Suppressor of Hairless


REGULATION

cis-Regulatory Sequences and Functions

Suppressor of Hairless [Su(H)]/Lag-1/RBP-Jkappa/CBF1 is the only known transducing transcription factor for Notch receptor signaling. Su(H) has three distinct functions in the development of external mechanosensory organs in Drosophila: Notch-dependent transcriptional activation and a novel auto-repression function (both of which direct cell fate decisions), and a novel auto-activation function required for normal socket cell differentiation. This third phase of activity, the first known Notch-independent activation function for Su(H) in development, depends on a cell type-specific autoregulatory enhancer that is active throughout adult life and is required for proper mechanoreception. These results establish a direct link between a broadly deployed cell signaling pathway and an essential physiological function of the nervous system (Barolo, 2000).

It is concluded that Su(H), a core component of the ubiquitous N signaling pathway, is recruited -- via a discrete transcriptional enhancer module in the Su(H) gene itself -- for a fundamental differentiative role specifically in the socket cell. It is suggested that the autoregulatory activity of Su(H) in the socket cells of external sensory organs represents an unusually direct link between initial cell fate specification and the physiological function of a differentiated cell type (Barolo, 2000).

In nearly all of the many developmental settings (both embryonic and post-embryonic) in which Su(H) acts as the key transducing transcription factor for the N pathway, Su(H) is expressed generally, at a similar low-to-moderate level, in all cells of each tissue in which it acts. The discovery that Su(H) transcript and protein levels are greatly elevated specifically in the socket cells of all external sensory organs of the larval and adult PNSs posed two questions. (1) What is the developmental function of this elevated expression, given that the much lower Su(H) protein levels in all other cell types of the fly are sufficient for transduction of the N signal? (2) How is this cell type-specific elevation of Su(H) transcript and protein accumulation achieved (Barolo, 2000)?

This study of the transcriptional regulation of Su(H) has identified two distinct cis-regulatory modules: a promoter-proximal region that drives moderate general and maternal Su(H) expression, and a downstream enhancer, the ASE, which is responsible for strong socket cell-specific transcriptional activation. Because these two regulatory modules are physically separable, the socket cell activation of Su(H) could be selectively eliminated by deleting or mutating the ASE, while retaining the general and maternal Su(H) expression that is essential for viability and fertility. The loss of socket cell-specific Su(H) activation has no significant effect on N-mediated specification of the socket cell fate, or on any other cell fate decision. Since Su(H) is genetically required for socket cell fate determination, it is concluded that moderate levels of Su(H) protein are both necessary and sufficient for transduction of the N signal in the socket cell (and all other N-responsive cell types of the fly) (Barolo, 2000).

The characteristic high level of Su(H) in the socket cell is not only dispensable for the initial specification of the tormogen fate -- several major aspects of tormogen differentiation, such as significant cell growth, envelopment of neighboring sensory organ cells, and generation of a cuticular socket structure, also proceed normally in its absence. However, in other respects, autoregulation-deficient tormogens appear to be defective, leading to a failure of mechanoreception. Taken together, these results support a relatively simple developmental model in which the moderate levels of Su(H) protein present in the newly born tormogen are sufficient to transduce the incoming N signal and implement the socket cell fate, while socket cell-specific upregulation of Su(H), initiated in response to N signaling, reflects a specific differentiative role for Su(H) in the socket cell that is required for normal mechanoreception (Barolo, 2000).

Socket cell-specific transcriptional elevation of Su(H) expression depends on direct auto-activation through eight Su(H) binding sites in the ASE. This is the first known autoregulatory activity for Su(H) in any species. It is suggested that the ASE-mediated Su(H) autoregulatory loop is initiated during the socket-shaft cell fate decision in the early pupa by the direct binding of NIC/Su(H) activating complexes. Maintenance of this loop does not require continued N signaling activity. When does Su(H) auto-activation become N signal-independent? Shifting Nts1/Nnull flies to the restrictive temperature after 72 hr of pupal development at 18°C has no detectable effect on either the cuticular morphology of the socket or ASE activity; since temperature-shifting 36 hr (at 18°C) pupae can inhibit both the socket fate and ASE expression, it can be estimated that Su(H) auto-activation becomes signal-independent within 36 hr (at 18°C) after the birth of the tormogen (Barolo, 2000).

It is conceivable that NIC generated during pupal-stage N signaling perdures in the tormogen and acts as a coactivator throughout adult life. While this remains a formal possibility, it would require that NIC molecules in sufficient numbers to strongly activate Su(H) persist in the tormogen for several weeks. At present, the notion that Su(H) utilizes a distinct coactivator in the adult tormogen is favored (Barolo, 2000).

CBF1, a mammalian Su(H) homolog, is well-documented as a transcriptional repressor. Two vertebrate corepressor proteins, Silencing Mediator for Retinoid and Thyroid receptor (SMRT) and CBF1 Interacting coRepressor (CIR), have been shown to act as bridges between CBF1 and a histone deacetylase (HDAC) complex, which exerts transcriptional repression through chromatin remodeling. Studies of CBF1 function in vertebrate cells have led to a 'transcriptional switch' model in which (1) CBF1 directly represses target genes by recruiting HDAC and (2) NIC (or the viral protein EBNA-2) binds to CBF1, displacing the corepressor complex and acting as a transcriptional coactivator. Mutating the autoregulatory Su(H) binding sites in the Su(H) gene causes a gain-of-function cell fate conversion phenotype in which shaft cells inappropriately assume the socket cell fate. This result, in which the loss of Su(H)-mediated repression leads to a cell fate transformation, lends support to the 'transcriptional switch' model for Su(H)/CBF1 activity, and suggests that this model applies to Drosophila Su(H), not only in gene activation at cell boundaries, but also in N-mediated asymmetric cell fate decisions (Barolo, 2000 and references therein).

When all eight Su(H) sites in the ASE have been mutated, the enhancer (ASEm) is no longer active in adult socket cells. One aspect of ASEm-GFP expression is potentially quite informative with respect to ASE regulation. ASEm is active in the tormogen in the early pupa, although its expression is substantially weaker than that of the wild-type enhancer, and unlike the wild-type ASE it is not active in adult tormogens. This early activity of the mutant enhancer indicates that other transcriptional activator(s), in addition to Su(H), bind directly to the ASE. These activator(s) are evidently present or active only in the socket and shaft cells, since these are the only cells in which ASEm-GFP is expressed. For the sake of brevity, the activity of this ASE binding factor or factors will be referred to as 'A', with the understanding that 'A' may stand for either expression or activity modulation (e.g., by phosphorylation) of one or more transcriptional activators. The activity 'A,' then, is sufficient to activate ASEm in the socket and shaft cells, but only during pupal stages. This provides an important clue to the necessity for Su(H) auto-repression. If 'A' is sufficient to activate Su(H) in the developing shaft cell (and perhaps to initiate a positive autoregulatory loop), the ASE must somehow be repressed in that cell in order to prevent inappropriate Su(H) activation. However, this repression must not occur in the socket cell, where Su(H) auto-activation is necessary for proper differentiation. Hence Su(H), which acts as a repressor only in the absence of N signaling, is an ideal repressor for the Su(H) ASE (Barolo, 2000).

The results obtained in this study lead to the following model for Su(H)-mediated transcriptional regulation during mechanosensory bristle development. Following the division of the precursor cell pIIA to give rise to the presumptive trichogen and tormogen, both daughter cells express the ligand Dl and the receptor N. However, the more anterior sister cell inherits from pIIA the N pathway inhibitor protein Numb, while the more posterior sister does not. In the absence of activated N (NIC), Su(H) in the anterior cell acts as an auto-repressor via the ASE, preventing activation of Su(H) by the activity 'A.' The posterior sister cell, however, contains nuclear NIC, which acts as a coactivator for Su(H) and induces the expression of socket-specific Su(H) target genes, including Su(H) itself. While this Su(H) auto-activation loop is initiated by N signaling, it is a consequence, rather than a determinant, of the socket cell fate. In the absence of N-stimulated activation of Su(H) target genes (and in the absence of N-dependent activities that repress the trichogen differentiation program), the anterior cell adopts the shaft cell fate. In the socket cell, Su(H) auto-activation becomes N signaling-independent during the course of pupal development, perhaps through an interaction with an alternative coactivator. By the time of eclosion of the adult fly, both Su(H) and 'A' are required to activate the ASE, but neither is sufficient. Su(H) auto-activation continues in the socket cell throughout pupal and adult life, where it is essential for specific aspects of late tormogen differentiation and for normal mechanoreception in the adult PNS (Barolo, 2000).

Transcriptional Regulation

Bre1 is required for Notch signaling and histone modification

Notch signaling controls numerous cell fate decisions during animal development. These typically involve a Notch-mediated switch in transcription of target genes, although the details of this molecular mechanism are poorly understood. dBre1 has been identified as a nuclear component required cell autonomously for the expression of Notch target genes in Drosophila development. dBre1 affects the levels of Su(H) in imaginal disc cells, and it stimulates the Su(H)-mediated transcription of a Notch-specific reporter in transfected Drosophila cells. Strikingly, dBre1 mutant clones show much reduced levels of methylated lysine 4 on histone 3 (H3K4m), a chromatin mark that has been implicated in transcriptional activation. Thus, dBre1 is the functional homolog of yeast Bre1p, an E3 ubiquitin ligase required for the monoubiquitination of histone H2B and, indirectly, for H3K4 methylation. These results indicate that histone modification is critical for the transcription of Notch target genes (Bray, 2005).

The lethal allele E132 was fortuitously identified among a collection of mutants that modify the wing notching phenotype caused by Armadillo depletion. Genetic mapping of the lethality associated with E132 placed this at 64E8, and it was found to be allelic to an existing mutation, l(3)01640, caused by the P element insertion P1541. Using plasmid rescue of the P element, the site of insertion was localized to the first intron of the open reading frame CG10542, which encodes a predicted protein of 1044 amino acids. The insertion site is 48 nucleotides upstream of the translation initiation codon. Precise excision of P1541 restores viability, confirming that the P element insertion and, by inference, E132 are lethal alleles of CG10542. In support of this, ubiquitous overexpression of the full-length protein encoded by CG10542 rescues the lethality of E132 or P1541 mutant embryos and sustains development to give essentially normal adult flies (with a few minor defects including slightly reduced bristles). CG10542 encodes a conserved protein with close relatives in mammals, C. elegans, plants, and fungi. The Drosophila protein has been named dBre1, after its relative Bre1p in the yeast S. cerevisiae (Bray, 2005).

The hallmarks of the Bre1 proteins are a C-terminal RING finger domain linked to an extensive N-terminal coiled-coil region. The 39 amino acid C3HC4 RING domain is flanked on both sides by ~15 conserved amino acids, suggesting that the fly and mammalian proteins are true orthologs of yeast Bre1p. RING domains are typically found in E3 ubiquitin ligases and frequently mediate the interaction with the E2 ubiquitin-activating enzyme while the other parts of the protein are involved in substrate recognition. The RING domains are therefore critical to catalyze the transfer of ubiquitin from the E2 to the substrate. To confirm the functional importance of the RING domain in dBre1, tests were performed to see whether an N-terminal fragment of dBre1 that lacks the RING domain (ΔRING) could rescue dBre1 mutants. No rescue was observed with any of the 4 transgenic lines (from a total of 814 flies scored), confirming that the RING domain is essential for the function of dBre1 as it is for yeast Bre1p (Bray, 2005).

To examine the subcellular location of full-length dBre1 and the derivative that lacks the RING domain, both forms of the protein were tagged with GFP at the N terminus. Both GFP-dBre1 and GFP-ΔRING are predominantly nuclear in embryonic and imaginal disc cells, although a low level of protein is also detectable in the cytoplasm. This nuclear-cytoplasmic distribution is similar to that of a ΔRING derivative of human Bre1-B when it is overexpressed in mammalian cells. Thus dBre1 appears to be a nuclear protein, like its mammalian counterpart, and deletion of the RING domain does not alter its subcellular distribution even though it abolishes its ability to rescue the mutants (Bray, 2005).

To investigate the role of dBre1 in the fly, homozygous mutant clones were generated in the imaginal disc precursors of the adult structures. Surprisingly, it was found that the majority of defects were similar to those caused by defects in Notch signaling. Thus, adult flies bearing E132 or P1541 mutant clones show notches in the wing margin and aberrant spacing of wing margin bristles, wing blistering and vein defects, fusions of leg segments, and loss of notal bristles and rough eyes. Most of these phenotypes are characteristic of reduced Notch signaling and are distinct from those produced by loss-of-function of other signaling pathways, such as Wingless, Dpp, or Hedgehog signaling that also operate during imaginal disc development. The phenotypic data suggest therefore that dBre1 has a role in promoting Notch signaling (Bray, 2005).

To confirm this, the expression of Notch target genes was examined in dBre1 mutant clones. Since dBre1 mutant clones are considerably smaller than their matched wild-type twin clones, the Minute technique was used to compensate for the growth defect of the mutant clones. In wing imaginal discs, cut and Enhancer of split [E(spl)] are expressed along the prospective wing margin, and their expression depends directly on Notch signaling. Cut expression is absent in large E132 mutant clones, and is lost (3/11) or reduced (6/11) in most P1541 mutant clones. Likewise, E(spl) expression is lost cell autonomously from all E132 mutant clones in the wing. Conversely, expression of spalt, a target of Dpp signaling in the wing, is not reduced in P1541 and E132 mutant cells, indicating that the effects of dBre1 mutation are relatively specific. Similar results are obtained in the eye, where E(spl) expression is also disrupted in E132 clones. Expression in the neurogenic region at the furrow is lost, and elsewhere it is absent or severely reduced, except in the basal layer of undifferentiated cells where expression is independent of Notch. In addition, a derepression of the neuronal cell marker Elav was observed in eye disc clones. The latter indicates excessive neuronal recruitment due to diminished Notch-mediated lateral inhibition (note, however, that the phenotypes are not identical to those produced by complete absence of Notch, which in the eye results in loss of neuronal markers because Notch is needed to promote neural development by alleviating Su(H)-mediated repression. These results demonstrate that dBre1 functions in multiple developmental contexts and, specifically, that it is required for the subset of Notch functions that involve Su(H)-dependent activation of Notch target genes (Bray, 2005).

To further confirm the importance of dBre1 during Notch signaling, it was asked whether any genetic interactions could be detected between overexpressed dBre1 or ΔRING and mutations in Notch (N) or its ligand Delta (Dl). Indeed, overexpression of either protein in the wing disc results in adult phenotypes. In each of 5 ΔRING-expressing lines, mild if consistent mutant phenotypes were observed in both males and females, namely upward-curved wings (due to stronger expression in the dorsal wing compartment), tiny vein deltas, and a significant decrease in wing size. These defects are more severe after overexpression of ΔRING in dBre1 heterozygotes, indicating that ΔRING acts as a weak dominant-negative. Consistent with this, excess ΔRING significantly enhances the phenotypes of N/+ and Dl/+ heterozygotes, resulting in increased vein thickening and additional vein material and, in the case of N/+, also in more frequent wing notching. These genetic interactions support the link between dBre1 and Notch signaling (Bray, 2005).

Excess full-length dBre1 in wing discs causes vein defects whose strength, however, varies considerably between different dBre1-expressing lines, and between males and females (probably because the ms1096.GAL4 driver produces higher expression levels in males). In most lines (4/6), vein thickening and additional vein material were observe only in males, while female wings appear normal. These vein defects in male wings are suppressed to almost normal in dBre1 heterozygotes, suggesting that they are due to increased levels of functional dBre1 protein. The remaining 2 lines produce similar vein defects also in females. Unexpectedly, these defects are enhanced in N/+ and Dl/+ heterozygotes, suggesting that the overexpressed dBre1 interferes with Notch signaling, rather than enhancing it as might have been expected. This anomalous result could be explained if dBre1 is part of a multiprotein complex, in which case its overexpression might interfere with the function of this complex by titrating one of its components. Nevertheless, the genetic interactions between overexpressed dBre1 and Notch and Delta further underscore the link between dBre1 and Notch signaling (Bray, 2005).

To test whether dBre1 directly influences Notch-dependent transcription, Drosophila S2 cells were transfected with Flag-tagged or untagged dBre1, and the activity of a Notch-specific reporter containing 4 Su(H) binding sites (NRE, a luciferase derivative of Gbe+Su(H)m8) was measured in the presence or absence of low levels of NICD. As a control, a reporter was used with mutant Su(H) binding sites [NME, or Gbe+Su(H)mut]. These experiments reveal a significant stimulation of the NRE reporter by dBre1, especially in the presence of NICD. The degree of stimulation is similar to that observed when the ubiquitin ligase Hdm2 is added to transcription assays of Tat activity. dBre1 also elicits a slight stimulation of NME. The fact that overexpressed dBre1 has stimulatory effects on Notch in the transfection assays but not in imaginal discs presumably reflects differences either in the levels of dBre1 or in the amounts of other limiting factors in the two cell contexts. Nevertheless, the transfection assays reveal an intrinsic potential of dBre1 in stimulating the transcription mediated by Su(H) and its coactivator NICD (Bray, 2005).

All these results point to a role of dBre1 in promoting Notch signaling. Since other ubiquitin ligases have been shown to influence the levels of specific protein components of the Notch pathway, whether there were any alterations to Notch, Delta, or Su(H) levels in dBre1 mutant clones was investigated. While there are no detectable changes in Notch or Delta staining in dBre1 mutant cells, the levels of Su(H) staining are enhanced slightly but consistently, and cell autonomously, in mutant clones of both dBre1 alleles, regardless of the location of these clones within the disc. This is also obvious in clones induced early in larval development in a non-Minute background in which the mutant dBre1 clones remain small. As an aside, these clones reveal that individual dBre1 mutant cells are enlarged, reminiscent of the yeast bre1p mutant which also shows a 'large cell'phenotype. This phenotype has not been observed in cells lacking Notch signaling, so this aspect of dBre1 function appears distinct from its role in the Notch pathway, and suggests that there are additional molecular targets. Nevertheless, the elevated levels of Su(H) in the dBre1 mutant clones identify Su(H) as one molecular target of dBre1 and suggest that, in the wild-type, dBre1 may expose Su(H) to ubiquitin-mediated degradation. The effects on Su(H) are consistent with the cell-autonomous action of dBre1 on Notch target gene expression, but the fact that removal of dBre1 has a stabilizing effect on Su(H) appears to contradict its stimulating effect on Notch-dependent transcription. Since Su(H) functions as both a repressor and an activator, this may be explained if loss of dBre1 specifically stabilizes the repressor complex. Alternatively, the effect of dBre1 mutations on Su(H) may reflect an indirect bystander activity of dBre1 (Bray, 2005).

Finally, it was asked whether dBre1 has a similar molecular function as its relative yeast Bre1p. The latter is required for the monoubiquitination of histone H2B, which is a prerequisite for the subsequent methylation of histone H3 at K4 by SET1-containing complexes. H3K4 methylation appears to be a chromatin mark for transcriptionally active genes, and yeast bre1p mutants show defects in the transcription of inducible genes that have been ascribed to the lack of H2B ubiquitination and H3K4 methylation at the promoters of these genes. Since there are no in vitro assays for H2B ubiquitination and no antibodies that detect this modified form of H2B, effects of dBre1 mutations on the linked H3K4 methylation were investigated. Wing discs bearing dBre1 mutant clones were stained with an antibody specific for trimethylated H3K4 (H3K4m). This revealed a significant reduction of H3K4m in P1541 mutant clones. More strikingly, in clones of the stronger E132 allele, H3K4m is barely detectable. In contrast, staining of these clones with an antibody against H3K9m does not show any changes in the mutant territory, indicating that the effect in dBre1 mutant clones on the methylation of H3K4 is relatively specific. It is noted that, in wild-type wing discs, there is slight modulation of trimethylated H3K4, with higher levels at the dorsoventral boundary where Notch is activated. However, Notch mutant cells retain robust H3K4m staining, although occasionally show slightly lowered levels compared to adjacent wild-type cells. Thus, the reduced H3K4m staining in dBre1 mutant cells is primarily due to an activity loss of dBre1 rather than due to loss of Notch signaling. Based on its effects on tri-methylated H3K4, it is concluded that dBre1 is indeed the functional homolog of yeast Bre1p. Furthermore, it appears that the activity of dBre1 is essential for the bulk of trimethylated H3K4 in imaginal disc cells (Bray, 2005).

In yeast, H2B ubiquitination and H3K4 methylation are associated with sites of active transcription, but the only identified natural target gene is GAL1. In Drosophila, the target genes of dBre1 evidently include genes regulated by Notch, given the requirement of dBre1 for their transcription. It is therefore conceivable that Su(H) may have a role in targeting dBre1 to their promoters (although it was not possible to detect direct binding or coimmunoprecipitation between dBre1 and Su(H). It is puzzling that dBre1 has a slight destabilizing effect on Su(H), despite being an activating component of Notch signaling. It is believed that this could be a bystander effect of dBre1: evidence suggests that the Bre1p-mediated monoubiquitination of H2B leads to a transient recruitment of proteasome subunits to chromatin, and that the subsequent methylation of H3K4 depends on the activity of these proteasome subunits. Their transient presence at specific target genes may have a destabilizing effect on nearby DNA binding proteins, and the mildly increased levels of Su(H) in dBre1 mutant cells could therefore reflect a failure of proteasome recruitment due to loss of H2B monoubiquitination (Bray, 2005).

Perhaps the most interesting implication of the results is that the dBre1-mediated monoubiquitination of H2B and methylation of H3K4 may be critical steps in the transcription of Notch target genes. Indeed, it appears that the Notch target genes belong to a group of genes whose transcription is particularly susceptible to the much reduced levels of H3K4m in dBre1 mutant cells. Based on the dBre1 mutant phenotypes, there are likely to be other genes in this group, including for example genes controlling cell survival and cell size. Nevertheless, it would appear that the transcription of Notch target genes is particularly reliant on the activity of dBre1. Other examples are emerging where the transcriptional activity of a subset of signal responsive genes is particularly sensitive to the function of a particular chromatin modifying and/or remodelling factor. This sensitivity presumably reflects the molecular mechanisms used by signaling pathways to activate transcription at their responsive enhancers. Understanding why Notch-induced transcription is particularly susceptible to loss of dBre1 function will require knowledge of these underlying molecular mechanisms (Bray, 2005).

Targets of Activity

The consensus binding sequence for Su(H) C/T GTGG/AGAAC/A mediates transcriptional activation of the Enhancer of split complex triggered by Notch signaling. Mutants in Su(H) fail to express m5 and m8 genes from the Enhancer of split complex (Lecourtois, 1995) (Bailey,1995). Enhancer of split complex genes regulate proneural genes achaete and scute.

Notch signal transduction appears to involve the ligand-induced intracellular processing of Notch, and the formation of a processed Notch-Suppressor of Hairless complex that binds DNA and activates the transcription of Notch target genes. This suggests that loss of either Notch or Su(H) activities should lead to similar cell fate changes. However, previous data indicate that, in the Drosophila blastoderm embryo, mesectoderm specification requires Notch but not Su(H) activity. The determination of the mesectodermal fate is specified by Single-minded (Sim), a transcription factor expressed in a single row of cells abutting the mesoderm. The molecular mechanisms by which the dorsoventral gradient of nuclear Dorsal establishes the single-cell wide territory of sim expression are not fully understood. Notch activity is required for sim expression in cellularizing embryos. In contrast, at this stage, Su(H) has a dual function. Su(H) activity is required to up-regulate sim expression in the mesectoderm, and to prevent the ectopic expression of sim dorsally in the neuroectoderm. Repression of sim transcription by Su(H) is direct and independent of Notch activity. Conversely, activation of sim transcription by Notch requires the Su(H)-binding sites. Thus, Notch signaling appears to relieve the repression exerted by Su(H) and to up-regulate sim transcription in the mesectoderm. A model is proposed in which repression by Su(H) and derepression by Notch are essential to allow for the definition of a single row of mesectodermal cells in the blastoderm embryo. This is the first demonstration of the functional switch for Su(H) from a repressor to an activator (Morel, 2000).

To gain insight into the molecular mechanisms by which Su(H) and Notch regulate sim expression, an examination was carried out to see whether Su(H) regulates sim expression in a direct manner. The regulatory elements necessary for mesectodermal expression of sim are contained within a 2.8-kb genomic DNA region. Sequence analysis has identified 10 putative Su(H)-binding sites, with 6 of these exactly matching the GTGRGAA consensus binding sites (Su4, Su5, Su7, Su8, Su9, and Su10). In gel shift experiments, Su(H) binds strongly to oligonucleotides corresponding to each of these sites. Two additional sites, Su2 and Su6, match the consensus RTGRGAR that accomodates nearly all sites that have been shown to bind Su(H) in vitro. These two sites bind weakly to Su(H), both in direct binding assays and in competition experiments. The ability of two noncanonical sites, Su1 and Su3, to bind Su(H) in vitro was also examined. Both Su1 and Su3 bind weakly to Su(H). Other sequences that differ from the RTGRGAR at a single position are not known to bind Su(H) in vitro. Thus, the sim regulatory sequences contain at least 10 binding sites for Su(H). Eight of these sites are clustered in a 500-bp region that contains functional binding sites for Dorsal, Twist, and Snail. Moreover, the organization of this regulatory region has been conserved throughout evolution between D. melanogaster and D. virilis. Together, these data strongly suggest that Su(H) regulates sim transcription directly (Morel, 2000).

Su(H) not only mediates the Notch-dependent activation of sim transcription, but also acts as a transcriptional repressor. This latter conclusion is supported by the following two findings: (1) a complete loss of Su(H) activity leads to weak ectopic expression of sim in the neuroectoderm; (2) the deletion of all of the Su(H)-binding sites from the sim regulatory region also results in ectopic activation of the sim promoter in the ventral neuroectoderm. In Notch mutant embryos, repression by Su(H) is observed not only in the neuroectoderm, but also in the mesectoderm. Because Su(H) is expressed maternally, it is speculated that uniformly localized Su(H) might repress the activation of sim transcription in all of the cells in which Notch is not activated (Morel, 2000).

This study provides the first evidence that Su(H) can act as a transcriptional repressor in Drosophila, and that its repression activity is inhibited by the activation of the Notch receptor. In mammals it has been suggested that the binding of processed Notch to CBF1 competes with the binding of corepressors to CBF1 to promote the formation of an activation complex. The results presented here suggest that Su(H) might mediate such a transcriptional switch at the sim promoter in mesectodermal cells (Morel, 2000).

This regulatory mechanism, in which transcriptional repression is inhibited by a signaling input, may be a general feature of Notch-mediated gene regulation. Consistent with this view, repression by Su(H) might contribute to the difference seen between Notch and Su(H) mutant cuticular phenotypes. Similarly, the cuticular phenotype associated with a deletion removing all of the bHLH-Enhancer of split genes, but not groucho, also appears to be more severe than the one associated with a complete loss of Su(H) function. Because the bHLH-Enhancer of split genes are direct transcriptional targets of Su(H) during neurogenesis, it has been suggested that Su(H) might also act as a transcriptional repressor of the Enhancer of split genes. The finding that Su(H) can repress a Notch target gene indicates that phenotypic differences between Notch and Su(H) mutations do not necessarily imply that Notch signals in a Su(H)-independent manner (Morel, 2000).

How is a single-cell wide territory of sim expression established on the basis of the nuclear gradient of Dorsal? The data presented here, together with previous studies, suggest the following model. In the mesoderm, transcriptional activation of sim by Dorsal and Twist is inhibited by Snail. Whether Su(H) and/or Notch play any role in these cells is not known. In more dorsal cells that do not accumulate Snail, it is proposed that positive regulation of sim by low levels of Dorsal and Twist is antagonized by Su(H). However, in cells bordering the mesoderm, negative regulation by Su(H) would be relieved locally by Notch signaling. This would lead to the specific expression of sim in these cells, which will then form the mesectoderm (Morel, 2000).

An important feature of this model is that Notch signaling overcomes repression by Su(H) only in the single row of cells abutting the mesoderm. One possible explanation for this is that Notch participates in the contact-dependent reception of a mesodermal signal. Results from nuclear transplantation experiments support the existence of a mesodermal signal. When transplanted into snail/twist double mutant embryos that do not express sim, wild-type nuclei can induce the expression of sim in neighboring mutant cells. This result suggests that, in wild-type embryos, mesodermal cells may produce an inductive signal that activates sim transcription in the mesectoderm. Although the molecular nature of this signal is not known, it is speculated that this mesodermal signal might participate in the activation of Notch (Morel, 2000).

Consistent with the view that Notch is specifically activated in ventral cells, changes in the subcellular distribution of both Notch and Delta have been observed ventrally in stage 5 embryos. (1) Lower levels of Notch are found in ventral cells as the ventral furrow forms. (2) In cellularized embryos, Delta is found at the cell membrane, except in ventral cells, in which it predominantly accumulates in vesicles. Both down-regulation of Notch and vesicular accumulation of Delta are consistent with Delta activating Notch in ventral cells in stage 5 embryos (because Snail represses sim transcription, activation of Notch in the mesoderm may have no effect on sim transcription). It will thus be of interest to determine whether these changes in the subcellular distribution of Notch and Delta can be observed in both mesodermal and mesectodermal cells, but not in the more dorsal neuroectodermal cells (Morel, 2000).

In conclusion, repression by Su(H) can be viewed as a refining mechanism ensuring that Notch target genes are expressed only in cells reaching a high threshold of Notch activation. In the early embryo, repression of sim expression allows for the definition of a single row of mesectodermal cells. In these cells, a high level of Notch activity might be induced by a juxtacrine (contact-dependent) inductive signal produced by the mesoderm. In view of this hypothesis, the sharp mesodermal boundary defined by snail expression would be shifted dorsally by one cell, thereby defining a single row of mesectodermal cells (Morel, 2000).

The activity of Notch is required for the transcriptional activation of the sim gene in the mesectoderm, and Su(H) directly regulates sim expression. However, both the sim gene and the simmut-lacZ construct that does not respond to activated Su(H) are expressed in mesectodermal cells in the complete absence of Su(H) activity. These results might suggest that Notch signals, at least in part, in a Su(H)-independent manner to activate sim expression in the mesectoderm. Alternatively, the observation that Su(H) acts to repress sim expression raises the possibility that Notch might be required to antagonize repression by Su(H). To distinguish between these two possibilities, the expression of simmut-lacZ was examined in Notch mutant embryos. simmut-lacZ is expressed at a low level both in the mesectoderm and ectopically in the dorsal neuroectoderm. This pattern is very similar to that observed for simmut-lacZ in wild-type embryos, and dramatically differs from the complete loss of sim-lacZ expression seen in Notch mutant embryos. This shows that the Su(H)-binding sites are required to repress sim transcription in the mesectoderm as well as in the neuroectoderm in the absence of Notch signaling. Furthermore, this demonstrates that repression of sim expression by Su(H), both in ventral neuroectodermal and mesectodermal cells, does not require Notch activity. It is concluded that Su(H) acts as a Notch-independent repressor. Thus, no evidence has been found for a Su(H)-independent function of Notch in the regulation of sim expression (Morel, 2000).

It has been suggested that wingless expression at the dorsal-ventral boundary of the wing disc depends on a signal from dorsal to ventral cells mediated by Serrate and Notch. Wingless expression is lost from the wing margin and the size of the wing is significantly reduced when Notch activity is removed from the third instar larva using a temperature sensitive allele of Notch. In addition, clones of cells mutant for Serrate can cause extensive non-autonomous loss of wing tissue, but only when the clone includes the cells that abut the dorsal compartment boundary. Therefore, it is likely that wingless is regulated by the Notch pathway acting through Suppressor of Hairless (Diaz-Benjumea, 1995).

Several different mechanisms have been proposed to account for the activation of Su(H) by Notch. To further investigate how Su(H) activity is regulated, misexpression assays were used with wild-type Su(H) and with modified forms of Su(H) containing either a nuclear localization signal [Su(H)NLS], a transcriptional activation domain [Su(H)VP16], or a deletion of the domain required for interaction with the antagonist Hairless [Su(H)DH]. Only Su(H)VP16 is able to mimic Notch activation effectively in the Drosophila wing, in agreement with the model that Notch activity normally confers coactivator function on Su(H). Neither nuclear localization nor elimination of Hairless binding is sufficient for activation. The phenotypes produced by overexpression of Su(H)wt and Su(H)NLS indicate a mixture of both increased and reduced Notch pathway activity and point to a role for Su(H) in both activation and repression of gene expression, as has been proposed for the mammalian homolog CBF1. Some phenotypes are equivalent to Notch loss-of-function, with wing-nicks and inhibition of a subset of target genes, which is most consistent with the ectopic proteins displacing a Su(H)-coactivator complex. Conversely, other phenotypes are equivalent to Notch gain-of-function, with wing-overgrowths and ectopic target-gene expression. These effects can be explained by the ectopic Su(H)/Su(H)NLS titrating a repressor complex. The wing-overgrowth phenotype is sensitive to the dose of Hairless and the phenotypes produced by coexpressing Su(H) and Hairless suggest that Hairless could form a component of this repressive complex (Furriols, 2000).

The phenotypes produced by misexpressing Su(H)wt and Su(H)NLS appear to combine activation and repression of Notch activity. To confirm whether this interpretation is correct, the effects on genes whose expression at the dorsal/ventral boundary is dependent on Notch were analyzed. Two assays used fragments that are directly responsive to Su(H) and Nicd: mbeta1.5 (a fusion between the E(spl)mbeta regulatory sequences and lacZ) and vgBE-lacZ (a fusion between the vestigial boundary enhancer and lacZ). Expression of wingless and the entire vestigial gene, which may involve indirect as well as direct regulation by the Notch pathway, were also examined. All are ectopically activated by Su(H)VP16, consistent with it mimicking the effects of Nicd. As anticipated, the wing-nick phenotypes produced by the modified Su(H) proteins in combination with ptc-Gal4 correlate with a reduction in the levels of mbeta1.5 and wingless expression at the d/v boundary. mbeta1.5 appears to be more sensitive and is strongly repressed by all three proteins, with Su(H)NLS the most effective as suggested by the wing-nicking phenotypes. The effects on wingless were milder and only Su-(H)NLS strongly represses expression. In contrast, vgBE-lacZ shows a very different response and is ectopically activated by both Su(H)wt and Su-(H)NLS. The activation of vgBE-lacZ is also observed when the levels of misexpressed proteins are lower (using dpp-Gal4) although in this combination Su(H)NLS and Su(H)wt still repress mbeta1.5. The two enhancers therefore appear intrinsically different in the way they respond to Su(H), suggesting that their regulation may involve different thresholds of activating and repressing Su(H) containing complexes (Furriols, 2000).

The mixed loss and gain of Notch function phenotypes produced in the wing by ectopic expression of Su(H)wt and Su(H)NLS suggests that Su(H) has a dual function, acting in some contexts as an activator and in others as a repressor. The simplest model is that Su(H) can exist in at least two complexes -- one where it interacts with a coactivator(s) and the other where it interacts with a corepressor(s). In cells where there is no/low Notch signaling the primary function of Su(H) would be to keep the target genes repressed by interacting with a corepressor complex. One of these corepressors could be Hairless; another could be the HDAC complex described in vertebrates. In contrast, in the cells where Notch is active, Su(H) would be complexed with coactivator(s) (e.g., Nicd so that the transcription of Notch target genes would be initiated). Depending on the relative levels or activity of the components, the equilibrium would shift in favor of one or the other complex (Furriols, 2000).

The formation of many complex structures is controlled by a special class of transcription factors encoded by selector genes. It has been shown that Scalloped, the DNA binding component of the selector protein complex for the Drosophila wing field, binds to and directly regulates the cis-regulatory elements of many individual target genes within the genetic regulatory network controlling wing development. Furthermore, combinations of binding sites for Scalloped and transcriptional effectors of signaling pathways are necessary and sufficient to specify wing-specific responses to different signaling pathways. The obligate integration of selector and signaling protein inputs on cis-regulatory DNA may be a general mechanism by which selector proteins control extensive genetic regulatory networks during development (Guss, 2001).

Each of the Sd targets analyzed is activated in only a portion of the wing field, in patterns controlled by specific signaling pathways. For instance, cut is a target of Notch signaling along the dorsoventral boundary, and the sal and vg quadrant enhancers are targets of Dpp signaling along the anteroposterior axis. Binding sites for the transcriptional effectors of the Notch- and Dpp-signaling pathways, Suppressor of Hairless [Su(H)], and Mothers Against Dpp (Mad), and Medea (Med), respectively, have been shown to be necessary for the activity of a number of wing-specific cis-regulatory elements, and occur in these elements. This observation, coupled with the data demonstrating a direct requirement for Sd binding, suggests that gene expression in the wing field requires two discrete inputs on the cis-regulatory DNA: one from the selector proteins that define the field, and one from the signaling pathway that patterns the field (Guss, 2001).

These findings also raised the possibility that the combination of selector and signal inputs may be sufficient to drive field-specific, patterned gene expression. To test this, there were built a number of synthetic regulatory elements comprised of combinations of Sd binding sites with binding sites for Su(H) or Mad/Med. The activity of these elements was compared with those composed of tandem arrays of just selector- or signal effector-binding sites, or combinations of different signal effector sites. Each of the binding sites used in these constructs was selected from sequences found in native Drosophila cis-regulatory elements that have been demonstrated to function in vivo (Guss, 2001).

Elements containing only single classes of binding sites for the selector or signal effectors were unable to drive reporter gene expression in the wing. In contrast, the synthetic elements in which binding sites for both selector and signal effector were combined drove field-specific expression restricted to the wing and haltere discs in patterns predicted by the specific signaling inputs to each element. That is, the [Sd]2 [Su(H)]2 element drove wing-specific expression along the dorsoventral margin, consistent with Notch activation along this boundary, and the [Sd]2 [Mad/Med] element drove expression in a broad domain oriented with respect to the anteroposterior axis of the disc, consistent with Dpp-signaling activity along this boundary. These patterns of expression are similar to those of the native cut and vg quadrant cis-regulatory elements that also respond to Notch- and Dpp-signaling inputs, respectively. However, regulatory elements containing a combination of Su(H) and Mad/Med sites were not active in vivo, demonstrating that combinatorial input in the absence of selector input is not sufficient to drive gene expression. These results suggest that the Vg-Sd complex provides a qualitatively distinct function required to generate a wing-specific response to signaling pathways (Guss, 2001).

Notch-dependent activation of wingless, cut and vestigial at the wing margin depends on the activity of Suppressor of Hairless. Su(H)-mutant cells lose expression of the vestigial early enhancer, of wingless and of cut in a cell autonomous manner. Clones of Su(H)-mutant cells cause loss of wing tissue and scalloping of the wing, but only in Notch mutant clones at the D/V boundary. vestigial expression at the D/V boundary does not depend on wingless, since misexpression of wild-type wg cDNA, which results in wing margin bristles, does not cause an expansion of vestigial expression. Likewise, wingless expression does not depend on an early function of vestigial. Both Notch and wingless cooperate to activate cut at the D/V boundary (Neumann, 1996).

The Notch receptor mediates cell interactions controlling the developmental fate of a broad spectrum of undifferentiated cells. By modulating Notch signaling in specific precursor cells during Drosophila imaginal disc development, it has been demonstrated that Notch activity can influence cell proliferation. The activation of the Notch receptor in the wing disc induces the expression of the wing margin patterning genes vestigial and wingless, and strong mitotic activity. However, the effect of Notch signaling on cell proliferation is not the simple consequence of the upregulation of either vestigial or wingless. On the contrary, Vestigial and Wingless display synergistic effects with Notch signaling, resulting in the stimulation of cell proliferation in imaginal discs (Go, 1998).

To explore the consequences of Notch signaling modulation during Drosophila development, the UAS-GAL4 system was used. Loss-of-function phenotypes were elicited through the expression of either a truncated, dominant negative form of the Notch receptor (d.n.N) lacking the intracellular domain, or the Hairless (H) protein, a negative regulator of Notch signaling. Gain-of-function phenotypes were induced by expressing a constitutively activated form of the Notch receptor (act.N) (Go, 1998).

To examine the link between the H misexpression phenotypes and Su(H)-dependent Notch activity, transgenic animals were generated carrying a lacZ reporter construct driven by the fusion between multimerized Su(H)-binding sites and an E(spl)m gamma promoter, a known Su(H) target. This construct consists almost exclusively of engineered Su(H)-binding sites. In a cell culture based reporter, the expression from the reporter construct is induced by the simultaneous expression of Su(H) and act.N, while the expression of any one construct alone fails to induce transcription. Strong lacZ expression is detected in the posterior part of the eye disc of late third instar transgenic larva. This expression is effectively suppressed by misexpression of H using the GAL4 line T113 and results in small eye discs, indicating that H overexpression can suppress Su(H)-dependent Notch signaling in vivo. The size of the eye is significantly affected and, in extreme cases, the eye is missing. In addition to small eyes, small wings and halteres are observed as well as more typical Notch loss-of-function phenotypes, such as extra thoracic bristles. The 'small eye' phenotype induced by H expression is not associated with severe eye roughness. This 'small eye' phenotype, together with the wing and haltere abnormalities, is reminiscent of Serrate loss-of-function mutations. To further explore the possibility that the observed eye phenotype reflects Ser-dependent Notch signaling, the genetic interactions were examined with Beaded of Goldschmidt (BdG), a dominant negative mutation of Ser known to affect wing margin development. In combination with BdG, strong synergistic effects are observed displaying phenotypes characteristic of Ser, such as small eyes, wings and halteres. Therefore, H misexpression can mimic Ser loss-of-function mutations, raising the possibility that Ser/Notch signaling may control eye morphogenesis (Go, 1998).

To further investigate the role Notch signaling plays in morphogenesis, the H and d.n.N transgenes were expressed at the d/v compartment boundary of the wing disc using the vestigial-GAL4 driver. Misexpression of either H or d.n.N results in similar phenotypes, which range from wing margin notches to rudimentary wings. The effect of H misexpression can be suppressed by expressing act.N and vice versa. For example, the lethality associated with misexpression of act.N is suppressed by simultaneous expression of H. Conversely, the phenotypes elicited by H misexpression are largely suppressed by act.N. This mutual suppression is observed with other GAL4 lines as well. Given that the actions of act.N and H seem to be manifested through Su(H), it is likely that the mutual suppression of act.N and H is also mediated by Su(H). It is noteworthy that, even though both H and d.n.N act as antagonists of Notch signaling and the phenotypes associated with their expression are similar, their interactions with act.N are different. While act.N is an effective suppressor of the phenotypes induced by H misexpression, it fails to suppress the effects of d.n.N (Go, 1998).

The relationship between Notch signaling and the expression of vg and wg was examined, since the induction of both genes is considered to be essential for wing morphogenesis. When either d.n.N or H is misexpressed along the anterior/posterior (a/p) boundary using the ptc-GAL4 line, expression from the vg d/v boundary enhancer, as well as the wg enhancer, is effectively repressed near the intersection between the a/p and d/v boundaries. In contrast, the vg quadrant enhancer, which is normally silent at the intersection between a/p and d/v boundaries, is induced by the identical constructs. Essentially the opposite effect is observed when act.N is misexpressed, demonstrating that Notch signaling has opposite effects on two distinct enhancers of vg (Go, 1998).

The wing phenotypes elicited by misexpression of act.N are similar to those induced by Abruptex (Ax) mutations, which are Notch gain-of-function alleles associated with point mutations in the extracellular domain of the protein. A heteroallelic Ax combination results in the activation of Notch signaling and the expression of Notch downstream genes is induced. For instance, ectopic wg expression is found around the d/v boundary. Induction of the vg d/v boundary enhancer and repression of the vg quadrant enhancer around the d/v boundary are found, similar to the effect of expression of act.N. Activation of Notch signaling around the d/v boundary of the wing disc through either misexpression of act.N or the Ax mutations results in a substantial enlargement of the disc. BrdU incorporation experiments indicate that these phenotypes are associated with an elevated mitotic activity. BrdU incorporation is stimulated and is particularly obvious in the peripheral region of the wing pouch, suggesting that the periphery Is more responsive than other regions. Misexpression of act.N in other parts of the wing disc also results in the stimulation of mitotic activity. When act.N is expressed in the wing pouch, the disc grows in such a way that the characteristic folded structures of the wing pouch are pushed to the periphery. Conversely, the same structures are 'pushed' toward the d/v boundary when act.N is expressed in the periphery. When act.N is misexpressed in a discrete pattern in the periphery using the GAL4 line 766, a regional correspondence is observed between Notch signaling activation and high mitotic activity, demonstrating a local effect of Notch activity on cell proliferation in the periphery. However, as is particularly evident when the Notch receptor is activated around the d/v boundary, the region of Notch signaling activation does not coincide with the region of the highest mitotic activity. It is therefore concluded that the effect of Notch signaling on cell proliferation must be indirect (Go, 1998).

The effect of Notch activity on cell proliferation is not the simple consequence of vg induction Since vg is a direct target of Su(H)-dependent Notch signaling, it is possible that the mitogenic effect of Notch is mediated by the upregulation of vg. In this case, misexpression of Vg would be expected to result in phenotypes similar to those elicited by act.N. Misexpression of act.N in the dorsal side of the wing pouch, using the GAL4 line A9, induces expression from the vg d/v boundary enhancer as well as the wg enhancer The dorsal side of the wing pouch region appears enlarged. In contrast, when Vg is misexpressed in the same region, the dorsal side of the wing pouch becomes much smaller than the ventral side, while wg expression in the periphery of the dorsal side was suppressed. The loss of dorsal wing pouch induced by Vg misexpression is significantly rescued by expressing Wg simultaneously. This is consistent with the notion that the observed phenotype caused by misexpression of Vg is due to the repression of wg, whose expression in the wing pouch is more uniform at earlier stages. Misexpression of Wg alone in the dorsal side, unlike the misexpression of act.N, does not have a significant effect on cell proliferation in the wing pouch. These results indicate that the effect of act.N expression on mitosis is separable from vg induction. In addition, they indicate that Vg is capable of repressing wg expression in the wing pouch, but not at the d/v boundary (Go, 1998).

Misexpression of Vg compared to act.N has opposite effects in the wing disc. Thus, Vg misexpression in the wing disc induces wg downregulation and small discs. In contrast, misexpression of Vg in the eye discs upregulates wg and results in a clear enlargement of the discs, demonstrating that Vg can either repress or induce wg expression in a context-dependent manner. The observed context-dependent effect of Vg on wg expression raises the possibility that Notch signaling may be capable of modulating the way Vg affects wg expression. This is of particular interest in view of the possibility that Vg does not suppress wg expression at the d/v boundary because of the existing high level of Notch signaling activity. In fact, the simultaneous expression of act.N and Vg reveals a striking synergistic effect on cell proliferation. The most notable effects are in the eye discs, where tissue expressing the two proteins shows striking overgrowth associated with strong wg induction. The other discs are also clearly affected, displaying cellular overgrowth, but the effects are far less dramatic than the eye discs. This overgrowth phenotype is also evident when act.N and Vg misexpression are driven by dpp-GAL4, even though the synergistic effect is less dramatic. In contrast, the effect of misexpression of Vg with dpp-GAL4 on wg induction and cell proliferation in the eye discs is, in some cases, significantly suppressed by simultaneous expression of H. These experiments demonstrate that the proliferative potential of certain tissues can be modulated by the synergistic action of Notch with other genes. Moreover, they identify Notch signaling as an important factor in the way Vg affects wg expression and cell proliferation at the d/v boundary during wing morphogenesis (Go, 1998).

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 4 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 in only a subset of the territories and cells in which these modes of regulation are operative (Nellesen, 1999).

Two different activities of Suppressor of Hairless during wing development in Drosophila

The Notch pathway plays a crucial and universal role in the assignation of cell fates during development. In Drosophila, Notch is a transmembrane protein that acts as a receptor of two ligands, Serrate and Delta. The current model of Notch signal transduction proposes that Notch is activated upon binding its ligands and that this leads to the cleavage and release of its intracellular domain (also called Nintra). Nintra translocates to the nucleus where it forms a dimeric transcription activator with the Su(H) protein. In contrast with this activation model, experiments with the vertebrate homolog of Su(H), CBF1, suggest that, in vertebrates, Nintra converts CBF1 from a repressor into an activator. The role of Su(H) in Notch signaling during the development of the wing of Drosophila has been assessed. The results show that, during this process, Su(H) can activate the expression of some Notch target genes and that it can do so without the activation of the Notch pathway or the presence of Nintra. In contrast, the activation of other Notch target genes requires both Su(H) and Nintra, and, in the absence of Nintra, Su(H) acts as a repressor. The Hairless protein interacts with Notch signaling during wing development and inhibits the activity of Su(H). These results suggest that, in Drosophila, the activation of Su(H) by Notch involves the release of Su(H) from an inhibitory complex, which contains the Hairless protein. After its release Su(H) can activate gene expression in the absence of Nintra (Klein, 2000).

Since Su(H) is expressed ubiquitously and continuously, the fact that Su(H) can promote transcription without the presence of Nintra suggests that the activity of Su(H) must be suppressed in the absence of Notch activation during normal development. One possible mechanism for this is the binding of an inhibitory factor. A candidate for this function is the Hairless (H) protein, which has been shown to interact with Su(H) and antagonize its DNA-binding activity. Furthermore, several reports show that H antagonizes Notch signaling during adult PNS development. To test whether H is an antagonist of Su(H) also during wing development, H mutant clones were induced in the wing pouch and it was asked whether genes dependent only on Su(H) activity are expressed in these clones. If H regulates the activity of Su(H), the removal of H might lead to the activation of Su(H) and result in the expression of its targets, e.g. the vestigial boundary enhancer (vgBE) and Serrate. Both are ectopically activated in H mutant clones. The ectopic expression of the vgBE in the clones varies and is strongest near the DV boundary. This graded expression is possibly due to the requirement of a diffusible factor coming from the DV boundary. One candidate for this is Wg, which seems to be required for the proper expression of the vgBE. The cells in the H mutant clones do not express cut or wg, which are dependent on the presence of Nintra, suggesting that Notch is not activated in these clones (Klein, 2000).

The loss of H function seems to elicit Su(H)-dependent target gene expression in the wing pouch, a region probably devoid of Notch activity. This suggests that the inactivation of H is sufficient to activate Su(H). To test further this conclusion, an examination was performed to see whether the activity of the vgBE is maintained in H mutant wing pouches if Notch is concomitantly removed. For this, Notch mutant clones were induced in H mutant wing discs. In H mutant wing pouches, weak ubiquitous expression of the vgBE is observed throughout the whole area of the wing, confirming the clonal analysis. vgBE is also active in several Notch mutant clones near the DV and anteroposterior (AP) boundary, but the activity is not maintained in all clones. One explanation for this might be again the requirement of other so far unidentified factors emanating from the two compartment boundaries. In agreement with this, the vgBE enhancer has a late expression domain along the AP boundary, suggesting an input from these areas for its proper expression. However this domain is also dependent on Notch during normal development. The removal of the Su(H)-binding site in the enhancer leads to the loss of all expression domains in the wing pouch, suggesting that Su(H) is required (Klein, 2000).

Therefore, the fact that the cells of several mutant clones do express the vgBE suggest that the vgBE can be activated in the complete absence of Notch activity and that the inactivation of H is sufficient to activate Su(H). No activation of the vgBE was ever found in Notch mutant clones induced in wild-type wing pouches, suggesting that during wild-type development, the activity of Notch is required to activate the vgBE. Hence, Notch probably activates Su(H) through inactivation of H. An examination was performed to see whether the degree of endogenous Su(H) activation that results from the removal of H is sufficient to elicit a biological effect. To assay this, it was asked whether or not removal of H activity can induce Su(H)-dependent development of the pouch in wing discs in which Notch signaling is absent, such as apterous and Presenilin mutant wing discs. Loss of H function rescues the loss of wing development of ap mutants: whereas ap mutants have no wing pouch, ap;H double mutants have large wing pouches with no margin structures. The enlarged pouch of the double mutant discs expresses spalt (sal) and the two vg reporters, vgQE and vgBE, all of which are expressed specifically in the wing pouch in a Notch/Su(H)-dependent manner and are not expressed in ap mutants. In contrast, no wg expression is induced in these double mutant discs, suggesting that the observed rescue is likely to be due to the activation of Su(H) in the double mutants. This is strongly supported by the fact that Su(H);H double mutants exhibit a small wing rudiment identical to that of Su(H) mutants. Expression of UAS-vg by dpp-Gal4 in ap mutant discs can recover the pouch-specific expression domain of sal, suggesting that the activation of vg expression by Su(H) is responsible for the recovered sal expression in the ap;H double mutant wing discs. Similar to overexpression of UAS-Su(H) in ap mutant wing discs, the pouch in ap;H mutant discs develops near the residual wg expression in the remaining hinge. As expected from the analysis of the wing discs, the pharate adult ap;H double mutants have large wing pouches, which are devoid of any margin like structure such as innervated bristles (Klein, 2000).

The effects on wing development of removing H in Psn mutants were examined. As in the case of ap, loss of function of H effects a strong rescue of the wing pouch in the Psn;H mutant discs in comparison to the Psn mutant discs. However, in this case, the morphology of the discs is more like wild type and, in contrast to ap;H mutant discs, the pouch develops at its normal place. Closer monitoring of double mutant discs reveals some expression of wg and the vgBE along the DV boundary. This suggests that, in contrast to the situation of ap mutants, in Psn mutants, there is some activation of Notch and it seems that the lack of H activity can enhance this residual signaling of Notch at the DV boundary. This is remarkable considering that the wing phenotype caused by the loss of Psn is stronger than that caused by loss of Su(H) function. Taken together, these results provide further evidence for a positive transcriptional activity of Su(H). They further show that H is an antagonist of Su(H) during early wing development and that it suppresses the activity of Su(H) in the absence of Notch signaling. The results also suggest that the inactivation of H is sufficient to activate Su(H) and that the activity of Notch is required to inactivate H during normal development (Klein, 2000).

Overexpression of Su(H) leads to three different responses: (1) activation, as is the case for vg, some E(spl) genes, Dl and Ser; (2) inactivation, as shown for cut and E(spl)m8; or (3) no effect, as is the case for wg. This differential behavior is, at least in some cases, a consequence of direct binding of Su(H) to the promoters: the vgBE as well as the E(spl) genes contain Su(H)-binding sites to which Su(H) binds; such sites are necessary for the activation of these genes in vivo. Despite that, they react differently towards Su(H) overexpression. Since E(spl)m8, which is suppressed by Su(H) overexpression, can be activated by expression of Su(H)VP16 or Nintra, it is concluded that Nintra is required in addition to Su(H) to activate E(spl)m8 expression. The results suggest that, in this case, Nintra probably acts as an activation domain of a dimeric transcription factor containing Su(H), as has been proposed. From this, it follows that Nintra might have two function during a Notch signaling event: first it inactivates H, which leads to the release of Su(H) and then, in some instances, it provides the transactivation domain for free Su(H) to activate the expression of target genes (Klein, 2000).

Flies carrying reporter lacZ constructs with up to 12 Su(H)-binding sites do not display any activity in the wing disc. This suggests that Su(H) (even in association with Nintra) is not sufficient to activate transcription and requires other collaborating factors. It further suggests that, even in promoters that can be activated by Su(H) in the absence of Nintra, Su(H) probably interacts with other factors to promote gene expression. This is confirmed by a study of the vgBE. Although the Su(H)-binding site is absolutely necessary for its activity, other sites are equally important. So far the factors that bind to these sites are not identified. The dependence of Su(H) on these others factors is probably the reason for the differential expression of Notch target genes in H and H/N mutant clones that have been observed (Klein, 2000).

Recently it has been shown that Su(H) acts as a suppressor of single minded transcription during the formation of the midline cells in the embryonic central nervous system of Drosophila. This observation provides the first evidence that Su(H), like its mammalian counterpart CBF1, can act as a suppressor of transcription. The inactivation of the cut and E(spl)m8 expression in absence of Nintra suggests that Su(H) can act as a suppressor of gene expression also during adult development and provides further evidence for a suppressing activity of Su(H). However, this suppression is context dependent and not a general feature of Su(H). This context dependency might also exist for CBF1, since only the reaction of a small number of genes towards its activity has been tested so far and it is possible that some target genes can be activated by CBF1 in the absence of Nintra in a similar way, as has been shown for Su(H). In summary, these results suggest that the consequence of the binding of Su(H) to a promoter is dependent on its local architecture and, therefore, Su(H) can at the same time activate and suppress gene expression, like many other transcription factors. The removal of both the maternal and zygotic expression of H during embryogenesis seems to be of no consequence for the embryo. Since the overactivation of Notch/Su(H) signaling during embryogenesis has deleterious consequences, this observation contradicts the conclusion that H is required to inactivate Su(H). However, the context dependency and differential reaction of the target genes observed during wing development offer two explanations for this discrepancy, without having to postulate an unknown factor, which can functionally replace H. First, it is likely that the interacting factors, which are required for gene expression in concert with Su(H), are different during embryogenesis and this could modulate the responsiveness of the target promoters. This conclusion is supported by the observation that the genes of E(spl)C, although probably all requiring Su(H) for their expression, are all very similarly expressed in the embryo, but their expression pattern in the wing imaginal disc is very different. Another explanation is that the target promoters of binding Su(H) during embryogenesis might be all of the type that require the additional activity of Nintra. Therefore they would stay inactive even in the presence of free Su(H) until Notch is activated (Klein, 2000).

The Enhancer of split complex of Drosophila melanogaster harbors three classes of Notch responsive genes

Many cell fate decisions in higher animals are based on intercellular communication governed by the Notch signaling pathway. Developmental signals received by the Notch receptor cause Suppressor of Hairless [Su(H)] mediate transcription of target genes. In Drosophila, the majority of Notch target genes known so far is located in the Enhancer of split complex [E(spl)-C], encoding small basic helix-loop-helix (bHLH) proteins that presumably act as transcriptional repressors. The E(spl)-C contains three additional Notch responsive, non-bHLH genes: E(spl) region transcript m4 (m4) and malpha are structurally related, whilst m2 encodes a novel protein. All three genes depend on Su(H) for initiation and/or maintenance of transcription. The two other non-bHLH genes within the locus, m1 and m6, are unrelated to the Notch pathway: m1 might code for a protease inhibitor of the Kazal family, and m6 for a novel peptide. The five genes described in this paper are arrayed between mbeta and m7, both coding for bHLH proteins. Two other bHLH genes, m3 and m5 are intermingled with the five. Bearded and M4 are 16% identical. Furthermore, in transcripts of both Brd and m4 there are three common regulatory sequence motifs within the 3' UTR. These are known as the 'Brd box', the 'GY box' and the 'K box'. As in m4, the sequence motif of the Brd box is found twice in the 3'-UTR of malpha mRNA at similar positions but without a GY box. None of the other four non-bHLH E(spl)-C genes contains either Brd or GY box. The K box appears to be more common. It is found twice in the 3'-UTR of malpha and once each in the 3' UTRs of m2 and m6 (Wurmbach, 1999).

malpha and m4 embyonic expression patterns are nearly indistinguishable, and appear very similar to those of E(spl)-C bHLH genes, particularly m5, m7 and m8. The expression patterns suggest that both genes are under the same regulatory conrol as are the differ E(spl) bHLH genes and thus, might serve a role in Notch mediated cell differentiation. Surprisingly, also m2 transcripts accumulate in a pattern reminiscent of the transcript distribution of E(spl) bHLH genes, although there are no structural similarites with either the bHLH or the m4/malpha genes. Therefore m2 might serve as a Notch target gene. Unlike the other E(spl)-C genes, the gene is expressed within neuronal cells in the embryo. m6 mRNA accumulates in the CNS, brain and PNS, and in imaginal tissues. m1 is expressed in the digestive tract. Su(H) is shown to be the transmitter of Notch signaling to malpha, m4 and m2. Thus there are three types of Notch responsive genes. The bHLH genes are represented by m8 and others. m4 and malpha share structural similarity with Bearded. These Bearded family proteins share a presumptive basic amphipatic alpha-helical domain but differ with regard to other conserved sequence elements. m2, coding for a novel protein, represents the third class of Notch responsive genes (Wurmbach, 1999).

Discrete enhancer elements mediate selective responsiveness of enhancer of split complex genes to common transcriptional activators

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 known to be 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. 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, which 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]. As originally defined by its structure in m4, m8 and mgamma, the Su(H) paired site (SPS) configuration consists of two high-affinity (YGTGRGAAM; M denotes A or C) Su(H) binding sites in an inerted repeat arrangement, with 300 bp between the first G of the two sites. In addition, the 'Y' of the upstream site is T, while that of the downstream site is C. Finally, the sequence between the two Su(H) sites includes the hexamer GAAAGT or its complement ACTTTC. The SPS motifs of m4 and mgamma in Drosophila hydei are remarkably conserved. A 43-nt block in m4 SPS contains the entire SPS motif and shows only a single varient position. Also all four bHLH activator binding sites in the two genes are conserved. A putative bHLH repressor binding site defined by the N box consensus sequence CACNAG does not appear to be as conserved as sites defined by CACGYG that have been shown to bind these proteins with high affinity. Thus of four distinct CACGYG sites (one in mgamma and three in m4 ), two are conserved, while apparently only one of seven distinct N box sites (three in mgamma and four in m4) is conserved. Conserved transcription factor binding sites are often accompanied by strongly conserved flanking sequences. 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 in only a subset of the territories and cells in which these modes of regulation are operative (Nellesen, 1999).

Positive and negative control of Serrate expression during early development of the Drosophila wing

The product of the Drosophila gene Serrate acts as a short-range signal during wing development to induce the organizing center at the dorsal/ventral compartment boundary, from which growth and patterning of the wing is controlled. Regulatory elements reflecting the early Serrate expression in the dorsal compartment of the wing disc have recently been confined to a genomic fragment in the 5'-upstream region of the gene (from -8 to -18 kb). This fragment, termed the dorsal wing regulator or DWR, responds to various positive and negative inputs required for the early Serrate expression. Activation and maintenance of expression in the dorsal compartment of the wing discs of second and early third instar larvae depend on apterous, as revealed by reporter gene expression in discs either lacking or ectopically expressing apterous. The DWR is not activated by ectopic fringe expression in the ventral compartment, suggesting that the observed induction of Serrate protein by ectopic Fringe is mediated by a different enhancer, which is active at later stages during wing development. The lack of Suppressor of Hairless results in a precocious repression of reporter gene expression along the margin, suggesting that the DWR of Ser responds to the postulated feedback loop mediated by the Notch signaling cascade to maintain expression in cells adjacent to the dorsal wing margin (Bachmann, 1998b).

scalloped and strawberry notch are target genes of Notch signaling in the context of wing margin formation in Drosophila

In the Drosophila wing the cut gene is activated by Notch signaling along the dorso-ventral boundary but not in other cell types. Additional regulatory components, scalloped and strawberry notch, that are targets of the Notch pathway, are expressed specifically within the wing anlagen. As suggested by physical interactions, these proteins could be co-factors of the cut trans-regulator Vestigial. Additional regulatory input comes from the Wingless pathway. These data support a model whereby context specific involvement of distinct co-regulators modulates Notch target gene activation (Nagel, 2001).

These data show that the complex regulation of ct along the D/V boundary is based on a bifurcation of the Notch signaling pathway. Most signals from the Notch pathway are mediated by Su(H), which seems to act as a repressor on its own that is converted to an activator by Nact. Since Su(H) has the capacity to bind directly to the ct wing margin enhancer, the repression of ct by Su(H) and the activation by Nact/Su(H) might be direct. However, although sufficient for the activation of ct along the D/V boundary, a number of additional factors downstream of Nact are required. These include the products of wing fate selector genes vg and sd, that seem to be, together with Sno, part of a multi-factor trans-activation complex that binds to the ct wing margin enhancer. Thereby, Sd binds directly to the ct promoter, presumably recruiting the other factors by protein-protein interactions. In agreement with this hypothesis, respective physical interactions are observed between Vg and Sd or Sno. However, all three genes are targets of the Notch signaling pathway and are activated upon the overexpression of Su(H) specifically within presumptive wing tissue. Activation of Vg is also observed also within the wing pouch, although Su(H) acts as a repressor on the vg quadrant enhancer, indicating that the isolated enhancer elements reveal only a subset of the normal pattern and might contribute differently in a wild type context (Nagel, 2001).

A combination of Sd and Su(H) binding sites is sufficient to drive expression along the D/V boundary within the wing anlagen. This synthetic enhancer is too simplified to faithfully model ct regulation. Since the overexpression of Su(H) affects the accumulation of all the important trans-activator components, Vg, Sd, Sno and Su(H) itself, ct expression would be expected. Instead, repression of ct is observed: this might be due to a lack of Nact as co-activator of Su(H). However, repression can be overcome by concurrent expression of Wg resulting in strong ct activation. It is concluded that factors downstream of the Wg signaling cascade are able to convert Su(H) from a repressor to an activator, maybe by supplying a respective co-activator or by a cooperative combinatorial activity, e.g. together with the Wg signaling mediator dTCF, in accordance with a presumptive dTCF binding site within the ct wing margin enhancer (Nagel, 2001).

These signaling events appear to be unique to the activation of ct along the D/V boundary of the wing disc. Another important role of ct is the specification of external sensory organ cells during embryogenesis and imaginal development alike. Although Notch signaling is essential for setting up the correct number of neuronal cells in the peripheral nervous system by lateral specification, it appears not to be involved in the transcriptional activation of ct within these cells. The complex mechanism of ct trans-activation from the wing margin enhancer is, therefore, not a general paradigm for ct gene regulation. Moreover, neither wg, sd nor sno are under the direct regulatory influence of the Notch pathway in various embryonic tissues suggesting that this remarkably complex control is strictly tissue specific (Nagel, 2001).

These data confirm and extend the model of context dependent activity of Notch signaling towards the regulation of ct expression along the presumptive wing margin. The regulation of ct requires the combined input of components downstream of Su(H) and Wg, including Vg, Sd and Sno. The latter three components have the potential to form a multi-protein complex which seems to be a pre-requisite for the trans-activation of the ct wing margin enhancer. Whether Su(H) is part of this specific complex or other, similar complexes has to be elucidated in the future. Although there are no indications for direct interactions between Su(H) and Sd, Vg or Sno, Su(H) has the capacity to bind to the ct wing margin enhancer and act in a combinatorial manner together with the Sd/Vg/Sno transactivation complex and components of the Wg pathway. Presumably, in many instances of Notch signaling, where Su(H) acts as a DNA-binding molecule and signal transducer, a number of additional positive or negative co-regulators confers tissue and cell specificity. Therefore, the identification of corresponding factors should help to further the understanding of the context dependent outcome of Notch signaling events (Nagel, 2001).

Notch activation of yan expression is antagonized by RTK/Pointed signaling in the Drosophila eye

Receptor tyrosine kinase (RTK) signaling plays an instructive role in cell fate decisions, whereas Notch signaling is often involved in restricting cellular competence for differentiation. Genetic interactions between these two evolutionarily conserved pathways have been extensively documented. The underlying molecular mechanisms, however, are not well understood. Yan, an Ets transcriptional repressor that blocks cellular potential for specification and differentiation, is a target of Notch signaling during Drosophila eye development. The Suppressor of Hairless (Su[H]) protein of the Notch pathway is required for activating yan expression, and Su(H) binds directly to an eye-specific yan enhancer in vitro. In contrast, yan expression is repressed by Pointed (Pnt), which is a key component of the RTK pathway. Pnt binds specifically to the yan enhancer and competes with Su(H) for DNA binding. This competition illustrates a potential mechanism for RTK and Notch signals to oppose each other. Thus, yan serves as a common target of Notch/Su(H) and RTK/Pointed signaling pathways during cell fate specification (Rohrbaugh, 2002).

To investigate how yan expression is regulated, DNA fragments comprising a 20-kb genomic sequence surrounding the first exon of yan were tested for regulatory potential in corresponding transgenic flies. Through this approach, a 122-bp eye-specific enhancer located approximately 3.5 kb upstream of the first exon was identified. In eight out of nine transgenic lines, this enhancer activated expression of a bacterial lacZ reporter gene within posterior undifferentiated cells of eye discs. This recapitulates the endogenous yan gene expression in eye discs with the exception of the MF region. Three putative Su(H) binding sites were found in the yan enhancer. When tested through an in vitro electrophoretic mobility shift assay (EMSA), the Su(H) protein was shown to specifically bind to these sequences. Further, the yan enhancer became inactive in most of the posterior undifferentiated retinal cells when the Su(H) function was removed. All together, these loss-of-function and DNA binding analyses support the notion that Su(H) is required to promote yan transcription and that yan is a target gene of Su(H) in the eye (Rohrbaugh, 2002).

A candidate factor that may be involved in this compensation and could cooperate with Nintra/Su(H) proteins might be a DNA binding protein capable of interacting with a 5'-GAAACC/A-3' sequence. Two direct repeats of a 5'-GAAACC-3' sequence (hexamer, HEX) were found between S1 and S2. The second half of the S2 site might be considered as a third HEX, since there is only one variant base (as 5'-GAAACA-3'). When clustered point mutations were introduced into the first and second HEX, expression of the reporter gene was abolished in all six transgenic lines. Therefore, the HEX element is essential for the yan enhancer activity. Expression analysis of the HEX repeats has provided further evidence supporting the finding that the hexamer is an activation element. The reporter gene expression can be detected over the entire eye disc of all six lines when a six-copy concatomer of a 22-bp sequence containing three HEX repeats is used; however, one copy of this 22-bp oligonucleotide is not sufficient to induce gene expression in eye discs. It is proposed that a putative HEX binding protein functions together with Su(H) and Nintra to activate the yan enhancer. The nature of the HEX binding factor remains to be investigated (Rohrbaugh, 2002).

An Ets domain binding site (EBS, 5'-GGAA/T-3') was found within the S2 site. Since Yan is an Ets domain protein and a transcriptional repressor, whether Yan could be involved in autoregulation was examined. When a constitutively activated Yan (YanAct) was overproduced in eye discs, the reporter gene expression was strongly reduced. Since Yan is capable of negatively regulating yan transcription, this autoinhibitory mechanism might be used to prevent overproduction of Yan in undifferentiated cells. DNA binding data suggests that Yan can be directly involved in this negative regulation. However, this Yan-mediated autoinhibitory feedback appears to play a minor role in regulating yan expression, because the yan enhancer activity is apparently not affected in yan mutant clones produced in eye discs (Rohrbaugh, 2002).

A role for RTK signaling in regulating yan transcription was investigated. When the RTK pathway is constitutively activated by torD-DER or Ras1V12, the yan enhancer activity is greatly reduced. Thus, RTK signaling appears to negatively regulate yan transcription, in addition to its effect on Yan protein stability. Evidence supports a view that the inhibitory effect of RTK/Ras1 signaling on yan expression is mediated through the pointed (pnt) gene. Taken together, the results demonstrated that Pnt negatively regulates yan expression, and it is likely that Pnt is directly involved in repressing yan transcription. Although a role for Pnt as a transcriptional repressor has not been extensively investigated, pnt has been shown to negatively regulate hid transcription in embryos. Interestingly, a P-DLS motif is present in the Pnt protein (amino acids 356–360 in PntP1), which might mediate interaction with the transcriptional corepressor dCtBP. At this point, the data does not exclude the possibility that Pnt might also activate expression of a repressor, which in turn switches off yan transcription (Rohrbaugh, 2002).

The nesting of an Ets binding site within the S2 site suggests a possible mechanism whereby the binding of Pnt could interfere with Su(H)'s DNA binding activity. Indeed, increasing the amount of Pnt effectively prevents Su(H) from DNA binding. Such competition provides a mechanism by which RTK/Pnt signaling directly antagonizes Notch-mediated lateral inhibition at the transcriptional level. Since Ets binding sites are nested in many Su(H) binding sites, competitive occupancy of the common sequence could be a general mechanism for regulating expression of genes targeted by both Notch and RTK pathways (Rohrbaugh, 2002).

It is proposed that spatially restricted yan expression in the developing eye is coordinated by actions of multiple regulatory factors that include Su(H) and Pnt. Consequently, the yan enhancer provides an interface for Notch and RTK signals to oppose one another. The DNA binding analysis and mutagenesis of yan Su(H) binding sites provide evidence that supports a cell-autonomous role of Notch and RTK signaling in the regulation of yan expression. Interestingly, Yan expression is reduced not only in Su(H)D47 clones but also in some Su(H)+ cells that surround the mutant clones in eye discs. This result implies that loss-of-Su(H) function might also cause a cell-nonautonomous effect on yan expression, possibly due to upregulation of RTK signaling in those Su(H)+ cells. This upregulation may occur via an increase of a diffusible activator of the RTK pathway due to the loss of Su(H). The model presented here illustrates a mechanism that should help explain how progenitor cells are maintained in an undifferentiated state by Notch-mediated inhibitory signals and how they can be effectively induced for cellular differentiation by RTK-mediated inductive signals (Rohrbaugh, 2002).

Notch signaling controls cell fate specification along the dorsoventral axis of the Drosophila gut

The genetic programs that control patterning along the gut dorsoventral (DV) axis have remained largely elusive. The activation of the Notch receptor occurs in a single row of boundary cells that separates dorsal from ventral cells in the Drosophila hindgut. rhomboid, which encodes a transmembrane protein, and knirps/knirps-related, which encode nuclear steroid receptors, are Notch target genes required for the expression of crumbs, which encodes a transmembrane protein involved in organizing apical-basal polarity. Notch receptor activation depends on the expression of its ligand Delta in ventral cells, and localizing the Notch receptor to the apical domain of the boundary cells may be required for proper signaling. The analysis of gene expression mediated by a Notch response element suggests that boundary cell-specific expression can be obtained by cooperation of Suppressor of Hairless and the transcription factor Grainyhead or a related factor. These results demonstrate that Notch signaling plays a pivotal role in determining cell fates along the DV axis of the Drosophila hindgut. The finding that Notch signaling results in the expression of an apical polarity organizer, one which, in turn, may be required for apical Notch receptor localization, suggests a simple mechanism by which the specification of a single cell row might be controlled (Fusse, 2002).

These results further demonstrate that Notch signaling induces the expression of the rho and kni/knrl genes and that both components are required, in turn, for the expression of Crb. It has been suggested recently that Su(H) functions as a core of a molecular switch by which the transcription of Notch target genes is regulated. In the absence of Notch signaling, Su(H) functions as a repressor, and, in the presence of Notch signaling, Su(H) can cooperate synergistically with other transcriptional activators to induce transcription of target genes. The finding that boundary cell-specific reporter gene expression can be induced in the hindgut by using a model Notch response element [composed of binding sites for Su(H) and the widely expressed activator Grainyhead] suggests the possibility that the localized activation of the rho and kni/knrl genes could rely on the same factors and the same molecular switch mechanism that has recently been proposed for this element and for Notch-dependent atonal and single minded expression. In evolutionary terms, the gut is most likely one of the most ancient organs that evolved in multicellular organisms. Consistently, the morphological processes involved in the development of the gastrointestinal tract of animals are highly similar. It remains to be shown whether or not the evolutionarily conserved regulators of the Notch signaling cascade also determine dorsoventral aspects of gut development in other animals, including vertebrates (Fusse, 2002).

Context-dependent utilization of Notch activity in Drosophila glial determination

During Drosophila neurogenesis, glial differentiation depends on the expression of glial cells missing. Understanding how glial fate is achieved thus requires knowledge of the temporal and spatial control mechanisms directing gcm expression. In the adult bristle lineage, gcm expression is negatively regulated by Notch signaling. The effect of Notch activation on gliogenesis is context-dependent. In the dorsal bipolar dendritic (dbd) sensory lineage in the embryonic peripheral nervous system (PNS), asymmetric cell division of the dbd precursor produces a neuron and a glial cell, where gcm expression is activated in the glial daughter. Within the dbd lineage, Notch is specifically activated in one of the daughter cells and is required for gcm expression and a glial fate. Thus Notch activity has opposite consequences on gcm expression in two PNS lineages. Ectopic Notch activation can direct gliogenesis in a subset of embryonic PNS lineages, suggesting that Notch-dependent gliogenesis is supported in certain developmental contexts. Evidence is presented that POU-domain protein Nubbin/PDM-1 is one of the factors that provides such context (Umesono, 2002).

Notch signaling promotes glial fate during asymmetric division in the embryonic dbd lineage. Notch is specifically activated in the presumptive DBD support glia cell (DBDG) owing to the negative regulation by Numb in the sibling cell, and provides instructive information to induce gcm transcription and glial development. Expression of gcm occurs quickly after the artificial activation of Notch, even in cells that have initiated neuronal development. In gcm mutants, DBDG are transformed into neurons, although the activation of Notch, visualized by the Su(H)-reporter, is normal in the presumptive glia. Likewise, ectopic expression of gcm in presumptive dbd neurons causes neuron-to-glia transformation without affecting Notch activity. These findings suggest that gcm expression appears to be the sole target of Notch activation in establishing glial fate in the dbd lineage. Within the 3.5 kb region upstream of the gcm gene, two sequences have been identified that perfectly match the consensus core sites for Su(H). Thus, gcm could be a direct target of Su(H), downstream of the Notch signaling pathway (Umesono, 2002).

Su(H) regulation of the vestigial boundary enhancer

The expression of vestigial during wing development is regulated through two enhancers: the second intron or boundary enhancer (vgBE), and the fourth intron quadrant enhancer (vgQE). These names reflect the patterns of expression directed by these regulatory regions: vgBE produces a thin stripe over the prospective wing margin, and vgQE produces a pattern in four quadrants that are complementary to the vgBE and which fill in the developing wing blade. Both, vgBE and vgQE, act as integrators of signaling systems that drive wing development and, in this manner, these regulatory regions determine the tempo and the mode of wing development (Klein, 1999 and references).

The vgBE is activated first during the second instar. Its expression pattern is very similar to that of the Vestigial protein at this stage suggesting that, in these early stages, the vgBE is responsible for the complete pattern of vg expression. Deletion analysis of the enhancer reveals two regions essential for its activity: a binding site for Suppressor of Hairless and sequences contained in the first 80 base pairs of the enhancer. In an attempt to map the nature and timing of the inputs into this enhancer, the activity of the wild-type vgBE and of deletions of the two essential regions have been compared during wing development. At the end of the second instar, lacZ reporter expression from the wild-type vgBE outlines a horseshoe over the ventral region of the wing disc, with weak expression in the ventral anterior region where it overlaps with the expression of wingless. lacZ expression increases in this region at the beginning of the third instar. This increase does not occur in Suppressor of Hairless [Su(H)] mutants or in wg mutants. The activity of the enhancer deleted for the Su(H)-binding site is different from the wild type. The activity of this enhancer is never initiated over the ventral region of the disc, where the wing primordium is established and remains absent during later stages. This result demonstrates that the activity of Notch is required not only for the maintenance, but also for the initiation of the expression of vg through the vgBE. The activity of the enhancer deleted from 0-80 is similar to that of the wild-type enhancer early on, but it never acquires the high levels of activity in the anterior ventral region. As the wing blade develops, a line of faint activity can be detected over the DV boundary, but it fades quickly and, by the end of the third instar, in most discs there is no activity over the developing wing blade. This enhancer still shows some activity in the flanking notal regions of the wing disc in wild-type and Su(H) mutant discs. This indicates the existence of additional inputs in the regulation of the vgBE in the notal region. These results suggest that the cells in which the expression of the vgBE is upregulated at the end of the second instar represent the anlage of the wing and require Notch/Su(H) signaling. These cells are located at the DV interface, on the domain of wg expression and overlap the expression of nubbin. The suggestion that these cells represent the primordium of the wing pouch can explain why a deletion of the vgBE results in the abolition of the development of the wing pouch; in such a mutant, the anlage would never be defined (Klein, 1999).

Notch signaling is also required for the initial activity of the Quadrant Enhancer (vgQE) The activity of the vgQE can be detected first at the beginning of the third instar, several hours after the upregulation of the vgBE, when it closely outlines the realm of the growing wing. This enhancer is only expressed in the growing wing blade and thus provides a unique and most specific marker for wing blade tissue. A variety of experiments have shown that the vgQE receives a negative input from Notch signaling and a positive one from Dpp. The presence of an E(spl)-binding site in the sequence of the vgQE has led to the suggestion that this suppression by Notch is mediated by the E(spl) protein. However, no strong suppression of the activity of the vgQE is found if E(spl)-m8 is ectopically expressed, suggesting that the effect of Notch requires other mediators. Although the vgQE is suppressed in the domain of Notch activity, Notch signaling plays a non-autonomous role in its activation. For example, the vgQE is never active in Serrate (Ser) mutants in which wing development initiates normally but is aborted very early. Ectopic expression of Delta rescues the wing pouch and leads to the activation of the vgQE. Interestingly, this activity arises in regions devoid of Notch signaling. This result suggests that Notch signaling influences the activity of the vgQE in two ways: it represses the activity of the vgQE autonomously but it is also required for its activity in a non-autonomous way (Klein, 1999).

Wingless is shown to acts synergistically with Vestigial to promote the activity of the vgQE. It is clear that the activity of the vgBE is required for the activation of the vgQE, but little is known about how this interaction takes place. The observation that vgQE is activated in Ser mutant discs in which vg is expressed ectopically suggests that the activation of the vgQE is mediated by Notch signaling through the activity of Vestigial on the vgQE. However, in this experiment, expression of wg is also restored and this raises the possibility that activation of the vgQE is mediated through the presumed organizing activity of Wingless. This is probably not the case since ectopic expression of wg alone does not lead to the activation of the vgQE in Ser mutants. The inability of Wingless to activate the vgQE in this experiment is not due to a general insensitivity of the cells to Wingless signaling, since ectopic wg is always capable of inducing hinge fate ectopically. These results clearly demonstrate a requirement for vg in the activation of the vgQE. However, clonal analysis has shown that vg acts cell autonomously and therefore, in the wild type, the non-autonomous effects of Notch on the vgQE must be mediated by another, diffusible molecule(s), which is under control of Notch signaling. A number of studies suggest that Wingless has an influence on the expression of vg in the wing pouch and that its expression at the wing margin is under control of Notch signaling. Therefore, it is possible that Wingless is mediating the non-autonomous effect of Notch on the vgQE. It might be that Wingless acts by acting on the vgQE to elevate and maintain the levels of vg expression that had been induced by Vestigial through the vgBE. In agreement with this proposal, it is found that the activity of the vgQE is elevated in response to the ectopic expression of wg and that expression of a dominant negative Wingless molecule suppresses the activity of the vgQE and reduces the size of the wing pouch. Altogether these results suggest that the upregulation of vg expression in response to wg is mediated by the vgQE. This conclusion is supported by the existence of several putative TCF-1 binding sites in the vgQE. However, the effects of Wingless are always restricted to the normal domain of vg expression, in agreement with the results presented above that Wingless alone is not sufficient to initiate ectopic expression of vg through the vgQE. These effects are likely to be mediated by Vestigial itself. The role of Wingless on this regulation is to maintain and modulate the levels of activity of the vgQE. Consistent with the conclusion that Wingless enhances the effects of Vestigial, coexpression of Wingless and Vestigial, which leads to the ectopic induction of pouch and hinge fate in the notal regions, triggers a widespread and stable expression of the vgQE throughout the wing disc (Klein, 1999).

Su(H) regulation of E(spl) genes

Expression of the Drosophila Enhancer of split [E(spl)] genes, and their homologs in other species, is dependent on Notch activation. The seven E(spl) genes are clustered in a single complex and their functions overlap significantly; however, the individual genes have distinct patterns of expression. To investigate how this regulation is achieved and to find out whether there is shared or cross regulation between E(spl) genes, the enhancer activity of sequences from the adjacent E(spl)mbeta, E(spl)mgamma and E(spl)mdelta genes were analyzed and comparisons to E(spl)m8 were made. Although regulatory elements can be shared, most aspects of the expression of each individual gene are recapitulated by small (400-500 bp) evolutionarily conserved enhancers. Activated Notch or a Suppressor of Hairless-VP16 fusion are only sufficient to elicit transcription from the E(spl) enhancers in a subset of locations, indicating a requirement for other factors. In tissue culture cells, proneural proteins synergise with Suppressor of Hairless and Notch to promote expression from E(spl)mgamma and E(spl)m8, but this synergy is only observed in vivo with E(spl)m8. It is concluded that additional factors besides the proneural proteins limit the response of E(spl)mgamma in vivo. In contrast to the other genes, E(spl)mbeta exhibits little response to proneural proteins and its high level of activity in the wing imaginal disc suggests that wing-specific factors cooperate with Notch to activate the E(spl)mbeta enhancer. These results demonstrate that Notch activity must be integrated with other transcriptional regulators; since the activation of target genes is critical in determining the developmental consequences of Notch activity, these results provide a framework for understanding Notch function in different developmental contexts (Cooper, 2000).

E(spl)m8 is transcribed in all sensory organ clusters: E(spl)mdelta and E(spl)mgamma in a subset of sensory clusters but strongly in the developing eye, and E(spl)mbeta in the intervein regions of the wing primordium, at the dorsal/ventral boundaries of the wing and eye, and in the presumptive leg joints. To identify the regions responsible for conferring the specific expression patterns, 1- to 2-kb fragments from the region encompassing E(spl)mdelta, E(spl)mgamma, E(spl)mbeta were fused to a minimal promoter upstream of the lacZ gene to test for enhancer activity. For each of the three genes, the fragment adjacent to the promoter (mdelta1.9, mgamma1.1, and mbeta1.5) confers a pattern of expression that largely recapitulates the endogenous genes, although there are some notable exceptions: (1) neither mdelta1.9 nor mgamma1.1 generates the strong expression associated with the morphogenetic furrow that is observed with both genes; (2) the mdelta1.9 fragment fails to confer the tegula expression normally associated with E(spl)mdelta. Given the close proximity of the genes in the complex, it is possible that adjacent genes could share regulatory elements. Because mgamma1.1 confers strong expression in the tegula domain, it might account for the tegula expression of E(spl)mdelta as well as E(spl)mgamma. To test whether there is an insulator within mgamma1.1 that would prevent it acting on the adjacent E(spl)mdelta transcription unit, mgamma1.1 was inserted between the lacZ and CD2 coding sequences. Both proteins have similar patterns of expression, indicating that mgamma1.1 is able to regulate an upstream transcription unit and so could mediate the tegula expression of the upstream E(spl)mdelta. Further indirect support for the hypothesis that enhancers can act on neighboring genes comes from analysis of a P-element (K33) inserted at the E(spl)mgamma locus. When the sequences proximal to the P-element are deleted, as occurs in Df(3R)NF1P1, the inserted lacZ gene is now expressed in a pattern weakly resembling the distal E(spl)mbeta gene, even though none of the intervening sequences have been altered. These results indicate that the E(spl)mbeta enhancer has the potential to act on the E(spl)mgamma region, but in the wild-type chromosome it must be prevented by the sequences adjacent to the E(spl)mgamma promoter (Cooper, 2000).

Thus E(spl)mbeta, E(spl)mgamma, and E(spl)mdelta patterns can largely be recapitulated by DNA fragments of ~400-500 bp located close to the transcription start site. As expected, these fragments contain Su(H) binding sites, consistent with their responsiveness to Notch signaling. However, they are also sufficient to generate quite diverse patterns of expression. The fact that this activity resides in such localized enhancers contrasts with the organization of other genes expressed in similar complex patterns in the disc, such as proneural and intervein genes. These are regulated by an array of enhancers, each of which responds to a different combination of patterning genes. The comparative simplicity of the identified E(spl) enhancers suggests that they are unlikely to be regulated by a similar array, but are more likely to be responding to the next level in the hierarchy, i.e., to the factors that are themselves expressed in complex patterns (Cooper, 2000).

The suggestion that E(spl) genes are regulated by intermediates in the patterning hierarchy is consistent with the proneural proteins contributing to their regulation. However, this also presents an inconsistency, because the E(spl) products are not detected in the neural precursor cells where proneural proteins accumulate to highest levels. This study demonstrates that proneural proteins work synergistically with Su(H)/Nicd (the complex between Suppressor of hairless protein and the intracellular domain of Notch) to activate transcription from E(spl)m8 and E(spl)mgamma enhancers in cultured cells. For E(spl)m8, this synergy can also be demonstrated in vivo, as a combination of proneural proteins and Nicd leads to higher levels of m8-lacZ expression than either component alone. This combined regulation can explain why E(spl) genes are activated in the cells surrounding the sensory organ precursors, since these are cells where both proneural proteins and Notch activity would be present. In this respect the regulation of some E(spl) genes, in particular E(spl)m8, fits with a combinatorial model, which suggests that the activation of genes in response to signaling pathways involves the transcriptional response factor for the signaling pathway acting in combination with specific patterning genes (Cooper, 2000).

The combinatorial synergy between Notch and proneural proteins may be sufficient to explain E(spl)m8 regulation, but it is not sufficient to account for the expression of some other E(spl) genes. Two key points are highlighted by the different enhancers and tissues that have been analysed. The first is that there must be factors equivalent to the proneural proteins that synergise with Notch on the E(spl)mbeta enhancer. The second is that the competence of the E(spl) enhancers to respond to Su(H)/Nicd is spatially restricted by more than just the availability of an appropriate synergising activator. Unlike the other enhancers analysed, mbeta1.5 is highly sensitive to activated Notch and Su(H)VP16 throughout the wing pouch. Intriguingly, the E(spl)mbeta fragments confer much higher levels of expression than any of the other fragments tested, even though one of the two Su(H) sites in mbeta1.5 does not conform fully to a consensus binding site. The widespread activation of mbeta1.5 in the wing pouch and its poor response to proneural proteins suggest that the E(spl)mbeta enhancer responds to other activators. This explains why it is still possible for ectopic Nicd to promote increased levels of E(spl) proteins in scute10-1 discs. Under these conditions transcription of E(spl)mbeta [and possibly E(spl)m3] could still be increased in the wing pouch, even if E(spl)m8, E(spl)mgamma and E(spl)mdelta could not. These investigations have not yet identified specific activators that account for the activity of mbeta1.5, although there are binding sites for a variety of factors including two proteins expressed in the wing, Scalloped and Caupolican (Cooper, 2000).

The differences in the responses of mgamma1.1 and mdelta1.9 compared to E(spl)m8 argue that there is an additional level of regulation that limits the accessibility of mgamma1.1 and mdelta1.9 proneural proteins/Su(H). Thus, although mgamma1.1 and mdelta1.9 are targets for proneural proteins and Su(H)/Nicd, based on effects in tissue culture and/or in vivo, they cannot be activated very effectively within the wing pouch even when high levels of certain proneural proteins and/or Nicd are expressed ectopically. Likewise, mgamma1.1 and mdelta1.9 are largely resistant to activation by Su(H) VP16 in the wing pouch, although weak activation of mdelta1.9 is sometimes detected. Similar restrictions have been observed when an E(spl)m5 enhancer, whose Su(H) binding sites had been replaced with Gal4 UAS sites, was exposed to ubiquitous Gal4. This transgene could only be activated in a limited domain, indicating that Gal4 activity can also be influenced by E(spl) regulatory sequences (Cooper, 2000).

The factors that modulate the responsiveness of the enhancers to Su(H)/Nicd and activators such as proneural proteins also act through the small 180- to 500-bp enhancer fragments, and several different mechanisms can be envisioned that might account for this modulation. One is that there is a 'prefactor' that is necessary to initially modify the chromatin and allow entry of Su(H) and proneural proteins. Recent analyses of the mechanisms involved in gene activation demonstrate that there may be sequential stages in chromatin remodelling. If an earlier step of chromatin modification is needed before Su(H) and other activators can access the enhancers, the differential response of E(spl)m8 and E(spl)mgamma fragments to Su(H) VP16 in the wing pouch would arise from a requirement for different factors to implement this initial step. An alternative model is that the enhancer fragments are also targets for specific repressors, for example, mdelta1.9 and mgamma1.1 could be specifically repressed throughout most of the wing pouch. However, none of the truncations or site-specific mutations of the mgamma1.1 and mdelta1.9 fragments have ever led to ectopic activity, as would be indicative of loss of a repressor binding region (Cooper, 2000).

Su(H)VP16 mimics phenotypes produced by activated Notch both in Drosophila and in Xenopus consistent with the evidence that Su(H) is essential for activation of target genes, via its association with Nicd. Results from mammalian tissue culture cells, however, indicate that CBF/Su(H) also functions as a repressor, interacting with histone deacetylase (HDAC). There is as yet no evidence to support this model in Drosophila, but the low levels of residual expression from E(spl) enhancers in Su(H) mutant discs might be explained by this mechanism. If in wild-type discs, Su(H) is bound to E(spl) enhancers in association with HDAC, it could prevent any activation from proneural proteins until Nicd is present. In animals that lack Su(H), this repression would no longer occur, so that high levels of proneural proteins could activate the enhancers. In support of this reasoning it is found that in tissue culture cells some activation is elicited by proneural proteins alone, particularly of the E(spl)m8 reporter. Furthermore, the residual expression from mdelta1.9 and mgamma1.1 enhancers is greatest in the oldest discs, where the levels of proneural proteins are highest and residual maternal Su(H) protein would be lowest. The dual repressor/activator roles proposed for Su(H) are like those put forward for TCF/Pangolin, which becomes a transcriptional activator of Wnt/Wingless responsive genes upon binding to beta-catenin, but appears to act as a repressor in the absence of Wnt signalling (Cooper, 2000).

Previous studies of E(spl) regulation in the embryo suggested an element of autoregulation since expression of m8-lacZ is elevated in E(spl) mutant embryos. Similar effects are also seen with HES expression in tissue culture cells, where the levels of transcription decline after their initial activation. The data suggest that this is likely to be a general mechanism, since all four E(spl) enhancers are responsive to ectopic E(spl) proteins in vivo, especially mbeta1.5. Furthermore, in cells where the repressive function of E(spl) proteins is compromised, their expression levels increase. Both these results are compatible with autoregulatory negative feedback by E(spl) proteins, so that once a critical amount is produced these proteins inhibit their own expression. This negative feedback regulation could help to keep cells in a pliable state, for example, during neurogenesis, when the balance between proneural and E(spl) proteins is critical in determining whether a cell adopts the neural fate (Cooper, 2000).

Several results indicate that the individual enhancers are able to influence more than one E(spl) gene. (1) The fragment between E(spl)mdelta and E(spl)mgamma (mgamma1.1) confers strong tegula cluster expression and contains no insulator to prevent it from acting on the 5' E(spl)mdelta gene, suggesting that it normally acts on both transcription units and accounts for the tegula expression of both genes (although the possibility that there is an insulator within E(spl)mdelta itself has not been ruled out). (2) In the Df(3R)NF1P1 deletion, the E(spl)mbeta enhancers acts on the lacZ gene inserted at E(spl)mgamma, demonstrating that the regulatory elements have the potential to act on adjacent genes. Other evidence suggests that the complex E(spl) expression patterns involve a combination of shared and redundant elements. For example, although E(spl)mgamma and E(spl)mdelta are both expressed in the ommatidial field, only mdelta1.9 confers a high level of ommatidial expression: mgamma1.1 is much less robust. In the native E(spl) complex, these two elements could act in concert to give strong E(spl)mgamma expression in ommatidia (Cooper, 2000).

The sharing of regulatory elements means that there is significant overlap in the expression patterns of adjacent genes, which accounts for some of their redundancy. In addition the effects of deleting one gene could be rescued by residual elements influencing the expression of neighboring genes. The fact that there is some interdigitation of regulatory elements may also help to explain the conservation of the E(spl) complex, as has been argued for the paralogous Hox clusters in mammals where sharing of regulatory elements has been documented and is proposed to have helped constrain the organization of the clusters (Cooper, 2000).

Su(H) regulation of shaven

How multifunctional signals combine to specify unique cell fates during pattern formation is not well understood. Together with the transcription factor Lozenge, the nuclear effectors of the Egfr and Notch signaling pathways directly regulate D-Pax2 (shaven) transcription in cone cells of the Drosophila eye disc. Moreover, the specificity of shaven expression can be altered upon genetic manipulation of these inputs. Thus, a relatively small number of temporally and spatially controlled signals received by a set of pluripotent cells can create the unique combinations of activated transcription factors required to regulate target genes and ultimately specify distinct cell fates within this group. It is expected that similar mechanisms may specify pattern formation in vertebrate developmental systems that involve intercellular communication (Flores, 2000).

shaven is the Drosophila homolog of the vertebrate Pax2 gene. This locus is represented by at least two classes of mutant alleles: shaven (sv) and sparkling (spa). spa mutants show cone cell defects resulting from mutations in the fourth intron of the gene, which have led to the identification of a 926 bp SpeI fragment within this intron that includes the eye-specific enhancer (Flores, 2000).

In Nts third-instar larvae raised at 29°C for 20 hr prior to dissection, Shaven expression is eliminated from cone cell precursors. Similarly, expression of a dominant-negative form of N under lz-Gal4 control causes a loss of Shaven expression in cone cell precursors without perturbing neuronal development. Shaven expression is also reduced in discs mutant for Delta (Dl), which encodes a N ligand. Moreover, expression of a dominant-negative form of Dl (DlDN) under lz-Gal4 control causes a loss of Shaven expression in cone cell precursors, while neuronal patterning occurs in a wild-type fashion. A further reduction in Shaven expression is seen when DlDN is driven by GMR-Gal4. A loss of Shaven expression is also seen upon ectopic expression of Hairless (H), a direct antagonist of Su(H) function. These results together suggest that N/Dl signaling via Su(H) is required for proper shaven expression in cone cell precursors. This is an inductive rather than lateral inhibitory function of the N signaling pathway in cone cell development that has not been previously analyzed with molecular markers. A reporter gene under the transcriptional control of Su(H) binding sites is expressed in cone cell precursors, which demonstrates that Su(H) is activated by the N pathway in cone cells (Flores, 2000).

The Su(H) binding sites in the minimal shaven eye promoter (SME) were altered to determine whether the N pathway directly regulates shaven transcription. The SME contains eight putative Su(H) binding sites. EMSAs show that the Su(H) consensus binding sequence is not strictly followed, since three sites with one mismatch can bind Su(H). Su(H) binding is eliminated when the central 5'-GRG-3' sequence is mutated to 5'-CCC-3' in all eight sites. A construct containing these mutations in the context of SME-lacZ was transformed into flies. In these transgenic flies, ß-galactosidase expression is lost in cone cell precursors. These in vitro and in vivo results together demonstrate that Su(H) directly controls shaven expression in cone cell precursors by binding to the SME (Flores, 2000).

Mutating Su(H) and ETS binding sites eliminates expression of the target gene in the cone cells, which demonstrates a direct role for these pathways in transcriptional activation of shaven. Clonal analysis was undertaken to establish the requirement of the Notch and Egfr pathways in shaven expression. Unfortunately, these pathways are necessary for proliferation and have many layers of function. Therefore a flip-out strategy was used to inhibit N and Egfr function in GFP-labeled single-cell clones. This was best achieved in clones induced by GMR-flp. The GMR enhancer is only active behind the furrow and only a single cell division takes place in this population of cells. As a result, the clone size is very small. In a wild-type background, single cells marked with GFP express Shaven. However, when these single cells also express EGFRDN or NECN, they do not express Shaven. Thus, cone cells need functional Notch and Egfr receptors in order to express Shaven (Flores, 2000).

The results described so far suggest that shaven expression is limited to cells which (1) express Lz; (2) receive a sufficiently strong Egfr signal to both alleviate Yan-imposed repression and stimulate PntP2 activation, and (3) receive a N signal able to stimulate Su(H) activation. The tripartite control of shaven expression in the cone cell precursors requires that they receive all three inputs at the proper time in their development. Lz expression in cone cell precursors has been documented. Consistent with their reception of the Egfr signal, activated MAPK is detected in cone cell precursors at the time when they initiate Shaven expression. Dl is expressed in developing photoreceptor clusters at the time when the cone cell precursors express Shaven. Thus, the neuronal clusters signal through an inductive Dl/N pathway to activate shaven expression in the neighboring cone cell precursors. These results suggest that, in addition to expressing Lz, the cone cell precursors receive the Egfr and N signals at the time of fate acquisition and Shaven expression. Presumably, at least one of these three activation mechanisms is lacking in cells that do not express shaven. This hypothesis was tested through genetic manipulation of the system (Flores, 2000).

Undifferentiated cells immediately posterior to the furrow receive the N signal and express Lz, but they do not express Shaven. It is hypothesized that the absence of Shaven expression in these cells is caused by a lack of the Egfr signal. This hypothesis is consistent with the observation that Egfr signaling causes these cells to differentiate. Indeed, Shaven is ectopically expressed in undifferentiated cells that express an activated form of Egfr. Loss-of-function yane2D/yanpokX8 discs also show ectopic expression of Shaven in undifferentiated cells. Similarly, in discs expressing SMEmETSx6-lacZ, in which the six ETS sites in the SME are mutated, ß-galactosidase is also expressed in undifferentiated cells. Presumably, relief of Yan repression is sufficient to activate some shaven in undifferentiated cells. In SMEmETS(1,6)-lacZ,where the Pnt binding sites are eliminated but two of the Yan binding sites are still intact, there is no expression of ß-galactosidase in the undifferentiated cells. These results suggest that while the undifferentiated cells posterior to the furrow express Lz and receive the N signal, they fail to express Shaven because they do not receive the Egfr signal and are therefore unable to relieve the Yan-imposed repression of shaven (Flores, 2000).

The R7 precursors express Lz and receive RTK signals, yet they do not express Shaven. It is hypothesized that this is due to the lack of the N signal at the time of R7 determination. Indeed, expression of an activated form of N (Nact), leads to ectopic Shaven expression in R7 precursors, which suggests that Shaven is not normally expressed in R7 because this cell does not receive the N signal. These results are consistent with the previous observation that the R7 cell loses its neuronal characteristics upon expression of Nact (Flores, 2000).

Thus far, this study has focused on cells that express Lz. However, the regulation of shaven expression can also be tested in cells that lack Lz, such as the R3/R4 precursors. These cells receive the Egfr signal but receive the N signal after their initial fate specification, during ommatidial rotation. Ectopic expression of either Lz or Nact in the R3/R4 precursors fails to activate shaven expression in these cells. However, when Lz and Nact are coexpressed in the R3/R4 precursors, Shaven is expressed in these cells. These results demonstrate that the lack of both N signaling and Lz during the proper time window prevents R3/R4 cells from expressing shaven (Flores, 2000).

Structural rules and complex regulatory circuitry constrain expression of a Notch- and EGFR-regulated eye enhancer of sparkling

Enhancers integrate spatiotemporal information to generate precise patterns of gene expression. How complex is the regulatory logic of a typical developmental enhancer, and how important is its internal organization? This study examined in detail the structure and function of sparkling, a Notch- and EGFR/MAPK-regulated, cone cell-specific enhancer of the Drosophila Pax2 gene, in vivo. In addition to its 12 previously identified protein-binding sites, sparkling is densely populated with previously unmapped regulatory sequences, which interact in complex ways to control gene expression. One segment is essential for activation at a distance, yet dispensable for other activation functions and for cell type patterning. Unexpectedly, rearranging sparkling's regulatory sites converts it into a robust photoreceptor-specific enhancer. These results show that a single combination of regulatory inputs can encode multiple outputs, and suggest that the enhancer's organization determines the correct expression pattern by facilitating certain short-range regulatory interactions at the expense of others (Swanson, 2010).

The goal of this study was to use a well-characterized, signal-regulated developmental enhancer to examine, in fine detail, the regulatory interactions and structural rules governing transcriptional activation in vivo. This study used functional in vivo assays to test the power of the proposed combinatorial code of 'Notch/Su(H) + Lz + MAPK/Ets' to explain the activity and cell type specificity of the spa cone cell enhancer of dPax2. In the course of this work, several surprising properties of spa were discovered that are not accounted for in current models of enhancer function (Swanson, 2010).

The spa enhancer for fine-scale analysis because (1) the known direct regulators and their binding sites are well defined, (2) they could, in theory, constitute the sum total of the patterning information received by the enhancer, and (3) the enhancer, at 362 bp, is relatively small, simplifying mutational analyses. Surprisingly, a large proportion of the previously uncharacterized sequence within spa is vital for normal enhancer activity in vivo, and of that subset, a large proportion directly influences cell type specificity (Swanson, 2010).

In addition to necessary inputs from Lz, Pnt, and Su(H), three segments of spa were identified, regions 4, 5, and 6, that make essential contributions to gene expression in cone cells. In addition, region 2 makes a relatively minor contribution. (Region 1, another essential domain, will be discussed separately.) Fine-scale mutagenesis reveals that within regions 4, 5, and 6, very little DNA is dispensable for cone cell activation. The previously uncharacterized regulatory sites in spa are very likely bound by factors other than Lz/Pnt/Su(H), for the following reasons: no sequences resembling Lz/Pnt/Su(H)-binding sites reside in these regions; mutations in the newly mapped sites have different effects than removing the defined TFBSs or the proteins that bind them; doubling the known TFBSs fails to compensate for the loss of the newly mapped sequences; and, most importantly, mutating the newly mapped regulatory regions does not significantly affect binding of the known activators to nearby binding sites in vitro. It is not known whether the proposed novel regulators are cone cell-specific, eye-specific, or ubiquitous in their expression. It is known that the newly mapped sites are necessary both for normal cone cell expression and ectopic PR expression. Cut, Prospero, and Tramtrack are expressed in cone cells, but are thought to act as transcriptional repressors. The transcription factor Hindsight is required for dPax2 expression and cone cell induction, but acts indirectly, activating Delta in R1/R6 to induce Notch signaling in cone cells (Swanson, 2010).

Unsurprisingly, placing the enhancer closer to the promoter boosts expression of spa(wt), as well as some of the impaired mutants. The spa enhancer is located at +7 kb in its native locus, and nearly all mutational studies place the enhancer immediately upstream of the promoter. If the entire analysis had been performed at −121 bp, the functional significance of several critical regulatory sequences would have been underrated, and region 1 would have been dismissed as nonregulatory DNA. Other well-characterized enhancers, which have been analyzed in a promoter-proximal position only, may therefore contain more critical regulatory sites than is currently realized (Swanson, 2010).

Like many transcriptional activators, all three known direct activators of spa (or their orthologs) recruit p300/CBP histone acetyltransferase coactivator complexes. Doubling the number of binding sites for these transcription factors (to 6 Lz, 8 Ets, and 10 Su(H) sites) does not suffice to drive cone cell expression in the absence of the newly mapped regulatory regions. It may be, then, that factors recruited to the newly mapped regulatory sites within spa employ mechanisms that are distinct from those of the known activators. The remote activity of spa, mediated by region 1, appears to be an example of such a mechanism (Swanson, 2010).

It was possible to convert spa into a R1/R6-specific enhancer in three ways: (1) by moving the defined TFBSs to one side of the enhancer in a tight cluster; (2) by placing Lz and Ets sites next to regions 1, 4, and 6a; and (3) by mutating regions 2, 3, 5, and 6b within spa while maintaining the native spacing of all other sites. From these experiments, it is concluded that spa contains short-range repressor sites that prevent ectopic activation in PRs by Lz + Pnt + regions 4 + 6a. spa contains at least two redundant repressor sites, because both region 5 and regions 2, 3, and 6b must be mutated to attain ectopic R1/R6 expression (Swanson, 2010).

klumpfuss, which encodes a putative transcriptional repressor, is directly activated by Lz in R1/R6/R7, but is also present in cone cells, making it an unlikely repressor of spa. seven-up, another known transcriptional repressor, is expressed in R3/R4/R1/R6 and could therefore act to repress spa in PRs. However, no putative Seven-up-binding sites were identified within spa. Phyllopod, an E3 ubiquitin ligase component, represses dPax2 and the cone cell fate in R1/R6/R7, but the transcription factor mediating this effect is not yet known (Shi, 2009). Perhaps the best candidate for a PR-specific direct repressor of spa is Bar, which encodes the closely related and redundant homeodomain transcription factors BarH1 and BarH2. Bar expression is activated by Lz in R1/R6 and is required for R1/R6 cell fates. Furthermore, misexpression of BarH1 in presumptive cone cells can transform them into PRs. It is unclear whether Bar-family proteins act as repressors, activators, or both. BarH1/2 can bind sequences containing the homeodomain-binding core consensus TAAT, and region 5 of spa contains two TAAT motifs. Future studies will explore the possibility that Bar directly represses spa in PRs (Swanson, 2010).

The combinatorial code of spa, then, requires multiple inputs in addition to Lz, MAPK/Ets, and Notch/Su(H). Indeed, the data suggest that the known regulators can contribute to expression in multiple cell types, depending on context. The newly mapped control elements identified within spa are necessary not only to facilitate transcriptional activation, but also to steer the Lz + Ets + Su(H) code toward cone cell-specific gene expression (Swanson, 2010).

Enhancers are often located many kilobases from the promoters they regulate. Enhancer-promoter interactions over such distances are very likely to require active facilitation. Even so, few studies have focused specifically on transcriptional activation at a distance, and the majority of this work involves locus control regions (LCRs) and/or complex multigenic loci, which are not part of the regulatory environment of most genes and enhancers. Like spa, many developmental enhancers act at a distance in their normal genomic context, yet can autonomously drive a heterologous promoter in the proper expression pattern, without requiring an LCR or other large-scale genomic regulatory apparatus. However, in nearly all assays of enhancer function, the element to be studied is placed immediately upstream of the promoter. In such cases, regulatory sites specifically mediating remote interactions cannot be identified. Because the initial mutational analysis of spa was performed on enhancers placed at a moderate distance from the promoter (−846 bp), it was possible to screen for sequences required only at a distance, by moving crippled enhancers to a promoter-proximal position. Only one segment of spa, region 1, was absolutely essential at a distance but completely dispensable near the promoter. This region, which contains the only block of extended sequence conservation within spa, plays no apparent role in patterning, or in basic activation at close range. Therefore this segment of spa is termed a 'remote control' element (RCE) (Swanson, 2010).

The remote enhancer regulatory activity described in this study differs from previously reported long-range regulatory mechanisms in two important ways. First, the remote function of spa does not require any sequences in or near the dPax2 promoter. This functionally distinguishes spa from enhancers in the Drosophila Hox complexes that require promoter-proximal 'tethering elements' and/or function by overcoming insulators. This distal activation mechanism also likely differs from enhancer-promoter interactions mediated by proteins that bind at both the enhancer and the promoter, as occurs in looping mediated by ER, AR, and Sp1. Second, studies of distant enhancers of the cut and Ultrabithorax genes have revealed a role for the cohesin-associated factor Nipped-B, especially with respect to bypassing insulators, but it has not been demonstrated that Nipped-B, or any other enhancer-binding regulator, is required only when the enhancer is remote (Swanson, 2010).

The spa RCE is the first enhancer subelement demonstrated to be essential for enhancer-promoter interactions at a distance, but unnecessary for proximal enhancer function and cell type specificity. However, the present work contains only a limited examination of this activity, as part of a broader study of enhancer function. These functional studies, testing for potential promoter preferences and distance limitations, and the identities of factors binding to the RCE are being persued(Swanson, 2010).

As discussed above, it is fairly easy to switch spa from cone cell expression to R1/R6 expression (though, curiously, a construct that is active in both cell types has yet to be constructed). The results show that multiple regions of spa mediate a repression activity in R1/R6, but not in cone cells. It is further concluded that these spa-binding repressors act in a short-range manner; that is, they must be located very near to relevant activator-binding sites, because moving Lz and Pnt sites to one side of spa, without removing the repressor sites (KO+synthCS), abolishes repression. Despite this failure of repression, synergistic interactions among Lz and Ets sites and the newly mapped sites still occur in this reorganized enhancer -- at least in R1/R6 cells. Cone cell-specific expression is lost, however, revealing (along with other experiments) that transcriptional activation in cone cells is highly sensitive to the organization of regulatory sites within spa. Slightly wider spacing of regulatory sites (KO+synthNS) kills the enhancer altogether, suggesting that synergistic positive interactions within spa, though apparently longer in range than repressive interactions, are severely limited in their range. The structural organization of spa, then, appears to be constrained by a complex network of short-range positive and negative interactions. Activator sites must be spaced closely enough to trigger synergistic activation in cone cells; at the same time, repressor sites must be positioned to disrupt this synergy in noncone cells, preventing ectopic activation (Swanson, 2010).

Recent work has shown that changes to enhancer organization can 'fine-tune' the output of a combinatorial code, subtly changing the sensitivity of the enhancer to a morphogen. Given the importance of the structure of the spa enhancer for its proper function, it is proposed that any combinatorial code model, no matter how complex, is insufficient to describe the regulation of spa, because the same components can be rearranged to produce drastically different patterns (Swanson, 2010).

One might expect that the regulatory and organizational complexity of the spa enhancer, and its extreme sensitivity to mutation, would be reflected in strict evolutionary constraints upon enhancer sequence and structure. Yet, very poor conservation of spa sequence was observed, both in the known TFBSs and in most of the newly mapped essential regulatory elements. The reduced presence of Lz/Ets/Su(H) sites in D. pseudoobscura could potentially be attributed to redundancy of those sites in D. melanogaster, or to compensatory gain of binding sites for alternate factors in the D. pse enhancer. Perhaps more difficult to understand is the apparent loss of critical regulatory sequences in regions 4, 5, and 6a in D. pse; the experiments in D. mel suggest that the absence of those inputs would result in loss of cone cell expression and/or ectopic activation. It remains possible that many of these inputs are in fact conserved, but that conservation is not obvious due to binding site degeneracy and/or rearrangement of elements within the enhancer. Fine-scale comparative studies are ongoing (Swanson, 2010).

spa is by no means the first example of an enhancer that is functionally maintained despite a lack of sequence conservation. The most thoroughly characterized example of this phenomenon is the eve stripe 2 enhancer; its function is conserved despite changes in binding site composition and organization. Note, however, that spa has undergone much more rapid sequence divergence than eve stripe 2, with no apparent change in function. In general, the ability of an enhancer to maintain its function in the face of rapid sequence evolution suggests that enhancer structure must be quite flexible. These observations support the 'billboard' model of enhancer structure, which proposes that as long as individual regulatory units within an enhancer remain intact, the organization of those units within the enhancer is flexible. Yet, the findings concerning the importance of local interactions among densely clustered, precisely positioned transcription factors are more consistent with the tightly structured 'enhanceosome' model. Further structure-function analysis will be necessary to fully understand the players and rules governing this regulatory element (Swanson, 2010).

Rapid evolutionary rewiring of a structurally constrained eye enhancer

Enhancers are genomic cis-regulatory sequences that integrate spatiotemporal signals to control gene expression. Enhancer activity depends on the combination of bound transcription factors as well as - in some cases - the arrangement and spacing of binding sites for these factors. This study examined evolutionary changes to the sequence and structure of sparkling, a Notch/EGFR/Runx-regulated enhancer that activates the dPax2 gene in cone cells of the developing Drosophila eye. Despite functional and structural constraints on its sequence, sparkling has undergone major reorganization in its recent evolutionary history. The data suggest that the relative strengths of the various regulatory inputs into sparkling change rapidly over evolutionary time, such that reduced input from some factors is compensated by increased input from different regulators. These gains and losses are at least partly responsible for the changes in enhancer structure that were observe. Furthermore, stereotypical spatial relationships between certain binding sites ('grammar elements') can be identified in all sparkling orthologs - although the sites themselves are often recently derived. It was also found that low binding affinity for the Notch-regulated transcription factor Su(H), a conserved property of sparkling, is required to prevent ectopic responses to Notch in non-cone cells. It is concluded that rapid DNA sequence turnover does not imply either the absence of critical cis-regulatory information or the absence of structural rules. These findings demonstrate that even a severely constrained cis-regulatory sequence can be significantly rewired over a short evolutionary timescale (Swanson, 2011).

Because of spa's rapid structural evolution and binding-site turnover, multispecies sequence alignments do not reveal many conserved features. Only the extreme 5' end of spa is unequivocally alignable across 12 Drosophila genomes. Given spa's complex regulatory circuitry and structure, its unusually rapid sequence divergence between D. mel and D. pse was surprising, especially because both orthologs of spa have identical cell-type specificities (Swanson, 2011).

This study demonstrated that even an enhancer that is subject to structural constraints can be evolutionarily flexible; therefore, an apparent lack of conserved cis-regulatory structure does not imply an absence of organizational rules within an enhancer (Swanson, 2011).

A model for the structural divergence of spa between the melanogaster and obscura groups is proposed, based on sequence analyses and experimental data. Although the remote control element (RCE) and its flanking Lz1-Ets1 pair are relatively stable, many other essential regulatory sites have been relocated. Within regions 4, 5, and 6a, putative novel regulatory motifs, essential for full-strength activation of both spa orthologs, have been identified whose movements are consistent with experimental data on spa's evolutionary restructuring (Swanson, 2011).

Important changes to the Lz/Ets/Su(H) inputs have also occurred: D. pse has fewer Su(H) and Lz sites, relative to the melanogaster group -- which can be compensated by newly acquired, functionally significant 5' Ets and epsilon (AGCCAG) sites. Meanwhile, the melanogaster group has gained a new Lz site and also has a relative abundance of Su(H) sites, which may compensate for relatively few epsilon and Ets sites (Swanson, 2011).

By tracking the reorganization of Su(H), Lz, Ets, and epsilon motifs across multiple species, a speculative phylogeny of the spa enhancer within the genus Drosophila is proposed and the cis-regulatory content of the last common ancestors (LCAs) of several species groups is predicted by reconstructing the gain and loss of sites, and the changing strengths of transregulatory inputs, in specific lineages. The main conclusions to be drawn from this evolutionary view of spa, informed by functional experiments, are: (1) significant enhancer rewiring has occurred since the divergence of the mel and pse lineages; (2) this rewiring involves the loss and gain of individual regulatory motifs, as well as compensatory changes in the overall strength of several trans-regulatory inputs through changes in binding-site number, position, and possibly affinity; (3) despite very rapid site turnover, characteristic configurations of sites ('grammar elements') can be identified; (4) these grammar elements can be relocated within the enhancer, suggesting that a specific arrangement of sites can be more ancient than the individual sites that compose it. These last two points, taken together, may explain how spa can continue to obey structural rules while being significantly reconfigured (Swanson, 2011).

A large proportion of the grammar elements that have been identified involve Lz/Runx and Ets motifs. Unlike the case of linked sites for Dorsal, Twist, and other factors in insect neurogenic enhancers, there is no single, clearly preferred arrangement of Lz and Ets sites within spa: seven distinct types of Lz/Ets grammar element were identified that are at least as ancient as the LCA of the melanogaster group (Swanson, 2011).

Perhaps Runx and Ets factors, which are known to directly interact and to cooperatively activate transcription in flies and vertebrates, can synergize productively in several different spatial configurations. This is consistent with mapped Runx and Ets sites in vertebrate genomes, which are frequently associated with one another in target enhancers, but not with a single rigid arrangement or spacing (Swanson, 2011).

A nonstructural constraint on the sequence of spa was discovered: a requirement for nonconsensus, low-affinity Su(H) sites for proper cone-specific patterning. Because ectopic dPax2 expression in photoreceptor precursors causes faulty cell fate specification and differentiation, resulting in defective eye morphology, it is reasonable to suppose that the expression pattern of spa[Su(H)-HiAff] would have negative fitness consequences for the fly. Taken together with previous work, the data presented in this study suggest that spa requires input from Notch/Su(H) but also requires that input to be attenuated at the cis-regulatory level, in order to generate the proper levels and cell-type specificity of dPax2 expression in a tissue with widespread Notch signaling. Like Notch/Su(H), EGFR/Ets signaling and Lz are also used to specify multiple cell types in the retina, which presents a challenge for combinatorial gene regulation: enhancers must be able to make fine qualitative distinctions in regulatory inputs and often must translate this information into relatively sharp on/off decisions. These pressures could result in a cis-regulatory logic for genes like dPax2 in which many weak inputs are independently tuned (and spatially arranged) to maximize activation in the proper cell type, while minimizing ectopic activation. Previous studies of spa present a picture of an enhancer operating just above a functional threshold, such that the loss of a single regulatory site, or a loss of proper grammar, can result in transcriptional failure in cone cells. One of the main conclusions from this study is that, over a relatively short evolutionary timescale, a cis-regulatory module can find multiple solutions to this complex computational problem (Swanson, 2011).

The presence of weak, nonconsensus binding sites for signal-regulated TFs is a common, but little remarked upon, feature of developmental enhancers. Low-affinity TF binding sites have well-documented functions in shaping a stripe of gene expression across a morphogen gradient and in determining temporal responses to developmental regulators. This study provides direct evidence supporting a role for weak signal response elements in preventing ectopic transcriptional responses to highly pleiotropic signaling pathways such as Notch (Swanson, 2011).

There is one striking question not addressed by this study: why is this enhancer evolving at an unusually high rate, given that its expression pattern is stable? Two plausible explanations are given for which supporting data exist. First, dPax2 is on chromosome 4, the 'dot' chromosome of Drosophila, which has a severely reduced recombination rate, resulting in inefficient selection and relaxed sequence constraint. No other cis-regulatory module on the fourth chromosome has been subjected to an extensive evolutionary analysis, nor are any as well-mapped as sparkling, but enhancers of the fourth-chromosome genes eyeless and toy contain fairly large blocks of sequence conservation, compared to spa. An alternative explanation for the rapid turnover observed within spa involves the presence of nonconsensus, predicted low-affinity sites for Su(H) and, in some cases, Lz and PntP2. For a typical TF, there are many more possible low-affinity binding sites than high-affinity sites: for example, the highest-affinity Su(H) consensus YGTGDGAAM encompasses only 12 variants (TGTGGGAAA, etc.), whereas the lower-affinity consensus of the same length nRTGDGWDn, which accommodates all of the known Su(H) sites within spa, contains 576 possible sequences. Accordingly, it is much more likely that an enhancer will acquire a low-affinity binding site via a single mutational event than a high-affinity site. Thus, an enhancer that does not require high-affinity binding sites for given trans-regulators may rapidly sample a variety of configurations of weak sites and may thereby undergo considerable sequence turnover without losing the input from that regulator. In other words, an enhancer such as spa, which must maintain a weak regulatory linkage with Notch/Su(H), may be less constrained than a high-affinity target with respect to the sequence, number, and position of its Su(H) binding sites. Whatever the reason for the rapid sequence divergence of spa, it provides an opportunity to examine in detail the evolutionary mechanisms by which a complex cis-regulatory module can be significantly reorganized, while still conforming to specific constraints of combinatorial logic and grammar (Swanson, 2011).

Function of Su(H) as a 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)

Su(H) regulates a Serrate enhancer

Drosophila wing development is a useful model to study organogenesis, which requires the input of selector genes that specify the identity of various morphogenetic fields and cell signaling molecules. In order to understand how the integration of multiple signaling pathways and selector proteins can be achieved during wing development, the regulatory network that controls the expression of Serrate (Ser), a ligand for the Notch (N) signaling pathway, which is essential for the development of the Drosophila wing, as well as vertebrate limbs, was examined. A 794 bp cis-regulatory element located in the 3' region of the Ser gene can recapitulate the dynamic patterns of endogenous Ser expression during wing development. Using this enhancer element, Apterous (Ap, a selector protein), and the Notch and Wingless (Wg) signaling pathways, are shown to sequentially control wing development through direct regulation of Ser expression in early, mid and late third instar stages, respectively. In addition, later Ser expression in the presumptive vein cells is controlled by the Egfr pathway. Thus, a cis-regulatory element is sequentially regulated by multiple signaling pathways and a selector protein during Drosophila wing development. Such a mechanism is possibly conserved in the appendage outgrowth of other arthropods and vertebrates (Yan, 2004).

The results reported here demonstrate that a 794 bp cis-acting regulatory module in the Ser locus can be temporally regulated by three distinct mechanisms that are employed for the proper establishment of the DV organizer during wing development. (1) The selector protein Ap directly activates Ser expression in the dorsal compartment during the early third instar, which sets up N activation for the next stage. (2) By the middle of the third instar, the N pathway maintains Ser expression by a positive-feedback loop along the DV boundary. This feedback loop maintains Ser and Dl expression, leading to the activation of N signaling at the DV boundary, which is essential for establishing the DV organizer. (3) At the end of the third instar, as a result of Wg signaling, Ser is expressed in two stripes flanking the DV boundary, which limits N activation to the DV border. In addition, Ser expression in provein cells is dependent on input from the Egfr pathway. These results indicate how tissue-specific selector and signaling molecules can work sequentially to achieve a complex developmental process, such as organogenesis, which involves a complex temporal and spatial regulation of genes. However, the conclusion that the Ser minimal wing enhancer is sequentially regulated by Ap, Notch, Wg and Egfr does not exclude the possibility that these molecules/signaling pathways may cooperate and synergistically stimulate gene expression at certain stages. In this case, mutations that specifically impair response to the intended factor would affect Ser-lacZ expression in other phases of disc development (Yan, 2004).

Around 24 hours after the L2/L3 molt, a transition occurs in Ser minimal enhancer expression from all dorsal cells to dorsal cells near the DV boundary [24 hours after the L2/L3 molt is defined as early third instar because 48-72 hours AEL (after egg laying) is generally taken as the early third instar, which is equal to 0-24 h after the L2/L3 molt]. During this transition, Ser expression in dorsal cells flanking the DV boundary may be regulated by Ap, as well as by the N pathway. At 24 hours after the L2/L3 molt, (mAp)Ser-lacZ displays no activity, and [mSu(H)]Ser-lacZ expression is evident in dorsal cells near the DV boundary. Although these data suggest that Ap regulates Ser expression in dorsal cells near the DV boundary, they do not exclude the possibility that Notch may still be involved in directly regulating Ser expression during this transition, since Su(H) may still be able to bind to and activate [mSu(H)]Ser-lacZ (Yan, 2004).

Activation of N signaling at the nascent DV boundary is essential for the formation of the DV boundary. Ser and Dl are highly expressed at the DV border in mid-third instar and their expression can be ectopically activated by a constitutively active form of N, which suggests a positive-feedback loop between N ligands and the receptor. The activation of such a feedback loop between N and its ligands is likely to be among the earliest events in the formation of the DV boundary. The finding that the Ser wing enhancer is regulated by the N pathway, and that two Su(H)-binding sites are required for the in vivo activity of this enhancer in the mid third instar, suggests that N signaling can directly regulate Ser expression through Su(H). Although these results are consistent with direct activation of the Ser gene by Su(H), they do not preclude the possibility that N signaling may regulate Ser through other transcription factors, possibly downstream of Su(H). This would explain why [mSu(H)]Ser-lacZ showed a significant, but not dramatic, loss of enhancer activity. Alternatively, it remains possible that Su(H) can still bind to and activate at least one of the two mutant Su(H) binding sites in [mSu(H)]Ser-lacZ (Yan, 2004).

Given that the Ser-Fng-N pathway is evolutionarily conserved in appendage development between insects and vertebrates, the mechanism by which Ser is sequentially regulated by Ap, N, Wg and Egfr may also be conserved in appendage outgrowth of other arthropods and vertebrates. Consistent with this hypothesis, the Ap, Wg/Wnt and Egfr/Fgf pathways are also involved in appendage development in vertebrates, as well as D. melanogaster. Indeed, a BLAST search of the Drosophila pseudoobscura genome identified a putative homolog of the Ser minimal wing enhancer. Interestingly, this enhancer region is also located less than 1 kb downstream of the putative D. pseudoobscura Ser 3'UTR. Sequence comparisons between the Ser minimal wing enhancer from D. melanogaster and the putative D. pseudoobscura enhancer show a significant degree of similarity, whereas the similarities in the 5' and 3' flanking regions are lower. Importantly, sequences of putative Ap, Su(H) and dTCF binding sites are highly conserved in D. pseudoobscura and D. melanogaster. Although the strong conservation of sequence and location suggests that the putative D. pseudoobscura Ser enhancer may be a functional homolog of the D. melanogaster Ser minimal wing enhancer, it remains to be tested whether this enhancer drives reporter gene expression at the identical time and location in the D. melanogaster wing discs (Yan, 2004).

Notch signaling patterns Drosophila mesodermal segments by regulating Twist

One of the first steps in embryonic mesodermal differentiation is allocation of cells to particular tissue fates. In Drosophila, this process of mesodermal subdivision requires regulation of the bHLH transcription factor Twist. During subdivision, Twist expression is modulated into stripes of low and high levels within each mesodermal segment. High Twist levels direct cells to the body wall muscle fate, whereas low levels are permissive for gut muscle and fat body fate. Su(H)-mediated Notch signaling represses Twist expression during subdivision and thus plays a critical role in patterning mesodermal segments. This work demonstrates that Notch acts as a transcriptional switch on mesodermal target genes, and it suggests that Notch/Su(H) directly regulates twist, as well as indirectly regulating twist by activating proteins that repress Twist. It is proposed that Notch signaling targets two distinct 'Repressors of twist' - the proteins encoded by the Enhancer of split complex [E(spl)C] and the HLH gene extra machrochaetae (emc). Hence, the patterning of Drosophila mesodermal segments relies on Notch signaling changing the activities of a network of bHLH transcriptional regulators, which, in turn, control mesodermal cell fate. Since this same cassette of Notch, Su(H) and bHLH regulators is active during vertebrate mesodermal segmentation and/or subdivision, this work suggests a conserved mechanism for Notch in early mesodermal patterning (Tapanes-Castillo, 2004).

Analysis of Notch mutant embryos revealed that Notch signaling is essential for Twist regulation at mesodermal subdivision. However, comparison of Notch and Su(H) mutant embryos indicated that Notch regulates Twist differently from Su(H). At stage 10, uniform high Twist expression was maintained in Nnull mutants; by contrast, Su(H)null mutants have a wild-type-like Twist pattern. Furthermore, while constitutive activation of Notch represses Twist expression at stage 10, constitutive expression of a transactivating form of Su(H) [Su(H)-VP16] increases Twist expression. Despite these differences, double mutant analysis and rescue experiments demonstrate that Notch requires Su(H) to repress Twist. Moreover, further rescue experiments show that Notch signaling acts as a transcriptional switch, which alleviates Su(H)-mediated repression and promotes transcription. In addition, genetics, combined with promoter analysis, suggest that Notch and Su(H) have multiple inputs into twist. Notch/Su(H) signaling both directly activates twist and indirectly represses twist expression by activating proteins that repress Twist. Finally, the data indicate that Notch targets two distinct 'Repressors of twist' - E(spl)-C genes and Emc. It is proposed that Notch signaling activates expression of E(spl)-C genes, which then act directly on the twist promoter to repress transcription. Since removing groucho enhances the phenotype of the E(spl)-C mutant embryos, it is suggested that the corepressor, Groucho, acts with E(spl)-C proteins and the Hairless/Su(H) repressive complex to mediate direct repression of twist. The second 'Repressor of twist', Emc, mediates repression of Twist in an alternative fashion. It is hypothesized Emc activity inhibits dimerization of Da with itself or another bHLH protein. This, in turn, prevents Da from binding DNA and activating twist transcription. Since Emc is expressed in the embryo prior to stage 10, it is likely that the transition from uniform high Twist expression to a modulated Twist pattern involves Emc inhibition of Da activity at stage 9. In conclusion, this work uncovers how Notch signaling impacts a network of mesodermal genes, and specifically Twist expression. Given that Notch signaling directs cell fate decisions in many Drosophila embryonic and adult tissues and that Notch regulates Twist in adult flight muscles, these data may suggest a more universal mode of Notch regulation (Tapanes-Castillo, 2004).

The distinct mesodermal phenotypes of Notch and Su(H) mutants can be explained by Notch acting as a transcriptional switch. This aspect of Notch signaling has been described in other systems, and the early Drosophila mesoderm appears no different in this regard. However, these data suggest that there is more to the phenotypes; that is, additional layers of Notch regulation in the transcriptional control of twist (Tapanes-Castillo, 2004).

Genetic experiments, as well as promoter analysis, raised the hypothesis that Notch signaling regulates twist directly, as well as indirectly by activating expression of a 'repressor of twist.' This indirect repression of twist concurs with the role of Notch in activating E(spl) transcriptional repressors. Moreover, a mechanism involving direct and indirect regulation is consistent with Su(H) mutant phenotypes. In Su(H)null embryos, neither twist nor repressor of twist (for example, emc) are repressed. The de-repression of both genes at the same time results in Twist expression appearing 'wild-type-like'. When a constitutively activating form of Su(H) is expressed, both twist and repressor of twist are activated. In these embryos, high Twist domains are expanded, but uniform high Twist expression is not observed because repressor of twist is expressed (Tapanes-Castillo, 2004).

However, simple direct and indirect regulation [through emc and E(spl)-C genes] by Notch still does not fully explain the phenotypes of Notch mutants. Both twist and repressor of twist should be repressed in Nnull embryos because Su(H) will remain in its repressor state. While the Nnull phenotype was consistent with repressor of twist being repressed, twist was still strongly expressed. Additionally, constitutive Notch activation should cause both twist and repressor of twist to be expressed. Consequently, Nintra was expected to cause a phenotype similar to that caused by Su(H)-VP16. Contrary to these predictions, panmesodermal expression of Nintra represses Twist, consistent with only repressor of twist being strongly expressed. Taken together, these results suggested that at stage 10, the twist promoter is less receptive to Notch/Su(H) activation than to Notch/Su(H) repression. As a result, constitutive activation of Notch represses twist, while loss of Notch activates twist ectopically (Tapanes-Castillo, 2004).

While Notch signaling has the ability to activate twist, Notch/Su(H) signaling ultimately leads to repression of twist at stage 10. This predominance of repression can be explained in two ways: (1) direct Notch activation of the twist promoter is overpowered by Notch activated repressors of twist; and (2) a repressor of twist gene, such as E(spl), is more responsive to Notch/Su(H) activation than twist. These ideas are discussed below in light of the results (Tapanes-Castillo, 2004).

The first model proposes that while Notch signaling might directly promote both twist and repressor of twist activation, repressors of twist might suppress an increase in twist transcription. The data suggest that Notch regulates multiple repressors of twist, including E(spl)-C genes and Emc. On the twist promoter, these multiple repressors could overwhelm Su(H) activation. Hence, twist would be transcriptionally repressed rather than activated. In Su(H)-VP16 embryos, the constitutive activating ability of Su(H) on the twist promoter might inhibit some of this repression. Consequently, Twist is ectopically expressed at high levels (Tapanes-Castillo, 2004).

The data are also consistent with the second model, which proposes that twist and a repressor of twist gene, such as E(spl), respond differently to Notch activation. The reason for this differential response is provided by the concept of Notch instructive and permissive genes. Transcription of Notch instructive genes requires the intracellular domain of Notch (Nicd) first to alleviate Su(H)-mediated repression and then to serve as a coactivator for Su(H). Transcription of Notch permissive target genes requires Nicd solely to de-repress Su(H); Su(H) bound to other coactivators and/or other transcriptional activators is necessary for permissive gene activation. Since panmesodermal expression of Nintra does not activate twist, it is concluded that simple de-repression of Su(H) is insufficient to activate twist expression and that other factors are required. Hence, Notch acts permissively on the twist promoter. By contrast, panmesodermal expression of Nintra is sufficient to activate a repressor of twist, resulting in the strong Twist repression. Since E(spl)-C genes have been categorized as Notch instructive target genes, it is suggested that E(spl)-C genes are the Notch instructive repressor of twist genes in this system. Although Notch can upregulate Emc expression, the inability to see a change in Emc expression in Nnull and Su(H)null mutants suggests Emc is not a Notch instructive target gene. Thus, based on all of this work, the instructive and permissive target gene regulation model is currently favored (Tapanes-Castillo, 2004).

In Drosophila, Notch signaling is activated by the Delta (Dl) and Serrate ligands. Delta is expressed throughout the mesoderm at late stage 9 and stage 10, while Serrate is not embryonically expressed until stage 11. While the germline requirement for Delta prevents germline clone embryos from being produced by recombination, embryos lacking zygotically expressed Dl exhibit a wild-type-like Twist pattern. In addition, expression of a full-length Notch protein missing the two EGF repeats critical for Dl binding (EGF repeats 11 and 12) rescues Twist modulation in Nnull mutant embryos. Thus Notch does not require EGF-like repeats 10-12 to repress Twist. These preliminary data suggest that Delta may use EGF-like repeats other than 10-12 to activate Notch. Alternatively, Notch may not be activated by canonical Delta signaling; a novel (non-DSL) ligand may activate Notch in the early mesoderm. Further experiments are required to evaluate whether the maternal component of Delta regulates Twist (Tapanes-Castillo, 2004).

While this work elucidates the molecular mechanism by which Notch represses Twist, how Notch signaling establishes a segmentally repeated pattern of low and high Twist domains -- that is, periodicity in Twist expression -- has yet to be understood. Two models, consistent with the data, are proposed to describe how Notch signaling contributes to a modulated Twist pattern. Model I proposes that during the transition from a uniform to a modulated Twist pattern, Notch signaling represses twist only in presumptive low Twist domains. Transcriptional activators, such as Da, maintain high Twist expression in presumptive high Twist domains. While Notch signaling components such as Notch, Su(H), and Delta are expressed throughout the mesoderm at late stage 9 and stage 10, this model predicts that Notch signaling is simply not activated in presumptive high Twist domains. Model II proposes that during the transition in Twist expression, Notch signaling represses twist throughout the mesoderm, but Notch independent transcriptional activators antagonize Notch repression in what will become high Twist domains, thereby promoting the formation of high Twist domains. For example, transcriptional effectors of Notch signaling [such as Su(H) and E(spl)] and an 'activator' that is only expressed in presumptive high Twist domains may converge and compete on the twist promoter (Tapanes-Castillo, 2004).

Consistent with model II, the segmentation gene sloppy-paired (slp) is a spatially regulated 'high Twist domain' activator. At stages 9-10, Slp is expressed in the mesoderm in transverse stripes that correspond to high Twist domains. Moreover, loss- and gain-of-function experiments indicate that Slp is required for high Twist expression. No change in Slp expression is found in Notch and Su(H) mutant embryos through mid-embryogenesis, indicating that slp is not regulated by Notch signaling at these stages. Mesodermal slp expression is activated by Wingless signaling; therefore, Wingless signaling is likely to alleviate Notch repression in high Twist domains. In the future, it will be important to establish the mechanism through which Notch signaling is antagonized in high Twist domains. Slp and Notch effectors may converge on the twist promoter to regulate expression. Additionally, Wingless signaling components may directly regulate and/or inhibit Notch (Tapanes-Castillo, 2004).

During vertebrate segmentation, mesodermal segments (called somites) are progressively segregated from a terminal undifferentiated growth zone called the presomitic mesoderm. Somites are then patterned though a process of subdivision, so that cells are allocated cells to distinct tissue fates. The first subdivision partitions each somite across the anterior-posterior axis into rostral and caudal halves. Later each somite is further subdivided across the dorsal-ventral axis into dermomyotome, which gives rise to dermis and skeletal muscle, and sclerotome, which develops into the axial skeleton. The Notch signal transduction pathway has been shown to play a central role in both somite segmentation and rostral/caudal subdivision (Tapanes-Castillo, 2004).

While Notch does not appear to be involved in fly segmentation, this work uncovers a previously uncharacterized role for Notch in the subdivision of Drosophila mesodermal segments. Notch repression is required to subdivide each mesodermal segment into a low and high Twist domain. Hence, Drosophila, like vertebrates, utilizes Notch and bHLH regulators to subdivide the mesoderm and transform uncommitted mesoderm into patterned segments. Since the homologs and/or family members of the bHLH regulators studied here -- Twist, Emc, Da and E(spl) -- are involved in vertebrate segmentation and/or somite subdivision, it will be interesting to determine whether these proteins are regulated in vertebrates in a manner similar to that governing their regulation in the fly (Tapanes-Castillo, 2004).

A regulatory code for neurogenic gene expression in the Drosophila embryo involves Dorsal, Twist and Su(H)

Bioinformatics methods have identified enhancers that mediate restricted expression in the Drosophila embryo. However, only a small fraction of the predicted enhancers actually work when tested in vivo. In the present study, co-regulated neurogenic enhancers that are activated by intermediate levels of the Dorsal regulatory gradient are shown to contain several shared sequence motifs. These motifs permit the identification of new neurogenic enhancers with high precision: five out of seven predicted enhancers direct restricted expression within ventral regions of the neurogenic ectoderm. Mutations in some of the shared motifs disrupt enhancer function, and evidence is presented that the Twist and Su(H) regulatory proteins are essential for the specification of the ventral neurogenic ectoderm prior to gastrulation. The regulatory model of neurogenic gene expression defined in this study permitted the identification of a neurogenic enhancer in the distant Anopheles genome. The prospects for deciphering regulatory codes that link primary DNA sequence information with predicted patterns of gene expression are discussed (Markstein, 2004).

Previous studies identified two enhancers, from the rho and vnd genes, that are activated by intermediate levels of the Dorsal gradient in ventral regions of the neurogenic ectoderm. The present study identified a third such enhancer from the brk gene. This newly identified brk enhancer corresponds to one of the 15 optimal Dorsal-binding clusters described in a previous survey of the Drosophila genome. Although one of these 15 clusters has been shown to define an intronic enhancer in the short gastrulation (sog) gene, the activities of the remaining 14 clusters were not tested. Genomic DNA fragments corresponding to these 14 clusters were placed 5' of a minimal eve-lacZ reporter gene, and separately expressed in transgenic embryos using P-element germline transformation. Four of the 14 genomic DNA fragments were found to direct restricted patterns of lacZ expression across the dorsoventral axis that are similar to the expression patterns seen for the associated endogenous genes (Markstein, 2004).

The four enhancers respond to different levels of the Dorsal nuclear gradient. Two direct expression within the presumptive mesoderm where there are high levels of the gradient. These are associated with the Phm and Ady43A genes. The third enhancer maps ~10 kb 5' of brk, and is activated by intermediate levels of the Dorsal gradient, similar to the vnd and rho enhancers. Finally, the fourth enhancer maps over 15 kb 5' of the predicted start site of the CG12443 gene, and directs broad lateral stripes throughout the neurogenic ectoderm in response to low levels of the Dorsal gradient. In terms of the dorsoventral limits, this staining pattern is similar to that produced by the sog intronic enhancer (Markstein, 2004).

The remaining ten clusters failed to direct robust patterns of expression and are thus referred to as 'false-positives'. Since analysis of spacing and orientation of the Dorsal sites alone did not reveal features that could discriminate between the false positives and the enhancers, whether additional sequence motifs could aid in this distinction was examined. A program called MERmaid was developed that identifies motifs over-represented in specified sets of sequences. MERmaid analysis identified a group of motifs, which was largely specific to the brk, vnd and rho enhancers, suggesting that the regulation of these coordinately expressed genes is distinct from the regulation of genes that respond to different levels of nuclear Dorsal (Markstein, 2004).

The rho, vnd and brk enhancers direct similar patterns of gene expression. The rho and vnd enhancers were previously shown to contain multiple copies of two different sequence motifs: CTGNCCY and CACATGT. A three-way comparison of minimal rho, vnd and brk enhancers permitted a more refined definition of the CTGNCCY motif (CTGWCCY), and also allowed for the identification of a third motif, YGTGDGAA. The CACATGT and YGTGDGAA motifs bind the known transcription factors, Twist and Suppressor of Hairless [Su(H)], respectively. All three motifs are over-represented in authentic Dorsal target enhancers directing expression in the ventral neurogenic ectoderm, as compared with the 10 false-positive Dorsal-binding clusters. Some of the false-positive clusters contain motifs matching either Twist or CTGWCCY; however, none of the false-positive clusters contain representatives of both of these motifs. The rho enhancer is repressed in the ventral mesoderm by the zinc-finger Snail protein. The four Snail-binding sites contained in the rho enhancer share the consensus sequence, MMMCWTGY; the vnd and brk enhancers contain multiple copies of this motif and are probably repressed by Snail as well (Markstein, 2004).

The functional significance of the shared sequence motifs was assessed by mutagenizing the sites in the context of otherwise normal lacZ transgenes. Previous studies have suggested that bHLH activators are important for the activation of rho expression, since rho-lacZ fusion genes containing point mutations in several different E-box motifs (CANNTG) exhibited severely impaired expression in transgenic embryos. However, it was not obvious that the CACATGT motif was particularly significant since it represents only one of five E-boxes contained in the rho enhancer. Yet, only this particular E-box motif is significantly over-represented in the rho, vnd and brk enhancers. vnd-lacZ and brk-lacZ fusion genes were mutagenized to eliminate each CACATGT motif, and analyzed in transgenic embryos. The loss of these sites causes a narrowing in the expression pattern of an otherwise normal vnd-lacZ fusion gene. By contrast, the brk pattern is narrower in central and posterior regions, but relatively unaffected in anterior regions. The brk enhancer contains two copies of an optimal Bicoid-binding site, and it is possible that the Bicoid activator can compensate for the loss of the CACATGT motifs in anterior regions (Markstein, 2004).

Similar experiments were performed to assess the activities of the Su(H)-binding sites (YGTGDGAA) and the CTGWCCY motif. Mutations in the latter sequence cause only a slight reduction and irregularity in the activity of the vnd enhancer, whereas similar mutations nearly abolish expression from the brk enhancer. Thus, CTGWCCY appears to be an essential regulatory element in the brk enhancer, but not in the vnd enhancer. Mutations in both Su(H) sites in the brk enhancer caused reduced staining of the lacZ reporter gene, suggesting that Su(H) normally activates expression. Further evidence that Su(H) mediates transcriptional activation was obtained by analyzing the endogenous rho expression pattern in transgenic embryos carrying an eve stripe 2 transgene with a constitutively activated form of the Notch receptor (NotchIC). rho expression is augmented and slightly expanded in the vicinity of the stripe2-NotchIC transgene. A similar expansion is observed for the sim expression pattern (Markstein, 2004).

To determine whether the shared motifs would help identify additional ventral neurogenic enhancers, the genome was surveyed for 250 bp regions containing an average density of one site per 50 bp and at least one occurrence of each of the four motifs for Dorsal, Twist, Su(H) and CTGWCCY. In total, only seven clusters were identified. Three of the seven clusters correspond to the rho, vnd and brk enhancers. Two of the remaining clusters are associated with genes that are known to be expressed in ventral regions of the neurogenic ectoderm: vein and sim. Both clusters were tested for enhancer activity by attaching appropriate genomic DNA fragments to a lacZ reporter gene and then analyzing lacZ expression in transgenic embryos. The cluster associated with vein is located in the first intron, about 7 kb downstream of the transcription start site. The vein cluster (497 bp) directs robust expression in the neurogenic ectoderm, similar to the pattern of the endogenous gene. The cluster located in the 5' flanking region of the sim gene (631 bp) directs expression in single lines of cells in the mesectoderm (the ventral-most region of the neurogenic ectoderm), just like the endogenous expression pattern. These results indicate that the computational methods define an accurate regulatory model for gene expression in ventral regions of the neurogenic ectoderm of D. melanogaster (Markstein, 2004).

To assay the generality of these findings, genomic regions encompassing putative sim orthologs from the distantly related dipteran Anopheles gambiae were scanned for clustering of Dorsal, Twist, Su(H), CTGWCCY and Snail motifs. One cluster located 865 bp 5' of a putative sim ortholog contains one putative Dorsal binding site, two Su(H) sites, three CTGWCCY motifs (or close matches to this motif), a CACATG E-box and several copies of the Snail repressor sequence MMMCWTGY. A genomic DNA fragment encompassing these sites (976 bp) was attached to a minimal eve-lacZ reporter gene and expressed in transgenic Drosophila embryos. The Anopheles enhancer directs weak lateral lines of lacZ expression that are similar to those obtained with the Drosophila sim enhancer. These results suggest that the clustering of Dorsal, Twist, Su(H) and CTGWCCY motifs constitutes an ancient and conserved code for neurogenic gene expression (Markstein, 2004).

This study defines a specific and predictive model for the activation of gene expression by intermediate levels of the Dorsal gradient in ventral regions of the neurogenic ectoderm. The model identified new enhancers for sim and vein in the Drosophila genome, as well as a sim enhancer in the distant Anopheles genome. Five of the seven composite Dorsal-Twist-Su(H)-CTGWCCY clusters in the Drosophila genome correspond to authentic enhancers that direct similar patterns of gene expression. This hit rate represents the highest precision so far obtained for the computational identification of Drosophila enhancers based on the clustering of regulatory elements. Nevertheless, it is still not a perfect code (Markstein, 2004).

Two of the seven composite clusters are likely to be false-positives: they are associated with genes that are not known to exhibit localized expression across the dorsoventral axis. It is possible that the order, spacing and/or orientation of the identified binding sites accounts for the distinction between authentic enhancers and false-positive clusters. For example, there is tight linkage of Dorsal and Twist sites in each of the five neurogenic enhancers. This linkage might reflect Dorsal-Twist protein-protein interactions that promote their cooperative binding and synergistic activities. Previous studies identified particularly strong interactions between Dorsal and Twist-Daughterless (Da) heterodimers. Da is ubiquitously expressed in the early embryo and is related to the E12/E47 bHLH proteins in mammals. Dorsal-Twist linkage is not seen in one of the two false-positive binding clusters (Markstein, 2004).

The regulatory model defined by this study probably fails to identify all enhancers responsive to intermediate levels of the Dorsal gradient. There are at least 30 Dorsal target enhancers in the Drosophila genome, and it is possible that 10 respond to intermediate levels of the Dorsal gradient. Thus, half of all such target enhancers might have been missed. Perhaps the present study defined just one of several 'codes' for neurogenic gene expression (Markstein, 2004).

The possibility of multiple codes is suggested by the different contributions of the same regulatory elements to the activities of the vnd and brk enhancers. Mutations in the CTGWCCY motifs nearly abolish the activity of the brk enhancer, but have virtually no effect on the vnd enhancer. Future studies will determine whether there are distinct codes for Dorsal target enhancers that respond to either high or low levels of the Dorsal gradient. Indeed, it is somewhat surprising that the sog and CG12443 enhancers essentially lack Twist, Su(H) and CTGWCCY motifs, even though they direct lateral stripes of gene expression that are quite similar (albeit broader) to those seen for the rho, vnd and brk enhancers (Markstein, 2004).

This study provides direct evidence that Twist and Su(H) are essential for the specification of the neurogenic ectoderm in early embryos. The Twist protein is transiently expressed at low levels in ventral regions of the neurogenic ectoderm. SELEX assays indicate that Twist binds the CACATGT motif quite well. The presence of this motif in the vnd, brk and sim enhancers, and the fact that it functions as an essential element in the vnd and brk enhancers, strongly suggests that Twist is not a dedicated mesoderm determinant, but that it is also required for the differentiation of the neurogenic ectoderm. However, it is currently unclear whether the CACATGT motif binds Twist-Twist homodimers, Twist-Da heterodimers or additional bHLH complexes in vivo. Su(H) is the sequence-specific transcriptional effector of Notch signaling. The restricted activation of sim expression within the mesectoderm depends on Notch signaling; however, the rho, vnd and brk enhancers direct expression in more lateral regions where Notch signaling has not been demonstrated. Nonetheless, mutations in the two Su(H) sites contained in the brk enhancer cause a severe impairment in its activity. This observation raises the possibility that Su(H) can function as an activator, at least in certain contexts, in the absence of an obvious Notch signal (Markstein, 2004).

The Dorsal gradient produces three distinct patterns of gene expression within the presumptive neurogenic ectoderm. It is proposed that these patterns arise from the differential usage of the Su(H) and Dorsal activators. Enhancers that direct progressively broader patterns of expression become increasingly more dependent on Dorsal and less dependent on Su(H). The sog and CG12443 enhancers mediate expression in both ventral and dorsal regions of the neurogenic ectoderm, and contain several optimal Dorsal sites but no Su(H) sites. By contrast, the sim enhancer is active only in the ventral-most regions of the neurogenic ectoderm, and contains just one high-affinity Dorsal site but five optimal Su(H) sites. The reliance of sim on Dorsal might be atypical for genes expressed in the mesectoderm. For example, the m8 gene within the Enhancer of split complex may be regulated solely by Su(H). The Anopheles sim enhancer might represent an intermediate between the Drosophila sim and m8 enhancers, since it contains optimal Su(H) sites but only one weak Dorsal site. This trend may reflect an evolutionary conversion of Su(H) sites to Dorsal sites, and the concomitant use of the Dorsal gradient to specify different neurogenic cell types. A testable prediction of this model is that basal arthropods use Dorsal solely for the specification of the mesoderm and Su(H) for the patterning of the ventral neurogenic ectoderm (Markstein, 2004).

Lateral inhibition in proneural clusters: cis-regulatory logic and default repression by Suppressor of Hairless

Lateral inhibition, wherein a single cell signals to its neighbors to prevent them from adopting its own fate, is the best-known setting for cell-cell communication via the Notch (N) pathway. During peripheral neurogenesis in Drosophila, sensory organ precursor (SOP) cells arise within proneural clusters (PNCs), small groups of cells endowed with SOP fate potential by their expression of proneural transcriptional activators. SOPs use N signaling to activate in neighboring PNC cells the expression of multiple genes that inhibit the SOP fate. These genes respond transcriptionally to direct regulation by both the proneural proteins and the N pathway transcription factor Suppressor of Hairless [Su(H)], and their activation is generally highly asymmetric; i.e., only in the inhibited (non-SOP) cells of the PNC, and not in SOPs. The substantially higher proneural protein levels in the SOP put this cell at risk of inappropriately activating the SOP-inhibitory genes, even without input from N-activated Su(H). This is prevented by direct 'default' repression of these genes by Su(H), acting through the same binding sites Su(H) uses for activation in non-SOPs. Derepression of even a single N pathway target gene in the SOP can extinguish the SOP cell fate. Finally, crucial roles are defined for the adaptor protein Hairless and the co-repressors Groucho and CtBP in conferring repressive activity on Su(H) in the SOP. This work elucidates the regulatory logic by which N signaling and the proneural proteins cooperate to create the neural precursor/epidermal cell fate distinction during lateral inhibition (Castro, 2005).

A 1.1 kb genomic DNA fragment that includes the promoter and proximal upstream region of E(spl)malpha drives strong reporter gene expression in the PNCs of late third-instar imaginal discs, but this expression is excluded from SOPs. A 1.0 kb subfragment lacking the E(spl)malpha promoter confers this same expression pattern on a heterologous promoter, thus defining a discrete PNC-specific cis-regulatory module for the gene. The mechanistic basis of the striking specificity of activation of the module is of particular interest; i.e., only in the inhibited (non-SOP) cells of the PNC (Castro, 2005).

Activation of N-regulated genes of the E(spl)-C in imaginal disc PNCs makes use of a combination of Su(H)-binding sites and binding sites for the proneural proteins Ac and Sc. Consistent with this 'Su(H) plus proneural' cisregulatory code, the E(spl)malpha module includes five high-affinity Su(H) sites and a single high-affinity proneural site. The effects on reporter gene activity of mutating only the proneural site (Em), only the five Su(H) sites (Sm) or all six sites (EmSm) were examined. It was first observed that the integrity of the 'E box' proneural protein binding site is strictly required for detectable reporter expression in nearly all wing disc PNCs; residual expression is observed along the entire wing margin and in a very small subset of PNCs. This result demonstrates that, as for other N pathway target genes, the proneural proteins make an essential input as direct transcriptional activators of E(spl)malpha in PNCs (Castro, 2005).

Mutation of the five Su(H)-binding sites in the E(spl)malpha PNC module yields a dramatic alteration in the spatial pattern of its activity. Reporter gene expression in non-SOP cells is drastically reduced or eliminated, and strong ectopic expression is now observed in SOPs. Direct comparison of the wildtype (RFP) and Sm (GFP) reporter transgenes in the same disc emphasizes the stark contrast in their specificities. This finding indicates (1) that Su(H) has an essential role as a direct transcriptional activator of E(spl)malpha in the N-responsive non-SOPs, and (2) that it acts as a direct transcriptional repressor of the gene in SOPs. Finally, it was observed that mutation of the proneural protein binding site in addition to the Su(H)-binding sites (EmSm) abolishes detectable PNC expression of the reporter gene. Most importantly, this result shows that both the residual non-SOP and the ectopic SOP expression of the Sm mutant is strictly dependent on direct proneural input. It also indicates that the residual activity of the Em proneural site mutant along the wing margin and in a few PNCs requires direct input from Su(H) (Castro, 2005).

The bHLH repressor-encoding genes of the E(spl)-C, exemplified by E(spl) itself [referred to as E(spl)m8 to distinguish it readily from E(spl)malpha], likewise make use of a 'Su(H) plus proneural' cis-regulatory code for their activation in PNCs during lateral inhibition. Attempts were made to determine whether direct repression by Su(H) in SOPs applies as well to this class of N pathway target genes. It was found that a 1.1 kb genomic DNA fragment from immediately upstream of E(spl)m8 confers PNC-specific expression on a heterologous promoter-reporter construct in late third-instar wing discs. Expression is also observed along the wing margin. As with E(spl)malpha, double labeling (using anti-Hnt to mark SOPs) reveals that the PNC activity of this fragment is predominantly in non-SOPs and excluded from SOPs. Mutation of the three Su(H) binding sites (Sm) abolishes most non-SOP expression and yields strong ectopic expression in SOPs. It is concluded that Su(H) normally acts as a direct repressor of both the Brd family genes and the bHLH repressor genes of the E(spl)-C in SOPs of the adult PNS (Castro, 2005).

The results with mutant enhancer-reporter constructs define crucial roles for both proneural protein and Su(H)-binding sites in generating the non-SOP-only expression patterns of E(spl)malpha and E(spl)m8 in wing disc PNCs. It would be expected, then, that these sites should be conserved in orthologous PNC cis-regulatory modules from other Drosophila species, and, further, that such modules should function appropriately when introduced into D. melanogaster. Initially, the upstream sequence regions corresponding to the E(spl)malpha PNC module from four species, D. melanogaster, D. pseudoobscura, D. hydei and D. virilis were compared and aligned. Consistent with the established phylogenetic relationships between these species, it was found that the D. melanogaster and D. pseudoobscura sequences are overall more related to each other than to the D. hydei and D. virilis sequences, and vice versa. Of particular note is the stability, over 40-60 million years, of the number, spacing and exact sequences of the Su(H) and proneural protein binding sites. With a single exception, all of these sites are precisely conserved in sequence, as is the spacing between the S2 site and the proneural site. Whether the activity of the E(spl)malpha cis-regulatory module in PNCs is likewise evolutionarily conserved was examined. The D. virilis version of the E(spl)malpha enhancer fragment drives reporter gene expression in the D. melanogaster wing disc in a pattern that largely recapitulates the activity of the D. melanogaster module; specifically, strong activity was observed in the non-SOP cells of PNCs but little or none in SOPs. Thus, the binding site composition, architecture and in vivo function of the E(spl)malpha PNC cis-regulatory module are all evolutionarily conserved, and hence clearly subject to strong selection (Castro, 2005).

The results described thus far support the conclusion that transcriptional repression by Su(H) is required to prevent inappropriate expression of N pathway target genes in SOPs. This raises the question of the developmental significance of such repression, particularly for the SOP cell fate. To investigate, an assay was designed based on previous observations that strong over- or mis-expression of either E(spl)m5 (another bHLH repressor gene) or E(spl)m8 leads to bristle loss in adult flies, the cellular basis of which is loss of SOPs. It was anticipated that a wild-type E(spl)m8 transgene might have minimal phenotypic effects because it would be expressed normally in non-SOP cells (reinforcing their commitment to the epidermal fate) and repressed normally in SOPs; thus, bristle development would be largely unaffected. By contrast, it was hypothesized that a mutant transgene not subject to direct repression by Su(H) might yield E(spl)m8 activity in the SOP sufficient to affect this development of the cell (Castro, 2005).

The bristle patterns of w1118 adults carrying two copies of either a wild-type E(spl)m8 transgene or the same transgene with its Su(H)-binding sites mutated [E(spl)m8 Sm] were compared. For two reasons, this is considered a very stringent assay of the requirement for Su(H)-mediated repression in the SOP: (1) the effects of derepressing a single N pathway target gene were compared, although there are several other such genes (both bHLH repressor and Brd family) residing in the E(spl)-C alone; (2) the level of ectopic E(spl)m8 expression generated by a de-repressed genomic DNA transgene is expected to be much lower than that achieved by a UAS-E(spl)m8 construct activated by strong GAL4 drivers. It was found that whereas flies carrying the wild-type E(spl)m8 transgene display only very mild bristle loss, flies carrying E(spl)m8 Sm exhibit a significantly more severe bristle-loss phenotype. Staining of late third-instar wing discs with anti-Senseless (Sens) antibody to visualize SOPs showed that this bristle loss was due to a failure of SOP specification. It is concluded that loss of direct Su(H)-mediated repression of a single N pathway target gene can be sufficient to extinguish the SOP fate, thus altering the adult bristle pattern (Castro, 2005).

Su(H) is known to act as a transcriptional repressor in another context during sensory organ development; namely, the socket/shaft sister cell fate decision in the bristle lineage. Auto-repression of Su(H) is necessary to prevent inappropriate high-level activation of the gene in the shaft cell, which in turn can cause this cell (which does not respond to N signaling) to adopt the N-responsive socket cell fate. The biochemical basis of transcriptional repression by Su(H) has been studied in some detail in this setting. Specifically, the Hairless (H) protein has been shown to act as an adaptor that recruits the transcriptional corepressor proteins Gro and CtBP to Su(H), thus conferring repressive activity (Castro, 2005).

Earlier work can be interpreted to suggest that a similar protein complex might mediate repression by Su(H) in the SOP. At several macrochaete and many microchaete positions on the adult fly, simultaneous reduction of the doses of Hairless and gro in an otherwise wild-type background leads to significant bristle loss; this is due to a failure of commitment to the SOP cell fate. A plausible interpretation of these findings is that H and Gro are normally part of a repressive Su(H)-containing complex in the SOP, and that reduction of their doses sufficiently compromises the repressive activity as to partially de-repress N pathway target genes like E(spl)m8, leading to failure of SOP specification. As a test of this model, it was thought that it might be possible to detect such de-repression of a suitable reporter gene. This expectation was borne out. Late third-instar wing discs from wild-type larvae or larvae heterozygous for null alleles of either Hairless or gro only rarely exhibit detectable activity of an E(spl)malpha-GFP reporter transgene in SOPs. By contrast, wing discs from larvae doubly heterozygous for null alleles of both Hairless and gro show substantial frequencies of ectopic GFP expression in SOPs. Moreover, the SOP expression observed in the double heterozygotes is considerably stronger than that detected rarely in a wild-type background. These results demonstrate that normal levels of Hairless and gro activity are required for the Su(H)-dependent repression of N pathway target genes in SOPs, and are consistent with the participation of a Su(H)-H-Gro-containing protein complex in this repression (Castro, 2005).

Broad overexpression of Hairless (including in proneural clusters) during lateral inhibition causes a 'neurogenic' phenotype; that is, the appearance of supernumerary bristles surrounding normal bristles. This phenotype is readily understood in light of the model described above; namely, that Hairless normally serves to recruit Gro and CtBP to Su(H) for its repressive activity in the SOP. Overexpression of Hairless in the N-responsive non-SOP cells of the PNC would be expected to elevate their levels of the repressive form of Su(H), causing repression of N pathway target genes that would normally be activated by the Su(H)-NIC-Mam complex. This in turn would result in a partial failure of lateral inhibition and the commitment of additional cells in the PNC to the SOP fate, giving rise to ectopic bristles in the adult (Castro, 2005).

A key prediction of the model is that the ability of Hairless to bind Gro (via the motif YSIHSLLG) and CtBP (via the motif PLNLSKH) should be required for the SOP fate-promoting activity of H. This prediction was tested by using an E(spl)malpha GAL4 driver to express different forms of H specifically in the non-SOP cells of the PNCs. The orbital region of the adult fly head is a particularly favorable territory in which to assay the production of supernumerary bristles by H overexpression. Expression of a wild-type UAS-Hairless transgene results in the appearance of an average of approximately four ectopic bristles in the orbital region. This activity is significantly impaired by mutating either the Gro recruitment motif (UAS-H[Gm]) or the CtBP-binding motif (UAS-H deltaC), suggesting that both co-repressors make a functional contribution. Loss of both motifs (UAS-H[Gm] deltaC) essentially abolishes the capacity of Hairless to promote ectopic bristle development in this assay. These results are strongly consistent with the interpretation that the SOP cell's requirement for Hairless activity is based on the recruitment by Hairless of Gro and CtBP to confer repressive activity on Su(H), thus preventing inappropriate expression of inhibitory N pathway target genes (Castro, 2005).

It is concluded that discrete transcriptional cis-regulatory modules, bearing binding sites for both Su(H) and the proneural proteins, direct the non-SOP-only expression pattern of E(spl)-C genes in PNCs. Mutation of the Su(H) sites in these modules results in an inversion of this pattern of activity, including both the loss of most non-SOP expression and the appearance of strong ectopic expression in SOPs. These observations reveal a dual role for Su(H) in the PNC: as a direct, N-activated transcriptional activator of E(spl)-C genes in non-SOP cells, and as a direct transcriptional repressor of the same genes in the SOP. The issue was addressed as to whether Su(H)-mediated repression of E(spl)-C genes in the SOP is important developmentally. The experiments with wild-type and Sm versions of an E(spl)m8 genomic DNA transgene demonstrate that it is. Failure to repress this single bHLH repressor gene is sufficient to extinguish the SOP fate (marked by Sens) at a frequency significantly greater than that observed with a repressible (wild-type) transgene. Evidence is provided that the Hairless protein is responsible for conferring repressive activity on Su(H) in the SOP, by recruiting the co-repressors Gro and CtBP. It is suggested that the Hairless null phenotype widespread, irreversible loss of the SOP fate in an E(spl)-C-dependent manner, offers the best indication of the developmental consequences of relieving Su(H)-mediated repression of all E(spl)-C genes in the SOP (Castro, 2005).

A specific configuration of Su(H)-binding sites known as the Suppressor of Hairless Paired Site (SPS) has been shown to be essential for transcriptional synergy between proneural proteins and Su(H) in driving specific expression in PNCs. The results reported in this study on transcriptional regulation of E(spl)malpha contradict this conclusion with regard to the function of the SPS. The strong expression of E(spl)malpha in the non-SOP cells of the PNC depends crucially on cooperation between proneural activators and Su(H), yet none of the Su(H) sites of this gene are in the SPS configuration. Thus, until the mechanistic basis for proneural/Su(H) synergy is more fully elucidated, it is thought that the term 'Su(H) plus proneural' remains the most accurate and most general description of the PNC cis-regulatory code (Castro, 2005).

Direct repression of E(spl)-C genes in the SOP during lateral inhibition is a conspicuous example of what has been termed 'default repression', a property of developmental signaling pathways whereby pathway target genes are repressed by a signal-regulated transcription factor in the absence of signaling. It is proposed that default repression has evolved in order to prevent inappropriate (signal-independent) activation of pathway target genes in cells that express local activators but do not respond to the signal. Indeed, the SOP is in particular need of default repression because it is characterized (perhaps unusually) by elevated accumulation of the local activators for the PNC, the proneural proteins. That Su(H) can keep N pathway target genes off in SOPs even in the face of exceptionally high local activator levels is testament to the efficacy of default repression as a regulatory strategy (Castro, 2005).

It is now clear that default repression by Su(H) is a crucial feature of the operation of the N pathway in all three of the developmental situations in which it is known to function: lateral inhibition (this study), binary cell fate decisions in lineages, and formation of tissue boundaries. This conclusion is based on an analysis, in all three cases, of the consequences of mutating Su(H)- binding sites in one or more N pathway-activated genes; it is emphasized that attribution of a default repression activity to a signal-regulated transcription factor can be made only after such cis-regulatory experiments have been performed. It is likely that default repression by Su(H) is an integral part of N pathway function during Drosophila development (Castro, 2005).

The studies presented here, when combined with earlier reports, illuminate a prominent feature of the transcriptional regulation of gene expression and cell fate during lateral inhibition in Drosophila. It is now clear that three key regulatory factors [the proneural proteins (Ac and Sc), Su(H) and Gro] each have dual, and oppositely directed, functions in the SOP versus the non-SOP cells of the PNC during lateral inhibition. The proneural proteins are strictly required for the SOP cell fate, at least in part because they directly activate genes that promote or execute this fate, such as sens, phyllopod and ac itself. But, proneural proteins also have a vital role in non-SOPs as direct activators of genes, including those of the E(spl)-C, that are involved in inhibiting the SOP fate. Su(H) also has crucial, but opposing, functions in the SOP [as a direct default repressor of SOP-inhibitory E(spl)-C genes] and in the non-SOPs (as an essential direct activator of these same genes in response to N signaling). Finally, evidence is presented strongly supporting the hypothesis that Gro is likewise a 'double agent' during lateral inhibition: in the non-SOPs, where it serves as the co-repressor for the E(spl)-C bHLH repressor proteins to inhibit the SOP fate (its traditional function in the process), whereas in the SOP it partners with Su(H) via H to effect default repression and thus protect the SOP fate. The regulatory machinery underlying lateral inhibition is all the more elegant for its versatility and economy (Castro, 2005).

Single-minded, Dmef2, Pointed, and Su(H) act on identified regulatory sequences of the roughest

Roughest (Rst) is a cell adhesion molecule of the immunoglobulin superfamily that has multiple and diverse functions during the development of Drosophila melanogaster. The pleiotropic action of Rst is reflected by its complex and dynamic expression during the development of Drosophila. By an enhancer detection screen, several cis-regulatory modules have been identified that mediate specific expression of the roughest gene in Drosophila developmental processes. To identify trans-regulators of rst expression, the Gal4/UAS system was used to screen for factors that were sufficient to activate Rst expression when ectopically expressed. By this method the transcription factors Single-minded, Pointed.P1, and Su(H)-VP16 were identified. Furthermore, these factors and, in addition, Dmef2 are able to ectopically activate rst expression via the previously described rst cis-regulatory modules. This fact and the use of mutant analysis allocates the action of the transcription factors to specific developmental contexts. In the case of Sim, it could be shown to regulate rst expression in the embryonic midline, but not in the optic lobes. Mutagenesis of Sim consensus binding sites in the regulatory module required for rst expression in the embryonic midline, abolishes rst expression; indicating that the regulation of rst by Sim is direct (Apitz, 2005).

Rst has complex and multifaceted functions throughout the development of the fly, which include myogenesis, eye development, as well as axonal pathfinding in the optic lobes. To gain a better understanding of these functions at the levels of gene regulation and signal transduction, a number of tests were designed to identify both the transcriptional activators and their respective targets surrounding the rst locus. In a preceding study (Apitz, 2004), a number of DNA segments upstream of rst were characterized and regulatory regions were discovered that mediate gene expression in myoblasts, midline, and eyes, respectively. In the present study these results were supplemented with an in vivo screen to identify regulators of rst expression using the Gal4/UAS system. Several factors were discovered that are able to induce ectopic Rst expression and to activate reporter gene expression via rst cis-regulatory sequences (Apitz, 2005).

The experimental route taken to identify protein factors involved in the regulation of the rst gene is based on the detection of their potential to induce ectopic Rst expression in vivo. The use of sca-Gal4 as a driver line in this experimental approach is based on the following criteria. sca-Gal4 mediates expression in neuroectodermal cells of the embryo. At embryonic stage 10, these cells can be examined for ectopic Rst expression because no endogenous Rst expression is found at this time in these cells; this allows operators to obtain clear-cut and unequivocal results. The use of alternative Gal4 driver lines did not prove suitable because of the dynamic expression of Rst during all developmental stages, and due to its subcellular localization. For example, when dll-Gal4 is used as a driver, it is difficult to distinguish between ectopic Rst expression induced in the apical tips of cells of the leg discs, and endogenous Rst expression in the overlaying ectodermal cells. Furthermore, the use of sca-Gal4 has the advantage that neuroectodermal cells are not fully differentiated cells. This may more closely resemble the developmental state of the cells in which Rst expression is normally induced endogenously, e.g., in undifferentiated cells of the developing eye disc. However, this approach generally fails to reveal transcription factors that need a coactivator for induction of rst expression, which is not present in the cells of the neuroectoderm at embryonic stage 10. This may explain the failure of Dmef2 to induce rst expression at this stage. It was shown, however, that Dmef2 is able to induce rst expression at later stages by the use of rst-lacZ constructs. The function of the different rst-lacZ constructs has been linked to specific developmental circumstances (Apitz, 2004) and their activation by corresponding factors is consistent with the known roles of these proteins in development (Apitz, 2005).

Ectopic expression of a constitutively active Pnt variant (Pnt.P1) mediates strong activation of Rst expression in neuroectodermal cells. Since Pnt.P1 recognizes the same target sequences as its splice variant Pnt.P2, the nuclear effector of the Ras-MAPK pathway, ectopic activation of Rst expression by Pnt.P1 is consistent with a regulation by the Ras-MAPK pathway. Similarly, the ectoptic activation of Rst expression in neuroectodermal cells by Su(H)-VP16 points to a regulation of rst by the Notch pathway (Apitz, 2005).

The Ras-MAPK and the Notch pathways display significant crosstalk during developmental processes in Drosophila, e.g., in cell fate specification of the eye disc. It is difficult to elucidate a possible regulation of a candidate gene by mutant analysis if it is activated by both pathways. In mutants, for one of the pathways, the activity of the other pathway will ensure residual expression of the candidate gene under scrutiny. rst expression is activated by both pathways and single mutant analysis did not reveal a significant loss of expression. Both pathways converge on regulatory elements contained within F6 and not in F5. Furthermore, a regulatory module is present in the nonoverlapping part of F6 that is activated in IOC before apoptotic decisions are made in these cells (Apitz, 2004). This module is located within an approximately 600-bp sequence and is active during several apoptotic decisions (Apitz, 2004). Consensus binding sites for Su(H) and Pnt in this module are conserved between D. melanogaster and D. pseudoobscura. Both the Ras-MAPK and Notch pathways are involved in apoptotic processes of IOC cells. Together, these data suggest that rst transcription is regulated by these pathways in the context of apoptotic decisions (Apitz, 2005).

An NRSF/REST-like repressor downstream of Ebi/SMRTER/Su(H) regulates eye development in Drosophila; charlatan is repressed by Ebi/SMRTER and promotes Dl expression

The corepressor complex that includes Ebi and SMRTER is a target of epidermal growth factor (EGF) and Notch signaling pathways and regulates Delta (Dl)-mediated induction of support cells adjacent to photoreceptor neurons of the Drosophila eye. A mechanism is described by which the Ebi/SMRTER corepressor complex maintains Dl expression. charlatan (chn) is repressed by Ebi/SMRTER corepressor complex by competing with the activation complex that includes the Notch intracellular domain (NICD). Chn represses Dl expression and is critical for the initiation of eye development. Thus, under EGF signaling, double negative regulation mediated by the Ebi/SMRTER corepressor complex and an NRSF/REST-like factor, Chn, maintains inductive activity in developing photoreceptor cells by promoting Dl expression (Tsuda, 2006).

The corepressor complex that includes Ebi, SMRTER and Su(H) is required for expression of Dl in Drosophila photoreceptor cells. To identify genetic loci that are transcriptionally repressed by the Ebi corepressor, a screen was set up using an ectopic gene expression system (Gene Search System). Insertion of a Gene Search (GS) vector, a modified P-element carrying the Gal4 upstream activating sequence (UASG) near its 3' end, causes overexpression of a nearby gene under the control of the Gal4-UASG system. GS insertions into the chn locus were identified, whose overexpression phenotype in the eye using an eye-specific Gal4 driver (GMR-Gal4) was modified by reducing ebi activity. Thus the regulation of chn by Ebi-dependent transcriptional repression was studied (Tsuda, 2006).

In third instar larval-stage eye discs, the chn transcript is highly expressed in the morphogenetic furrow (MF), where photoreceptor differentiation initiates, but is downregulated in cells in the later stage photoreceptor development. In ebi mutant eye discs, however, chn expression becomes detectable in differentiating photoreceptor cells, and its expression in the MF is increased, suggesting that Su(H) in association with Ebi and SMRTER represses chn transcription in the eye disc (Tsuda, 2006).

To reveal the role of Su(H) as an activator, chn expression was examined when the level of Su(H) expression was reduced. Removing one copy of Su(H) suppresses the loss-of-Dl expression phenotype in ebi mutants. It was found that reducing one copy of Su(H) suppresses ectopic chn expression in ebi mutants, suggesting that ectopic expression of chn in ebi mutants is Su(H)-dependent. RT-PCR analysis of chn expression in ebi- eye discs differing in the dosage of Su(H) gene also supported these results. Strong reduction of Su(H) expression alone reduced expression of chn in the MF; this expression became weaker and was slightly broader. The phenotype of ebi, Su(H) double mutants is almost the same as Su(H) single mutants , suggesting that Su(H) acts as an activator in the absence of Ebi. This might be due to dual functions of Su(H) as an activator or repressor. Hence, reducing the amount of Ebi in the corepressor complex involving Su(H) might convert Su(H) to an activator by permitting the replacement of the corepressor complex with NICD (Tsuda, 2006).

To reveal the molecular nature of transcriptional regulation of chn by Su(H), Su(H) target sites were sought in the genomic region of chn. Since Su(H) binds slightly degenerate sequences, it was not easy to identify the functional Su(H) binding region from a simple genomic search. An alternative approach was taken to map the chn genomic region, which is regulated by Su(H) in the normal chromosomal context. Ebi-mediated repression involves SMRTER, a corepressor that recruits histone deacetylases and induces the formation of inactive chromatin, which spreads from the site where Su(H) recruits the corepressor complex. Promoters near the Su(H)-binding site are thus expected to be downregulated in an Ebi-dependent manner. Four insertion lines of the GS vector were identified in the chn promoter region. All these GS lines caused ectopic expression of chn with consequent abnormal eye morphology when they were crossed with GMR-Gal4. If the effect of the Ebi/SMRTER corepressor complex reaches the UASG in those insertions, reduction of Ebi activity will derepress UASG and further enhance activation by GMR-Gal4. One copy of a dominant-negative construct of ebi (GMR-ebiDN) caused only a mild defect in eye morphology and weak, if any, ectopic expression of chn. GMR-ebiDN strongly enhanced the overexpression phenotype of chnGS17605 and chnGS11450, which contained GS vector insertions (-474 and -734, respectively) upstream of the transcriptional start site. However, GMR-ebiDN failed to enhance the overexpression phenotype of other GS lines (chnGS2112 and chnGS17892) that were inserted downstream (+773 and +1040, respectively) of the first exon. From these results, it is concluded that Ebi-dependent transcriptional repression is targeted to the proximity of the transcriptional initiation site of the chn promoter (Tsuda, 2006).

Chn is a 1108-amino-acid protein with multiple C2H2-type zinc-finger motifs. Although no highly homologous gene within the mammalian genome could not be detected using BLAST, a small sequence of similarity between the N-terminal zinc-finger motif of Chn and the fifth zinc-finger of human NRSF/REST was found. Chn has several structural and functional similarities to human NRSF/REST, as follows. First, Chn and NRSF/REST each contain an N-terminal region with multiple zinc-finger motifs (five motifs in 264 residues in Chn and eight motifs in 251 residues in NRSF/REST), followed by a cluster of S/T-P motifs (serine or threonine followed by a proline) and a single zinc-finger motif at the C terminus. Second, the C-terminal region of NRSF/REST binds a corepressor, CoREST, which serves as an adaptor molecule to recruit a complex that imposes silencing activities. The Drosophila homolog of CoREST (dCoREST) (Andres, 1999; Dallman, 2004) can associate with the C-terminal half of Chn in cultured S2 cells. Finally, NRSF/REST binds to NRSE/RE1, a 21-bp sequence located in the promoter region of many types of neuron-restricted genes, via the N-terminal zinc-finger motifs. It was found that a recombinant protein containing the N-terminal zinc-fingers of Chn bound specifically to the NRSE/RE1 sequence in vitro. Thus, the structural similarity to NRSF/REST, binding to dCoREST and the DNA-binding specificity of Chn suggest that it is a candidate for a functional Drosophila homolog of NRSF/REST (Tsuda, 2006).

If Chn acts as a regulator of neural-related functions, as suggested for NRSF/REST, then Chn would be expected to bind to a regulatory region common to many types of neural-related genes in Drosophila. Numerous sequences similar to NRSE/RE1 were identified in the Drosophila genome, and their binding to Chn was assessed by EMSA. Using these sequences, a consensus binding sequence for Chn (Chn-binding element (CBE), 5'-BBHASMVMMVCNGACVKNNCC-3') was derived. 26 CBEs were identified within 10 kb of annotated genes from the Drosophila genome. Binding to Chn was confirmed for 18 CBEs using EMSA competition assay. Genes containing the CBE include dopamine receptor 2 (DopR2) and the potassium channel, ether-a-go-go, for which the mammalian homologs are target genes of NRSF/REST. These results suggest that the CBE is a good indicator of Chn binding sites and that Chn regulates many types of neural-related genes, as is implicated for NRSF/REST. However, it was found that divergent forms of CBE adjacent to hairy and extramacrochaetae were bound specifically by Chn. Likewise, some of the CBE sites failed to bind to Chn. Thus, a further refinement will be necessary to predict a definitive set of Chn binding sites in the Drosophila genome (Tsuda, 2006).

Although it has been established that mammalian NRSF/REST is a key regulator of neuron-specific genes, attempts to isolate invertebrate homolog of NRSF/REST have so far failed to identify a true homologous factor in invertebrates. The properties of Chn, including the similarity in DNA-binding specificity, association with CoREST and transcriptional repressor activity, suggest that Chn is a strong candidate for a functional Drosophila homolog of NRSF/REST. chn was originally identified by its requirement in the development of the PNS. This study identified a number of candidate target genes of Chn, a large fraction of which is implicated in neural function and/or gene expression. It is expected that further analysis of these candidates will provide valuable information about chn function in vivo, which may be extended to the understanding of NRSF/REST (Tsuda, 2006).

The Chn mutation blocks eye development by preventing the initiation of MF, a process requiring Notch signaling. This phenotype is likely owing to a loss of Notch function, because elevated Dl expression is known to block Notch signaling. The function of Chn during the early stage of eye development might be to regulate Notch signaling at an appropriate level by downregulating Dl. It is possible that Chn-mediated modulation places a variety of Notch functions in eye under the influence of EGFR signaling and provides flexibility in its regulation (Tsuda, 2006).

Although chn is expressed in the MF, genetic analyses show that small clones of chn mutant cells permit progression of the MF and photoreceptor differentiation. It is speculated that the repressive effect of Chn is overcome by other signals in the MF, such as hedgehog signaling, which strongly induces Dl (Tsuda, 2006).

Developing photoreceptor cells are exposed to the EGFR ligand, Spitz, and the Notch ligand, Dl, and each cell must assess the level of the two signals and respond appropriately to perform each task of photoreceptor cell specification and induction of non-neural cone cells. This question was investigated by studying the expression of Dl in photoreceptor cells. chn was identified as a direct target of Ebi/SMRTER-dependent transcriptional repression and as a repressor of Dl expression. The abrogated expression of Dl in ebi mutants was recovered by reducing one copy of chn, suggesting that the negative regulation of chn by ebi is indeed prerequisite for photoreceptor cell development (Tsuda, 2006).

Genetic data suggest that Su(H) may activate or repress chn expression. This idea is supported by data showing that Ebi/SMRTER and NICD are recruited to the promoter region of chn. The Ebi/SMRTER complex formed in this region did not contain any detectable level of the intracellular domain of Notch (NICD), suggesting that the binding of Ebi/SMRTER and NICD to this region may be mutually exclusive, and therefore it is expected that a regulatory system controls the balance between the active and repressive states of Su(H). Taken together, these results suggest that chn is a key factor in the crosstalk between two major signal transduction pathways: the EGFR-dependent pathway and the Notch/Delta-dependent pathway (Tsuda, 2006).

In the mammalian system, competition between SMRT and NICD for interaction with RBPJkappa determines the state of RBPJkappa-dependent transcriptional activity. Extracellular signaling may modulate this competition; diverse signaling pathways modulate the functions of N-CoR/SMRT. The current findings would prompt investigations of potential interaction of two repression systems of NRSF/REST and N-CoR/SMRT, and their regulation by Notch and EGF signaling in mammalian neuronal differentiation (Tsuda, 2006).

Evolution of the ventral midline in insect embryos; Regulatioon of Sim by the Notch pathway

The ventral midline is a source of signals that pattern the nerve cord of insect embryos. In dipterans such as the fruitfly Drosophila melanogaster (D.mel.) and the mosquito Anopheles gambiae (A.gam.), the midline is narrow and spans just 1–2 cells. However, in the honeybee, Apis mellifera (A.mel.), the ventral midline is broad and encompasses 5–6 cells. slit and other midline-patterning genes display a corresponding expansion in expression. Evidence is presented that this difference is due to divergent cis regulation of the single-minded (sim) gene, which encodes a bHLH-PAS transcription factor essential for midline differentiation. sim is regulated by a combination of Notch signaling and a Twist (Twi) activator gradient in D.mel., but it is activated solely by Twi in A.mel. It is suggested that the Twi-only mode of regulation—and the broad ventral midline—represents the ancestral form of CNS patterning in Holometabolous insects (Zinzen, 2006).

Dorsoventral (DV) patterning of the D.mel. embryo is initiated by a nuclear gradient of the Dorsal (Dl) transcription factor, which differentially regulates at least 50 target genes in a concentration-dependent manner. Most of these genes encode sequence-specific transcription factors and components of cell signaling pathways that control gastrulation. Genetic analyses, microarray screens, and DNA-binding assays with defined DV enhancers have elucidated a gene network of functional interconnections among 40 Dl target genes. The goal of this study is to use this information to understand the evolution of DV patterning among divergent insects (Zinzen, 2006).

In D.mel., the Dl gradient leads to localized activation of Notch signaling in single rows of cells straddling the presumptive mesoderm (Bardin, 2006; De Renzis, 2006). This localized Notch signal works together with the bHLH factor Twi to activate sim expression. After invagination of the ventral furrow, the sim-expressing cells converge at the ventral midline, and the bHLH-PAS Sim transcription factor activates target genes required for midline differentiation (Zinzen, 2006).

The ventral midline is a source of localized signals that help pattern the nerve cord. For example, a transmembrane protease encoded by rhomboid (rho) produces a secreted source of the EGF ligand Spitz. Sim also leads to the expression of slit, which encodes a secreted repellant that binds the Roundabout receptor and inhibits the growth of axonal projections across the midline (Zinzen, 2006).

Sim target genes are highly conserved in A.mel., and in situ hybridization assays reveal that they are similarly expressed in the ventral midline of the developing honeybee nerve cord. However, their expression is significantly broader in A.mel. than in D.mel., 5–6 cells versus 1–2 cells, respectively. Evidence is presented that this broader midline is due to divergent regulation of sim expression. In A.mel., sim is regulated solely by Twi and does not depend on Notch signaling, whereas Notch is responsible for restricting sim to single rows of cells in the early D.mel. embryo (e.g., Bardin, 2006; De Renzis, 2006). It is proposed that the acquisition of Notch dependence at the sim locus is sufficient to account for restricted expression of sim and the narrow midline in D.mel (Zinzen, 2006).

The ventral midline in D.mel. embryos encompasses just 1–2 cells that express signaling molecules such as rho and slit. In contrast, orthologous genes are expressed in 5–6 cells in the honeybee embryo. Notably, the initial expression pattern of A.mel. sim is expanded, and the sim-staining pattern remains broad after convergence of the midline following the spreading of neurogenic ectoderm over the mesoderm. In addition to expression in the ventral midline, sim staining is also detected in more lateral clusters of cells exhibiting segmental periodicity in A.mel. embryos; these might be neurons or glial cells migrating away from the midline (Zinzen, 2006).

Previous studies suggest that sim functions as a 'master control gene' to direct differentiation of the ventral midline in D.mel. To determine whether the expanded sim pattern in honeybees can account for the broadening of the midline, whether ectopic sim expression is sufficient to induce transcription of target genes such as slit and rho. The D.mel. sim-coding sequence was placed under the control of the eve stripe 2 enhancer (eve.2) and expressed in transgenic embryos. There is transient sim expression in the stripe 2 domain of early (stages 5–7) embryos in addition to the endogenous pattern (mesectoderm) in the presumptive ventral midline (Zinzen, 2006).

The initial sim expression pattern is established by a distal 5′ enhancer that contains linked Dl-, Twi-, and Suppressor of Hairless [Su(H)]-binding sites. Expression is maintained by a separate autoregulatory enhancer containing Sim/Tango-binding sites; Tango is a ubiquitous bHLH-PAS transcription factor that forms heterodimers with Sim. Though the eve stripe 2 enhancer mediates transient activation, autoregulation maintains expression of the endogenous sim gene in the ventral neurogenic ectoderm of advanced-stage embryos, but not in the mesoderm or dorsal ectoderm (Zinzen, 2006).

Ectopic sim expression leads to the induction of various target genes, including rho, slit, sog, and the transcription factor otd. These results provide evidence that ectopic sim expression is sufficient to expand the ventral midline in D.mel. In principle, the altered midline seen in the honeybee embryo could be explained by a change in sim regulation. The distal 5′ enhancer that establishes sim expression is the most likely site of change, since the autoregulatory enhancer merely maintains expression within the limits of the established pattern (Zinzen, 2006).

To determine the basis for the distinct sim expression patterns in flies and honeybees, it was necessary to isolate the early sim enhancer from A.mel. However, the identification of homologous enhancers is complicated by the rapid turnover of noncoding DNA sequences in insect genomes. For example, the 5′ flanking regions of the sim loci in D.mel. and A.gam. lack simple sequence homology, even though they belong to the same order (Diptera). Nonetheless, it was possible to identify the early sim enhancer in A.gam. based on the clustering of Dl-, Twi-, and Su(H)-binding sites. The D.mel. and A.gam. enhancers are located in similar positions relative to the sim transcription unit (Zinzen, 2006).

A.mel. is a member of the order Hymenoptera and is highly divergent from D.mel. Computational methods used for the in silico identification of the A.gam. sim enhancer were further developed to ensure the accurate identification of the sim enhancer in A.mel. The current method (ClusterDraw2) employs position-weighted matrices (PWMs) to identify binding motif clusters (Zinzen, 2006).

The efficacy of the method was tested by surveying ~50 kb genomic intervals encompassing the sim loci of D.mel. and A.gam.. PWMs of Dl, Twi, Snail, and Su(H) were used in various combinations and individually. The best binding site clusters coincide exactly with the known sim enhancers (Zinzen, 2006).

ClusterDraw2 was used to survey a ~50 kb genomic DNA interval encompassing the sim locus of A.mel.. The best prediction occurs in the 5′ flanking region of the gene, similar to the locations of the fly and mosquito enhancers. However, while the D.mel. and A.gam. sim enhancers contain several optimal Su(H)-binding sites, the A.mel. cluster lacks such sites, but contains several high-scoring Twi sites. This is consistent with the possibility that A.mel. sim is regulated by Twi alone, rather than by the combination of Twi+Notch (Zinzen, 2006).

A 2.2 kb genomic DNA fragment encompassing the predicted A.mel. sim enhancer directs lateral stripes of lacZ expression in transgenic D.mel. embryos. A similar pattern was obtained with a 471 bp fragment containing the predicted Twi-binding sites. This pattern encompasses 3–4 cells on either side of the presumptive mesoderm, similar to the expression of the endogenous A.mel. sim gene, but distinct from the single-row sim patterns in D.mel. and A.gam. (Zinzen, 2006).

The fly, honeybee, and mosquito sim enhancers were crossed into various genetic backgrounds to determine the basis for their distinct expression patterns. The D.mel. m5/8 enhancer was also examined. It is located within the Enhancer of split (E(spl)) complex, where it controls the expression of the m5 and m8 genes within the mesectoderm. The m5/8 enhancer directs lacZ expression in a pattern that is virtually identical to that produced by the D.mel. sim enhance (Zinzen, 2006).

Transgenic D.mel. embryos carrying an eve.2::NICD fusion gene exhibit ectopic Notch signaling in the eve stripe 2 domain. The m5/8-lacZ transgene is strongly induced in the neurogenic ectoderm and dorsal ectoderm, but not in the mesoderm, where the Sna repressor is present. The D.mel. sim-lacZ transgene displays only modest ectopic induction by the eve.2::NICD transgene; this induction appears as a 'pyramid' limited to ventral regions of the neurogenic ectoderm. This pyramid coincides with the intersection of ectopic Notch signaling and the endogenous Twi gradient. The different patterns ('pyramid' versus 'column') seen for the sim and m5/8 enhancers appear to reflect activation by Notch+Twi or regulation by Notch alone, respectively. The m5/8 enhancer contains an SPS (Su(H) Paired Site) motif, and it has been suggested that the endogenous m8 gene is activated solely by Notch signaling (Zinzen, 2006).

The A.mel. sim enhancer is not activated by the eve.2::NICD transgene, consistent with the absence of Su(H) sites in this enhancer. To determine whether it is activated by Twi, the lacZ fusion gene was crossed into embryos carrying an hsp83::twi-bcd-3′UTR transgene that produces high levels of Twi transcripts at the anterior pole. The resulting ectopic anteroposterior Twi protein gradient induces intense expression of the lacZ reporter gene directed by the A.mel. sim enhancer. In contrast, neither the D.mel. sim enhancer nor the m5/8 enhancer is induced by this ectopic gradient. Finally, the D.mel. sim enhancer is inactive in mutant embryos derived from germline clones lacking Su(H) activity, whereas the honeybee sim enhancer is fully active. Thus, unlike the D.mel. sim enhancer, the A.mel. enhancer does not rely on Notch signaling (Zinzen, 2006).

The preceding analysis suggests that the D.mel. sim enhancer is activated by Twi and Notch signaling, whereas the A.mel. sim enhancer is activated solely by Twi. These distinct modes of regulation are reflected by the composition of binding sites in the different enhancers. The A.mel. enhancer contains several optimal Twi sites, but it lacks unambiguous Su(H) sites. In contrast, the D.mel. enhancer contains several optimal Su(H) sites, but just one optimal Twi site. Both enhancers contain binding sites for the Sna repressor, which inhibits expression in the mesoderm (Zinzen, 2006).

sim regulation was examined in the mosquito, A.gam., to determine whether the midline of ancestral dipterans might have been regulated solely by Notch signaling, as seen for the fly m5/8 enhancer. The A.gam. genome contains a clear ortholog of the sim gene, expressed in a single row of cells in the mesectoderm, similar to the pattern seen in D.mel. The A.gam. sim enhancer directs sporadic expression within the mesectoderm of transgenic D.mel. embryos, but it is strongly induced by the eve.2::NICD transgene. This response is similar to that obtained with the D.mel. m5/8 enhancer, but it is distinct from the 'pyramid' pattern seen for the D.mel. sim enhancer (Zinzen, 2006).

To determine whether the sim loci of other drosophilids are regulated by Twi+Notch, as seen in D.mel., or Notch alone, sim enhancers from D. pseudoobscura (D.pse.) and D. virilis (D.vir.) were tested in transgenic eve.2::NICD D.mel. embryos. Surprisingly, these enhancers behave like the A.gam. sim enhancer: they are expressed throughout the neurogenic ectoderm and dorsal ectoderm (“column”) in response to Notch signaling, rather than the “pyramid” pattern indicative of Notch+Twi regulation. These observations suggest that the evolution of sim regulation is highly dynamic, although there is no obvious difference in the number or quality of Su(H) and Twi sites in the different drosophilid enhancers. Perhaps a subtle shift in the organization of binding sites distinguishes regulation by Notch alone versus Notch+Twi (Zinzen, 2006).

Notch activation stimulates transient and selective binding of Su(H)/CSL to target enhancers

The CSL [CBF1/Su(H)/Lag2] proteins [Su(H) in Drosophila] are implicated in repression and activation of Notch target loci. Prevailing models imply a static association of these DNA-binding transcription factors with their target enhancers. Analysis of Su(H) binding and chromatin-associated features at 11 E(spl) Notch target genes before and after Notch revealed large differences in Su(H) occupancy at target loci that correlated with the presence of polymerase II and other marks of transcriptional activity. Unexpectedly, Su(H) occupancy was significantly and transiently increased following Notch activation, suggesting a more dynamic interaction with targets than hitherto proposed (Krejcí, 2007).

To investigate changes in chromatin that accompany Notch activation, it was necessary to establish conditions where receptor activation could be temporally controlled. It has been reported that exposing cells to EDTA stimulates shedding of the Notch ectodomain. This renders the residual transmembrane fragment a substrate for γ-secretase cleavage and, hence, results in Notch activation. Despite results suggesting that cell surface Notch in Drosophila would not be susceptible, it was found that EDTA causes robust activation of E(spl) Notch-target genes in a Notch-expressing Drosophila S2 cell line (S2-N). No effect was seen when S2 cells that do not express Notch were treated with EDTA (Krejcí, 2007).

All 11 E(spl) genes were induced following EDTA treatment. Most were expressed at very low levels before activation and, although stimulated following EDTA treatment, their absolute levels of expression remained low. One gene, m3, was expressed at intermediate levels prior to activation and was induced 50 times by EDTA treatment to expression levels that were ~100 times higher than other E(spl) genes. There was no change in expression of the housekeeping genes or nontarget loci analyzed. A qualitatively similar effect on E(spl) gene expression was obtained in S2 cells transfected with a plasmid expressing Nicd (Krejcí, 2007).

To ascertain whether EDTA activates E(spl) genes through its effects on Notch, cells were treated in the presence of γ-secretase inhibitors (e.g., DFK-167). This compromised the induction of m3 and m7 expression by EDTA. In addition, by immunoprecipitating Su(H) from cell extracts and probing for coprecipitation of Nicd, it was confirmed that there is a robust association of Nicd with Su(H) in EDTA-treated, but not in control cells (Krejcí, 2007).

In summary, EDTA treatment provides a method to rapidly activate Notch in a temporally controlled manner throughout a cell population (and is subsequently referred to here as Notch activation). By activating Notch in this a larger, more concerted burst of Notch activity may taking planc than occurs during normal signaling in the animal. Nevertheless, this makes it possible to analyze the chromatin state before and after Notch activation under carefully timed conditions (Krejcí, 2007).

The results demonstrate that there is a significant increase in Su(H) occupancy at target genes following EDTA/Notch activation. This increase is transient and correlates with the presence of Nicd, implying that the kinetics of binding differ when Su(H) is complexed with Nicd, and that the association between Su(H) and its cognate sites is much more dynamic than expected. It was also noted that there are differences in Su(H) occupancy between genes prior to activation, suggesting the possibility of gene-specific modes of regulation [i.e., at some there may be constitutive recruitment of Su(H), whereas at others Su(H) binding is only signaling induced] (Krejcí, 2007).

Several mechanisms could account for the increased Su(H) occupancy after Notch activation. One possibility is that the activation complex has a higher affinity for DNA. Structural analysis of the CSL/Nicd/Mam tertiary complex did not reveal any novel interactions between CSL and DNA that could account for an increase in affinity per se. However, the affinity could be increased by cooperative interactions between two activation complexes on the DNA. Recent analysis demonstrates that Nicd-containing complexes can bind cooperatively to DNA with appropriately arranged paired sites. This may therefore be a significant factor in the enhanced Su(H) occupancy that is detected after activation. However, not all the enhancer fragments analyzed contain paired Su(H) sites, suggesting that additional mechanisms are involved (Krejcí, 2007).

A second explanation is that interactions with other cofactors help to stabilize the Su(H) activation complex on the DNA. Studies of nuclear receptors suggest that in the resting state they rapidly exchange on and off the DNA, and that the formation of transcriptionally competent complexes slows this exchange. It is envisaged that the interactions of Su(H)/CSL with its cognate sites have similar change in dynamics; a fast exchange and low residency occurring when Su(H)/CSL is complexed with corepressors, a slow exchange and longer residency occurring when it is complexed with Nicd and competent to recruit productive transcription complexes and/or to make cooperative interactions (Krejcí, 2007).

Direct response to Notch activation: signaling crosstalk and incoherent logic

Notch is the receptor in one of a small group of conserved signaling pathways that are essential at multiple stages in development. Although the mechanism of transduction impinges directly on the nucleus to regulate transcription through the CSL [CBF-1/Su(H)/LAG-1] DNA binding protein, there are few known direct target genes. Thus, relatively little is known about the immediate cellular consequences of Notch activation. This study set out to determine the genome-wide response to Notch activation by analyzing the changes in messenger RNA (mRNA) expression and the sites of CSL occupancy within 30 minutes of activating Notch in Drosophila cells. Through combining these data, high-confidence direct targets of Notch were identified that are implicated in the maintenance of adult muscle progenitors in vivo. These targets are enriched in cell morphogenesis genes and in components of other cell signaling pathways, especially the epidermal growth factor receptor (EGFR) pathway. Also evident are examples of incoherent network logic, where Notch stimulates the expression of both a gene and the repressor of that gene, which may result in a transient window of competence after Notch activation. Furthermore, because targets comprise both positive and negative regulators, cells become poised for both outcomes, suggesting one mechanism through which Notch activation can lead to opposite effects in different contexts (Krejci, 2009).

Specificity of Notch pathway activation: twist controls the transcriptional output in adult muscle progenitors

Cell-cell signalling mediated by Notch regulates many different developmental and physiological processes and is involved in a variety of human diseases. Activation of Notch impinges directly on gene expression through the Suppressor of Hairless [Su(H)] DNA-binding protein. A major question that remains to be elucidated is how the same Notch signalling pathway can result in different transcriptional responses depending on the cellular context and environment. This study investigated the factors required to confer this specific response in Drosophila adult myogenic progenitor-related cells. This analysis identifies Twist (Twi) as a crucial co-operating factor. Enhancers from several direct Notch targets require a combination of Twi and Notch activities for expression in vivo; neither alone is sufficient. Twi is bound at target enhancers prior to Notch activation and enhances Su(H) binding to these regulatory regions. To determine the breadth of the combinatorial regulation Twi occupancy was mapped on a genome-wide level in DmD8 myogenic progenitor-related cells by chromatin immunoprecipitation. Comparing the sites bound by Su(H) and by Twi in these cells revealed a strong association, identifying a large spectrum of co-regulated genes. It is concluded that Twi is an essential Notch co-regulator in myogenic progenitor cells and has the potential to confer specificity on Notch signalling at over 170 genes, showing that a single factor can have a profound effect on the output of the pathway (Bernard, 2010).

To determine whether the Twi co-regulation could be extrapolated to a broad spectrum of Notch targets in muscle progenitors, whether there was a significant association between Su(H) and Twi binding in the muscle progenitor-related DmD8 cells was assessed. Comparison of the binding regions genome-wide revealed a strong association of Twi and Su(H) among these targets: 71% of Su(H) peaks directly overlapped with Twi peaks. This association was highly significant based on random models that constrained the positions of peaks across the genome to take account of the non-random distribution of transcription factor binding sites and in comparison to several other ChIP data sets. Expression of putative Notch-Twi targets in myogenic precursors was dependent on Twi, as predicted by the association. Together, these data indicate that one transcription factor, Twi, has the potential to co-ordinate the expression of a broad cross-section of Notch targets in muscle progenitors (84% of previously assigned Notch targets are associated with a Twi peak) and thus to confer a specific context on the Notch response (Bernard, 2010).

Further evidence in support of the instructive role of Twi comes from its ability to confer Notch responsiveness on some muscle precursor targets when expressed in a heterologous cell line. Twi itself was found to occupy sites on the target enhancers prior to Notch activation, and in the heterologous cells it was accompanied by increased Su(H) binding after Notch activation. This suggests that Twi binding precedes Su(H) recruitment. However, the co-regulation does not appear to require the Twi partner Da [the E47 (TCF3) homologue], which was previously reported to contact Notch. Furthermore, there does not appear to be any specific organisation or spacing of Twi and Su(H) motifs among the co-regulated targets, in contrast to the conserved motif, consisting of paired Su(H) sites closely linked to an A-class bHLH binding site, that is thought to underlie Notch-proneural bHLH synergy. Nevertheless, in most of the co-regulated targets, the Su(H) and Twi mid-peaks are separated by less than 500 bp, suggesting that the interaction operates over a limited range. The Twi-Su(H) co-regulation appears, therefore, more in keeping with models in which binding sites for transcription factors are flexibly disposed and act independently with targets in the basal transcriptional machinery (the so-called 'billboard' model). However, mutation of a single Twi binding motif in the aos enhancer is sufficient to compromise activity, despite the fact that there are several matches to the Twi consensus site, suggesting that only a subset of the possible binding motifs are crucial. In addition, as the characterised Notch-Twi-dependent enhancers do not have identical patterns of expression, it is likely that their activity is further constrained by others factors. This is most evident for E(spl)m6, which is only expressed in a small patch of the AMPs but nevertheless responds very robustly to Twi and NICD. What are the likely characteristics conferred on cells by the Twi-Notch combination (Bernard, 2010)?

The AMPs have the capacity for self-renewal and are not committed to a particular muscle lineage, characteristics similar to those of mammalian muscle satellite cells. The genes regulated by the combination of Twi and Notch might therefore be important for maintaining these cells as progenitors with myogenic potential. Normally, Twi expression declines as the muscles differentiate. Interfering with this regulation by persistent expression of Twi or Notch inhibits the development of mature fibres. Conversely, ablating Notch results in premature differentiation. The genes regulated by the combination of Twi and Notch include those with proven roles in myogenic regulation that are relevant to the maintenance of muscle progenitors. These include twi itself, Him [an inhibitor of Mef2 (Liotta, 2007)] and zfh1 [a repressor of myogenesis. As Notch signalling and Twi homologues also inhibit vertebrate myogenic differentiation, and overexpression of Twi in terminally differentiated myotubes can induce reversal of cell differentiation, it will be interesting to test whether homologues of the identified co-regulated targets of Notch and Twi are similarly regulated (Bernard, 2010).

The Notch-Twi combination might also be required to confer properties on the adult myogenic precursors, such as differential adhesion, migration and proliferation. Besides the genes with proven roles in myogenic regulation, many of the Notch-Twi co-regulated genes are implicated in morphogenesis. These include the Ig-domain proteins Roughest, Kirre and Dscam, the netrin receptor Unc-5 and leucine repeat protein Capricious. Notch and Twi have both been found to contribute to the regulation of the epithelial-mesenchymal transition (EMT), and Twi is proposed to affect malignant progression by inducing EMT and suppressing the senescence response. Therefore, it is possible that the co-regulated genes might also confer specialised behaviours that are required in the adult precursors and in cells undergoing EMT (Bernard, 2010).

In conclusion, this study found that Notch and Twi potentially co-regulate a broad spectrum of genes required for the maintenance of muscle progenitors. This suggests that a single co-regulatory relationship can account for a significant component (>170 genes) of the Notch output in one cell type (Bernard, 2010).

Transcriptional control of stem cell maintenance in the Drosophila intestine

Adult stem cells maintain tissue homeostasis by controlling the proper balance of stem cell self-renewal and differentiation. The adult midgut of Drosophila contains multipotent intestinal stem cells (ISCs) that self-renew and produce differentiated progeny. Control of ISC identity and maintenance is poorly understood. This study found that transcriptional repression of Notch target genes by a Hairless-Suppressor of Hairless complex is required for ISC maintenance, and genes of the Enhancer of split complex [E(spl)-C] were identified as the major targets of this repression. In addition, it was found that the bHLH transcription factor Daughterless is essential to maintain ISC identity and that bHLH binding sites promote ISC-specific enhancer activity. It is proposed that Daughterless-dependent bHLH activity is important for the ISC fate and that E(spl)-C factors inhibit this activity to promote differentiation (Bardin, 2010).

Adult stem cells self-renew and, at the same time, give rise to progeny that eventually differentiate. This work provides evidence that one of the strategies used to maintain the identity of ISCs in Drosophila is to repress the expression of Notch target genes. Consistent with this finding, the loss of a general regulator of transcriptional repression, the Histone H2B ubiquitin protease Scrawny, gives a similar phenotype to Hairless (Buszczak, 2009). Additionally, several recent studies indicate that transcriptional repression of differentiation genes may be a central hallmark of stem cells in general (Bardin, 2010).

Two models have been proposed for Hairless activity. One proposes that Hairless competes with NICD for interaction with Su(H), thereby preventing transcriptional activation of Notch target genes by low-level Notch receptor activation. A second, non-exclusive, model proposes that Hairless antagonizes the transcriptional activation of Notch target genes by tissue-specific transcription factors other than Notch. Since the loss of Su(H) can suppress the phenotype of Hairless on ISC clone growth, it is proposed that Hairless promotes ISC maintenance by repressing the transcription of genes that would otherwise be activated by Notch signaling in ISCs. Thus, Hairless appears to set a threshold level to buffer Notch signaling in ISCs. In the absence of this repression, the expression of E(spl)-C genes and other Notch targets would lead to loss of the ISC fate (see Model for ISC maintenance). Importantly, these findings suggest a mechanism for how the transcriptionally repressed state is turned off and activation of the differentiation program is initiated: high activation of Notch in enteroblasts (EBs) displaces Hairless from Su(H) and leads to expression of the E(spl)-C genes (Bardin, 2010).

It is proposed that Hairless prevents ISC loss by repressing expression of Notch target genes, including the E(spl)-C genes. It is further proposed that Da-dependent bHLH activity promotes ISC identity, including the ability to self-renew and to express Delta. Delta, in turn, activates Notch in the adjacent EB, releasing the intracellular domain of Notch (NICD). It is speculated that, in response to Notch activation, the E(spl)-bHLH repressors downregulate Da-dependent bHLH activity in EBs as described in other systems, thereby shutting off ISC identity and promoting differentiation (Bardin, 2010).

E(spl)-C bHLH repressors act in part through their ability to inhibit bHLH activators. The data demonstrate that Da is also essential to maintain ISC fate and that E-box Da-binding sites are required to promote ISC-specific enhancer activity. Thus, it is proposed that activation of E(spl)-C genes by Notch in EBs downregulates Da bHLH activity and thereby contributes to turning off ISC identity in the differentiating cell (see Model for ISC maintenance). The specificity of ISC-specific E-box expression might be due to the ISC-specific expression of a bHLH family member. Although an array analysis raised the possibility that Scute may be specifically expressed in ISCs, genetic analysis indicates that scute function is not essential for ISC maintenance. Alternatively, specificity of gene expression might result from inhibition of bHLH activity in the EB and differentiating daughters, possibly by E(spl)-bHLH factors, rather than by the ISC-specific expression of a Da partner. It is also possible that a non-bHLH, ISC-specific factor restricts the Da-dependent bHLH activity to ISCs in a manner similar to the synergism observed in wing margin sensory organ precursors (SOPs) between the Zn-finger transcription factor Senseless and Da (Bardin, 2010).

Recently, a role for the Da homologs E2A (Tcf3) and HEB (Tcf12) has been found in mammalian ISCs marked by the expression of Lgr5 and, in this context, E2A and HEB are thought to heterodimerize with achaete-scute like 2 (Ascl2), which is essential for the maintenance and/or identity of Lgr5+ ISCs (van der Flier, 2009). In Drosophila, however, AS-C genes are not essential for ISC maintenance, but appear to play a role in enteroendocrine fate specification. The observation that Da bHLH activity is required for the identity of both Drosophila ISCs and mammalian Lgr5+ ISCs suggests that there might be conservation at the level of the gene expression program. Additionally, the bHLH genes Atoh1 (Math1) and Neurog3 are both important for differentiation of secretory cells in the mammalian intestine. Clearly, further analysis of the control of Da/E2A bHLH activity, as well as of the gene networks downstream of Da/E2A, will be of great interest (Bardin, 2010).

The data suggest that ISC fate is promoted both by inhibition of Notch target genes through Hairless/Su(H) repression and by activation of ISC-specific genes through bHLH activity. How then is asymmetry in Notch activity eventually established between the two ISC daughters to allow one cell to remain an ISC and one cell to differentiate? Three types of mechanisms can be envisioned that would allow for asymmetry of Notch signaling (Bardin, 2010).

First, the binary decision between the ISC and EB fates might result from a competition process akin to lateral inhibition for the selection of SOPs. In this process, feedback loops establish directionality by amplifying stochastic fluctuations in signaling between equivalent cells into a robust unidirectional signal. The finding that the Da activator and E(spl)-bHLH repressors are important to properly resolve ISC/EB fate is consistent with this type of model. Activation of the Notch pathway in one of the daughter cells may then lead to the changes in nuclear position (Bardin, 2010).

Second, the asymmetric segregation of determinants could bias Notch-mediated cell fate decisions. The cell fate determinants Numb and Neur are asymmetrically segregated in neural progenitor cells to control Notch signaling. However, no evidence was found for the asymmetric segregation of these proteins in dividing ISCs. Additionally, the data indicate that Numb is not important to maintain ISC fate. It cannot be excluded, however, that another, unknown Notch regulator is asymmetrically segregated to regulate the fate of the two ISC daughters (Bardin, 2010).

A third possibility is that after ISC division, one of the two daughter cells receives a signal that promotes differential regulation of Notch. Indeed, it has been noted that the axis of ISC division is tilted relative to the basement membrane, resulting in one of the progeny maintaining greater basal contact than the other. An extracellular signal coming either basally or apically could bias the Notch-mediated ISC versus EB fate decision. For instance, Wg secreted by muscle cells could act as a basal signal to counteract Notch receptor signaling activity in presumptive ISCs. This could be accomplished by Wg promoting bHLH activity or gene expression. Indeed, Wg has been demonstrated to promote proneural bHLH activity in Drosophila (Bardin, 2010 and references therein).

These models are not mutually exclusive, however, and proper control of ISC and differentiated cell fates during tissue homeostasis might involve multiple mechanisms (Bardin, 2010).

Serpent, suppressor of hairless and U-shaped are crucial regulators of hedgehog niche expression and prohemocyte maintenance during Drosophila larval hematopoiesis

The lymph gland is a specialized organ for hematopoiesis, utilized during larval development in Drosophila. This tissue is composed of distinct cellular domains populated by blood cell progenitors (the medullary zone), niche cells that regulate the choice between progenitor quiescence and hemocyte differentiation [the posterior signaling center (PSC)], and mature blood cells of distinct lineages (the cortical zone). Cells of the PSC express the Hedgehog (Hh) signaling molecule, which instructs cells within the neighboring medullary zone to maintain a hematopoietic precursor state while preventing hemocyte differentiation. As a means to understand the regulatory mechanisms controlling Hh production, a PSC-active transcriptional enhancer was characterized that drives hh expression in supportive niche cells. The findings indicate that a combination of positive and negative transcriptional inputs program the precise PSC expression of the instructive Hh signal. The GATA factor Serpent (Srp) is essential for hh activation in niche cells, whereas the Suppressor of Hairless [Su(H)] and U-shaped (Ush) transcriptional regulators prevent hh expression in blood cell progenitors and differentiated hemocytes. Furthermore, Srp function is required for the proper differentiation of niche cells. Phenotypic analyses also indicated that the normal activity of all three transcriptional regulators is essential for maintaining the progenitor population and preventing premature hemocyte differentiation. Together, these studies provide mechanistic insights into hh transcriptional regulation in hematopoietic progenitor niche cells, and demonstrate the requirement of the Srp, Su(H) and Ush proteins in the control of niche cell differentiation and blood cell precursor maintenance (Tokusumi, 2010).

The lymph gland hematopoietic organ is formed near the end of embryogenesis from two clusters of cells derived from anterior cardiogenic mesoderm (Crozatier, 2004; Mandal, 2004). About 20 pairs of hemangioblast-like cells give rise to three distinct lineages that will form the lymph glands and anterior part of the dorsal vessel. Notch (N) pathway signaling serves as the genetic switch that differentially programs these progenitors towards cell fates that generate the lymph glands (blood lineage), heart tube (vascular lineage), or heart tube-associated pericardial cells (nephrocytic lineage). An essential requirement has also been proven for Tailup (Islet1) in lymph gland formation, in which it functions as an early-acting regulator of serpent, odd-skipped and Hand hematopoietic transcription factor gene expression (Tokusumi, 2010).

By the end of the third larval instar, each anterior lymph gland is composed of three morphologically and molecularly distinct regions (Jung, 2005). The posterior signaling center (PSC) is a cellular domain formed during late embryogenesis due to the specification function of the homeotic gene Antennapedia (Antp) (Mandal, 2007) and the maintenance function of Collier, the Drosophila ortholog of the vertebrate transcription factor early B-cell factor. PSC cells selectively express the Hedgehog (Hh) and Serrate (Ser) signaling molecules and extend numerous thin filopodia into the neighboring medullary zone. This latter lymph gland domain is populated by undifferentiated and slowly proliferating blood cell progenitors (Mandal, 2007). Prohemocytes within the medullary zone express the Hh receptor Patched (Ptc) and the Hh pathway transcriptional effector Cubitus interruptus (Ci). Medullary zone cells also express components of the Jak/Stat signaling pathway. By contrast, the third lymph gland domain -- the cortical zone -- solely contains differentiating and mature hemocytes, such as plasmatocytes and crystal cells. Upon wasp parasitization, or in certain altered genetic backgrounds, lamellocytes will also appear in the cortical zone as a third type of differentiated hemocyte (Tokusumi, 2010).

Two independent studies have provided compelling data to support the contention that the PSC functions as a hematopoietic progenitor niche within the lymph gland, with this cellular domain being essential for maintaining normal hemocyte homeostasis (Krzemien, 2007; Mandal, 2007). These investigations showed that communication between the PSC and prohemocytes present in the medullary zone is crucial for the preservation of the progenitor population and to prevent these cells from becoming abnormally programmed to differentiate into mature hemocytes. Seminal findings from these studies can be summarized as follows: Col expression must be restricted to the PSC by the localized expression of Ser; Hh must be expressed selectively in the PSC, coupled with the non-autonomous activation of the Hh signaling pathway in prohemocytes of the medullary zone; and the PSC triggers activation of the Jak/Stat pathway within cells of the medullary zone. With the perturbation of any of these molecular events, the precursor population of the medullary zone is lost owing to the premature differentiation of hemocytes, which swell the cortical zone. Although the exact interrelationship of Ser, Hh and Jak/Stat signaling within the lymph gland is currently unknown, the cytoplasmic extensions emanating from PSC cells might facilitate instructive signaling between these niche cells and hematopoietic progenitors present in the medullary zone (Krzemien, 2007; Mandal, 2007). A more recent study showed that components of the Wingless (Wg) signaling pathway are expressed in the stem-like prohemocytes to reciprocally regulate the proliferation and maintenance of cells within the supportive PSC niche (Sinenko, 2009). The cellular organization and molecular signaling of the Drosophila lymph gland are remarkably similar to those of the hematopoietic stem cell niches of vertebrate animals, including several mammals (Tokusumi, 2010 and references therein).

Through detailed molecular and gene expression analyses this study has identified the PSC-active transcriptional enhancer within hh intron 1 and delimited its location to a minimal 190 bp region. The hh enhancer-GFP transgene faithfully recapitulates the niche cell expression of Hh derived from the endogenous gene, as double-labeling experiments with the GFP marker and Antp or Hh show a clear co-expression in PSC domain cells. Appropriately, GFP expression is not detected in Antp loss-of-function or TCFDN genetic backgrounds, which culminate in an absence of niche cells from the lymph gland. The hematopoietic GATA factor Srp serves as a positive activator of hh PSC expression, as mutation of two evolutionarily conserved GATA elements in the enhancer abrogates its function and Srp functional knockdown via srp RNAi results in hh enhancer-GFP transgene inactivity and the absence of Hh protein expression. An additional intriguing phenotype was observed in lymph glands expressing the srp RNAi transgene, that being a strong reduction in the number of filopodial extensions emerging from cells of the PSC. This phenotype suggests a functional role for Srp in the correct differentiation of niche cells, via a requirement for normal Hh presentation from these cells and/or the transcriptional regulation by Srp of additional genes needed for the formation of filopodia (Tokusumi, 2010).

As Srp accumulates in all cells of the lymph gland, a question arose as to how hh expression is restricted to cells of the PSC. This paradox could be explained by a mechanism in which hh expression is also under some means of negative transcriptional control in non-PSC cells of the lymph gland. This possibility proved to be correct, with the analyses identifying two negative regulators of hh lymph gland expression. The first is Su(H). Mutation of the evolutionarily conserved GTGGGAA element, a predicted recognition sequence for this transcriptional repressor, resulted in an expanded activity of the hh PSC enhancer-GFP transgene; that is, the de novo appearance of GFP was observed in prohemocytes of the medullary zone. Likewise, ectopic medullary zone expression of the wild-type PSC enhancer-GFP transgene and of Hh protein was seen in lymph glands mutant for Su(H). These findings, coupled with the detection of Su(H) in blood cell progenitors, strongly implicate this factor as a transcriptional repressor of the hh PSC enhancer, restricting its expression to niche cells (Tokusumi, 2010).

Additional studies identified Ush as a second negative regulator of hh expression. Ush is expressed in most cells of the lymph gland, with the exception of those cells resident within the PSC domain. Previous research demonstrated that ush expression in the lymph gland is under the positive control of both Srp. Why Ush protein fails to be expressed in the PSC remains to be determined. Forced expression of ush in niche cells resulted in inactivation of the hh PSC enhancer and reduced the formation of filopodia. It was hypothesized that Ush might be forming an inhibitory complex with the SrpNC protein, changing Srp from a positive transcriptional activator to a negative regulator of hh lymph gland expression. Such a mechanism has been demonstrated previously in the negative regulation by Ush of crystal cell lineage commitment. The expansion of wild-type hh enhancer-GFP transgene and Hh protein expression to prohemocytes within the medullary zone and to differentiated hemocytes within the cortical zone in lymph glands mutant for ush is also supportive of Ush functioning as a negative regulator of hh expression (Tokusumi, 2010).

Bringing these results together, a model can be proposed for the regulatory events that culminate in the precise expression of the vital Hh signaling molecule in niche cells. Srp is a direct transcriptional activator of hh in the lymph gland and Hh protein is detected in niche cells due to this activity. hh expression is inhibited in prohemocytes of the medullary zone by Su(H) action, while a repressive SrpNC-Ush transcriptional complex prevents Srp from activating hh expression in prohemocytes and in differentiated hemocytes of the medullary zone and cortical zone. Together, these positive and negative modes of regulation would allow for the niche cell-specific expression of Hh and facilitate the localized presentation of this crucial signaling molecule to neighboring hematopoietic progenitors (Tokusumi, 2010).

The identification of Srp and Su(H) as key regulators of Hh expression in the larval hematopoietic organ prompted an investigation into the functional requirement of these proteins in the control of blood cell homeostasis. Since Srp knockdown by RNAi leads to an absence of the crucial Hh signal, it was not surprising to find that normal Srp function is required for prohemocyte maintenance and the control of hemocyte differentiation within the lymph gland; that is, a severe reduction of Ptc-positive hematopoietic progenitors and a strong increase in differentiated plasmatocytes and crystal cells was observed in srp mutant tissue (Tokusumi, 2010).

Likewise, Ptc-positive prohemocytes were lost and large numbers of plasmatocytes were prematurely formed in Su(H) mutant lymph glands. This disruption of prohemocyte maintenance occurred even though Hh protein expression was expanded throughout the medullary zone. This raised the question as to why expanded Hh protein and possible Hh pathway activation did not increase the progenitor population in Su(H) mutant lymph glands, instead of the observed loss of prohemocytes and appearance of differentiated plasmatocytes. One explanation might be that the PSC niche is not expanded in Su(H) mutant lymph glands and Hh might only function in promoting blood cell precursor maintenance within the context of the highly ordered progenitor-niche microenvironment. It has been hypothesized that the filopodial extensions that emanate from differentiated niche cells are crucial for Hh signal transduction from the PSC to progenitor cells of the medullary zone. The possibility exists that ectopic Hh protein, which is not produced or presented by niche cells, is unable to positively regulate prohemocyte homeostasis. An experimental result consistent with this hypothesis is that expression of UAS-hh under the control of the medullary zone-specific tepIV-Gal4 driver failed to expand the blood cell progenitor population. A second possibility is that the Hh pathway transcriptional effector Ci might require the co-function of Su(H) in its control of prohemocyte maintenance. This model would predict that, in the absence of Su(H) function, Hh signaling would be less (or non) effective in controlling the genetic and cellular events needed for the maintenance of the prohemocyte state. Third, Su(H) might regulate additional target genes, the expression (or repression) of which is crucial for normal blood cell precursor maintenance and the prevention of premature hemocyte differentiation. Finally, it cannot be ruled out that the expression of ectopic Hh in medullary zone cells, in the context of the adverse effects of Su(H) loss of function in these cells, culminates in the disruption of normal Hh pathway signaling due to an unforeseen dominant-negative effect (Tokusumi, 2010).

In summary, these findings add significantly to knowledge of hematopoietic transcription factors that function to control stem-like progenitor maintenance and blood cell differentiation in the lymph gland. An additional conclusion from these studies is that the hh enhancer-GFP transgene can serve as a beneficial reagent to identify and characterize genes and physiological conditions that control the cellular organization of the hematopoietic progenitor-niche cell microenvironment. RNAi-based genetic screens could be undertaken using this high-precision marker to determine signaling pathways and/or environmental stress conditions that might alter niche cell number and function, leading to an alteration in hematopoietic progenitor maintenance coupled with the robust production of differentiated blood cells. Much remains to be determined about the regulated control of these critical hematopoietic changes and their likely relevance to hematopoietic stem cell-niche interactions in mammals (Tokusumi, 2010).

Insensitive is a corepressor for Suppressor of Hairless and regulates Notch signalling during neural development

The Notch intracellular domain functions as a co-activator for the DNA-binding protein Suppressor of Hairless [Su(H)] to mediate myriad cell fate decisions. Notch pathway activity is balanced by transcriptional repression, mediated by Su(H) in concert with its Drosophila corepressor Hairless. This study demonstrates that the Drosophila neural BEN-solo protein Insensitive (Insv) is a nuclear factor that inhibits Notch signalling during multiple peripheral nervous system cell fate decisions. Endogenous Insv was particularly critical when repressor activity of Su(H) was compromised. Reciprocally, ectopic Insv generated several Notch loss-of-function phenotypes, repressed most Notch targets in the E(spl)-C, and opposed Notch-mediated activation of an E(spl)m3-luc reporter. A direct role for Insv in transcriptional repression was indicated by binding of Insv to Su(H), and by strong chromatin immunoprecipitation of endogenous Insv to most E(spl)-C loci. Strikingly, ectopic Insv fully rescued sensory organ precursors in Hairless null clones, indicating that Insv can antagonize Notch independently of Hairless. These data shed first light on the in vivo function for a BEN-solo protein as an Su(H) corepressor in the Notch pathway regulating neural development (Duan, 2011).

The peripheral nervous system (PNS) of Drosophila includes hundreds of mechanosensory organs arranged in characteristic patterns. Major aspects of the developmental progression of peripheral sensory organs are well understood. Within an initially undifferentiated ectodermal field, groups of cells termed proneural clusters (PNCs) selectively express basic helix-loop-helix (bHLH) activators, whose patterned activity defines territories of neural competence. Cell interactions among PNC cells, mediated by the Notch receptor and its associated signalling cascade, restrict neural potential to singular cells known as sensory organ precursors (SOPs); the remaining PNC cells eventually adopt an ordinary epidermal fate. At this stage, a loss of Notch signalling results in multiple SOPs emerging from a PNC, while a gain of Notch signalling extinguishes the SOP fate (Duan, 2011).

Once stably selected, each SOP executes a stereotyped series of asymmetric cell divisions. The first SOP division produces two cells termed pIIA and pIIB. pIIA generates socket and shaft cells, which are visible on the fly exterior. pIIB undergoes two sets of divisions yielding several internal cells, a glial cell, a sheath cell, and the neuron; the glial cell is apoptotic in mechanosensory organ lineages. Notch signalling operates at each division to guarantee the distinct developmental choices of each pair of daughter cells. The neuron escapes Notch activation throughout the sensory lineage, while the socket cell derives from cells that consistently activate the pathway. Consequently in Notch mutant clones, all cells of peripheral sensory lineages adopt the neural fate, while hyperactivation of Notch activity within the sensory lineage can yield mutant organs composed exclusively of sockets (Duan, 2011).

Upon activation by ligand, the Notch receptor undergoes a series of proteolytic cleavages, resulting in the release and nuclear translocation of its intracellular domain (NICD). This fragment binds directly to members of the CSL (for vertebrate CBF1, Drosophila Suppressor of Hairless (Su(H)), and nematode LAG-1) family of transcription factors, which mediate most if not all of the nuclear aspects of Notch signalling. Although originally recognized as a transcriptional repressor in cultured cells, CSL proteins were subsequently found to mediate activation of Notch target genes in vivo. These opposing activities have been reconciled by a 'switch; model in which CSL proteins repress target genes in the absence of signalling via associated corepressor molecules, but activate target genes via NICD and associated co-activator molecules (Duan, 2011).

The specific roles of CSL-mediated repression can be difficult to recognize owing to the massive and pleiotropic defects induced by loss of Notch signalling. Nevertheless, substantial mutant phenotypes have been observed in the appropriate genetic contexts. For example, Drosophila mutants of the dedicated Su(H) corepressor encoded by Hairless reveal many phenotypes in both inhibitory and inductive contexts of Notch signalling that reflect elevated Notch signalling. The asymmetry of pIIa division is particularly sensitive to Su(H) repressor function, since Hairless heterozygotes exhibit a number of double-socket organs that reflect Notch pathway gain-of-function (Duan, 2011).

This study characterizes Drosophila insensitive (insv) that encodes a novel protein containing a BEN domain. Null mutants of insv were earlier reported to be lethal and to exhibit Notch gain-of-function phenotypes in notum clones. These phenotypes were confounded by simultaneous loss of the Notch antagonist lethal giant larvae from available alleles). Nevertheless, upon cleaning of these stocks, viable insv mutant animals maintained detectable Notch gain-of-function PNS phenotypes that were fully rescued by insv genomic DNA. Detailed genetic interaction analysis revealed the endogenous role of Insv to restrain Notch signalling during multiple cell fate decisions, including SOP specification, pIIA-pIIB decision, and socket-shaft decision. The nuclear localization of Insv suggested that it might regulate Notch target gene expression. Consistent with this hypothesis, ectopic Insv generated multiple Notch loss-of-function phenotypes, strongly repressed the expression of an array of Notch target genes across the Enhancer of split-Complex, and suppressed Notch-mediated activation of an E(spl)m3-luc reporter in cultured cells (Duan, 2011).

It was determined that Insv is a direct corepressor for Su(H), as revealed by protein-protein interactions in vitro and strong binding of endogenous Insv to multiple Su(H) target genes by chromatin immunoprecipitation (ChIP). While both Insv and Hairless bind Su(H), ectopic Insv supported SOP specification in null clones of Hairless, and could in fact generate a lateral inhibition defect in Hairless clones, as in wild-type. Therefore, Insv is capable of inhibiting Notch signalling independently of Hairless. Altogether, these findings shed first light on a member of the BEN-solo protein family as an Su(H) corepressor that regulates multiple Notch-mediated cell fate decisions during neural development (Duan, 2011).

Insensitive (insv) is an SOP-specific gene product of novel structure, containing only the domain of unknown function 1172 (DUF1172). An extended version of DUF1172 was recently recognized across a set of >100 animal and viral proteins, and renamed the BEN domain. BEN domains are often found in association with other domains with chromatin-relevant functions (e.g., POZ, SCML1, or MCAF N-terminal domains). However, Insv belongs to a family of invertebrate and vertebrate proteins containing only the BEN domain ('BEN-solo' proteins), which have been little studied to date (Duan, 2011).

Insv, as detected using an antiserum, accumulates in pupal SOP/pI cells at 14 h after puparium formation (APF), colocalizing with the nuclear transcription factor Senseless (Sens). Notably, Insv appears exclusively nuclear, potentially reflecting a chromatin-associated role (Duan, 2011).

Insv expression was traced through the bristle lineage. Insv was detected in both pIIA and pIIB, and was later seen in their daughters at the 4-cell stage. However, Insv was strongly downregulated in all but one of the lineage cells. Insv was extinguished in this cell before expression of typical markers of terminal PNS cell fates, such as Prospero (marking the sheath cell) or Elav (marking the neuron). However, weak co-expression of Insv and Elav was seen at a number of positions, while Insv never colocalized with Pros. This identified the last Insv+ cell in the microchaete lineage as the neuron. The accumulation of Insv in SOPs and nascent neurons is analogous in the sense that neither of these cells activates Notch signalling during PNS development (Duan, 2011).

Default repression by members of the conserved CSL transcription factor family is critical for proper cell fate decisions mediated by Notch signalling. Curiously, while activation of Notch target genes involves a conserved N[ICD]-Mastermind-CSL complex, a diversity of corepressor complexes have been defined in invertebrate and vertebrate systems. The major corepressor for the Drosophila CSL protein Su(H) is Hairless, an adaptor protein that recruits both CtBP and Groucho repressor complexes. Mammalian Hairless proteins have not been identified; however, it should be noted that Hairless is extremely rapidly evolving and not trivial to identify even in other insects. Therefore, the absence of mammalian proteins aligning to Hairless is not necessarily conclusive. On the other hand, mammalian SHARP and CIR were reported to bind the mammalian CSL protein CBF1, and recruit SMRT/N-CoR and HDAC repressor complexes. Recently, the histone demethylase KDM5A/Lid was reported to be a direct partner of both CBF1 and Su(H) corepressor complexes, although KDM5A/Lid is also documented to have pleiotropic functions involving diverse DNA binding partners such as Rb, Myc, and PRC2 (Duan, 2011 and references therein).

Genetic and biochemical studies show that Insv is a neural nuclear protein that functions as a direct Su(H) partner to antagonize Notch pathway activity during multiple steps of Drosophila peripheral neurogenesis. These data shed first light on the in vivo function for a BEN-solo protein as a neural corepressor in the Notch pathway. Although the phenotypes of insv mutants are mild, they were seen in multiple allelic combinations and were fully rescued by insv genomic DNA. More substantially, insv mutants exhibited strong genetic interactions with several Notch pathway alterations. This genetic situation is not unique to insv, as other critical components of the Notch pathway exhibit redundancy in the nervous system (e.g., multiple E(spl)bHLH-encoding genes must be removed to reveal strong neurogenic defect, and both Notch ligands must be removed to reveal PNS lineage defects. Perhaps most striking is the fact that shaft cell specification completely fails in insv mutants where Su(H) corepressor function is reduced by heterozygosity of Hairless, the major direct Su(H) corepressor identified to date. Reciprocally, elevation of Insv level completely compensates for the null condition of Hairless during SOP specification, and can partially rescue the specification of internal cells including neurons. In fact, ectopic Insv can still generate a Notch loss-of-function lateral inhibition defect without Hairless. These data do not rule out the possibility of a trimeric Su(H)-Hairless-Insv repression complex, but they indicate that Insv does not require Hairless to mediate in vivo repression by Su(H). Preliminary tests indicate that Insv may not bind directly to Groucho, as shown for Hairless. However, now that a molecular function has been assigned to Insv, future studies can be aimed at understanding how it interfaces with other silencing proteins and perhaps eventually to chromatin modifying enzymes (Duan, 2011).

The Drosophila genome contains other loci encoding BEN domains, including other BEN-solo factors (CG9883 and CG12205) and mod(mdg4), a highly alternatively spliced locus that encodes proteins with BEN and POZ domains. It remains to be seen whether the Drosophila BEN proteins exhibit any functional overlap. More generally, the data shed light on the in vivo function for a BEN-solo protein as a corepressor in the Notch pathway. Other BEN domain proteins containing BTB/POZ domains have been linked to transcriptional repression (vertebrate NAC1) and enhancer blocking [Drosophila mod(mdg4)] activities, and the mammalian BEN-solo protein SMAR1/BANP recruits the SIN3/HDAC1 repressor complex. The data add to a growing theme for BEN factor involvement in transcriptional repression. While there are not clear mammalian orthologues of Insv, they do express several BEN-solo proteins. In light of the relatively specific effects of Insv in Notch-mediated cell fate decisions in both endogenous and ectopic contexts, these studies generate hypotheses to direct the study of mammalian BEN-solo proteins (Duan, 2011).

Finally, it is noted that BEN domains are also encoded by viral genomes, including the BEN-solo protein Chordopox E5R_VVC_137623. Viral proteins such as Epstein Barr viral oncoprotein EBNA2 and the adenoviral oncoprotein 13S E1A bind CBF1 and function as NICD mimics. This elucidation of a BEN-solo protein as a CSL corepressor raises the possibility that viruses may have co-opted cellular proteins to dominantly repress Notch signalling (Duan, 2011).

The BEN domain is a novel sequence-specific DNA-binding domain conserved in neural transcriptional repressors

Drosophila Insensitive (Insv) has been shown to promote sensory organ development and has activity as a nuclear corepressor for the Notch transcription factor Suppressor of Hairless [Su(H)]. Insv lacks domains of known biochemical function but contains a single BEN domain (i.e., a 'BEN-solo' protein). Chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq) analysis confirmed binding of Insensitive to Su(H) target genes in the Enhancer of split gene complex [E(spl)-C]; however, de novo motif analysis revealed a novel site strongly enriched in Insv peaks (TCYAATHRGAA). Binding was validated of endogenous Insv to genomic regions bearing such sites, whose associated genes are enriched for neural functions and are functionally repressed by Insv. Unexpectedly, it was found that the Insv BEN domain binds specifically to this sequence motif and that Insv directly regulates transcription via this motif. The crystal structure of the BEN-DNA target complex was determined, revealing homodimeric binding of the BEN domain and extensive nucleotide contacts via alpha helices and a C-terminal loop. Point mutations in key DNA-contacting residues severely impair DNA binding in vitro and capacity for transcriptional regulation in vivo. DNA-binding and repression activities was further demonstrated by the mammalian neural BEN-solo protein BEND5. Altogether, this study defines novel DNA-binding activity in a conserved family of transcriptional repressors, opening a molecular window on this extensive gene family (Dai, 2013).

This study used molecular genetics and ChIP-seq analysis of the neural regulator Insv to delineate two strategies by which it associates with chromatin to mediate transcriptional repression. First, Insv functions as a corepressor for the transcription factor Su(H). In this mode of action, it is recruited to Notch target genes and can oppose the action of Notch signaling in activating these targets (Duan, 2011). Second, it was found that the Insv BEN domain comprises a novel DNA-binding fold that directly recruits Insv homodimers to hundreds of target genes via a palindromic binding site. These dual activities permit Insv to act as a transcriptional repressor during neurogenesis by Su(H)-dependent and Su(H)-independent mechanisms, endowing it with functional versatility. As a corepressor tethered to DNA via Su(H), it tunes the appropriate transcriptional output of Notch target genes (Duan, 2011). It has long been appreciated that cell fate decisions regulated by the Notch pathway are exquisitely sensitive to the precise level of signaling. This appears to be manifest not only by the direct mechanism by which the Notch receptor transduces an extracellular signal into the nucleus, and is in fact obligately destroyed during this process, but also by the fact that transcriptional activation by Notch is balanced by multiple mechanisms of transcriptional repression actively mediated by Notch transcription factors of the CSL family. In Drosophila, Hairless serves as a major direct corepressor for Su(H), and multiple stages of peripheral nervous system development require Hairless function, including during specification of the SOP and at multiple steps in the SOP lineage. However, misexpression of Insv is sufficient to fully rescue SOP specification in Hairless-null clones and can partially rescue their lineage divisions, attesting to its identity as a genuine Su(H) corepressor (Dai, 2013).

This study shows that Notch target genes comprise only a minor fraction of the totality of endogenous Insv target genes. Nevertheless, one of the most prominent categories of genes that contain Insv-occupied target sites are those involved in nervous system development. Indeed, Insv binding and functional repression of a host of genes that regulate neural development was confirmed, including vestigial, mir-263a, repo, fringe, grainyhead, hamlet, tramtrack, Synaptotagmin1, fne, and chinmo, among others. Direct transcriptional regulation by Insv provides a strategy for it to regulate neurogenesis independently of Notch signaling. In fact, the specific expression of Insv in SOPs (and within the lineage), its requirement for fully stable commitment to the SOP fate, and its capacity to promote ectopic peripheral sensory organs when misexpressed (Duan, 2011) are all consistent with the viewpoint that Insv plays an active role in directing neurogenesis upstream of and/or in parallel to its role in inhibiting Notch signaling (Dai, 2013).

The experimental data extensively support that Insv functions as a transcriptional repressor. Consistent with this, the catalog of validated Insv repression targets includes many genes that oppose neurogenesis (e.g., tramtrack), promote late temporal events in neural lineages (e.g., hamlet, grainyhead, and chinmo), or explicitly promote nonneural cell fates in the nervous system (e.g., the proglial factor repo). These observations support the notion that Insv-mediated repression helps guide proper neural specification and cell fate determination within the neural lineage (Dai, 2013).

It is clear that the BEN consensus is modest at best, with no amino acids actually shared across all instances of this domain. Nevertheless, structural studies draw particular attention to Insv residues that participate in direct contacts with the DNA backbone or that recognize specific DNA base side chains. This knowledge allows identification of certain BEN domains as candidates for Insv-like binding specificity. For example, the BEN domain in the Drosophila Mod(mdg4)-PC (Abhiman, 2008) isoform retains few of the base-specific contacts of Insv, but two other Drosophila BEN-solo domain proteins bear striking homology with Insv across multiple residues that were defined as functionally critical for the DNA-binding activity of Insv (Dai, 2013).

It is predicted that CG9883 and Bsg25A are good candidates for harboring sequence-specific DNA-binding activity. Mammalian genomes encode many BEN domain proteins (Abhiman, 2008), but BEN domains per se have been little studied to date. This study has found that mammalian BEND5 is noticeably related to Insv across several functionally important DNA-binding residues, including at many positions within the C-terminal tail region. Although it is possible that BEND5 may have a different optimal binding site, it was possible to demonstrate specific binding of BEND5 to the Insv target site. Therefore, it can be concluded that the BEN domain is a conserved DNA-binding domain. Other functional parallels between Insv and BEND5 have been identified, including their specific expression in neurons and their capacity to function as transcriptional repressors. Given that Insv and BEND5 are fairly unrelated in primary sequence, these findings suggest that some of the other >100 BEN proteins are likely to be transcription factors. Curiously, some other BEN proteins, including the BTB/POZ-BEN factor NAC-1 and the quadruple BEN protein BEND3, have previously been linked to transcriptional repression (Korutla, 2009; Sathyan, 2011). Potentially, their BEN domains are involved in recruiting these factors to their appropriate chromatin targets (Dai, 2013).

Finally, not only are BEN domains widely distributed across metazoans, they are also encoded by several viral genomes (Abhiman, 2008). The data suggest that BEN proteins may potentially prove to add to the catalog of cellular transcription factors that have been co-opted by viruses. Overall, our findings now provide molecular direction to the study of this large and enigmatic family of proteins (Dai, 2013).

Notch regulates numb: integration of conditional and autonomous cell fate specification

The Notch cell-cell signaling pathway is used extensively in cell fate specification during metazoan development. In many cell lineages, the conditional role of Notch signaling is integrated with the autonomous action of the Numb protein, a Notch pathway antagonist. During Drosophila sensory bristle development, precursor cells segregate Numb asymmetrically to one of their progeny cells, rendering it unresponsive to reciprocal Notch signaling between the two daughters. This ensures that one daughter adopts a Notch-independent, and the other a Notch-dependent, cell fate. In a genome-wide survey for potential Notch pathway targets, the second intron of the numb gene was found to contain a statistically significant cluster of binding sites for Suppressor of Hairless, the transducing transcription factor for the pathway. This region contains a Notch-responsive cis-regulatory module that directs numb transcription in the pIIa and pIIIb cells of the bristle lineage. These are the two precursor cells that do not inherit Numb, yet must make Numb to segregate to one daughter during their own division. These findings reveal a new mechanism by which conditional and autonomous modes of fate specification are integrated within cell lineages (Rebeiz, 2011).

The transcriptional regulation of the numb gene has not previously received much attention because most experimental efforts have been focused on Numb protein localization, asymmetric segregation and function as a Notch pathway inhibitor. The motivation for the present study originated in a computational search of the fly genome for new Notch pathway target genes based on statistically significant clustering of Su(H) binding sites. Although it has been suggested that homotypic site clustering is not a general property of cis-regulatory modules in Drosophila, and therefore that this parameter is of limited utility in computational prediction of enhancers, the data presented in this study and in other reports indicate that this approach can be quite effective in the case of Su(H) and other transcription factors. One beneficial feature of the SCORE method (Rebeiz, 2002) is the use of a largely unbiased window size (100-5000 bp) for the identification of statistically significant binding site clusters. This wide range allows the detection of local maxima that do not necessarily conform to the size expected for a canonical cis-regulatory module. Judging from the present study, the unbiased window-size approach might permit functional enhancer elements to be detected owing to the proximity of multiple enhancers with similar binding inputs. In any case, the SCORE technique successfully identified a functional cis-regulatory module within the ~50 kb of non-coding DNA within and surrounding numb (Rebeiz, 2011).

This study has shown here that a 20 kb genomic DNA fragment is capable of nearly complete phenotypic rescue of two different numb loss-of-function genotypes, and that deletion of the intronic numb CD2 enhancer from this fragment results in widespread 'double socket' and 'double sheath' phenotypes, reflecting a failure to specify the numb-dependent shaft and neuron cell fates. Thus, transcriptional activation of numb in the pIIa and pIIIb precursor cells, in response to the Notch signaling events that specify their respective fates, plays an important role in the proper specification of the Notch-independent progeny cell fate (Rebeiz, 2011).

Given the high proportion of sensory organs in which the shaft and neuron cell fates are correctly specified in the absence of the CD2 enhancer, it seems clear that CD2 is not the only source of Numb for pIIa and pIIIb. This inference was confirmed directly by detecting Numb crescents in dividing pIIa cells in tissue lacking CD2 function, having first demonstrated that the numb796 allele is protein-null (Rebeiz, 2011).

What might be the source of this additional Numb protein? It is, of course, possible that numb is served by a second enhancer module that also contributes to the transcriptional activation of the gene in pIIa and pIIIb in response to Notch signaling; there is substantial precedent for such 'shadow' or 'secondary' enhancers in insects. However, it is very likely that the basal level of Numb protein that is detected in all cells in the epidermis also accumulates in developing sensory organ cells, including pIIa and pIIIb, independently of the CD2 enhancer. This protein would presumably be segregated by the two precursor cells to their shaft and neuron daughter cells, respectively, and might suffice, in most cases, to inhibit Notch signaling in those cells (Rebeiz, 2011).

What, then, would generate the need for the numb CD2 enhancer activity? Integrating all of the current findings, the following evolutionary scenario is favored. Among the cells in the bristle lineage, the pIIa and pIIIb precursors face a unique challenge: because their own fates are specified by Notch signaling, it is crucial that they do not inherit Numb, yet each must make sufficient Numb to distribute asymmetrically to one of their progeny cells. In an ancestral sensory organ lineage, the ubiquitous basal level of Numb accumulation might have been adequate to supply the needs of pIIa and pIIIb. But, perhaps as the execution of the lineage became faster in some rapidly developing insects [the time from birth to division for pIIa and pIIIb is only 3-4 hours in Drosophila, Numb accumulation in these cells failed to meet the required threshold, resulting in unacceptably high failure rates in shaft cell and neuron specification. The emergence of the CD2 enhancer would then have offered the selective advantage of supplementing the basal Numb specifically in these two Notch-dependent precursor cells, without elevating the global activity of the gene. In this scenario, CD2 represents an evolutionary adaptation for ensuring the fidelity of two cell fate decisions during mechanosensory organ development (Rebeiz, 2011).

The Drosophila external sensory organ lineage has stood for many years as an elegant example of the integration of conditional and autonomous mechanisms of cell fate specification. The repeated use of a combination of bi-directional Notch signaling between sister cells and asymmetric segregation of the Notch pathway antagonist Numb is a highly effective strategy for ensuring the proper specification of cell fates in a succession of asymmetric cell divisions. This is particularly so because the orientation of the mitotic spindles and the segregation of Numb are tied to the planar polarity system, such that the appropriate fate is assigned to the appropriate daughter with extremely high fidelity. The results reported in this study bring this Notch-Numb partnership full circle by demonstrating that a reciprocal regulatory linkage also exists: Notch signaling regulates numb (see Model for the Notch-stimulated activation of numb transcription in the pIIa precursor cell) (Rebeiz, 2011).

This study has shown that, although Notch signaling is essential to the activation of the numb bristle enhancer, the transcriptional activation function of Su(H) is not strictly required for enhancer activity. Accordingly, it is suggested that Notch signaling acts here in large part as a trigger, relieving Su(H)-mediated 'default repression' and permitting other activators bound to the enhancer to drive numb transcription. Some or all of these activators are likely to be expressed in both pIIa and pIIb, as implied by the nearly equivalent level of reporter gene activity observed in the two cells when the Su(H) binding sites of the enhancer are mutated. It is further suggested that this regulatory strategy is relevant to the question of timing. Having Notch signaling act as a trigger for the action of a pre-assembled complex of other activators might help to ensure that the transcriptional response is very rapid, allowing sufficient numb mRNA to be accumulated and translated in pIIa and pIIIb before they divide (Rebeiz, 2011).

Differential regulation of transcription through distinct Suppressor of Hairless DNA binding site architectures during Notch signaling in proneural clusters

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)

Chromatin modification of Notch targets in olfactory receptor neuron diversification

Neuronal-class diversification is central during neurogenesis. This requirement is exemplified in the olfactory system, which utilizes a large array of olfactory receptor neuron (ORN) classes. An epigenetic mechanism was discovered in which neuron diversity is maximized via locus-specific chromatin modifications that generate context-dependent responses from a single, generally used intracellular signal. Each ORN in Drosophila acquires one of three basic identities defined by the compound outcome of three iterated Notch signaling events during neurogenesis. Hamlet, the Drosophila Evi1 and Prdm16 proto-oncogene homolog, modifies cellular responses to these iteratively used Notch signals in a context-dependent manner, and controls odorant receptor gene choice and ORN axon targeting specificity. In nascent ORNs, Hamlet erases the Notch state inherited from the parental cell, enabling a modified response in a subsequent round of Notch signaling. Hamlet directs locus-specific modifications of histone methylation and histone density and controls accessibility of the DNA-binding protein Suppressor of Hairless at the Notch target promoter (Endo, 2011).

This study analyzed the ORN lineage history that gives rise to three primary ORN identities (Naa, Nab and Nba). These three identities arise in a sensillum via iterated rounds of Notch-mediated binary cell-fate decisions. Together with previous findings, these results suggest that diversification of Drosophila ORN classes is the result of the combined output of two predominantly hardwired mechanisms; spatially localized factors determine at least 21 types of sensilla, and Notch and Ham then act in each sensillum to maximize ORN class variety (Endo, 2011).

Biochemical and molecular analyses of Ham function indicate that it can repress Notch target enhancers. In the ORN lineage, Notch signaling is used in consecutive cell fate decisions, and it was found that Ham acts to turn off Notch targets before a subsequent a round of selective reactivation. Ham is expressed specifically in pNa, the neuronal-intermediate precursor with high Notch activity, and inherited by both of the pNa daughter cells. In addition to the current findings, studies in other contexts have observed that some Notch targets require a Notch signal for their transcriptional induction, but not for maintaining their expression. These Notch targets could aberrantly persist in both pNa progeny without the intervention of a mechanism to erase the effects of the preceding Notch signal (Endo, 2011).

ham mutants showed an unusual ORN fate switch. They not only transformed ORN fate with respect to Notch state, but also altered sublineage-specific identity (low-Notch Nab to high-Notch Nba identity). This phenotype suggests that, in addition to suppressing the previous round of Notch activation, Ham may delineate the selection of the next round of targets. As Ham activity resulted in altered chromatin modifications at Notch targets, this suggests that Ham could set an epigenetic context in which the terminal round of Notch signaling occurs (Endo, 2011).

Although this was demonstrated with respect to Ham, it is suggested that this approach to modifying the transcriptional outputs of a signaling pathway may have widespread importance in other lineages that utilize iterative signals. Notch signaling iteration is a widespread phenomenon. One important example is in the maintenance of neural and other stem cells, and it is now known that some chromatin-modifying factors promote stem cell self-renewal. Notably, several Prdm factors have regionalized expression in neural precursor domains of the embryonic mouse spinal cord and could modify and diversify stem cell identity during mammalian CNS development (Endo, 2011).

In Drosophila, Notch signaling and Ham expression are transient in nascent ORNs. Thus, Notch- and Ham-mediated fate choices must be perpetuated during the later selection of alternative axon guidance factors and odorant receptors. It is possible that chromatin methylation not only sets the context of immediate Notch signaling outcomes, but also maintains initial fate choice by priming or silencing promoters for readout during differentiation. The existence of such mechanisms in neural development is now beginning to emerge. It was recently shown that, in mouse cortical precursor cells, the trithorax factor Mixed-lineage leukemia1 (Mll1) prevents epigenetic silencing of the neural differentiation gene Distal-less homeobox 2 (Dlx2), enabling it to be properly upregulated during differentiation stages. In contrast, in the mammalian olfactory system, epigenetic repression is used during the transition from multipotent precursor to immature ORN to silence all ~2,800 odorant receptor genes before subsequent de-repression of a single odorant receptor per neuron (Endo, 2011).

To determine how chromatin modifications create a context-dependent outcome from signaling and how resultant cell-fate choices are perpetuated during Drosophila ORN differentiation, it will be necessary to elucidate the components and action of the chromatin-modification complex targeted by Ham. Furthermore, genome-wide identification of the promoters targeted by Su(H) and Ham will reveal the genes regulated by these factors to confer specific ORN fates (Endo, 2011).

Genetic characterization of ebi reveals its critical role in Drosophila wing growth.

The ebi gene of Drosophila has been implicated in diverse signalling pathways, cellular functions and developmental processes. However, a thorough genetic analysis of this gene has been lacking and the true extent of its biological roles is unclear. This study characterize eleven ebi mutations and found that ebi has a novel role in promoting growth of the wing imaginal disc: viable combinations of mutant alleles give rise to adults with small wings. Wing discs with reduced EBI levels are correspondingly small and exhibit down-regulation of Notch target genes. Furthermore, EBI was shown to colocalize on polytene chromosomes with Smrter (SMR), a transcriptional corepressor, and Suppressor of Hairless (SU(H)), the primary transcription factor involved in Notch signalling. Interestingly, the mammalian orthologs of ebi, transducin β-like 1 (TBL1) and TBL-related 1 (TBLR1), function as corepressor/coactivator exchange factors and are required for transcriptional activation of Notch target genes. It is hypothesized that EBI acts to activate (de-repress) transcription of Notch target genes important for Drosophila wing growth by functioning as a corepressor/coactivator exchange factor for SU(H) (Marygold, 2011; full text of article).

Notch and Mef2 synergize to promote proliferation and metastasis through JNK signal activation in Drosophila

Genetic analyses in Drosophila revealed a synergy between Notch and the pleiotropic transcription factor Mef2 (myocyte enhancer factor 2), which profoundly influences proliferation and metastasis. This study shows that these hyperproliferative and invasive Drosophila phenotypes are attributed to upregulation of eiger, a member of the tumour necrosis factor superfamily of ligands, and the consequent activation of Jun N-terminal kinase signalling, which in turn triggers the expression of the invasive marker MMP1. Expression studies in human breast tumour samples demonstrate correlation between Notch and Mef2 paralogues and support the notion that Notch-MEF2 synergy may be significant for modulating human mammary oncogenesis (Pallavi, 2012).

A genetic modifier screen was undertaken in Drosophila and a number of genetic modifiers of Notch signals were identified that affect proliferation. Further examination of one of these modifiers, Mef2, established that its synergy with Notch signals directly triggers expression of the Drosophila JNK pathway ligand eiger, consequently activating JNK signalling that profoundly influences proliferation and metastatic behaviour. It might perhaps be worth noting that metastatic behaviour in Drosophila may not be completely equivalent to mammalian metastasis, notwithstanding the fact that they share molecular signatures, for example, MMP activation (Pallavi, 2012).

Cancer is characterized by the deregulation of the balance between differentiation, proliferation and apoptosis; thus, it is not surprising that the Notch signalling pathway, which plays a central role in all these developmental events, is increasingly implicated in oncogenic events. The rationale of this study is based on the fact that synergy between Notch and other genes is key in understanding how Notch signals contribute to oncogenesis. It remains a remarkable fact that while activating mutations in the Notch receptor have been associated with >50% of T-cell lymphoblastic leukemias (T-ALLs), a search for mutations in other cancers, despite a few suggestive reports, remains essentially unfruitful. Yet, correlative studies have linked Notch activity with a broad spectrum of human cancers and work in mice suggests that while Notch activation promotes proliferation, it is the synergy between Notch and other factors that eventually leads to cancer. Similar synergies have been identified before but the extraordinary complexity of the gene circuitry that modulates the Notch pathway suggests that more such relationships will be uncovered as exemplified by the discovery of Mef2 as a Notch synergistic partner affecting proliferation (Pallavi, 2012).

The transcription factor Mef2 plays an essential role in myogenic differentiation, but several studies have also shown a broad pleiotropic role of Mef2. Mef2 can integrate signals from several signalling cascades through chromatin remodelling factors and other transcriptional regulators to control differentiation events. This study extends the functionality of Mef2 by uncovering the profound effect it can have on proliferation and metastatic cell migration in synergy with Notch signals (Pallavi, 2012).

This is not the first study to link Mef2 with Notch. They have been linked before in the context of myogenesis both in Drosophila and in vertebrates. A ChIP-on-chip analysis of Mef2 target regions identified several Notch pathway components as potential Mef2 targets during Drosophila myogenesis. In human myoblasts, Mef2C was suggested to bind directly to the intracellular domain of Notch via the ankyrin repeat region, suppressing Mef2C-induced myogenic differentiation. Mef2 has also been reported to interact with the Notch coactivator MAML1 and suppress differentiation (Pallavi, 2012).

While upregulation of Mef2 alone does not show overt proliferation effects, these analyses demonstrate that in vivo it can activate MMP1. Even though Mef2 was ectopically expressed in the whole wing pouch, MMP1 expression was confined around the D/V boundary, where endogenous Notch signals are active. This effect of Mef2 overexpression depends on Notch signals, a notion corroborated by the fact that inhibiting Notch activity by RNAi reverses the effects of Mef2 on MMP1 (Pallavi, 2012).

The polarity gene scribble cooperates with Ras signalling to upregulate the JNK pathway, promoting invasiveness and hyperplasticity. However, the synergy seen in this study appears to be scribble independent. The fact that both the scribbled/Ras and the Notch/Mef2 metastatic pathways converge at the level of JNK signal activation suggests that JNK is a crucial regulator of oncogenic behaviour, which is controlled by inputs from multiple signals. Even though there is little evidence that twist activates JNK signalling, it is a crucial regulator of epithelial-to-mesenchymal transition and metastasis and has also been independently linked to both Mef2 and Notch in myogenesis. However, it is noted that the Notch-Mef2 synergy seems to be independent of twist, as Twist cannot replace Mef2 in the synergistic relationship (Pallavi, 2012).

Numerous reports link JNK signalling to normal developmental events requiring cell movement and to metastatic phenomena both in Drosophila and in vertebrates. JNK signals seem to be crucial for controlling gene activities involved in epithelial integrity and the observations from Drosophila suggest that the Nact and Mef2 synergy may be important in JNK-linked carcinogenesis. A role for Notch in controlling JNK signals has been reported previously. While studies carried out using breast cancer samples can only be correlative now, the observations suggest that metastatic breast tumours harbour higher levels of Notch and Mef2 paralogue pairs, consistent with observations in Drosophila (Pallavi, 2012).

Although the majority of studies on Mef2 are focused on muscle development/differentiation, some intriguing links between Mef2C and leukaemias are noteworthy. MEF2C and Sox4 synergize to cause myeloid leukaemia in mice. Analysis of T-ALL patient samples revealed increased levels of Mef2C; however, Mef2C alone could not cause cellular transformation of NIH3T3 cells, but it could do so in the presence of RAS or myc. Given the role of Notch in T-ALL it will be important to examine how activated Notch mutations, often the causative oncogenic mutation, correlate with Mef2 family members. The functional differences between the different Mef2 homologues in humans are not well understood and the specific role each may play in the Notch synergy remains to be elucidated (Pallavi, 2012).

This analysis clearly indicates that, in Drosophila, the underlying molecular mechanism of the Notch/Mef2 synergy relies on the direct upregulation of expression of the prototypical TNF ligand egr through the binding of Mef2 and Su(H), the effector of Notch signals, to regulatory sequences on the egr promoter. In Drosophila, egr is the only JNK ligand while in humans, the superfamily is large and includes the cytokines TNFα (TNF), TRAIL and RANKL which have been associated with tumour progression in numerous human cancers including breast. RANKL plays a key role in bone metastasis of breast cancer, and is the target of a therapeutically effective monoclonal antibody. In breast cancer cells, TNFα, which can signal through several pathways, including JNK and NF-κB, affects proliferation and promotes invasion and metastasis (Pallavi, 2012).

In human breast cancer, clinical relapse after initial treatment is almost always accompanied by metastatic spread and it is almost invariably lethal. ER- tumours tend to respond well to first-line chemotherapy, but a significant subset of these tumours recur. Recurrent ER- tumours are typically resistant to chemotherapy and radiation, and are highly lethal. The current data suggest that ER- tumours that recur but not ER- tumours that do not recur show significant positive correlation between NOTCH1 and all four MEF2 paralogues. Further, the data show that even within the recurrent subset, NOTCH1 expression predicts poor survival but MEF2 expression does not. While these observations do not establish causality, they are consistent with the hypothesis that NOTCH1/MEF2 coexpression identifies a set of breast cancers that are more likely to relapse, and that MEF2 genes act as NOTCH cofactors rather than independently of NOTCH (Pallavi, 2012).

In conclusion, this study in Drosophila uncovers a new functional role for Mef2, which in synergy with Notch affects proliferation and metastasis. Mechanistically, this synergy relies on the direct upregulation of the JNK pathway ligand eiger. The correlation analysis and tumour staining of human cancer samples suggests that the observations in Drosophila may well be valid in humans, defining Notch-Mef2 synergy as a critical oncogenic parameter, one that may be associated with metastatic behaviour, emphasizing the value of model systems in gaining insight into human pathobiology (Pallavi, 2012).

Role of architecture in the function and specificity of two Notch-regulated transcriptional enhancer modules

In Drosophila melanogaster, cis-regulatory modules that are activated by the Notch cell-cell signaling pathway all contain two types of transcription factor binding sites: those for the pathway's transducing factor Suppressor of Hairless [Su(H)] and those for one or more tissue- or cell type-specific factors called 'local activators.' The use of different 'Su(H) plus local activator' motif combinations, or codes, is critical to ensure that only the correct subset of the broadly utilized Notch pathway's target genes are activated in each developmental context. However, much less is known about the role of enhancer "architecture"--the number, order, spacing, and orientation of its component transcription factor binding motifs--in determining the module's specificity. This study investigated the relationship between architecture and function for two Notch-regulated enhancers with spatially distinct activities, each of which includes five high-affinity Su(H) sites. The first, which is active specifically in the socket cells of external sensory organs, is largely resistant to perturbations of its architecture. By contrast, the second enhancer, active in the 'non-SOP' cells of the proneural clusters from which neural precursors arise, is sensitive to even simple rearrangements of its transcription factor binding sites, responding with both loss of normal specificity and striking ectopic activity. Thus, diverse cryptic specificities can be inherent in an enhancer's particular combination of transcription factor binding motifs. It is proposed that for certain types of enhancer, architecture plays an essential role in determining specificity, not only by permitting factor-factor synergies necessary to generate the desired activity, but also by preventing other activator synergies that would otherwise lead to unwanted specificities (Liu, 2012).

Detailed analysis of two different Notch-regulated transcriptional enhancer modules has revealed that they are very differently dependent on a particular architecture for their activity and specificity. The socket cell-specific ASE5 enhancer tolerates a variety of rearrangements of its required motifs without appreciable alteration of function in either nascent or mature sockets. Even when ASE5 is impaired quantitatively as a result of mutating all of its non-essential sequences, motif rearrangement generally has only modest effects on activity level, and never modifies the enhancer's specificity. In contrast, it was found that the mα enhancer is sensitive to simple exchanges in the positions of transcription factor binding motifs, responding with both loss of normal spatial specificity and ectopic activity (Liu, 2012).

Broadly speaking, then, one might say that ASE5 is more representative of a 'billboard' model of enhancer architecture (which posits that transcription factor binding motifs contribute to enhancer function largely independently of how they are organized), while the mα enhancer might be thought of as conforming more closely to an 'enhanceosome' model (which suggests that a module's function is crucially dependent on a particular configuration of transcription factor binding sites in order to create synergy between their inputs) (Liu, 2012).

It is useful to consider the characteristics that may determine whether a given module is more likely to lie at the 'billboard' or the 'enhanceosome' end of the spectrum. Though ASE5 and the mα enhancer are both Notch-activated, they function in different biological contexts, and it is suggested that this may be relevant to their respective architectural constraints. ASE5 acts in a single post-mitotic, differentiated cell type to establish and maintain autoregulation of Su(H) for several days. In this instance, due to the availability of cell type-specific 'local activators' such as Vvl, and the strong contribution that high Su(H) levels alone can make to the enhancer's activity, the need for a constrained architecture may be quite minimal. The mα enhancer, on the other hand, is faced with the challenging task of rapidly and transiently (over a period of hours) activating expression of the E(spl)mα gene in multiple non-SOP cells per PNC, while at the same time repressing its expression in each SOP. This might be expected to create a stringent requirement for constrained spacing between the lone proneural protein binding site and one or more Su(H) sites. At the same time, other aspects of the enhancer's normal specificity rely on inputs via POU-HD and/or homeodomain binding sites -- yet these must not be permitted to promote inappropriate activity in socket cells. Again, particular binding motif configurations may be called for as a preventative. The overall point is that two parameters -- an enhancer's specific biological task and context, and its particular combination of factor binding sites -- are likely to play a major role in determining the architectural constraints to which it may be subject (Liu, 2012).

The case of the mα enhancer serves to underscore the insufficiency, in many instances, of a transcription factor binding site 'code' in predicting the specificity of a cis-regulatory module. Despite the presence of five Su(H) sites and two motifs that can be bound by Vvl, the native mα enhancer shows no meaningful activity even in adult socket cells. Yet the mα-shuffle1 and mα-shuffle2 variants, in which the positions of the Vvl motifs are altered, do exhibit substantial adult socket cell activity. Thus, it is specifically the wild-type enhancer's architecture that normally prevents this from happening. A similar conclusion derives from examining the functionality of the proneural (E) plus Su(H) (S) 'code' embodied in the mα enhancer. When the lone E box site is in its native and evolutionarily conserved position 14 bp away from one of the Su(H) sites, it provides sufficient input to drive robust expression in all wing disc PNCs. But when it is moved instead to the location of one of the Vvl sites, the module's PNC activity is severely reduced. Again, the simple presence of Su(H) and proneural binding motifs in the mα enhancer does not suffice to predict its specificity; rather, the specific arrangement of these sites has a profound effect on its ability to generate the PNC specificity (Liu, 2012).

The critical role of binding site spacing and organization in generating the transcription factor synergies necessary for the normal activity of many enhancers is becoming increasingly clear. But the mutational analyses of both ASE5 and the mα enhancer demonstrate an equally important role for architecture in preventing inappropriate synergies and hence inappropriate specificities (Liu, 2012).

Two ASE5 variants are particularly informative in illuminating the importance of motif spacing in restraining enhancer activity. ASE5M2, in which only the five Su(H) sites are intact but spacing is preserved, is completely inactive in both pupal and adult socket cells. By contrast, the ABm version of ASE5-shrink, which likewise retains only the five Su(H) sites but now places them much closer together, is strongly active in adult sockets. Thus, ASE5's native architecture serves in part to prevent the Su(H) sites from responding on their own, and in this way maintains the enhancer's dependence on inputs from the box A and/or box B sequence elements, even in adult socket cells (Liu, 2012).

Next, the wholly ectopic responsiveness of mα-shrink in both pupal and adult socket cells demonstrates clearly that the potential for unrelated and unwanted specificities can be inherent in an enhancer's particular combination of transcription factor binding motifs. Even as it functions in an inappropriate cell type, mα-shrink follows a recognizable regulatory logic. Its activity in nascent socket cells is fully dependent, as expected from ASE5, on its POU-HD and/or homeodomain sites (and not on its 'E box' proneural protein binding site), while its robust adult socket activity -- as in the case of the ABm version of ASE5-shrink -- requires only the five Su(H) sites (Liu, 2012).

Finally, the far more modest alterations represented by the 'shuffle' versions of the mα enhancer explicitly demonstrate the critical role that motif placement and spacing may have in suppressing inappropriate specificities. Simply exchanging the position of one of the module's 'Vvl' sites with that of the E box proneural site creates novel activities in both the wing imaginal disc and the socket cell (Liu, 2012).

In a recent report, Swanson (2011) identified short-range transcriptional repression as the mechanism that prevents the cone cell-specific sparkling (spa) eye enhancer, which serves the Drosophila dPax2 gene, from being ectopically active in nearby photoreceptor cells. In this instance, moving the repression-mediating sequences out of their native context apparently eliminated their ability to exert a repressive effect, permitting the module to be active in an inappropriate cell type (Liu, 2012).

It is believed that these results with the mα enhancer are most simply consistent with a different mechanism for restraining unwanted enhancer specificities. In this model, the relative positions and spacings of transcription factor binding sites are organized so as to promote functional synergies between activators that generate the desired specificity, while at the same time preventing different activator synergies that would otherwise create undesirable specificities. Note that, while this mechanism places definite constraints on the allowable motif locations in the module, it does not require that the enhancer be transcriptionally repressed in the incorrect cell type(s) (Liu, 2012).

The possibility that, despite their simplicity, both of the 'site switches' embodied in the mα-shuffle1 and mα-shuffle2 constructs have disrupted the interaction of a short-range repressor with its target activator(s) cannot strictly rulef out. However, this is thought unlikely for a number of reasons. For example, such a repressor would have to be active in both a broad zone of wing disc tissue and in socket cells — two very different settings. It is suggested instead that the most parsimonious explanation for these findings is the synergy promotion/prevention model described above (Liu, 2012).

What might determine whether a given enhancer makes use of active repression to limit its specificity, or instead utilizes a simpler synergy prevention mechanism? One reasonable possibility is that repression is required, or more common, when the ectopic specificity that must be prevented consists of a cell or cells that are very closely related developmentally to those in which expression is wanted. Such inappropriate cells may be spatially very close to the correct cells, and/or may have a high degree of similarity in their developmental histories and gene expression profiles. In such cases, it may be difficult or impossible to evolve a motif architecture that simultaneously allows the proper activity and prevents the improper. On the other hand, when the ectopic specificity is a very different cell type or tissue, distant both temporally and spatially from the correct one, and sharing very little developmental history, perhaps motif arrangements that act to prevent inappropriate synergies are easier to evolve. Under this rubric, the use of repression by modules as different as the eve stripe 2 and spa enhancers is readily understood, just as the mα enhancer might instead be expected to inhibit socket cell activity by prevention of the necessary activator synergy. Indeed, the mα module appears to make use of both mechanisms: Activity of this enhancer in the SOP cell is antagonized by repression mediated by Su(H). As a member of the PNC, the SOP is of course surrounded by, and very closely related to, the non-SOPs (Liu, 2012).

Finally, it is interesting to consider what characteristics of an enhancer might put it particularly at risk for ectopic activity, which in turn would require the use of the preventive mechanisms that this study consider. Certainly utilizing transcription factors that are broadly expressed and active [such as Su(H)] would contribute to such a need, as would using inputs from factors that are members of paralogous families with very similar DNA-binding specificities (e.g., POU-HD proteins) (Liu, 2012).

The results described in this study, have important implications for understanding of enhancer evolution. It appears that, due to the specific combination of transcription factor binding motifs they employ, some (perhaps most) enhancers harbor the hidden potential to generate certain novel specificities that can be revealed through comparatively simple sequence changes. In a sense, such enhancers are 'poised' to express these silent specificities. Depending on how widespread this phenomenon is among enhancers in the whole genome, a tremendous potential may exist to explore a vast 'specificity space' through modest mutational events. Moreover, when applied to an individual enhancer, this perspective suggests that a particular novel specificity -- one that requires only relatively minor changes in motif placement to be expressed -- might be seen to evolve independently in more than one lineage (Liu, 2012).

These results also suggest that the minimum size of a given enhancer module may be subject to significant constraints, due to the need to prevent unwanted activator synergies through motif spacing. Thus, even if not all sequences in the enhancer mediate transcription factor inputs, some may be preserved evolutionarily in order to maintain distance between transcription factor binding sites (Liu, 2012).

Context-dependent enhancer selection confers alternate modes of Notch regulation on argos

Wiring between signaling pathways differs according to context, as exemplified by interactions between Notch and epidermal growth factor receptor (EGFR) pathways, which are cooperative in some contexts but antagonistic in others. To investigate mechanisms that underlie different modes of cross talk, this study has focused on argos, an EGFR pathway regulator in Drosophila melanogaster which is upregulated by Notch in adult muscle progenitors but is repressed in the wing. Results show that the alternate modes of cross talk depend on the engagement of enhancers with opposite regulatory logic, which are selected by context-determining factors. This is likely to be a general mechanism for enabling the wiring between these pathways to switch according to context (Housden, 2014).

Analysis of tissue-dependent responses to Notch demonstrates that, in argos, these are determined at the level of specific enhancers. These respond either to Su(H) or to the bHLH repressors downstream of Notch, giving rise to different consequences on argos expression and explaining how the logic of signaling pathway cross talk can be switched. Indeed, the different modes of argos regulation correlate with the relationship between Notch and EGFR pathways, with cooperative cross talk occurring in the adult muscle progenitors (AMPs), where the enhancer directly regulated by Su(H) is active, and antagonistic cross talk taking place in the wing pouch, where the repressive enhancer regulated by bHLH operates. Similar distinctive enhancers may also operate at different stages in development, where Notch first activates and then represses the expression of a gene via independent regulatory elements. In both cases it is likely that context-determining factors will alter the ability of specific enhancers to respond to distinct Notch inputs. These will then dictate how signaling pathways will act on the cognate gene, depending on which regulatory elements they make available (Housden, 2014).

Several observations, such as the inability of HLHmβ or HLHmβ-VP16 to alter expression of argos(p)-lacZ when expressed in the AMPs, suggest that, like Su(H), HLHmβ can occupy its binding sites only when the enhancer becomes accessible. Consistent with this possibility, another HLH family transcriptional repressor, Hairy, was shown to bind and repress only those enhancers that had been rendered accessible by prior binding of other factors. Alternatively, HLHmβ may still be capable of binding to its site in enhancer fragment argos2 but lacks the ability to mediate long-range repression, restricting its effects to transcription factors bound within the same vicinity, as observed for short-range repressors regulating even-skipped enhancers. Given that Hairy bHLH repressors can mediate long-range as well as short-range repression, this explanation seems unlikely. Furthermore, as studies of other bHLH factors, such as Myc, argue that they can only bind to chromatin in open conformations, the model in which enhancer accessibility is regulated seems the more probable explanation (Housden, 2014).

Thus, the context-dependent response of argos to Notch could be explained by a two-stage model. Key determining factors, such as Twist in the AMPs or Vvl in the wing pouch, would first regulate the accessibility of different enhancers in the argos intron. This would enable the second stage, which integrates the effects of Notch and EGFR. For example, in the wing pouch, multiple binding sites for the repressor Cic keep the gene repressed, except in regions where EGFR is active. Superimposed on this is the additional regulation from the E(spl)bHLH repressors, acting downstream of Notch to fine-tune the expression patterning within this active domain. Such a model is broadly consistent with two general principles proposed previously for gene regulation by signaling pathways. The first is the reliance on cooperation with context-determining transcription factors, fulfilled here by the requirements for Twist or Vvl. The second is the pivotal role played by repressors, which prevent enhancer activity in appropriate places, as seen here for Cic and E(spl)bHLH (Housden, 2014).

The disparate activities of the argos enhancers suggests that correct modes of response will also require functional boundaries to enable the enhancers to function independently. As no insulator elements have been reported within the argos intron, based on the binding of known factors such as Su(Hw) and CTCF, the mechanism that separates the different functions remains to be elucidated. Other examples of independently functioning enhancers that lack clearly defined insulator elements include the even-skipped stripe enhancers. In this context, the activators and repressors bound to each enhancer act only over short distances, and the spacer sequences between the enhancers prevent cross-regulation. As spacers of a few hundred base pairs were sufficient to enable the even-skipped enhancers to function independently, it is possible that a similar mechanism enables the argos enhancers to operate properly. Such independent operation of these context-dependent enhancers is pivotal for their alternate modes of Notch regulation, and it is likely that similar mechanisms operate when genes are required to adopt different response modes to other widely active signaling pathways (Housden, 2014).

Notch directly regulates cell morphogenesis genes, Reck, talin and trio, in adult muscle progenitors

There is growing evidence that Notch pathway activation can result in consequences on cell morphogenesis and behaviour, both during embryonic development and cancer progression. In general, Notch is proposed to co-ordinate these processes by regulating expression of key transcription factors. However, many Notch-regulated genes identified in genome-wide studies are involved in fundamental aspects of cell behaviour, suggesting a more direct influence on cellular properties. By testing the functions of 25 such genes it was confirmed that 12 are required in developing adult muscles consistent with roles downstream of Notch. Focusing on three, Reck, rhea/talin and trio, their expression was varified in adult muscle progenitors, and Notch-regulated enhancers in each were identified. Full activity of these enhancers requires functional binding sites for Su(H), the DNA-binding transcription factor in the Notch pathway, validating their direct regulation. Thus, besides its well-known roles in regulating the expression of cell-fate determining transcription factors, Notch signalling also has the potential to directly affect cell morphology/behaviour by modulating expression of genes such as Reck, rhea/talin and trio. This sheds new light on functional outputs of Notch activation in morphogenetic processes (Pezeron, 2014).

The bHLH-PAS transcription factor Dysfusion regulates tarsal joint formation in response to Notch activity during Drosophila leg development

A characteristic of all arthropods is the presence of flexible structures called joints that connect all leg segments. Drosophila legs include two types of joints: the proximal or 'true' joints that are motile due to the presence of muscle attachment and the distal joints that lack musculature. These joints are not only morphologically, functionally and evolutionarily different, but also the morphogenetic program that forms them is distinct. Development of both proximal and distal joints requires Notch activity; however, it is still unknown how this pathway can control the development of such homologous although distinct structures. This study shows that the bHLH-PAS transcription factor encoded by the gene dysfusion (dys), is expressed and absolutely required for tarsal joint development while it is dispensable for proximal joints. In the presumptive tarsal joints, Dys regulates the expression of the pro-apoptotic genes reaper and head involution defective and the expression of the RhoGTPases modulators, RhoGEf2 and RhoGap71E, thus directing key morphogenetic events required for tarsal joint development. When ectopically expressed, dys is able to induce some aspects of the morphogenetic program necessary for distal joint development such as fold formation and programmed cell death. This novel Dys function depends on its obligated partner Tango to activate the transcription of target genes. A dedicated dys cis-regulatory module was identified that regulates dys expression in the tarsal presumptive leg joints through direct Su(H) binding. All these data place dys as a key player downstream of Notch, directing distal versus proximal joint morphogenesis (Cordoba, 2014: PubMed).

MAPK-dependent phosphorylation modulates the activity of Suppressor of Hairless in Drosophila

Cell differentiation strictly depends on the epidermal growth factor receptor (EGFR)- and Notch-signalling pathways, which are closely intertwined. This study addresses the molecular cross talk at the level of Suppressor of Hairless [Su(H)]. The Drosophila transcription factor Su(H) mediates Notch signalling at the DNA level: in the presence of signalling input Su(H) assembles an activator complex on Notch target genes and a repressor complex in its absence. Su(H) contains a highly conserved mitogen activated protein kinase (MAPK) target sequence. Evidence is provided that Su(H) is phosphorylated in response to MAPK activity. Mutation of the Su(H) MAPK-site modulated the Notch signalling output: whereas a phospho-deficient Su(H)MAPK-ko isoform provoked a stronger Notch signalling activity, a phospho-mimetic Su(H)MAPK-ac mutant resulted in its attenuation. In vivo assays in Drosophila cell culture as well as in flies support the idea that Su(H) phosphorylation affects the dynamics of repressor or activator complex formation or the transition from the one into the other complex. In summary, the phosphorylation of Su(H) attenuates Notch signalling in vivo in several developmental settings. Consequently, a decrease of EGFR signal causes an increase of Notch signalling intensity. Hence, the antagonistic relationship between EGFR- and Notch-signalling pathways may involve a direct modification of Su(H) by MAPK in several developmental contexts of fly development. The high sequence conservation of the MAPK target site in the mammalian Su(H) homologues supports the idea that EGFR signalling impacts on Notch activity in a similar way in humans as well (Auer, 2014).

Chromatin signatures at Notch-regulated enhancers reveal large-scale changes in H3K56ac upon activation

The conserved Notch pathway functions in diverse developmental and disease-related processes, requiring mechanisms to ensure appropriate target selection and gene activation in each context. To investigate the influence of chromatin organisation and dynamics on the response to Notch signalling, this study partitioned Drosophila chromatin using histone modifications and established the preferred chromatin conditions for binding of Su(H), the Notch pathway transcription factor. Manipulating activity of a co-operating factor, Lozenge/Runx, showed that it can help facilitate these conditions. While many histone modifications were unchanged by Su(H) binding or Notch activation, rapid changes were detected in acetylation of H3K56 at Notch-regulated enhancers. This modification extended over large regions, required the histone acetyl-transferase CBP and was independent of transcription. Such rapid changes in H3K56 acetylation appear to be a conserved indicator of enhancer activation as they also occurred at the mammalian Notch-regulated Hey1 gene and at Drosophila ecdysone-regulated genes. This intriguing example of a core histone modification increasing over short timescales may therefore underpin changes in chromatin accessibility needed to promote transcription following signalling activation (Skalska, 2015).

Signalling pathways such as Notch have diverse functions depending on the context in which they are activated and on the specific subsets of genes that are regulated in each context. This specificity necessitates mechanisms that enable Su(H) to recognise and bind to appropriate enhancers and effect relevant gene expression changes. By utilising the comprehensive collection of chromatin modifications gathered by the modENCODE project, this study has generated maps of chromatin states (see The full list of signal tracks) in two Drosophila cell types and related those to the loci that are bound by Su(H). In doing so, the profile of H3K56ac across the genome was also analysed, and that this core histone modification was found to be present at enhancers, and at transcription start sites, similar to the reported distribution in mammalian ES cells. Significantly, the inclusion of H3K56ac-binding data in the computational model helped to discriminate the active enhancers. Even more striking was the robust increase in this core nucleosome modification in response to Notch activation. Such changes were also detected in mammalian cells and at ecdysone-regulated genes in Drosophila, arguing that H3K56ac is likely to be a widespread modification associated with enhancer activation (Skalska, 2015).

Unlike the modifications to exposed histone tails, which primarily provide docking sites for further chromatin modifying proteins, H3K56ac can directly alter nucleosomal DNA accessibility by increasing DNA breathing and unwrapping rate. As a consequence, this modification can influence transcription factor (TF) occupancy within the nucleosome and it has been argued that H3K56ac drives chromatin towards the disassembled state during transcriptional activation. As the increase in H3K56ac appears to precede transcription elongation, it fits with the latter model. Furthermore, as mammalian CSL has been found to bind preferentially to motifs at the nucleosome exit point\, H3K56ac may enhance recruitment, giving a feed-forward benefit that could potentially explain the increase in occupancy following Notch activation. In addition, H3K56ac facilitates divergent transcription by promoting rapid nucleosome turnover and also promotes small RNA production in neurospora, which is consistent with the detection of intergenic enhancer-templated RNAs in the modified regions following Notch activation (Skalska, 2015).

The increase in H3K56ac appears to require CBP-HAT activity, which is also essential for catalysing this modification on free histones. It is plausible therefore that the increase in H3K56ac could occur through the incorporation of pre-modified nucleosomes. The modification of histone dimers requires interaction with the chaperones CAF1 and ASF1, and while genetic evidence that the chaperone subunit dCAF-1-p105 can help promote Notch signalling favours such a model, the current results suggest this is less likely. First, it was found that CBP is required at the time of activation, making it improbable that the increase in H3K56ac is a consequence of loading pre-modified histones. Second, an inhibitor of the CBP bromodomain, which plays an important role in enabling H3K56ac on histone dimers via its interaction with chaperones, had no effect on the increase in H3K56ac. Thus, it seems more likely that the modification occurs at the time of enhancer activation, although it may nevertheless involve nucleosome exchange. For example, SWI/SNF nucleosome remodellers have been found to act in combination with H3K56ac to promote nucleosome turnover and gene activity in yeasts. At several loci where changes were detected in H3K56ac, the modification extended broadly from the site of Su(H)/NICD binding, correlating with domains that already possessed H3K4me1. Along with data from other studies of enhancer activation, and the observation that levels of H3K56ac are affected by mutation of H3K4, this suggests that H3K4me1 is likely to be one of the earliest modifications, prefiguring sites of active enhancer. It may also facilitate the spread of H3K56ac across the regulated regions (Skalska, 2015).

Analysis of the relationship between chromatin states and regions occupied by Su(H) suggests that the pre-existing chromatin environment is likely to make an important contribution to recruitment. First, Su(H)-occupied motifs were almost exclusively located in highly accessible chromatin, with modifications such as H3K4me1 characteristic of enhancer states. Second, expression of the cooperating transcription factor Lz converted enhancers towards this preferred chromatin state where additional Su(H) was recruited. By having a preference for a particular chromatin signature, the vast majority (>91%) of potential Su(H) binding motifs will be masked by unfavourable chromatin. Indeed, the small fraction of sites that do not fit with this pattern may reflect false positives in the ChIP data or in chromatin assignment. The greater paradox is that only 7%-10% of CSL motifs within the favourable Enh chromatin were bound. Furthermore, many of the positions that were differentially bound in two cell types existed in Enh chromatin in both cell types examined. These observations suggest that additional factors restrict CSL binding to a subset of sites located within favourable chromatin. Such factors might include currently unknown histone modifications, protein-protein interactions, 3D organisation and/or DNA sequence properties around the CSL motif (Skalska, 2015).

Once bound, Su(H) itself also helps to shape the local chromatin environment. Depleting cells of Su(H) resulted in an increase in local histone acetylation (H3K27ac, H3K56ac), suggesting that, in the absence of NICD, Su(H) helps to suppress enhancer activity through its association with co-repressors. Thus, a model emerges in which Su(H) is recruited to regions that have already acquired regulatory competence and that it keeps these in a transitional state with low levels of H3K56ac. As there is considerable variability between enhancers, this suggests that each attains an activity that reflects the balance between the transcription factors promoting enhancer activity and those, such as Su(H), that can antagonise it. In those instances where Su(H)-corepressor complexes win out, then the enhancer is suppressed until the complimentary activity of NICD converts it from a transitional to an active state, a conversion that is associated with a large-scale increase in H3K56ac (Skalska, 2015).

The extent that the principles observed in this study will be of general relevance for other signalling pathways remains to be established, although it seems likely that their target gene specificity will be similarly dependant on the pre-existing chromatin substrate. However, it is possible that the inferred transitional enhancer states may be particularly relevant for those pathways/contexts where there is a fine-scale switch between repression and activation, as occurs for Notch and ecdysone signalling. Nevertheless, the correlation of H3K56ac with H3K4me1 suggests that H3K56ac is likely to be of widespread importance in enhancer activation. Whether this will be mediated through its direct effects on DNA-histone core interactions or through intermediate bromodomain containing proteins that link to the core transcription machinery, such as Brd4, remains to be determined (Skalska, 2015).


Suppressor of Hairless: Biological Overview | Evolutionary Homologs | Protein Interactions | Developmental Biology | Effects of Mutation | References

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