BIOLOGICAL OVERVIEW

mastermind is a neurogenic gene. Neurogenic gene mutants produce neural hyperplasia (too many nerves). The distribution of mastermind protein (Mam) has been examined. Mam is expressed through all germlayers during early embryogenesis, including ectodermal precursors to both neuroblasts and epidermoblasts. In early stage 6 embryos, the presumptive mesoderm expresses Mam at lower levels than other blastoderm cells. At late stage 7 and early stage 8 there is a higher level of staining in the nuclei of cells along the ventral midline and cephalic furrow. The mesoderm expresses Mam at lower levels than does the ectoderm. The delaminating neuroblasts stain similarly to the ectodermal layer, however, it is apparent that some neuroblasts do not stain as intensely as the ectoderm. Mam is subsequently down-regulated within the nervous system and then reexpressed. Mam is expressed in the segmental cells of the midline; there is overlap between Prospero and Mam at regular intervals at the midline. Neuroblasts express Mam after delamination and apparently after division. Numerous Prospero-expressing cells do not contain significant levels of Mam. These small cells are presumably GMCs, immature neurons and/or glial cells that have down-regulated Mam. Cells that express Mam strongly may derive from a late wave of neuroblast delamination. Mam persists in the nervous system through late embryogenesis and postembryonically. The mesoderm, anterior midgut, and posterior midgut express Mam at low levels, whereas the proventriculus, pharynx, Malpighian tubules and hindgut maintain high levels of Mam. Expression in the nervous system continues postembryonically in second and third instar larvae. Mam is detected throughout the brain and is expressed strongly within the optic lobe and within thoracic regions of the CNS known to contain clusters of neuroblasts and their progeny. Mam is ubiquitously expressed in wing and leg imaginal discs and is not down-regulated in sensory organ precursor cells of the wing margin or notum. In the eye disc, Mam shows most prominent expression posterior to the morphogenetic furrow. Expression of the protein during oogenesis appears limited to follicle cells. Immunohistochemical detection of Mam on polytene chromosomes reveals binding at >100 sites. Chromosome colocalization studies with RNA polymerase and the Groucho corepressor protein implicate Mam in transcriptional regulation (Bettler, 1996).

If Mam functions late in the neurogenic pathway as a nuclear regulatory protein, there are two principal roles to consider: activation of products of the E(spl) complex and/or repression of the proneural loci. The genetic interaction between mam and Suppressor of Hairless points to the former possibility. Based on its similarity to CBF1, it has been suggested that Su(H) protein may need to recruit a coactivator for E(spl) induction; it is conceivable that Mam performs this function (Bettler, 1996).

Mam has been shown to be an evolutionarily conserved protein, with identifiable homologs in C. elegans and humans. Mam has been shown to interact with Su(H) in the presence of the fly Notch intracellular domain, but not in its absence (Petcherski, 2000).

Mastermind acts downstream of Notch to determine alternate cell fates in neural lineages. Neural precursors (or neuroblasts) divide in a stem cell lineage to generate a series of ganglion mother cells, each of which divides once to produce a pair of postmitotic neurons or glial cells. An exception to this rule is the MP2 neuroblast, which divides only once to generate two neurons. A screen was carried out for genes expressed in the MP2 neuroblast and its progeny as a means of identifying the factors that specify cell fate in the MP2 lineage. A P-element insertion line was identified that expresses the reporter gene tau-beta-galactosidase in the MP2 precursor and its progeny, the vMP2 and dMP2 neurons. The transposon disrupts the neurogenic gene mastermind, but does not lead to neural hyperplasia. However, the vMP2 neuron is transformed into its sibling cell, dMP2. By contrast, expression of a dominant activated form of the Notch receptor in the MP2 lineage transforms dMP2 to vMP2. Notch signaling requires Mastermind, suggesting that Mastermind acts downstream of Notch to determine the vMP2 cell fate. Mastermind plays a similar role in the neurons derived from ganglion mother cells 1-1a and 4-2a, where it specifies the pCC and RP2sib fates, respectively. This suggests that Notch signaling through Mastermind plays a wider role in specifying neuronal identity in the Drosophila central nervous system. Notch is expressed in both MP2 progeny. Notch signaling is blocked by Numb, which segregates exclusively to dMP2 when the MP2 precursor divides. Numb interacts directly with the intracellular domain of Notch. By antagonizing Notch, Numb promotes the dMP2 cell fate. Thus it is likely that Numb antagonism of Notch signaling in dMP2 confines Mastermind function, acting downstream of Notch, to the vMP2 neuron (Schuldt, 1998).

Studies of mammalian homolog of Mastermind provide 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).

Mastermind mutations generate a unique constellation of midline cells within the Drosophila CNS

The Notch pathway functions repeatedly during the development of the central nervous system in metazoan organisms to control cell fate and regulate cell proliferation and asymmetric cell divisions. Within the Drosophila midline cell lineage, which bisects the two symmetrical halves of the central nervous system, Notch is required for initial cell specification and subsequent differentiation of many midline lineages. This study provides the first description of the role of the Notch co-factor, mastermind, in the central nervous system midline of Drosophila. Overall, zygotic mastermind mutations cause an increase in midline cell number and decrease in midline cell diversity. Compared to mutations in other components of the Notch signaling pathway, such as Notch itself and Delta, zygotic mutations in mastermind cause the production of a unique constellation of midline cell types. The major difference is that midline glia form normally in zygotic mastermind mutants, but not in Notch and Delta mutants. Moreover, during late embryogenesis, extra anterior midline glia survive in zygotic mastermind mutants compared to wild type embryos. This is an example of a mutation in a signaling pathway cofactor producing a distinct central nervous system phenotype compared to mutations in major components of the pathway (Zhang, 2011).

Notch has been shown to play multiple developmental roles in the CNS of several organisms. The Drosophila midline, with its easy to identify neural and glial lineages, has provided examples of multiple and reiterative roles of the Notch pathway within a single CNS lineage. In this study, the characterization of mam^ΔC mutants indicates how a co-factor within a signaling pathway contributes to the development of different midline cell types and adds to understanding of Notch signaling complexity (Zhang, 2011).

Initial activation of sim in the mesectoderm depends on maternal Notch expression, as N^55e11 germline clones lack most sim expression and therefore, contain few midline cells. Likewise, mam^ΔC germline clones also show a reduction in sim expression. Thus, maternal contributions of both mam and Notch appear to act in the same pathway to activate sim early in development. Similarly, many midline neural phenotypes in zygotic mam^ΔC mutant embryos are largely consistent with those of N^55e11 and Dl³, suggesting mam and Notch act together during the development of these neurons. Notch is required for formation of neurons expressing en and may be needed to maintain en expression in midline cells that develop in the posterior compartment of each CNS segment. The results described in this study suggest mam is also required for the formation of the midline neurons that express en and develop into the iVUMs, the MNB and its progeny. While these cells of the posterior compartment were absent, the H cell and mVUM midline neurons were expanded in mam^ΔC mutants, similar to N^55e11 and Dl³ mutants, suggesting that mam function is needed within the Notch signaling pathway to obtain the variety of midline neurons found in wild type embryos (Zhang, 2011).

The major difference observed between zygotic mam^ΔC and N^55e11 mutants was the presence of midline glia in mam^ΔC, but not N^55e11 mutant embryos during mid to late embryogenesis. Not only were AMG present, but additional AMG survived in the mature CNS midline in mam^ΔC mutants compared to wild type embryos (and N^55e11 mutants). The presence of AMG in mam^ΔC mutants suggests either (1) the mam^ΔC mutation is hypomorphic, (2) mam is not required within the Notch pathway for midline glial differentiation or (3) maternally deposited mam transcripts are stable and functional during the Notch signaling event needed for midline glial formation. Results with mam deficiency embryos indicated that midline glia formed and persisted in the complete absence of zygotic mam activity, suggesting it is not the hypomorphic nature of the mam^ΔC allele that allows the midline glia to form. Currently, it is not possible to distinguish between the other two possibilities, although the last hypothesis is favored due to the timing of midline cell divisions. At gastrulation, each segment contains 8 mesectodermal cells, which each divide, resulting in 16 MPs per segment at stage 10. Cells that give rise to AMG and PMG do not divide again, whereas MPs that develop into neurons each divide once at stage 11. Because MPs that give rise to glia undergo their last division earlier than MPs that give rise to neurons, the Notch signaling event needed for midline glial differentiation may occur prior to Notch events that dictate midline neural fates at stage 11. Maternal Mam protein may linger just long enough to allow midline glia to form, but not long enough to function when MPs divide to give rise to midline neurons slightly later. It is thought that this is the reason N^55e11 mutants contain more midline cells per segment than wild type (and mam^ΔC). In N^55e11 mutants, MPs that would normally form glia and not divide, instead take on neural fates and do divide. The data are consistent with this hypothesis, but future, additional experiments are required to properly test it (Zhang, 2011).

In addition to this temporal sensitivity, mam may also be sensitive to spatially restricted events within the midline. Existing evidence suggests the 16 MPs fall into 3 equivalence groups at stage 10: the MP1s, MP3s and MP4s. MP1s are in the anterior, MP3s in the middle and MP4s in the posterior of each CNS segment and effects of mam^ΔC vary according to these positions. The results indicate that neurons derived from the anterior MP1s are sensitive to N^55e11, but not mam^ΔC; the middle MP3s are more sensitive to N^55e11 than mam^ΔC; while the posterior MP4s are equally sensitive to N^55e11 and mam^ΔC. In other words, mam^ΔC mutants 1) differ with N^55e11 mutants in neurons derived from MP1s (MP1 neurons), 2) have similar, less severe effects compared to N^55e11 mutants in cells derived from the MP3s (the H cell and H cell sib) and 3) the same effects as N^55e11 mutants in cells derived from the posterior MP4s (mVUMs, iVUMS and MNB). These differences may be due to region specific differences in expression of other midline regulators that combine with Notch and/or Mam to control cell fate specification during embryogenesis. Possible candidates include hedgehog and wingless, which are expressed in the midline, affect cell fate and both interact with mam in a Notch-independent manner in other tissues. In any case, clear differences in zygotic mam and Notch mutations within the midline exist and demonstrate that variations in different Notch signaling components can alter the cellular composition of the CNS in unique ways (Zhang, 2011).

Close examination of mam^ΔC and N^55e11 mutants during mid embryogenesis indicates they also differ in sim expression. After stage 10, sim diminishes in N^55e11 mutants, but persists in mam^ΔC mutants. Likewise, midline glia, which are known to require sim expression to differentiate, do not develop in N^55e11 mutants, but do develop in mam^ΔC mutants. The data indicate that all midline lineages that normally express sim are absent in N^55e11 mutants, while midline lineages that do not normally express sim are present and expanded in zygotic mutants of N^55e11. Therefore, similar to the initiation of sim expression early, the maintenance of sim expression at this later time also appears to require zygotic Notch activity. In contrast, the results suggest sim expression persists in zygotic mam^ΔC mutants (Zhang, 2011).

In the canonical Notch pathway, Mam normally functions as a co-factor and collaborates with both the NICD and Su(H) to activate target genes. Consistent with this role, overexpression of mam alone does not affect the number of AMG generated at mid embryogenesis, whereas the overexpression of the NICD in wild type embryos increases AMG cell number. Overexpression of the NICD in a mam^ΔC mutant background still increased the number of AMG during this stage, further supporting the idea that zygotic mam is not needed at this time. During late embryogenesis, mam^ΔC mutants contained extra AMG. Mutations in mam are known to promote neural tissue at the expense of ectoderm and this may result in the production of additional Spi, which inhibits apoptosis and allows extra midline glia to survive (Zhang, 2011).

Altogether, the data suggest a high level of complexity in the regulation of CNS target genes of Notch. Notch likely interacts with additional cell-lineage specific co-activators other than, or in addition to, Mam in certain cells. In this way, combinatorial interactions between components of Notch signaling and other signaling pathways can lead to different outputs in various cell types, increasing cell diversity and function. These result indicate mam^ΔC mutants contain AMG and PMG, whereas N^55e11 mutants do not. While this report describes major disruptions in mam, less severe mutations, such as small deletions, insertions or polymorphisms could also affect the midline and modify its cellular composition. Because mam mutations have more subtle effects on the midline compared to mutations in Notch or Delta, they may be tolerated more than mutations in major components of the pathway and actually contribute to CNS cellular variation in natural populations. Future experiments are needed to fully explore these functional differences between mam and Notch in the midline, as well as other tissues. Such differences can then be exploited to develop progressively specific research and clinical tools to regulate Notch signaling and the cellular composition of tissues from the different sensitivities (Zhang, 2011).

GENE STRUCTURE

Genomic DNA length - 67 kb

cDNA clone length - 6.3 kb. There are four zygotic transcripts, differing in their times of expression, and one maternal transcript. There are two sites at which transcription can start, and multiple lengths of the 3' UTR (Smoller, 1990).

Bases in 5' UTR - 754

Exons - seven

Bases in 3' UTR - 1791 and longer

PROTEIN STRUCTURE

Amino Acids - 1596

Structural Domains and Evolutionary Homologs

The protein has several homopolymeric amino acid runs: these include one polyalanine stretch, three polyasparagine, 21 polyglutamine, and four polyglycine stretches. The protein has few charged residues. There is a signal for nuclear import (Smoller, 1990). Mam has been shown to be an evolutionarily conserved protein, with identifiable homologs in C. elegans and humans (Petcherski, 2000).

Comparison of the D. melanogaster sequence with that of D. virulus indicates a significant conservation in a major cluster of charged amino acids. In contrast, extensive variation is noted in homopolymeric domains that immediately flank the acidic cluster. The repetitive areas show a high rate of amino acid substitution and insertion/deletions (Newfeld, 1991).

Database comparisons to Mam do not reveal string similarities in nonrepetitive domains, however, limited similarities between Mam and some leucine zipper proteins have been noted. The basic DNA binding domain that flanks the leucine zipper of the proteins encoded by cap 'n' collar, junD, fos and ATF-3 exhibits some features in common with Mam, although Mam does not contain a leucine zipper. The similarity exends to Skn-1, a C. elegans protein that likewise does not contain a leucine zipper, but shows more significant similarity to the zipper class of proteins. Skn-1 binds DNA as a monomer, in a sequence-specific fashion. Two leucine zipper class proteins, ATF-2/ATF-1 and ACR1, contain an additional small block of sequence similarity to Mam. Thus, it is conceivable that Mam represents a DNA-binding protein that is related to, but highly diverged from the leucine zipper class (Bettler, 1996).

Notch receptors are involved in cell-fate determination in organisms as diverse as flies, frogs and humans. In Drosophila, loss-of-function mutations of Notch produce a 'neurogenic' phenotype in which cells destined to become epidermis switch fate and differentiate to neural cells. Upon ligand activation, the intracellular domain of Notch (ICN) translocates to the nucleus, and interacts directly with the DNA-binding protein Suppressor of hairless [Su(H)] in flies, or recombination signal binding protein Jkappa (RBP-Jkappa) in mammals, to activate gene transcription. But the precise mechanisms of Notch-induced gene expression are not completely understood. The gene mastermind has been identified in multiple genetic screens for modifiers of Notch mutations in Drosophila. MAML1, a human homolog of the Drosophila gene Mastermind, has been cloned; it encodes a protein of 130 kD localizing to nuclear bodies. MAML1 binds to the ankyrin repeat domain of all four mammalian NOTCH receptors, forms a DNA-binding complex with ICN and RBP-Jkappa, and amplifies NOTCH-induced transcription of HES1. These studies provide a molecular mechanism to explain the genetic links between mastermind and Notch in Drosophila and indicate that MAML1 functions as a transcriptional co-activator for NOTCH signaling (Wu, 2000).

The Lin12/Notch receptors regulate cell fate during embryogenesis by activating the expression of downstream target genes. These receptors signal via their intracellular domain (ICD), which is released from the plasma membrane by proteolytic processing and associates in the nucleus with the CSL family of DNA-binding proteins to form a transcriptional activator. How the CSL/ICD complex activates transcription and how this complex is regulated during development remains poorly understood. Nrarp is a new intracellular component of the Notch signaling pathway in Xenopus embryos. Nrarp is a member of the Delta-Notch synexpression group and encodes a small protein containing two ankyrin repeats. Nrarp expression is activated in Xenopus embryos by the CSL-dependent Notch pathway. Conversely, overexpression of Nrarp in embryos blocks Notch signaling and inhibits the activation of Notch target genes by ICD. Nrarp forms a ternary complex with the ICD of XNotch1 and the CSL protein XSu(H) and in embryos Nrarp promotes the loss of ICD. By down-regulating ICD levels, Nrarp could function as a negative feedback regulator of Notch signaling that attenuates ICD-mediated transcription (Lamar, 2001).

Both Nrarp and Mastermind form ternary complexes with the CSL proteins and ICD. It was asked, therefore, whether the binding of Nrarp and Mastermind to Su(H) and ICD is mutually exclusive or whether these proteins can exist in a complex together. Binding of human Mastermind (hMM) to XSu(H) and ICD from XNotch1 was examined first by co-IP analysis in which extracts were prepared from embryos injected with RNA encoding Flag-tagged XSu(H), myc-tagged hMM, and myc-tagged ICDDeltaC. Flag-tagged XSu(H) was recovered from total extracts, and associated proteins were analyzed by Western analysis. The results show that hMM is co-IPed detectably with XSu(H), but only in the presence of ICDDeltaC. Moreover, the amounts of ICDDeltaC associated with XSu(H) in a co-IP complex increase markedly in the presence of hMM. Both of these results are consistent with the idea that hMM binds to XSu(H) and ICD in a ternary complex, as reported by others. Myc-tagged Nrarp is also co-IPed with XSu(H) in the presence of hMM and ICDDeltaC, consistent with the formation of multimeric complexes. However, this finding is inconclusive as to whether these proteins can form a quaternary complex. To address this issue, embryos were injected with RNA encoding Flag-tagged Nrarp along with myc-tagged hMM, XSu(H), and ICD. Flag-tagged Nrarp was recovered from extracts by immunoprecipitation, and associated proteins were analyzed by Western analysis using an alpha-myc antibody. The results show that the immunoprecipitation of Nrarp recovers not only XSu(H) and ICD, but hMM as well, indicating that Nrarp and hMM can bind in tandem to XSu(H)/ICD to form a quaternary complex (Lamar, 2001).

This paper describes a biochemical mechanism of action of Mam within the Notch signaling pathway. Expression of a human sequence related to Drosophila Mam (hMam-1) in mammalian cells augments induction of Hairy Enhancer of split (HES) promoters by Notch signaling. hMam-1 stabilizes and participates in the DNA binding complex of the intracellular domain of human Notch1 and a CSL protein. Truncated versions of hMam-1 that can maintain an association with the complex behave in a dominant negative fashion and depress transactivation. Furthermore, Drosophila Mam forms a similar complex with the intracellular domain of Drosophila Notch and Drosophila CSL protein during activation of Enhancer of split, the Drosophila counterpart of HES. These results indicate that Mam is an essential component of the transcriptional apparatus of Notch signaling (Kitagawa, 2001).

If hMam-1 is a homolog or ortholog of DMam, its expression should have effects on the mammalian Notch signaling pathway. Ligand binding to Notch on cell surfaces induces cleavage of the receptor in or near its transmembrane domain (site 3), releasing the IC domain of the receptor from the membrane. The Notch1-mediated activation of the HES-1 promoter has been shown to require this cleavage, and this can be mimicked experimentally by expression of the IC domain of Notch. Whether hMam-1 acts synergistically with Notch1IC in this system was examined by cotransfecting the expression vector of hMam-1 with that of the IC domain of Notch1 into NIH 3T3 cells. The HES-5 promoter is activated by the expression of Notch1IC alone but not by the expression of hMam-1 alone. Coexpression of Notch1IC and hMam-1 augments the activation of the HES-5 promoter. More modest effects were observed using the HES-1 promoter (Kitagawa, 2001).

To map the domain(s) of hMam-1 required for physical association with RBP-J and Notch, an examination was made of the effects of C-terminal truncations of hMam-1 on the DNA binding complexes involving RBP-J and Notch1IC. The truncations included those exhibiting dominant negative effects on the transcription activation assay. All the truncations up to amino acid 103 greatly diminish the complexes involving RBP-J only. Thus, the N-terminal region of hMam-1 is necessary and sufficient to mediate the physical association (Kitagawa, 2001).

Coimmunoprecipitation assays have revealed that hMam-1 associates with Notch1IC and RBP-J even in the absence of the binding site of DNA. This assay also reveals that hMam-1 associates with Notch1IC only in the presence of RBP-J. Expression of hMam-1 without Notch1IC does not significantly alter the DNA binding activity of RBP-J, indicating that hMam-1 associates with RBP-J only in the presence of Notch1IC. These results suggest that hMam-1 associates with the complex of the two proteins but not with the single protein species. The coimmunoprecipitation assays further reveal that expression of hMam-1 enhances the physical association of Notch1IC and RBP-J. More carboxyl portions of the hMam-1 protein presumably contain a domain(s) necessary for transcriptional activation, because overexpression of the N-terminal region hampers the transactivation induced by Notch signaling (Kitagawa, 2001).

There is substantial genetic evidence implicating DMam as a positive effector in Notch signaling. However, no genetic information is yet available for hMam-1, and additionally, hMam-1 has diverged significantly from the DMam sequence outside the charged amino acid clusters. Therefore, additional evidence to link the functions of these two proteins was sought. Whether DMam can form a complex with DNotch and Su(H) (Drosophila CSL) proteins in Drosophila cells was examined (Kitagawa, 2001).

Drosophila S2 cells endogenously express DMam. S2 cells were cotransfected with either Myc epitope-tagged Su(H) [Su(H)-Myc] and DNotch1IC or Su(H)-Myc, DNotchIC, and DMam. Coimmunoprecipitations show that endogenous DMam or transfected DMam exists in a complex with Su(H) and DNotchIC. These data also reveal that DNotchIC is required for DMam's association with the complex. Cotransfection of a Notch IC domain and Su(H) activates expression of an E(spl) reporter. Consistent with the hMam-1 data, cotransfection of DMam augments the activation levels of the E(spl) reporter (Kitagawa, 2001).

The results presented here suggest that Mam is an element involved in orchestrating the formation of transactivating complexes on the promoters of the target genes. In the absence of Notch receptor activation, the CSL protein associates with histone deacetylases and binds the promoter, effecting transcription repression. Under these conditions, Mam may not be associated with the complex. After Notch signaling is evoked, Mam can interact with the nuclear forms of Notch and CSL, thereby contributing to an activation complex. This complex likely recruits histone acetylases (Kitagawa, 2001).

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).

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)

The ventral spinal cord generates multiple inhibitory and excitatory interneuron subtypes from four cardinal progenitor domains (p0, p1, p2, p3). Cell-cell interactions mediated by the Notch receptor play a critical evolutionarily conserved role in the generation of excitatory v2aIN and inhibitory v2bIN interneurons. Lineage-tracing experiments show that the v2aIN and v2bIN develop from genetically identical p2 progenitors. The p2 daughter cell fate is controlled by Delta4 activation of Notch receptors together with MAML factors. Cells receiving Notch signals activate a transcription factor code that specifies the v2bIN fate, whereas cells deprived of Notch signaling express another code for v2aIN formation. Thus, this study provides insight into the cell-extrinsic signaling that controls combinatorial transcription factor profiles involved in regulating the process of interneuron subtype diversification (Peng, 2007).

Mastermind recruits CycC:CDK8 to phosphorylate the Notch ICD and coordinate activation with turnover

Notch signaling releases the Notch receptor intracellular domain (ICD), which complexes with CBF1 and Mastermind (MAM) to activate responsive genes. It has previously been reported that MAM interacts with CBP/p300 and promotes hyperphosphorylation and degradation of the Notch ICD in vivo. This study shows, in cultured HeLa cells, that CycC:CDK8 and CycT1:CDK9/P-TEFb are recruited with Notch and associated coactivators (MAM, SKIP) to the HES1 promoter in signaling cells. MAM interacts directly with CDK8 and can cause it to localize to subnuclear foci. Purified recombinant CycC:CDK8 phosphorylates the Notch ICD within the TAD and PEST domains, and expression of CycC:CDK8 strongly enhances Notch ICD hyperphosphorylation and PEST-dependent degradation by the Fbw7/Sel10 ubiquitin ligase in vivo. Point mutations affecting conserved Ser residues within the ICD PEST motif prevent hyperphosphorylation by CycC:CDK8 and stabilize the ICD in vivo. These findings suggest a role for MAM and CycC:CDK8 in the turnover of the Notch enhancer complex at target genes (Fryer, 2004).

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).

Mastermind-like 1 (MamL1) and mastermind-like 3 (MamL3) are essential for Notch signaling in vivo

Mastermind (Mam) is one of the elements of Notch signaling, a system that plays a pivotal role in metazoan development. Mam proteins form transcriptionally activating complexes with the intracellular domains of Notch, which are generated in response to the ligand-receptor interaction, and CSL DNA-binding proteins. In mammals, three structurally divergent Mam isoforms (MamL1, MamL2 and MamL3) have been identified. There have also been indications that Mam interacts functionally with various other transcription factors, including the p53 tumor suppressor, β-catenin and NF-kappaB. It has been demonstrated that disruption of MamL1 causes partial deficiency of Notch signaling in vivo. However, MamL1-deficient mice did not recapitulate total loss of Notch signaling, suggesting that other members could compensate for the loss or that Notch signaling could proceed in the absence of Mam in certain contexts. This study reports the generation of lines of mice null for MamL3. Although MamL3-null mice showed no apparent abnormalities, mice null for both MamL1 and MamL3 died during the early organogenic period with classic pan-Notch defects. Furthermore, expression of the lunatic fringe gene, which is strictly controlled by Notch signaling in the posterior presomitic mesoderm, was undetectable in this tissue of the double-null embryos. Neither of the single-null embryos exhibited any of these phenotypes. These various roles of the three Mam proteins could be due to their differential physical characteristics and/or their spatiotemporal distributions. These results indicate that engagement of Mam is essential for Notch signaling, and that the three Mam isoforms have distinct roles in vivo (Oyama, 2011).

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 (N^ICD) from the plasma membrane results in the accumulation of deregulated nuclear N^ICD that has been linked to human cancers, notably T-cell acute lymphoblastic leukemia (T-ALL). Nuclear N^ICD 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 N^ICD 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 N^ICD 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 N^ICD forms two distinct protein complexes in cells. One complex is predominately localized in the nucleus and is composed of N^ICD, 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, N^ICD 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 N^ICD multimers. This intermediate complex is then resolved by interaction with CSL, resulting in the formation of the Notch activation complex consisting of monomeric N^ICD, Maml1, and CSL (Vasquez-Del Carpio, 2011).

Maml1 is an essential component in Notch signaling, forming a stable complex with N^ICD 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 N^ICD 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 N^ICD 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 N^ICD multimers, and since N^ICD 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 N^ICD multimer, Maml1 and Skip can be detected. Therefore, it appears that the N^ICD multimer-Skip complex is assembled to provide a docking site for Maml1 to form a preactivation complex. The data indicating that N^ICD, 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 N^ICD is present. Based on these data, it is predicted that by preventing the N^ICD 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 N^ICD 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 N^ICD molecules. Previous studies demonstrated that CSL interacts with a 4-amino-acid motif (PhiWPhiP) found within the RAM domain of N^ICD. This study shows that the RAM domain also interacts with a region between amino acids 2203 and 2216 of N^ICD, 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 N^ICD 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 N^ICD. However, these studies did not detect multimeric forms of N^ICD. Why were Notch multimers not detected? This study has demonstrated that the C-terminal region of N^ICD is required for multimer formation. Deletion of the C-terminal region of N^ICD impairs the formation of N^ICD 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 N^ICD utilized in these biophysical studies did not contain the RAM domain or the C-terminal region of N^ICD, which this study has demonstrated to be necessary for the formation of N^ICD multimers (Vasquez-Del Carpio, 2011).

N^ICD 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 N^ICD with the ankyrin domain of another N^ICD. 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 N^ICD plays a role in multimerization, the residue was mutated, and it was determined if N^ICD still formed multimers. The N^ICDR1985A 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 N^ICDR1985A forms multimers, 293T cells were cotransfected with N^ICDR1985A carrying Flag (N^ICDR1985AF) and Myc (N^ICDR1985AM) epitope tags. When N^ICDR1985AF was immunoprecipitated, N^ICDR1985AM 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 N^ICD forms multimers. This establishes the initial step in regulation, because multimerization is a function of free (monomeric) N^ICD concentration. N^ICD multimer formation is necessary to form a complex with Skip. This provides a second step of regulation in Notch activation, because the interaction between N^ICD 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 N^ICD and Maml1 to form the activation complex with CSL and concomitantly the release of N^ICD and Skip. In this step, the N^ICD multimer is disassociated by CSL, resulting in the retention of only one N^ICD molecule in the activation complex. The released N^ICD monomer is then free to multimerize and initiate another round of activation complex assembly. Once in the activation complex, N^ICD 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 N^ICD monomers and multimers may provide a mechanism for the oscillation of Notch transcriptional activity (Vasquez-Del Carpio, 2011).

Olfactory Sensory Neurons Control Dendritic Complexity of Mitral Cells via Notch Signaling

Mitral cells (MCs) of the mammalian olfactory bulb have a single primary dendrite extending into a single glomerulus, where they receive odor information from olfactory sensory neurons (OSNs). Molecular mechanisms for controlling dendritic arbors of MCs, which dynamically change during development, are largely unknown. This study found that MCs displayed more complex dendritic morphologies in mouse mutants of Maml1 (see Drosophila Mastermind), a crucial gene in Notch signaling. Similar phenotypes were observed by conditionally misexpressing a dominant negative form of MAML1 (dnMAML1) in MCs after their migration. Conversely, conditional misexpression of a constitutively active form of Notch reduced their dendritic complexity. Furthermore, the intracellular domain of Notch1 (NICD1) was localized to nuclei of MCs. These findings suggest that Notch signaling at embryonic stages is involved in the dendritic complexity of MCs. After the embryonic misexpression of dnMAML1, many MCs aberrantly extended dendrites to more than one glomerulus at postnatal stages, suggesting that Notch signaling is essential for proper formation of olfactory circuits. Moreover, dendrites in cultured MCs were shortened by Jag1-expressing cells. Finally, blocking the activity of Notch ligands in OSNs led to an increase in dendritic complexity as well as a decrease in NICD1 signals in MCs. These results demonstrate that the dendritic complexity of MCs is controlled by their presynaptic partners, OSNs (Muroyama, 2016).

Coordination of neuron production in mouse and human cerebral cortex by the homolog of Drosophila Mastermind protein

The coordination of progenitor self-renewal, neuronal production, and migration is essential to the normal development and evolution of the cerebral cortex. Numerous studies have shown that the Notch, Wnt/beta-catenin, and Neurogenin pathways contribute separately to progenitor expansion, neurogenesis, and neuronal migration, but it is unknown how these signals are coordinated. In vitro studies suggested that the mastermind-like 1 (MAML1) gene, homologue of the Drosophila mastermind, plays a role in coordinating the aforementioned signaling pathways, yet its role during cortical development remains largely unknown. This study shows that ectopic expression of dominant-negative MAML (dnMAML) causes exuberant neuronal production in the mouse cortex without disrupting neuronal migration. Comparing the transcriptional consequences of dnMAML and Neurog2 ectopic expression revealed a complex genetic network controlling the balance of progenitor expansion versus neuronal production. Manipulation of MAML and Neurog2 in cultured human cerebral stem cells exposed interactions with the same set of signaling pathways. Thus, these data suggest that evolutionary changes that affect the timing, tempo, and density of successive neuronal layers of the small lissencephalic rodent and large convoluted primate cerebral cortex depend on similar molecular mechanisms that act from the earliest developmental stages (Ayoub, 2019).

date revised: 12 January 2022

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