Table of contents

Constitutively active human Notch1 binds to the transcription factor CBF1 and stimulates transcription through a promoter containing a CBF1-responsive element

A truncated active form of Notch1 binds CBF1, also known as JkappaRBP, the mammalian homolog of Suppressor of Hairless and activates transcription through a CBF1 response element-containing promoter. One model for the function of mammalian Notch assumes that Notch is cleaved by a membrane protease, and the released membrane domain is translocated to the nucleus. This model is supported by the observation that the untethered intracellular domain of Notch is as active as truncated Notch and is located predominantly in the nucleus, and by the observation that small amounts of CBF1 are associated with Notch1 in the nucleus (Lu, 1996 and references).

Epstein-Barr virus immortalization: Notch2 interacts with CBF1 and blocks differentiation

The ability of Epstein-Barr virus (EBV) latent infection nuclear protein EBNA3C to activate transcription of two EBNA2-responsive genes and to inhibit EBNA2 activation of transcription in transient-transfection assays appears to be due to its ability to interact with RBPJkappa, a cell protein that links EBNA2 to its response elements. EBNA3A and EBNA3B are similar to EBNA3C in binding to RBPJkappa. EBNA3A and EBNA3B can also inhibit the interaction of RBPJkappa with cognate DNA in vitro. Although EBNA3 open reading frames are each close to 1,000 codons long, EBNA3A amino acids 1 to 138, EBNA3B amino acids 1 to 311, and EBNA3C amino acids 1 to 183 are sufficient for RBPJkappa interaction, while EBNA3B amino acids I to 109 have less or no binding. The RBPJkappa interacting domains overlap with the most highly conserved domain (amino acids 90 to 320) among the EBNA3 proteins. Thus, the EBNA3 gene family appears to have evolved to differentially regulate promoters with RBPJkappa binding sites. EBNA2, EBNA3A, and EBNA3C are important in EBV transformation of primary human B lymphocytes. Their interaction with RBPJkappa links EBV transformation to the notch signaling pathway and the effects of activated notch in T-cell leukemogenesis (Robertson, 1966).

Mammalian Notch intracellular domain (NotchIC) interacts with the transcriptional repressor CBF1, which is the human homolog of Drosophila Suppressor of Hairless. The N-terminal 114-amino-acid region of mouse NotchIC contains the CBF1 interactive domain and the cdc10/ankyrin repeats are not essential for this interaction. This result was confirmed in immunoprecipation assays in which the N-terminal 114-amino-acid segment of NotchIC, but not the ankyrin repeat region, coprecipitated with CBF1. Mouse NotchIC itself is targeted to the transcriptional repression domain (aa179 to 361) of CBF1. NotchIC transactivates gene expression via CBF1 tethering to DNA. Transactivation by NotchIC occurs partially through abolition of CBF1-mediated repession. This same mechanism is used by Epstein-Barr virus EBNA2. Thus, mimicry of Notch signal transduction is involved in Epstein-Barr virus-driven immortalization (Hsieh, 1996).

Epstein-Barr virus immortalization: Notch2 interacts with CBF1 and blocks differentiation

Notch is involved in the cell fate determination of many cell lineages. The intracellular region (RAMIC) of Notch1 transactivates genes by interaction with a DNA binding protein RBP-J. The activities of mouse RAMIC and its derivatives were compared in transactivation and differentiation suppression of myogenic precursor cells. RAMIC comprises two separate domains: IC for transactivation (the IC domain includes the whole intracellular domain exclusive of the RAM domain) and RAM (immediately C-terminal to the transmembrane region) for RBP-J binding. Although the physical interaction of ankyrin repeats within IC with RBP-J is much weaker than is RAM interaction with RBP-J, transactivation activity of IC is shown to involve RBP-J by using an RBP-J null mutant cell line. IC shows differentiation suppression activity that is generally comparable to its transactivation activity. The RBP-J-VP16 fusion protein, which has strong transactivation activity, also suppresses myogenesis of C2C12 myogenic precursor cells. The RAM domain, which has no other activity than binding to RBP-J, synergistically stimulates transactivation activity of IC to the level of RAMIC. The RAM domain is proposed to compete with a putative co-repressor for binding to RBP-J because the RAM domain can also stimulate the activity of RBP-J-VP16. Taken together, these results indicate that differentiation suppression of myogenic precursor cells by Notch signalling is due to the transactivation of genes carrying RBP-J binding motifs (Kato, 1997).

Oncogenic forms of NOTCH1 lacking either the primary binding site for RBP-Jkappa or nuclear localization sequences retain the ability to associate with RBP-Jkappa and activate transcription

Truncated forms of the NOTCH1 transmembrane receptor engineered to resemble mutant forms of NOTCH1 found in certain cases of human T cell leukemia/lymphoma (T-ALL) efficiently induce T-ALL when expressed in the bone marrow of mice. Unlike full-sized NOTCH1, two such truncated forms of the protein either lacking a major portion of the extracellular domain (DeltaE) or consisting only of the intracellular domain (ICN) are found to activate transcription in cultured cells, presumably through RBP-Jkappa response elements within DNA. Both truncated forms also bind to the transcription factor RBP-Jkappa in extracts prepared from human and murine T-ALL cell lines. Transcriptional activation requires the presence of a weak RBP-Jkappa-binding site within the NOTCH1 ankyrin repeat region of the intracellular domain. Unexpectedly, a second, stronger RBP-Jkappa-binding site, which lies within the intracellular domain close to the transmembrane region and significantly augments association with RBP-Jkappa, is not needed for oncogenesis or for transcriptional activation. While ICN appears primarily in the nucleus, DeltaE localizes to cytoplasmic and nuclear membranes, suggesting that intranuclear localization is not essential for oncogenesis or transcriptional activation. In support of this interpretation, mutation of putative nuclear localization sequences decreases nuclear localization and increases transcriptional activation by membrane-bound DeltaE. Transcriptional activation by this mutant form of membrane-bound DeltaE is approximately equivalent to that produced by intranuclear ICN. These data are most consistent with NOTCH1 oncogenesis and transcriptional activation being independent of association with RBP-Jkappa at promoter sites (Aster, 1997).

Mastermind mediates chromatin-specific transcription and turnover of the Notch enhancer complex

Studies of mammalian homolog of Mastermind prove insight into the molecular interactions of Mastermind as a co-activator in the Notch pathway. Signaling through the Notch pathway activates the proteolytic release of the Notch intracellular domain (ICD), a dedicated transcriptional coactivator of CSL (CBF-1, Suppressor of Hairless, and Lag-1) enhancer-binding proteins. Chromatin-dependent transactivation by the recombinant Notch ICD-CBF1 enhancer complex in vitro requires an additional coactivator, Mastermind (MAM). MAM provides two activation domains necessary for Notch signaling in mammalian cells and in Xenopus embryos. The central MAM activation domain (TAD1) recruits CBP/p300 (Drosophila homolog: Nejire) to promote nucleosome acetylation at Notch enhancers and activate transcription in vitro. MAM expression induces phosphorylation and relocalization of endogenous CBP/p300 proteins to nuclear foci in vivo. Moreover, coexpression with MAM and CBF1 strongly enhances phosphorylation and proteolytic turnover of the Notch ICD in vivo. Enhanced phosphorylation of the ICD and p300 requires a glutamine-rich region of MAM (TAD2) that is essential for Notch transcription in vivo. Thus MAM may function as a timer to couple transcription activation with disassembly of the Notch enhancer complex on chromatin (Fryer, 2002).

Unexpectedly, expression of MAM induces endogenous CBP/p300 proteins to accumulate in multiple nuclear foci in vivo. These structures do not form upon expression of a mutant MAM protein lacking the C-terminal TAD2 region (1-301MM). Thus, binding of MAM to CBP/p300, which is mediated through TAD1, is not sufficient to cause CBP/p300 to accumulate in these structures. Expression of other Notch components (ICD, CBF1) did not affect the subnuclear localization of CBP/p300, indicating that these foci are not a consequence of high levels of Notch signaling in the nucleus. One possibility is that MAM may regulate the expression or modification of CBP/p300 independently of Notch signaling. Indeed, the MAM-induced foci are accompanied by increased phosphorylation of CBP, and this phosphorylation requires the C-terminal TAD2 domain of MAM. Consequently, overexpression of MAM in the nucleus may promote widespread phosphorylation of CBP, which may cause the CBP/p300 proteins to concentrate in these structures. Changes in CBP/p300 phosphorylation have been shown to alter its activity and differentially affect its interactions with other transcription factors. It will therefore be important to assess whether MAM promotes CBP/p300 phosphorylation within the Notch enhancer complex, and whether phosphorylation of CBP/p300 is important for transcriptional activation by Notch (Fryer, 2002).

The timing of Notch signaling is tightly controlled in developmental processes such as somite formation, during which Notch target genes such as cHairy1 and mHES1 undergo periodic cycles of expression at the direction of a molecular oscillator, or vertebrate segmentation clock. This clock may be established through the intrinsic timing of Notch signaling as well as the half-life of Notch-induced transcriptional repressors. The Notch ICD is subject to proteolytic degradation in the nucleus through the action of the ubiquitin ligases such as Sel-10. Rapid turnover of the ICD may be required to allow genes to respond rapidly to subsequent cycles of Notch signaling. Coexpression with MAM and CBF1 promotes the phosphorylation and proteolytic turnover of the ICD in vivo, indicating that MAM couples transcription activation with degradation of the ICD. In this respect, MAM may act as a timer to control the length of time that the Notch complex remains associated with the enhancer. By extension, MAM might contribute to the periodic expression of Notch target genes during somitogenesis through its potential effects on the disassembly of the Notch enhancer complex (Fryer, 2002).

The data indicate that CBF1 acts in concert with MAM to control the proteolytic turnover of the ICD in vivo. Importantly, both MAM and CBF1 appear to be stable upon coexpression with the ICD, and thus it appears that the ICD can be destabilized independently of its interacting partners. The requirement for CBF1 may reflect its ability to enhance binding of MAM to the ICD, or alternatively CBF1 might be needed to target the Notch enhancer complex to DNA. Stability of a mutant ICD protein lacking the PEST domain is unaffected by coexpression with MAM and CBF1, and turnover is accompanied by increased phosphorylation of the ICD. Importantly, the MAM TAD2 domain is necessary for both enhanced phosphorylation and turnover of the ICD. Because p300 has been shown to be critical for the regulated turnover of the p53 transactivator by MDM2, it will be important to assess whether recruitment of p300 by MAM may similarly be required for proteolytic degradation of the ICD. Nevertheless, it is clear that recruitment of CBP/p300 through the MAM TAD1 region is not sufficient to couple activation with turnover of the Notch ICD under the conditions examined in this study (Fryer, 2002).

Thus the TAD2 region is required for MAM to promote the phosphorylation of its two associated factors, CBP/p300 and the Notch ICD. Because MAM does not possess intrinsic ICD protein kinase activity, it is attractive to consider that the Notch ICD and CBP/p300 may instead be targeted for phosphorylation by cyclin-dependent kinases that associate with the transcription complex and are recruited to the promoter by MAM. Phosphorylation events mediated by CDK7 and Srb10 (the CDK8 homolog in yeast) have been implicated in the proteolytic destruction of other enhancer factors. The CDK9 subunit of the positive transcription elongation factor, P-TEFb, also associates with RNAPII, whereas CDK8 interacts with RNAPII as a component of human and yeast mediator complexes that have been variously implicated in activation and repression of transcription. Another possibility is that the ICD is phosphorylated by a protein kinase that associates with MAM directly. It remains to be determined whether the MAM-induced phosphorylation is accompanied by increased ubiquitination of the ICD, and whether the degradation of the ICD observed is caused by ubiquitin-dependent proteolysis such as that described for the nuclear Sel-10 ubiquitin ligase. It will also be important to learn whether modification of the ICD regulates its transcriptional activity, as has been observed for other transcription factors, and whether these steps may ultimately be coupled to disassembly of the Notch enhancer complex and turnover of the Notch ICD (Fryer, 2002).

In summary, MAM is an essential component of the Notch enhancer complex in vitro as well as in vivo. The human MAM protein recruits p300/CBP to the Notch enhancer complex and controls the stability of the Notch ICD through the action of its unique C-terminal activation domain. Further studies will be needed to evaluate whether these properties are shared among the various MAM proteins in different species, and to learn how MAM-induced phosphorylation of the ICD and CBP/p300 proteins is coordinated with the regulation of Notch transcription (Fryer, 2002).

Crystal structure of the CSL-Notch-Mastermind ternary complex bound to DNA

Notch signaling mediates communication between cells and is essential for proper embryonic patterning and development. CSL is a DNA binding transcription factor that regulates transcription of Notch target genes by interacting with coregulators. Transcriptional activation requires the displacement of corepressors from CSL by the intracellular portion of the receptor Notch (NotchIC) and the recruitment of the coactivator protein Mastermind to the complex. This study reports the 3.1 Å structure of the ternary complex formed by CSL, NotchIC, and Mastermind bound to DNA. As expected, the RAM domain of Notch interacts with the beta trefoil domain of CSL; however, the C-terminal domain of CSL has an unanticipated central role in the interface formed with the Notch ankyrin repeats and Mastermind. Ternary complex formation induces a substantial conformational change within CSL, suggesting a molecular mechanism for the conversion of CSL from a repressor to an activator (Wilson, 2006).

Structural basis for cooperativity in recruitment of MAML coactivators to Notch transcription complexes

Notch receptors transduce essential developmental signals between neighboring cells by forming a complex that leads to transcription of target genes upon activation. This study reports 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 (Nam, 2006).

Cooperative assembly of higher-order Notch complexes functions as a switch to induce transcription

Notch receptors control differentiation and contribute to pathologic states such as cancer by interacting directly with a transcription factor called CSL (for CBF-1/Suppressor of Hairless/Lag-1) to induce expression of target genes. A number of Notch-regulated targets, including genes of the hairy/enhancer-of-split family in organisms ranging from Drosophila to humans, are characterized by paired CSL-binding sites in a characteristic head-to-head arrangement. Using a combination of structural and molecular approaches, it has been establish that cooperative formation of dimeric Notch transcription complexes on promoters with paired sites is required to activate transcription. These findings identify a mechanistic step that can account for the exquisite sensitivity of Notch target genes to variation in signal strength and developmental context, enable new strategies for sensitive and reliable identification of Notch target genes, and lay the groundwork for the development of Notch pathway inhibitors that are active on target genes containing paired sites (Nam, 2007).

Cocrystals of a human Notch transcriptional activation complex (NTC) core, which consists of an N-terminal MAML-1 peptide, the ANK domain of human Notch1, and CSL on a DNA duplex derived from the HES-1 promoter, contain contacts between the convex surfaces of ANK domains from adjacent unit cells that also are seen in crystals of the ANK domain solved in isolation in several different crystallization conditions (Nam, 2006). These contacts lie near a twofold symmetry axis in the crystals, such that the interacting complexes are positioned head-to-head at a distance roughly equal to that needed to occupy both recognition elements of an SPS. Primary sequence alignment of Notch ANK domains from different homologs shows that the key contacts are evolutionarily conserved. These conserved residues are not engaged in contacts within an individual MAML1/ANK/CSL/DNA complex, suggesting that the observed conservation reflects functional importance in mediating dimerization at SPS sites. The conservation among the four mammalian Notch receptors also predicts that each receptor should be capable of making interactions like those between the adjacent Notch1 complexes (Nam, 2007).

The ANK-ANK contacts primarily are electrostatic and lie in the second and third ankyrin repeats. Key interactions consist of contacts between the guanidino group of Arg-1985 and at least three backbone carbonyl oxygen atoms, as well as interactions between Glu-1950 and Lys-1946. Arg-1983 also forms hydrogen bonds with Ser-1952 and a backbone carbonyl. In addition to homotypic interactions between the ANK domains, unmodeled electron density in the MAML-1/ANK/CSL/DNA complex also suggests the existence of interactions between the ANK domain of one complex and the N-terminal end of MAML-1 in the second complex. Based on the architecture of the complex, and the evolutionary conservation of SPSs and the crystal contact residues, it is postulated that the ANK domains of Notch receptors mediate dimerization of ternary complexes on SPSs found in Notch target gene promoters (Nam, 2007).

To test whether residues engaged in ANK-ANK contacts in the crystal contribute to transcriptional activation of SPS-bearing promoters, the ability of different forms of ICN to induce transcription of a luciferase gene under control of the HES-1 promoter, which has a functionally important SPS element, was tested. In contrast to normal ICN1, mutations that disrupt the predicted dimerization interface either abrogate (R1985A) or diminish (K1946E and E1950K) the ability of ICN1 to induce expression of the HES-1 reporter gene. Combining the K1946E and E1950K mutations in cis, however, rescues the defect in transcriptional activation, indicating that the putative dimerization interface is functionally important in regulating transcriptional activity at a promoter that contains an SPS. In addition, when coexpressed with ICN1, the R1985A mutation dominantly interferes with activation of the HES-1 promoter element by normal ICN1. Importantly, when these mutants are scored on an artificial reporter that contains four CSL-binding sites oriented in the same direction and in tandem, there is no change in the ability of the mutants to activate transcription. Moreover, in cotransfected cells, all ICN1 polypeptides with mutations that disrupt the predicted dimerization interface are expressed at similar levels to normal ICN1, and they coimmunoprecipitate in similar amounts with CSL and MAML-1. Together, these findings indicate that the ability to form monomeric ternary complexes with MAML-1 and CSL is not affected by these mutations (Nam, 2007).

To establish directly whether NTCs (consisting of one molecule each of MAML-1, ICN, and CSL) can cooperatively dimerize on DNA, electrophoretic mobility shift assays (EMSAs) were carried on an oligonucleotide probe containing the HES-1 promoter SPS. Without Notch or MAML-1, CSL binds to each of the two sites independently. When present in excess, most probes bind a single CSL molecule, a finding consistent with previous studies showing that CSL binds its recognition element as a monomer without detectable cooperativity at paired sites. Adding RAMANK from Notch1 does not change the stoichiometric distribution of complexes bound per probe molecule. However, when MAML-1 is added, the stoichiometric distribution of the complexes changes dramatically: all of the probe is either free or bound by NTC dimers, indicating that NTC loading at one site leads to cooperative loading of the second site. As predicted, cooperative loading is abrogated by the R1985A mutation, which instead produces a smear corresponding to an ensemble of species that likely results from a weak residual tendency to self-associate. In contrast, the R1985A mutation does not detectably affect ternary complex formation on a probe containing only a single CSL-binding site, indicating that the R1985A mutation is a cooperativity mutant that specifically interferes with dimerization. The partial loss of activity of the K1946E and E1950K mutants in the HES-1 reporter assays is echoed in EMSA titrations, where the proteins undergo a cooperative transition to form dimers at a concentration ~4-fold greater than normal ICN1 or the K1946E/E1950K double mutant (Nam, 2007).

To test whether higher-order complexes exhibit specificity for the SPS architecture, additional EMSA assays were carried out on variant DNA sequences that eliminate the integrity of one of the SPS sites, flip the site orientation, or alter the spacing between the sites by a half-turn of helix. When either site A or site B is mutated so that it no longer corresponds to a CSL consensus site (YGTGDGAA), cooperative assembly of the dimer is no longer observed. Moreover, cooperative dimerization is no longer detected when the second site is inverted, and it is dramatically diminished when the second site is moved by a 5-base insertion. Because the intrinsic affinity of a single ternary complex for DNA is not altered under the conditions of inversion or insertion, these studies show that the proper spatial arrangement of the two individual binding sites is needed for cooperative dimerization to occur (Nam, 2007).

It was next asked what range of spacer lengths between sites is compatible with cooperative loading of dimeric complexes. The optimal spacing between consensus sites for cooperative dimerization is 16 bp, but cooperative dimerization still can occur on templates with spacers varying from 15 to 19 bp, implying that two NTCs can adjust their positions relative to each other to accommodate a modest range of spacer lengths between sites. This inferred flexibility is consistent with the different conformations of CSL seen in the crystal structures of the Notch ternary complexes formed with the human and worm proteins and with the enrichment of adenosine and thymidine in the spacer between the paired sites (Nam, 2007).

To determine whether the assembly of NTCs and their cooperative dimers is general among the human Notch homologues, the ability of the RAMANK domains of Notch1-4 to form complexes on single and sequence-paired sites was tested. Despite qualitative differences in mobility on the EMSA, all four purified RAMANK polypeptides bind to CSL independent of MAML-1 and then recruit MAML-1 to ternary complexes on a single site probe. When the longer, paired site probe is provided, all RAMANK polypeptides mediate cooperative dimerization, as predicted from the conservation in primary sequence at the dimerization interface. Thus, a similar series of events takes place to assemble single and dimeric NTCs in all four mammalian Notch homologues (Nam, 2007).

Notch dimerization is required for leukemogenesis and T-cell development

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. This study shows 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 α (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 higher-order 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) (Liu, 2010).

Structural and mechanistic insights into cooperative assembly of dimeric Notch transcription complexes

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. This study reports 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 (Arnett, 2010; full text of article).

Assembly of a Notch transcriptional activation complex requires multimerization

Notch transmembrane receptors direct essential cellular processes, such as proliferation and differentiation, through direct cell-to-cell interactions. Inappropriate release of the intracellular domain of Notch (NICD) from the plasma membrane results in the accumulation of deregulated nuclear NICD that has been linked to human cancers, notably T-cell acute lymphoblastic leukemia (T-ALL). Nuclear NICD forms a transcriptional activation complex by interacting with the coactivator protein Mastermind-like 1 and the DNA binding protein CSL (for CBF-1/Suppressor of Hairless/Lag-1) to regulate target gene expression. Although it is well understood that NICD forms a transcriptional activation complex, little is known about how the complex is assembled. This study demonstrates that ICD multimerizes and that these multimers function as precursors for the stepwise assembly of the Notch activation complex. Importantly, it was demonstrated that the assembly is mediated by NICD multimers interacting with Skip (the human homolog of Drosophila Bx42). and Mastermind. These interactions form a preactivation complex that is then resolved by CSL to form the Notch transcriptional activation complex on DNA (Vasquez-Del Carpio, 2011).

Previous studies have demonstrated that NICD forms two distinct protein complexes in cells. One complex is predominately localized in the nucleus and is composed of NICD, Maml1, and CSL. This complex is thought to be the transcriptionally active form of Notch on DNA. In addition, a smaller Notch-containing complex is also detected in cells. This complex is primarily localized in the cytoplasm and does not contain either Maml1 or CSL. The nature and function of this complex has remained unclear. This study demonstrates that prior to forming a transcriptionally active complex, NICD forms multimers, and these multimers serve as precursors to the assembly of Notch activation complexes. Evidence is provided for a stepwise assembly of the Notch activation complex that is mediated by Skip and Maml1. It includes the formation of a preactivation complex composed of Skip, Maml1, and NICD multimers. This intermediate complex is then resolved by interaction with CSL, resulting in the formation of the Notch activation complex consisting of monomeric NICD, Maml1, and CSL (Vasquez-Del Carpio, 2011).

Maml1 is an essential component in Notch signaling, forming a stable complex with NICD and CSL and functioning as a 'coactivator'. A critical role of this protein in the active complex was observed when Maml1 deletion mutants that bind NICD but lack the C-terminal region inhibit Notch transactivation and can act as dominant negatives in Notch signaling. Therefore, it is thought that Maml1 functions to recruit other factors to drive Notch function. Although it is clear from the crystal structure that Maml1 makes formal contacts with Notch and CSL, purified Notch and Maml1 do not interact. In fact, using purified proteins, Maml1 does not interact with either NICD or CSL alone. Maml1 can do so only in the presence of all three proteins. Therefore, a question that remains to be resolved is how Maml1 is incorporated into the Notch activation complex (Vasquez-Del Carpio, 2011).

Skip was initially identified as a cofactor for the Ski oncoprotein. Subsequently, Skip has been reported to act both as a corepressor in association with the CSL corepressors SMRT/NCoR and Sharp and as an enhancer or coactivator of the Notch signaling pathway. The mechanistic details of how Skip works in Notch transcriptional activation are not known. How does Skip potentiate Notch signaling? Based on the current results, it is proposed that the role of Skip in Notch transactivation is to initiate complex assembly by binding to Notch multimers and to recruit Maml1 to form a preactivation complex. It was demonstrated that Skip preferentially binds to NICD multimers, and since NICD monomers and not multimers are in the Notch activation complex, it is suggested that Skip is likely involved in the early events of Notch activation complex assembly. Moreover, it was shown that in the presence of Skip, a protein complex containing NICD multimer, Maml1 and Skip can be detected. Therefore, it appears that the NICD multimer-Skip complex is assembled to provide a docking site for Maml1 to form a preactivation complex. The data indicating that NICD, Maml1, and Skip assemble into a complex prior to interacting with CSL provide a mechanism for previous observations showing that both Maml1 and Skip are found at the HES-1 promoter only when NICD is present. Based on these data, it is predicted that by preventing the NICD multimer-Skip interaction, Maml1 would not be efficiently recruited to the activation complex and thus the intensity of Notch signaling would be decreased (Vasquez-Del Carpio, 2011).

CSL appears to be the mediator involved in the conversion of a preactivation complex to the Notch transcriptional activation complex. The interaction between the preactivation complex and CSL essentially loads NICD and Maml1 onto CSL bound to DNA and initiates transcriptional activation. How does CSL perform this conversion? It appears that CSL is involved in destabilizing the interaction between the NICD molecules. Previous studies demonstrated that CSL interacts with a 4-amino-acid motif (PhiWPhiP) found within the RAM domain of NICD. This study shows that the RAM domain also interacts with a region between amino acids 2203 and 2216 of NICD, which is here defined as the C-terminal multimerization region (CTM). C-terminal deletion mutants of Notch that terminate at amino acid 2240 still form multimers, but deletion mutants that terminate at amino acid 2202 are monomeric. Furthermore, a deletion mutant that is monomeric is severely compromised for transcriptional activation. This is not simply due to the loss of a transactivation domain, since activity can be restored by rescue with a multimerization-competent, transactivation-deficient mutant of Notch. The interaction between the RAM domain and CTM does not require a functional PhiWPhiP motif. Therefore, it is possible to physically separate the RAM domain of NICD into an N-terminal multimerization (NTM), amino acids 1820 to 1847, and a CSL binding region (PhiWPhiP motif). Since the RAM domain has two distinct components, it is proposed that the RAM domain functions as a switch between the preactivation complex and the Notch activation complex. In this model, a region of the RAM domain (NTM) C-terminal to the PhiWPhiP motif interacts with the CTM. Thus, the NTM and CTM are the main sites of interaction for multimer formation, although other low-affinity sites might be present and contribute to the overall stability of the multimer, like the ankyrin repeats. Later, when CSL interacts with the PhiWPhiP motif, a conformational change likely occurs in the RAM domain. This results in the RAM domain no longer interacting with the CTM, which drives the conversion from the preactivation complex to the Notch activation complex. Moreover, during the transition from preactivation complex to activation complex, it is not difficult to envision how CSL can displace Skip by steric hindrance from the complex, since both interact with the ankyrin repeat of Notch. Depending on Notch presence or absence, it has been reported that Skip can be found forming part of a CSL-repressor complex or a transcriptional activation complex. The data that support the model in which Skip is associated with the Notch activation complex come from chromatin immunoprecipitation (ChIP) analysis, in which Skip and other proteins can be detected sitting on the chromatin when Notch is present. In this model, Skip will be displaced from the repressor complex and be recruited again once the Notch activation complex is formed. In the current model, Skip is displaced during the transition from the preactivation complex to the transcriptional activation complex by CSL. Considering the results provided, the possibility cannot be excluded that Skip may form part of the final Notch activation complex on DNA, either by staying in the complex upon CSL interaction or by rerecruitment after the transcriptional activation complex is formed. This issue cannot be resolved by ChIP assay, since the technique provides a snapshot in a certain time frame of proteins interacting and not the dynamics of complex formation or transcription (Vasquez-Del Carpio, 2011).

Biochemical and biophysical studies have been mostly focused on the ankyrin repeat domain of NICD. However, these studies did not detect multimeric forms of NICD. Why were Notch multimers not detected? This study has demonstrated that the C-terminal region of NICD is required for multimer formation. Deletion of the C-terminal region of NICD impairs the formation of NICD multimers. Moreover experiments using only the ankyrin repeats showed that this domain is not sufficient for multimerization, although its contribution to the stability of the multimer may be important. Furthermore, the truncated forms of NICD utilized in these biophysical studies did not contain the RAM domain or the C-terminal region of NICD, which this study has demonstrated to be necessary for the formation of NICD multimers (Vasquez-Del Carpio, 2011).

NICD has been shown to form dimeric activation complexes with Mastermind and CSL on DNA that contained two CSL binding sites positioned in a head-to-head arrangement. In this crystal structure, the dimeric complexes are stabilized by the interaction of the ankyrin domain from one NICD with the ankyrin domain of another NICD. The mutation of a residue within the ankyrin domain involved in the dimer formation into alanine (R1985A) prevented the formation of this dimeric structure on DNA. Is this dimerization on DNA similar to the multimerization that was observe? To determine if arginine at position 1985 of NICD plays a role in multimerization, the residue was mutated, and it was determined if NICD still formed multimers. The NICDR1985A mutant still retains reporter activity on a promoter that contains a tandem array of CSL binding sites, 8x CSL promoter, but not on a promoter that contains two CSL binding sites positioned head to head, Hes-1 promoter. These data indicate that the mutant is functioning similarly to the published mutant. To determine if NICDR1985A forms multimers, 293T cells were cotransfected with NICDR1985A carrying Flag (NICDR1985AF) and Myc (NICDR1985AM) epitope tags. When NICDR1985AF was immunoprecipitated, NICDR1985AM coimmunoprecipitated. These data indicated that the multimerization observed does not involve the R1985 residue found within the ankyrin domain of Notch. Furthermore the observed dimeric complexes in the crystal structure result from cooperative binding of transcriptional complexes on DNA and not from an intrinsic multimerization property of Notch that was described for complex assembly (Vasquez-Del Carpio, 2011).

Why do Notch proteins multimerize with each other? It is proposed that multimerization has evolved to regulate Notch function by controlling the timing/duration of Notch signaling (Vasquez-Del Carpio, 2011).

How does multimerization regulate the timing/duration of Notch signaling? Once released from the plasma membrane, it is proposed that NICD forms multimers. This establishes the initial step in regulation, because multimerization is a function of free (monomeric) NICD concentration. NICD multimer formation is necessary to form a complex with Skip. This provides a second step of regulation in Notch activation, because the interaction between NICD multimers and Skip is required to recruit Maml1 to form the preactivation complex. Therefore, Skip availability is predicted to be a limiting factor in Notch signaling. Once the preactivation complex is assembled, the formation of an activation complex with DNA-bound CSL is thought to be rapid. Interaction of the preactivation complex with CSL triggers the loading of NICD and Maml1 to form the activation complex with CSL and concomitantly the release of NICD and Skip. In this step, the NICD multimer is disassociated by CSL, resulting in the retention of only one NICD molecule in the activation complex. The released NICD monomer is then free to multimerize and initiate another round of activation complex assembly. Once in the activation complex, NICD is rapidly degraded following the initiation of transcription. Therefore, it is proposed that the duration of Notch signaling is a function of the rate of assembly and subsequent destruction of the Notch activation complex and that the cycling of NICD monomers and multimers may provide a mechanism for the oscillation of Notch transcriptional activity (Vasquez-Del Carpio, 2011).

The Notch1 transcriptional activation domain is required for development and reveals a novel role for Notch1 signaling in fetal hematopoietic stem cells

Notch1 is required to generate the earliest embryonic hematopoietic stem cells (HSCs); however since Notch-deficient embryos die early in gestation, additional functions for Notch in embryonic HSC biology have not been described. This study used two complementary genetic models to address this important biological question. Unlike Notch1-deficient mice, mice lacking the conserved Notch1 transcriptional activation domain (TAD) show attenuated Notch1 function in vivo and survive until late gestation, succumbing to multiple cardiac abnormalities. Notch1 TAD-deficient HSCs emerge and successfully migrate to the fetal liver but are decreased in frequency by embryonic day 14.5. In addition, TAD-deficient fetal liver HSCs fail to compete with wild-type HSCs in bone marrow transplant experiments. This phenotype is independently recapitulated by conditional knockout of Rbpj, a core Notch pathway component. In vitro analysis of Notch1 TAD-deficient cells shows that the Notch1 TAD is important to properly assemble the Notch1/Rbpj/Maml trimolecular transcription complex. Together, these studies reveal an essential role for the Notch1 TAD in fetal development and identify important cell-autonomous functions for Notch1 signaling in fetal HSC homeostasis (Gerhardt, 2014).

Table of contents

Notch continued: Biological Overview | Regulation | Protein Interactions | Post-transcriptional regulation of Notch mRNA | Developmental Biology | Effects of Mutation | References

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