Transcriptional Regulation

Studies of antineurogenic phenotypes induced by Notch proteins indicate that mastermind appears to function in an elaboration of a signal upstream of Notch to elaborate a signal that activates the receptor (Lieber, 1993). Suppressor of Hairless exhibits specific interactions with mastermind, indicating that the Notch pathway may regulate nuclear events by controlling mastermind transcription by means of Suppressor of Hairless (Fortini, 1994).

Targets of Activity

In embryos homozygotic for mutations in numb, the achaete-scute complex, daughterless and mastermind, the calmodulin transcription pattern is altered for each of these loci (Kovalick, 1992).

Hedgehog-stimulated stem cells depend on non-canonical activity of the Notch co-activator Mastermind

Normal self-renewal of follicle stem cells (FSCs) in the Drosophila ovary requires Hedgehog (Hh) signaling. Excess Hh signaling, induced by loss of patched (ptc), causes cell-autonomous duplication of FSCs. A genetic screen was used to identify Mastermind (Mam), the Notch pathway transcriptional co-activator, as a rare dose-dependent modifier of aberrant FSC expansion induced by excess Hh. Complete loss of Mam activity severely compromises the persistence of both normal and ptc mutant FSCs, but does not affect the maintenance of ovarian germline stem cells. Thus, Mam, like Hh, is a crucial stem cell factor that acts selectively on FSCs in the ovary. Surprisingly, other Notch pathway components, including Notch itself, are not similarly required for FSC maintenance. Furthermore, excess Notch pathway activity alone accelerates FSC loss and cannot ameliorate the more severe defects of mam mutant FSCs. This suggests an unconventional role for Mam in FSCs that is independent of Notch signaling. Loss of Mam reduces the expression of the Hh pathway reporter ptc-lacZ in FSCs but not in wing discs, suggesting that Mam might enhance Hh signaling specifically in stem cells of the Drosophila ovary (Vied, 2009).

Drosophila follicle stem cells (FSCs) provide a paradigm for stem cell behavior that includes the basic attributes of visualizing a defined stem cell and its environment, coupled with the potential to manipulate stem cell genotypes extensively and measure stem cell function. Nevertheless, only a limited number of factors have so far been defined as being essential to FSC function. Among these are the Hh, Wnt and BMP signaling pathways, adhesion molecules, a chromatin-remodeling factor and a histone ubiquitin protease. This study defines Mastermind as an essential FSC factor. Mam is not generally required for cell proliferation or survival in follicle cells or other Drosophila tissues. Mam is also not required for GSC function, assayed under exactly the same conditions and in the same animals that reveal its role in FSCs. However, in the absence of Mam, FSCs are lost as rapidly from adult ovaries as FSCs that cannot transduce a Hh signal (Vied, 2009).

These experiments show that Mam function is required cell-autonomously within the FSC lineage and it is assumed that this reflects a function in the FSC itself. More evidence of increased apoptosis of mam mutant FSCs or of prolonged quiescence of such cells (positive-marking studies) was seen, suggesting that mam FSCs are prematurely lost from their characteristic position in the germarium, taking on the fate of non-stem FSC daughter cells. Whether loss of Mam primarily affects a fundamental stem-daughter cell decision or adhesive properties contributing to niche retention is, as for most other FSC factors, unknown (Vied, 2009).

Mam has long been considered to be a dedicated co-activator in the Notch signaling pathway, because genetic analyses in Drosophila and other model organisms generally show a congruence between Notch and Mam loss-of-function phenotypes. Biochemically, Mam binds to a composite surface contributed by the cleaved intracellular domain of activated Notch and the DNA-binding protein Su(H), and provides an essential transcriptional activation function that includes the recruitment of CREB-Binding Protein (CBP). In ovaries, characteristic Notch mutant phenotypes were seen in response to mam mutations, showing that Mam does indeed act as an essential co-activator for Notch signaling in the follicle cell lineage. However, that function of Mam cannot account for its role in FSCs because FSC function is not impaired by null mutations affecting Notch and Su(H), the direct binding partners of Mam. That assertion is also consistent with the finding that expression of a dominant-negative Mam derivative inhibited the Notch-dependent behaviors of FSC derivatives without impairing FSC maintenance. To determine whether loss of Notch signaling contributed even partially to the mam mutant FSC phenotype, attempts were made to ameliorate the mam phenotype by activating Notch signaling in a Mam-independent manner. It was found that a synthetic Su(H)-VP16 activator could not rescue mam or ptc mam mutant FSC loss. Furthermore, increased activity of the Notch pathway by itself [using Su(H)-VP16 or Nintra] caused moderate FSC loss. Thus, the essential activity of Mam in FSCs appears to be entirely independent of the well-known role of Mam as a co-activator for Notch signaling (Vied, 2009).

The finding that FSC maintenance is not markedly impaired by the elimination of Notch signaling is notable in itself, because it contrasts with a requirement for each of the three other pathways (Hh, BMP and Wnt) that have been investigated to date. Notch signaling is important in the germarium for the earliest known decision of FSC progeny to adopt polar and stalk cell fates, and for the specification and maintenance of cap cells, which are themselves essential for maintaining GSCs (Vied, 2009).

The loss of FSCs in response to increased Notch activity might also be informative, although the heightened Notch activity induced by Nintra or Su(H)-VP16 is likely to be beyond physiological levels. The FSC loss induced by Nintra cannot be explained by titration of Mam away from other essential partners, because Su(H)-VP16 induces a similar phenotype but cannot bind to Mam in the absence of activated Notch (Vied, 2009).

The Notch ligand Delta is known to be expressed in terminal filament, cap, follicle and germline cells, and a clear increase in Delta signaling from the germline to overlying follicle cells at stage 6 triggers a switch from mitosis to follicle cell endocycles. Interestingly, that switch is still imposed on ptc mutant follicle cells that contact the germline, and is accompanied by Notch-dependent inhibition of ci expression and Hh pathway activity, which is mediated by the transcription factors Hindsight (Pebbled in FlyBase) and Tramtrack. FSC loss induced by Notch hyperactivity is also seen for ptc mutant cells and might conceivably involve an analogous mechanism, although Hindsight expression is not normally observed prior to stage 6. Moreover, it is possible that FSCs normally evade Notch-induced repression of Hh signaling by minimizing contact with the germline, while non-stem cell daughters embrace passing germline cysts (Vied, 2009).

There are currently very few reports of Notch-independent roles of Mam proteins (McElhinny, 2008). In two cases, the mammalian Mam homolog MAML1 was shown to bind and collaborate with DNA-binding proteins (p53 and MEF2C) other than those of the Su(H) family. In the third case, MAML1 was shown to bind β-catenin and to contribute to TCF-dependent induction of Wnt target genes. It seems likely from these examples, and from the established role of Mam in recruiting Mediator and histone acetyltransferase complexes, that the essential action of Mam in stem cells is as a transcriptional co-activator (Vied, 2009).

Mam function in FSCs has a notable dosage-sensitive interaction with the Hh signaling pathway. Mam was first identified in this context because a heterozygous mam mutation strongly suppressed ovarian somatic cell overproliferation induced by excess Hh signaling. This suppression was partially reproduced by controlling the level of Mam expression from a UAS-Mam transgene and was shown under those circumstances to suppress the duplication of FSCs normally induced by excessive Hh pathway activity. The initial genetic screen suggests that dose-dependent suppressors of Hh-induced FSC expansion are rare. Complete loss of mam was fully epistatic over ptc mutations with regard to FSC duplication and FSC maintenance (Vied, 2009).

Several mechanisms might theoretically account for the observed interactions of Mam with the Hh pathway. However, it was also seen that loss of Mam inhibited expression of the Hh pathway reporter ptc-lacZ in FSCs, focusing attention on the idea that Mam might act as a co-activator in the Hh pathway. Some further observations are relevant to this hypothesis (Vied, 2009).

First, no evidence was found of Mam affecting Hh signaling output in wing discs. Thus, any effect of Mam on FSC Hh signaling is tissue specific. Very little is known of the mechanisms underlying tissue-specific responses to Hh signaling, but tissue-specific interactions of Ci with other transcription factors and co-activators are likely conduits. Second, loss of Mam limited the induction of ptc-lacZ in ptc mutant FSC clones, but only to levels seen in normal FSCs. Thus, if Mam does indeed act in FSCs to potentiate Hh signaling, it could only be crucial for target genes induced by strong Hh pathway activity, for which ptc-lacZ is an insufficient marker. There is a precedent for exactly this situation in wing discs. There, loss of Fu kinase activity in ptc mutant clones completely eliminates the expression of Engrailed (which responds only to strong pathway activity) and substantially alters the resulting wing phenotype without reducing ptc-lacZ expression (Vied, 2009).

In summary, epistasis of mam over ptc and the specific requirement for Mam in FSCs, which experience higher Hh signaling than their progeny, are consistent with a role for Mam as a co-activator of crucial FSC target genes induced only by strong Hh pathway activity. However, it is also possible that Mam contributes to FSC function independently of the Hh pathway, affecting ptc-lacZ expression in FSCs only indirectly. Further investigation would benefit greatly from the identification of crucial FSC Hh target genes and detailed examination of the chromatin localization of Mam, Ci and other transcription factors in the FSC lineage (Vied, 2009).

Protein Interactions

During signaling by the Notch receptor, Notch's intracellular domain is cleaved, moves to the nucleus and associates with a DNA-binding protein of the CSL class (CSL for CBF1, Suppressor of Hairless [Su(H)], LAG-1); as a result, target genes are transcriptionally activated. In C. elegans, a glutamine-rich protein called LAG-3 forms a ternary complex with the Notch intracellular domain and LAG-1 and appears to serve as a transcriptional activator that is critical for signaling. Although database searches have failed to identify a LAG-3-related protein, it has been surmised that Notch signaling in other organisms might involve an analogous activity. To search for a LAG-3-like activity in mice, a modified yeast two-hybrid screen was used, similar to that used to identify LAG-3. Briefly, a complex bait was used to screen a library of mouse cDNAs fused to the Gal4 activation domain (Clontech). That bait included mouse CBF1 fused to the Gal4 DNA-binding domain (GD) as well as the intracellular domain of mouse Notch1. The bait proteins were co-expressed from a pBridge vector. Out of 6 million transformants, one positive with similarity to Drosophila Mastermind and human KIAA0200 was recovered. A focus was placed on this clone because Drosophila Mastermind is known to be critical for Notch signaling. The murine ortholog of Mastermind is called mMam1, and the human one hMam1. The mMam1 fragment recovered in the two-hybrid screen consists of 62 amino acids and included a conserved region present in both fly and human Mastermind proteins (Petcherski, 2000).

To explore the idea that Mastermind might have a role similar to LAG-3 in Notch signaling, a series of two-hybrid assays was conducted. mMam1 binds mCBF1-GD in the presence of either Notch1 or Notch3, but not in their absence. It was next asked whether Drosophila Mastermind might participate in a similar complex in flies. A fusion protein was used carrying the Gal4 activation domain and the amino-terminal 198 amino acids of fly Mastermind (dMam [1-198]; henceforth called dMam), which includes the conserved region of Mastermind that is critical for complex formation among mouse components. dMam was found to bind Su(H) strongly in the presence of the fly Notch intracellular domain, but not in its absence (Petcherski, 2000).

The interchangeability of proteins from different species was examined. Remarkably, the fly protein, dMam, interacts with murine Notch1 or Notch3 and murine CBF1, and mMam1 interacts with fly Notch and Su(H). In contrast, C. elegans LAG-3 does not form a complex with either murine or fly components, and mMam and dMam do not complex with worm components. It is concluded that both fly and murine Mastermind proteins form a ternary complex with either fly or murine receptors and CSL proteins. This interchangeability underscores the similarity between the fly and murine Notch pathways. Although murine Mastermind is not described, a full-length cDNA sequence for human Mastermind is available. Comparison of human and fly Mastermind sequences reveals only one short region of significant similarity that is limited to 60 amino acids at the amino terminus. Therefore, despite a low overall sequence similarity between mouse and Drosophila Mastermind proteins, the region crucial for complex formation is conserved (Petcherski, 2000).

The importance of Notch's ankyrin repeats for complex formation was examined. In C. elegans, formation of the ternary complex is dependent on the ankyrin repeats of the Notch-related receptor GLP-1. To ask whether the same situation holds for the murine complex, two missense mutants, M1 and M2, were used, each of which bears amino-acid substitutions in the fourth ankyrin repeat of mNotch1. Consistent with results in C. elegans, both M1 and M2 compromise interactions among Notch1, CBF1 and either mMam1 or dMam (Petcherski, 2000).

What is the role of Mastermind in Notch signaling? Previous studies have suggested a role in transcriptional control. In Drosophila, Mastermind is a nuclear protein and is bound to chromatin. Furthermore, in Drosophila, Mastermind acts downstream of Notch in signaling. The amino-acid sequences of both human and fly Mastermind proteins are rich in glutamine and proline, a common feature in transcriptional activators. In the work reported here, a physical link between Mastermind and the major CSL transcription factor of the Notch pathway is described. The interaction of both mMam and dMam with the Notch intracellular domain and CBF1 relies on the receptor's ankyrin repeats. These repeats are essential for Notch signaling and the transcriptional response. In C. elegans, point mutations in the ankyrin repeats severely compromise signaling by the Notch-related receptor GLP-1. In tissue culture cells, the M1 and M2 point mutations abolish receptor function and compromise the activation of transcription by Notch signaling. The simplest explanation for all these findings is that Mastermind functions as a transcriptional activator for Notch signaling (Petcherski, 2000).

Important parallels exist between LAG-3 in C. elegans and Mastermind in Drosophila and mammals. (1) All of these proteins form a ternary complex with an intracellular fragment of Notch and a CSL DNA-binding protein. (2) Mutations in the fourth ankyrin repeat of the receptor compromise ternary complex formation for C. elegans and mouse proteins, as is reported here. (3) All three proteins are rich in glutamine and proline: 27.6% in LAG-3, 29.4% in dMam and 22% in hMam1. (4) LAG-3 and Mastermind function downstream of Notch in C. elegans and Drosophila, respectively. It is proposed that LAG-3 and Mastermind perform analogous functions as activators for Notch (Petcherski, 2000).

What is the evolutionary relationship between LAG-3 and Mastermind? An intriguing idea is that LAG-3 and Mastermind share a common ancestor. The conservation in amino-acid sequence between Mastermind orthologs is much lower than is found for other components of the pathway: whereas hMam1 and dMam share similarity only in a stretch of 60 amino acids within a much larger protein, Notch and CSL proteins show high similarity (44.8% and 74.5% identity for hNotch1/dNotch and hCBF1/Su[H], respectively) over most of their length between these same species. It therefore seems plausible that the absence of similarity between LAG-3 and Mastermind may reflect a high rate of amino-acid substitution in these proteins rather than a distinct evolutionary origin (Petcherski, 2000).

The mastermind locus encodes a nuclear protein required in the Notch signaling pathway. In a screen for genes affecting wing pattern, an EP element was identified that directs expression of an alternatively spliced form of the mastermind transcript that has been called mam[DN]. Unlike the conventional mam transcript, mam[DN] is spatially regulated in the developing embryonic nervous system and eye imaginal disc. mam[DN] corresponds to an endogenous transcript and encodes an alternate form of the Mam protein that dominantly interferes with activity of the conventional Mam protein. Mam[DN] blocks Notch signaling downstream from the activated form of Notch but cannot interfere with an activated form of Su(H), suggesting that Mam[DN] may interfere with the activity of a ternary complex involving Mam, Notch and Su(H) (Giraldez, 2002).

The mam[DN] transcript shares one exon with the conventional mastermind transcript. mam[DN] has two short exons before the shared exon and one short exon after it. The predicted open reading frame of mam[DN] begins in its second exon, which encodes only four amino acids, continues through the common exon in the same reading frame as Mam and ends after an additional four amino acids encoded by its fourth exon. Thus, the alternate transcript is predicted to produce a short form of the Mam protein that differs at both the N- and C- termini, but which is identical to the conventional Mam protein through the shared exon. The alternate form of Mam protein encoded by GH07841 would lack the conserved N-terminal domain that is needed for interaction with the Ankyrin repeats of Notch and Su(H) (Giraldez, 2002).

The conventional form of Mam is ubiquitously expressed in the embryo and in the imaginal discs. To analyse the pattern of expression of mam[DN] an exon-specific RNA probe was used. mam[DN] is first detected by in situ hybridization in two pairs of cells adjacent to the midline in each segment at stage 10-11. These cells are the first to be labelled with Mab22C10, suggesting that they are vMP2 and dMP2. Later, expression of mam[DN] concentrates in vMP2. Between stages 13 and 15 mam[DN] accumulates in the CNS and in the salivary glands. During larval stages, mam[DN] is not detectably expressed in the leg or wing imaginal discs. mam[DN] is expressed in developing photoreceptors behind the morphogenetic furrow in the eye disc. These observations suggest that spatial regulation of mam[DN] expression could be used to modulate Notch activity levels (Giraldez, 2002).

A DNA transcription code for cell-specific gene activation by notch signaling

Cell-specific gene regulation is often controlled by specific combinations of DNA binding sites in target enhancers or promoters. A key question is whether these sites are randomly arranged or if there is an organizational pattern or 'architecture' within such regulatory modules. During Notch signaling in Drosophila proneural clusters, cell-specific activation of certain Notch target genes is known to require transcriptional synergy between the Notch intracellular domain (NICD) complexed with CSL proteins bound to 'S' DNA sites and proneural bHLH activator proteins bound to nearby 'A' DNA sites. Previous studies have implied that arbitrary combinations of S and A DNA binding sites (an 'S+A' transcription code) can mediate the Notch-proneural transcriptional synergy. By contrast, this study shows that the Notch-proneural transcriptional synergy critically requires a particular DNA site architecture ('SPS'), which consists of a pair of specifically-oriented S binding sites. Native and synthetic promoter analysis shows that the SPS architecture in combination with proneural A sites creates a minimal DNA regulatory code, 'SPS+A', that is both sufficient and critical for mediating the Notch-proneural synergy. Transgenic Drosophila analysis confirms the SPS orientation requirement during Notch signaling in proneural clusters. Evidence that CSL interacts directly with the proneural Daughterless protein, thus providing a molecular mechanism for this synergy. It is concluded that the SPS architecture functions to mediate or enable the Notch-proneural transcriptional synergy which drives Notch target gene activation in specific cells. Thus, SPS+A is an architectural DNA transcription code that programs a cell-specific pattern of gene expression (Cave, 2005).

The functional significance of the SPS element has not been determined, but initially, it was proposed that the arrangement of the S binding sites in the SPS may function to mediate cooperative DNA binding by CSL proteins, or it may be necessary for the recruitment of other proteins to the promoter. Subsequent studies, though, showed that CSL, NICD, and Mam "ternary complexes" can assemble on single S sites. To date, no studies have experimentally addressed whether there are significant functional differences between SPS elements and single S or other non-SPS binding site configurations, and the mechanistic function of the SPS element is not known (Cave, 2005).

In Drosophila, five of the seven bHLH repressor genes in the E(spl)-Complex contain an SPS element in their promoter regions, and four of these bHLH R genes contain both SPS and proneural bHLH A protein binding (A) sites. These four bHLH R genes (the m7, m8, , and genes, collectively referred to as the 'SPS+A bHLH R' genes have been shown genetically to depend upon proneural bHLH A genes for expression. In addition, transcription assays in Drosophila cells with at least two of these four genes (m8 and ) have shown that there is strong transcriptional synergy when NICD and proneural proteins are expressed in combination. These SPS+A bHLH R genes also have similar patterns of cell-specific expression within proneural clusters. Following determination of the neural precursor cell from within a proneural cluster of cells, Notch-mediated lateral inhibition is initiated and these SPS+A bHLH R genes are specifically upregulated in all of the nonprecursor cells but not in the precursor cell. The absence of NICD, and the presence of specific repressor proteins such as Senseless, prevent upregulation of SPS+A bHLH R genes in the precursor cells (Cave, 2005).

This study shows that there are important functional differences between the SPS architecture and non-SPS configurations of S binding sites. The SPS architecture is critical for synergistic activation of the m8 SPS+A bHLH R gene by Notch pathway and proneural proteins. Whereas previous studies have focused on which regulatory genes and proteins function combinatorially to activate SPS+A bHLH R gene expression, this study focuses on the underlying DNA transcription code that programs the Notch-proneural transcriptional synergy that drives cell-specific gene transcription. The results of previous studies have implied that an apparently arbitrary combination of S and A binding sites (S+A transcription code) is sufficient for transcriptional activation of SPS+A bHLH R genes. By contrast, this study shows that a minimal transcription code, SPS+A, is sufficient and critical for mediating Notch-proneural synergistic activation of these genes. The SPS+A code is composed of the specific SPS binding site architecture in combination with proneural A binding sites. Furthermore, evidence is presented that direct physical interactions between the Drosophila Su(H) and Daughterless protein mediate the transcriptional synergy, thus providing a molecular mechanism for the Notch-proneural synergy. Together, these studies show that the SPS architecture functions to mediate or enable the transcriptional synergy between Notch pathway and proneural proteins and that SPS+A is an architectural transcription code sufficient for cell-specific target gene activation during Notch signaling (Cave, 2005).

To test whether the SPS binding site architecture is important for Notch-proneural synergy, the ability of Drosophila NICD (dNICD) and proneural bHLH A proteins, such as Achaete and Daughterless (Ac/Da) to synergistically activate the wild-type native m8 promoter and SPS architecture variants was examined. Whereas the native m8 promoter carries the wild-type SPS architecture of S binding sites, the m8 promoter variants contain either a disrupted S site, leaving a single functional S site (SF-X or X-SR), or orientation variants in which the orientation of one or both S sites have been reversed (SR-SF, SF- SF, and SR-SR) (Cave, 2005).

The native m8 promoter is synergistically activated in transcription assays by coexpression of dNICD and Ac/Da, but it is only weakly activated by expression of dNICD or proneural Ac/Da proteins alone. However, neither promoter with a single S binding site (SF-X or X-SR) can mediate synergistic interactions between dNICD and proneural proteins. In fact, both single S site promoters are only weakly activated when proneural and dNICD proteins are expressed individually or together. Thus, single S sites are not sufficient to mediate Notch-proneural synergy in these contexts, even though they are in the same position as the SPS in the wild-type m8 promoter (Cave, 2005).

When the number of S binding sites are maintained, but the orientation of these sites within the SPS is varied (SR-SF, SF-SF, and SR-SR), only the wild-type (SF-SR) SPS orientation is synergistically activated by coexpression of dNICD and proneural Ac/Da proteins. Thus, the wild-type SPS architecture of S binding sites is clearly necessary for the m8 promoter to mediate transcriptional synergy between NICD and the proneural protein complexes assembled on the SPS and A sites, respectively (Cave, 2005).

The transcriptional synergy between NICD and proneural proteins mediated by the SPS element is crucial for the coactivation by the Mastermind (Mam) protein. Coexpression of Mam with both dNICD and proneural proteins provides a strong coactivation of transcription of the wild-type m8 promoter. However, this strong coactivation is not observed with any of the non-wild-type m8 SPS variants, which also cannot mediate Notch-proneural synergy. Thus, coactivation by both the NICD and Mam cofactors is strongly dependent on synergistic interactions with proneural combinatorial cofactors, and the specific SPS architecture is critical for mediating this synergy (Cave, 2005).

The native m8 promoter studies tested whether the organization of the S binding sites in the SPS are necessary to mediate the Notch-proneural synergy. In order to test which of these architectural features are sufficient to mediate that synergy, a set of synthetic promoters was created carrying the same SPS variants mentioned above in combination with A sites (SPS-4A reporter). These synthetic promoters thus contain the sites predicted to mediate the synergy but lack the other sites present in the native m8 promoter, which might also be necessary. This reductionist approach allows for the identification of a minimal promoter that contains only those sites that are necessary and sufficient to mediate the Notch-proneural synergy. All of these synthetic reporters are modestly activated by expression of proneural proteins alone, but expression of dNICD alone gives no activation. By contrast, only the SPS-4A reporter containing the wild-type SPS (SF-SR) mediates clear synergistic activation when dNICD and proneural proteins are coexpressed, and none of the SPS variants do so (Cave, 2005).

Given that functional CSL/NICD/Mam ternary complexes have been shown to assemble on single S sites and activate transcription, it was expected that promoters with single S sites could be activated at low levels by expression of dNICD in the absence of the proneural proteins and that promoters with two S sites might have more activity than single S sites. However, it was surprising to observe that all of the m8 and synthetic promoters, even with the wild-type SPS element, have very low or no activity when dNICD is expressed alone. Thus, the SPS binding site architecture does not appear to facilitate recruitment of functional NICD coactivator. This argues against previous proposals that suggested that the SPS architecture might function to recruit other proteins to the promoter. Thus, given that the wild-type SPS architecture is necessary and sufficient for Notch-proneural synergy, these results indicate that the function of the SPS element is to enable synergistic interactions with proneural proteins (Cave, 2005).

The synthetic promoters do not carry bHLH R sites, which are present in all E(spl)-C gene promoters. Thus, these sites clearly are not necessary for Notch-proneural synergy, although they may modulate it in vivo. It has been proposed that other repressor proteins bind the and SPS+A bHLH R gene promoters to restrict their expression to a subset of proneural clusters. Although these hypothetical repressor binding sites may be necessary to program the full and gene expression pattern, the current results indicate that they are not necessary for the Notch-proneural synergy that drives nonprecursor cell-specific upregulation (Cave, 2005).

Both the m8 and SPS-4A synthetic reporter contain a hexamer sequence that has been coconserved with the SPS element. Elimination of that hexamer site in a synthetic promoter does not disrupt Notch-proneural, suggesting that Notch-proneural synergy in vivo is not dependent on the hexamer site (Cave, 2005).

Together, the synthetic and m8 promoter results indicate that SPS+A is a minimal transcription code that is both necessary and sufficient for Notch-proneural synergy in Drosophila. The results with the promoters that were tested show that Notch-proneural transcriptional synergy requires the specific organization or architecture of the SPS element, in addition to its combination with proneural A binding sites. All of the promoters with SPS variants failed to mediate this synergy. This clearly indicates that arbitrary combinations of S and A binding sites are not sufficient to mediate Notch-proneural synergy (Cave, 2005).

An important question is whether there are other DNA binding transcription factors that can combinatorially synergize with CSL/NICD transcription complexes. Previous studies have shown that Notch pathway factors can synergize with a nonproneural transcription factor, Grainyhead, suggesting that synergy with the CSL/NICD transcription complexes could be very general or nonspecific. To test whether a general coactivator, the VP16 transcription activation domain, can synergistically interact with dNICD, an essentially identical wild-type SPS-containing synthetic promoter was created in which the A sites were replaced by UAS binding sites for the yeast Gal4 transcription factor (SPS-5U). Expression of a fusion protein containing the Gal4 DNA binding domain and the constitutively active VP16 activation domain can activate the synthetic SPS-5U promoter. However, the Gal4-VP16 fusion protein does not synergize with NICD. Thus, CSL/NICD complexes do not synergize with every nearby DNA bound transcription factor, and there is at least some specificity to the synergy with bHLH A proteins. This interaction specificity could contribute significantly to selective activation of Notch target genes. Further studies will be required to determine whether other DNA binding transcription factors can combinatorially synergize with Notch signaling and whether such factors fall into distinct classes (Cave, 2005).

Given that Notch signaling and neural bHLH A proteins have been conserved between Drosophila and mammals, it was next asked whether the transcriptional synergy between these proteins is also conserved in mammalian cells. Using the same set of synthetic promoters as mentioned above, activation following expression of the mammalian NICD and neural bHLH A protein homologs (Notch-1 ICD [mNICD] and MASH1/E47, respectively) was tested in murine NIH 3T3 cells. As in the Drosophila system, expression of MASH1/E47 proteins alone produces modest activation of the wild-type (SF-SR) SPS-4A promoter, and mNICD alone does not produce any significant activation of the promoter. However, clear transcriptional synergy is observed with the wild-type SPS promoter when both mNICD and neural bHLH A proteins are coexpressed. Moreover, SPS-mediated synergy requires nearly the same organizational features of S binding sites as observed in Drosophila. Neither of the single S site promoters can mediate that synergy, nor can most of the orientation variants. Although the SR-SR promoter is activated following coexpression of both the mNICD and bHLH A proteins, it is not activated by mNICD alone (Cave, 2005).

These results indicate that the potential for transcriptional synergy between NICD and neural bHLH A proteins has been conserved in a mammalian cell system and that the SPS+A code is sufficient and critical for mediating that transcriptional synergy. This raises the possibility that there may be mammalian genes that are regulated by neural bHLH A proteins and Notch signaling via this code. Although there is an SPS element conserved in the HES-1 promoter, HES-1 does not have an A site in its proximal promoter region, and HES-1 is not activated by expression of bHLH A genes. Thus, HES-1 appears to be similar to the Drosophila E(spl)-C m3 bHLH R gene, which also has an SPS but no obvious nearby A site. Whole-genome searches are being performed for genes in mammalian systems that may be regulated by the SPS+A code (Cave, 2005).

It has been proposed that the architecture of the SPS element may mediate cooperative binding of a second CSL protein once an initial CSL protein binds the DNA. Using electromobility gel shift assays to test for cooperative binding, the ability was compared of bacterially expressed and partially purified Drosophila Su(H) protein to bind DNA probes containing either the wild-type m8 SPS or an m8 SPS with one S site mutated. If there is cooperativity, one would expect to observe the band corresponding to two DNA bound CSL proteins to be as strong or stronger than the band corresponding to a single CSL protein bound to DNA. The single S site probe serves as a control because it cannot be cooperatively bound by two Su(H) proteins, and it also serves to identify the band corresponding to a single Su(H) protein bound to the wild-type SPS probe. Similar amounts of Su(H) protein bind strongly to the wild-type probe and to the single-site probe. In particular, because single protein binding to the wild-type DNA probe did not facilitate or stabilize simultaneous binding of two S proteins, Su(H) does not appear to bind cooperatively to the two S sites in the wild-type probe. These results suggest that CSL proteins do not bind cooperatively to the SPS in vivo, although posttranslational modifications in vivo could affect these binding properties Cave, 2005).

In addition, the protein binding affinity for the SF-SR and SR-SF probes appears to be comparable, although the reversed orientation of the two S sites would have likely disrupted cooperative binding if it were present. This result strongly suggests that the complete lack of activation by SR-SF sites in all of the promoters tested is not due simply to decreased ability of Su(H) protein to bind to the SR-SF orientation variant Cave, 2005).

To test the in vivo relevance of the conserved S binding site orientation in SPS elements, transgenic flies were created carrying β-galactosidase reporter genes driven by native m8 promoters containing either the wild-type (SF-SR) or SR-SF variant SPS elements. Wing and eye imaginal discs containing m8 promoters with the wild-type SPS element produced strong expression in proneural cluster regions, similar to the pattern described for endogenous m8. By contrast, comparably stained wing and eye discs carrying the m8 promoter reporters with the SR-SF SPS variant showed no expression or very low levels of expression, respectively. Extended staining of discs containing the SR-SF element revealed clear but weak expression in a pattern of single cells that resembles the distribution of neural precursors in the wing discs and eye discs. This is likely due to activation via the A site by proneural proteins because proneural levels are highest in the precursor cells. However, there was no expression in the surrounding nonprecursor cells within the proneural clusters even though Notch signaling is activated in these cells. Similar neural precursor-specific m8 reporter expression patterns have been observed when the S binding sites are eliminated, indicating that reversal of the S binding site orientations is functionally equivalent to eliminating them for this aspect of Notch target gene expression. These in vivo results confirm that the conserved orientation of the S binding sites in the wild-type SPS element is essential for nonprecursor cell specific upregulation of the SPS+A bHLH R m8 genes in response to Notch signaling in proneural clusters (Cave, 2005).

To gain an insight into the molecular mechanism underlying the strong transcriptional synergy between Notch signaling and bHLH A proteins on the m8 and SPS-4A promoters, whether this synergy involves a direct physical interaction was tested by using yeast two-hybrid assays with the Drosophila proteins. These experiments revealed that the Daughterless N-terminal domain directly and specifically interacts with the Su(H) protein in the absence of the bHLH domain and C terminus (Cave, 2005).

Using transcription assays in Drosophila cells, whether the Da N terminus (DaN construct), which contains a transcription activation domain, can synergistically activate the m8 promoter was tested in the absence of both its bHLH DNA binding domain and a heterodimerization partner, like Ac. The Da N-terminal protein synergistically activates the m8 promoter when dNICD is coexpressed, apparently by direct binding of the DaN protein to endogenous CSL bound to the SPS element. These results indicate that the Notch-proneural transcriptional synergy is not mediated by cooperative DNA binding interactions between the Su(H) and proneural proteins, although such cooperative binding may mediate transcriptional synergy between some combinatorial cofactors. These results suggest that a direct interaction between Su(H) and the Da N-terminal fragment, which can occur independent of NICD, facilitates the formation of an active transcription complex when NICD is also present during Notch signaling (Cave, 2005).

These results suggest that the SPS architecture functions to enable a direct physical interaction between Su(H) and Da proteins, thus providing a molecular mechanism for the observed Notch-proneural synergy that is mediated by the SPS element. This interaction could stabilize the recruitment or functional activity of NICD, which then recruits Mam, and could explain the strong dependence of both NICD and Mam coactivation functions on the presence of proneural proteins (Cave, 2005).

In previous studies, it has been proposed that neither the synergistic activation nor the transcriptional repression mediated by CSL protein complexes imply direct interactions between CSL and DNA bound combinatorial cofactors; rather, it is likely that CSL proteins exert their effects through the recruitment of non-DNA binding cofactors, such as chromatin modifying enzymes. While this might be the case for some Notch target gene promoters, in the case of m8, the results indicate that the mechanism underlying the synergistic interactions between CSL/NICD and bHLH A proteins does involve direct physical interactions (Cave, 2005).

A mechanistic model is proposed for programming Notch-proneural synergy with the SPS+A transcription code. These studies demonstrate that there are important functional differences between SPS and non-SPS organizations of S binding sites. The critical role of the SPS binding site architecture is not predicted or explained by the previous models for Notch target gene transcription. Previous models suggest that transcription is promoted by the binding of NICD to CSL, which displaces CSL bound corepressors, thus allowing transcriptional synergy with other DNA bound combinatorial cofactors. These models have not distinguished between Notch target genes with regulatory modules that contain SPS or non-SPS configurations of S binding sites, nor do they explain or predict the critical function of the SPS binding site architecture in mediating Notch-proneural transcriptional synergy (Cave, 2005).

A revised model is proposed that incorporates the essential requirement for the specific SPS binding site architecture in combination with the proneural A binding sites for transcriptional activation of m8 and the other SPS+A bHLH R genes. These genes each contain an SPS+A module and exhibit similar cell-specific upregulation in nonprecursor cells in proneural clusters. In this new model, the specific architecture of the S sites in the SPS element directs the oriented binding of Su(H) so that it is in the proper orientation and/or conformation to enable a direct interaction with Da. This interaction is an essential prerequisite for subsequent recruitment and/or functional coactivation by NICD during Notch signaling. This Notch-proneural complex is then further activated by subsequent recruitment of Mam (Cave, 2005).

It is interesting to note that the mammalian homologs of each of the Su(H), NICD, and Da proteins have been shown to interact with the p300 coactivator; thus, when complexed together, these proteins could potentially function combinatorially to recruit p300 or a related coactivator (Cave, 2005).

In Drosophila and mammals, Notch signaling is used throughout development to activate many different target genes, and in multiple developmental pathways. Thus, it is of paramount importance that the proper target genes are selectively activated in the proper cell-specific patterns. It is known that Notch signaling can activate genes through non-SPS configurations of S sites in certain other target genes. For example, expression of the Drosophila genes single minded, Su(H), and vestigal have all been shown to be regulated by Notch signaling, and all have single S sites or multiple unpaired S sites but no SPS elements in their promoter and/or enhancer regions (Cave, 2005).

The results show that for essentially every promoter tested, NICD cannot activate in the absence of neural bHLH A combinatorial cofactors, suggesting that NICD may always require a combinatorial cofactor to activate target genes. If so, the non-SPS Notch target genes are likely also to have specific combinatorial cofactors. The results also clearly show that the Notch-proneural combinatorial synergy requires a specific configuration of S sites, the SPS. There may be other specific configurations of S binding sites that mediate synergy for different classes of combinatorial cofactors for Notch signaling (Cave, 2005).

Together, these observations suggest that specific, but unknown, non-SPS configurations of sites may program the interactions between Notch complexes and the proper combinatorial cofactors. It is speculated that these non-SPS configurations might be unique to each target gene, or it is possible that there are specific patterns or classes of S binding site configurations -- an 'S binding site subcode' -- that determine cofactor specificity. Thus, the results suggest that selective Notch target gene activation may be programmed by distinct Notch transcription codes in which specific configurations of S binding sites mediate selective interactions with specific combinatorial cofactors (Cave, 2005).

Elucidating the various transcription codes controlling target gene activation during Notch signaling will be an important goal for future studies. The results have clearly shown that the architecture of transcription factor binding sites can be crucial for control of cell-specific Notch target gene activation. The studies presented here give a glimpse into the molecular mechanisms by which a one dimensional pattern of DNA binding sites can program cell-specific patterns of gene expression (Cave, 2005).

Nipped-A, the Tra1/TRRAP subunit of the Drosophila SAGA and Tip60 complexes, has multiple roles in Notch signaling during wing development

The Notch receptor controls development by activating transcription of specific target genes in response to extracellular signals. The factors that control assembly of the Notch activator complex on target genes and its ability to activate transcription are not fully known. This study shows, through genetic and molecular analysis, that the Drosophila Nipped-A protein is required for activity of Notch and its coactivator protein, Mastermind, during wing development. Nipped-A and Mastermind also colocalize extensively on salivary gland polytene chromosomes, and reducing Nipped-A activity decreases mastermind binding. Nipped-A is the fly homologue of the yeast Tra1 and human TRRAP proteins and is a key component of both the SAGA and Tip60 (NuA4) chromatin-modifying complexes (see Tip60). Like Nipped-A, the Ada2b component of SAGA and the Domino subunit of Tip60 are also required for Mastermind function during wing development. Based on these results, it is proposed that Nipped-A, through the action of the SAGA and Tip60 complexes, facilitates assembly of the Notch activator complex and target gene transcription (Gause, 2006).

Nipped-A mutations were isolated in a genetic screen for factors that regulate activation of cut by the wing margin enhancer, and it was found that they reduce Notch activity both at the wing margin and in the developing wing veins. Heterozygous Nipped-A mutations increase the severity of the mutant wing margin and blade reduction phenotype caused by the weak loss-of-function Notch (Nnd-1) mutation and decrease the severity of the vein-shortening phenotype caused by a gain-of-function Notch mutation (NAx-E2)(Gause, 2006).

Other genetic data also indicate that Nipped-A is important for Notch signaling. Mastermind is a coactivator protein required for transcriptional activation by Notch, and heterozygous Nipped-A mutations dramatically increase the weak wing-nicking phenotype caused by heterozygous mastermind mutations. The vestigial gene is directly activated by Notch, and flies heterozygous for both Nipped-A and vestigial mutations display wing margin defects. The Notch intracellular fragment binds to the Suppressor of Hairless [Su(H)] protein on target genes, and a Nipped-A Su(H) double mutant displays a dominant wing-nicking phenotype. Together, the effects that the Nipped-A dosage has on the mutant phenotypes displayed by Notch, mastermind, and vestigial mutants indicate that Nipped-A encodes a factor critical for Notch activity in the developing wing (Gause, 2006).

Two Nipped-A mutants have point mutations in the gene encoding the Drosophila homologue of the yeast Tra1 and mammalian TRRAP proteins. Tra1/TRRAP is a key component of the SAGA and Tip60 (NuA4) chromatin-remodeling complexes in yeast, flies, and humans (Gause, 2006).

Tra1/TRRAP is a direct target of transcriptional activators and helps them recruit the SAGA and Tip60 chromatin modification complexes to aid in gene activation. Mammalian Tra1/TRRAP was first identified as a coactivator that interacts directly with the Myc and E2F activators. Tra1/TRRAP is also a target of several other activators in yeast and mammalian cells, including Gal4, E1A, VP16, nuclear receptors, and p53. Tra1/TRRAP contains an ATM-phosphatidylinositol-3 (PI-3) kinase-like domain near the C terminus that is important for recruitment of histone acetyltransferase (HAT) activity in mammalian cells. The C terminus is also critical for interaction of yeast Tra1 with acidic activators (Gause, 2006 and references therein).

There is evidence that SAGA, which contains Tra1/TRRAP and the Gcn5/PCAF HAT, may be involved in transcriptional activation by the Notch complex. Several components of the Notch activator complex are known and functionally identical in worms, flies, and mammals. Upon binding of ligands such as Serrate or Delta to the extracellular EGF repeats of Notch, an intracellular fragment of Notch (NICD) is proteolytically released, allowing it to enter the nucleus, where it interacts with a DNA-bound CSL [CBF1/Su(H)/Lag-1] protein. NICD helps recruit the Mastermind coactivator. An N-terminal region of Mastermind interacts with both the CSL protein and an ankyrin repeat domain of NICD. The p300/CBP (CREB-binding protein) HAT coactivator is recruited by interactions with both the NICD ankyrin repeats and a specific region in the N-terminal half of Mastermind. The Gcn5/PCAF HAT is also recruited by the Notch activator complex in cultured mouse cells; this requires the ankyrin repeat region of NICD. The NICD ankyrin repeats bind other proteins, such as Mastermind and CBP, and thus it is possible that these proteins are also required to recruit Gcn5/PCAF. Because Tra1/TRRAP is the SAGA subunit targeted by several transcriptional activators, it is a distinct possibility that it is required for recruitment of Gcn5/PCAF by the Notch activator complex (Gause, 2006).

This study presents a molecular genetic analysis of several Nipped-A mutations that provides new insights into the roles of the Tra1/TRRAP protein and its complexes in Notch signaling. Reducing the Nipped-A gene dosage by half reduces both Mastermind and Notch activities during wing development and, surprisingly, certain mutant alleles can replace one copy of wild-type Nipped-A. These data also show that other subunits of the SAGA and Tip60 complexes that contain Nipped-A are required for Mastermind and Notch function in wing development and that Nipped-A is required for binding of Mastermind to chromosomes. Taken together, the results indicate that Nipped-A plays multiple roles in Notch signaling (Gause, 2006).

The evidence provided here, combined with the finding that two Nipped-A mutants have point mutations in the Tra1/TRRAP gene, demonstrates conclusively that Nipped-A encodes Tra1/TRRAP. All EMS-induced Nipped-A alleles sequenced to date have point mutations in the Tra1/TRRAP gene that affect the protein coding sequence or, in one case, the 3' UTR. A seventh allele generated by gamma rays, Nipped-A323, does not produce Tra1/TRRAP mRNA. Additional Nipped-A mutant alleles have been sequenced, and all contain point mutations that alter the protein coding sequence (Gause, 2006).

The results show that the major Nipped-A transcript differs from a previously reported splicing pattern, which appears to be a rare variant. Antibodies against a polypeptide encoded largely by the rare exons detect a weak Tra1/TRRAP signal in Western blot assays of concentrated nuclear extracts or purified complexes, confirming that the variant produces Tra1/TRRAP protein in vivo. The rare transcript does not, however, support at least one essential function of Nipped-A and Notch signaling in the wing margin because mutation of a splice site in Nipped-ANC106 for an exon that is not included in the rare variant is lethal and causes defects in Notch signaling. Nipped-ANC106, however, had little effect on the NAx-E2 wing vein phenotype, raising the possibility that the alternatively spliced product can support Notch function in developing wing veins (Gause, 2006).

An unexpected finding is that the Nipped-ANC105 allele, which encodes the N-terminal 2,048 residues of Tra1/TRRAP, suffices to replace one wild-type copy of Nipped-A to support Notch and Mastermind function in vivo. This was unexpected because the protein encoded by Nipped-ANC105 lacks the ATM-PI3 kinase motif which, in mammalian cell culture experiments, is required for Tra1/TRRAP to associate with Gcn5 and Tip60. One possible explanation is that the C terminus of the Nipped-A protein is not required for Notch and Mastermind function and that the truncated protein can replace the full-length protein. Because the effects of the Nipped-A mutations on Notch functions in wing development could only be studied in the presence of a wild-type allele, it is also possible that a truncated protein somehow increases the activity of the remaining full-length Nipped-A protein. The truncated protein could not be detected in Western blot assays of extracts or by immunostaining, suggesting that if this is the case, only a small amount of the mutant protein is sufficient. It is considered improbable that linked second-site mutations are masking effects of Nipped-ANC105 on both Notch mutant phenotypes and the mastermind phenotype. Many mutations have effects similar to Nipped-A, and few have opposing effects, and it would likely require multiple mutations to counteract the effects of Nipped-ANC105 on all three phenotypes. It is also unlikely that there is a linked second-site mutation that counteracts the effects of Nipped-ANC105 by increasing the expression of wild-type Nipped-A, because mutant embryos and larvae show the expected decrease in full-length Nipped-A protein (Gause, 2006).

The Nipped-ANC194 allele, which encodes residues 1 to 1500, had a significant effect on both of the Notch mutant phenotypes but did not increase the severity of the wing-nicking phenotype displayed by mamg2. Again, this differs from null alleles of Nipped-A, which affect all three phenotypes, suggesting that Nipped-ANC194 retains sufficient activity to replace one copy of the wild type in support of Mastermind activity. Again, one possible explanation is that Nipped-A residues 1 to 1500 are sufficient to support Mastermind function, although it is conceivable that the truncated protein somehow increases the activity of the remaining wild-type Nipped-A protein. It was not possible to detect this truncated protein, suggesting that if a truncated protein is responsible, only low levels are required. Despite extensive screens with a deficiency collection and candidate genes, no mutations that suppress mastermind mutant phenotypes have been mapped to chromosome 2. Thus, it is unlikely that a linked second-site mutation masks an effect of Nipped-ANC194 on the mastermind phenotype. Similar to Nipped-ANC105, heterozygous Nipped-ANC194 mutants display the expected reduced levels of full-length protein, although the possibility cannot be excluded of a subtle increase in the expression of the wild-type Nipped-A allele that is sufficient to rescue the mastermind phenotype but not the Notch mutant phenotypes (Gause, 2006).

Isolation and analysis of additional Nipped-A truncation alleles and development of more sensitive biochemical assays will lead to a fuller understanding of how Nipped-A alleles encoding truncated proteins support Notch signaling (Gause, 2006).

The experiments presented in this study indicate that the roles of Nipped-A in supporting Mastermind function likely involve both the SAGA and Tip60 complexes. The Ada2b protein is specific to SAGA, and Ada2b mutations affect the mastermind phenotype but not the two Notch mutant phenotypes. It is thought unlikely that the effect of the Ada2b mutations is more specific than Nipped-A mutations because the mastermind phenotype is more sensitive. As shown by the Nipped-ANC96 hypomorph, the Nnd-1 phenotype is more sensitive to the Nipped-A dosage than is mastermind. Moreover, the Nipped-ANC194 allele has a specificity opposite that of the Ada2b mutations and affects the Notch mutant phenotypes but not the mastermind phenotype. Combined, the contrasts in the effects of Ada2b and various Nipped-A mutations show that Nipped-A and its complexes play multiple roles in Notch signaling. They suggest that the SAGA complex, or at least the Ada2b subunit, is more specific for Mastermind function and that Nipped-A has additional functions (Gause, 2006).

Another possibility raised by the specificity of the effects of Ada2b mutations for effects on Mastermind activity in wing margin development is that Mastermind may have functions in margin development independent of Notch. For example, Mastermind could conceivably function as a coactivator for other activator proteins in addition to Notch. This possibility is consistent with the binding of Mastermind to several sites in polytene chromosomes, including the ecdysone-dependent puffs (Gause, 2006).

The Domino protein, a putative ATPase remodeling enzyme, is a subunit of the Tip60 complex. The Nnd-1 and NAx-E2 phenotypes and the Mastermind phenotype are modified by domino mutations, although the effect on NAx-E2 is modest. These effects are similar to those of the Nipped-ANC106 allele and thus suggest that the Tip60 complex also supports Mastermind function and Notch signaling during wing development. It is possible, however, that Domino functions independently of Tip60 and Nipped-A because the human Domino homologue SRCAP interacts directly with the CBP HAT enzyme that interacts with Mastermind. Nevertheless, the likely involvement of the Tip60 complex raises the possibility that histone exchange could facilitate transcriptional activation by Notch because, in addition to acetylating histone H4, Tip60 exchanges histone H2 variants during DNA repair (Gause, 2006).

As revealed by immunostaining of salivary gland polytene chromosomes, at least one function of Nipped-A is to regulate the binding of Mastermind to chromosomes. The reduction in binding of Mastermind to polytene chromosomes caused by the hypomorphic Nipped-ANC96 and Nipped-ANC186 alleles is dramatic. Supporting the idea that Nipped-A directly regulates Mastermind binding, virtually all sites on polytene chromosomes that bind Mastermind also bind Nipped-A. A few possible explanations for these results are envisioned. The SAGA and Tip60 complexes that contain Nipped-A could acetylate Mastermind, proteins in the Notch activator complex, and/or possibly histones to facilitate binding of the Notch activator complex to chromatin. These modifications could be made by Gcn5 and/or Tip60, which acetylate histones H3 and H4, respectively. Alternatively, Nipped-A or its complexes could bind to chromosomes cooperatively with Mastermind. This would be consistent with the published observation that the ankyrin repeats of the NICD fragment of Notch, which help recruit Mastermind to the Notch activator complex, are also required to recruit Gcn5/PCAF SAGA subunit in transfected mouse cells. Both the Ada2b component of SAGA and the Domino subunit of Tip60 affect Mastermind function, so it is likely that Nipped-A supports Mastermind function in more than one way (Gause, 2006).

Because the evidence suggests that Nipped-A supports Mastermind function through both the SAGA and Tip60 chromatin-modifying complexes, it is theorized that, in addition to controlling the binding of Mastermind to chromosomes, Nipped-A could also cooperate with Mastermind to recruit these complexes to facilitate transcriptional activation through chromatin modification (Gause, 2006).

The data indicate that the SAGA complex, or at least its Ada2b subunit, is not required for some functions of Nipped-A in Notch signaling. Unlike Nipped-A and domino mutations, Ada2b mutations did not affect Notch mutant phenotypes, while they did enhance the phenotype caused by a mastermind mutation. It is postulated, therefore, that the Tip60 complex is also required for functions of Nipped-A beyond controlling the binding of Mastermind to chromosomes. The Tip60 complex could affect the expression of Notch activator complex components, or it could modify proteins in the Notch activator complex. It is also possible that Tip60 modifies chromatin to either aid binding of the Su(H) protein to the Notch target genes or, as mentioned above, to aid transcriptional activation by the Notch activator complex. In any case, the evidence indicates that two subunits of Tip60, Nipped-A and Domino, play more than one role in Notch signaling during wing development (Gause, 2006).

Structural and functional analysis of the repressor complex in the Notch signaling pathway of Drosophila melanogaster

CSL is the nuclear effector of the Notch signaling pathway and is required for both repression and activation of transcription from Notch target genes. In the absence of a signal, CSL functions as a transcriptional repressor by interacting with corepressor proteins, such as SHARP, SMRT/NCoR, KyoT2, and CIR. CSL-corepressor interactions function to localize histone deacetylase and histone demethylase activity at Notch target genes, which converts the local chromatin into a condensed, transcriptionally silent state. On pathway activation, the ICN binds CSL, and together with Mam, forms a transcriptionally active ternary complex that ultimately displaces corepressors from CSL and upregulates transcription from Notch target genes. In mammals, a number of corepressors have been shown to interact with the BTD of CSL, similar to ICN, which provides a model in which ICN displaces or outcompetes corepressors for binding to CSL. Thus, there are potentially two modes of repression mediated by corepressors: 1) at the transcription or chromatin level, in which the recruitment of HDAC/HDM-containing complexes by corepressors silences gene expression -- this mode of transcriptional repression is independent of Notch; and 2) at the protein level, by which corepressors and ICN compete for binding to CSL (Maier, 2011).

Although several of the mammalian corepressors have fly orthologues, these molecules do not seem to be generally involved in repressing transcription from Notch target genes in flies. A complex involving the SMRT homologue SMRTER negatively regulates Notch signaling during the specification of a subset of nonneuronal cell types in the developing Drosophila retina. However, mammalian SMRT is believed to contact CBF1 directly, whereas SMRTER does not bind Su(H) on its own. The Drosophila orthologue of SHARP/MINT, termed Spen, which genetically inhibits Notch signaling in the context of eye development, is presumably not a transcriptional repressor of Notch target genes in this process. Recently it was shown that Spen is required for the activation rather than the repression of Notch target genes during the development of hemocytes. Moreover, the region of SHARP/MINT that has been defined to interact with CSL is not conserved in Spen. Although formally SHARP/MINT might act as a functional Hairless analogue in mammals, the role of the structurally related Spen proteins seems largely diverse in different organisms (Maier, 2011).

In flies, the major antagonist of Notch signaling is the transcriptional corepressor Hairless, which is ubiquitously expressed in all tissues. Hairless binds the transcription factor Su(H), as well as the corepressors Groucho and CtBP, which serves to localize the transcriptional repression machinery in the nucleus to Notch target genes, thereby repressing gene expression. Removal of the Groucho and CtBP-binding sites from Hairless does not completely eliminate its activity as a repressor, suggesting that, similar to other corepressors, Hairless might compet.e with ICN for binding Su(H). However, the molecular mechanism by which Su(H) is converted from a repressor to an activator complex is unclear (Maier, 2011).

This work investigated the molecular details of the Notch repressor complex in Drosophila. The analysis was multidisciplinary in nature, using biophysical, biochemical, cellular, and in vivo assays to characterize the protein-protein interface between Hairless and Su(H). Hairless was shown to forms a high-affinity 1:1 complex with Su(H) (~1 nM Kd) but only interacts with the CTD, which is in stark contrast to mammalian CSL-corepressor interactions, which are largely mediated through BTD contacts. Previous ITC binding studies of the mammalian Notch components Notch1 (ICN) and RBP-J showed that the Kd of the ICN/RBP-J complex is ~10 nM, suggesting that Su(H)-Hairless and Su(H)-Notch interactions are likely of comparable affinity (Maier, 2011).

Given the similar affinities of ICN and Hairless for Su(H), the question arises whether ICN and Hairless compete for binding to CSL. On one hand, gel-shift assays with purified protein components showed that ICN can displace Hairless from Su(H) independent of Mastermind. On the other hand, it was shown that residues on Su(H) that are important for Notch ANK and Mam binding to CTD do not affect interactions with Hairless. These data suggest that the ICN- and Hairless-binding sites on Su(H) do not overlap. If the ANK domain of ICN and Hairless are competing for binding to the CTD of Su(H), then there is an additional factor to consider: based on binding studies of the mammalian proteins, the vast majority of the binding energy for the Su(H)-ICN complex comes from the RAM domain interaction with the BTD of Su(H), whereas the isolated CTD-ANK interaction is of very low affinity. This represents at least a 10,000-fold difference in the affinities of ANK and Hairless for CTD, which suggests that the ANK domain of ICN would seem to be a very poor competitor for removing Hairless from Su(H) (Maier, 2011).

How then is ICN able to supplant Hairless from Su(H) in order to activate transcription from Notch target genes? Certainly additional experiments will be required to fully address this question; however, at present the following hypothesis is favored: the binding of ICN to Su(H), that is, the RAM-BTD interaction, results in allosteric changes in Su(H) that decreases its overall affinity for Hairless, thereby making ANK a more effective competitor for CTD. Consistent with this notion, gel-shift experiments showed that Hairless was far less effective at removing ICN from Su(H), even when Hairless was present in vast excess (Maier, 2011).

The studies also analyzed two absolutely conserved residues in Hairless (L235 and F243) for their contribution to binding Su(H). Whereas F243 was dispensable for binding, L235 was absolutely required for binding Su(H) in vitro. Mutation of this site to aspartate abrogated binding but did not change the secondary structure content of Hairless, which suggests that L235 lies at the Su(H)-Hairless interface. Given the conservation of F243 but its dispensability for Su(H) binding, this perhaps suggests that this residue is important for interacting with other nuclear factors. Consistent with in vitro binding results, the Hairless mutant L235D failed to assemble a repressor complex with Su(H) in cellular and in vivo assays in the fly. In fact, the L235D mutant was as deficient in repression as the Hairless deletion mutant ΔNT, which removes residues 232-270, emphasizing the importance of this contact in forming the Su(H)-Hairless complex (Maier, 2011).

In conclusion, the fly Notch repressor complex shows similarities and differences compared with the mammalian complex. Despite the high degree of sequence and likely structural conservation, Su(H) in Drosophila differs from mammalian CBF1/RBP-J in that it has no repressor activity on its own; overexpression of Su(H) in cell culture and in vivo results in a Notch gain-of-function phenotype. It is not until the binding of Hairless that Su(H) is transformed into a repressor. Of interest, this study showed that Hairless bound the mammalian CSL orthologue CBF1 nearly as avidly as Su(H), which suggests that the Hairless-binding site on the CTD has been conserved in mammals. In accordance, a potent repression of Notch transcriptional activity was found in cultured mammalian cells by the Hairless NT construct. This raises the possibility of identifying Hairless homologues in other organisms or potentially other transcriptional coregulators that use the Hairless-binding site on CTD, which may be indicative of an as-yet-unidentified mode in mammals to repress Notch signaling. Nonetheless, detailed knowledge of Su(H)-Hairless interactions can now be used to develop molecules that target Notch transcription complexes and either enforce or disrupt their activity, thereby opening new therapeutic avenues (Maier, 2011).



mastermind and neuralized, another neurogenic locus, are expressed early in the ventral mesoderm anlage, prior to gastrulation (Bettler, 1991). The boundaries of its dorsal expression are probably involved in structuring the mesectoderm, as is the case with neuralized and Notch (Martin-Bermudo, 1995). mam expression is seen on either side of the cephalic furrow. During early embryogenesis, mam is expressed ubiquitously; during gastrulation, the predominant domain of MAM RNA and protein accumulation is found along the ventral longitutinal surface, including cells of the mesoderm, endoderm, mesectoderm and neuroectoderm. Thus regions of elevated MAM accumulation appears to be under control of dorsoventral patterning genes (Bettler, 1991).

Effects of Mutation or Deletion

mastermind mutants are lethal to the embryo. A neural hyperplasia is observed, caused by failure of the most ventral ectodermal cells to differentiate as epidermal cells rather than neuroblasts. Neural hyperplasia can be suppressed by mutation in proneural genes (Brand, 1988).

mastermind has a similar profile of embryonic defects as those produced by other neurogenic mutations, including Notch and Delta (Hartenstein, 1992), except that defects do not include the endoderm. A full list of tissues affected by neurogenic mutations is found at the Notch pathway.

The phenotypes and genetic interactions associated with mutations in the Drosophila mastermind(mam) gene have implicated mam as a component of the Notch signaling pathway. However, its function and site of action within many tissues requiring Notch signaling have not been thoroughly investigated. To address these questions, truncated versions of the Mam protein have been constructed that elicit dominant phenotypes when expressed in imaginal tissues under GAL4-UAS regulation. By several criteria, these effects appear to phenocopy loss of function for the Notch pathway. When expressed in the notum, truncated Mam results in failure of lateral inhibition within proneural clusters and perturbations in cell fate specification within the sensory organ precursor cell lineage. Expression in the wing is associated with vein thickening and margin defects, including nicking and bristle loss. The truncation-associated wing margin phenotypes are modified by mutations in Notch and Wg pathway genes and are correlated with depressed expression of wg, cut, and vg. These data support the idea that Mam truncations have lost key effector domains and therefore behave as dominant-negative proteins. Coexpression of Delta or an activated form of Notch suppresses the effects of the Mam truncation, suggesting that Mam can function upstream of ligand-receptor interaction in the Notch pathway. This system should prove useful for the investigation of the role of Mam within the Notch pathway (Helms, 1999).

Chip may encode an enhancer-facilitator, acting to facilitate the activity of distal enhancers. The mechanisms allowing remote enhancers to regulate promoters several kilobase pairs away are unknown but are blocked by the Drosophila suppressor of Hairy-wing protein [su(Hw)] that binds to gypsy retrovirus insertions between enhancers and promoters. su(Hw) bound to a gypsy insertion in the cut gene also appears to act interchromosomally to antagonize enhancer-promoter interactions on the homologous chromosome when activity of the Chip gene is reduced. Chip is needed for the wing margin enhancer of cut. The Chip mutation dominantly enhances the mutant phenotypes displayed by partially suppressed gypsy insertions in both cut and Ultrabithorax and is a homozygous larval lethal, indicating that Chip regulates multiple genes. Chip is normally required for wing margin enhancer function of cut because Chip mutations also enhance the cut wing phenotype of a cut mutation and heterozygotes for Chip display cut wing phenotypes when either scalloped or mastermind (mam) are also heterozygous mutant. Both Sc and Mam are known to regulate the cut distal enhancer, but in contrast to sd and mam mutants, Chip mutants display stronger genetic interactions with gypsy insertions than with wing margin enhancer deletions. Thus, in a heterozygous Chip mutant, a heterozygous gypsy insertion in cut displays a cut wing phenotype, whereas a heterozygous enhancer deletion does not. Dependence on the nature of the heterozygous lesion in the regulatory region strongly suggests that Chip directly regulates cut. More strikingly, it indicates that in a Chip heterozygote, a gypsy insertion is more deleterious to enhancer function than deletion of the enhancer. The simplest explanation is that su(Hw) bound to gypsy in one cut allele acts in a transvection-like manner (interchromosomally) to block the wing enhancer in the wild-type cut allele on a second chromosome. This implicates Chip in enhancer-promoter communication (Morcillo, 1997 and references).

Chip was cloned and found to encode a homolog of the recently discovered mouse Nli/Ldb1/Clim-2 and Xenopus Xldb1 proteins, which bind nuclear LIM domain proteins. Chip protein interacts with the LIM domains in the Apterous homeodomain protein, and Chip interacts genetically with apterous, showing that these interactions are important for Apterous function in vivo. Importantly, Chip also appears to have broad functions beyond interactions with LIM domain proteins. Chip is a ubiquitous chromosomal factor required for normal expression of diverse genes at many stages of development. It is suggested that Chip cooperates with different LIM domain proteins and other factors to structurally support remote enhancer-promoter interactions (Morcillo, 1997).

Integrins are evolutionarily conserved transmembrane alpha,beta heterodimeric receptors involved in cell-to-matrix and cell-to-cell adhesions. In Drosophila, the position-specific (PS) integrins (see Myospheroid) mediate the formation and maintenance of junctions between muscle and epidermis and between the two epidermal wing surfaces. Besides integrins, other proteins are implicated in integrin-dependent adhesion. In Drosophila, somatic clones of mutations in PS integrin genes disrupt adhesion between wing surfaces to produce wing blisters. To identify other genes whose products function in adhesion between wing surfaces, a screen was conducted for autosomal mutations that produce blisters in somatic wing clones. 76 independent mutations were isolated in 25 complementation groups, 15 of which contained more than one allele. Chromosomal sites were determined by deficiency mapping, and genetic interactions with mutations in the beta PS integrin gene myospheroid were investigated. Mutations in four known genes (blistered [Drosophila's Serum response factor implicated in the specification of intervein cells], Delta, dumpy and mastermind) were isolated. Mutations were isolated in three new genes (piopio, rhea and steamer duck) that affect myo-epidermal junctions or muscle function in embryos. Mutations in three other genes (kakapo, kiwi and moa) may also affect cell adhesion or muscle function at hatching. These new mutants provide valuable material for the study of integrin-dependent cell-to-cell adhesion. It is thought that blisters arise in Delta and mastermind clones because of a failure to maintain the normal properties of ectodermal cells within the clonal boundaries (Prout, 1997).

The mechanisms that allow enhancers to activate promoters from thousands of base pairs away are disrupted by the Drosophila Suppressor of Hairy-wing protein (Su[Hw]). Su[Hw] binds a DNA sequence in the gypsy retrotransposon and prevents activation of promoter-enhancers that are distal to a gypsy insertion in a gene without affecting proximal promoter-enhancers. Several observations indicate that SUHW does not affect enhancer-binding activators. Instead, SUHW may interfere with factors that structurally facilitate interactions between an enhancer and promoter. To identify putative enhancer facilitators, a screen for mutations that reduce activity of the remote wing margin enhancer in the cut gene was performed. Mutations in scalloped, mastermind, and a previously unknown gene, Chip, were isolated. A TEA DNA-binding domain in the Scalloped protein binds the wing margin enhancer. Interactions among scalloped, mastermind and Chip mutations indicate that Mastermind and Chip act synergistically with Scalloped to regulate the wing margin enhancer. Chip is essential and also affects expression of a gypsy insertion in Ultrabithorax. Relative to mutations in either scalloped or mastermind, a Chip mutation hypersensitizes the wing margin enhancer in cut to gypsy insertions. Therefore, Chip might encode a target of su(Hw) enhancer-blocking activity (Morcillo, 1996).

Sanpodo regulates Notch-mediated sibling cell fate decisions but is not involved in Notch-mediated lateral inhibition. Notch functions in the neurogenic ectoderm to limit the number of cells adopting a neural fate. spdo mutation does not alter the number of neuroblasts that delaminate from the ectoderm, but instead is involved only in regulating sibling cell fate in the progeny of neuroblasts. Although the spdo sibling neuron phenotype is identical to the Notch sibling neuron phenotype, none of the 11 spdo alleles show the excess neuroblast formation characteristic of Notch mutations. Mutations in two other genes, Delta (10 alleles) and mastermind (1 allele) have been identified that yield similar equalization of sibling neuron fates. Because both Delta and mastermind are in the well-characterized Notch signaling pathway, null and hypomorphic alleles of several 'Notch pathway' genes have been tested: Delta, Notch, mam, neuralized and E(spl). Mutations in all these genes result in an excess of neuroblasts due to failure of lateral inhibition within the neuroectoderm. However, mutations in neuralized and E(spl) have no effect on the identity of the sibling neurons that were assayed, despite strong defects in the earlier process of neuroblast formation. In contrast, Delta, Notch and mam mutations all yield similar sibling neuron phenotypes, in addition to excessive neuroblast formation. These results can be illustrated using embryos homozygous for a hypomorphic mam allele in which neuroblast formation is essentially normal but sibling neuron fates are equalized. Loss of mam does not affect eve expression in GMCs, but leads to the duplication of RP2, Usib, aCC and dMP2 fates at the expense of the RP2sib, U, pCC and vMP2 fates, respectively. Thus, mutations in three genes (Delta, Notch and mam) have precisely the same sibling neuron phenotype as spdo mutations, suggesting that spdo, Delta, Notch and mam act together to specify asymmetric sibling neuron fate (Skeath, 1998).

Asymmetric cell division is a widespread mechanism in developing tissues that leads to the generation of cell diversity. For the most part the basis of asymmetric cell division has been analyzed in neuroblasts in the process by which neuroblast division yields another neuroblast and a secondary precursor cell: the ganglion mother cell (GMC). In the embryonic central nervous system of Drosophila melanogaster, GMCs divide and produce postmitotic neurons that take on different cell fates. The current study analyses the process of binary fate decision of two pairs of sibling neurons that occurs during cell division in GMCs. This process is accomplished through the intrinsic fate determinant, Numb. GMCs have apical-basal polarity; Numb localization and the orientation of division are coordinated to segregate Numb to only one sibling cell. The correct positioning of Numb and the proper orientation of division require Inscuteable (Insc). Loss of insc results in the generation of equivalent sibling cells. These results provide evidence that sibling neuron fate decision is nonstochastic and normally depends on the presence of Numb in one of the two siblings. Moreover, the data suggest that the fate of some sibling neurons may be regulated by signals that do not require lateral interaction between the sibling cells (Buescher, 1998).

The focus for the analysis of the roles of insc, numb, and components of the N-signaling pathway in fate specification, was on the only two pairs of GMC-derived neurons for which sibling relationships have been established: the RP2/RP2sib and the aCC/pCC neurons. These neurons are derived from two GMCs that can be identified unambigously by their specific expression of the nuclear protein Even-skipped (Eve). GMC1-1a divides into the aCC/pCC neurons that have approximately equal size and continue to express Eve. However, at later stages of development, aCC is distinguished from pCC by the expression of Zfh-1 and 22C10 (a membrane associated antigen). aCC is a motoneuron and forms an ipsilateral projection that pioneers the intersegmental nerve. GMC4-2a divides to form the sibling neurons RP2/RP2sib that are morphologically distinguishable. In 88% of the hemisegments, the newborn siblings show a significant difference in the size of their nuclei and cell bodies. This asymmetry appears to be initiated during cell division. In GA1019 mutant embryos, in which GMC4-2a fails to complete cytokinesis, cells are formed that contain one large and one small nucleus. This strongly suggests that the difference in size is generated early, prior to the completion of cytokinesis. The larger cell always adopts the RP2 fate, which is characterized by the expression of Eve, Zfh-1, and 22C10. RP2 forms an antero-ipsilateral projection. The smaller sibling always adopts the RP2sib fate, which is characterized by a further decrease in cell and nuclear size and the loss of Eve immunreactivity. Zfh-1 and 22C10 expression have not been shown in RP2sib. These observations suggest that the cell and nuclear size difference may serve as an early physical marker that will allow one to differentiate between the two progeny of GMC4-2a, irrespective of the molecular markers they express later (Buescher, 1998).

Mutations in mastermind (mam),sanpodo, and Notch equalize aspects of sibling cell fate but retain the difference in cell and nuclear size of sibling neurons. In mam mutant embryos, both progeny of GMC4-2a can adopt the RP2 fate with respect to Eve, Zfh-1, and 22C10 expression. However, despite this apparent change from the RP2sib to the RP2 cell fate, the unequal sizes of the GMC4-2a daughter cells remain; that is, their sizes are unaffected. mam is required for the correct fate specification of RP2sib and pCC but not for that of RP2 and aCC. The requirement for mam suggests that N signaling may be involved in the resolution of distinct sibling neuron cell fate. Mutations in mam and N result in similar defects and support the notion that N signaling is required for the resolution of sibling neuron fate. In inscuteable mutant embryos, GMC1-1a and GMC4-2a are correctly formed and express normal levels of Eve (and in the case of GMC4-2a, also Pdm-1). However, GMC1-1a divides to form two sibling neurons that both adopt the aCC fate (94%) with respect to marker gene expression. Similarly, GMC4-2a division results in two sibling cells, both of which adopt the RP2 fate (96%) with respect to expression of Eve, Zfh-1, and 22C10, as well as axon morphology. This strongly suggests that in wild-type embryos, the divisions of GMC1-1a and GMC4-2a are asymmetric in an insc-dependent manner and produce sibling cells that are intrinsically different; loss of insc function leads to the generation of sibling neurons with equivalent cellular identities. Moreover, in contrast to mam, sanpodo, and Notch mutant embryos, the duplicated RP2s seen in insc mutants are equal with respect to their cell and nuclear size. These observations are consistent with the idea that the size difference seen in wild-type embryos is generated by an insc-dependent process during the GMC cell division and occurs prior to the events mediated by mam, spdo, and N that presumably act at the level of the postmitotic sibling cells. No size asymmetry between the sibling neurons should be generated in an insc background regardless of whether the other functions (e.g., spdo) are present or not (Buescher, 1998).

The Notch receptor is the central element in a cell signaling mechanism controlling a broad spectrum of cell fate choices. Genetic modifier screens in Drosophila and subsequent molecular studies have identified several Notch pathway components, but the biochemical nature of signaling is still elusive. The results are described of a genetic modifier screen of the bristle phenotype of a gain-of-function Notch allele, Abruptex16. Abruptex mutations interfere with lateral inhibition/specification events that control the segregation of epidermal and sensory organ precursor lineages, thus inhibiting bristle formation. Mutations that reduce Notch signaling suppress this phenotype. This screen of approximately 50,000 flies led to the identification of a small number of dominant suppressors in seven complementation groups. These include Notch, mastermind, Delta, and Hairless , known components in the pathway, as well as two novel mutations: A122 and M285. A122, appears to interact with Notch only during bristle development. M285, displays extensive genetic interactions with the Notch pathway elements and appears, in general, capable of suppressing Notch gain-of-function phenotypes while enhancing Notch loss-of-function phenotypes, suggesting that it plays an important role in Notch signaling. The profile of the genetic interactions documented with M285 is quite similar to that of mutations in other known components of the Notch pathway. Three kismet alleles were isolated as weak suppressors of the Ax16 bristle phenotype. Interestingly, mutations in kismet have been isolated independently as enhancers of the eye phenotype associated with the expression of constitutively activated forms of the Notch receptor. kismet, which may encode a structural component of chromatin, does not display broad genetic interactions with Notch. It has therefore been suspected that the identification of these alleles through the eye screen may reflect its effect on the expression of the transgene by perturbing normal chromatin function rather than significant interactions with Notch signaling. The fact that such alleles were isolated in the bristle screen may be indicative of a link between Notch signaling and kismet function; however, further analysis is necessary before such a relationship can be established (Go, 1998).

In the mesoderm of Drosophila embryos, a defined number of cells segregate as progenitors of individual body wall muscles. Progenitors and their progeny founder cells display lineage-specific expression of transcription factors but the mechanisms that regulate their unique identities are poorly understood. The homeobox genes ladybird early and ladybird late are shown to be expressed in only one muscle progenitor and its progeny: the segmental border muscle (SBM) founder cell and two precursors of adult muscles. lb activity is associated with all stages of SBM formation, namely the promuscular cluster, progenitor cell, founder cell, fusing myoblasts and syncytial fiber. The segregation of the ladybird-positive progenitor requires coordinate action of neurogenic genes and an interplay of inductive Hedgehog and Wingless signals from the overlying ectoderm. The SBM progenitor corresponds to the most superficial cell from the promuscular cluster, thus suggesting a role for the overlying ectoderm during its segregation. . Since epidermal Wg and Hedgehog (Hh) signaling has been shown to influence muscle formation, the SBM-associated lb expression was examined in embryos carrying hh and wg thermosensitive mutations. Wg and Hh signalings, mutually dependent at this time, are shown to be required for the promuscular lb activity and/or the segregation of SBM progenitors. The initial influence of these signals is no longer observed later in development. In addition to signals from the epidermis, the activity of the mesodermal gene tinman, initially expressed in the whole trunk mesoderm, is involved in the early events of myogenesis. In tin - embryos, the formation of SBM promuscular clusters and segregation of lb-positive progenitor cells are strongly affected, leading to the absence of the majority of SBM fibers. During promuscular cluster formation, since tin expression becomes restricted to the dorsal mesoderm, its influence on ventrolaterally located SBMs is likely to be indirect and mediated via an unknown factor. The lack of neurogenic gene function, known to be involved in cell-cell interactions during lateral inhibition, generates the opposite phenotype. Mastermind - and Enhancer of split - embryos fail to restrict promuscular lb expression to only one cell; in consequence, they display a hyperplastic lb pattern in later stages (Jagla, 1998).

The Notch pathway plays a key role in the formation of many tissues and cell types in Metazoans. Notch acts in two pathways to determine muscle precursor fates. The first is the 'standard' Notch pathway, in which Delta activates the Notch receptor, which then translocates into the nucleus in conjunction with Su(H) to reprogram transcription patterns and bring about changes in cell fates. The second pathway is poorly defined, but known to be independent of the ligands and downstream effectors of the standard pathway. The standard pathway is required in many different developmental contexts; it was of interest to determine if there is a general requirement for the novel pathway. The novel Notch pathway is required for the development of each of five examined cell types. Holonull Notch mutants (mutants null for maternal and zygotic Notch) have a more extreme phenotype than null mutants for Su(H), Delta, neuralized or mastermind. In Notch holonull embryos, clusters of 10 or 15 eve expressing RP2-like cells are found in place of a normal single RP2. The phenotype for the other neurogenic genes is far less severe. Notch and other neurogenic genes are involved in the determination of the mesectoderm and the visceral mesoderm. The Notch holonull phenotype is more severe in both cases than that of other holonull embryos. These results indicate that the novel pathway is a widespread and fundamental component of Notch function (Rusconi, 1999).

In the Drosophila CNS glial cells are known to be generated from glioblasts, which produce exclusively glia or neuroglioblasts that bifurcate to produce both neuronal and glial sublineages. The genesis of a subset of glial cells, the subperineurial glia (SPGs), involves a new mechanism and requires Notch. SPGs share direct sibling relationships with neurons and are the products of asymmetric divisions. This mechanism of specifying glial cell fates within the CNS is novel and provides further insight into regulatory interactions leading to glial cell fate determination. Furthermore, Notch signaling positively regulates glial cells missing expression in the context of SPG development (Udolph, 2001).

In order to better understand how a complete lineage of a specific NGB with all its progeny, including its glial cells, might be created, NB1-1 was chosen for a detailed analysis. NB1- 1 has been extensively used for cell fate specification studies and a sound basis of information about this NB lineage is available. NB1-1 is a NB that develops differential lineages in the thoracic versus the abdominal segments. Focus was placed on the abdominal NB1-1A because only these abdominal NB1-1 lineages contain glia. In addition to the aCC/pCC sibling neurons, which are the progeny of the first GMC produced from this lineage, NB1-1A generates 2 to 3 glial cells and 4 to 5 clustered interneurons (cN), yielding a total of 9 to 10 cells. The three glial cells belong to the group of subperineurial glia (SPG) that lie at the periphery of the nerve cord and enwrap the entire ventral nervous system. Two of the glia, the A- and B-SPGs, can be found in dorsal positions, with a third glia, the LV-SPG, located at ventral positions of the nerve cord. All SPGs, including the A- and B-SPG and LV-SPG of NB1-1A, are specifically labelled by two enhancer trap lines, M84 and P101 (Udolph, 2001 and references therein).

As a first step toward elucidating the origin of the glial cells of the NB1-1A lineage, the effects of loss of function mutants in several genes, Notch, mastermind (mam) and numb, which are known to affect the resolution of distinct sibling cell fates, were tested for their effect on the development of A-, B- and LV-SPGs. Embryos hemizygous/homozygous for a conditional Notch allele, Nts1, and also carrying one copy each of M84 and P101 (Nts1/M84/P101) were subjected to the non-permissive temperature of 29°>C after 6 hours of development. This regime allows Notch to function during the singling out of NBs and removes Notch during the crucial period when it is required for sibling cell fate resolution. Double staining with anti-Eve and anti-ß-gal was performed. As expected, in most hemisegments, Nts1/M84/P101 embryos duplicate the RP2 neuron at the expense of its sibling cell. Moreover, in 96% of the hemisegments, M84/P101+ cells could not be found in typical dorsal or ventral positions. It is concluded that Notch function is required for the specification of the M84/P101 positive A-, B- and LV-SPGs. In wild-type embryos, M84/P101 is expressed in about eight SPGs per hemisegment, including the A- and B-SPGs and the LV-SPG (Udolph, 2001).

mastermind, which has been linked to the Notch signaling pathway by its genetic interactions with Notch and its strikingly similar phenotype in early and late neurogenesis, was tested. mam acts downstream of Notch during sibling cell fate specification in the embryonic nervous system. The hypomorphic mam345 allele used in this study shows only a mild hypertrophy of the nervous system but clearly has an effect on sibling cell fate specification. A severe reduction (94%) of P101+ cells was observed in mam345;P101 embryos similar to that seen with Nts1/M84/P101 embryos. These data suggest that both genes are strictly required for the specification of SPGs, most likely in a linear pathway. However, it is unclear how Notch acts in the specification of the SPGs. The possibility is considered that SPG glial cells could arise from a series of asymmetric cell divisions, with Notch being required to specify the glial daughters of these divisions (Udolph, 2001).

Based on its function as a negative regulator of Notch signaling, the expected numb phenotype is opposite that of Notch in terms of sibling cell fate transformation. The P101 expression pattern was tested in the background of a strong numb mutation. In contrast to Notch and mam, additional P101+ cells were found in the vicinity of the aCC/pCC position. In most of the examined hemi-neuromeres, up to four ß-gal-positive cells were detected in dorsal positions close to aCC/pCC. This is indicative of a duplication of the A- and B-SPGs. Additional P101+ cells with glial morphology were found in lateral and ventral positions of the nerve cord, presumably duplications of other SPGs. These findings are consistent with an asymmetric cell division model for the genesis of the SPGs (Udolph, 2001).

Differential effects of Mastermind on asymmetric cell fate specification and neuroblast formation

During neurogenesis in the ventral nerve cord of the Drosophila embryo, Notch signaling participates in the pathway that mediates asymmetric fate specification to daughters of secondary neuronal precursor cells. In the NB4-2 --> GMC-1 --> RP2/sib lineage, a well-studied neuronal lineage in the ventral nerve cord, Notch signaling specifies sib fate to one of the daughter cells of GMC-1. Notch mediates this process via Mastermind (Mam). Loss of function for mam, similar to loss of function for Notch, results in GMC-1 symmetrically dividing to generate two RP2 neurons. Loss of function for mam also results in a severe neurogenic phenotype. In this study, a functional analysis has been undertaken of the Mam protein. While ectopic expression of a truncated Mam protein induces a dominant-negative neurogenic phenotype, it has no effect on asymmetric fate specification. This truncated Mam protein rescues the loss of asymmetric specification phenotype in mam in an allele-specific manner: an interallelic complementation of the loss-of-asymmetry defect is demonstrated. These results suggest that Mam proteins might associate during the asymmetric specification of cell fates and that the N-terminal region of the protein plays a role in this process (Yedvobnick, 2004).

In the ventral nerve cord of the Drosophila embryo, the Notch pathway mediates terminal asymmetric division of secondary neuronal precursor cells. The secondary precursor cells, GMCs, in the nerve cord generally divide by asymmetric mitosis to generate two different daughter cells. For example, in the GMC-1 --> RP2/sib lineage during GMC-1 division, the Inscuteable protein asymmetrically localizes to the apical end, which forces Numb to localize to the basal end. Basally localized Numb then segregates to the future RP2. The function of Numb is to prevent the cleaving of the intracellular domain of Notch. In the absence of Numb, the intracellular domain of Notch gets cleaved and then translocated into the nucleus where it specifies a sib fate by complexing with Su(H) and Mam and activating downstream target genes. Previous results also show that for the specification of an RP2 identity Numb is not required, but it is required to prevent that cell from becoming a sib in the presence of an intact Notch pathway (Yedvobnick, 2004).

Mam exerts differential effects on asymmetric cell fate specification vs. neuroblast formation in the ventral nerve cord of the Drosophila embryo. A Mam truncation, which has the basic and the first acidic domain (MamN), rescues the asymmetric cell fate specification defect in an allele-specific manner. These conclusions are based on several lines of evidence. (1) A transgene that encodes this truncated Mam protein causes a dominant-negative neurogenic defect, but it does not cause a dominant-negative effect on asymmetric division. Thus, expression of this transgene during the asymmetric division of GMC-1 does not cause a duplication of RP2 as one would expect if this transgene functions as a dominant negative. The same transgene when expressed earlier when NBs are formed causes a neurogenic defect. This indicates that the truncated transgene functions as a dominant negative but only during the earlier neurogenic process. (2) MamN rescues the asymmetry defect in one of the mam mutant alleles, mamHD10/6. This is a hypomorphic P-element insertion allele, which causes the loss of asymmetric division defect but does not cause a neurogenic defect except in combination with strong alleles of mam. These results and the fact that the P element is inserted in the untranslated first exon suggest that low levels of wild-type Mam are produced by this allele. However, the finding that MamN does not rescue the asymmetry defect in another mam mutant allele, mamIL42, which is predicted to produce a truncated Mam protein similar to MamN, indicates that this rescue is allele specific. Thus, some wild-type Mam protein appears to be necessary for the rescue by MamN and it is possible that the two proteins interact to provide the rescue function (Yedvobnick, 2004).

Sequence analysis of mamIL42 suggests that this allele encodes a Mam protein that is similar to MamN (although it is seven amino acids shorter). The inability of MamN to rescue mamIL42 argues that this truncated protein in combination with MamN is not sufficient to rescue the asymmetry defect. However, the interallelic complementation between mamHD10/6 and mamIL42 (a situation very similar to the mamHD10/6;MamN combination) also suggests that MamIL42 and MamHD10/6 proteins (which are expected to be wild type, but present at reduced levels) interact to rescue the loss of asymmetric division of GMC-1. These results raise the question as to whether or not MamN (which is similar to the Mam protein in the mamIL42 allele) has all the necessary function for generating asymmetry. Since it does not rescue the asymmetry defect in mamIL42, clearly it does not have all the necessary information. However, it does have the required function in the presence of some presumably wild-type protein (i.e., in mamHD10/6 background). This is consistent with the fact that MamN does not function as a dominant negative during the asymmetric division of GMC-1 but only at earlier stages during the formation of NBs (Yedvobnick, 2004).

There might be some difference between MamN and MamIL42 in their ability to complement loss of asymmetric division in mamHD10/6. This is indicated by the findings that while MamN can rescue the asymmetry defect in mamHD10/6, the interallelic complementation of the asymmetry phenotype between mamIL42 and mamHD10/6 is not as complete as rescue of mamHD10/6 by MamN. This may, in part, be due to the seven-amino-acid difference between MamN and MamIL42. Alternatively, there may be a protein-level difference between the two cases; in the former, MamN is expressed at high levels under Hs-GAL4, whereas in the latter mamIL42 is under the control of the mam promoter. Yet, the seven-amino-acid residues could make some difference, given that these amino acids are mostly glutamine residues, which can be involved in multimerization of proteins. It is possible that the region of the Mam polypeptide defined by MamN (and MamIL42) is required to interact efficiently with the full-length Mam during the asymmetric fate specification. The requirement of some wild-type Mam protein for the rescue activity of MamN or MamIL42 also suggests that the remaining portions of Mam are also required for generating asymmetry. The most likely scenario would be that this is a protein-protein interaction, although some other possibilities cannot be excluded. Since the available antibody against Mam recognizes multiple bands on a Western blot of proteins from embryo, no immunoprecipitation experiments have been performed to address protein-protein interaction between Mam molecules (Yedvobnick, 2004).

The results show that mamIL42 carries a partial suppressor of neurogenic defect since a strong neurogenic defect can be restored to this allele upon recombination. This is consistent with the result that expression of MamN elicits a strong dominant-negative neurogenic defect. However, this suppressor in mamIL42 has no modifying effect on the loss-of-asymmetry phenotype of mamIL42, as indicated by the fact that there was no change in the penetrance of this defect between the original and the recombinant mamIL42. The location of this suppressor(s) has not been mapped beyond its tentative assignment to chromosome 2 (Yedvobnick, 2004).

Previous studies utilizing MamH and MamN have demonstrated that both truncations elicited dominant-negative effects when overexpressed in imaginal tissues. It was later shown that the basic region of Mam is conserved in fly, mouse, and human and that the region physically interacts with the processed intracellular segment of Notch. Mam, Nintra, and Su(H)/CSL proteins associate in a ternary complex that binds to HES/E(spl) promoters and activates gene expression. The expression of MamH and MamN presumably leads to transcription complexes containing a defective form of Mam in a complex with Su(H) and Nintra. The current results, however, indicate that these interactions may be distinct during the generation of asymmetry. For instance, the MamN polypeptide may lack sequences required for interaction with factors necessary for NB formation but not for asymmetric division (Yedvobnick, 2004).

Finally, the results indicate that the mam phenotype in the RP2/sib lineage (symmetrical division of GMC-1 into RP2 and sib) is epistatic to (functions downstream of) the numb phenotype (symmetrical division of GMC-1 into two sibs). During the division of GMC-1, Insc localizes to the apical end of GMC-1, which in turn segregates Nb to the basal end. The cell that inherits Nb is specified as RP2 due to the ability of Nb to block Notch signaling, whereas the cell that does not inherit Nb (but inherits Insc) is specified as sib by Notch. Thus, in insc mutants, both daughters of the GMC-1 adopt an RP2 fate whereas in nb mutants they assume a sib fate. The sib cell adopts an RP2 fate in Notch; nb double mutants. This indicates that Nb is needed to specify RP2 fate only when there is intact Notch. The mam, numb double mutant result is consistent with the above result. That is, Numb is needed only when there is intact Mam. This result further indicates that Mam functions downstream of Notch during the asymmetric specification of RP2 and sib, an observation consistent with the prevailing view of the Notch signal transduction pathway (Yedvobnick, 2004).

Notch signal organizes the Drosophila olfactory circuitry by diversifying the sensory neuronal lineages

An essential feature of the organization and function of the vertebrate and insect olfactory systems is the generation of a variety of olfactory receptor neurons (ORNs) that have different specificities in regard to both odorant receptor expression and axonal targeting. Yet the underlying mechanisms that generate this neuronal diversity remain elusive. This study demonstrates that the Notch signal is involved in the diversification of ORNs in Drosophila. A systematic clonal analysis showed that a cluster of ORNs housed in each sensillum were differentiated into two classes, depending on the level of Notch activity in their sibling precursors. Notably, ORNs of different classes segregated their axonal projections into distinct domains in the antennal lobes. In addition, both the odorant receptor expression and the axonal targeting of ORNs were specified according to their Notch-mediated identities. Thus, Notch signaling contributes to the diversification of ORNs, thereby regulating multiple developmental events that establish the olfactory map in Drosophila (Endo, 2007).

In the Drosophila olfactory system there are about 50 different types of ORNs, each of which is characterized by the expression of a specific odorant receptor. Yet little is known about the molecular mechanisms underlying the generation of this diverse array of ORNs. By analyzing the projection patterns of ORNs and the cell lineage of the olfactory organ in mastermind (mam) and numb (nb) clones, it was shown that differential Notch activity in the two sibling ORN precursors leads to ORN diversification and thereby regulates multiple developmental events in the organization of the olfactory system (Endo, 2007).

There are several types of sensory organs in Drosophila, and the component cells in each organ are generally derived from a single SOP. All of the neuronal and non-neuronal cells that constitute the olfactory sensilla are derived from three precursors that arise before early pupal stages. MARCM-clone analyses was used to examine developmental events in the olfactory sensory lineage that precede these stages, and it was found that all the cells in the olfactory sensillum are generated from a single SOP that arises as early as 30 h BPF. Therefore, the olfactory sensilla seem to adopt developmental mechanisms similar to those seen in other sensory organs. However, although only one or two neurons are produced in the canonical, or ancestral, peripheral nervous system lineage, 1 to 4 ORNs are generated in the lineages of the olfactory sensilla. These findings reveal that this difference in the number of generated neurons is a consequence of the generation of more inner-cell precursors in the olfactory sensilla lineage. Interestingly, the gustatory sensillum is also innervated by 2 to 4 gustatory receptor neurons. It is proposed that the gustatory and olfactory systems share similar developmental mechanisms for producing a variety of neurons (Endo, 2007).

Although Notch signaling asymmetrically differentiates the ORN precursors, lineage analysis of nb clones in the antenna suggests that Notch does not diversify the Notch-ON-class ORN siblings (referring to a class with activate Notch signaling) on their generation from a common precursor. This notion is consistent with the results of systematic mam- and nb-clone analyses in the antennal lobe, where most ORNs are categorized into either Notch-ON or Notch-OFF classes. Therefore, the Notch-ON-class ORN siblings seem to be diversified by Notch-independent mechanisms. The asymmetric distribution of other proteins may mediate this process in ORN siblings. Thus, the olfactory sensory lineages use both Notch-dependent and Notch-independent mechanisms to generate a variety of neurons as well as non-neuronal cells (Endo, 2007).

The data in this study indicate that the genetic information that specifies the characteristics of the olfactory organ, such as sensilla and ORN type, is derived from SOPs in the developing antennal disc. Part of the information in SOPs is possibly encoded by the proneural genes atonal and amos, because they are essential for the differentiation of SOPs that produce different types of sensilla. It is proposed that the identities of SOPs arise, in part, from positional information allocated along the axes of the disc. This hypothesis is supported by the observation that ORNs expressing different types of odorant receptors are roughly segregated into distinct domains that are distributed along the proximo-distal axis. Positional information along other axes is also likely to refine genetic information in SOPs, as evidenced by a correlation between the regional localization of sensilla types on the antenna and the patterned projections of the corresponding ORNs in the antennal lobe. The data of lineage analysis indicate that Notch signaling further diversifies the genetic information of SOPs to produce the outer and inner cell lineages, and subsequently differentiates ORN precursors to generate two Notch-mediated classes of ORNs. Importantly, Notch signaling diversifies not only the genetic information specific to each SOP, but also the information shared by a subset of SOPs, as evidenced by the observation that a subset of ORNs of a given Notch-mediated class share their axonal pathways, target domains or both in the antennal lobe. Thus, Notch signaling controls multiple aspects of ORN development that contribute to the organization and function of the olfactory circuit (Endo, 2007).

In the adult olfactory system, odor information is first represented as a spatial map of activated glomeruli in the antennal lobes. Recent optical recording studies in vertebrates have shown that distinct chemical classes activate distinct regions of glomeruli, forming a chemotopic map. In Drosophila, although the larval olfactory system has a glomerular organization, in which two distinct domains represent specific classes of chemicals, the chemotopic organization of glomeruli in the adult brain is less obvious than that in the fly larvae and vertebrates. The glomerular domains identified in this study may be correlated with the chemical properties of some odorants, but no specific chemical classes have been assigned to these domains, and there are no significant sequence similarities among odorant receptors expressed in ORNs that project to the same glomerular domains compared with those expressed in ORNs that project to distinct domains. One possible implication of this domain organization is that glomeruli are physically segregated, as is typically found in the posterior group, and the sensory information from ORNs is independently processed in distinct domains. Alternatively, these domains may evoke different behavioral responses. In the Drosophila gustatory system, the axonal targets of the gustatory receptor neurons are segregated by taste category: neurons that recognize sugars project to a different region from those that recognize noxious substances. Currently, little is known about the relationship between the olfactory circuitry and behavior. Further functional studies would be necessary to assess whether Notch-mediated glomerular domains function in olfactory coding and behavior (Endo, 2007).

Requirements for mediator complex subunits distinguish three classes of notch target genes at the Drosophila wing margin

Spatial and temporal gene regulation relies on a combinatorial code of sequence-specific transcription factors that must be integrated by the general transcriptional machinery. A key link between the two is the mediator complex, which consists of a core complex that reversibly associates with the accessory kinase module. Genes activated by Notch signaling at the dorsal-ventral boundary of the Drosophila wing disc fall into three classes that are affected differently by the loss of kinase module subunits. One class requires all four kinase module subunits for activation, while the others require only Med12 and Med13, either for activation or for repression. These distinctions do not result from different requirements for the Notch coactivator Mastermind or the corepressors Hairless and Groucho. It is proposed that interactions with the kinase module through distinct cofactors allow the DNA-binding protein Suppressor of Hairless to carry out both its activator and repressor functions (Janody, 2011).

Intercellular signaling pathways drive many processes during development. Their activation results in changes in transcription factor activity that lead to the activation or repression of specific target genes. An important goal is to understand the transcriptional regulatory codes that allow the combinations of proteins bound to enhancer elements to direct precise patterns of gene expression. One well-characterized developmental paradigm is the specification of the Drosophila wing margin by Notch signaling. The Notch receptor is specifically activated at the dorsal-ventral boundary of the larval wing imaginal disc, due to the restricted expression of its ligands Delta and Serrate and of the glycosyltransferase Fringe. Notch activation results in expression of the target genes Enhancer of split m8 (E(spl)m8), cut, wingless (wg), and vestigial (vg), the last through a specific enhancer element known as the boundary enhancer (vgBE). Wg signaling then leads to the differentiation of characteristic sensory bristles adjacent to the margin of the adult wing (Janody, 2011).

Upon ligand binding, Notch is cleaved by the γ-secretase complex, and its intracellular domain (Nintra) enters the nucleus, where it interacts with the DNA-binding protein Suppressor of Hairless (Su(H)). In the absence of Notch activation, Su(H) represses target gene expression through interactions with the corepressor Hairless (H), which binds to Groucho (Gro) and C-terminal binding protein (CtBP). Nintra displaces these corepressors from Su(H) and recruits coactivators such as Mastermind (Mam). It has been proposed that only a subset of Notch target genes require Su(H) to recruit coactivators, while others require Notch signaling only to relieve Su(H)-mediated repression, allowing transcription to be activated by other factors. However, the mechanisms by which Su(H) directs both activation and repression are not fully understood (Janody, 2011).

The mediator complex is thought to promote transcriptional activation by recruiting RNA polymerase II (Pol II), the general transcriptional machinery, and the histone acetyltransferase p300 to promoters, and by stimulating transcriptional elongation by Pol II molecules paused downstream of the promoter. The 'head' and 'middle' modules of the core complex bind to Pol II and general transcription factors, while the 'tail' module consists largely of adaptor subunits that bind to sequence-specific transcription factors. This core complex reversibly associates with a fourth 'kinase' module that consists of the four subunits Med12, Med13, Cdk8, and Cyclin C (CycC). Several studies have implicated the kinase module in transcriptional repression, which can be mediated by phosphorylation of Pol II and other factors by Cdk8, by histone methyltransferase recruitment, and by occlusion of the Pol II binding site. However, this module also appears to function in activation in some contexts; for example, it promotes Wnt target gene expression during Drosophila and mouse development, in mammalian cells, and in colon cancer. Although all four subunits have very similar mutant phenotypes in yeast, loss of Med12 or Med13 has more severe effects on Drosophila development than loss of Cdk8 or CycC, suggesting that Med12 and Med13 have evolved additional functions in higher eukaryotes (Janody, 2011).

This study shows that Notch target genes at the wing margin can be divided into three classes based on their requirements for kinase module subunits. An E(spl)m8 reporter requires all four subunits for its activation, cut requires only Med12 and Med13 (known as Kohtalo [Kto] and Skuld [Skd], respectively, in Drosophila) for its activation, and wg and the vgBE enhancer require Med12 and Med13 for their repression in cells close to the wing margin. Because Med12 and Med13 coimmunoprecipitate with Su(H), regulate an artificial reporter driven by Su(H) binding sites, and can be replaced by a VP16 activation domain or a WRPW repression signal fused to Su(H), it is proposed that the kinase module directly regulates Notch target genes. All four Notch target genes fail to be expressed in the absence of Mam and are similarly affected by the loss of Hairless or Gro, suggesting that other more specific cofactors might recruit kinase module subunits to these genes (Janody, 2011).

The kinase module of the mediator complex is conserved throughout eukaryotes, yet its functions in transcription remain poorly understood. In yeast, loss of any of the four subunits has a very similar effect. In Drosophila, however, loss of Med12 or Med13 has more dramatic effects than loss of Cdk8 or CycC. The kinase module was originally thought to be primarily important for transcriptional repression, mediated by the kinase activity of Cdk8. However, Med12 and Med13 appear to directly activate genes regulated by Wnt signaling in Drosophila and mammalian systems, and also play a positive role in gene activation by the Gli3 and Nanog transcription factors. The data presented in this study confirm that Med12 and Med13 have functions distinct from Cdk8 and CycC. In addition, evidence is provided that all four kinase module subunits contribute to the activation of E(spl)m8 (Janody, 2011).

The human Mastermind homologue MAM has been shown to recruit Cdk8 and CycC to promoters of Notch target genes, where Cdk8 phosphorylates the intracellular domain of Notch, leading to its ubiquitination by the Fbw7 ligase and degradation (Fryer, 2004). This mechanism would be expected to reduce Notch target gene expression, consistent with the increase in E(spl)mβ expression seen in clones lacking the Drosophila Fbw7 homologue Archipelago (Nicholson, 2011); thus it cannot explain the positive effects of Cdk8 and CycC on E(spl)m8. A function for Cdk8 and CycC in Notch-mediated activation would be analogous to recent findings showing that Cdk8 phosphorylation of Smad transcription factors and of histone H3 promotes activation. Cdk8 phosphorylation of RNA polymerase II (Pol II) is also important for transcriptional elongation (Janody, 2011).

Of interest, the current data also suggest that Med12 and Med13 are involved in the repression of wg and the vgBE enhancer in the absence of Notch signaling. The kinase module has been proposed to inhibit transcription through steric hindrance of Pol II binding, independently of Cdk8 kinase activity. Removal of this module on the C/EBP promoter is thought to convert the mediator complex to its active form. In contrast, this study find that wg and vgBE require Med12 and Med13 for their repression but not their activation, while cut and E(spl)m8 require Med12 and Med13 only for their activation, arguing that the two functions occur on different promoters. It cannot be ruled out that Med12 and Med13 have only indirect effects on some of the genes examined; however, their physical association with Su(H) and the requirement for Su(H) binding sites for misexpression of an artificial reporter in skd and kto mutant clones are consistent with a direct effect of Med12 and Med13 on the Su(H) complex (Janody, 2011).

Med12 and Med13 are found associated with both active and inactive promoters in genome-wide chromatin immunoprecipitation studies, suggesting that they can have different effects on transcription when bound to distinct interaction partners. Although both are very large proteins, they contain no domains predicted to have enzymatic activity, and may instead act as scaffolds for the assembly of transcriptional complexes (Janody, 2011).

It has been proposed that Notch target genes could be categorized into two classes: permissive genes, for which the primary function of Notch is to relieve repression by the Su(H) complex, and instructive genes, for which Notch plays an essential role in activation by recruiting specific coactivators. These differences presumably depend on the combinatorial code of transcription factors that regulate each promoter. This study shows that vgBE, an enhancer previously placed in the permissive category, as well as wg, require Med12 and Med13 for their repression but not their activation. During eye development, the proneural gene atonal is likewise regulated permissively by Notch, and ectopically expressed in skd or kto mutant clones. Unexpectedly, this study found that Gro, previously thought to be a cofactor through which Hairless mediates repression, is not required for the repression of vgBE or wg. Hairless may repress target genes at the wing margin through CtBP, its other binding partner. Alternatively, Gro may affect the expression of other upstream regulators of wing margin fate, masking its repressive effect on the genes that were examined (Janody, 2011).

It was also show in this study that instructive Notch target genes can be further subdivided into two classes based on their requirement for kinase module subunits; E(spl)m8 requires all four subunits, while cut requires Med12 and Med13, but not Cdk8 and CycC. Cdk8 and CycC may simply increase the ability of the mediator complex to recruit Pol II or promote transcriptional initiation; this model would suggest that E(spl)m8 has a higher activation threshold than cut. Alternatively, Cdk8 and CycC might enhance the function of a transcription factor that is specifically required for the expression of E(spl)m8 but not cut. Good candidates for such factors would be the proneural proteins Achaete or Scute or their partner Daughterless (Janody, 2011).

The mechanism by which the kinase module is recruited to promote the activation of instructive target genes is not yet clear. Although Mam proteins are well-characterized coactivators for Nintra, this study found that Mam is necessary for the activation of both instructive and permissive genes. It may thus have a general function in transcriptional activation, such as recruiting histone acetyltransferases or stabilizing the Notch-Su(H) complex. A coactivator that recruits Med12 and Med13 specifically to instructive target genes to promote activation may remain to be identified. The current results, like recent reports demonstrating that the arrangement of Su(H) binding sites can affect the interactions between Notch and its coactivators, highlight the complexity in the mechanisms through which promoter elements respond to Notch signaling (Janody, 2011).

A rapid one-generation genetic screen in a Drosophila model to capture rhabdomyosarcoma effectors and therapeutic targets

Rhabdomyosarcoma (RMS; see Drosophila as a Model for Human Diseases - Rhabdomyosarcoma) is an aggressive childhood malignancy of neoplastic muscle-lineage precursors that fail to terminally differentiate into syncytial muscle. The most aggressive form of RMS, Alveolar-RMS (A-RMS), is driven by misexpression of the PAX-FOXO1 oncoprotein, which is generated by recurrent chromosomal translocations that fuse either the PAX3 or PAX7 gene (homologs of Drosophila Paired) to FOXO1 (homolog of Drosophila Foxo). The molecular underpinnings of PAX-FOXO1-mediated RMS pathogenesis remain unclear, however, and clinical outcomes poor. This study reports a new approach to dissect RMS, exploiting a highly efficient Drosophila PAX7-FOXO1 model uniquely configured to uncover PAX-FOXO1 RMS genetic effectors in only one generation. With this system, a comprehensive deletion screen was performed against the Drosophila autosomes, and mutation of Mef2, a myogenesis lynchpin in both flies and mammals, was demonstrated to dominantly suppresses PAX7-FOXO1 pathogenicity and act as a PAX7-FOXO1 gene target. Additionally, mutation of mastermind, a gene encoding a MEF2 transcriptional co-activator, was shown to similarly suppress PAX7-FOXO1, further pointing towards MEF2 transcriptional activity as a PAX-FOXO1 underpinning. These studies show the utility of the PAX-FOXO1 Drosophila system as a robust one-generation (F1) RMS gene discovery platform and demonstrate how Drosophila transgenic conditional expression models can be configured for the rapid dissection of human disease (Galindo, 2014: PubMed).

Given the critical role that the PAX-FOXO1 fusion oncoprotein plays in RMS, this study focuses on PAX-FOXO1 as an entry-point for designing a transgenic Drosophila RMS-related model that would be amenable to forward genetic screening and RMS gene discovery. To bypass the issue of cumbersome multigenerational screening schemes that would normally be required, a Gal80 X-linked chromosomal transgene was incorporated to generate a viable screening Gal4/UAS-PAX-FOXO1 master stock that allows for the rapid identification of PAX-FOXO1 genetic modifiers in a single genetic cross (Galindo, 2014).

With this platform, new PAX-FOXO1 pathogenesis underpinnings were probed. Though very similar in molecular structure, PAX3-FOXO1− and PAX7-FOXO1−positive RMS demonstrate differing clinical behaviors, as PAX3-FOXO1 tumors are more common and notoriously aggressive. Consequently, PAX3-FOXO1 is the PAX-FOXO1 fusion most commonly investigated in vertebrate models. This study focuses on PAX7-FOXO1 in the Drosophila system, which demonstrates phenotypes that are better penetrant and experimentally tractable due to the fact that human PAX7 demonstrates slightly greater sequence identity to fly PAX3/7 than does human PAX3. Additionally, as no other animal models of PAX7-FOXO1 presently exist, the fly PAX7-FOXO1 model also conveniently serves as a complement to vertebrate PAX3-FOXO1 models (Galindo, 2014).

The extent to which observations from the PAX7-FOXO1 fly model would impact the clinically more aggressive PAX3-FOXO1 RMS subtype, as well as PAX-FOXO1-negative (embryonal) RMS, is unknown. Notably, previous studies show that genetic modifiers identified from the Drosophila system impact PAX3-FOXO1 RMS oncogenesis and tumorigenesis. Furthermore, unpublished studies suggest that fly PAX7-FOXO1 genetic modifiers are similarly involved in Embryonal RMS. These findings provide marked validation for the applicability and value of this genetic fly system to human RMS (Galindo, 2014).

Interestingly, though PAX7-FOXO1 induces expression of the late myogenic differentiation marker MHC, PAX-FOXO1 RMS myoblasts in culture and in vivo demonstrate only partial differentiation with little-to-no MHC expression. In considering this discrepancy, it should be first noted that PAX-FOXO1 is a relatively weak driver of RMS in culture and in vivo and requires additional/sequential genetic aberrations to induce oncogenic transformation. Thus, secondary mutations might be necessary to force the strength of RMS myoblast differentiation-arrest seen in human RMS tumors; by contrast, the PAX7-FOXO1 model of this study differs in that the system is free of any additional background mutations. Second, earlier studies show that expression of PAX3-FOXO1 in mouse embryonic cultured cells induces the formation of MHC-positive myocytes and myotube, similar to the Drosophila system in this study as the da-Gal4/UAS-PAX7-FOXO1 expression system targets undifferentiated embryonic primordia. Uncovering of the genetic/molecular sequence of RMS pathogenesis and the cell(s) origin will shed further insight into the underlying mechanisms that account for the myoblast differentiation arrest phenotypes seen in RMS in vivo (Galindo, 2014).

The differentiation and fusion of myoblasts into postmitotic, syncytial muscle requires that the bHLH myogenic regulatory factors (MRFs: Myf5, Mrf4, MyoD, and Myogenin) interact with E-proteins, which drive and regulate critical aspects of myogenic fate determination. The MRFs subsequently interact with the MEF2 transcription factors that, although lacking intrinsic myogenic activity, cooperate with the MRFs to synergistically activate muscle-specific genes and the downstream myogenic terminal differentiation program. Vertebrates possess four MEF2 family member genes (-A, -B, -C, -D), which demonstrate complex overlapping spatial and temporal expression patterns in embryonic and adult tissues, with greatest expression levels seen in striated muscle and brain. Because of genetic redundancy and overlapping expression patterns of the MEF2 genes, interrogating individual MEF2 gene activity in mammals is experimentally challenging, with loss-of-function mutation studies revealing only limited insights into MEF2 gene function in tissues in which the MEF2 genes do not overlap/compensate. Conveniently, flies possess only one Mef2 gene (D-Mef2) and serve as an excellent model system to delineate MEF2’s critical role in myogenesis. The study speculates that the lack of Mef2 redundancy in flies provides a marked experimental advantage in isolating D-Mef2 as a PAX7-FOXO1 effector. Similarly, the identification of mam was also likely facilitated by the fact that flies possess one mam gene, whereas mammals contain three mam orthologs. Thus, the study proposes that the comparative lack of genetic compensation/redundancy is an attractive advantage to Drosophila as a disease model system (Galindo, 2014).

The study suggests that further interrogation of MEF2 in RMS will open new avenues for RMS chemotherapy, which for high-risk disease has not improved for decades. For example, since MEF2 activity is tightly governed by class IIa histone deacetylases, histone deacetylase inhibitors are now ripe for preclinical testing as new anti-RMS agents. Additionally, it was found that the MEF2 cofactor Mastermind, which interacts with MEF2C and mediates crosstalk between Notch signals during myogenic differentiation, similarly influences PAX-FOXO1 pathogenicity in flies. Interestingly, Mastermind-specific, cell-permeable peptide inhibitors have been shown to block the progression of T-cell acute lymphoblastic leukemia in mice in vivo and thus are also new agents available for RMS preclinical testing. Further characterization of MEF2 in RMS cell and mouse models will continue to refine both our understanding and the potential targeting of MEF2 activity in RMS (Galindo, 2014).

In conclusion, the study postulates that: 1) The Drosophila PAX7-FOXO1 model is uniquely configured for the quick uncovering of new RMS genetic effectors with one simple genetic screening cross; 2) a putative PAX-FOXO1-to-MEF2/MASTERMIND axis underlies A-RMS; and 3) Drosophila conditional expression models are an efficient and powerful gene discovery platform for the rapid dissection of human disease (Galindo, 2014).

mastermind: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | References

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