Mastermind

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 N^intra] 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 N^intra or Su(H)-VP16 is likely to be beyond physiological levels. The FSC loss induced by N^intra 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, mγ, and mδ 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 mγ) 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 (S_F-X or X-S_R), or orientation variants in which the orientation of one or both S sites have been reversed (S_R-S_F, S_F- S_F, and S_R-S_R) (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 (S_F-X or X-S_R) 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 (S_R-S_F, S_F-S_F, and S_R-S_R), only the wild-type (S_F-S_R) 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 (S_F-S_R) 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 mγ and mδ 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 mγ and mδ 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 (S_F-S_R) 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 S_R-S_R 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 S_F-S_R and S_R-S_F 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 S_R-S_F sites in all of the promoters tested is not due simply to decreased ability of Su(H) protein to bind to the S_R-S_F 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 (S_F-S_R) or S_R-S_F 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 S_R-S_F SPS variant showed no expression or very low levels of expression, respectively. Extended staining of discs containing the S_R-S_F 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 (N^nd-1) mutation and decrease the severity of the vein-shortening phenotype caused by a gain-of-function Notch mutation (N^Ax-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-A^NC106 for an exon that is not included in the rare variant is lethal and causes defects in Notch signaling. Nipped-A^NC106, however, had little effect on the N^Ax-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-A^NC105 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-A^NC105 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-A^NC105 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-A^NC105 on all three phenotypes. It is also unlikely that there is a linked second-site mutation that counteracts the effects of Nipped-A^NC105 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-A^NC194 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 mam^g2. Again, this differs from null alleles of Nipped-A, which affect all three phenotypes, suggesting that Nipped-A^NC194 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-A^NC194 on the mastermind phenotype. Similar to Nipped-A^NC105, heterozygous Nipped-A^NC194 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-A^NC96 hypomorph, the N^nd-1 phenotype is more sensitive to the Nipped-A dosage than is mastermind. Moreover, the Nipped-A^NC194 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 N^nd-1 and N^Ax-E2 phenotypes and the Mastermind phenotype are modified by domino mutations, although the effect on N^Ax-E2 is modest. These effects are similar to those of the Nipped-A^NC106 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-A^NC96 and Nipped-A^NC186 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).

DEVELOPMENTAL BIOLOGY

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

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