Enhancer of split


Promoter Structure and Transcriptional Regulation

Suppressor of Hairless [Su(H)] directly activates transcription of E(spl)-C genes in response to Notch receptor activity. The proximal upstream regions of three E(spl)-C genes contain multiple specific binding sites for Su(H). The integrity of these sites, as well as Su(H) gene activity, is required for both normal levels of expression of E(spl)-C genes in imaginal disc proneural clusters and their transcriptional response to hyperactivity of the N receptor. Su(H) is thus a direct regulatory link between N receptor activity and the expression of E(spl)-C genes (Bailey, 1995 and Lecourtois, 1995).

Enhancer of split is a direct downstream target of Achaete and Scute as judged by the following two criteria: first, E(spl) is expressed in proneural clusters of the wing imaginal disc in an AC-SC-dependent manner; second, the proximal promoter contains high-affinity AC-SC binding sites, which define the novel consensus GCAGGTG(T/G)NNNYY (Singson, 1994).

Activation sites in the promoters of E(spl) genes have been identified. About 0.46 kb of E(spl) and 1.9 kb of HLH-m5 upstream sequences are necessary to reproduce the normal transcription pattern of these genes. The gene products of achaete, scute and lethal of scute, together with that of ventral nervous system condensation defective, act synergistically to specify the neuroectodermal E(spl) and HLH-m5 expression domains. Negative cross- and autoregulatory interactions of the E(SPL)-C on E(spl) contribute, directly or indirectly, to this regulation (Kramatschek, 1994).

It is now clear that E(spl)-C gene expression is totally dependent on lateral inhibition and the Notch pathway acting through Suppressor of Hairless. If this is true, then the role of E-boxes in the transcriptional activation of E(spl)-C genes is currently unclear. Perhaps VND activate proneural genes which in turn activate E(spl)-C genes through the Notch pathway, although the possibility of direct interaction of VND with E(spl)-C genes has not been excluded. Achaete and Scute upregulate E(spl)m7 and Enhancer of split in a wing disc pattern very similar to that achaete and scute expression. This is surprising since the wild function of E(spl)-C genes is to antagonize the SOP cell fate within the proneural cluster. It is thought that other mechanisms (Notch signaling for example) normally operate to regulate the SOP expression or activity of E(spl)-C genes (Singson, 1994).

In Drosophila, genes of the Enhancer of split Complex [E(spl)-C] are important components of the Notch (N) cell-cell signaling pathway, which is utilized in imaginal discs to effect a series of cell fate decisions during adult peripheral nervous system development. Seven genes in the complex encode basic helix-loop-helix (bHLH) transcriptional repressors, while four others encode members of the Bearded family of small proteins. A striking diversity is observed in the imaginal disc expression patterns of the various E(spl)-C genes, suggestive of a diversity of function, but the mechanistic basis of this variety has not been elucidated. Strong evidence is presented from promoter-reporter transgene experiments that regulation at the transcriptional level is primarily responsible. Certain E(spl)-C genes are direct targets of transcriptional activation both by the N-signal-dependent activator Suppressor of Hairless [Su(H)] and by the proneural bHLH proteins Achaete and Scute. An extensive sequence analysis of the promoter-proximal upstream regions of 12 transcription units in the E(spl)-C reveals that such dual transcriptional activation is likely to be the rule for at least 10 of the 12 genes. The very different wing imaginal disc expression patterns of E(spl)m4 and E(spl)mgamma are a property of small (200-300 bp), evolutionarily conserved transcriptional enhancer elements that can confer these distinct patterns on a heterologous promoter despite their considerable structural similarity [each having three Su(H) and two proneural protein binding sites]. The characteristic inactivity of the E(spl)mgamma enhancer in the notum and margin territories of the wing disc can be overcome by elevated activity of the N receptor. It is concluded that the distinctive expression patterns of E(spl)-C genes in imaginal tissues depend to a significant degree on the capacity of their transcriptional cis-regulatory apparatus to respond selectively to direct proneural- and Su(H)-mediated activation, often only in a subset of the territories and cells in which these modes of regulation are operative (Nellesen, 1999).

Others argue that achaete and scute do not regulate E(spl). In the developing sensory precursor cell, expression of E(spl) is dependent on the activity of Notch and does not directly correlate with expression of achaete (Jennings, 1995).

Human RBP-J kappa protein is the homolog of Drosophila Suppressor of hairless. Database search identified the best naturally occurring binding motif (CACTGTGGGAACGG) for the RBP-J kappa protein in the promoter region of the E(spl) gene in the Enhancer of split gene cluster of Drosophila (Tun, 1994).

Is it possible to estimate the number of target genes of the homeoproteins Eve and Ftz? Eve and Ftz have been shown to bind with similar specificities to many genes, including four genes chosen because they were thought to be unlikely targets of Eve and Ftz. Eve and Ftz bind at the highest levels to DNA fragments throughout the length of three probable target genes: eve, ftz and Ubx. However, Eve and Ftz also bind at only two- to ten-fold lower levels to four genes chosen in an attempt to find non targets: Adh, hsp70, rosy and actin 5C, suggesting that Eve and Ftz bind at significant levels to a majority of genes. The expression of these four unexpected targets is controlled by Eve and probably by the other selector homeoproteins as well. A correlation is observed between the level of DNA binding and the degree to which gene expression is regulated by Eve (Liang, 1998).

At stages 10-14, 87% of cDNAs in the 8-12 hour library are likely to be directly or indirectly regulated by Eve, Ftz, Engrailed and all of the Hox proteins. These downstream genes are each expressed in unique, segmentally repeating patterns. Some are expressed at dramatically altered levels between segments. Most vary from segment to segment in the number and position of cells in which they are most prominently expressed. This is not simply because expression follows the distribution of a particular cell type. Between segments, the majority of genes are most highly expressed in differently positioned subsets of the same cell types, indicating that these patterns cannot result solely from the action of cell-type specific transcription factors. Eve, Ftz and Engrailed establish the segmentally repeating structure of the embryo. Therefore, all genes expressed in segmentally repeated patterns by stage 11 should be downstream of these three genes. This has been experimentally confirmed for eve and ftz. The expression of all 14 segmentally expressed genes tested is altered in eve and ftz mutant embryos at stage 11. These and other downstream genes can be divided into three classes: genes expressed in strong, moderate or weak segmentally repeated patterns. 33% of cDNAs fall into the strongly repeated class. For this class, staining levels vary five fold or more between cells across a transverse section of a segment along the anterior/posterior axis of the embryo. 24% of clones belong to the moderately regulated class. These genes show two- to five-fold variations in staining across the width of a segment. Finally, the weak segmentally repeated genes vary only 1.2 to 2 fold in staining between cells across a segment. Thus, most downstream genes are expressed in all cells, but each are still subject to specific and precise control by the selector homeoproteins. The more strongly regulated genes include many developmental control genes such as Enhancer of split [E(spl)] , tramtrack, division abnormally delayed (dally), and Dwnt4. A high proportion of the moderate and weakly regulated genes are involved in essential cellular functions such as splicing (e.g. RNA helicases), translation (e.g. met tRNA synthetase), general signal transduction (e.g. G-protein beta13F) and cytoskeletal structure (e.g. alpha tubulin 84B). This raises the question of whether or not modest changes in the expression of essential enzymes and structural proteins are important for morphogenesis. It is argued that they probably are. 11% of the genes picked from the 8-12 hour cDNA library do not appear to be downstream of the selector homeoproteins. Most of these genes are expressed relatively uniformly in all cells. But even these genes show some differences in expression pattern (Liang, 1998).

In the developing Drosophila retina, the proneural gene for photoreceptor neurons is atonal, a basic helix-loop-helix transcription factor. Using atonal as a marker for proneural maturation, the stepwise resolution of proneural clusters was examined during the initiation of ommatidial differentiation in the developing eye disc. In addition, evidence is provided that atonal is negatively regulated by rough, a homeobox-containing transcription factor expressed exclusively in the retina. This interaction leads to the refinement of proneural clusters to specify R8, the first neuron to emerge in the retinal neuroepithelium. Either ectopic expression of atonal or the removal of rough results in the transformation of a discrete 'equivalence group' of cells into R8s. In addition, ectopic expression of rough blocks atonal expression and proneural cluster formation within the morphogenetic furrow. Thus, rough provides retina-specific regulation to the more general atonal-mediated proneural differentiation pathway. Expression of Rough and Atonal is mutually exclusive: as atonal expression resolves from an initial ubiquitous stripe to individual proneural clusters, rough expression emerges in the intervening cells. The opposing roles of atonal and rough are not mediated through the Notch pathway, as their expression remains complementary when Notch activity is reduced. These observations suggest that homeobox-containing genes can serve a function of tissue-specific repression for bHLH factors. Rough is not likely to be a direct negative regulator of Enhancer of split expression since their expression patterns show extensive overlap. Instead, Rough-induced loss of E(spl) expression may be due to loss of atonal expression in a manner analogous to E(spl) requirement for achaete and scute activity. Notch signaling is also presumably required for E(spl) expression in this system (Dokucu, 1997).

Strawberry notch is a nuclear protein that functions downstream of Notch. Subjecting temperature sensitive strawberry notch to heat shock results in a down regulation of wg at the wing margin. Expression of wg in other regions of the wing disc as well as in other imaginal discs is unaffected by the loss of sno function. Likewise sno is required for the expression of vestigial, cut and E(spl)-m8 at the wing margin (Majumdar, 1997).

Immunohistochemical detection of Mastermind on polytene chromosomes reveals binding at >100 sites. Chromosome colocalization studies with RNA polymerase and the Groucho corepressor protein implicate Mam in transcriptional regulation (Bettler, 1996).

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

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

The transcriptional effect of E(spl) region transcript m4 (m4/alpha) is observed in S2 cultured cells, where two different luciferase reporter genes, one driven by a promoter fragment of E(spl)- mgamma and one by a promoter fragment of E(spl)-m8 were tested. These promoters are induced to 6x and 12x, respectively, after cotransfection with plasmids expressing Su(H) and an intra-cellular activated form of Notch (RICN). Addition of expression plasmid for m4 or malpha gives a small yet consistent ~2x repression of both reporters. In contrast, m4/alpha has no effect on an unrelated luciferase reporter gene driven by the achaete proximal promoter. A specific repressive effect of m4/alpha is thus observed on E(spl)bHLH transcription both in imaginal discs and in cultured cells. The ability of m4/alpha to downregulate E(spl)bHLH levels, could account for their promotion of SOP fate. If this is the sole activity of m4/alpha proteins, then restoring the expression of E(spl)bHLH genes should abolish the m4/alpha gain-of-function (GOF) phenotype. Scutellum specific GAL4 lines were used to ectopically co-express m4 or malpha with one of the E(spl)bHLH genes. Whereas m4/alpha alone produces 6+/-13 scutellar bristles, coexpression of E(spl)bHLH eliminates scutellars, a phenotype indistinguishable from the gain-of-function phenotype of E(spl)bHLH alone. This epistatic relation of UAS-E(spl) over UAS-m4/alpha led to the conclusion that m4/alpha cannot antagonize E(spl) bHLH factors at the protein level; rather, they must act upstream of E(spl) bHLH accumulation. Consistent with this conclusion, no interactions is observed for m4 or malpha with a number of E(spl) bHLH or with their co-repressor Groucho in a yeast two-hybrid assay. Furthermore, m4/alpha does not interact with the proneural proteins Da, Ac, Sc or Ato. In fact m4 appears to be a cytoplasmic protein, at least as far as can be judged from the subcellular localization of an m4- green fluorescent protein (GFP) fusion, making interaction with these nuclear factors unlikely (Apidianakis, 1999).

Experiments by Nagel (2000), suggest that the overexpression phenotype of E(spl) m4 and E(spl) malpha obtained by Apidianakis (1999) is likely to be due to a dominant negative effect and does not reflect the biological function of these two genes. In order to elucidate m4/malpha gene function directly, RNAi, which causes sequence-specific transcript degradation, was carried out by injecting either m4 or malpha double-stranded RNA or a mixture of both into pre-blastoderm embryos. In agreement with genetic data, RNAi causes a high incidence of lethality (~50%). Dead embryos develop intermediate to strong neurogenic phenotypes (too many neurons) typical of loss of E(spl) bHLH activity. Surviving embryos hatch into wild type appearing larvae that develop normally to adult flies. From this it is concluded that the m4/malpha genes are required to positively transduce the Notch signal during neurogenesis, and presumably during bristle development as well. Therefore, suppression of lateral inhibition observed after overexpression of either m4/malpha family member must be due to a dominant-negative effect, presumably by titrating out other important Notch pathway components (Nagel, 2000b).

Composite signalling from Serrate and Delta establishes leg segments in Drosophila through Notch

The expression of the Serrate and Delta genes patterns the segments of the leg in Drosophila by a combination of their signaling activities. Coincident stripes of Serrate and Delta expressing cells activate Enhancer of split expression in adjacent cells through Notch signaling. These cells form a patterning boundary from which a putative secondary signal leads to the development of leg joints. Elsewhere in the tarsal segments, signaling by Dl and N is necessary for the development of non-joint parts of the leg. It is proposed that these two effects result from different thresholds of N activation, which are translated into different downstream gene expression effects. A general mechanism is proposed for creation of boundaries by Notch signaling (Bishop, 1999).

As a marker of N activity, the expression of members of the E(spl) complex has been monitored. Using reporter constructs with the regulatory regions of E(spl), which reproduce the endogenous E(spl) expression in the leg discs, it can be seen that E(spl)m8 expression is related to joints while m5 and presumably m6 are not. Expression of the E(spl)m8 reporter construct in third instar discs is initially strong in regions undergoing PNS development. In the legs, these correspond to the chordotonal organs in the femur and the tibia. In addition, expression near the presumptive joints is seen to appear, and then resolve in the pupa into one-cell wide stripes proximal to the leg constrictions, in positions that correlate with cells with maximum levels of disco expression. A similar although much weaker pattern of expression of E(spl)mdelta is seen as revealed by the mAb323 antibody. Another marker of N activity is the expression of N itself, which becomes upregulated in cells where N signaling is being received. Using an anti-N antibody, upregulated expression of N is seen immediately proximal to constrictions in pupal legs. This upregulation is restricted to a single row of cells at this position, thus confirming that Ser and Dl are triggering N signaling in these cells (Bishop, 1999).

These results suggest a model in which the co-expression of Ser and high levels of Dl in a stripe of cells activate N in cells adjacent but distal to this stripe. Activation of N promotes expression of members of the E(spl) complex and leads to joint formation and disco expression. Loss of Dl eliminates first the regions between disco/Ser-expressing rings, but also, secondly, joints. Since loss of interjoint regions is also seen both in N mutants and following expression of a dominant-negative form of N, it is postulated that Dl expression in the interjoint regions produces low levels of activation of N that do not lead to E(spl) expression but which allow cell survival and/or cell proliferation. Joint loss in Dl mutants is presumably less severe than interjoint loss because Ser and Dl expression could be synergistic and partially redundant. The combined and potentially synergistic effects of Ser and Dl would produce a high level of activation of N that would lead to expression of members of the E(spl) complex, upregulation of N expression, and to joint development and disco expression. Thus, it is believed that combinations of signaling by Ser and Dl could produce different levels of activation of N, which in turn are translated into different downstream effects. As noted in other systems these downstream effects of N signaling should be mediated by more factors than just E(spl), since E(spl) mutant legs have been reported as having a wild-type phenotype (Bishop, 1999 and references).

The width of the final joint region is wider than the single row of cells activated by the membrane-tethered Ser and Dl proteins and visualised by E(spl) expression. In principle it is possible that the cells of the whole final joint all descend from the E(spl) expressing cells, but previous studies have shown that only one or two cell divisions occur in the legs after puparium formation. Thus it is likely that in the E(spl) expressing cells another cell signaling molecule is activated, which in a secondary event would define a wider joint presumptive region, just as N-induced expression of the secreted signaling wingless protein defines the presumptive wing margin. A reflection of this putative second signaling event in the joints can be seen in the expression of disco. disco expression is dependent on Ser but it is wider than the single row of cells where N is activated and thus it cannot be directly reflecting N signaling at the joint. However, the 'bell-shaped' distribution of disco might reflect this putative secondary signaling event, with a maximum in cells at the edge of the Ser-Dl stripe. The nature of the joint-promoting putative secondary signal is unknown at the moment, but one possible component is the product of the four-jointed (fj) gene. The fj protein is a putative signaling molecule that is expressed and required at the joints. fj expression has recently been shown to depend on fng and N signaling during eye development, and it is lost in N mutant legs (Bishop, 1999 and references).

An autonomous negative effect of Dl and Ser does not explain why cells adjacent but proximal to the Ser-Dl stripe do not seem to be signaled. A possible explanation would be either an asymmetric distribution of Ser and Dl, forming gradients like those seen in the late third instar wing margin and in ectopic expression situations, or a downregulation of N expression as has been noted in the developing wing veins. The Ser and Dl stripes in legs show no apparent asymmetry but N distribution, although ubiquitous and initially uniform, becomes upregulated in cells distal to the Ser-Dl stripes. Low availability of N protein could have an effect on the intensity of N signaling, but since upregulation of N is in itself a consequence of N signaling, some other factor must polarize the signaling initially. Another explanation would rely on the action of a repressor acting upon cells proximal to the stripe. The phenotypes obtained after ectopic expression of fng are consistent with such a role for fng, as postulated in the wing. The expression of fng in the leg, which has been described as complementary to that of E(spl), that is, present in non-signaled cells but excluded from joint forming ones, is also consistent with this hypothesis. Such a function of fng could also repress Ser and Dl signaling in the stripe without recourse, or in addition, to putative autonomous dominant negative effects of Ser and Dl. However, other factors could also be involved, such as the cell polarity pathway. Mutant phenotypes for dsh and other members of the cell polarity pathway produce ectopic joints with reversed polarity, which appear just proximal to the position of Ser and Dl stripes. Furthermore, in dsh mutants ectopic N activation is seen proximal to the Ser-Dl stripe. Since the Dsh protein has been shown to interact with N, and Dsh has been postulated to inhibit N signaling in this manner, the cell polarity pathway could be involved in repressing Ser and Dl signaling to cells proximal to the Ser and Dl stripe (Bishop, 1999 and references).

The Snail repressor positions Notch signaling in the Drosophila embryo

The maternal Dorsal nuclear gradient initiates the differentiation of the mesoderm, neurogenic ectoderm and dorsal ectoderm in the precellular Drosophila embryo. Each tissue is subsequently subdivided into multiple cell types during gastrulation. This study investigates the formation of the mesectoderm within the ventral-most region of the neurogenic ectoderm. Previous studies suggest that the Dorsal gradient works in concert with Notch signaling to specify the mesectoderm through the activation of the regulatory gene sim within single lines of cells that straddle the presumptive mesoderm. This model was confirmed by misexpressing a constitutively activated form of the Notch receptor, NotchIC, in transgenic embryos using the eve stripe2 enhancer. The NotchIC stripe induces ectopic expression of sim in the neurogenic ectoderm where there are low levels of the Dorsal gradient. sim is not activated in the ventral mesoderm, due to inhibition by the localized zinc-finger Snail repressor, which is selectively expressed in the ventral mesoderm. Additional studies suggest that the Snail repressor can also stimulate Notch signaling. A stripe2-snail transgene appears to induce Notch signaling in 'naïve' embryos that contain low uniform levels of Dorsal. It is suggested that these dual activities of Snail -- repression of Notch target genes and stimulation of Notch signaling -- help define precise lines of sim expression within the neurogenic ectoderm (Cowden, 2002).

This study provides further evidence that Notch signaling is essential for the formation of the mesectoderm at the boundary between the mesoderm and neurogenic ectoderm. Two different Notch target genes were examined: m8 expression appears to depend almost exclusively on Notch signaling, whereas sim is a conditional Notch target gene that is activated only in cells containing Dorsal. Evidence is presented that Snail functions as both a repressor and an indirect activator of Notch signaling. In particular, a transient stripe of the Snail repressor creates a domain of Notch signaling in apolar embryos that contain low, uniform levels of Dorsal (Cowden, 2002).

A crucial finding of this study is that a stripe2-snail transgene induces ectopic expression of m8 and sim in both wild-type and Tollrm9/Tollrm10 mutant embryos, suggesting that the Snail repressor is actually playing a positive role in Notch signaling. Importantly, this stimulatory activity depends on the ability of Snail to function as a transcriptional repressor. Mutant forms of the stripe2-snail transgene that contain single amino acid substitutions in the two repression domains (PxDLSxK and PxDLSxR) fail to induce sim and m8 expression in either wild-type or Tollrm9/Tollrm10 mutant embryos. By contrast, a stripe2-snail/hairy transgene that contains the Hairy repression domain continues to activate both sim and m8 in mutant embryos (Cowden, 2002).

The localized Snail repressor restricts Notch signaling to the mesectoderm of early embryos, presumably by directly repressing Notch target genes. Indeed, the sim 5' regulatory region contains a series of high-affinity Snail repressor sites. It is conceivable that Snail restricts Notch signaling in other developmental processes. For example, after its transient expression in the ventral mesoderm of early embryos, snail is reactivated in delaminating neuroblasts at the completion of germ band elongation. At this stage, Notch signaling subdivides the neurogenic ectoderm into neurons and ventral epidermis. Notch is selectively activated in epidermal cells, where it induces the expression of E(spl) repressors that silence Achaete-Scute proneural genes. The localized expression of the Snail repressor in delaminating neuroblasts might help ensure neuronal differentiation by inhibiting Notch-specific target genes. Removal of snail along with two related linked zinc-finger repressors (Worniu and Escargot) leads to a reduction in the number of CNS neuroblasts (Cowden, 2002).

It is proposed that Snail functions as a gradient repressor to restrict Notch signaling. In precellular embryos, the initial snail expression pattern is broad and extends into the future mesectoderm. During cellularization, the pattern is refined and snail expression is lost in the mesectoderm and restricted to the mesoderm. The early, broad snail pattern might create a broad domain of potential Notch signaling by repressing components of the Notch pathway, such as Delta and lethal of scute. After cellularization, Notch signaling is blocked in the presumptive mesoderm by sustained, high levels of the Snail repressor. However, Notch can be activated in the mesectoderm because of the loss of Notch inhibitors repressed by transient expression of the Snail repressor. According to this model, the dynamic snail expression pattern determines both the timing and limits of Notch signaling (Cowden, 2002).

The analysis of Tollrm9/Tollrm10 embryos suggests that Dorsal functions synergistically with Notch signaling to activate sim expression. A stripe2-NotchIC transgene induces strong sim expression in these embryos, even though they contain low levels of Dorsal and lack Twist. However, the same transgene barely activates sim when crossed into embryos that lack both Dorsal and Twist. By contrast, m8 is strongly expressed in these mutants, indicating m8 is primarily activated by Su(H)-NotchIC and does not require Dorsal (Cowden, 2002).

Perhaps the low levels of Dorsal present in the presumptive mesectoderm are not sufficient to activate sim. Instead, activation might rely on protein-protein interactions between Dorsal and the Su(H)-NotchIC complex within the sim 5' cis-regulatory region. sim contains a number of optimal Su(H) recognition sequences; these might help recruit Dorsal to adjacent sites. By contrast, the stripe2-NotchIC transgene appears to be sufficient to activate m8, even though it contains fewer optimal Su(H) binding sites than the sim 5' cis-regulatory region. Perhaps m8 is ‘poised’ for activation by ubiquitous bHLH activators that are maternally expressed and present throughout early embryos (e.g. Daughterless and Scute). Notch signaling might trigger expression upon binding of the Su(H)-NotchIC complex. By relying on ubiquitous bHLH ‘co-factors’, Notch signaling may be sufficient to activate m8 in diverse cellular contexts. Accordingly, the differential regulation of sim and m8 by Notch signaling is combinatorial and depends on the distribution of distinct co-factors (Cowden, 2002).

A model Notch response element detects Suppressor of Hairless-dependent molecular switch

Cell-cell signaling mediated by Notch is critical during many different developmental processes for the specification or restriction of cell fates. Currently, the only known transduction pathway involves a DNA binding protein, Suppressor of Hairless [Su(H)] in Drosophila and CBF1 in mammals, and results in the direct activation of target genes. It has been proposed that in the absence of Notch, Su(H)/CBF1 acts as a repressor and is converted into an activator through interactions with the Notch intracellular domain. It has also been suggested that the activation of specific target genes requires synergy between Su(H) and other transcriptional activators. An assay has been designed that allows a direct test of these hypotheses in vivo. The results clearly demonstrate that Su(H) is able to function as the core of a molecular switch, repressing transcription in the absence of Notch and activating transcription in the presence of Notch. In its capacity as an activator, Su(H) can cooperate synergistically with a DNA-bound transcription factor, Grainyhead. These interactions indicate a simple model for Notch target-gene regulation that could explain the precision of gene activation elicited by Notch signaling in different developmental fate decisions (Furriols, 2001).

Activation of Notch by its ligands promotes proteolytic processing, releasing an intracellular fragment (Nicd) that embodies most functions of the activated receptor. There is substantial in vivo and in vitro evidence demonstrating that Su(H) and its homologs in other species are required for the activation of Notch target genes, such as the Enhancer of split/HES genes, and it is proposed that Su(H) DNA binding proteins cooperate with Nicd to promote transcription. However, Su(H) and Nicd are relatively ineffectual at activating Enhancer of split [E(spl)] genes in ectopic locations and it appears that their capacity to promote transcription of specific target genes requires synergistic interactions with other enhancer-specific factors. Cell transfection assays have also revealed a potential repressive role for the mammalian homolog of Su(H), CBF1, and indicate that in the absence of Nicd, Su(H)/CBF1 could recruit a corepressor complex to shut off target genes. Recent work in Drosophila has supported this model through the analysis of single-minded, one target gene whose expression is derepressed in animals that lack Su(H) function (Furriols, 2001).

An assay has been designed that allows investigation of whether this is a general mechanism by first testing whether Su(H) can mediate repression of a heterologous activator, and second, whether it can synergize with the same activator in the presence of Nicd to promote transcription (Furriols, 2001).

In order to assess whether Su(H) is able to function as a repressor as well as an activator, it was necessary to target it to a well-defined enhancer that independently confers widespread expression. Through work on the Grainyhead (Grh) transcription factor, a palindromic binding site (Gbe) has been defined that, when combined in three copies with a minimal promoter, confers expression throughout the imaginal discs, epidermis, and trachea of the Drosophila larvae. Since Su(H) is expressed ubiquitously, it was anticipated that when Su(H) sites are combined with Gbe, Su(H) would cooperate with Grh to yield high levels of expression in the cells where Notch is active (e.g., dorsal/ventral boundary, interveins in the wing imaginal disc) and would prevent Grh-mediated activation in cells where Notch is inactive (e.g., larval epidemis, where there is no evidence for Notch activity based on expression patterns of known Notch target genes (Furriols, 2001).

The Su(H) binding sites used were the paired sites derived from the regulatory region of the Enhancer of split m8 gene, which is primarily expressed in association with proneural clusters in the imaginal discs. On their own, two pairs of Su(H)m8 sites only give extremely limited activity; patchy expression was detected at the wing disc dorsal/ventral boundary and the tracheal branchpoints [Su(H)m8]. In contrast, the Su(H)m8 sites have a dramatic effect when combined with three copies of Gbe [Gbe+ Su(H)m8]. In the imaginal discs, strong activation is detected in a pattern reminiscent of the most widely expressed Notch target gene, Enhancer of split mß [E(spl)mß]. Expression also occurs at tracheal branchpoints in a similar manner to E(spl)mß suggesting that this, too, is a site of Notch activity (Furriols, 2001).

The activation was coupled with apparent inhibition of Gbe-driven expression in some patches in the discs that correspond to the places where E(spl)mß is also silent. More definitive, however, is the effect in the epidermis and the trachea. The widespread expression throughout these tissues that is normally elicited by Gbe is shut off, while the activation at the tracheal branchpoints is enhanced. Similar results were obtained using a single copy of the paired Su(H)m8 site. This construct has virtually no expression on its own but give an E(spl)mß-like pattern in the discs with Gbe and in two out of six lines represses Gbe-derived expression in the epidermis and trachea. Overall, the patterns obtained with Gbe+ Su(H)m8 indicate first that Grh and Su(H) can cooperate synergistically to confer high levels of transcription in places known to have Notch activity and second that Su(H) is able to repress the Grh activation function in regions without Notch activity. Intriguingly, the resulting pattern strongly resembles that of E(spl)mß, although, since no evidence is as yet available that Grh normally confers this expression, Su(H) may synergize with a different activator on this E(spl)mß enhancer. It is important to note, however, that neither the synergy nor the repressive effects imply direct interactions between Su(H) and the DNA-bound activators. Based on the experiments with CBF-1, it is likely that Su(H) exerts its effects through the recruitment of cofactors, which probably include chromatin-modifying enzymes such as histone deacetylases (Furriols, 2001).

To confirm that the effects of adding the paired Su(H)m8 sites to Gbe are due to the activity and not simply to the length of the Su(H) sequences inserted, a similar construct was generated in which the Su(H)m8 sites had been mutated by substituting critical bases in the recognition sequence. The resulting transgene [Gbe+ Su(H)MUT] has an expression pattern similar to the parental Gbe sites alone, although the levels of expression are reduced. Since these mutations restore the widespread activity of the enhancer, it must be the Su(H) sequence per se that confers the activation and repression detected with Gbe+ Su(H)m8 (Furriols, 2001).

If this interpretation is correct and the E(spl)mß -like expression from Gbe+ Su(H)m8 reflects a synergistic interaction between Su(H)/Nicd and Grh, this expression should be dependent on Notch and Su(H). Reducing Su(H) activity [Su(H)SF8] in clones of cells in the wing disc leads to an autonomous loss of the high levels of expression from the mutant cells. Likewise, reducing Notch activity using a temperature sensitive combination (Nts1/N55e11) at the nonpermissive temperature also eliminates the intervein pattern and reduces the dorsal/ventral boundary expression of Gbe+ Su(H)m8. Thus, the strong disc expression requires Notch and Su(H) and, as it is not seen with the Su(H)m8 sites alone, must involve cooperation with Gbe-bound protein (Furriols, 2001).

Similar experiments were carried out to assess whether repression depends on Su(H). In this case, it would be anticipated that mutations in Su(H) should cause derepression, restoring Gbe-mediated epidermal expression, whereas mutations in Notch should not. In Su(H) mutant animals [Su(H)SF8/Su(H)AR9], there is widespread expression from Gbe+ Su(H)m8 throughout the epidermis and tracheal cells, and the strong activation at tracheal branchpoints is lost. In contrast, there is no expression in the epidermal cells or most tracheal cells when Notch function is reduced. In the latter case, the activity at the branchpoints is reduced, as in Su(H) mutants, consistent with this being Notch-dependent activation, but there is no derepression in the other tracheal cells. Clearly, the transgene can be expressed in a similar pattern to the parental Gbe when there is little or no Su(H) protein present, confirming, therefore, that Su(H) is critical for the repression. In contrast, reducing Notch activity has no effect on repression. This differential highlights the fact that mutations in Notch and Su(H) are unlikely to have the same consequences on many target genes, as shown recently for singleminded. Since nonconsonance in phenotypes has been taken to indicate that certain Notch functions are independent of Su(H), it will be important to reevaluate these phenotypes, taking into consideration the possibility that Su(H) mutations can lead to derepression of target genes (Furriols, 2001).

If Su(H) has the ability to function as a molecular switch, the silencing of Gbe+ Su(H)m8 expression in the epidermis should be alleviated by ectopic activation of Notch in this tissue. To test this, hsNicd flies, which have the intracellular domain of Notch (Nicd) under the control of the heat-shock promoter, were used. Exposure to 37°C induces ubiquitous expression of Nicd, which is a constitutively active fragment of Notch, and under these conditions Gbe+ Su(H)m8 confers expression throughout the epidermis and the trachea. In the presence of Nicd, therefore, the silencing is alleviated, and the transgene becomes activated in all the places where Grh is present (Furriols, 2001).

These data indicate that in the absence of Notch activation, Su(H) is capable of binding to its cognate sites and repressing transcription. Notch activation can alleviate the repression so that Su(H) is able to cooperate with other DNA-bound activators, like Grh, to promote transcription. These results are in agreement with recent models and strongly suggest that this is a general mechanism through which Su(H) acts at native targets. Thus, Su(H) is capable of acting as the pivot in a sensitive switch that would ensure that Notch target genes can be poised but silent until Notch is activated. For example, the E(spl) genes, which mediate the inhibitory effect of Notch during lateral inhibition, appear to be targets of proneural proteins. However, E(spl) genes are not expressed in the cells that are selected to be neural, even though proneural proteins accumulate at highest levels in these cells. According to the model, Su(H) would be able to suppress activators like the proneural proteins until Notch is activated. As soon as levels of Notch are sufficient to overcome Su(H)-mediated repression, the synergistic interactions with activators would lead to a sharp transition in the expression of E(spl) genes. The potent effect of combining Su(H) and Grh also gives a precedent for the way that individual target genes might respond to Notch in specific contexts, if each involves a different transregulator cooperating with Notch. This demonstrates the potential for designing specific molecular assays for Notch activity in different cellular contexts. By replacing the Gbe sites with elements that respond to other activators, it should be possible to generate a transcriptional readout for Notch activity in any cell type (Furriols, 2001)

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).

The Daughterless N-terminus directly mediates synergistic interactions with Notch transcription complexes via the SPS+A DNA transcription code

Cell-specific expression of a subset of Enhancer of split (E(spl)-C) genes in proneural clusters is mediated by synergistic interactions between bHLH A (basic Helix-Loop-Helix Activator) and Notch-signalling transcription complex (NTC) proteins. For a some of these E(spl)-C genes, such as m8, these synergistic interactions are programmed by an "SPS+A" transcription code in the cis-regulatory regions. However, the molecular mechanisms underlying this synergistic interaction between NTCs and proneural bHLH A proteins are not fully understood. Using cell transcription assays, it was shown that the N-terminal region of the Daughterless (Da) bHLH A protein is critical for synergistic interactions with NTCs that activate the E(spl)-C m8 promoter. These assays also show that this interaction is dependent on the specific inverted repeat architecture of Suppressor of Hairless (Su(H)) binding sites in the SPS+A transcription code. Using protein-protein interaction assays, it was shown that two distinct regions within the Da N-terminus make a direct physical interaction with the NTC protein Su(H). Deletion of these interaction domains in Da creates a dominant negative protein that eliminates NTC-bHLH A transcriptional synergy on the m8 promoter. In addition, over-expression of this dominant negative Da protein disrupts Notch-mediated lateral inhibition during mechanosensory bristle neurogenesis in vivo. These findings indicate that direct physical interactions between Da-N and Su(H) are critical for the transcriptional synergy between NTC and bHLH A proteins on the m8 promoter. These results also indicate that the orientation of the Su(H) binding sites in the SPS+A transcription code are critical for programming the interaction between Da-N and Su(H) proteins. Together, these findings provide insight into the molecular mechanisms by which the NTC synergistically interacts with bHLH A proteins to mediate Notch target gene expression in proneural clusters (Cave, 2009).

Senseless, a Zn finger transcription factor, is necessary and sufficient for sensory organ development in Drosophila

Induction of Senseless (Sens) expression using the dpp-GAL4 driver alters Delta expression. The domain that normally gives rise to the third wing vein, is altered in Sens-overexpressing discs. Overexpression of Sens induces Delta expression ectopically in the dpp domain, broadening and intensifying the endogenous Delta domain. In addition, a consistent reduction of expression in the fourth wing vein domain is observed. This ectopic Delta expression is likely to be mediated by Scute/Asense overexpression (Nolo, 2000 and references therein).

To determine the relationship between Sens expression and the proteins of the Enhancer of Split complex, wild-type discs were stained for both proteins. There is little overlap between the two nuclear proteins. Cells that express Sens are intermingled with E(spl) expressing cells, but the majority of cells that express Sens do not express E(spl). Similar observations were also made with E(spl)m8-lacZ and with E(spl)m4-lacZ. These data indicate that Sens expression in cells fated to develop into SOPs is concomitant with the presence of E(spl) proteins, but that elevation of Sens expression and cell enlargement during SOP specification accompanies a rapid removal of the E(spl) protein. These data are also in agreement with the proposition that E(spl) is a negative regulator of proneural gene expression and that its downregulation permits SOP development (Nolo, 2000 and references therein).

Ectopic expression of Sens may not only activate the proneural genes and Delta but may recreate an ectopic proneural field. Expression of several E(spl) proteins depends on the presence of the proneural genes. Therefore Sens was overexpressed using the dpp-GAL4 driver in E(spl)m8-lacZ and E(spl)m4-lacZ imaginal discs. Wild-type discs contain proneural clusters that express cytoplasmic ßgalactosidase [E(spl)] in which few cells are Sens positive. Overexpression of Sens causes a strong induction of ßgalactosidase staining associated with E(spl)m4-lacZ and E(spl)m8-lacZ. This induction is not restricted to cells in which Sens is expressed but can be detected in adjacent cells as well. This indicates that Sens can induce in a cell-nonautonomous fashion E(spl) expression, probably by activating Delta expression. A more detailed cellular analysis shows that when Sens expression is elevated in a particular cell, ßgalactosidase levels are consistently low or absent. It is inferred that ectopic Sens leads to expression of the essential components required to establish a proneural domain in some areas of the wing discs. This ability is most likely mediated by its ability to activate the proneural genes. The wing hinge region is, however, refractory to induction of Scute, Delta, and E(spl) upon overexpression of Sens (Nolo, 2000).

Since ectopic Sens is able to induce E(spl) expression and since elevated Sens levels are associated with low levels or absence of E(spl) protein during SOP specification in normal and ectopic conditions, it was of interest to enquire how ectopic expression of both proteins in the same cells would affect PNS organ development. Since overexpression of E(spl) causes a loss of external sensory organs, the component that is most downstream in the pathway should be epistatic to the more upstream component. The dorsal portion of the thorax of a dpp-GAL4; UAS-sens fly has extra bristles. Scutellar bristles are lost in dpp-GAL4; UAS-E(spl)m8 flies. Coexpression of both Sens and E(spl)m8 proteins always leads to a very strong reduction in supernumerary bristles in most areas, occasionally loss of bristles. Hence, ectopic E(spl), counteracting neurogenesis, is epistatic to ectopic Sens, stimulating neurogenesis, in the pathway that specifies the SOP (Nolo, 2000).

The ability of ectopic Sens to induce external sensory organ formation is most likely due to its ability to cause expression of many key players that are normally expressed in the proneural cluster. Proneural genes activate the transcription of the E(spl) genes and sens. The Sens protein may then act via two pathways in the SOP. (1) It may directly activate proneural gene expression participating in the initiation and/or maintenance of an autoregulatory loop. This mode of action is supported by the observation that ectopic Sens can induce proneural gene expression in the absence of endogenous proneural proteins or E(spl) proteins. In addition, the proneural genes contain consensus binding sites for the Sens protein, suggesting that the interaction may be direct. (2) Sens may first enhance and subsequently inhibit transcription of E(spl) genes. Expression of the genes of the E(spl) complex is clearly reduced in the SOPs to permit their specification. It is proposed that Sens also plays a role in this process by inhibiting transcription of the E(spl) genes in the SOPs. This in turn may allow further upregulation of proneural gene expresssion, followed by ectopic expression of E(spl) in neighboring cells that do not express Sens, suggesting that they receive a signal from the proneural proteins expressing cells. This signal is most likely Delta, since Delta expression is clearly upregulated in the cells that express Sens. Cells that do not express or express very low levels of Sens then accumulate more E(spl) than those that do express higher levels of Sens. Hence, the reduction of Notch signaling in the SOP may be strongly enhanced by the presence of Sens to help specify SOPs. Indeed, ectopic coexpression of E(spl)m8 and Sens dramatically reduces the action of Sens and in some areas of the notum creates a phenotype that is typically associated with overexpression of E(spl)m8 alone, i.e., loss of bristles. This suggests that E(spl)m8 acts downstream of Sens. In ectodermal cells, Sens normally does not play a role because none of these cells acquire enough proneural gene expression to activate Sens at a level that is sufficient to activate proneural gene expression above a required threshold. The latter statement is supported by the expression pattern of Sens, which is restricted to those cells that express the highest levels of proneural proteins and by the observation that robust levels of ectopic proneural gene expression must be attained to induce ectopic Sens expression. In summary, it is proposed that the function of Sens is to integrate proneural gene expression into the Notch signaling pathway to promote proper SOP development in the Drosophila PNS (Nolo, 2000).

Mitotic G2-arrest is required for neural cell fate determination in Drosophila: regulation of Enhancer of split expression

In the wing discs of Drosophila, the mechanosensory precursor cells are singled out from clusters of cells blocked at the G2 phase of the cell cycle. This mitotic quiescence and the selection of the precursors are under strict spatio-temporal control. G2 cells were forced to enter mitosis by overexpression of string, the Drosophila homolog of the cdc25 gene. Premature entrance in the cell cycle is associated with a loss of precursor cells. Precursors are lost consecutively to a transcriptional down-regulation of the determinant proneural achaete/scute genes. This down-regulation results from an over-activation of the Enhancer of Split genes, known as effectors of the Notch signalling pathway. It is concluded that exit from the cell cycle is required for proper neural cell fate determination (Nègre, 2003).

Thus, forcing G2 arrested cells into mitosis results in a loss of adult sense organs. The corresponding precursors are also lost. This result was obtained by using two distinct transgenic systems to control the timing and spatial location of stg-overexpression. In both cases, precursors are not selected because ac/sc proneural expression is repressed. This repression occurs at a transcriptional level. Noteworthy is the fact that bristles are lost using either the sca-Gal4 driver to overexpress stg, or the klu-Gal4 driver; this demonstrates that overexpression of stg not only prevents the early accumulation of Ac/Sc (klu-Gal4 driver), but can also downregulate Ac/Sc after the levels of these proteins have started to rise (sca-Gal4 driver). Thus, it is concluded that the arrest in G2 is necessary for proper determination of precursor cells. The complexity of the 5' regulatory sequences of stg indicates that this mitotic regulator might itself integrate information from patterning genes. For instance, the regulatory regions of the stg gene possess putative recognition sites for Achaete and Scute transcription factors. Here, it has been shown that stg can itself control the expression of developmental genes. The effect of stg on cell determination is unlikely to be direct, however, since the only known function of stg is to dephosphorylate the CDK1- cyclin B mitotic kinase. Future genetic approaches may reveal whether or not String has other biochemical targets (Nègre, 2003).

After stg overexpression using the klu-Gal4 driver, it was observed that E(Spl) expression is maintained in proneural regions in absence of Ac/Sc. It was also observed that stg can cause accumulation of the E(Spl) bHLH genes outside of proneural clusters, in a cell-autonomous mode. Maintenance of expression of E(Spl), a transcriptional repressor of the ac/sc expression, is relevant. It can functionally justify the loss of precursor cells. Nevertheless, it has been reported that E(Spl) transcription is dependent on the ac-sc genes in the proneural clusters. One explanation could be that deregulation of the cell cycle directly or indirectly increases transcription of the E(Spl) genes by modifying activity of upstream activators of the E(Spl) expression. Considering this hypothesis, E(Spl) should sometimes be expressed in incorrect positions compared to its wild-type expression. On the contrary, because E(Spl) genes are expressed at the exact positions for proneural clusters, it is suggested that forcing cell cycle more likely affects E(Spl) expression at a post-translational level rather than at a transcriptional level. In the mutants, initial transcription of E(Spl) genes would still have been dependent on Ac/Sc, which begin to accumulate in proneural domains. But, it is known that at least E(Spl) m5, m7 and m8 isoforms contain a PEST-rich motif that harbors an invariant Serine residue, which is phosphorylated by the casein kinase II. Casein kinase II is a ubiquitous serine/threonine kinase whose activity fluctuates with cell cycle progression. Phosphorylation usually regulates protein stability via activation of PEST motifs. Modification in the phosphorylation status of some E(Spl) proteins could exhibit a longer half-life in vivo, thus leading to their predominance over the proneural proteins, and therefore to an inhibition of neurogenesis. In other words, premature entry in the cell cycle would introduce an external bias in the highly dynamic process that opposes the antagonistic E(Spl) and Ac/Sc proteins and which normally occurs in cells of proneural clusters. It would confer an advantage to E(Spl) over proneural activity and would explain persistence of E(Spl) proteins after proneural products have disappeared (Nègre, 2003).

Altogether, these results suggest that proneural competence can only develop in mitotically arrested cells. The programmed incompatibility between cell cycling and proneural product accumulation may have several general, and not mutually exclusive, functional correlates. In proneural clusters, keeping cells together in a continuous group may be necessary. Indeed, cell interactions could be required to maintain Ac/Sc levels via indirect autoregulation through cell- cell signalling. Furthermore, a G2 arrest may be necessary to preserve a balance between the levels and/or activities of E(Spl) and Ac/Sc products that could directly or indirectly be dependent on post-transductional modifications. The relative strength of the signal impinging on a given cell determines whether products of the proneural genes or products of the E(Spl) become finally predominant. Changing the cell cycle phase could disrupt this equilibrium. Finally, divisions that underly normal cell proliferation and those involved in the fixed lineage of the precursor cell, make different demands on the cytoskeletal machinery. The asymmetric divisions of the precursor cell are strictly controlled in orientation and in time. These controls are presumably essential to realize a correct lineage. A period of mitotic quiescence may give the precursor cell the time and/or conditions required to reorganize its cytoskeleton in order to shift to an asymmetric mode of division. Although a quiescent period systematically precedes the emergence of neural precursors, re-entry into mitosis is independently controlled in the precursor and the surrounding epidermis. This suggests that quiescence is a necessary step preceding the lineage of the precursor. Moreover, the decision of the precursor to enter in its lineage is made independently of the mitotic state of its surrounding cells (Nègre, 2003).

In this study, causal relationship has been demonstrated to exist between cell cycle and neural determination in an endogenous system: the Drosophila wing imaginal discs, in which E(Spl) effectors of the Notch pathway behave as integrative sensors of the cell cycle status (Nègre, 2003).

Scabrous complexes with Notch to mediate boundary formation

The mechanisms that establish and sharpen pattern across epithelia are poorly understood. In the developing nervous system, the first pattern elements appear as 'proneural clusters'. In the morphogenetic furrow of the immature Drosophila retina, proneural clusters emerge in a wave as a patterned array of 6 to 10 cell groups, which are recognizable by expression of Atonal, a basic helix-loop-helix transcription factor that is required to establish and pattern the first cell fate. The establishment and subsequent patterning of Atonal expression requires activity of the signaling transmembrane receptor Notch. In vivo and biochemical evidence is presented that the secreted protein Scabrous associates with Notch, and can stabilize Notch protein at the surface. The result is a regulation of Notch activity that sharpens proneural cluster boundaries and ensures establishment of single pioneer neurons (Powell, 2001).

In the morphogenetic furrow, Atonal's expression can be divided into four steps:(1) it is expressed as a broad, unpatterned stripe; (2) expression is then upregulated into evenly spaced proneural clusters;(3) in these proneural clusters, a 2 to 3 cell 'R8 equivalence group' emerges, and (4) expression narrows to identify a single cell in this group as the R8 photoreceptor neuron, the first cell type of the developing retina. In each step, as cells lose Atonal expression they concurrently gain expression of negative regulators, such as members of the E(spl) (Enhancer of Split) complex. Expression of E(spl) initially requires the presence of Atonal, and is subsequently amplified by the Notch signaling pathway to downregulate proneural bHLH expression and function. E(spl), therefore, represents one reporter of Notch activity (Powell, 2001).

Patterning of Atonal and E(spl) expression requires the normal activity of Scabrous, a secreted fibrinogen-related protein with a potential for association with components of the extracellular matrix. In the retina, Scabrous protein first appears in the proneural clusters, mirroring Atonal expression by narrowing to the R8 equivalence group, and eventually R8 alone. This expression is dependent on Atonal activity, which indicates that the scabrous locus may be a direct target of Atonal (Powell, 2001).

Genotypically null scaBP2 proneural clusters are poorly spaced with poorly defined borders. Broadened E(spl) expression throughout much of the proneural cluster region is one potential cause of this imprecision, suggesting that Notch activity is altered in sca BP2 mutants. These observations suggest that initial broad, low-level Atonal expression activates broad, low-level E(spl) expression, and that Scabrous is required to refine the complementary Atonal and E(spl) expression in the proneural cluster region -- events also associated with Notch activity (Powell, 2001).

A 1 hour pulse of ectopic Scabrous results in rapid loss of Atonal within 2 h; E(spl) shows low, diffuse expression that is lost within 4 h. This ectopic expression of Scabrous leads to aberrant patterning of R8s in a manner similar to that of the phenotypes observed in scabrous loss-of-function alleles. The loss of the initial broad stripe of Atonal, a Notch-dependent step, suggests that ectopic Scabrous can lead to a disruption of Notch function, a result consistent with overexpression studies in the Drosophila wing (Powell, 2001).

A novel Pzg-NURF complex regulates Notch target gene activity

Drosophila putzig was identified as a member of the TRF2-DREF complex that is involved in core promoter selection. Additionally, putzig regulates Notch signaling, however independently of DREF. This study shows that Putzig associates with the NURF complex. Loss of any NURF component including the NURF-specific subunit Nurf 301 impedes binding of Putzig to Notch target genes, including cut, Enhancer of split and vestigial, suggesting that NURF recruits Putzig to these sites. Accordingly, Putzig can be copurified with any NURF member. Moreover, Nurf 301 mutants show reduced Notch target gene activity and enhance Notch mutant phenotypes. These data suggest a novel Putzig-NURF chromatin complex required for epigenetic activation of Notch targets (Kugler, 2010).

Putzig is a component of a large multiprotein complex that includes the TATA-box-binding-protein-related factor 2 (TRF2) and the DNA-replication related element (DRE) binding factor DREF. The TRF2-DREF complex has been associated with the transcriptional regulation of replication-related genes that contain DREF binding sites. Accordingly, Pzg acts as a positive regulator of cell cycle and replication-related genes. In addition to this, Pzg is also required for Notch target gene activation in a DREF-independent manner. Presumably, Pzg functions at the level of chromatin activation, because the open chromatin structure typical of active Notch target genes is no longer detectable in a pzg mutant background (Kugler, 2010).

The TRF2-DREF complex consists of more than a dozen of proteins and the biochemical function of most of them remains still elusive. Interestingly, it also contains three members of the nucleosome remodeling factor (NURF), imitation switch (ISWI), Nurf 55 and Nurf 38. NURF is a multisubunit complex that has been associated with chromatin activation and repression. NURF triggers nucleosome sliding thereby provoking changes in the dynamic properties of the chromatin. The subunit ISWI is a member of the SWI2/SNF ATPase family and is thought to provide energy for nucleosome remodeling. Nurf 38 encodes an inorganic pyrophosphatase, which catalyzes the incorporation of nucleotides into a growing nucleic acid chain during transcription, replication, and DNA repair mechanisms. Nurf 55 harbors WD-40 repeats, which allow interaction with other proteins and protein complexes. The fourth and largest subunit Nurf 301 is specific to the NURF complex, whereas all other members are shared with other chromatin modifying complexes. Accordingly, Nurf 301 is not a component of the TRF2-DREF complex. Nurf 301 exhibits a number of protein motifs that typify transcription factors and other chromatin modifying proteins. In addition, the N-terminal region of Nurf 301 shows homology to the DNA-binding protein HMGA (high mobility group A) implying that Nurf 301 mediates the contact with the DNA or provides a platform to recruit other transcription factors. In this context it has already been shown that Nurf 301 is required for the transcriptional activation for example of homeotic genes and notably of Ecdyson-receptor (EcR) and Wingless target genes (Kugler, 2010).

The DREF independence of Pzg during the activation of Notch target genes raised the possibility that it may instead involve the NURF complex for chromatin activation. This study provides evidence for a functional interplay between Pzg and the NURF complex with regard to Notch target gene activation. Coimmunoprecipitations revealed that Pzg is present in protein complexes containing the known NURF subunits. Moreover, Pzg binding on Notch target genes is neither detectable in mutants of the NURF-specific subunit Nurf301, nor in mutants affecting other subunits of NURF. In addition, Nurf301 is required for Notch target gene expression, which is impaired in Nurf301 mutant cell clones. Consistent with this, Nurf301 mutants enhance the Notch mutant wing phenotype, strongly arguing for an involvement of the NURF complex in Pzg-mediated epigenetic Notch target gene activation (Kugler, 2010).

This work shows that Pzg is associated with at least two different types of protein complexes that are involved in transcriptional activation: the TRF2-DREF complex and the NURF complex. Interestingly, these two complexes share several members apart from Pzg despite their different roles in core promoter selection versus nucleosome sliding and chromatin activation. However, the specific role for Pzg in the promotion of Notch target gene transcription involves NURF and not the TRF2-DREF complex. Notably, NURF also promotes efficient expression of a subset of Wingless target genes. In this case, a direct interaction between ISWI and Armadillo, the major transcriptional coactivator of Wingless targets, was shown. There is no indication however, that pzg is involved in the regulation of wg, suggesting that the NURF complex recruits Pzg only onto specific promotors. Furthermore, the NURF subunit Nurf 301 contacts the Ecdysone receptor (EcR), thereby modulating the activity of ecdysone signaling during the larval and pupal stages of Drosophila development. How is NURF recruited to Notch target sites? Notch target gene activation involves a ternary complex containing the DNA-binding protein Suppressor of Hairless [Su(H)], intracellular Notch, and Mastermind, plus other more general coactivators. There is no indication of a direct contact of Pzg to either Notch or Su(H), tested by coimmunoprecipitations as well as yeast two-hybrid assays. However, contacts between the other components, notably Mastermind or ISWI cannot be excluded. Mastermind has been shown to interact with several chromatin modifying proteins, for example, with the histone acetyltransferase p300 or with cyclin-dependent kinase 8 (Kugler, 2010).

Several studies in Drosophila and vertebrates have shown that many Notch-responsive target genes are regulated by combinatorial signal inputs, which need the Notch ternary complex and additional cooperators bound to sites nearby. In contrast to cofactors within the transactivation complex, these other factors do not physically interact with the Notch ternary complex but instead synergize during transcriptional activation at Notch target gene promoters. It is conceivable, that a Pzg-NURF complex is likewise needed in conjunction with the Notch transactivator complex for full Notch target gene expression (Kugler, 2010).

It is well established, that chromatin modification complexes share several components. For example, ISWI is not only contained in NURF and TRF2-DREF complexes but also in chromatin-remodeling and assembly factor (CHRAC) and ATP-utilizing chromatin-remodeling and assembly factor (ACF) in Drosophila, where it serves to increase the accessibility of nucleosomal DNA. Nurf 55, also known as CAF-1, forms a stable complex with Drosophila Myb and E2F2/RBf and regulates the transcription of several developmentally important genes. Like ISWI and Nurf 55, also Nurf 38 is present in the TRF2-DREF complex. Pzg is contained within the TRF2-DREF and within the NURF complex serving the activation of proliferation related genes and N target genes, respectively. Not all NURF complexes, however, require pzg, for example, as during the activation of Wg target genes. Sharing components raises the question, how specificity of the different complexes is achieved. Obviously, specificity is mediated either by unique subunits or by certain combinations of shared subunits. These subunits may specifically modulate the activity of the ATPase subunit or, more likely, may help to target the remodeling complexes to particular promoters. Two members of the NURF complex, ISWI and Nurf 301, have been shown to directly target transcription factors. It is tempting to speculate, that Pzg might be a specific cofactor needed to realize some of the operation spectrum of NURF, notably during the epigenetic regulation of Notch target genes (Kugler, 2010).

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).

In Drosophila, achaete (ac) and m8 are model basic helix-loop-helix activator (bHLH A) and repressor genes, respectively, that have the opposite cell expression pattern in proneural clusters during Notch signaling. Previous studies have shown that activation of m8 transcription in specific cells within proneural clusters by Notch signaling is programmed by a 'combinatorial' and 'architectural' DNA transcription code containing binding sites for the Su(H) and proneural bHLH A proteins. The study shows that the ac promoter contains a similar combinatorial code of Su(H) and bHLH A binding sites but contains a different Su(H) site architectural code that does not mediate activation during Notch signaling, thus programming a cell expression pattern opposite that of m8 in proneural clusters (Cave, 2011).

The results provide important new insights into the DNA transcription codes that program cell-specific gene expression in response to Notch signaling. The ac promoter contains an S-site architecture that mediates repression, not activation, during Notch signaling in proneural clusters. Given that there are unpaired S sites in the promoters of many other proneural and panneural genes, it is predicted that some, or potentially all, of these S sites could mediate repression in cells where Notch is activated. This differential activation versus repression of gene transcription programmed by distinct S-site architectures greatly expands the potential regulatory complexity of pathways mediated by Notch signaling. Previous studies suggested that specific S-site architectures (S-site 'subcodes') programmed specific interactions between Notch complexes on S sites and specific combinatorial coactivator proteins bound to nearby DNA sites. Together with these previous findings, the current study provides an important and novel understanding of the role that S-site architecture plays in mediating differential transcriptional responses to Notch signaling. Given that at least some aspects of the S-site architectural codes are functionally conserved in mammals, it will be interesting and important to test whether the same differential regulation mechanisms are conserved in mammals (Cave, 2011)

bHLH-O proteins are crucial for Drosophila neuroblast self-renewal and mediate Notch-induced overproliferation

Drosophila larval neurogenesis is an excellent system for studying the balance between self-renewal and differentiation of a somatic stem cell (neuroblast). Neuroblasts (NBs) give rise to differentiated neurons and glia via intermediate precursors called GMCs or INPs. E(spl)mγ, E(spl)mβ, E(spl)m8 and Deadpan (Dpn), members of the basic helix-loop-helix-Orange protein family, are expressed in NBs but not in differentiated cells. Double mutation for the E(spl) complex and dpn severely affects the ability of NBs to self-renew, causing premature termination of proliferation. Single mutations produce only minor defects, which points to functional redundancy between E(spl) proteins and Dpn. Expression of E(spl)mγ and m8, but not of dpn, depends on Notch signalling from the GMC/INP daughter to the NB. When Notch is abnormally activated in NB progeny cells, overproliferation defects are seen. This depends on the abnormal induction of E(spl) genes. In fact E(spl) overexpression can partly mimic Notch-induced overproliferation. Therefore, E(spl) and Dpn act together to maintain the NB in a self-renewing state, a process in which they are assisted by Notch, which sustains expression of the E(spl) subset (Zacharioudaki, 2012).

This paper presents an analysis of the expression and function of two types of bHLH-O proteins expressed in Drosophila neuroblasts, Dpn and the E(spl) family. The main conclusions are that (1) these two types of factors have distinct expression modalities: E(spl)mγ and m8 are targets of Notch signalling, whereas Dpn is not; (2) these factors have redundant functions to maintain NBs in a self-renewing state in normal development, yet (3) in a pathological NB hyperproliferation context, Dpn and E(spl) have distinct functions (Zacharioudaki, 2012).

It was heretofore thought that embryonic NBs are cells that escape from Notch signalling, which is only perceived in the surrounding neuroectoderm. Only in the later post-embryonic period, were the NBs thought to respond to Notch. This study has shown that this happens much earlier, already in the embryonic NBs. Soon after the NB delaminates, a time when it sends, but does not receive, a Notch signal, it starts asymmetrically dividing. The results are consistent with the daughter GMCs sending a Delta signal back to their sister NBs, thereby initiating E(spl) expression. E(spl)mγ expression ceases when the NB enters quiescence, only to restart when proliferation resumes (Zacharioudaki, 2012).

Dpn, another bHLH-O protein, is also expressed in NBs, but much less dynamically. Its expression initiates upon NB delamination from the neuroepithelium and persists throughout its life. dpn does display some degree of dynamic expression, as it is rapidly turned off in the immature intermediate progenitors (iINPs), only to be reactivated upon maturation. Loss-of-function data clearly indicate that it is not a target of Notch in the NB, in contrast to E(spl). Paradoxically, dpn is induced upon Notch hyperactivation. This could be an indirect effect mediated through E(spl). Indeed, E(spl)mγ overexpression can induce ectopic dpn expression. Still, dpn does harbour a Notch-responsive enhancer that drives expression in larval NBs. This same region scored positively for Su(H) binding in a ChIP-chip approach in a cell line of mesodermal origin. How this enhancer contributes to the overall expression pattern of dpn will be a matter of future analysis (Zacharioudaki, 2012).

Despite their different expression modalities, Dpn and E(spl) have redundant functions in the larval NBs, as only double mutant clones show proliferation defects. These mutant NBs do not stop proliferating immediately, rather gradually terminate their cycling within a few days following homozygosing of the mutant alleles. It is proposed that Dpn/E(spl) keep the NB in an undifferentiated state and proliferation is a consequence of the ability of these cells to respond to mitogens. Upon Dpn/E(spl) loss, this state becomes unstable and prone to switch to a terminally differentiated state. This transition takes a few days, probably reflecting the time needed to accumulate pro-differentiation factors. A redundant role of Dpn/E(spl) in maintaining the undifferentiated state also during quiescence transpired from the genetic analysis of NB re-activation after embryogenesis. Whereas dpn–/– NBs quite successfully re-entered the cell cycle, dpn–/–;E(spl)+/– NBs were unable to do so, despite trophic growth factor stimulation (Zacharioudaki, 2012).

E(spl) expression has been associated with the less differentiated of two alternative outcomes in other instances. For example, during NB formation, E(spl) genes are expressed in the undifferentiated embryonic neuroectoderm and not in the NBs. The same happens in the optic lobe neuroepithelium. This work has presented evidence for a similar role for Dpn/E(spl) in the NB. Excessive Dpn/E(spl) activity in GMCs/INPs can revert these partially differentiated cells back to a NB-like fate. For this reason, NB asymmetric divisions must ensure that Dpn and E(spl) are never expressed in the GMC or iINP. Regarding E(spl), it is proposed that this is ensured by the directionality of Notch signalling (GMC to NB). Dpn is also never seen to accumulate in the GMCs/iINPs, suggesting a repression mechanism at work in these cells, e.g., via Pros. These modes of transcriptional control are probably combined with active protein clearance by degradation (Zacharioudaki, 2012).

An anti-differentiation role has also been proposed for vertebrate homologues of Dpn/E(spl), the Hes proteins. Hes1, Hes5 and Hes3 are all expressed in proliferating neural stem cells of the embryonic CNS. Upon Hes knockout, neural stem cells prematurely differentiate resulting in a hypoplastic nervous system, with increasing severity as more Hes genes are lost. In an interesting analogy, only Hes1 and Hes5 are direct targets of Notch signalling. Another example where anti-differentiation during quiescence is mediated by high Hes1 expression are cultured fibroblasts and rhabdomyosarcoma cells. Similar to what was observed with Dpn/E(spl)-mutant embryos, a quiescence trigger, like serum depletion, can result in irreversible cell-cycle withdrawal, if Hes1 activity is compromised (Zacharioudaki, 2012).

The results have shed light on the paradox of why Notch loss of function has only minor effects in larval neurogenesis, whereas its hyperactivation causes significant overproliferation. Notch loss of function decreases E(spl) expression, leaving Dpn levels unaffected. Furthermore, Notch pathway disruption does not seem to directly affect NB proliferation, as Cyclin E expression is not eliminated. Therefore, Notch signalling from the GMC/iINP to the NB acts to ensure robustness in NB maintenance, in collaboration with Dpn (Zacharioudaki, 2012).

When Notch signalling is aberrantly activated in the GMCs/iINPs, both type I and type II lineages overproliferate, although the former do so with lower penetrance (fewer lineages) and expressivity (smaller clones). Yet, for both types of lineages, E(spl) genes are necessary to implement overproliferation. This is consistent with the hypothesis that ectopic E(spl)/Dpn activity in the GMCs/iINPs inhibits their differentiation and makes them competent to respond to mitogenic stimuli (Zacharioudaki, 2012).

Why are Type II lineages more sensitive than type I lineages to Notch gain of function? A crucial difference between these NBs is the lack of expression of Ase in type II, as its artificial reinstatement can revert the latter to type I-like behaviour. It was recently demonstrated that Ase downregulates E(spl) expression. It is even possible that Ase antagonizes E(spl) proteins post-transcriptionally, as the two can interact and extensive antagonistic interactions. Thus, N hyperactivation will probably cause a smaller increase in E(spl) levels/activity in type I cells, compared with type II. If resistance to differentiation stimuli depends on the level of E(spl)/Dpn activity, this would account for the relative resilience of type I lineages to Notch-induced overproliferation (Zacharioudaki, 2012).

Regulation of other Enhancer of Split Complex Genes

Continued: Enhancer of Split Regulation part 2/2

Enhancer of split: Biological Overview | Evolutionary Homologs | Targets of Activity | Protein Interactions and Post-transcriptional Regulation | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.