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SUMMARY Notch is a conserved signaling pathway that is essential for metazoan development and homeostasis; dysregulated signaling underlies the pathophysiology of numerous human diseases. Receptor-ligand interactions result in gene expression changes, which are regulated by the transcription factor RBPJ. RBPJ forms a complex with the intracellular domain of the Notch receptor and the coactivator Mastermind to activate transcription, but it can also function as a repressor by interacting with corepressor proteins. Here, we determine the structure of RBPJ bound to the corepressor SHARP and DNA, revealing its mode of binding to RBPJ. We tested structure-based mutants in biophysical and biochemical-cellular as-says to characterize the role of RBPJ as a repressor, clearly demonstrating that RBPJ mutants deficient for SHARP binding are incapable of repressing transcription of genes responsive to Notch signaling in cells. Altogether, our structure-function studies provide significant insights into the repressor function of RBPJ., Graphical Abstract, In Brief Yuan et al. determine the X-ray structure of the corepressor SHARP bound to RBPJ, the nuclear effector of the Notch pathway. The structure-function analysis provides insights into corepressor binding to RBPJ and how RBPJ functions as a repressor of transcription of Notch target genes.

CD1d-restricted invariant NKT (iNKT) cells are a unique lineage of T lymphocytes that regulate both innate and adaptive immunity. The Mediator complex forms the bridge between transcriptional activators and the general transcription machinery. Med1/TRAP220 (also called DRIP205) is a key component of Mediator that interacts with ligand-bound hormone receptors, such as the vitamin D receptor. Here, we show that T-cell–specific Med1 deficiency results in a specific block in iNKT cell development but the development of conventional αβ T cells remains grossly normal. The defect is cell-intrinsic and depends neither on apoptosis, cell-cycle control, nor on CD1d expression of CD4 + CD8 + double-positive thymocytes. Surprisingly, ectopic expression of a Vα14-Jα18 T-cell receptor transgene completely rescues the defect caused by Med1 deficiency. At the molecular level, thymic iNKT cells in Med1 −/− animals display reduced levels of IL-2Rβ and T-bet expression and could not complete terminal maturation. Thus, Med1 is essential for a complete intrathymic development of iNKT cells.

The evolutionarily conserved Notch signal transduction pathway regulates fundamental cellular processes during embryonic development and in the adult. Ligand binding induces presenilin-dependent cleavage of the receptor and a subsequent nuclear translocation of the Notch intracellular domain (NICD). In the nucleus, NICD binds to the recombination signal sequence-binding protein J (RBP-J)/CBF-1 transcription factor to induce expression of Notch target genes. Here, we report the identification and functional characterization of RBP-J interacting and tubulin associated (RITA) (C12ORF52) as a novel RBP-J/CBF-1-interacting protein. RITA is a highly conserved 36 kDa protein that, most interestingly, binds to tubulin in the cytoplasm and shuttles rapidly between cytoplasm and nucleus. This shuttling RITA exports RBP-J/CBF-1 from the nucleus. Functionally, we show that RITA can reverse a Notch-induced loss of primary neurogenesis in Xenopus laevis. Furthermore, RITA is able to downregulate Notch-mediated transcription. Thus, we propose that RITA acts as a negative modulator of the Notch signalling pathway, controlling the level of nuclear RBP-J/CBF-1, where its amounts are limiting.

The Mediator complex forms the bridge between transcriptional activators and the RNA polymerase II. Med1 (also known as PBP or TRAP220) is a key component of Mediator that interacts with nuclear hormone receptors and GATA transcription factors. Here, we show dynamic recruitment of GATA-1, TFIIB, Mediator, and RNA polymerase II to the β-globin locus in induced mouse erythoid leukemia cells and in an erythropoietin-inducible hematopoietic progenitor cell line. Using Med1 conditional knockout mice, we demonstrate a specific block in erythroid development but not in myeloid or lymphoid development, highlighted by the complete absence of β-globin gene expression. Thus, Mediator subunit Med1 plays a pivotal role in erythroid development and in β-globin gene activation.

Timely acquisition of cell fates and the elaborate control of growth in numerous organs depend on Notch signaling. Upon ligand binding, the core transcription factor RBP-J activates transcription of Notch target genes. In the absence of signaling, RBP-J switches off target gene expression, assuring the tight spatiotemporal control of the response by a mechanism incompletely understood. Here we show that the histone demethylase KDM5A is an integral, conserved component of Notch/RBP-J gene silencing. Methylation of histone H3 Lys 4 is dynamically erased and re-established at RBP-J sites upon inhibition and reactivation of Notch signaling. KDM5A interacts physically with RBP-J; this interaction is conserved in Drosophila and is crucial for Notch-induced growth and tumorigenesis responses. © 2010 by Cold Spring Harbor Laboratory Press.

This Podcast features an interview with Tilman Borggrefe, author of a Research Article that appears in the 24 March 2015 issue of Science Signaling , about a posttranslational modification of the receptor Notch that targets the receptor for degradation. The Notch signaling pathway is involved in many developmental processes, and its misregulation has been implicated in several types of cancer. Upon ligand binding, Notch is proteolytically cleaved, and the Notch intracellular domain (NICD) is released into the cytoplasm. NICD then translocates into the nucleus, where it cooperates with transcription factors to activate gene expression. Several posttranslational modifications that affect the stability of Notch, and therefore its ability to transduce signaling, have been characterized. Hein et al . discovered that methylation promotes degradation of Notch and that this modification is specifically required to mediate short periods of transient Notch signaling during embryonic development. Mutant forms of Notch that cannot be methylated may contribute to dysregulation of Notch signaling in cancer.

Physiologically, Notch signal transduction plays a pivotal role in differentiation; pathologically, Notch signaling contributes to the development of cancer. Transcriptional activation of Notch target genes involves cleavage of the Notch receptor in response to ligand binding, production of the Notch intracellular domain (NICD), and NICD migration into the nucleus and assembly of a coactivator complex. Posttranslational modifications of the NICD are important for its transcriptional activity and protein turnover. Deregulation of Notch signaling and stabilizing mutations of Notch1 have been linked to leukemia development. We found that the methyltransferase CARM1 (coactivator-associated arginine methyltransferase 1; also known as PRMT4) methylated NICD at five conserved arginine residues within the C-terminal transactivation domain. CARM1 physically and functionally interacted with the NICD-coactivator complex and was found at gene enhancers in a Notch-dependent manner. Although a methylation-defective NICD mutant was biochemically more stable, this mutant was biologically less active as measured with Notch assays in embryos of Xenopus laevis and Danio rerio. Mathematical modeling indicated that full but short and transient Notch signaling required methylation of NICD.

Marginal zones (MZs) are microdomains in the spleen that contain various types of immune cells, including MZ B cells, MOMA1(+) metallophilic macrophages, and mucosal addressin cell adhesion molecule 1 (MAdCAM-1)(+) endothelial cells. MAdCAM-1(+) and MOMA1(+) cells line the sinus, that separates MZs from splenic follicles. Here we show that a receptor for the lysophospholipid sphingosine-1-phosphate (S1P), S1P(3), is required for normal numbers of splenic immature and MZ B cells, and for S1P-induced chemotaxis of MZ B cells. S1P(3) is also essential for proper alignment of MOMA1(+) macrophages and MAdCAM-1(+) endothelial cells along the marginal sinus. The lack of cohesion of the marginal sinus in S1P(3)(-/-) mice affects MZ B cell functions, as wild-type (WT) MZ B cells migrate more into S1P(3)(-/-) follicles than into WT follicles after treatment with lipopolysaccharide. Additionally, short-term homing experiments demonstrate that WT MZ B cells home to the S1P(3)(-/-) spleen in increased numbers, suggesting a role for the marginal sinus in regulating MZ B cells numbers. Moreover, S1P(3)(-/-) mice are defective in mounting immune responses to thymus-independent antigen type 2 due to defects in radiation-resistant cells in the spleen. These data identify lysophospholipids and the S1P(3) receptor as essential regulators of the MZ sinus and its role as a barrier to the follicle.

The Srb8, -9, -10, and -11 proteins of yeast have been isolated as a discrete, stoichiometric complex. The isolated complex phosphorylates the C-terminal domain (CTD) of the largest subunit of RNA polymerase II at serines 2 and 5. In addition to the previously reported human homologs of Srb10 and 11, we have identified TRAP230/ARC240 and TRAP240/ARC250 as the human homologs of Srb8 and Srb9, showing the entire Srb8/9/10/11 complex is conserved from yeast to humans.

Cross-linking of the high-affinity IgE receptor (FcepsilonRI) on mast cell activates signaling pathways that trigger degranulation and the release of multiple pro-inflammatory mediators. Mature,immature and precursor mast cells are degranulation competent. We show here that the signaling protein SWAP-70 has a function in mast cell biology. While not found in many cell types, we find that apart from B cells, mast cells also express SWAP-70. In activated B cells, SWAP-70 shuttles between cytoplasm and nucleus, but in mast cells it is confined to the cytoplasm. SWAP-70(ko/ko) (double knockout) mice have reduced numbers of mature mast cells, and these are degranulation competent. However, although immature mast cells from SWAP-70(ko/ko) mice respond normally to SCF and IL-3 and have functional granules, their FcepsilonRI-mediated degranulation is inhibited. Thus, in mast cells SWAP-70 plays a role both in establishing the initial competence to degranulate and to develop into mature mast cells.

Chromatin-based mechanisms significantly contribute to the regulation of many developmentally regulated genes, including Notch target genes. After specific ligand binding, the intracellular part of the Notch receptor is cleaved off and translocates to the nucleus, where it binds to the transcription factor CSL (encoded by the RBPJ gene in mammals), in order to activate transcription. In the absence of a Notch signal, CSL represses Notch target genes by recruiting a co-repressor complex. Both NICD co-activator and CSL co-repressor complexes contain chromatin modifiers such as histone acetyltransferases and methyltransferases, which dynamically regulate chromatin marks at Notch target genes. Here we provide protocols for ChIP (chromatin immunoprecipitation) to analyze the chromatin status of dynamically regulated Notch target genes. Furthermore, an example is presented how to perform a primary analysis of ChIP-Seq data at Notch target genes using the Cistrome platform.

The Notch signaling pathway is activated in many cell types, but its effects are cell type- and stage-specific. In the immune system, Notch activity is required for the differentiation of T cell progenitors, but it is reduced in more mature thymocytes, in which Notch is oncogenic. Studies based on single-gene models have suggested that the tumor suppressor protein Ikaros plays an important role in repressing the transcription of Notch target genes. We used genome-wide analyses, including chromatin immunoprecipitation sequencing, to identify genes controlled by Notch and Ikaros in gain- and loss-of-function experiments. We found that Ikaros bound to and directly repressed the expression of most genes that are activated by Notch. Specific deletion of Ikaros in thymocytes led to the persistent expression of Notch target genes that are essential for T cell maturation, as well as the rapid development of T cell leukemias in mice. Expression of Notch target genes that are normally silent in T cells, but are activated by Notch in other cell types, occurred in T cells of mice genetically deficient in Ikaros. We propose that Ikaros shapes the timing and repertoire of the Notch transcriptional response in T cells through widespread targeting of elements adjacent to Notch regulatory sequences. These results provide a molecular framework for understanding the regulation of tissue-specific and tumor-related Notch responses.

Notch refers to a highly conserved cell-to-cell signaling pathway with essential roles in embryonic development and tissue maintenance. Dysfunctional signaling causes human disease, highlighting the importance of pathway regulation. Notch signaling ultimately results in the activation of target genes, which is regulated by the nuclear effector CSL. CSL dually functions as an activator and repressor of transcription through differential interactions with coactivator or corepressor proteins, respectively. While the structures of CSL-coactivator complexes have been determined, the structures of CSL-corepressor complexes are unknown. Here, using a combination of structural, biophysical, and cellular approaches, we characterize the structure and function of CSL in complex with the corepressor KyoT2. Collectively, our studies provide molecular insights into how KyoT2 binds CSL with high affinity and competes with coactivators, such as Notch, for binding CSL. These studies are important for understanding how CSL functions as both an activator and repressor of transcription of Notch target genes.

Chromatin-based mechanisms significantly contribute to the regulation of many developmentally regulated genes, including Notch target genes. After specific ligand binding, the intracellular part of the Notch receptor is cleaved off and translocates to the nucleus, where it binds to the transcription factor CSL (encoded by the RBPJ gene in mammals), in order to activate transcription. In the absence of a Notch signal, CSL represses Notch target genes by recruiting a co-repressor complex. Both NICD co-activator and CSL co-repressor complexes contain chromatin modifiers such as histone acetyltransferases and methyltransferases, which dynamically regulate chromatin marks at Notch target genes. Here we provide protocols for ChIP (chromatin immunoprecipitation) to analyze the chromatin status of dynamically regulated Notch target genes. Furthermore, an example is presented how to perform a primary analysis of ChIP-Seq data at Notch target genes using the Cistrome platform.

TAP tags and dot blot analysis have been used to measure the amounts of RNA polymerase II transcription proteins in crude yeast extracts. The measurements showed comparable amounts of RNA polymerase II, TFIIE, and TFIIF, lower levels of TBP and TFIIB, and still lower levels of Mediator and TFIIH. These findings are consistent with the presumed roles of the transcription proteins, but do not support the idea of their recruitment in a single large complex to RNA polymerase II promoters. The approach employed here can be readily extended to quantitative analysis of the entire yeast proteome.

SWAP-70 is part of a protein complex that catalyzes cell-free DNA recombination between immunoglobulin heavy chain gene switch region substrates. This report studies the expression pattern of SWAP-70 in mouse tissues, sorted cells, and cultured primary cells. SWAP-70 RNA is strongly increased upon switch-induction of spleen cells, and very weakly expressed in thymus and bone marrow. SWAP-70 protein is specifically expressed in B cells, and levels increase rapidly after stimulation. Tissue staining shows strong expression in germinal center B cells, while macrophages and T lymphocytes do not stain. SWAP-70 is not detected in early B cells in the bone marrow. Its expression during mouse ontogeny after birth correlates with the appearance of non-IgM isotypes. While SWAP-70 localizes to the cell nucleus in activated B cells, it is not tightly associated with the chromatin and is found in the cytoplasm as well. SWAP-70 expression is not increased by gamma or UV irradiation of spleen cells, nor does it depend on p53. These characteristics are consistent with the putative role of SWAP-70 in immunoglobulin class switching.

We have purified and biochemically characterized a multiprotein complex designated SWAP. In a DNA transfer assay, SWAP preferentially recombines ("swaps") sequences derived from Ig heavy chain switch regions. We identified four of the proteins in the SWAP complex: B23 (nucleophosmin), C23 (nucleolin), poly(ADP-ribose) polymerase (PARP), and SWAP-70. The first three are proteins known to be present in most cells. B23 promotes single-strand DNA reannealing and the formation of joint molecules in a D-loop assay between homologous, but also between Smu and Sgamma sequences. SWAP-70 is a novel protein of 70 kDa. Its cDNA was cloned and sequenced, and the protein was overexpressed in Escherichia coli. SWAP-70 protein expression was found only in B lymphocytes that had been induced to switch to various Ig isotypes and in switching B-cell lines. SWAP-70 is a nuclear protein, has a weak affinity for DNA, binds ATP, and forms specific, high affinity complexes with B23, C23, and poly(ADP-ribose) polymerase. These findings are consistent with SWAP being the long elusive "switch recombinase" and with SWAP-70 being the specific recruiting element that assembles the switch recombinase from universal components.

Notch signaling plays a pivotal role in embryonic and postnatal development.Upon binding of a Notch ligand, proteolytic cleavage events liberate the Notch-intracellular domain (NICD) that migrates into the nucleus. In order to activate target genes, NICD associates with the transcription factor RBP-J (also known as CSL), Mastermind and the acetyltransferase p300.Here, we identify the DEAD-box RNA helicase Ddx5 as a novel component of the RBP-J/NICD complex utilizing a biotinylation-tagging approach followed by mass-spectrometry. Biochemical assays confirm a direct interaction of Ddx5 with RBP-J. We show that Ddx5 localizes at RBP-J binding sites within the Notch target genes preTCRα, Hes1 and CD25 in a Notch-dependent manner. Moreover, knockdown of Ddx5 also downregulates a subset of Notch target genes in a murine pre T-cell model. Interestingly, also knockdown/overexpression of the RNA coactivator SRA, a cofactor of Ddx5, downregulates Hes1 and preTCRα. Using Chromatin-IP, we show that this effect is accompanied with a loss of p300 occupancy at Notch target genes and decreased histone acetylation. Together, our data demonstrate that Ddx5 and SRA function as coactivators of Notch signaling.

The evolutionarily conserved Notch signal transduction pathway regulates fundamental cellular processes during embryonic development and in the adult. Ligand binding induces presenilin-dependent cleavage of the receptor and a subsequent nuclear translocation of the Notch intracellular domain (NICD). In the nucleus, NICD binds to the recombination signal sequence-binding protein J (RBP-J)/CBF-1 transcription factor to induce expression of Notch target genes. Here, we report the identification and functional characterization of RBP-J interacting and tubulin associated (RITA) (C12ORF52) as a novel RBP-J/CBF-1-interacting protein. RITA is a highly conserved 36 kDa protein that, most interestingly, binds to tubulin in the cytoplasm and shuttles rapidly between cytoplasm and nucleus. This shuttling RITA exports RBP-J/CBF-1 from the nucleus. Functionally, we show that RITA can reverse a Notch-induced loss of primary neurogenesis in Xenopus laevis. Furthermore, RITA is able to downregulate Notch-mediated transcription. Thus, we propose that RITA acts as a negative modulator of the Notch signalling pathway, controlling the level of nuclear RBP-J/CBF-1, where its amounts are limiting.

Notch is a transmembrane receptor that determines cell fates and pattern formation in all animal species. After specific ligand binding, the intracellular part of Notch is cleaved off and translocates to the nucleus, where it targets the DNA binding protein RBP-Jkappa. In the absence of Notch, RBP-Jkappa represses Notch target genes by recruiting a corepressor complex. We and others have previously identified SHARP as one component of this complex. Here, we show that the corepressor ETO as well as the leukemogenic fusion protein AML1/ETO directly interacts with SHARP, that ETO is part of the endogenous RBP-Jkappa-containing corepressor complex, and that ETO is found at Notch target gene promoters. In functional assays, corepressor ETO, but not AML1/ETO, augments SHARP-mediated repression in an histone deacetylase-dependent manner. Furthermore, either the knockdown of ETO or the overexpression of AML1/ETO activates Notch target genes. Therefore, we propose that AML1/ETO can disturb the normal, repressive function of ETO at Notch target genes. This activating (or derepressing) effect of AML1/ETO may contribute to its oncogenic potential in myeloid leukemia.

The Mediator complex forms the bridge between transcriptional activators and RNA polymerase II. Mediator subunit Med1/TRAP220 is a key component of Mediator originally found to associate with nuclear hormone receptors. Med1 deficiency causes lethality at embryonic day 11.5 because of defects in heart and placenta development. Here we show that Med1-deficient 10.5 days postcoitum embryos are anemic but have normal numbers of hematopoietic progenitor cells. Med1-deficient progenitor cells have a defect in forming erythroid burst-forming units (BFU-E) and colony-forming units (CFU-E), but not in forming myeloid colonies. At the molecular level, we demonstrate that Med1 interacts physically with the erythroid master regulator GATA-1. In transcription assays, Med1 deficiency leads to a defect in GATA-1-mediated transactivation. In chromatin immunoprecipitation experiments, we find Mediator components at GATA-1-occupied enhancer sites. Thus, we conclude that Mediator subunit Med1 acts as a pivotal coactivator for GATA-1 in erythroid development.

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.

A unified nomenclature for protein subunits of mediator complexes linking transcriptional regulators to RNA polymerase II.

The multiprotein Mediator complex has been shown to interact with gene-specific regulatory proteins and RNA polymerase II in vitro . Here, we use chromatin immunoprecipitation to analyze the recruitment of Mediator to GAL genes of yeast in vivo . We find that Mediator associates exclusively with transcriptionally active and not inactive GAL genes. This association maps to the upstream activating sequence, rather than the core promoter, and is independent of RNA polymerase II, general transcription factors, and core promoter sequences. These findings support the idea of Mediator as a primary conduit of regulatory information from enhancers to promoters in eukaryotic cells.

Protein SWAP-70 was initially isolated from nuclei of activated B cells and was implicated in the immunoglobulin class switch process. After B cell activation the protein translocates from the cytoplasm to the nucleus, and may serve to signal nuclear processes. We have generated mice deficient in SWAP-70 and found three main differences when compared to wild-type mice: (i) their B lymphocytes are two- to threefold more sensitive to gamma-irradiation than B cells of wild type; (ii) SWAP-70-deficient mice developed autoantibodies at a much higher frequency; and (iii) the CD40 signaling pathway is compromised in the mutant mice. CD40-dependent switching to the IgE isotype is reduced five- to eightfold in vitro. In SWAP-70-deficient mice, IgE levels prior to immunization were six- to sevenfold lower than in wild-type mice, and after immunization three- to fourfold lower. CD40-induced proliferation was transiently increased in the mutant. LPS-induced switching to other isotypes, however, and LPS-induced proliferation were normal. We propose that SWAP-70 serves a specific role in the CD40 signaling pathway, in particular in the IgE response.

Higher eukaryotes produce immunoglobulins (lg) of different classes, which are defined by the constant region (C) of the heavy chain. In the mouse the heavy chains are designated μ, δ, γ3, γ1, γ2b, γ2a, e and α. Upon stimulation by antigen, expression of the early IgM class changes to that of another class (lgG3, lgG1, lgG2b, lgG2a, lgE, or lgA) can occur in B lymphocytes. Because the variable region is retained during this switch, the antigen specificity is unaltered. At the DNA level the process, named simply “class switching” from hereon, occurs through a DNA recombination event (for reviews see Esser and Radbruch 1990; Harriman et al. 1993).

GATA transcription factors are essential for diverse developmental processes including erythropoiesis. Med1/TRAP220 was shown to interact with GATA factors in vitro (Crawford et al. (2002): J Biol Chem. 277(5):3585-92.). Med1/TRAP220 is part of the multiprotein Mediator complex that forms a bridge between gene-specific regulatory proteins and the RNA polymerase II (Borggrefe (2004): Research Adv. in Biol. Chem. (2: 9-20), Bourbon et al. (2004) Mol. Cell 14(5):553-7). Mediator subunit Med1/TRAP220 deficient mice die during mid-embryonic development (day 11.5dpc) due to defects in placental, cardiac and hepatic development (Ito et al. (2000): Mol Cell. 5 (4):683–93., Landles et al. (2004): Mol Endocrinol. 17(12):2418–35). In order to analyze hematopoiesis in TRAP220-deficient embryos, AGMs (aorta-gonad-mesonephros) and Yolk-sacs were isolated from day 10.5 dpc embryos and analyzed by FACS and methylcellulose assays. TRAP220-deficient embryos contain c-Kit positive cells and BFU-E (Burst forming units erythrocytes) activity. However, CFU-E (Colony-forming units erythrocytes) activity was 10-fold reduced. This correlated with severe decrease of the CD71/Ter119 double-positive cells, the CFU-E containing fraction, in FACS-analysis. Our data shows that Mediator subunit Med1/TRAP220 is required for erythropoiesis just before the CFU-E stage. Because GATA factors affect early erythropoiesis and because MED1/TRAP220 binds to GATA factors in vitro, our data suggest that the Mediator of transcriptional regulation forms the bridge between GATA factors and the RNA polymerase II machinery.

The Notch gene encodes a transmembrane receptor that gave the name to the evolutionary highly conserved Notch signaling cascade. It plays a pivotal role in the regulation of many fundamental cellular processes such as proliferation, stem cell maintenance and differentiation during embryonic and adult development. After specific ligand binding, the intracellular part of the Notch receptor is cleaved off and translocates to the nucleus, where it binds to the transcription factor RBP-J. In the absence of activated Notch, RBP-J represses Notch target genes by recruiting a corepressor complex. Here, we review Notch signaling with a focus on gene regulatory events at Notch target genes. This is of utmost importance to understand Notch signaling since certain RBP-J associated cofactors and particular epigenetic marks determine the specificity of Notch target gene expression in different cell types. We subsequently summarize the current knowledge about Notch target genes and the physiological significance of Notch signaling in development and cancer.