed in the external granule cell layer of the developing cerebellum. In several brain areas, Notch1 and Notch2 mRNAs are relatively concentrated in white matter, whereas Notch3 mRNA is not. Neurosphere cultures (which contain CNS stem cells), purified astrocyte cultures, and striatal neuron-enriched cultures express Notch1 mRNA. However, in these latter cultures, Notch1 mRNA is produced by nestin-containing cells, rather than by postmitotic neurons. Taken together, these results support multiple roles for Notch1, 2, and 3 receptor activation during CNS development, particularly during gliogenesis.

Distinct mechanisms contribute to generate and change the CD4:CD8 cell ratio during thymus development: a role for the Notch ligand, Jagged1.

In adult life, the high CD4:CD8 cell ratio observed in peripheral lymphoid organs originates in the thymus. Our results show that the low peripheral CD4:CD8 cell ratio seen during fetal life also has an intrathymic origin. This distinct production of CD4(+)CD8(-) and CD4(-)CD8(+) thymocytes is regulated by the developmental age of the thymic stroma. The differential expression of Notch receptors and their ligands, especially Jagged1, throughout thymus development plays a key role in the generation of the different CD4:CD8 cell ratios. We also show that the intrathymic CD4:CD8 cell ratio sharply changes from fetal to adult values around birth. Differences in the proliferation and emigration rates of the mature thymocyte subsets contribute to this change.

The neural RNA-binding protein Musashi1 translationally regulates mammalian numb gene expression by interacting with its mRNA.

Musashi1 (Msi1) is an RNA-binding protein that is highly expressed in neural progenitor cells, including neural stem cells. In this study, the RNA-binding sequences for Msi1 were determined by in vitro selection using a pool of degenerate 50-mer sequences. All of the selected RNA species contained repeats of (G/A)U(n)AGU (n = 1 to 3) sequences which were essential for Msi1 binding. These consensus elements were identified in some neural mRNAs. One of these, mammalian numb (m-numb), which encodes a membrane-associated antagonist of Notch signaling, is a likely target of Msi1. Msi1 protein binds in vitro-transcribed m-numb RNA in its 3'-untranslated region (UTR) and binds endogenous m-numb mRNA in vivo, as shown by affinity precipitation followed by reverse transcription-PCR. Furthermore, adenovirus-induced Msi1 expression resulted in the down-regulation of endogenous m-Numb protein expression. Reporter assays using a chimeric mRNA that combined luciferase and the 3'-UTR of m-numb demonstrated that Msi1 decreased the reporter activity without altering the reporter mRNA level. Thus, our results suggested that Msi1 could regulate the expression of its target gene at the translational level. Furthermore, we found that Notch signaling activity was increased by Msi1 expression in connection with the posttranscriptional down-regulation of the m-numb gene.

Epstein-Barr virus BamHi-a rightward transcript-encoded RPMS protein interacts with the CBF1-associated corepressor CIR to negatively regulate the activity of EBNA2 and NotchIC.

The Epstein-Barr virus (EBV) BamHI-A rightward transcripts (BARTs) are expressed in all EBV-associated tumors as well as in latently infected B cells in vivo and cultured B-cell lines. One of the BART family transcripts contains an open reading frame, RPMS1, that encodes a nuclear protein termed RPMS. Reverse transcription-PCR analysis revealed that BART transcripts with the splicing pattern that generates the RPMS1 open reading frame are commonly expressed in EBV-positive lymphoblastoid cell lines and are also detected in Hodgkin's disease tissues. Experiments undertaken to determine the function of RPMS revealed that RPMS interacts with both CBF1 and components of the CBF1-associated corepressor complex. RPMS interaction with CBF1 was demonstrated in a glutathione S-transferase (GST) affinity assay and by the ability of RPMS to alter the intracellular localization of a mutant CBF1. A Gal4-RPMS fusion protein mediated transcriptional repression, suggesting an additional interaction between RPMS and corepressor proteins. GST affinity assays revealed interaction between RPMS and the corepressor Sin3A and CIR. The RPMS-CIR interaction was further substantiated in mammalian two-hybrid, coimmunoprecipitation, and colocalization experiments. RPMS has been shown to interfere with NotchIC and EBNA2 activation of CBF1-containing promoters in reporter assays. Consistent with this function, immunofluorescence assays performed on cotransfected cells showed that there was colocalization of RPMS with NotchIC and with EBNA2 in intranuclear punctate speckles. The effect of RPMS on NotchIC function was further examined in a muscle cell differentiation assay where RPMS was found to partially reverse NotchIC-mediated inhibition of differentiation. The mechanism of RPMS action was examined in cotransfection and mammalian two-hybrid assays. The results revealed that RPMS blocked relief of CBF1-mediated repression and interfered with SKIP-CIR interactions. We conclude that RPMS acts as a negative regulator of EBNA2 and Notch activity through its interactions with the CBF1-associated corepressor complex.

Defects in development of the kidney, heart and eye vasculature in mice homozygous for a hypomorphic Notch2 mutation.

The Notch gene family encodes large transmembrane receptors that are components of an evolutionarily conserved intercellular signaling mechanism. To assess the in vivo role of the Notch2 gene, we constructed a targeted mutation, Notch2(del1). Unexpectedly, we found that alternative splicing of the Notch2(del1) mutant allele leads to the production of two different in-frame transcripts that delete either one or two EGF repeats of the Notch2 protein, suggesting that this allele is a hypomorphic Notch2 mutation. Mice homozygous for the Notch2(del1) mutation died perinatally from defects in glomerular development in the kidney. Notch2(del1)/Notch2(del1 )mutant kidneys were hypoplastic and mutant glomeruli lacked a normal capillary tuft. The Notch ligand encoded by the Jag1 gene was expressed in developing glomeruli in cells adjacent to Notch2-expressing cells. We show that mice heterozygous for both the Notch2(del1) and Jag1(dDSL) mutations exhibit a glomerular defect similar to, but less severe than, that of Notch2(del1)/Notch2(del1 )homozygotes. The co-localization and genetic interaction of Jag1 and Notch2 imply that this ligand and receptor physically interact, forming part of the signal transduction pathway required for glomerular differentiation and patterning. Notch2(del1)/Notch2(del1 )homozygotes also display myocardial hypoplasia, edema and hyperplasia of cells associated with the hyaloid vasculature of the eye. These data identify novel developmental roles for Notch2 in kidney, heart and eye development.

Ligand-induced signaling in the absence of furin processing of Notch1.

Notch is a conserved cell surface receptor that is activated through direct contact with neighboring ligand-expressing cells. The primary 300-kDa translation product of the Notch1 gene (p300) is cleaved by a furin-like convertase to generate a heterodimeric, cell-surface receptor composed of 180- (p180) and 120- (p120) kDa polypeptides. Heterodimeric Notch is thought to be the only form of the receptor which is both present on the cell surface and able to generate an intracellular signal in response to ligand. Consistent with previous reports, we found that disruption of furin processing of Notch1, either by coexpression of a furin inhibitor or by mutation of furin target sequences within Notch1 itself, perturbed ligand-dependent signaling through the well-characterized mediator of Notch signal transduction, CSL (CBF1, Su(H), and LAG-1). Yet contrary to these reports, we could detect the full-length p300 Notch1 product on the cell surface. Moreover, this uncleaved form of Notch1 could suppress the differentiation of C2C12 myoblasts in response to ligand. Taken together, these data support our previous studies characterizing a CSL-independent Notch signaling pathway and identify this uncleaved isoform of Notch as a potential mediator of this pathway. Our results suggest a novel paradigm in signal transduction, one in which two isoforms of the same cell-surface receptor could mediate two distinct signaling pathways in response to ligand.

Notch signaling enhances survival and alters differentiation of 32D myeloblasts.

The Notch transmembrane receptors play important roles in precursor survival and cell fate specification during hematopoiesis. To investigate the function of Notch and the signaling events activated by Notch in myeloid development, we expressed truncated forms of Notch1 or Notch2 proteins that either can or cannot activate the core binding factor 1 (CBF1) in 32D (clone 3) myeloblasts. 32D cells proliferate as blasts in the presence of the cytokines, GM-CSF or IL-3, but they initiate differentiation and undergo granulopoiesis in the presence of granulocyte CSF (G-CSF). 32D cells expressing constitutively active forms of Notch1 or Notch2 proteins that signal through the CBF1 pathway maintained significantly higher numbers of viable cells and exhibited less cell death during G-CSF induction compared with controls. They also displayed enhanced entry into granulopoiesis, and inhibited postmitotic terminal differentiation. In contrast, Notch1 constructs that either lacked sequences necessary for CBF1 binding or that failed to localize to the nucleus had little effect. Elevated numbers of viable cells during G-CSF treatment were also observed in 32D cells overexpressing the basic helix-loop-helix protein (bHLH), HES1, consistent with activation of the CBF1 pathway. Taken together, our data suggest that Notch signaling enhances 32D cell survival, promotes entry into granulopoiesis, and inhibits postmitotic differentiation through a CBF1-dependent pathway.

Fringe differentially modulates Jagged1 and Delta1 signalling through Notch1 and Notch2.

Proteins encoded by the fringe family of genes are required to modulate Notch signalling in a wide range of developmental contexts. Using a cell co-culture assay, we find that mammalian Lunatic fringe (Lfng) inhibits Jagged1-mediated signalling and potentiates Delta1-mediated signalling through Notch1. Lfng localizes to the Golgi, and Lfng-dependent modulation of Notch signalling requires both expression of Lfng in the Notch-responsive cell and the Notch extracellular domain. Lfng does not prevent binding of soluble Jagged1 or Delta1 to Notch1-expressing cells. Lfng potentiates both Jagged1- and Delta1-mediated signalling via Notch2, in contrast to its actions with Notch1. Our data suggest that Fringe-dependent differential modulation of the interaction of Delta/Serrate/Lag2 (DSL) ligands with their Notch receptors is likely to have a significant role in the combinatorial repertoire of Notch signalling in mammals.

Notch4 and Jagged-1 induce microvessel differentiation of rat brain endothelial cells.

The mouse Notch4 gene is expressed specifically in endothelial cells. Notch4/int-3, a truncated form of Notch4, acts as a constitutive activated Notch receptor. We used rat brain microvessel endothelial cells (RBE4) to study the role of Notch4 and Jagged-1 in endothelial cell differentiation. Both Notch4/int-3 and Jagged-1 were able to induce microvessel-like structures with morphological and biochemical properties similar to brain endothelial microvessels. Ectopic expression of full-length Notch4 did not effect RBE4 cells. Activation of the Notch signal transduction pathway was measured by the induction of endogenous Notch4 and Jagged-1 genes and of Jagged-1 proteins. The observed morphological changes to RBE4 cells correlated with endogenous Notch4 and Jagged-1 gene activation. Our observations demonstrate that Notch signaling can promote endothelial cell differentiation and morphogenesis.

Aspartate mutations in presenilin and gamma-secretase inhibitors both impair notch1 proteolysis and nuclear translocation with relative preservation of notch1 signaling.

It has been hypothesized that a presenilin 1 (PS1)-related enzymatic activity is responsible for proteolytic cleavage of the C-terminal intracellular protein of Notch1, in addition to its role in beta-amyloid protein (Abeta) formation from the amyloid precursor protein (APP). We developed an assay to monitor ligand-induced Notch1 proteolysis and nuclear translocation in individual cells : Treatment of full-length Notch1-enhanced green fluorescent protein-transfected Chinese hamster ovary (CHO) cells with a soluble preclustered form of the physiologic ligand Delta leads to rapid accumulation of the C terminus of Notch1 in the nucleus and to transcriptional activation of a C-promoter binding factor 1 (CBF1) reporter construct. Nuclear translocation was blocked by cotransfection with Notch's physiologic inhibitor Numb. Using this assay, we now confirm and extend the observation that PS1 is involved in Notch1 nuclear translocation and signaling in mammalian cells. We demonstrate that the D257A and the D385A PS1 mutations, which had been shown previously to block APP gamma-secretase activity, also prevent Notch1 cleavage and translocation to the nucleus but do not alter Notch1 trafficking to the cell surface. We also show that two APP gamma-secretase inhibitors block Notch1 nuclear translocation with an IC(50) similar to that reported for APP gamma-secretase. Notch1 signaling, assessed by measuring the activity of CBF1, a downstream transcription factor, was impaired but not abolished by the PS1 aspartate mutations or gamma-secretase inhibitors. Our results support the hypotheses that (a) PS1-dependent APP gamma-secretase-like enzymatic activity is critical for both APP and Notch processing and (b) the Notch1 signaling pathway remains partially activated even when Notch1 proteolytic processing and nuclear translocation are markedly inhibited. The latter is an important finding from the perspective of therapeutic treatment of Alzheimer's disease by targeting gamma-secretase processing of APP to reduce Abeta production.

Notch signal transduction: a real rip and more.

The Notch signaling pathway functions in a wide variety of processes that regulate tissue patterning and morphogenesis in developing vertebrates and invertebrates. Research on the mechanism of ligand-induced Notch signal transduction has revealed a novel and essential element in the signal cascade. Some recent findings support a model in which sequential proteolytic cleavage serves to regulate Notch signal transduction.

Transient Notch activation initiates an irreversible switch from neurogenesis to gliogenesis by neural crest stem cells.

The genesis of vertebrate peripheral ganglia poses the problem of how multipotent neural crest stem cells (NCSCs) can sequentially generate neurons and then glia in a local environment containing strong instructive neurogenic factors, such as BMP2. Here we show that Notch ligands, which are normally expressed on differentiating neuroblasts, can inhibit neurogenesis in NCSCs in a manner that is completely dominant to BMP2. Contrary to expectation, Notch activation did not maintain these stem cells in an uncommitted state or promote their self-renewal. Rather, even a transient activation of Notch was sufficient to cause a rapid and irreversible loss of neurogenic capacity accompanied by accelerated glial differentiation. These data suggest that Notch ligands expressed by neuroblasts may act positively to instruct a cell-heritable switch to gliogenesis in neighboring stem cells.

Independent development of pancreatic alpha- and beta-cells from neurogenin3-expressing precursors: a role for the notch pathway in repression of premature differentiation.

The nature and identity of the pancreatic beta-cell precursor has remained elusive for many years. One model envisions an early multihormonal precursor that gives rise to both alpha- and beta-cells and the other endocrine cell types. Alternatively, beta-cells have been suggested to arise late, directly from the GLUT2- and pancreatic duodenal homeobox factor-1 (PDX1)-expressing epithelium, which gives rise also to the acinar cells during this stage. In this study, we have identified a subset of the PDX1+ epithelial cells that are marked by expression of Neurogenin3 (Ngn3). Ngn3, a member of the basic helix-loop-helix (bHLH) family of transcription factors, is suggested to act upstream of NeuroD in a bHLH cascade. Detailed analysis of Ngn3/paired box factor 6 (PAX6) and NeuroD/PAX6 co-expression shows that the two bHLH factors are expressed in a largely nonoverlapping set of cells, but such analysis also suggests that the NeuroD+ cells arise from cells expressing Ngn3 transiently. NeuroD+ cells do not express Ki-67, a marker of proliferating cells, which shows that these cells are postmitotic. In contrast, Ki-67 is readily detected in Ngn3+ cells. Thus, Ngn3+ cells fulfill the criteria for an endocrine precursor cell. These expression patterns support the notion that both alpha- and beta-cells develop independently from PDX1+/Ngn3+ epithelial cells, rather than from GLU+/INS+ intermediate stages. The earliest sign of alpha-cell development appears to be Brain4 expression, which apparently precedes Islet-1 (ISL1) expression. Based on our expression analysis, we propose a temporal sequence of gene activation and inactivation for developing alpha- and beta-cells beginning with activation of NeuroD expression. Endocrine cells leave the cell cycle before NeuroD activation, but re-enter the cell cycle at perinatal stages. Dynamic expression of Notch1 in PDX+ epithelial cells suggests that Notch signaling could inhibit a Ngn-NeuroD cascade as seen in the nervous system and thus prevent premature differentiation of endocrine cells.

SKIP, a CBF1-associated protein, interacts with the ankyrin repeat domain of NotchIC To facilitate NotchIC function.

Notch proteins are transmembrane receptors that mediate intercell communication and direct individual cell fate decisions. The activated intracellular form of Notch, NotchIC, translocates to the nucleus, where it targets the DNA binding protein CBF1. CBF1 mediates transcriptional repression through the recruitment of an SMRT-histone deacetylase-containing corepressor complex. We have examined the mechanism whereby NotchIC overcomes CBF1-mediated transcriptional repression. We identified SKIP (Ski-interacting protein) as a CBF1 binding protein in a yeast two-hybrid screen. Both CBF1 and SKIP are highly conserved evolutionarily, and the SKIP-CBF1 interaction is also conserved in assays using the Caenorhabditis elegans and Drosophila melanogaster SKIP homologs. Protein-protein interaction assays demonstrated interaction between SKIP and the corepressor SMRT. More surprisingly, SKIP also interacted with NotchIC. The SMRT and NotchIC interactions were mutually exclusive. In competition binding experiments SMRT displaced NotchIC from CBF1 and from SKIP. Contact with SKIP is required for biological activity of NotchIC. A mutation in the fourth ankyrin repeat that abolished Notch signal transduction did not affect interaction with CBF1 but abolished interaction with SKIP. Further, NotchIC was unable to block muscle cell differentiation in myoblasts expressing antisense SKIP. The results suggest a model in which NotchIC activates responsive promoters by competing with the SMRT-corepressor complex for contacts on both CBF1 and SKIP.