InteractiveFly: GeneBrief

Enhancer of split m7, helix-loop-helix: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

The decision of ectodermal cells to adopt the sensory organ precursor fate in Drosophila is controlled by two classes of basic-helix-loop-helix transcription factors: the proneural Achaete (Ac) and Scute (Sc) activators promote neural fate, whereas the E(spl) repressors suppress it. E(spl) proteins m7 and mgamma are potent inhibitors of neural fate, even in the presence of excess Sc activity and even when their DNA-binding basic domain has been inactivated. Furthermore, these E(spl) proteins can efficiently repress target genes that lack cognate DNA binding sites, as long as these genes are bound by Ac/Sc activators. This activity of E(spl)m7 and mgamma correlates with their ability to interact with proneural activators, through which they are probably tethered on target enhancers. Analysis of reporter genes and sensory organ (bristle) patterns reveals that, in addition to this indirect recruitment of E(spl) onto enhancers via protein-protein interaction with bound Ac/Sc factors, direct DNA binding of target genes by E(spl) also takes place. Irrespective of whether E(spl) are recruited via direct DNA binding or interaction with proneural proteins, the co-repressor Groucho is always needed for target gene repression (Giagtzoglou, 2003).

Basic helix-loop-helix (bHLH) proteins constitute a large family of transcriptional regulators that are characterized by a basic DNA-binding domain contiguous with a dimerization domain consisting of two amphipathic alpha-helices separated by a loop. Members of this family are implicated in a multitude of biological functions, from proliferation to response to toxic stress. Most notable are members of a class of bHLH proteins, termed Class II, that are capable of directing cells towards specific fates; well-studied examples are the myogenic and the proneural factors. These bHLH proteins dimerize (via their HLH domains) with ubiquitous bHLH Class I co-factors, also known as E-proteins, as a prerequisite to DNA binding. The heterodimer acts as a transcriptional activator of multiple target genes, some of which encode transcription factors, thus setting off a cascade of gene regulation that implements the particular developmental program. Ac, Sc and L'sc are among the proneural bHLH proteins in Drosophila and together with the E-protein Daughterless (Da) are responsible for specifying most CNS and external sensory neural precursors (Giagtzoglou, 2003 and references therein).

Within the anlagen of the CNS and PNS, proneural genes are initially expressed in groups of cells termed proneural clusters. From these broad domains, only a subset of cells will commit to the neural fate. These neural precursors transiently upregulate proneural gene expression and activate a number of neural differentiation genes, such as asense, senseless/Lyra, deadpan and others, which are direct transcriptional targets of proneural bHLH activators. The remaining cells of the proneural cluster are inhibited from embarking into a neural pathway and will either continue proliferation or differentiate to alternative cell types, such as epidermis. This is the outcome of intercellular signaling within the proneural cluster, which is mediated by the Notch pathway and is termed lateral inhibition. Cells that receive a high level of Notch signal cannot turn on the proneural target genes (such as ase, dpn, etc.); this block requires the activity of members of yet another class of bHLH proteins, named Class VI or HES proteins. The seven clustered E(spl) genes in Drosophila, m8, m7, m5, m3, mß, mgamma and mdelta, encode Class VI bHLH proteins and are directly turned on (transcriptionally) by Notch signaling. Their products accumulate in all cells of the proneural cluster, but are minimal within the neural precursors; they can be therefore considered 'anti-neural' proteins. Indeed, deletion of the entire E(spl) locus results in severe overcommitment of neural precursors. However, mutations in individual E(spl) genes display no phenotypic defects, as a result of partial functional redundancy, a fact that prohibits forward genetic dissection of E(spl) protein function (Giagtzoglou, 2003 and references therein).

The link between proneural bHLH proteins, Notch signaling and HES proteins is evolutionarily conserved, since it is also encountered in vertebrates, where the cellular events of neurogenesis are very distinct from those in insects. In both phylogenetic groups, allocation of neural versus non-neural fates is the outcome of two antagonistic bHLH activities: proneural proteins that promote neurogenesis and HES proteins that inhibit it. As in Drosophila, in vertebrates some HES genes are direct transcriptional targets of Notch. Despite the central importance of these bHLH transcription factors in early neural commitment, there are many gaps in knowledge of the regulatory circuits underlying neurogenesis, both in terms of the target genes of proneural and HES genes, and in terms of the mechanisms of gene activation and repression by these bHLH proteins. It was originally proposed that E(spl) proteins might block neurogenesis in Drosophila by repressing proneural genes. More recent data suggest that this is true only for specific enhancers of the proneural genes that are autostimulatory and sensory organ precursor (SOP) specific, while the major function of E(spl) proteins is to repress downstream target genes of the proneural proteins. HES proteins are indeed transcriptional repressors. Key amino acid differences between the basic domains of HES and Ac/Sc proteins endow these different bHLH factors with distinct target site specificities: Da-Ac/Sc heterodimers bind the E_A box GCAGSTG (Singson, 1994), whereas E(spl) homodimers preferentially bind to E_B-boxes (CACGTG) and variants thereof, the C and N boxes (CACGCG and CACNAG, respectively) (Jennings, 1999; Oellers, 1994; Ohsako, 1994; Tietze, 1992; Van Doren, 1994). E_A, E_B, C and N boxes are encountered clustered in enhancers of proneural target genes, such as ase and dpn, which are expressed strongly in the neural precursor and repressed in the remaining proneural cluster cells. The importance of E_A sites in such enhancers has been confirmed by mutagenesis; ablation of E_A boxes leads to loss of transcriptional activity (Culi, 1998; Jarman, 1993). The same does not hold true, however, for E_B/C/N boxes; mutation of these does not lead to derepression of reporter genes -- mutant versions of the sc SMC enhancer lacking all E(spl) binding sites are still expressed only in the SOPs (Culi, 1998). Furthermore, E(spl) proteins retain residual activity after disruption of their DNA-binding basic domain, although this is still somewhat controversial. As a result, alternative models regarding the mechanism of target gene repression by E(spl) have been suggested. One proposes that E(spl) can sequester activator complexes away from DNA. A second model proposes that E(spl) proteins may be recruited to target enhancers indirectly, via interactions with other uncharacterized DNA bound factors (Giagtzoglou, 2003 and references therein).

The simplest explanation for the fact that proneural target enhancers can be repressed by E(spl) in the absence of cognate DNA-binding sites is that E(spl) proteins use a DNA-binding-independent mechanism for proneural target gene repression, instead of, or in addition to, a DNA-binding-dependent one. In the present work, it was asked if this is indeed the case. In vivo data is presented that strongly support protein-tether-mediated recruitment of some E(spl) repressors onto DNA -- interestingly, this is achieved via protein-protein interactions with proneural activators. Direct DNA binding also contributes significantly to E(spl) activity, while activator sequestering is unlikely to be used by E(spl) proteins to counteract proneural function (Giagtzoglou, 2003).

The first indication that E_B/C sites are dispensable for E(spl)-mediated repression came from reporter gene analysis in transfected Schneider S2 cells. T5-0.9wt/luc, a luciferase reporter driven by the proximal 5' regulatory region of the ac gene was used. This fragment probably constitutes an autoregulatory element, since it contains three E_A boxes and can be activated by Da/Sc or Da/Ac; it also contains one C-box needed for repression by the E(spl)-related protein Hairy. When an E(spl) expression plasmid (expressing either E(spl)m7, mgamma or mdelta) is included in addition to those expressing da and sc in a transient transfection experiment, repression of T5-0.9wt/luc was observed. A mutant version of the same reporter, T5-0.9mut/luc, was also used -- in this reporter the C box had been mutated, disabling repression by Hairy. E(spl)m7 and mgamma were still capable of repressing the mutant reporter, whereas E(spl)mdelta had lost the ability to repress, in fact it somewhat activated transcription [an unexplained result, also observed with Hairy. It thus appears that different members of the HES family of repressors may use different mechanisms of repression, with Hairy and E(spl)mdelta being strictly dependent on a DNA target site, versus E(spl)m7 and mgamma retaining activity in the absence of direct DNA binding (Giagtzoglou, 2003).

To gain more insight into this novel repression mechanism of E(spl)m7 and mgamma, an in vivo system was used. An artificial reporter gene in the fly driven solely by E_A boxes was used to avoid the possibility of E(spl) proteins binding to atypical sites, a behavior for which there is ample precedent (Chen, 1997; Culi, 1998; Yang, 2001), and may have been the cause of repression of T5m-luc in transfection experiments. The EE4-lacZ reporter, consisting of eight tandem E_A boxes in front of a minimal promoter was shown (Culi, 1998) to respond to proneural proteins by turning on in all proneural cluster cells in the wing disk. The response of EE4-lacZ was assayed in larval imaginal disks in response to E(spl) proteins expressed using the Gal4/UAS system. Overexpression of E(spl)m7 abolishes EE4-lacZ activity, whereas E(spl)mdelta only moderately reduces expression. This was somewhat surprising, given that E(spl) proteins do not recognize the E_A target site (Culi, 1998; Jennings, 1999; Oellers, 1994). Thus, the possibility was entertained that the repression by E(spl) was not a direct effect on the EE4 enhancer, rather it could have arisen from the fact that overexpression of E(spl) represses endogenous proneural genes, which in turn are needed to activate EE4. Therefore Ac protein was visualized in wing disks overexpressing E(spl)m7. The overall proneural pattern of Ac was not altered, but expression levels were variably reduced within the overexpression domain. Strongly expressing SOP cells within proneural clusters were never seen, in agreement with the well-established sensory-organ suppressive activity of E(spl) proteins (Giagtzoglou, 2003).

In order to test more rigorously the mechanism of E(spl)-mediated repression of EE4-lacZ and to avoid the fluctuation of endogenous proneural protein levels caused by E(spl) overexpression, the need for endogenous proneural proteins was bypassed altogether by providing excess Sc exogenously. A UAS-sc transgene was expressed alone or together with UAS-E(spl) transgenes. Ectopic Sc gave the expected broad, yet patchy, ectopic activation of EE4-lacZ. Patchy activation of proneural target genes has been observed before and apparently reflects stochastic damping of Sc activity, at least partly because of induction of endogenous E(spl) genes. Co-expression of E(spl)m7 results in strong repression of the EE4 enhancer, whereas E(spl)mdelta did not affect activation by UAS-sc. The same effects were observed using two different GAL4 lines, pnr-GAL4 and omb-GAL4, which drive expression in a central wing pouch region. It thus appears that E(spl)m7, but not mdelta, can repress transcription of EE4-lacZ without directly binding to DNA, consistent with the different behavior of these proteins in transfection assays. mdelta still weakly represses EE4-lacZ transcription, most probably through repression of activators, such as sc. Another UAS-E(spl) transgene, E(spl)mgamma, was able to repress UAS-sc-driven activation of EE4-lacZ, similar to E(spl)m7 (Giagtzoglou, 2003).

If direct DNA binding is dispensable for the repression by E(spl)m7 and mgamma of EE4-lacZ, mutant versions that lack the DNA-binding basic domain should be functional. E(spl)m7KNEQ, a double point mutation in the basic domain, which abolishes DNA binding, was generated, and it was tested in transgenic flies. UAS-E(spl)m7KNEQ has strong repressive activity on EE4-lacZ when expressed either alone or together with UAS-sc, confirming the dispensability of the basic domain in this assay. UAS-mgammaKNEQ, which bears the same basic domain inactivating mutations as m7KNEQ, was also capable of repressing EE4-lacZ, even in the presence of exogenous UAS-sc (Giagtzoglou, 2003).

In a converse experiment, the activity of the EE4-lacZ reporter was examined in loss-of-function backgrounds for E(spl). EE4-lacZ was consistently more active in a mutant background lacking E(spl)m7 and m8, as compared with wild type. This happens even though the number and pattern of SOPs in this mutant background is identical to wild type, presumably owing to the activity of the remaining E(spl) genes. It is concluded that activity of E(spl)m7 and m8, the two most highly expressed E(spl) genes in wing disk proneural clusters, attenuates EE4-lacZ expression. Since E(spl) genes other than m7 and m8 were still present in the above genetic background, the response of EE4-lacZ was tested in homozygous clones of a deficiency removing the entire E(spl) locus. Increased levels of ß-galactosidase expression were again observed within mutant patches, confirming the response of EE4-lacZ to E(spl) activity, despite the absence of E(spl) binding sites on this reporter. A caveat in interpreting these experiments is that E(spl) loss-of-function may increase sc expression, which would then act on the EE4-lacZ reporter (Giagtzoglou, 2003).

Ectopic expression of sc in flies is known to induce formation of supernumerary chetae, reflecting induction of endogenous Sc target genes. Individual UAS-E(spl) transgenes were tested for their ability to block ectopic cheta production by sc. When expressed alone by pnr-Gal4, all UAS-E(spl) genes inhibited formation of both macro- and mirco-chetae, resulting in a bald stripe in the center of the thorax. This was even true for UAS-E(spl)m7KNEQ and E(spl)mgammaKNEQ, suggesting that, under the conditions of this assay, direct DNA binding (to presumably natural target genes controlling SOP fate) is dispensable. When co-expressed with UAS-sc, UAS-E(spl)m7 and mgamma, as well as E(spl)m7KNEQ and mgammaKNEQ, still produced completely bald thoracic stripes, indicating that these proteins can inhibit the activity of both endogenous and overexpressed Sc on (endogenous) target genes very effectively. By contrast, UAS-E(spl)mdelta only partially suppresses the ectopic bristle phenotype of UAS-sc. This behavior was essentially the same as that documented above using EE4-lacZ and was further confirmed by assaying the expression of two target genes, SMC-lacZ and ase. The sole difference was that E(spl)mdelta could partially decrease the number of ectopic bristles, while having no effect on EE4-lacZ activation. The bristle/SOP suppressive activity of E(spl)mdelta is attributed to DNA-binding-dependent repression of proneural target genes. Taken together, reporter and bristle repression assays demonstrate that E(spl)m7 and mgamma, but not mdelta, can repress an E_A-driven artificial reporter gene, as well as endogenous target genes, despite the overexpression of sc. Based on the fact that basic domain mutated versions of E(spl)m7 and mgamma are much more potent repressors than mdelta, it is concluded that in this assay some activity of E(spl) proteins other than their direct DNA binding ability is most important in target gene repression (Giagtzoglou, 2003).

The most important conclusions from this study are the following: (1) targets of E(spl) repression are the target genes of the proneurals, and to a lesser extent the proneural genes themselves; (2) E(spl) recruitment onto target genes can occur via direct DNA binding (to E_B/C/N boxes), but also via interactions with E_A-box-bound proneural activators; (3) sequestration of the proneural activators off DNA, a mechanism employed by the Emc/Id family of HLH proteins, does not seem to operate in the case of E(spl), and (4) in both DNA-mediated and activator-mediated modes of E(spl) tethering to target genes Groucho recruitment is required for repression (Giagtzoglou, 2003).

It is sometimes assumed that E(spl) proteins suppress neurogenesis solely by repressing proneural gene transcription. This is not the case, since E(spl)m7 and mgamma can completely block sensory organ commitment in a background of exogenously (transgenically) provided high levels of Sc. Target genes (genuine and artificial) that are activated by Da/Sc are still repressed by E(spl)m7 and mgamma in the above genetic background. This is consistent with the earlier observation that E(spl) overexpression has only a moderate effect on ac expression, whereas it completely represses downstream targets, such as SMC-lacZ (Culi, 1998), ase or EE4-lacZ. Even though ac and sc are not the main targets of E(spl), some of their enhancers are repressible by E(spl). ac and sc genes elaborate expression pattern is dependent on a number of prepattern enhancers, which are controlled by patterning systems and are weakly, if at all, repressible by E(spl) (Gomez-Skarmeta, 1995). One enhancer each of ac [the proximal 900bp (Martinez, 1993) and sc (the SMC enhancer) (Culi, 1998) has been described that is repressible by E(spl). Both of these are autoregulatory inasmuch as they contain E_A boxes and are activated by Da/Sc or Ac, hence they act to boost ac/sc levels after transcription has been initiated via the prepattern enhancers; in this context the SMC and ac-proximal enhancers can be viewed as 'target genes' of the proneural proteins (Giagtzoglou, 2003).

Another piece of evidence in favor of regulation of proneural target genes (rather than proneural genes themselves) by E(spl) is that E(spl)m7VP16 can activate the neural pathway in genetic backgrounds mutant for ac and sc. Other than displaying aberrant spacing, bristles produced in such a background are normal, at least in external appearance. This is consistent with E(spl)m7VP16 binding and activating many, perhaps all, target genes of Ac/Sc (not just the autoregulatory ac/sc enhancers), bypassing the need for proneural proteins. One should be aware, however, that there are other bHLH proneural genes, besides ac and sc, in the fly genome; e.g. l'sc is not affected by the sc^10-1 allele used in this study. Although l'sc is not normally expressed in the larval wing disk, it is conceivable that it is turned on by E(spl)VP16 activators and then takes over the task of activating the panel of downstream genes. Another potential candidate that might single-handedly mediate the sensory-organ promoting activity of E(spl)VP16 is ase, a SOP-specific gene that bears homology to the proneural genes of the ac/sc family and can act as a proneural gene itself. Thus, it is a matter of further research whether the bristle-induction ability of E(spl)VP16 in a sc^10-1 background is channeled through activation of a single E(spl) target gene or of a number of target genes (Giagtzoglou, 2003).

All proneural target genes contain E_A boxes, via which the Da/proneural activators exert their effect. Analysis of the EE4-lacZ enhancer has revealed that the same E_A boxes are sufficient for E(spl)-mediated repression, even though the latter bind a different class of target sites, the E_B/C/N-boxes. Based on the data presented in this work, it is proposed that this is achieved by enhancer recruitment of E(spl) proteins via protein-protein interactions with proneural activators. This study has focused on three E(spl) proteins. Two, m7 and mgamma, have been shown to interact with both Da and Ac/Sc (Alifragis, 1997) and in the present study display an equivalent ability to be indirectly recruited onto DNA by Da/Sc. The third, E(spl)mdelta, shows no proneural-mediated recruitment activity, apparently because of its inability to interact with either Da or Sc. Perhaps this Da/Sc-binding activity of some of the E(spl) proteins has evolved to enable them to repress all proneural target genes effectively without the need for direct DNA binding. Ac and Sc seem to play a central role in this repression mechanism, since the ubiquitous Da is not sufficient to recruit E(spl)-VP16 proteins to EE4-lacZ and other proneural target enhancers (Giagtzoglou, 2003).

Even though E(spl) proteins can be recruited onto their target genes via proneural complexes, all characterized proneural target enhancers (e.g. SMC, ac-proximal, ase, dpn, neur) do bear E_B/C/N-boxes in addition to E_A-sites. Likewise, all E(spl) proteins possess well-conserved DNA-contacting basic domains. Two observations from this work strongly suggest that direct DNA binding is also used in the repression of target genes by E(spl). (1) A significant suppression of bristle formation by E(spl)mdelta is observed upon co-expression with Sc. This can only be interpreted as repression of Sc targets by E(spl)mdelta by direct binding to their E_B/C/N-boxes, since it has been established that E(spl)mdelta is incapable of proneural-mediated enhancer binding. (2) E(spl)m7VP16, but not a basic region mutant version, turns on bristle commitment in the absence of proneural genes, pointing towards DNA-binding-dependent recruitment onto proneural target genes (Giagtzoglou, 2003).

The realization that some E(spl) proteins can act as both repressors and co-repressors of the proneurals prompts reconsideration of the proneural proteins as dedicated transcriptional activators; they seem to be equally important in effecting repression of their target genes. Other transcriptional activators, such as Dorsal and HNF4 can act as repressors in certain contexts, suggesting that this may be quite a widespread mechanism (Giagtzoglou, 2003).

This study has used a transgenic approach to establish the ability of E(spl) proteins to be recruited onto target genes by the two mechanisms discussed above. It cannot be predicted whether in a wild-type background the two mechanisms are used exclusively of one another or simultaneously. The presence of E_B/C/N-boxes in close proximity to E_A-boxes in enhancers of proneural target genes favors the latter possibility, namely that proneural and E(spl) proteins each bind their cognate target sites and subsequently also interact at the protein level. Protein-protein interaction concomitant with DNA binding may enable cooperative enhancer binding, which would ensure a rapid response of target genes to changes in concentration of proneural and E(spl) proteins (Giagtzoglou, 2003).

Having realized the plausibility for two (alternative or simultaneous) modes of E(spl) recruitment onto target enhancers, a complete picture still does not exist of what it takes (in terms of transcriptional regulation) to achieve a robustly laterally inhibited response to proneural activity; in other words, to turn on a proneural target gene solely in the neural precursor. The artificial EE4-lacZ enhancer, though responsive to wild-type levels of E(spl) is still not fully repressed, and is expressed in most cells of a wild-type proneural cluster. By contrast, another enhancer that also lacks E_B/C/N boxes has been reported to be fully repressible by wild-type levels of E(spl): SMCN-delta147-181 is a mutant version of the SMC enhancer lacking all E(spl)-binding sites, but containing two E_A-boxes -- this enhancer expresses solely in the neural precursor (SOP) and not in surrounding proneural cluster cells (Culi, 1998). One can hypothesize that additional factors binding SMCN-delta-147-181 favor the formation of a repressive DNA-protein complex in the E(spl)-containing non-SOPs. Indeed this enhancer contains two copies each of conserved alpha and ß boxes (bound by unknown factors) interspersed with the E_A boxes (Culi, 1998). One or both of these factors may cooperate with low (wild-type) levels of E(spl) (bound to E_A via interaction with the proneural complex) to stabilize Gro binding to this enhancer; indeed Gro often has to simultaneously interact with more than one DNA bound factor to gain access to an enhancer (Giagtzoglou, 2003).

Natural proneural target enhancers contain E_A, E_B, C, N, alpha and ß boxes, in addition to binding sites for other factors, such as the Zn-finger protein Senseless. Some of these enhancers (e.g. SMC, ase, dpn, neur) are expressed solely in the neural precursor, whereas others [ac proximal, sca, various E(spl) enhancers] are expressed more widely within the proneural cluster, apparently not responding (or less responsive) to lateral inhibition. Yet, the two types of enhancers are not obviously different with respect to types of target sites contained. Perhaps it is the exact number and arrangement of the various target sites and DNA-bound factors that defines the threshold level of lateral inhibition that each enhancer is responsive to. Seen in this light, it is conceivable that interaction of E(spl) with proneural factors (and perhaps other factors within a large protein-DNA complex) may bring about conformational changes, which are needed to fine-tune crosstalk of these transcription factors with co-activators, co-repressors and other components of the transcriptional machinery. Characterizing these regulatory interactions will improve insight on the transcriptional mechanisms that mediate neural fate acquisition and will be a major challenge for the future (Giagtzoglou, 2003).

REGULATION Promoter Structure

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 from promoter-reporter transgene experiments is presented that regulation at the transcriptional level is primarily responsible. Certain E(spl)-C genes were known previously 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 is presented that reveals 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]. 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).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Transcriptional Regulation

In Drosophila imaginal discs, the spatially restricted activities of the achaete (ac) and scute (sc) proteins, which are transcriptional activators of the basic-helix-loop-helix class, define proneural clusters (PNCs) of potential sensory organ precursor (SOP) cells. Several genes are direct downstream targets of ac-sc activation, as judged by the following criteria. The genes are expressed in the PNCs of the wing imaginal disc in an ac-sc-dependent manner; the proximal promoter regions of all of these genes contain one or two high-affinity ac-sc binding sites, which define the novel consensus GCAGGTG(T/G)NNNYY; where tested, these binding sites are required in vivo for PNC expression of promoter-reporter fusion genes. Interestingly, these ac-sc target genes, including Bearded, Enhancer of split m7, Enhancer of split m8, and scabrous, are all known or believed to function in the selection of a single SOP from each PNC, a process mediated by inhibitory cell-cell interactions. Thus, one of the earliest steps in adult peripheral neurogenesis is the direct activation by proneural proteins of genes involved in restricting the expression of the SOP cell fate (Singson, 1994).

The Notch signaling pathway is involved in many processes where cell fate is decided. Previous work has shown that Notch is required at successive steps during R8 specification in the Drosophila eye. Initially, Notch enhances atonal expression and promotes atonal function. After atonal autoregulation has been established, Notch signaling represses atonal expression during lateral specification. Once ato autoregulation is established, lateral specification starts to limit ato expression to R8 precursor cells. Thus Notch signaling is required at successive steps during R8 specification, initially to promote neural potential and later to suppress it through lateral specification. Consequently the phenotype of loss of Notch gene function varies with time. If Notch function is removed conditionally once ato expression has been enhanced, supernumerary R8 cells differentiate because lateral specification is affected. If N function is absent from the outset, such as in a clone of cells lacking N, little R8 specification can occur. For this reason clones of N null mutant cells in the eye disc almost completely lack neural differentiation, contrasting with the neurogenic phenotype of null mutant embryos. Using clonal analysis it is shown that Delta, a ligand of Notch, is required along with Notch for both proneural enhancement and lateral specification (Ligoxygakis, 1998).

E(spl) bHLH genes have been shown to be transcriptionally activated as a direct consequence of Notch signaling and, along with the corepressor protein Groucho, to mediate inhibition of proneural genes in the nucleus. In the eye, Notch-dependent expression of mdelta and mgamma accompanies repression of ato expression, suggesting that at least these two of the E(spl) bHLH genes contribute to R8 patterning during lateral specification. In addition, mdelta and perhaps mgamma are also transiently expressed prior to lateral specification, and the m7, m8 and mbeta genes are transcribed in distinct patterns that remain uncharacterized in detail for lack of specific antibodies. Thus, particular E(spl) bHLH proteins might mediate proneural Notch signaling as well as or instead of lateral specification. Clones of cells deleted for portions of the E(spl) complex were used to define its role more precisely. The E(spl)b32.2 deficiency deletes all seven bHLH genes and m4. Partial gro function was supplemented in the experiment by a linked gro + transgene. E(spl)b32.2 gro + homozygous cells display a cell autonomous neurogenic phenotype quite unlike that of N or Dl mutant clones. Antibodies against Boss or Elav proteins each label a much greater number of cells within the clone than in the surrounding wild-type tissue. Some clones were difficult to photograph because neurogenic regions often seem to fold in on themselves and crease the eye disc. Because neurogenesis can still occur, it appears that the proneural function of Notch can proceed without any E(spl)-C bHLH genes, whereas N function in lateral specification is severely impaired. Clones of cells homozygous for E(spl)BX22 are also neurogenic in phenotype. E(spl)BX22 affects gro and the bHLH genes m5, m7 and m8. It follows that gro is also dispensable for the proneural function of Notch, although it is probably required in lateral specification (Ligoxygakis, 1998).

Midline repression by Sim functions by activating transcription of one or more genes that, in turn, repress transcription of genes normally expressed in the lateral CNS. The nature of these repressive factor genes and how they function are unknown, although plausible candidate genes exist. Since E(spl) proteins repress lateral CNS expression, members of this family are candidates for midline repressors, and several are expressed in the CNS midline cells early in development (m5, m7, and m8). In this scheme, Sim:Tgo would activate factors that would modify or interact with E(spl) proteins to repress proneural gene activity in the midline. The vnd upstream regulatory region contains numerous E(spl) consensus binding sites, although none of the sites lie within the 0.5-kb fragment shown to be important for repression. Since the E(spl) proteins reside in midline cells well before repression occurs, it is unlikely that Sim is required for initial E(spl) transcription, although maintenance of their expression is a possibility. Other potential repressors have not yet been identified (Estes, 2001).

Targets of Activity

To learn about the acquisition of neural fate by ectodermal cells, a very early sign of neural commitment in Drosophila has been analyzed, namely the specific accumulation of achaete-scute complex (AS-C) proneural proteins in the cell that becomes a sensory organ mother cell (SMC). An AS-C enhancer has been analyzed that directs expression specifically in SMCs. To delimit the sequences responsible for expression in SMCs, subfragments of a 3.7-kb fragment immediately upstream of scute were assayed for their ability to drive lacZ expression in wing discs. The necessary sequences are within a 356-bp fragment. This fragment specifically directed expression in SMCs. It also promotes expression in SMCs of other imaginal discs and of the embryonic PNS, but not in neuroectoderm neuroblasts. The SMC enhancer is shown to promote macrochaetae formation. Interspecific sequence comparisons and site-directed mutagenesis show the presence of several conserved motifs necessary for enhancer action, some of them binding sites for proneural proteins. The conserved sequences contain three E boxes: these are putative binding sites for bHLH proteins of the Achaete, Scute, and Daughterless type. The most proximal of the three is adjacent to an N box, a site that can be recognized by the E(spl)-C bHLH proteins. In addition, there are three copies of a motif reminiscent of a consensus binding site for the NF-kappaB family of transcription factors (named alpha1, alpha2, and alpha3), and three copies of a T-rich motif (termed beta1, beta2 and beta3) that does not fit with known protein-binding sequences. In spite of considerable effort, the NF-kappaB family member binding to the alpha motifs has not been identified (Culi, 1998).

N signaling, triggered by Ac-Sc in the emitter cell, promotes in the receptor cell the accumulation of E(spl)-C proteins, the main effectors of this signal. E(spl)-C proteins are detectable in proneural cluster cells, except for the SMCs. This correlates with the SMCs being the cells that signal maximally and inhibit their neighbors from acquiring the neural fate, while the SMCs themselves are not inhibited. Ectopic accumulation of E(spl)-C protein prevents SMCs from emerging, as detected by a neuralized enhancer trap line and the consequent absence of SOs in the adult fly. Overexpression of UAS-E(spl)-m8 or UAS-E(spl)-m7 transgenes driven by da-GAL4 or the C-253 GAL4 lines block the activity of the SMC enhancer and the development of the corresponding SOs. In contrast, either of these overexpressions allowed normal accumulation of Ac and Sc in proneural clusters despite the high levels of ectopic E(spl)-m8 mRNA, which are severalfold higher than those in the wild type. However, overexpression with presumably stronger GAL4 drivers does interfere with ac-sc expression in proneural clusters. Taken together these results indicate that the function of the SMC enhancer is more sensitive to E(spl)-C inhibition than are the proneural cluster enhancers, and suggest that the SMC enhancer is the main target of lateral inhibition mediated by the N pathway (Culi, 1998).

Does E(spl)-m8 bind to the SMC enhancer, given the inhibition of SMC enhancer function by E(spl)-C? E(spl)-m8 binds to the N box and, unexpectedly, also protects a broad region of the enhancer (nucleotides 142-182), which does not contain sequences that fit the E(spl)-C consensus binding site. Binding to an enhancer with a mutated N box is weaker, and binding to an enhancer without the N box and the second E(spl)-m8-binding site is undetectable. Remarkably, the removal of one or both binding sites does not modify the SMC specificity of the enhancer, as might be expected if these binding sites mediated the repression of enhancer function in response to N signaling. E(spl)-m8 is unable to bind to the synthetic SMC-specific minienhancer. These results were extended to other E(spl)-C proteins by verifying that [similar to E(spl)-m8] E(spl)-m5 binds to an oligonucleotide with the E1-N sequence, but not to oligonucleotides containing only E2 or E3 boxes. Thus, it is concluded that the E(spl)-C proteins restrict enhancer function to SMCs by a mechanism that does not require direct interaction with enhancer DNA. Thus the Enhancer of split bHLH proteins block the proneural gene self-stimulatory loop, possibly by antagonizing the action on the enhancer of the NF-kappaB-like factors or the proneural proteins. These data suggest a mechanism for SMC committment (Culi, 1998).

The overall similarity in the binding of different E(spl) proteins in vitro suggests that they are capable of recognizing the same targets in vivo and is consistent with the phenotypes observed when the individual proteins are expressed ectopically. Ectopic expression of M8, M5, Mbeta, Mdelta, and M7 all produce phenotypes of vein and bristle loss. Both Mbeta and M7 are able to interact with DNA sequences regulating achaete. The ability to recognize the same DNA target sequences could explain the apparent redundancy between the E(spl) genes, as they would all have the potential to act in the same processes. The observation that specific E(spl)bHLH proteins are more or less efficient in regulating different processes (e.g., Mbeta more effective at suppressing veins and M8 more effective at suppressing bristles) is thus more likely to be consequence of differences in protein:protein interactions than of differences in target recognition (Jennings, 1999 and references).

In the absence of E(spl)bHLH proteins, proneural protein expression persists at high levels in all cells of a proneural cluster. Thus, one action of E(spl)bHLH proteins is to antagonize the proneural proteins, with the ultimate consequence that proneural gene expression is repressed. It has been proposed that E(spl)bHLH proteins exert their influence by binding to regulatory regions within the AS-C and repressing transcription of the proneural genes. This hypothesis is supported by the observations that expression of Achaete is induced by M7^ACT and Mbeta^ACT and that induction of ectopic bristles in the Drosophila wing and notum by M7^ACT is abolished in the absence of proneural proteins. One putative binding site for the E(spl)bHLH proteins, that upstream of the achaete gene, has the sequence 5'-CGGCACGCGACA-3' (Hairy site). Mgamma will bind this site in vitro, and M7 can bind this sequence and repress transcription in a cotransfection assay in Drosophila S2 cultured cells. However, mutation of this site in vivo results in a phenotype resembling that caused by mutations in hairy rather than in the E(spl)-C. This fits with the observation that this sequence conforms to an optimal Hairy DNA binding site but is a suboptimal site for the E(spl) proteins and indicates that the E(spl) proteins do not recognize this sequence in vivo. Thus, if E(spl) proteins are directly repressing achaete expression, there should be more optimal target sites elsewhere within the AS-C. Indeed, a search of recently available AS-C genomic sequence identifies >10 sequences with good matches to ESE boxes, in addition to the sites that have been identified by in vitro binding assays (Jennings, 1999 and references).

An alternative hypothesis is that the primary function of the E(spl)bHLH proteins is to antagonize the actions of proneural proteins posttranscriptionally. Evidence in support of this comes from experiments in which L'sc is ectopically expressed using a heterologous promoter that is not subject to direct regulation by E(spl)bHLH proteins. Under these conditions L'sc expression results in isolated ectopic bristles, rather than clusters of bristles, demonstrating that lateral inhibition is still able to restrict neural fate to a single cell even though l'sc transcription is insensitive to Notch signaling. This implies that E(spl)bHLH proteins are able to antagonize proneural genes in ways other than by repressing their transcription. One possibility is that the E(spl) proteins can interact with the same targets as proneural proteins, but that they repress rather than activate transcription. The ability of E(spl) proteins to bind to the B1 and A1 sequences and repress transcription from a heterologous promoter is consistent with this model, as is the observation that M7^ACT can induce certain ectopic leg bristles in the absence of the achaete and scute genes. In the latter context, M7^ACT is likely to be acting on genes with functions downstream of the proneural proteins to cause neural differentiation. In addition, the E(spl)bHLH proteins are involved with developmental processes that do not involve the proneural proteins, e.g. wing vein development; thus, they cannot act solely to repress proneural gene transcription during development (Jennings, 1999 and references).

E(spl) proteins are basic helix-loop-helix (bHLH) repressors, and most of them (m7, m8, mß, mdelta, and mgamma) are expressed in the posterior undifferentiated cells in eye discs. When E(spl) proteins (e.g., m7 and m8) are overproduced in eye discs, the yan enhancer activity is strongly reduced. Similarly, the level of Yan protein is also reduced. These results show that yan expression can be negatively regulated by E(spl) proteins. E(spl) proteins might act through an N box (5'-CACAAG-3') in the enhancer. Interestingly, mutations of the N box didn't cause upregulation of the reporter gene, but, instead, the reporter expression was abolished in all three transgenic lines. One explanation for this result is that the N box sequence might be shared by an activation element located in the region. Indeed, a Runt domain binding site (RBS) (5'-RACCRCA-3', R = purine) overlaps with the N box, which could mediate an effect by the Runt domain protein Lozenge (Lz), which has previously been shown to act as a transcriptional activator in the developing eye. Supporting this idea, the yan enhancer was completely inactivated in lz^r15 mutant eye discs. However, the level of Yan protein was not apparently affected by the lz mutation. This result suggests that Lz is not essential for the expression of the endogenous yan gene and that the loss of lz function could be compensated by other molecules so that yan expression is unaffected in lz mutants (Rohrbaugh, 2002).

Protein Interactions

The basic HLH domain of the proteins coded for by the Enhancer of split and achaete-scute complexes differ in their ability to form homo- and heterodimers. The bHLH domains of E(spl)C proteins m5, m7 and m8 interact with bHLH domains of the Achaete and Scute proteins. These bHLH domains form an interaction network which may represent the molecular mechanism whereby the competent state of proneural genes is maintained until the terminal determination to neuroblast identity occurs (Gigliani, 1996).

Neural fate specification in Drosophila is promoted by the products of the proneural genes, such as those of the achaete-scute complex, and is antagonized by the products of the Enhancer of split [E(spl)] complex, hairy, and extramacrochaetae. Since all these proteins bear a helix-loop-helix (HLH) dimerization domain, their potential pairwise interactions were investigated using the yeast two-hybrid system. The fidelity of the system was established by its ability to closely reproduce the already documented interactions among Daughterless, Achaete, Scute, and Extramacrochaetae. The seven E(spl) basic HLH proteins can form homo- and heterodimers inter-se with distinct preferences. A subset of E(spl) proteins (Mß and M5) can heterodimerize with Da, another subset (M3) can heterodimerize with proneural proteins, and yet another (Mbeta, Mgamma and M7) with both, indicating specialization within the E(spl) family. Hairy displays no interactions with any of the HLH proteins tested. It does interact with the non-HLH protein Groucho, which itself interacts with all E(spl) basic HLH proteins, but with none of the proneural proteins or Da. An investigation was carried out of the structural requirements for some of these interactions, using site-specific and deletion mutagenesis. Deletion analysis of M3 and Scute is consistent with their interaction being mediated by their respective bHLH domains. The dependence of the E(spl)-activator HLH interactions on the HLH domain is nicely reflected in the fact that the functional grouping of the E(spl) proteins correlates well with the amino acid sequences of their bHLH domains, e.g., M5 and M8 have highly similar bHLH regions, different from those of the M7/Mbeta/ and Mgamma group, which also display high intragroup similarity. The strong interactions observed between E(spl) proteins and proneural proteins might lead one to hypothesize the E(spl) proteins act like Extramachrochaetae, i.e., by sequestering HLH activators. This is unlikely, since residual activities of E(spl) proteins with mutated basic domains have only weak residual activities (Alifragis, 1997).

Drosophila melanogaster casein kinase II interacts with and phosphorylates the basic helix-loop-helix proteins m5, m7, and m8 derived from the Enhancer of split complex

Drosophila Casein kinase II (DmCKII) is composed of catalytic (alpha) and regulatory (beta) subunits associated as an alpha2beta2 heterotetramer. Using the two-hybrid system, a D. melanogaster embryo cDNA library has been screened for proteins that interact with DmCKIIalpha. One of the cDNAs isolated in this screen encodes m7, a basic helix-loop-helix (bHLH)-type transcription factor encoded by the Enhancer of split complex [E(spl)C], which regulates neurogenesis. m7 interacts with DmCKIIalpha but not with DmCKIIbeta, suggesting that this interaction is specific for the catalytic subunit of DmCKII. In addition to m7, DmCKIIalpha also interacts with two other E(spl)C-derived bHLH proteins, m5 and m8, but not with other members, such as m3 and mC. Consistent with the specificity observed for the interaction of DmCKIIalpha with these bHLH proteins, sequence alignment suggests that only m5, m7, and m8 contain a consensus site for phosphorylation by CKII within a subdomain unique to these three proteins. Accordingly, these three proteins are phosphorylated by DmCKIIalpha, as well as by the alpha2beta2 holoenzyme purified from Drosophila embryos. In line with the prediction of a single consensus site for CKII, replacement of Ser(159) of m8 with either Ala or Asp abolishes phosphorylation, identifying this residue as the site of phosphorylation. m8 forms a direct physical complex with purified DmCKII, corroborating the observed two-hybrid interaction between these proteins. Substitution of Ser(159) of m8 with Ala attenuates interaction with DmCKIIalpha, whereas substitution with Asp abolishes the interaction. These studies constitute the first demonstration that DmCKII interacts with and phosphorylates m5, m7, and m8 and suggest a biochemical and/or structural basis for the functional equivalency of these bHLH proteins that is observed in the context of neurogenesis (Trott, 2001).

All E(spl)C-derived proteins are structurally conserved. The sequence alignment emphasizes conservation of the HLH domain, helices III and IV (also known as Orange domain), a motif in the vicinity of the C terminus with a high PEST score, and the WRPW motif. The seven E(spl) proteins have been aligned with emphasis on residues N-terminal to the basic domain and those comprising the region from helix IV to the C terminus (C-domain) to determine whether some structural features were unique only to m5, m7, and m8. No sequences in the N terminus were found that were conserved among and/or unique to these three proteins. In contrast, analysis of the C-domain indicates that only these three proteins contain a consensus site for phosphorylation by CKII, ¹⁵⁶SDNE in m5, ¹⁶⁸SDNE in m7, and ¹⁵⁹SDCD in m8, immediately following the highly conserved sequence, (I/L)SP(V/A)SSGY, in a region that is characterized by a high PEST score. Although PEST-rich sequences act as cis-acting signals that regulate protein turnover and have been suggested to be activated via phosphorylation, the role of this motif in m5/7/8 is currently unknown. This conserved Ser in m5/7/8 conforms to the requirement that it must contain an acidic residue at the n+1 and n+3 positions to be a target for CKII. It should be noted, that although mß also contains a site for phosphorylation by CKII (¹⁹⁵SEDE), m is neither preceded by the (I/L)SP(V/A)SSGY sequence nor contained within its PEST motif. Interestingly, the cytology and spatial organization of the E(spl)C locus of Drosophila hydei exhibits an extraordinary level of conservation relative to that of D. melanogaster (Maier, 1993). Because the DNA sequence of only D. hydei m8 is currently available, this protein was compared with m5, m7, and m8 from D. melanogaster. Remarkably, D. hydei m8 also contains the CKII site following the (I/L)SP(V/A)SSGY sequence, both of which are contained within a region with a high PEST score (Trott, 2001).

One question raised by the sequence alignment was whether the presence of the consensus CKII site in m5, m7, and m8 correlates with their phosphorylation. Therefore GST-m5, GST-m7, GST-m8, and GST-mC, a noninteracting member, were subjected to phosphorylation using two isoforms of CKII, i.e. the monomeric alpha subunit purified from a yeast expression system, and the alpha₂ß₂ holoenzyme purified from embryos. The former isoform mimics the two-hybrid analysis, whereas the latter mimics the in vivo environment. The results demonstrate that m5, m7, and m8 are phosphorylated by both isoforms of CKII and corroborate their observed two-hybrid interaction with DmCKIIalpha. At a quantitative level, however, the rates of phosphorylation of the three E(spl) proteins are different for both enzyme isoforms, such that m5 > m7 = m8. What mechanism can account for the observed differences? Detailed kinetic analysis of CKII suggests that whereas DmCKIIalpha and the holoenzyme display virtually identical k_m values for the protein substrate, the K_cat can differ 5-50-fold in a substrate-dependent manner. Furthermore, studies with peptides suggest that whereas the acidic residues at n+1 and n+3 are absolutely required for phosphorylation, additional acidic residues C-terminal to the n+3 position further increase the K_cat with marginal effects on the k_m. These criteria, therefore, make it possible to predict the relative rates of phosphorylation of m5/7/8. In this regard, although m7 and m8 fit the consensus, m5 is probably the best because it contains an additional Asp at the n+4 position. The rank order for phosphorylation is, therefore, predicted to be m5 > m7 = m8. The analysis presented in this study essentially reflects this prediction. Because the gel analysis described in this study inherently reflects a semiquantitative assessment of phosphorylation, kinetic analysis will be necessary to determine whether the observed differences in phosphorylation of m5 versus m7/8 are due to differing catalytic efficiencies. That CKII interacts with and phosphorylates these proteins is consistent with the observation that this kinase has been found to exist in a complex with some of its in vivo substrates, such as Topoisomerase II, HSP90, ANTP, and Dishevelled, to name a few (Trott, 2001).

These results raise the likely prospect that DmCKII interacts with m5/7/8 when these proteins are in the nonphosphorylated state and that the complexes dissociate upon phosphorylation. It is not possible to extrapolate the two-hybrid and biochemical results to the situation in the epidermal precursors in the developing Drosophila embryo with certainty. However, given the requirements of CKII for cell cycle progression, it is likely that epidermal progenitors, which are expressing E(spl) proteins, also contain CKII. A direct test of this proposal in the developing embryo still remains a difficult task due to restricted expression of m5/7/8 and the absence of isoform-specific antibodies. At a functional level, the data indicate that interaction and/or phosphorylation of m5/7/8 is unlikely to affect their DNA binding properties (which require the basic region), their ability to heterodimerize with proneural proteins (which requires the HLH domain), or their ability to interact with Groucho (which requires the WRPW motif). What function could then be ascribed to interaction and/or phosphorylation? The structural and functional properties common to m5/7/8, and by extension those in D. hydei m8, provide the basis for a likely possibility. As mentioned above, all three proteins contain a PEST-rich motif that harbors an invariant Ser residue that is phosphorylated by CKII. In this regard, a mutation that removes sequences encompassing the PEST-rich region and the resident CKII site acts as a dominant-negative allele with regard to suppression of bristle development (Giebel, 1997). That this variant of m8 behaves as a dominant-negative, rather than a loss-of-function (as one would have predicted), suggests that the mutant protein might sequester endogenous wild-type m8, and possibly m5 and m7 as well, thus leading to enhanced neurogenesis. Thus, this region negatively regulates the activity of m8, in line with its ability to homodimerize or heterodimerize with m5 and m7 (Alifragis, 1997; Gigliani, 1996). These results and their interpretations are consistent with the proposal that this region of m5/7/8 may influence the stability of these proteins in vivo. A collective theme that emerges is that phosphorylation, in at least a restricted class of proteins, regulates protein stability via activation of PEST motifs. These studies further implicate the PEST motif in m5, m7, and m8 as a target for regulation via CKII-mediated phosphorylation. Future studies employing expression of epitope-tagged m8 and its nonphosphorylatable and/or constitutively phosphorylated variants in transgenic flies will be needed to clarify the role of this motif (Trott, 2001).

In summary, these data demonstrate that select members of the E(spl)C, i.e. m5, m7, and m8, physically interact with DmCKII and are phosphorylated by this enzyme at an invariant Ser residue that is contained within a motif unique to these three isoforms. The suggestion that these three proteins are more functionally related and that the C-terminal domain of m8 acts to negatively regulate function in vivo implicates the PEST motif and its resident CKII phosphorylation site. These data strengthen the contention for the presence of a new functional motif in these transcriptional repressors and raise the possibility that CKII may regulate neurogenesis via posttranslational modification of these proteins (Trott, 2001).

Senseless acts as a binary switch during sensory organ precursor selection

During sensory organ precursor (SOP) specification, a single cell is selected from a proneural cluster of cells. Evidence is presented that Senseless (Sens), a zinc-finger transcription factor, plays an important role in this process. Sens is directly activated by proneural proteins in the presumptive SOPs and a few cells surrounding the SOP in most tissues. In the cells that express Sens low levels, Sens acts in a DNA-binding-dependent manner to repress transcription of proneural genes. In the presumptive SOPs that express Sens at high levels, Sens acts as a transcriptional activator and synergizes with proneural proteins. It is therefore proposed that Sens acts as a binary switch that is fundamental to SOP selection (Jafar-Nejad, 2003).

Proneural genes have been shown to be required for sens expression. To determine whether proneurals directly activate sens expression, the putative enhancers of sens were identified and were scanned for proneural protein-binding sites (E boxes). An 11-kb genomic fragment containing the sens locus is able to rescue the sens mutant phenotype. To identify the embryonic and imaginal disc enhancers, three genomic DNA fragments were used to create lacZ reporter transgenes. Both 5.9-kb and 3.4-kb fragments are sufficient to drive expression in the embryonic PNS in a pattern similar to endogenous sens. To refine sens enhancers, the 3.4-kb enhancer was divided into nine overlapping fragments. Fragments 8 and 9 induced lacZ expression in a pattern similar to the original 3.4-lacZ line, indicating that both contain regulatory elements sufficient for sens expression in the embryonic PNS. Fragments 8 and 9 were further divided into overlapping fragments. Only 9-1-lacZ expresses the reporter in a pattern similar to the 3.4-lacZ. Inspection of the 9-1 sequence showed that it contains a single E box. The recently sequenced genome of Drosophila pseudoobscura, a species 25-30 myr divergent from Drosophila melanogaster was used to align the genomic regions. The alignment showed that the E box, as well as several other elements in the 9-1 enhancer, is fully conserved. Upon mutation of this E box from CAGGTG to CCGGTG, most of the PNS cells failed to express lacZ, and staining in other cells was much weaker than for the wild-type transgene. These data indicate that proneural genes directly regulate the transcription of sens (Jafar-Nejad, 2003).

It is thought that the two core nucleotides of the E box as well as its flanking sequences are involved in the specificity of each E box for its cognate bHLH transcription factor. It was intriguing that expression of the lacZ marker is almost abolished in chordotonal organs that are dependent on atonal (ato) as well as in external organs and multiple dendritic organs that are dependent on ac, sc, and amos. Because the 9-1 fragment contains only a single E box, the data suggest that different proneural proteins can bind the same E box in vivo. Therefore EMSA was performed to determine whether a variety of Da-proneural heterodimers can shift a wild-type or an E box-mutated probe taken from the 9-1 sequence. Da homodimer, Ato/Da heterodimer, Ac/Da heterodimer, and Sc/Da heterodimer were all able to bind to this E box. Mutation from A to C in the second position of the E box abolished binding for all protein combinations tested, suggesting that these interactions are sequence specific. It is concluded that at least three proneural proteins (Ac, Sc, and Ato) directly regulate sens expression in the embryonic PNS, and that they may bind the same site in vivo (Jafar-Nejad, 2003).

To examine whether sens regulation in the precursors of the adult PNS is also under direct proneural regulation, the 9-1-lacZ and 9-1-mut-lacZ expression patterns were compared in the SOPs of the thoracic microchaetae. Similar to what was observed in embryos, a single-nucleotide change in the 9-1 E box abolishes most of the lacZ expression in pupae of the same age, again suggesting direct regulation of sens by proneurals (Jafar-Nejad, 2003).

The effects of loss- and gain-of-function of proneural genes on sens expression were assessed in the imaginal discs of third instar larvae. Because fragments 9 and 9-1 do not drive lacZ at this stage, enhancer 8 was used. The 8-lacZ transgene drives lacZ expression in several wing SOPs in late third instar larvae. To determine whether proneural genes are able to control 8-lacZ expression, Sc was overexpressed in the wing pouch using the C5-GAL4 driver. Many more cells express lacZ in the wing pouch than in wild type, indicating that the Sc protein is able to induce lacZ expression ectopically. However, removal of the activity of both ac and sc genes results in loss of lacZ expression in all of the ac/sc-dependent SOPs. The precursors of the ventral radius and the femoral chordotonal organs still express lacZ, since these cells are dependent on Ato expression. Moreover, upon Ato overexpression driven by dpp-GAL4, 8-lacZ is strongly induced at the A/P boundary. Together, these data indicate that proneural proteins regulate sens expression in the precursors of the adult PNS. Fragment 8 contains two E boxes, one of which is fully conserved between D. pseudoobscura and D. melanogaster. Band-shift experiments show that the Ac/Da heterodimer can bind to a radioactive probe that contains the conserved E box of fragment 8, further suggesting that proneurals directly regulate sens expression (Jafar-Nejad, 2003).

E(spl) proteins are known to prevent SOP formation through transcriptional repression of proneural gene expression. Whether they affect sens expression was examined. scabrous (sca)-GAL4 was used to express E(spl)m8 in the SOPs and a few cells around the SOPs in third instar imaginal discs. lacZ expression is abolished in most or all cells. Moreover, misexpression of an 'activator' version of E(spl)m7 (m7^ACT), in which the Gro-binding motif is replaced with the VP16 transactivator domain, caused numerous extra lacZ-positive cells when driven in the wing pouch. These observations suggest that E(spl)m7 and E(spl)m8 proteins are also involved in the transcriptional regulation of sens and that proneural proteins and E(spl) proteins have an antagonistic relationship in transcriptional control of sens. E(spl) proteins are known to bind to proneural gene enhancers and m7^ACT is able to activate ac and sc transcription. Therefore, it is formally possible that m7^ACT is indirectly activating the sens enhancer through its up-regulation of proneural proteins. However, it has been shown that even in the absence of endogenous ac and sc, overexpression of m7^ACT causes extra bristle formation, suggesting that the E(spl) proteins not only regulate proneural gene expression, but also regulate the expression of one or more of proneural target genes. Is m7^ACT able to induce sens expression in the absence of ac and sc? To address this question, it was confirmed that overexpression of m7^ACT can produce several extra bristles in a sc^10-1 background. Staining of the imaginal wing discs of these flies shows that there are many Sens-positive cells in the anterior part of the presumptive notum, where the Eq-GAL4 driver used in this experiment is expressed. The data suggest that sens is one of the targets of the E(spl) proteins. Altogether, sens enhancers seem to be able to integrate the positive and negative inputs from proneural and E(spl) proteins, respectively (Jafar-Nejad, 2003).

Protein-protein interactions play a significant role in determining how a transcription factor regulates its target genes. To identify proteins that bind Sens, a yeast two-hybrid (YTH) screen was performed. Of 38 positives sequenced from the screen, seven correspond to members of the E(spl) complex. To confirm the interactions identified in yeast, coimmunoprecipitation (co-IP) assays were performed using in vitro-translated E(spl) proteins and myc-tagged Sens. A monoclonal anti-myc antibody can precipitate E(spl)m7, E(spl)m8, and E(spl)m5 only in the presence of myc-Sens. The yeast two-hybrid assay was used to identify the interaction motif in each partner. Testing a series of Sens deletion constructs showed that a 25-amino acid fragment of Sens (amino acids 276-300) is necessary and sufficient for Sens/E(spl) interaction. To further delineate the interaction motif, the 25 amino acids were mutated to alanines five at a time and five mutant sens constructs were generated. The YTH assays suggested that a 15-amino acid deletion (Sens-del) would abrogate the interaction for all three members of the E(spl) complex. This was indeed observed (Jafar-Nejad, 2003).

Proteins of the E(spl) complex have several conserved motifs, for example, a basic domain, a Helix Loop Helix (HLH) domain, an Orange domain, a caseine kinase-binding motif (CK), and a WRPW or Gro interaction domain (W). To find the interaction motif in the E(spl) proteins, deletion constructs of E(spl)m8 were created and their ability to bind Sens was tested in yeast. The Orange domain was necessary and sufficient for the Sens/E(spl)m8 interaction. The 25-amino acid motif of Sens in isolation interacts with the Orange domain of E(spl)m8 in isolation in the yeast two-hybrid assay. The Orange domain is conserved in all members of the Hairy-E(spl) family of proteins and there is evidence that this domain is functionally important. Alignment of the Orange domains of E(spl)m5, E(spl)m7, and E(spl)m8 prompted mutation of three amino acids in each of the two conserved motifs to alanine and the ability of mutant m8 proteins to interact with Sens was tested. Replacement of EVS with AAA or THL with AAA is sufficient to abolish the interaction of E(spl)m8 with Sens in the yeast assay. In summary, the data indicate that Sens and E(spl) proteins interact in yeast and in vitro (Jafar-Nejad, 2003).

Two modes of recruitment of E(spl) repressors onto target genes

E(spl) proteins interact selectively with proneural ones in a yeast two-hybrid assay (Alifragis, 1997); E(spl)m7 and mgamma interact with Ac, Sc and Da, whereas mdelta interacts with none. Tests with mutant E(spl) proteins indicate that some activity of E(spl) proteins other than their direct DNA binding ability is most important in target gene repression. In the light of these results, it is possible that the ability of E(spl) proteins to interact with activator bHLH proteins might underlie the ability of the former to repress target genes in the absence of direct DNA binding and enhance their potency in neural fate suppression. The question arises as to how interaction with proneural proteins might help realize this potent repressive activity: do E(spl) proteins sequester proneural activators off the target DNA or do they use the proneural complexes as tethers to bind to DNA? A way to approach the question of whether a repressor works on or off DNA has been devised by Jiménez (1997), whereby a fusion of a strong transcriptional activation domain (VP16) to a repressor is tested for its ability to activate transcription, which can only happen if the VP16 domain is tethered to the DNA. If, however, the repressor works by sequestering activators off DNA, the VP16-tagged repressor should still be able to repress (rather than activate) target genes (Giagtzoglou, 2003).

A hybrid E(spl)m7VP16 protein was expressed in wing disks and its effect on EE4-lacZ was assayed. In both pnr-Gal4 and omb-Gal4 expression domains, strong activation of EE4-lacZ was observed, suggesting that E(spl)m7VP16 is somehow tethered to this artificial enhancer. Rather than being ubiquitous, activation by E(spl)m7VP16 was patterned in a way that strongly resembles the proneural pattern, suggesting that E(spl)m7VP16 is tethered to EE4-lacZ via proneural complexes. To demonstrate this, the same effector-reporter combination was assayed in both loss-of-function and gain-of-function backgrounds for proneural genes. sc^10-1 is a null allele for both ac and sc, the only proneural proteins expressed in the wing disk. In sc^10-1 wing disks, EE4-lacZ was not expressed and could not be activated by E(spl)m7VP16. In the converse experiment, ectopic Sc was supplied by co-expressing UAS-sc with UAS-m7VP16; in this case, the pattern of EE4-lacZ activation was broadened to encompass the whole expression domain and was not restricted to proneural clusters. It therefore appears that it is the availability and spatial distribution of proneural proteins that determines the pattern of activation of EE4-lacZ by E(spl)m7VP16. The simplest way to account for this finding is to propose that E(spl)m7VP16 is recruited onto DNA using the proneural complexes (and not some other DNA-bound factor) as tethers. This was confirmed by testing the ability of two other E(spl)VP16 variants: E(spl)mgammaVP16 and mdeltaVP16. Whereas the former behaves identically to E(spl)m7VP16, E(spl)mdeltaVP16 has no effect on EE4-lacZ expression. The inability of E(spl)mdeltaVP16 to become recruited onto EE4-lacZ is attributed to its inability to interact with the proneural protein-tethering factors (Giagtzoglou, 2003).

It is possible that proneural cluster restriction of EE4-lacZ activation by E(spl)m7VP16 and mgammaVP16 was due to some regional inactivation (by protein modification) of the VP16 effector itself, and not to recruitment onto DNA via proneural complexes. It was therefore asked whether the E(spl)-VP16 variants are inherently capable of transcriptional activation in all cells by assaying their ability to activate another artificial enhancer (Gbe-B1-lacZ) that bears three E_B boxes (recognized by HES-family proteins) in addition to binding sites for Grh, an activator ubiquitously present in wing disk cells. In a wild-type background, Gbe-B1-lacZ is expressed very weakly and cannot be activated by UAS-sc, since Sc only weakly binds the B1 E_B box. In the presence of UAS-E(spl)m7VP16, mgammaVP16 or mdeltaVP16, strong ubiquitous activation was observed, indicating that all three E(spl)VP16 variants are strong activators when directly tethered to DNA and their activity does not seem to be spatially modulated. It is therefore thought that the variable activation of EE4-lacZ reflects selective recruitment of the VP16 proteins onto the EE4 enhancer and is not a result of post-translational modulation of their transactivation ability. This result also strengthens the conclusion from that E(spl)mdeltaVP16 cannot become recruited onto EE4-lacZ (Giagtzoglou, 2003).

An E(spl)m7VP16 variant with mutated basic region should behave in a manner complementary to E(spl)mdeltaVP16, since it should lack direct DNA-binding activity but should retain the ability to be indirectly tethered to targets via proneural proteins. The behavior of a UAS-E(spl)m7KNEQ-VP16 transgene showed that this was indeed the case. This effector was unable to activate the Gbe-B1-lacZ reporter, confirming disruption of its basic region. By contrast, it was able to activate the EE4-lacZ reporter to the same extent as wild type E(spl)m7VP16. One interesting difference was that the activity of E(spl)m7KNEQ-VP16 was restricted to proneural clusters (where ac and sc are expressed), whereas E(spl)m7VP16 gave additional patchy activation of EE4-lacZ in non-proneural cells of the pnr-Gal4 domain. This was accompanied by marked ectopic accumulation of the Ac proneural protein, something not seen with E(spl)m7KNEQ-VP16. Ectopic activation of endogenous proneural genes by E(spl)m7VP16 is probably achieved by directly binding to enhancers that contain E_B/C/N boxes (such as the autoregulatory ones), because it is abolished by mutation of the basic region. The resulting ectopic proneural protein is subsequently used as a tether to bring E(spl)m7VP16 onto the EE4-lacZ reporter. To bypass this feedback loop involving endogenous proneural genes, Sc was supplied via co-expression of a UAS-sc transgene. UAS-sc alone results in patchy activation of EE4-lacZ. However, in the presence of E(spl)m7VP16 or m7KNEQ-VP16 activation become ubiquitous and much stronger, reflecting ubiquitous tethering of the E(spl)m7VP16 effector regardless of the integrity of its basic domain (Giagtzoglou, 2003).

The data presented so far have highlighted a novel mechanism of target gene repression by E(spl), one that requires recruitment on DNA via protein-protein interactions with proneural proteins. What role, if any, does direct DNA binding play in the activity of E(spl) proteins? This question was addressed by assaying the ability of E(spl)VP16 variants to activate endogenous target genes in the absence of ac and sc, which eliminates the possibility of proneural-protein-mediated recruitment. All E(spl)m7VP16, mgammaVP16 and mdeltaVP16 induced bristles when driven by pnr-Gal4 in a sc^10-1 background. This suggests that these E(spl)VP16 variants can bypass the requirement for endogenous proneural genes and trigger the sensory organ pathway, presumably by directly activating one or more proneural target genes. Indeed direct binding of target genes must be involved, since cheta production in a sc^10-1 background was abolished by mutating the basic domain of E(spl)m7VP16. In a wild-type background, E(spl)m7KNEQ-VP16 induces fewer ectopic bristles than its wild-type counterpart, which suggests a lower activity, consistent with its ability to activate target genes only via protein-mediated recruitment, whereas E(spl)m7VP16 can also directly bind to its target genes. mgammaKNEQ-VP16 behaved identically to m7KNEQ-VP16. Therefore, both mechanisms, direct DNA contact and interaction with the pre-bound proneural activators, seem to play a role in the recruitment of E(spl) proteins to their target genes. It should be noted that in a wild-type background both E(spl)m7- and mdelta-VP16 variants produce a larger number of excess bristles than is produced in a sc^10-1 background, indicating synergy between the hybrid E(spl) activators and the proneural ones, which is in part due to protein-mediated recruitment of the former onto the latter (Giagtzoglou, 2003).

E(spl) proteins are known to recruit the co-repressor Groucho in order to silence target genes (Fisher, 1998). It is conceivable that when E(spl) proteins exert their repressive effect by interacting with proneural proteins, a different mechanism might be at play, such as occlusion of the transcriptional activation domain of proneural activators. It was therefore of interest to address whether Gro is needed to mediate repression when E(spl) proteins are indirectly bound to DNA. To this end, expression of UAS-sc was driven together with UAS-E(spl)m7 in a mosaic background containing patches homozygous for the severe gro^E48 allele and the response of the EE4-lacZ reporter was assayed. This reporter is repressed by E(spl)m7 exclusively via protein-mediated recruitment. Indeed in gro⁺ territory little or no expression was observed, as expected; however, within mutant clones EE4-lacZ was strongly expressed. Therefore, E(spl) proteins employ a Gro-dependent repression mechanism regardless of mode of recruitment on target genes (Giagtzoglou, 2003).

The requirement for Gro was corroborated by cuticle phenotype: gro^E48 clones produce tufts of bristles on the notum, a result of the breakdown of lateral inhibition during SOP commitment. Although ubiquitous expression of E(spl)m7 abolishes bristles, when it was induced in gro^E48 clones in an ap-Gal4; UAS-E(spl)m7 background (which abolishes bristles throughout the notum), patches of high bristle density were recovered in a bald notum. This suggests that ectopic (as well as normally expressed) E(spl)m7 cannot repress endogenous target genes in the absence of Gro, just as it cannot repress the artificial EE4-lacZ target. Finally, a UAS-E(spl)m7deltaW transgene, which lacks the C-terminal tryptophane of the Gro-binding WRPW motif, was completely inactive in both bristle suppression and reporter gene repression. A corollary from these experiments is that E(spl)m7 does not function by sequestering proneural activators off DNA. The latter activity should have no requirement for a co-repressor like Gro, since physical removal of activators should suffice to turn target genes off (Giagtzoglou, 2003).

Role of the Scute C terminus in transcriptional activation and E(spl) repressor recruitment

Neurogenesis in all animals is triggered by the activity of a group of basic helix-loop-helix transcription factors, the proneural proteins, whose expression endows ectodermal regions with neural potential. The eventual commitment to a neural precursor fate involves the interplay of these proneural transcriptional activators with a number of other transcription factors that fine tune transcriptional responses at target genes. Most prominent among the factors antagonizing proneural protein activity are the HES basic helix-loop-helix proteins. Two HES proteins of Drosophila, E(spl)m gamma and E(spl)m7, interact with the proneural protein Sc and thereby get recruited onto Sc target genes to repress transcription. Using in vivo and in vitro assays an important dual role has been discovered for the Sc C-terminal domain: (1) it acts as a transcription activation domain, and (2) it is used to recruit E(spl) proteins. In vivo, the Sc C-terminal domain is required for E(spl) recruitment in an enhancer context-dependent fashion, suggesting that in some enhancers alternative interaction surfaces can be used to recruit E(spl) proteins (Giagtzoglou, 2005).

The fact that proneural and E(spl) bHLH proteins have mutually antagonistic activities has long been accepted. This study describes a molecular basis for this antagonism of the Sc-E(spl)m7 pair, which relies on the ability of the latter to interact and inhibit the activity of the TAD of the former. Sc and E(spl)m7 were dissected and in the process the following were identified: (1) the TAD of Sc, which resides in its 25 C-terminal amino acids; (2) the E(spl)m7 interaction domain of Sc, which is identical to or overlaps with the Sc TAD, and (3) the Sc interaction domain of E(spl)m7, which is contained within the N-terminal 80 amino acids. Three more of the seven E(spl) proteins, E(spl)mgamma., E(spl)mbeta, and E(spl)m3, share the ability E(spl)m7 to inhibit the Sc TAD, consistent with an increased structural similarity among these four E(spl) proteins (Giagtzoglou, 2005).

To address a possible in vivo role for this interaction between Sc and E(spl) proteins, several points have to be taken into consideration. Natural enhancers recruit a number of transcription factors and co-factors to assemble an enhanceosome, which regulates transcription initiation. For example, Da-Sc target enhancers may variably contain additional activators, such as a putative NFkappaB-like alpha-factor, Sens, or Sis-a. While affording robustness in gene regulation, the multifactorial nature of the enhanceosome and its ability to assemble itself using multiple alternative macromolecular interactions may cause frustration to the researcher trying to dissect out the function of individual components. Artificial enhancers, in contrast, can reveal functions of individual domains, because they rely on a small number of transcription factors because of the very simplicity of their design. Using the artificial enhancers UAS-tk-luc and EE4-lacZ, it was shown that the Sc C terminus is necessary for both transcriptional activation and recruitment of E(spl) proteins. Already, when assayed on a more complex natural enhancer, ac-lacZ, the role of the Sc C terminus starts becoming blurry. Equally good activation and E(spl)m7KNEQ-VP16 recruitment appears to take place whether the Sc C terminus is present or not. This is attributed to the presence of alternative TADs and alternative contact surfaces that are able to recruit E(spl)m7 onto this enhanceosome but not onto the simpler EE4-lacZ. Other than Sc, transcription factors that have been reported in the literature to interact with E(spl)m7 are Da and Sens. Although the presence of Da (predicted to bind on EE4-lacZ) can only weakly sustain transcription and E(spl)m7 recruitment in the absence of Sc TAD, the presence of Sens or some other yet-to-be-identified E(spl) interaction surface on the ac-lacZ appears to render the Sc TAD dispensable in some assays. It is noteworthy that E(spl) uses different domains to contact Sc (the N-terminal region) versus Sens (the middle Orange region). The existence of more than one protein-protein interaction domain on any given factor is likely to be advantageous in complex formation. Establishing contacts via both the N terminus and the Orange domain would likely result in cooperative recruitment, allowing an E(spl) protein to repress a Sc+Sens-containing enhanceosome more effectively. Further functional characterization of the E(spl) proteins will determine the relative contribution of each documented (or yet-to-be documented) protein-protein interaction, as well as of direct DNA binding, to recruitment onto target genes (Giagtzoglou, 2005).

The C terminus of Sc is conserved in other Sc family proneural proteins in Drosophila (Ac and L'sc), as well as homologues from other phyla,which in itself argues for some important function. Its role had been overlooked so far; in fact an earlier report had proposed that it is dispensable for the proneural activity of Lethal of Scute (L'sc). In that work, a transgene essentially consisting of only the bHLH domain of L'sc (l'scDelta) was able to promote ectopic sensory organ (bristle) production, only slightly more weakly than full-length L'sc. Because that transgene was not tested against specific reporter genes such as the ones used in this study, caution is required in drawing conclusions about the function of the L'sc C terminus for the reasons described above. Namely, bristle production is the outcome of the activation of a (still ill-defined) number of Sc (L'sc) target genes driven by complex enhancers and multiple factors, the presence of which might compensate for the lack of the L'sc C-terminal domain. So, in a transgenic assay, the presence of the Sc (or L'sc) TAD may be dispensable, whereas its bHLH domain is sufficient to recruit Da to the bristle-promoting target genes to nucleate the assembly of complex enhanceosomes. Indeed the behavior of C-terminally truncated transgenes sc_1-260 and sc_1-290 is entirely consistent with that of l'scDeltaNDeltaC. Adult flies expressing the UAS-sc transgene have approximately the same number of ectopic bristles as those expressing UAS-sc_1-260 or UAS-sc_1-290. These very same genotypes, however, display a dramatic difference in the activation of the EE4-lacZ reporter (Giagtzoglou, 2005).

Does phylogenetic conservation of the C terminus imply that both functions, TAD and HES repressor recruitment, have also been conserved? Preliminary data suggest that at least the TAD function has been conserved in Mash1. Additionally, some evidence exists in the literature, consistent with protein interaction-mediated antagonism between Mash1 and HES1. Mash1 promotes and HES1 inhibits neuronal differentiation of rat hippocampal neural precursors in culture. Importantly, transfection of Mash1 together with HES1 also inhibits neuronal differentiation, suggesting that HES1 antagonizes Mash1 post-translationally. In a reporter assay, this ability of HES1 to antagonize Mash1 was retained by a basic region mutant of HES1. This would be consistent with HES1 interacting with the TAD of Mash1 to block its activity, independently of the ability of HES1 to bind DNA. Further dissection of these and related vertebrate bHLH proteins will reveal the extent to which the present documented mechanism has been conserved through evolution (Giagtzoglou, 2005).

Another interesting question raised by this work regards the remaining proneural proteins. The second subclass of proneural proteins, the Ato/Ngn subclass, is equally important in neural precursor commitment but has a bHLH domain different from that of the achaete-scute proteins and, most importantly for the present discussion, lacks the conserved C-terminal TAD. In fact the TADs of Ato/Ngn proteins remain to be identified. It will be interesting to determine whether the Ato/Ngn TADs have also evolved to be inhibited by HES proteins. Preliminary analysis has shown that Ato can interact with two E(spl) proteins in yeast two-hybrid, so an analogous mechanism for this proneural subclass is conceivable (Giagtzoglou, 2005).

Pervasive regulation of Drosophila Notch target genes by GY-box-, Brd-box-, and K-box-class microRNAs

Although hundreds of distinct animal microRNAs (miRNAs) are known, the specific biological functions of only a handful are understood at present. Three different families of Drosophila miRNAs directly regulate two large families of Notch target genes, including basic helix-loop-helix (bHLH) repressor and Bearded family genes. These miRNAs regulate Notch target gene activity via GY-box (GUCUUCC), Brd-box (AGCUUUA), and K-box (cUGUGAUa) motifs. These are conserved sites in target 3'-untranslated regions (3'-UTRs) that are complementary to the 5'-ends of miRNAs, or 'seed' regions. Collectively, these motifs represent >40 miRNA-binding sites in Notch target genes, and all three classes of motif are shown to be necessary and sufficient for miRNA-mediated regulation in vivo. Importantly, many of the validated miRNA-binding sites have limited pairing to miRNAs outside of the "box:seed" region. Consistent with this, it was found that seed-related miRNAs that are otherwise quite divergent can regulate the same target sequences. Finally, it is demonstrated that ectopic expression of several Notch-regulating miRNAs induces mutant phenotypes that are characteristic of Notch pathway loss of function, including loss of wing margin, thickened wing veins, increased bristle density, and tufted bristles. Collectively, these data establish insights into miRNA target recognition and demonstrate that the Notch signaling pathway is a major target of miRNA-mediated regulation in Drosophila (Lai, 2005).

The E(spl)-C and Brd-C of Drosophila melanogaster (Dm) contain two large families of direct Notch target genes, including seven bHLH repressor-encoding genes and 10 Bearded family genes. With the exception of E(spl)mbeta and Ocho, all of these genes contain GY-box (GUCUUCC), Brd-box (AGCUUUA), and/or K-box (UGUGAU) motifs in their 3'-UTRs, which are propose to be miRNA-binding sites. Nine of these genes contain three or more box sites, a density that is especially remarkable when one considers how short their 3'-UTRs are (often <350 nt in length). The conservation of these sites were systematically assessed in their orthologs from Drosophila pseudoobscura (Dp) and Drosophila virilis (Dv), species that are ~30 million and 60 million years diverged from Dm, respectively. 33/51 Brd-boxes, GY-boxes, and K-boxes have been perfectly conserved and reside in syntenic locations among all three species; 11 additional sites are identical in two of the three species. This indicates that all three motifs are under strong selective constraint (Lai, 2005).

Closer examination of nucleotide divergence surrounding these boxes has revealed some unexpected features that are germane to the proposition that these boxes represent miRNA-binding sites. These features are best illustrated by comparing rapidly evolving genes. Notably, Bearded is an unusually rapidly evolving protein, with only 15 residues preserved between Dm and Dv orthologs (out of 81 and 66 amino acids, respectively), and Dv Bearded has a significantly different arrangement of these 3'-UTR motifs. The 3'-UTR of Dv E(spl)m5 is also quite different from its counterparts in Dm/Dp. Alignment of Dm/Dp orthologs of Bearded and E(spl)m5 reveals that sequences upstream of most GY-boxes are well conserved; these regions include most sequences presumed to pair with miR-7. Similar patterns are seen for many other GY-boxes in other Notch target genes. However, the sequence upstream of many Brd- and K-boxes is strongly diverged, so that only 'box'-pairing is often preserved. In fact, many Brd- and K-boxes generally lack extensive pairing to miRNAs outside of the 'box' sequence. These factors likely preclude their identification by various published computational algorithms for miRNA-binding sites. Indeed, Brd- and K-boxes in Notch target genes have been deemed unlikely to represent miRNA-binding sites. In contrast, rapid divergence of the upstream portion of miRNA-binding sites is consistent with the idea that pairing between the miRNA "seed" (positions ~2-8) and the 3'-UTR 'box' (approximately the last one-third of the miRNA-binding site) is most critical for miRNA-mediated regulation (Lai, 2005).

It is also noted that precise spacing of several motif occurrences that are closely paired is also conserved, even though orthologous 3'-UTRs otherwise display significant insertions and deletions. In these cases, one would presume that simultaneous binding of miRNAs to their respective sites would not be possible unless the 3'-end of the downstream miRNA was unpaired, a configuration that unexpectedly proved functional in vitro. Finally, there are a few nonconserved boxes in these 3'-UTRs (7/51 total sites). In several cases, the nonconserved site is highly related to a neighboring conserved site [i.e., the first and second GY-boxes of Dp E(spl)m4 are equally similar to the first GY-box in Dm E(spl)m4; the third and fourth Brd-boxes in Dp E(spl)m5 are highly related to the third Brd-box in Dm E(spl)m5], implying that these nonconserved sites may be functional, newly evolved miRNA-binding sites (Lai, 2005).

GY-box-, Brd-box-, and K-box-class miRNAs are highly conserved among diverse insects, and many are, indeed, identical. Therefore Brd-boxes, GY-boxes, and K-boxes were sought in the predicted 3'-UTRs of E(spl)bHLH and Brd genes from mosquitoes, bees, and moths; these species cover ~350 million years of divergence from Drosophila. Impressively, homologs of both E(spl)bHLH and Brd genes in these highly diverged species all contain multiple copies and multiple classes of 'box' motifs in their 3'-UTRs. This strongly suggests that regulation by all three families of miRNAs is an ancient feature of Notch target gene regulation in insects (Lai, 2005).

To directly test the capacity of miRNAs to regulate the 3'-UTRs of these Notch target genes, an in vivo assay was used. The target in this assay is a ubiquitously expressed reporter (tub>GFP or arm>lacZ) fused to an endogenous 3'-UTR (a 3'-UTR sensor). The reporter transgene is introduced into a genetic background in which a UAS-DsRed-miRNA transgene is activated with dpp-Gal4 or ptc-Gal4. This results in ectopic miRNA production in a stripe of red-fluorescing cells at the anterior–posterior boundary of imaginal discs. Inhibition of the green reporter within the red miRNA-misexpressing domain reflects direct miRNA-mediated negative regulation. Focus was placed on the central wing pouch region of the wing imaginal disc (Lai, 2005).

The ability of sensor transgenes for most Bearded family genes [Bob, Bearded, Tom, Ocho, E(spl)malpha, and E(spl)m4] and most E(spl)bHLH repressor genes [E(spl)mgamma, E(spl)mdelta, E(spl)m3, E(spl)m5, and E(spl)m8] to be regulated by ectopic GY-box-, Brd-box-, and K-box-class miRNAs was extensively analyzed. Sensor expression is influenced by the level to which it is negatively regulated by endogenous factors, including miRNAs. In this assay, the disc sensor must be expressed at sufficient levels before one can observe its knock-down by ectopic miRNAs. 3'-UTR sensor constructs for different Notch target genes accumulate to different levels in vivo, consistent with variable amounts of endogenous miRNA-mediated regulation. Nevertheless, it was possible to reliably detect expression of all sensors excepting E(spl)m8. As detailed in the following three sections, these sensors were used to unequivocally demonstrate GY-boxes, Brd-boxes, and K-boxes to be sites of miRNA-mediated negative regulation by corresponding families of complementary miRNAs in vivo (Lai, 2005).

miR-7 is the only known Drosophila miRNA whose 5'-end is complementary to the GY-box (GUCUUCC). miR-7 has been shown to regulate three GY-box targets, including two members of the E(spl)-C, E(spl)m3 and E(spl)m4. While these two genes scored well in a genome-wide prediction of miR-7 targets, many other members of the Brd-C and E(spl)-C also contain between one and three GY-boxes in their 3'-UTRs [Bob, Bearded, Tom, E(spl)mgamma, E(spl)m5]. Of these, only Tom was computationally identified as a compelling candidate for miR-7 (Lai, 2005).

The specificity of the disc sensor assay was assayed by showing that neither an empty tub-GFP sensor nor an Ocho sensor were affected by miR-7. The previous experiments done with E(spl)m3 and E(spl)m4 were repeated and it was observed that both were, indeed, inhibited by ectopic miR-7. This assay was used to demonstrate that miR-7 negatively regulates all seven GY-box-containing members of the Brd-C and E(spl)-C, including those with single sites [E(spl)m3, E(spl)mgamma, and Bearded], those with two sites [E(spl)m4, Tom, Bob], and those with three sites [E(spl)m5]. These data convincingly support the hypothesis that GY-boxes are general signatures of miR-7-binding sites in Notch target genes, irrespective of the overall amount of pairing between miR-7 and sequences outside of the GY-box. In order to more definitively demonstrate that miR-7-mediated regulation occurs through identified GY-boxes, mutant sensors bearing point mutations in the GY-boxes were tested. A Bearded sensor carrying five point mutations in its single GY-box no longer responded to miR-7. In a more stringent test, an E(spl)m5 sensor carrying 2-nt mutations in each of its three GY-boxes was generated. These targeted changes also abolished the ability of miR-7 to negatively regulate E(spl)m5. Therefore, ~7 continuous base pairs between the 'box' motif and its cognate miRNA seed are critical for in vivo target regulation. It is also noted that when mutant 3'-UTRs are tested, a mild increase in reporter activity in miRNA-misexpressing cells was sometimes observed, the reason for which has not been determined (Lai, 2005).

Previous work has suggested synergism between miRNA-binding sites on the same transcript. Multiple GY-box 3'-UTRs were generally subject to greater regulation than single-site 3'-UTRs, even though the amount of miR-7 pairing to individual GY-boxes in multiple-site 3'-UTRs is often less than its pairing with single GY-box 3'-UTRs. Indeed, negative regulation of E(spl)m4, Tom, Bob, and E(spl)m5 by miR-7 was qualitatively indistinguishable from an artificial sensor containing two perfectly miR-7-complementary sites, even though many sites in these genes display relaxed pairing with miR-7 outside of GY-boxes. This suggests that as little as 7–8 nt of complementarity may suffice for miRNA target recognition, especially where multiple sites are present. However, since all three single GY-box-containing 3'-UTRs were also regulated by miR-7, synergism is not required for biologically significant regulation by miRNAs (Lai, 2005).

There are two Drosophila miRNAs, miR-4 and miR-79, whose 5'-ends are complementary to the Brd-box (AGCUUUA). Both miRNAs are resident in miRNA clusters, and miR-4 resides in particularly dense clusters containing several unrelated miRNAs. Use was made of a UAS-DsRed-miR-286, miR-4, miR-5 transgene that is referred to as "UAS-miR-4" and a UAS-DsRed-miR-79 transgene. miR-4 and miR-79 have only limited similarity outside of their Brd-box seed, and there is little indication from pairwise alignments that these miRNAs are specifically "tuned" to different Brd-box sites in Notch target genes. In fact, all of these Brd-boxes lack the extended complementarity to miRNAs that is typical of miR-7:GY-box pairs, and no Notch target genes were previously predicted computationally as targets of miR-4 or miR-79 (Lai, 2005).

Seven Brd-box-containing Notch target genes were validated as being regulated by Brd-box-family miRNAs, including those with single sites [Tom, E(spl)mdelta, E(spl)mgamma] and those with multiple sites [Bearded, E(spl)malpha, E(spl)m4, and E(spl)m5]. Curiously, the negative regulatory effects of miR-4 on E(spl)mgamma, E(spl)malpha, E(spl)m4, and E(spl)m5 were greater than those of miR-79 on these same 3'-UTRs, even though miR-4 is no more complementary to these sites than is miR-79. Nevertheless, the common ability of miR-4 and miR-79 to down-regulate individual sensors indicates that cross-regulation of individual sites by multiple members of a given miRNA family may occur. Notably, both miRNAs are expressed at high levels during embryonic development (Lai, 2005).

The specificity of miR-4 and miR-79 was tested using two mutant Bearded sensors, one bearing several point mutations in each of its three Brd-boxes and another containing mutations in the Brd-boxes and the GY-box. In both cases, the mutant transgenes accumulate to higher levels, consistent with relief from negative regulation by endogenous Brd-box-class miRNAs in the wing disc. In addition, they are no longer responsive to ectopic Brd-box-class miRNAs, indicating that the observed regulation occurs directly via Brd-boxes. As well, this experiment demonstrates that regulation by the miR-4 transgene is not attributable to miR-286 and miR-5 carried on this construct. Nevertheless, this miRNA construct efficiently down-regulates a miR-5 sensor containing two miR-5 sites, indicating that the other miRNAs carried on this construct are functional. As a final test of the specificity of this assay, it was observed that this three-miRNA construct fails to inhibit the expression of an empty tub-GFP sensor (Lai, 2005).

Having demonstrated that Brd-boxes are bona fide miRNA-binding sites, it was asked whether regulation of the Bearded 3'-UTR by miR-7 requires the presence of Brd-boxes. This might be the case, for example, if negative regulation of a given 3'-UTR required synergism between different types of miRNA-binding sites. A Bearded 3'-UTR carrying mutations in each of the three Brd-boxes was observed to be still strongly inhibited by miR-7, indicating that individual types of miRNA-binding sites suffice for regulation in this assay (Lai, 2005).

The largest family of Drosophila miRNAs includes those whose 5'-ends are complementary to the K-box (cUGUGAUa, where the lowercase nucleotides represent positions of strong bias). The K-box is also the most pervasive motif within these Notch target genes; it is present in almost every member of the Brd-C and E(spl)-C [excepting E(spl)mbeta and Ocho, which lack any box motifs]. The maximum overall site complementarity of any given K-box site to any K-box family miRNAs is generally modest, and less than that seen with other demonstrated targets of the K-box family miRNA miR-2, namely, the proapoptotic genes grim, reaper, and sickle. In fact, the sole Notch target gene that was predicted informatically as a target of a K-box family miRNA in any study was E(spl)m8: miR-11 , and this pair ranked only 46^th (Lai, 2005).

The ability was tested of two quite distinct K-box family miRNAs, those of the miR-2 cluster (miR-2a-1, miR-2a-2, and miR-2b-2) and miR-11, to regulate K-box-containing 3'-UTRs. Given the abundance of K-box complementary miRNAs (as a class, they are among the more frequently cloned fly miRNAs), the occupancy of K-box sites by endogenous K-box-class miRNAs may be near-saturating in some cases. In fact, negative regulation of E(spl)m8, whose K-boxes mediate 10-fold negative regulation and nearly eliminate expression of this sensor, could not be convincingly demonstrated. In spite of this, positive evidence was obtained that four other K-box-containing 3'-UTRs, E(spl)m4, Bob, E(spl)malpha, and E(spl)mdelta, are directly regulated by K-box-family miRNAs, although the amount of regulation observed was weaker than that seen with GY-box- or Brd-box-class miRNAs. As was the case with the two Brd-box-class miRNAs, both miR-2 and miR-11 are capable of regulating some of the same K-box-containing targets. This constitutes further evidence for the possibility of cross-regulation of miRNA-binding sites, even where the miRNAs in question display very little similarity outside of their seeds (Lai, 2005).

In performing pairwise tests of these miRNAs with Notch target gene sensors, two instances were observed of miRNA-mediated regulation of sensors lacking canonical boxes. (1) It was observed that the E(spl)mdelta sensor was inhibited by miR-7. Although E(spl)mdelta lacks a canonical GY-box, it does contain a GY-box-like site that would have a single G:U base pair with the miR-7 seed. The nucleotides that are 5' and 3' to the box are also paired with miR-7, and there is a significant region of pairing to the 3'-end of the miRNA. These factors may allow this site to be recognized by miR-7. The 9-mer AGUUUUCCA is found in both Dp and Dv orthologs of E(spl)mdelta, indicating that this site is under selection and therefore is likely important for regulation of E(spl)mdelta. (2) It was observed that the Bob sensor was negatively regulated by both Brd-box-class miRNAs, miR-4 and miR-79. Although Bob lacks a canonical Brd-box, it does contain two matches to positions 2-7 of the Brd-box, which would pair to positions 2-7 of the miR-4/79. In this regard, this type of site is reminiscent of the 6-mer K-box, which pairs to positions 2-7 of K-box miRNAs. One of these Brd-box-like sites is conserved in Dp, and the syntenic site in Dv is, in fact, a canonical Brd-box, further indicating a functional relationship between Bob and miRNAs of the Brd-box family (Lai, 2005).

The apparent functionality of these noncanonical sites led to a search for other such sites in Notch target 3'-UTRs. Although one might expect to find many-fold more copies of degenerate sites relative to canonical sites, instead only a few additional examples of relaxed GY-box-like or Brd-box-like sites were found. For comparison, there are 28 canonical sites of these classes in Notch target 3'-UTRs (16 Brd-boxes and 12 GY-boxes), but only three additional examples of a 7-mer box-like site with a G:U base-pair to a miRNA seed [all are GY-box-like sites in E(spl)mdelta, E(spl)m3, and E(spl)m7]. In addition, there are only five additional examples of sites that match only positions 2-7 of the GY-box or the Brd-box [all of which are Brd-box-like sites: the two in Bob, one in E(spl)m7, one in E(spl)malpha, and one in E(spl)mdelta]. These considerations strongly suggest that the much more restricted, canonical sites are actively selected for function in these Notch target 3'-UTRs, a conclusion that is bolstered by the patterns of evolutionary conservation of these sites (Lai, 2005).

These experiments presented thus far demonstrate that target gene 3'-UTRs harboring sequence elements with Watson-Crick complementarity to the 5'-ends of miRNAs are, indeed, regulated by these miRNAs in vivo, and that such sites are necessary for miRNA-mediated regulation. Are these sites sufficient for regulation by complementary miRNAs? Although a variety of studies of model sites in tissue culture assays indicate site sufficiency, tests in animals suggest that miRNA site context can be less forgiving in vivo. For example, certain reporters containing multimers of six lin-4 or three let-7 sites are not appropriately regulated by lin-4 or let-7 in nematodes. In addition, mutation of sequences outside of the let-7-binding sites in lin-41 abolishes regulation by let-7 in vivo. Therefore, it was of interest to test the functionality of GY-boxes, Brd-boxes, and K-boxes when abstracted from endogenous 3'-UTR context (Lai, 2005).

To do so, a tandem of isolated GY-box, Brd-box, and K-box elements were cloned from Bob, Bearded, and E(spl)m8, respectively, into tub-GFP transgenes. Also mutant versions were cloned containing single changes in the Brd-box sites or dual changes in the GY-boxes. The ability of these 'box' sensors to respond to exogenously expressed miRNAs was tested. It was found that wild-type GY-box, Brd-box, and K-box sensors are all negatively regulated by corresponding miRNAs. These data directly demonstrate that all three types of box sites are sufficient for miRNA-mediated negative regulation. In contrast, mutant box sensors are nonfunctional in this assay. Since the mutant box sensors contain only one or two changes in each site, these data provide strong in vivo support for the idea that Watson-Crick pairing to the 5'-end of the miRNA (the "seed") is the key essential feature of miRNA target recognition. As a further test of this idea, the ability of the three different K-box miRNAs, miR-6, miR-2, and miR-11, to down-regulate a miR-6 sensor was tested. All three inhibited miR-6 sensor expression, consistent with the ability of seed-pairing to mediate regulation by miRNAs (Lai, 2005).

With these UAS-miRNA transgenic lines in hand, the consequences of ectopically expressing miRNAs on Drosophila development were tested. It should be noted that Notch target-regulating miRNAs were fully expected to regulate other functionally unrelated targets in vivo. For example, it has been established that K-box-family miRNAs also negatively regulate the proapoptotic genes reaper, sickle, and grim via K-boxes in their 3'-UTRs, while Brd-box-family miRNAs target the mesodermal determinant bagpipe via a Brd-box in its 3'-UTR. Therefore, even if ectopic miRNAs are able to affect normal development, they would not necessarily be expected to affect Notch signaling exclusively. Nevertheless, it has been previously reported that ectopic miR-7 induces loss of molecular markers of wing margin development, resulting in wing notching. This indicates that phenotypic characterization of miRNA misexpression can be informative (Lai, 2005).

Using an independently derived UAS-miR-7 construct lacking DsRed, it was verified that dpp-Gal4>miR-7 wings display notching and loss of Cut expression at the developing wing margin of wing imaginal discs; the size of the L3-L4 intervein domain was also reduced. It was next observed that ectopic K-box miRNAs of the miR-2a-1, miR-2a-2, miR-2b-2 cluster or miR-6-1, miR-6-2, miR-6-3 cluster had similar effects on wing margin development, although two UAS-transgenes were necessary to produce this effect. Also loss of anterior crossvein and occasional L3 vein breaks was observed, although these are not indicative of loss of N signaling. More generalized expression of miR-7 using bx-Gal4 induced strong thickening of wing veins, which is indicative of compromised Notch signaling during lateral inhibition of wing veins. Expression of K-box miRNAs using bx-Gal4 had severe effects on wing development, resulting in tiny, crumpled wings. It is suspected that this results from misregulation of non-Notch-pathway-related targets. The Brd-box miRNAs miR-4 and miR-79 and the K-box miRNA miR-11 did not affect wing margin development, even when these transgenes were present in two copies, indicating that this phenotype is not generally due to misexpression of miRNAs. However, miR-79 induced strong wing curling at high levels, potentially due to misregulation of non-Notch-pathway-related targets (Lai, 2005).

Next, focus was placed on development of the adult peripheral nervous system, as exemplified by the bristle sensory organs that decorate the body surface. A classic role for Notch signaling is to restrict the number of sensory organ precursors. It was found that misexpression of miR-6 using bx-Gal4 results in a strong increase in microchaete bristle density and clustered dorsocentral macrochaetes, phenotypes that are consistent with loss of Notch signaling during lateral inhibition of sensory organ precursors. Ectopic miR-2 had a similar, but milder, effect and mostly induced clustered dorsocentral and scutellar macrochaetes. Therefore, divergent members of the K-box miRNA family have similar effects on sensory organ development, consistent with data indicating that seed-related miRNAs can regulate overlapping sets of target genes. Ectopic miR-7 also induces macrochaete tufting, which correlates with the differentiation of supernumerary sensory organ precursors in wing imaginal discs. Finally, occasional duplication of bristles was observed upon misexpression of the Brd-box miRNA mir-79, although this construct also induced occasional bristle loss. Ectopic expression of miRNAs does not in itself induce bristle defects per se, since misexpression of miR-4 or miR-11 does not interfere with bristle development (Lai, 2005).

Overall, the ability of different classes of Notch-regulating miRNAs to specifically induce phenotypes that are characteristic of Notch pathway loss of function in multiple developmental settings is a strong indication that Notch pathway targets validated in this study are key endogenous targets of these miRNAs (Lai, 2005).

It appears, therefore, that cells go through a significant amount of trouble to actively inhibit Notch signaling. Core components of the Notch pathway are subject to significant negative regulation at every step in their life cycle, including at the transcriptional, post-transcriptional, and post-translational levels. For example, in the absence of activated nuclear Notch, CSL proteins are transcriptional repressors that actively repress Notch target gene activity. In addition, multiple dedicated ubiquitin ligases promote degradation of Notch pathway components, including the receptor Notch itself. To this list, may be added transcripts of most direct Notch target genes in Drosophila that are negatively regulated by multiple families of miRNAs (Lai, 2005).

The evidence provided in this study to support this conclusion is that (1) three different classes of miRNA-binding sites (GY-boxes, Brd-boxes, and K-boxes) are pervasive among two major classes of Notch target genes; (2) all three classes of motif are selectively constrained in 3'-UTRs during evolution; (3) transcripts bearing these box sites are negatively regulated by complementary miRNAs in vivo; (4) all three classes of sites are both necessary and sufficient for miRNA-mediated regulation in vivo; and (5) ectopic expression of Notch target-regulating miRNAs phenocopies Notch pathway loss of function during multiple developmental settings. Perhaps most importantly, it has been shown that genomic transgenes specifically mutated for miRNA-binding sites are sufficiently hyperactive so as to perturb normal development of the peripheral nervous system. This places these Drosophila Notch target genes in a relatively select group of miRNA targets for which miRNA-mediated regulation is phenotypically essential for normal development (Lai, 2005).

While most of the previously characterized in vivo targets of miRNAs are of the 'extensive pairing' variety, many of the validated targets in this study display much more limited 'box:seed'-pairing to miRNAs. In fact, within the context of the set of Notch target gene 3'-UTRs, the presence of conserved GY-boxes, Brd-boxes, and K-boxes allowed for highly effective prediction of miRNA:target relationships. This is the case even without first taking into account the extent of miRNA-pairing outside of box motifs. Rapid divergence of sequences upstream of box motifs, particularly those of the Brd-box and K-box classes, further indicates that extensive pairing is not selected for in these bona fide target sites. Consistent with this, multiple lines of evidence are presented that show that divergent seed-related miRNAs can regulate overlapping sets of target in vivo. Conversely, the importance of pairing between 3'-UTR boxes to miRNA seeds was demonstrated by endogenous 3'-UTR and box sufficiency tests, where even single-nucleotide disruption of seed-pairing abolishes regulation by miRNAs in vivo (Lai, 2005).

Identification and characterization of miRNA-binding sites in these Notch target 3'-UTRs mesh well with other recent bioinformatics and experimental studies that together help to define the 'look' of miRNA-binding sites. The concept of using conserved 'boxes' with Watson-Crick complementarity to miRNA seeds to identify miRNA targets is at the heart of the TargetScanS approach. A recent study has identify statistically significant signal not only for conserved 3'-UTR sites that match positions 2-8 of the miRNA (as is characteristic of the Brd-box and GY-box), but also for matches to positions 2-7 of the miRNA (as is characteristic of the K-box). In addition, a significant bias was identified for the nucleotide corresponding to position one of the miRNA to be an adenosine in predicted target sites. Interestingly, 27/42 (64%) of GY-boxes, Brd-boxes, and K-boxes in Dm Notch target genes also have an adenosine in this position, consistent with the notion that this feature can help to identify genuine target sites. These results are also consistent with directed tests of model sites using an imaginal disc sensor assay. Together with the recent observation that miRNAs can down-regulate large numbers of transcripts that contain box:seed matches in their 3'-UTRs, a current view emerges that conserved 3'-UTR boxes that are 6-7 nt in length and complementary to the 5'-ends of miRNAs need to be considered seriously as functional regulatory sites. While seed-pairings with G:U base pairs are evidently not generally selected for, evidence is shown that rare sites of this class are functional. This is consistent with other studies that demonstrate that G:U seed-pairing impairs, but does not necessarily abolish target site function (Lai, 2005).

Finally, the presence of multiple classes of miRNA-binding sites in most Notch target gene 3'-UTRs raises the possibility of combinatorial regulation. Although this has been widely suggested as a formal possibility, extensive evidence has been provided that 3'-UTRs can bear multiple classes of functional sites. Phylogenetic considerations indicate that 10 different Notch target genes are likely regulated by multiple classes of miRNAs, and direct experimental support of this was provided for six Notch target genes. Multiple Brd-box-, K-box-, and GY-box-class miRNAs are present at high levels in the Drosophila embryo, and the Brd-box miRNA miR-4 is co-transcribed with the K-box miRNAs miR-6-1, miR-2, miR-3, suggesting that combinatorial control of Notch target genes actually occurs during normal development. Future studies are aimed at examining how different miRNA-binding sites collectively contribute to overall regulation of an individual gene (Lai, 2005).

Of the few animal miRNAs whose in vivo functions and targets are well understood, most act as genetic switches that determine binary, on/off states of target gene activity. For example, lin-4 and let-7 are temporal switches that control progression through nematode larval stages by inhibiting their targets at designated times in development. lsy-6 and miR-273 are spatial switches whose extremely restricted cell-type-specific expression patterns control neuronal identity. In these cases, temporally or spatially restricted miRNA expression is central to their control of specific processes, and each of these miRNAs appears to have a small number of key targets (Lai, 2005).

A different rationale is proposed for Brd-box and K-box miRNAs during Drosophila development. Although endogenous territories of GY-box-mediated regulation are not known, negative regulation by Brd-boxes and K-boxes appears spatially and temporally ubiquitous. Thus, Notch target transcripts of the Brd family and E(spl)bHLH families are subject to modes of miRNA-mediated regulation that operate in all cells, even though the genes themselves display highly restricted patterns of spatial expression. This suggests that these miRNAs are not dedicated to regulating Notch signal transduction, but may 'tune' the expression of many target genes. Indeed, the K-box-family miRNAs miR-2, miR-6, and miR-11 also directly regulate K-box-containing proapoptotic genes, and the Brd-box-family miRNAs miR-4 and miR-79 regulate the mesodermal determinant bagpipe. One prediction is that even though mutation of Brd-boxes and K-boxes in individual Notch target genes results in specific defects in Notch-mediated cell fate decisions, mutation of Brd-box and K-box miRNAs would have more general developmental consequences. This is supported by the observation that many, but not all, of the phenotypes induced by ectopic expression of Notch-regulating miRNAs appear to be obviously related to repression of Notch pathway activity (Lai, 2005).

An important advance of this study is the in vivo validation of a large number of biologically relevant miRNA targets that are minimally paired to miRNAs outside of the 'box:seed' region. Since modestly complementary sites are both necessary and sufficient for miRNA-mediated regulation, it might be relatively easy for novel miRNA-binding sites to arise in 'tuning' targets. Indeed, a subset of box sites has apparently newly evolved during Drosophilid radiation. In the greater context of insect Notch target genes, it appears to have been important that they be negatively regulated by miRNAs, although the precise numbers and arrangement of different sites is variable. These features of tuning targets seem to allow for highly customized regulation of individual genes (Lai, 2005).

The experimental validation of many tuning targets may be challenging or impossible to obtain where quantitative regulation is subtle. Nevertheless, minor changes in gene activity, even of a fraction of a percent, could become highly significant when selecting the fitness of individuals at the population level. Deep evolutionary profiling of related species will therefore be key to revealing the full complement of biologically important miRNA-binding sites. The data suggest that multiple classes of miRNA-binding sites can be recognized with confidence as highly conserved 3'-UTR 'boxes' complementary to miRNA seeds, and this approach has been applied to the analysis of mammalian genomes. By mid-2005, 12 Drosophila genomes will be completed, which should enable high-confidence identification of miRNA-binding sites on the genome-wide scale -- even in cases in which only 7 nt of the target are paired to a miRNA (Lai, 2005).

Recent computational work pointed to regulation of vertebrate Notch and Delta by miR-34; however, no Notch target genes were similarly singled out in various bioinformatics efforts. miR-34 is conserved in flies; however, inspection of fly Notch or its ligands Delta and Serrate failed to reveal 'boxes' that might indicate similar regulation by miR-34. Brd-box-, GY-box-, and K-box-complementary miRNAs are likewise conserved between flies and vertebrates. Are any vertebrate Notch target genes predicted to be targeted by these miRNAs by virtue of 'boxes'? Although Brd proteins have thus far been found only in insects, E(spl)bHLH proteins are conserved in and are primary effectors of Notch signaling in all vertebrates. No enrichment for Brd-boxes, GY-boxes, and K-boxes is observed across the set of vertebrate E(spl)bHLH 3'-UTRs as a whole. However, members of a specific subset of E(spl)-related repressors, named the Hey genes, contain a preponderance of these boxes in their 3'-UTRs. This appears to be the case in a variety of mammals (human, mouse, and rat) and fish (fugu and zebrafish). Therefore, miRNA-mediated regulation may be a conserved feature of Notch target genes, a scenario that is under current experimental investigation (Lai, 2005).

Drosophila CK2 regulates lateral-inhibition during eye and bristle development by targeting proteins of the E(spl) complex

Lateral inhibition is critical for cell fate determination and involves the functions of Notch (N) and its effectors, the Enhancer of Split Complex, E(spl)C repressors. Although E(spl) proteins mediate the repressive effects of N in diverse contexts, the role of phosphorylation has been unclear. This study implicates a common role for the highly conserved Ser/Thr protein kinase CK2 during eye and bristle development. Compromising the functions of the catalytic (α) subunit of CK2 elicits a rough eye and defects in the interommatidial bristles (IOBs). These phenotypes are exacerbated by mutations in CK2 and suppressed by an increase in the dosage of this protein kinase. The appearance of the rough eye correlates, in time and space, to the specification and refinement of the ‘founding’ R8 photoreceptor. Consistent with this observation, compromising CK2 elicits supernumerary R8’s at the posterior margin of the morphogenetic furrow (MF), a phenotype characteristic of loss of E(spl)C and impaired lateral inhibition. Compromising CK2 elicits ectopic and split bristles. The former reflects the specification of excess bristle SOPs, while the latter suggests roles during asymmetric divisions that drive morphogenesis of this sensory organ. In addition, these phenotypes are exacerbated by mutations in CK2 or E(spl), indicating genetic interactions between these two loci. Given the centrality of E(spl) to the repressive effects of N, these studies suggest conserved roles for this protein kinase during lateral inhibition. Candidates for this regulation are the E(spl) repressors, the terminal effectors of this pathway (Bose, 2006).

Neurogenesis reflects the outcome of a complex balance between the activities of transcription factors that favor this cell fate (ASC/Ato) and those that oppose it (E(spl)). It is increasingly apparent that formation of the eye and bristle are predicated on a similar mechanistic framework, even though the proneurals that participate in these two developmental programs are distinct. For example, the PNCs in the eye (the R8 cell) require ato, while those in the bristle (macrochaetes and IOBs) require ASC. Nevertheless, one common feature of the resolution of PNCs in the eye and the bristle is the centrality of E(spl)C, since loss of E(spl)C leads to exaggerated neurogenesis in both contexts. In the eye it leads to excess R8’s, rough eyes, and duplicated IOBs, while in the bristles this manifests as ectopic, split and missing bristles. Extensive analyses have identified the genes involved with these developmental programs, the feedback loops that reinforce proneural expression in R8’s/SOPs, and the role of E(spl)C for lateral inhibition. In contrast, it has remained unclear how phosphorylation contributes to the dynamics of this process (Bose, 2006).

It has been thought that transcription of E(spl) is, by itself, necessary and sufficient for lateral inhibition. This model emerged from studies on bristle development, where ectopic E(spl) proteins extinguished the SOPs, whereas loss of E(spl) favored this cell fate. This model needs qualification because similar outcomes have not been recapitulated in the eye. In this context, loss of E(spl) demonstrably compromises lateral inhibition and elicits excess R8’s. However, ectopic expression of E(spl) members does not block the R8 fate, and consequently the eye displays the normal hexagonal packing of the ommatidia; the only defect is loss of the IOBs whose developmental program bears similarities to that of the macrochaetes. In contrast, R8 formation is blocked by the truncated M8* protein encoded by the E(spl)D allele, or by the CK2 phophomimetic variant M8SD. It is important to note that the eye defect of E(spl)D requires N^spl, a recessive allele that attenuates ato, but not E(spl), expression. The inability of M8* to recruit Gro, which compromises repression, thus necessitates a sensitized background, one conferred by N^spl. Accordingly, M8SD (which binds Gro) elicits eye defects independent of N^spl (Karandikar, 2004). Based on the observation that both M8* and M8SD display exacerbated and equivalent interactions with Ato, it was proposed that CK2 phosphorylation switches M8 into an active repressor by uncovering the Orange domain, and it is this regulation that is bypassed by the E(spl)D mutation (Karandikar, 2004). Given that the Orange domain mediates binding to other proneurals as well, this regulation by CK2 should have been more general to lateral inhibition. These studies suggest just such a role in the eye and the bristle (Bose, 2006).

This work supports the notion that CK2 is a participant in lateral inhibition. Compromising CK2 by a number of independent routes, i.e., in wild type and backgrounds mutant for CK2 and E(spl), elicits neural defects in the eye and bristle. These include rough eyes due to the specification of excess 'founding' R8 cells, and ectopic bristles (macrochaetes and IOBs) due to the specification of excess SOP’s. These phenotypes are hallmarks of impaired lateral inhibition, and have been previously described for loss of function of the E(spl)C. Evidence is provided for genetic interactions between CK2 and E(spl). While these studies provide multiple lines of evidence, the absence of suitable antibodies have precluded a formal demonstration that E(spl) repressors are, in fact, phosphorylated in cells undergoing lateral inhibition. Nevertheless, the congruence of the results utilizing CK2-RNAi or CK2-DN in conjunction with extant mutants and cell fate in imaginal discs, together, constitute a plausible argument supporting a role for this protein kinase (Bose, 2006).

These studies also suggest secondary roles for CK2 in the bristle lineage. In contrast to R8 patterning, the roles of N and E(spl) are different during bristle morphogenesis. In the case of the macrochaete or the IOB, N and E(spl) are re-deployed following SOP selection. Specifically, the SOP gives rise to the pI neuroblast that undergoes two asymmetric divisions to generate four cell types characteristic of the sensillum; socket, shaft, sheath and neuron, and these divisions are dependent on N- and E(spl)-inhibitory signaling. Thus loss of E(spl) following SOP selection manifests as split bristles (aberrant division of the pIIa cell) or missing bristles (aberrant division of the pI cell). The split bristles described thus suggest a role for CK2 during the socket-to-shaft sister cell fate. In contrast, while the missing bristles suggest a role for CK2 during the pIIa-vs-pIIb fates, this phenotype could result from loss of the SOP itself, a possibility if CK2 levels become rate limiting for cell division. Despite the fact that the timing of the asymmetric divisions of pI, pIIa and pIIb are well known, CK2-RNAi or CK2-DN are not suitable for dissecting the roles of CK2 at these later steps of bristle development. Conditional alleles of CK2, e.g., temperature-sensitives, will be necessary to better define its roles during specification of these sister-cell fates. One major question that emerges from these studies is why is phosphorylation necessary, given that not all members of the E(spl)C are targets of CK2. It is thought that evolutionary principles, the diversities and/or affinities of interactions between E(spl)C and ASC/ato, and their spatial expression patterns, perhaps, offer insights (Bose, 2006).

Of the seven E(spl) proteins, three (M8, M5, and M7) are targeted by CK2, and these are also the most closely related. Among all E(spl) members, two regions largely account for length heterogeneity and divergence. These are sequences between HLH and Orange and those between Orange and WRPW, the CtD. However, within the CtD of M8 (and M5 and M7 as well) is a highly invariant sequence, the phosphorylation domain (P-domain) that harbors the CK2 site. Given the phylogenetic relationships of these species, it is noteworthy that over a period of ~50 million years the P-domain and the CK2 site have been remarkably conserved. For example, of all M8 homologs, only D. pseudoobscura, D. grimshawii and D. hydei harbor a Glu residue, in place of Asp, at the n + 3 position of the CK2 phosphoacceptor. While it has not been experimentally confirmed that these homologs are phosphorylated, the possibility is high because this change still conforms to the consensus (S/T-D/E-x-D/E) for recognition by CK2. The virtually identical consensus site that is present in mammalian Hes6 is targeted by CK2 (Gratton, 2003) in vivo (Bose, 2006).

The mechanisms by which E(spl) proteins mediate repression have been intensely studied. In essence, E(spl) proteins repress ASC/Ato. Repression was initially thought to involve binding to a DNA sequence, the N-box. This, however, is not the case, because E(spl) proteins neutralized for DNA-binding still function as potent repressors. Furthermore, no N-box has been found in the regulatory region of ato, while that in sc is dispensable for repression in non-SOPs. It is now thought that direct (protein–protein) interactions between E(spl) and proneurals are more critical for repression, the protein–tether model (Giagtzoglou, 2003). In this model, repression by E(spl) occurs via direct interactions with enhancer bound proneurals, rather than by activator sequestration. This model is consistent with direct interactions between E(spl) and ASC/Ato proteins. It was, in fact, the analyses of various binary combinations that were the first to suggest that these interactions are regulated and non-redundant, two aspects that appear relevant to the current findings (Bose, 2006).

Analysis of M8 and its E(spl)D encoded variant, M8* provided the first hint that these antagonistic interactions are regulated. For example, it has been reported that, in addition to Ato, M8* interacts with a much higher affinity with Ac, Sc, and Ase. A similar case is described for M8SD, which interacts with Ato or L’sc with affinities significantly higher than M8 or its non-phosphorylatable variant M8SA (Karandikar, 2004). It is noteworthy that phosphorylation of mammalian Hes6 by CK2 is also a pre-requisite for its interactions with Hes1 (Gratton, 2003). Thus CK2 phosphorylation influences antagonistic interactions between the E(spl) and the ASC/Ato. Because these studies have employed two hybrid, instead of direct protein, approaches the possibility that these are kinetic effects remains open. This interpretation is consistent with the observations that a 2× dosage of a UAS-mδ construct interferes with Ato and blocks eye development in the wild type, whereas that of m7, m5 or m8 requires N^spl. Together, these findings argue that E(spl)-ASC interactions are of variable strengths and are isoform-specific. Given that only a subset of E(spl) and ASC members are expressed in the eye and wing disc, the possibility thus arises that distinct domains of ASC define sub-regions of the proneural field. In this context, E(spl) members might have been selected based on their affinities and/or specificities for these proneural factors. Thus the type of E(spl) repressors that are deployed might reflect the combinations and levels of proneurals, with CK2 playing an integrative role. The currently available techniques preclude a distinction between these possibilities (Bose, 2006).

It is presently unclear if/how CK2 activity is modulated during neurogenesis. Expression of this enzyme appears to be constitutive in the eye and wing disc (Karandikar, 2004). Holoenzyme formation, proposed to be a dynamic process in vivo, represents an attractive regulatory mechanism, given that CK2β modulates substrate recognition and that the fly CK2β gene encodes for non-redundant isoforms of this regulatory subunit. Alternatively, CK2 might be regulated by assembly into multiprotein complexes and/or via interactions with protein phosphatases. Such a coordinated function has been described for regulation of Period, the central component of the circadian clock, by CK2 and the phosphatase PP2A. Future studies aimed at the identification of protein phosphatase(s) that counteract the phosphorylation of E(spl)m8/5/7 by CK2, or multiprotein complexes containing E(spl) and/or CK2 will be required to better define the regulatory dynamics of this process during eye and bristle development (Bose, 2006).

Essential roles of Da transactivation domains in neurogenesis and in E(spl)-mediated repression

E proteins are a special class of basic helix-loop-helix (bHLH) proteins that heterodimerize with many bHLH activators to regulate developmental decisions, such as myogenesis and neurogenesis. Daughterless (Da) is the sole E protein in Drosophila and is ubiquitously expressed. This study has characterized two transcription activation domains (TADs) in Da, called activation domain 1 (AD1) and loop-helix (LH), and has evaluated their roles in promoting peripheral neurogenesis. In this context, Da heterodimerizes with proneural proteins, such as Scute (Sc), which is dynamically expressed and also contributes a TAD. Either one of the Da TADs in the Da/Sc complex is sufficient to promote neurogenesis, whereas the Sc TAD is incapable of doing so. Besides its transcriptional activation role, the Da AD1 domain serves as an interaction platform for E(spl) proteins, bHLH-Orange family repressors which antagonize Da/Sc function. The E(spl) Orange domain is needed for this interaction and strongly contributes to the antiproneural activity of E(spl) proteins. A mechanistic model on the interplay of these bHLH factors in the context of neural fate assignment is presented (Zarifi, 2012).

It was hitherto believed that the main role of Da is to heterodimerize with other group A bHLH factors to enable them to bind their targets. This work has characterized two additional important functions of Da. First, its two transcriptional activation domains are critically needed for peripheral neurogenesis, whereas the TAD of the partner (Sc) is inactive in this context. Second, Da AD1 serves to recruit E(spl) proteins, which inhibit Da/Sc activity. This ability to recruit E(spl) is shared by the Sc TAD and contributes toward moderating the activity of proneural proteins, to restrict the number of SOPs formed in any given proneural field of cells. As AD1 and Sc TAD interact with different domains of E(spl) proteins, the Orange domain and the N terminus/bHLH, respectively, Da and Sc recruit a different subset of the seven E(spl) proteins (Zarifi, 2012).

One difference noted among the three Da/Sc TADs concerns their relative strengths, which seems to be tissue context sensitive. It is therefore possible that these conserved TADs have evolved for specific functions in different tissues. In vertebrate systems, there is a precedent for this; for example, the LH domain seems to be selectively active in myogenesis. Even within the same lineage, B lymphogenesis, the AD1 and LH of E2A are redundantly needed for some processes and differentially needed for others. In Drosophila, either of the two Da TADs (but not that of Sc) was able to sustain peripheral neurogenesis in the wing disk, antenna, and retina, despite the fact that when tested in isolation or in a simple reporter, they displayed disparate strengths (Zarifi, 2012).

The Da TADs could play a special role in neurogenesis if they recruit crucial coactivators for the induction of SOP-specific genes, such as ase, sens, and phyl, as these coactivators are dispensable for the EE4 enhancer. One characterized Da/Sc coactivator is Senseless. It is not thought that the recruitment of Sens is what distinguishes the Da TADs from the Sc TAD, as both Da and Sc have been shown to recruit Sens in glutathione S-transferase pull downs in vitro, whereas the current result would predict that only Da should be able to do so. Another transcription factor that can interact with each of the N-terminal two-thirds of Da (the AD1 and LH regions) is Su(H) (Cave, 2009). Although this interaction is important for the activation of E(spl)m8, which is broadly expressed in proneural territories, SOP-restricted Da/Sc target genes do not rely on Su(H) binding for activation. For these reasons, it is considered unlikely that either of the Da-Sens or Da-Su(H) interactions is what makes AD1 and LH necessary for SOP formation (Zarifi, 2012).

An alternative scenario for the necessity of the Da versus the Sc TADs in neurogenesis invokes the role of the Da REP domain. The latter consists of aa 503 to 535 and can downregulate the TAD function in cis, i.e., when juxtaposed to the AD1 or LH domains of the mammalian E-protein E2A/E12 (Markus, 2002), but also in trans, namely, on the Da partner Twist. If the latter is generally true, then perhaps Da/Sc dimerization inactivates the Sc TAD on some enhancers, making it unable to promote SOP-specific target gene transcription. This is consistent with the current results showing a dominant negative function of DaΔTADs (aa 415 to 710, which retain the REP domain) on processes that need either Ac-Sc or Ato activity. Cave (2009) constructed a more severely truncated Da molecule (DabHLH, aa 546 to 710), which lacks the REP domain, and did not report any dominant negative effects using the same C253-Gal4 driver as was used for the current. It should be stressed that the inhibitory effect of the REP domain appears to be target enhancer specific, although the mechanism behind this is still unexplored (Zarifi, 2012).

What is the role of the Da-E(spl) interaction? E(spl) proteins constitute a seven-member protein family of bHLH-O repressors that have arisen from recent gene duplication events in drosophilids and show partially redundant activities, with the most notable being the suppression of neural commitment, where they antagonize proneural proteins. This study showed that Da AD1 is an interaction surface for E(spl) proteins. A similar function has been shown for the Sc TAD. Both in vitro and in vivo suggest that these interactions serve to recruit E(spl) proteins onto Da/Sc-occupied enhancers rather than to prevent Da and Sc from binding onto DNA, which had earlier been proposed to be a mechanism of action for some of the vertebrate Hes proteins, homologues of E(spl) (Zarifi, 2012).

Besides interacting with Da and Sc, E(spl) proteins form potent DNA binding dimers that recognize binding sites distinct from those bound by Da/Sc. What, then, is the role of proneural-E(spl) protein interactions? Early insight on this was gained upon comparing bristle suppression by E(spl)m7 versus E(spl)mδ upon Sc overexpression. E(spl)m7, which interacts with both Da and Sc, can potently suppress bristle formation even at high Sc levels, whereas mδ (which does not interact with Da/Sc) can do so only at low (endogenous) Sc levels. The present work presents evidence that mutating the Orange domain of E(spl)m7 can compromise its bristle suppression activity without blocking its DNA binding-dependent repression activity. These results strongly suggest that most E(spl) proteins have evolved the ability to contact all three, DNA, Da, and Sc, in order to improve their recruitment onto enhancers. This is probably due to the extreme instability of E(spl) proteins, which prevents them from accumulating to high levels. In order for low E(spl) levels to have any biological significance, they would need to bind to target enhancers with high affinity. This would best be achieved by cooperative contributions from both DNA and protein contacts and would ensure robust repression of target genes over a broad range of Da/Sc levels. The efficiency of neural suppression will probably be the net result of DNA binding, Da binding, and Sc binding, although other factors such as intramolecular interactions may also contribute. This multiparameter system makes it hard to predict from only one feature (e.g., Orange domain integrity) the potency of bristle suppression by any given E(spl) variant (Zarifi, 2012).

The demonstration that Da AD1 interacts with the Orange domain of E(spl) proteins adds a new function to that elusive domain, which defines the bHLH-O subfamily and to date has been implicated in dimerization and Sens binding. The recently proposed structure of the Orange domain as a dimeric hairpin of two α helices is consistent with both its role in dimerization and its role as a protein interaction surface. In fact, both Sens and Da interactions have been mapped to the two extremes of the Orange domain, which are predicted to be in close proximity due to the folding back of the hairpin. Given its dual role in the selectivity of the bHLH-O dimer partner as well as in the binding to non-bHLH-O factors (such as Sens or Da), it comes as no surprise that the Orange domain had originally been recognized to be a specificity determinant for the function of bHLH-O repressors on the early Sxl enhancer (Zarifi, 2012).

E(spl) proteins are one (in fact, seven) out of a few negative regulators of Da/Sc and perhaps more group A bHLH activators, with others being Id/Emc and Eto/Nvy. E(spl) recruitment can account for some of the instances where Da has been shown to act repressively; e.g., E(spl) and Da have been shown to act interdependently to repress ato at the eye morphogenetic furrow. It should be noted that E(spl) uses a different mechanism from either of the other two negative regulators. Emc prevents Da DNA binding, whereas E(spl) does not; in fact, it seems to cooperatively enhance it. Nvy is a cofactor that is recruited onto Da but cannot bind DNA on its own. Given the nonconservation of Da AD1 with vertebrate E proteins but at the same time the conserved antagonism between E proteins and bHLH-O/Hes proteins in many contexts, it would be interesting to determine whether E-protein/Hes interactions can be detected in vertebrate systems and which mechanisms they use (Zarifi, 2012).

DEVELOPMENTAL BIOLOGY

Embryonic

The peak of HLHm7 expression during embryogenesis occurs at 6-10 hours. In the late blastoderm, expression is detected in a 2-3 cell-wide stripe on each side of the embryo, in groups of cells over the dorsal half of the poles, in the vitellophage, and in a dorsomedian band spanning the anterioposterior axis. During germ band extension, ectodermal expression is detected, and at the extended germ band stage, epidermal expression is abundant. In late stage 11, epidermal expression becomes patchy. At stage 10, the primordia of the supraoesophageal ganglion and the posterior midgut express HLHm7. From stage 11 through late stage 12, expression is detected in the entire mesodermal layer. From late stage 11 through stage 14, expression is also detected in the primordia of the stomatogastric nervous system and in the optic lobes (Knust, 1987).

Larval

Enhancer of split complex genes manifest distinct patterns of expression in the wing imaginal disc. m8 and m7 mRNAs are detected in clusters of cells that correspond to the locations where sensory organ precursors (SOPs) develop. In addition m8 is also detected in cells at the dorsal/ventral boundary throughout the third instar. The expression of mgamma and mdelta is at times associated with the same SOPs, and at other times, with different SOPs. However mdelta and mgamma mRNAs are only detected in a subset of proneural clusters. Like m8, mgamma is also present at high levels at the dorsal/ventral boundary in early stages. The domain of mß is the most distinctive. It is expressed in the wing blade associated with developing veins, and is also present at the dorsal/ventral boundary and wing margin, and is expressed in a complex pattern elsewhere in the disc, with no simple association with developing sensory organs (de Celis, 1996). In the eye disc, m8 and m7 are expressed spanning the morphogenetic furrow, whereas mgamma and mdelta are expressed just posterior to the furrow. mgamma, mdelta and mß are expressed in the more posterior portions of the disc, where the recruitment of undifferentiated cells into ommatidial units occurs; there is little expression of m8 and m7 in this region (de Celis, 1996).

A common consequence of Notch signaling in Drosophila is the transcriptional activation of seven Enhancer of split [E(spl)] genes, which encode a family of closely related basic-helix-loop-helix transcriptional repressors. Different E(spl) proteins can functionally substitute for each other, hampering loss-of-function genetic analysis and raising the question of whether any specialization exists within the family. Each individual E(spl) gene was expressed using the GAL4-UAS system in order to analyse each gene's effect in a number of cell fate decisions taking place in the wing imaginal disk. A focus was placed on sensory organ precursor determination, wing vein determination and wing pattern formation. All of the E(spl) proteins affect the first two processes in the same way: they antagonize neural precursor and vein fates. Yet the efficacy of this antagonism is quite distinct: E(spl)mbeta, which is normally expressed in intervein regions, has the strongest vein suppression effect, whereas E(spl)m8 and E(spl)m7 are the most active bristle suppressors. While E(spl)m8 is more effective in abolishing the notum microchaeta fate, E(spl)m7 is most active against wing margin bristles (Ligoxygakis, 1999).

EFFECTS OF MUTATION

Drosophila neurogenesis requires the opposing activities of two sets of basic helix-loop-helix (bHLH) proteins: proneural proteins, which confer on cells the ability to become neural precursors, and the Enhancer-of-split [E(spl)] proteins, which restrict such potential as part of the lateral inhibition process. Do E(spl) proteins function as promoter-bound repressors? The answer was sought by examining the effects on neurogenesis of an E(spl) derivative containing a heterologous transcriptional activation domain [E(spl) m7Act (m7Act)]. The activator domain is derived from VP16. m7Act contains the bHLH and adjacent putative helical domains from m7 but lacks the last 43 amino acids of the protein, including the C-terminal WRPW tetrapeptide required for repressor activity of the related Hairy protein. In contrast to the wild-type E(spl) proteins, m7Act efficiently induces neural development, indicating that it binds to and activates target genes normally repressed by E(spl). Persistent expression of wild-type E(spl) proteins causes loss of neural precursors and sensory bristles and also suppresses wing vein formation. By contrast, m7Act efficiently induces supernumerary external sense organs, as predicted if E(spl) proteins function as direct repressors. An equivalent m7 truncation lacking the VP16 activation domain has no phenotypic effects, indicating that m7Act does not function by passively interfering with endogenous E(spl) activity, but instead acts as a transcriptional activatior. Mutations in the basic domain disrupt m7Act activity, suggesting that its effects are mediated through direct DNA binding. m7Act causes ectopic transcription of the proneural achaete and scute genes. These results support a model in which E(spl) proteins normally regulate neurogenesis by direct repression of genes at the top of the neural determination pathway (Jiménez, 1997).

echinoid (ed) encodes a cell-adhesion molecule (CAM) that contains immunoglobulin domains and regulates the Egfr signaling pathway during Drosophila eye development. Genetic mosaic and epistatic analysis, has suggested that Ed, via homotypic interactions, activates a novel, as yet unknown pathway that antagonizes Egfr signaling. Alternatively, later studies indicate that Ed inhibits Egfr through direct interactions. Another body of work suggests that Ed functions as a homophilic adhesion molecule, and also engages in a heterophilic trans-interaction with Drosophila Neuroglian (Nrg), an L1-type CAM. Co-expression of ed and nrg in the eye exhibits a strong genetic synergy in inhibiting Egfr signaling. This synergistic effect requires the intracellular domain of Ed, but not that of Nrg. A model for this interaction suggest that Nrg acts as a heterophilic ligand and activator of Ed, which in turn antagonizes Egfr signaling (Spencer, 2003 and references therein; Islam, 2003 and references therein).

Complicating the picture even further is an analysis of a paralogue of Ed termed friend of echinoid (fred). ed and fred transcription units are adjacent to one another, approximately 100 kilobases apart on chromosome arm 2L, but they are divergently transcribed in opposite directions. Fred acts in close concert with the Notch signaling pathway. Suppression of fred function results in specification of ectopic SOPs in the wing disc and a rough eye phenotype. Overexpression of N, Su(H), and E(spl)m7 suppresses the fred RNAi phenotypes. Accordingly, decreasing Su(H) or overexpression of Hairless enhances the fred RNAi phenotypes. Thus fred, a paralogue of ed, shows close genetic interaction with the Notch signaling pathway. The weak genetic interaction observed between fred and components of the Egfr pathway also links fred to the Egfr pathway; however, analysis of additional components of the Egfr pathway are necessary to determine Fred's role in the Egfr signaling (Chandra, 2003).

In order to study the function of fred, the heritable and inducible double-stranded RNA-mediated interference (RNAi) method was used. For this study, transcript sequence of fred was cloned as a dyad symmetric molecule in the pUAST vector and transgenic lines established. Expression of the construct was induced by crossing the transgenic lines to tissue- and/or stage-specific GAL4 driver lines. Transcription of a dyad symmetric molecule results in a RNA that snaps back to give rise to a dsRNA with a hairpin loop; this mediates the degradation of the corresponding endogenous mRNA. A 638-bp region of fred was selected for this analysis based on minimal similarity to ed sequence (Chandra, 2003).

The Notch signaling pathway is involved in limiting the SOP fate to a single cell within each proneural cluster. Since degradation of fred mRNA leads to formation of ectopic SOPs, it was of interest to see if the Notch signaling pathway genes functionally interact with fred in this process and, thus, may modulate the fred RNAi phenotype. To this end, four Notch pathway genes, Notch (N), Suppressor of Hairless [Su(H)], Hairless (H), and E (spl) m7 were tested for genetic interactions with fred (Chandra, 2003).

Among the best characterized targets of Notch signaling in Drosophila are the seven Enhancer of split [E (spl)] complex genes. Activation of the Notch signaling pathway results in the activation of the expression of various E(spl) complex genes. Overexpression of E (spl)m8, E (spl)m7, E (spl)m&#223;, E (spl)mgamma, E (spl)m 3, and E (spl)mDelta in wing discs results in loss of sensory organs. To determine whether the phenotype associated with suppression of fred could be modulated by expression of an E(spl) complex gene, E (spl) m7 was expressed simultaneously with fred dsRNA using the pnr-GAL4 driver. Flies that overexpress both fred dsRNA and E (spl) m7 are indistinguishable from those expressing only E (spl) m7. Third instar larval wing discs from these crosses were also analyzed for A101-lacZ expression. Ectopic expression of E(spl)m7 by pnr-GAL4 results in the loss of dorsocentral and scutellar SOPs, while suppression of fred activity results in large domains of A101-positive cells. Notably, wing discs of UAS-E(spl)m7; UAS-fred RNAi/pnr-GAL4: A101-lacZ larvae show the same SOP pattern as UAS-E(spl) m7; pnr-GAL4: A101-lacZ larvae. Therefore, ectopic expression of E (spl)m7 suppresses the phenotype associated with the reduction of fred in the wing disc (Chandra, 2003).

Whether this is also the case in the developing eye was also tested. Degradation of fred mRNA in the eye with GMR-GAL4 results in a rough eye phenotype with missing or duplicated bristles and fused ommatidia. Ectopic expression of E (spl) m7 by GMR-GAL4 results in the loss of most of the bristles in the eye. While the ommatidia remain highly organized, bristle sockets are present only infrequently or are entirely missing. If present, sockets are mispositioned and sometimes duplicated. The phenotype of eyes of animals expressing both E (spl) m7 and fred ds RNA under the control of GMR-GAL4 is very similar to the phenotype of UAS-E(spl)m7/GMR-GAL4 flies, with the exception of a few fused ommatidia that can still be observed in the posterior part of the eye (Chandra, 2003).

The observations that changes in the activity of four genes of the Notch signaling pathway can either suppress or enhance the phenotypes associated with the suppression of fred function suggest that fred is functioning in close concert with the Notch signaling pathway. Reduction in the activity of a Notch signaling pathway gene, Su(H) results in an enhancement of the fred RNAi phenotype. In contrast, ectopic expression of Notch signaling pathway genes, Notch, Su(H), and E(spl)m7 suppresses, to different degrees, different aspects of the fred RNAi phenotype in the developing wing, thorax, and eye. In contrast, overexpression of Hairless (a negative regulator of the Notch pathway) enhances the phenotypes induced by Fred suppression. It is presently not clear whether Fred defines a separate pathway for SOP determination or if it shares downstream components of the Notch signaling pathway. The remarkable degree to which ectopic expression of an E(spl) complex bHLH transcription factor results in a nearly complete suppression of phenotypes associated with fred degradation strongly supports the idea of very close functional interactions. These observations, furthermore, raise the possibility that E(spl) complex genes and/or other genes of the Notch signaling pathway act downstream of fred function (Chandra, 2003).

During neurogenesis in Drosophila, groups of ectodermal cells are endowed with the capacity to become neuronal precursors. The Notch signaling pathway is required to limit the neuronal potential to a single cell within each group. Loss of genes of the Notch signaling pathway results in a neurogenic phenotype: hyperplasia of the nervous system accompanied by a parallel loss of epidermis. Echinoid (Ed), a cell membrane associated Immunoglobulin C2-type protein, has been shown to be a negative regulator of the EGFR pathway during eye and wing vein development. Using in situ hybridization and antibody staining of whole-mount embryos, Ed has been shown to have a dynamic expression pattern during embryogenesis. Embryonic lethal alleles of ed reveal a role of Ed in restricting neurogenic potential during embryonic neurogenesis, and result in a phenotype similar to that of loss-of-function mutations of Notch signaling pathway genes. In this process Ed interacts closely with the Notch signaling pathway. Loss of ed suppresses the loss of neuronal elements caused by ectopic activation of the Notch signaling pathway. Using a temperature-sensitive allele of ed it has been shown that Ed is required to suppress sensory bristles and for proper wing vein specification during adult development. In these processes also, ed acts in close concert with genes of the Notch signaling pathway. Thus the extra wing vein phenotype of ed is enhanced upon reduction of Delta (Dl) or Enhancer of split [E(spl)] proteins. Overexpression of the membrane-tethered extracellular region of Ed results in a dominant-negative phenotype. This phenotype is suppressed by overexpression of E(spl)m7 and enhanced by overexpression of Dl. This work establishes a role for Ed during embryonic nervous system development, as well as adult sensory bristle specification and shows that Ed interacts synergistically with the Notch signaling pathway (Ahmed, 2003).

Regulation of Notch output dynamics via specific E(spl)-HLH factors during bristle patterning in Drosophila

The stereotyped arrangement of sensory bristles on the adult fly thorax arises from a self-organized process, in which inhibitory Notch signaling both delimits proneural stripes and singles out sensory organ precursor cells (SOPs). A dynamic balance between proneural factors and Enhancer of split-HLH (E(spl)-HLH) Notch targets underlies patterning, but how this is regulated is unclear. This study identified two classes of E(spl)-HLH factors, whose expression both precedes and delimits proneural activity, and is dependent on proneural activity and required for proper SOP spacing within the stripes, respectively. These two classes are partially redundant, since a member of the second class, that is normally cross-repressed by members of the first class, can functionally compensate for their absence. The regulation of specific E(spl)-HLH genes by proneural factors amplifies the response to Notch as SOPs are being selected, contributing to patterning dynamics in the notum, and likely operates in other developmental contexts (Couturier, 2019).

Pattern formation is a central question in developmental biology. Patterns of differentiated cells that are invariant across individuals can be observed in many species. These can be generated by interpreting a fixed pre-pattern or via self-organization, possibly guided by fixed initial conditions. The patterns of sensory organs in Drosophila are model systems to study how simple patterns dynamically emerge during development. On the dorsal thorax, or notum, macrochaetae are found at fixed positions and microchaetae are regularly distributed in five dorso-central rows in each hemi-notum. As each of these sensory bristles develops from a single sensory organ precursor cell (SOP), their pattern in the adult results from the pattern of SOPs in imaginal tissues in late third instar larvae and early pupae. SOPs emerge from groups of cells that express one or more transcriptional activators of the bHLH proneural family, e.g., Achaete (Ac) and Scute (Sc). These factors confer these cells with the ability to become SOPs and these groups of cells are known as proneural clusters. Thus, the position-specific expression of Ac and Sc determine where sensory bristles can develop. Two models have been proposed to explain how proneural clusters develop. In a first model, positional cues govern early proneural activity, and thereby the stereotyped layout of sensory organs whereas inhibitory Notch signaling acts downstream of proneural activity to select SOPs within each proneural cluster. This model applies well for the macrochateae. In a second model, it is instead Notch activity that negatively defines where proneural activity can emerge, and self-organization involving cell-cell interactions mediated by Notch directs patterning dynamics at the tissue scale. In this model, the role of positional cues is limited to defining the initial and/or boundary conditions that guide self-organized Notch dynamics. This model is proposed to apply for the five rows of microchaetae in the dorsal-central notum, which arise from a series of proneural stripes that emerge in a defined sequence (Couturier, 2019).

Once proneural clusters or stripes have formed, inhibitory cell-cell interactions mediated by Notch restrict the potential to become an SOP to one or a few cells per cluster (or stripe). Notch inhibits the competence to become neural via the E(spl)-HLH family of transcriptional repressors which act redundantly to antagonize the activity and expression of Ac and Sc. Therefore, adoption of the SOP fate depends on a balance between the activity of Ac and Sc, acting synergistically with Senseless (Sens), and the anti-proneural activity of the E(spl)-HLH proteins. How this balance is dynamically regulated during patterning in the notum is not clear, in part because the dynamic expression of individual E(spl)-HLH factors is not known. While all seven E(spl)-HLH genes are directly regulated by Notch, each of these genes has, however, a unique expression pattern. The transcriptional response of the E(spl)-HLH genes to Notch is therefore context-specific, and spatially restricted factors, including Ac and Sc, appear to cooperate with Notch for their regulation in embryos and imaginal tissues. Thus, whether and how different E(spl)-HLH factors contribute to the evolution of the proneural pattern and the emergence of isolated SOPs remains to be studied (Couturier, 2019).

This study examined the role of Notch signaling in early stripe patterning and characterized the expression and function of the different E(spl)-HLH factors in the developing notum. Using reporters for each of the seven E(spl)-HLH proteins, it was found that a subset of E(spl)-HLH factors are expressed early, prior to the onset of proneural activity, and that additional E(spl)-HLH factors become expressed late, in a proneural-dependent manner. Early-onset factors define where the first stripes emerge while late-onset factors contribute to SOP selection. It is proposed that the regulation of specific E(spl)-HLH factors by Ac and Sc promotes mutual inhibition through a proneural-dependent increase in the number of E(spl)-HLH genes responding to Notch in cells with intermediate levels of Ac and Sc (Couturier, 2019).

An early, widespread and evolutionarily conserved response of the genome to Notch activation is the CSL-dependent transcription of the HES family genes. In Drosophila, the seven HES family genes encoded by the E(spl)-C act redundantly and the relative contribution of individual E(spl)-HLH factors to the overall Notch output has remained unclear. For this study a complete set of GFP-tagged reporters were generated, and the expression dynamics of all E(spl)-HLH factors were examined in the developing notum. m3 and mβ are expressed early in response to Mib1-dependent Notch signaling, and additional E(spl)-HLH factors, notably mδ, m7, and m8, become expressed later, once proneural stripes are established and in a proneural-dependent manner. Early-onset factors appeared to mediate the negative template activity of Notch for early stripe patterning, while late-onset factors, notably m7 and m8, are essential to reach sufficient Notch signaling output for the proper spacing of sensory bristles. Thus, different E(spl)-HLH factors contribute at distinct steps of this patterning process and the regulation of specific E(spl)-HLH genes by Ac and Sc plays an important role by raising the level of E(spl)-HLH activity, produced in response to Notch, in groups of cells progressing towards the SOP fate. This upregulation is transient, and once proneural stripes have resolved, the same early-onset factors that mediate stripe patterning are expressed in non-SOP cells to lock down their fate (Couturier, 2019).

The regulation of the E(spl)-C genes by a dual Notch/proneural input has been well studied before. This regulation was previously interpreted to suggest that Ac and Sc set up the initial conditions for a regulatory feed-back loop operating between proneural cluster cells. In this model, Ac and Sc initiate the conditions for both signaling, via the regulation of the Dl and neur genes, and responding to Notch, via the regulation of the E(spl)-C genes. Detailed analysis of the expression, regulation and function of the E(spl)-HLH factors did not support this model. Indeed, the proneural-independent expression of m3 and mβ, downstream of Mib1-dependent Notch signaling, appeared to provide the initial conditions for patterning. A different model is proposed different in this paper, whereby the proneural-dependent regulation of specific E(spl)-HLH factors serves to modulate mutual inhibition within the proneural stripes during SOP selection, and thereby to shape the dynamics of patterning. Cells with intermediate levels of Ac and Sc, because they activate an increasing number of Notch-responsive E(spl)-HLH genes, cannot evade mutual inhibition as readily as they would otherwise. Such a regulatory logic may favor the robust emergence of regularly spaced sensory organs (Couturier, 2019).

A role for the modulation of mutual inhibition was anticipated by an abstract mathematical model that recapitulates the temporal and spatial dynamics of fate patterning in the notum, and the current findings suggest a molecular basis for several features of the model's dynamics. In brief, this simple model represents the state of each cell by a single variable, u. This variable varies in time as a function of an inhibitory signal, s, representing the level of Notch ligands to which the cell is exposed, produced by other cells according to their own state u. Cells in the model have bistable dynamics, tending to one of two stable cell states, a high u/low s state (SOP fate; high proneural activity and low inhibitory signal) and a low u/high s state (non-SOP fate; low proneural activity and high inhibitory signal). Given appropriate initial conditions, the model recapitulates the sequential emergence of proneural stripes and their resolution into SOP rows. In simulations of this mathematical model, the balance between activation and inhibition is such that cells located at the center of the proneural stripes and progressing towards the SOP fate show increasing levels of both proneural activity and inhibitory signal (that is, of u and s). Interestingly, these simulations predicted that, once a proneural stripe emerges, the inhibitory signal is strongest in cells at the center of the stripe. This prediction was, however, not verified using GFPm3 as a Notch activity reporter: high levels of GFPm3 were observed in cells flanking the proneural stripes, not at the center of the stripes where SOPs develop. While cis-inhibition of Notch by Dl could account for a discrepancy between the inhibitory signal in the model (representing ligand levels) and Notch activity itself, detailed analysis of E(spl)-HLH expression suggests a different explanation: this particular Notch target, m3, does not fully reflect the in vivo activity of Notch, and other E(spl)-HLH factors, notably mδ, m7, and m8, contribute to the Notch output, particularly at the center of the proneural stripes. The contribution of cis-inhibition, if any, may thus be limited to emergent SOPs, which exhibit the highest levels of proneural factors and low levels of all E(spl)-HLH factors. The model further tied a gradual narrowing of neural competence, as observed experimentally, to a progressive increase in the strength of mutual inhibition. While this may result from a modulation of ligand activity, e.g., by Neur, in signal-sending cells, the findings identify another contribution, from the regulation of Notch target expression in receiving cells (Couturier, 2019).

This analysis also raises additional questions about how Notch and Ac/Sc regulate gene expression dynamics in the notum. In particular, two observations deserve consideration. First, how is it that the late-onset genes (mδ, m7, m8 etc.) do not respond to Notch signaling prior to the proneural onset? Second, why are m3 and mβ are not maximally expressed at the center of the stripes, where inhibitory signaling is predicted to be maximal? Several models can be proposed. In a first accessibility model (see Simulation of a mathematical model for patterning in the notum), only a subset of the CSL-binding sites is accessible for binding by CSL/NICD complexes in the absence of Ac and Sc, so that only a fraction of the E(spl)-HLH genes, i.e., mβ and m3, is activated by Notch; upon expression of Ac and Sc, additional binding sites become accessible, possibly through a change in chromatin structure induced by Ac and Sc, resulting in the expression of additional E(spl)-HLH genes. In support of this model, the proneural factor Ascl1 was shown to bind both closed and open chromatin in mouse neural progenitors, and binding to closed chromatin appeared to promote accessibility. In a second cooperativity model, early-onset genes contain high-affinity CSL binding sites, whereas late-onset genes have low-affinity CSL binding sites, so that only early-onset genes, i.e., m3 and mβ, respond to a Notch-only input; but the low-affinity CSL binding sites of the late-onset genes would be located close to E-boxes, such that Ac and Sc promote cooperative binding, hence gene expression. While these two models could explain the temporal sequence of gene activation, they do not, however, explain why m3 and mβ are not maximally expressed at the center of the stripes. Two possible mechanisms might account for this observation. One possibility is that the nuclear concentration of NICD is limiting such that not all CSL binding sites can be occupied, even when Notch signaling is maximal. If so, binding sites would effectively compete for the binding of CSL/NICD. If CSL/NICD complexes preferentially bind the regulatory sites of late-onset genes, this should then result in lower levels of m3 and mβ expression when and where these sites become accessible (accessibility model) or bound by Ac and Sc (cooperativity model). A second possibility is that the expression of the m3 and mβ genes is inhibited by late-onset E(spl)-HLH factors. Further studies will address these different models (Couturier, 2019).

Auto-repression and cross-repression is seen within the HES gene family in vertebrates. By contrast, self/cross-inhibition by E(spl)-HLH factors had not been observed in Drosophila, prior to this study. This study showed that the mδ gene is de-repressed in mβ m3 double mutant pupae, and ectopic expression in mutant nota appeared strikingly similar to those of the mβ and m3 genes in wild-type pupae. This finding may help resolve a long-held paradox which is that E(spl)-HLH genes have very distinct expression yet are functionally redundant. Obviously, ectopic expression of one (or several) factor upon loss of one (or several) others would account for redundancy despite specificity in expression (Couturier, 2019).

This analysis provides no evidence for functional specificity at the molecular level amongst the E(spl)-HLH factors. For instance, m3 and mβ appeared to be functionally replaced by mδ. Similarly, SOP selection could be achieved, at least to some extent, by m7 and m8 alone, as well as by the other five factors, at the exclusion of m7 and m8. Thus, non-overlapping sets of E(spl)-HLH factors can provide a proper Notch output for stripe patterning and SOP selection. These observations, together with earlier findings, indicate that the E(spl)-HLH factors have very similar molecular activities. Thus, the proneural-dependent expression of additional E(spl)-HLH factors may simply result in a global increase of an anti-proneural activity that would be provided by any of these factors. Therefore, describing Notch output dynamics and understanding its regulatory logic in a given context may require the analysis of all E(spl)-HLH factors that collectively contribute to this output. The tools generated in this study will help to achieve this (Couturier, 2019).

The patterning logic uncovered in this study may be of general relevance. While earlier studies viewed SOP selection in larvae as a multi-step process of proneural cluster resolution, the current data suggest that the progressive transition from mutual inhibition among proneural cells to lateral inhibition from SOPs is dynamically shaped by the regulation of a specific subset of E(spl)-HLH factors by Ac and Sc, with no need for specific mechanisms to restrict competence to a subgroup of proneural cells. In the adult fly gut, a subset of E(spl)-HLH genes, including mδ, m7, and m8, are also regulated by Sc and this regulation may be functionally relevant as Sc is a key cell fate regulator within the Intestinal Stem Cell (ISC) lineage. Interestingly, ISCs that are in a low Sc state may respond to Notch via mβ and m3 factors, which may suffice to remain in a low Sc state, whereas ISCs that are in a high Sc state may revert to a low Sc state in response to a stronger Notch output that would be produced in part through the mδ, m7, and m8 factors. Thus, the regulatory logic unraveled by this study may regulate cell fate within the ISC lineage (Couturier, 2019).

The regulation of the HES family genes by a dual Notch/proneural input is evolutionarily conserved. In Xenopus, an enhancer integrates the Notch and proneural inputs to regulate the expression of two Hes5-like genes. In the vertebrate brain, Ascl1 regulates the expression of Hey1, a direct Notch3 target. In this context, it is interesting to note that neural stem cells cycle between a quiescence state, regulated by high Notch3 activity and an activated state, also involving Notch3 but in the context of Ascl1 expression, a combination that might lead to expression of different HES family genes. Actually, in numerous contexts in development, Notch receptor activation intersects with the expression of proneural transcription factors. Thus, the regulatory logic uncovered here for the patterning of sensory organs, involving a proneural-dependent increase in E(spl)-HLH gene expression as a mean to upregulate the Notch output, may similarly operate in mammals (Couturier, 2019).

REFERENCES

Search PubMed for articles about Drosophila Enhancer of split m7, helix-loop-helix

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date revised: 15 July 2013

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