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).
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 (SF-X or X-SR), or orientation variants in which the orientation of one or both S sites have been reversed (SR-SF, SF- SF, and SR-SR) (Cave, 2005).
The native m8 promoter is synergistically activated in transcription assays by coexpression of dNICD and Ac/Da, but it is only weakly activated by expression of dNICD or proneural Ac/Da proteins alone. However, neither promoter with a single S binding site (SF-X or X-SR) can mediate synergistic interactions between dNICD and proneural proteins. In fact, both single S site promoters are only weakly activated when proneural and dNICD proteins are expressed individually or together. Thus, single S sites are not sufficient to mediate Notch-proneural synergy in these contexts, even though they are in the same position as the SPS in the wild-type m8 promoter (Cave, 2005).
When the number of S binding sites are maintained, but the orientation of these sites within the SPS is varied (SR-SF, SF-SF, and SR-SR), only the wild-type (SF-SR) SPS orientation is synergistically activated by coexpression of dNICD and proneural Ac/Da proteins. Thus, the wild-type SPS architecture of S binding sites is clearly necessary for the m8 promoter to mediate transcriptional synergy between NICD and the proneural protein complexes assembled on the SPS and A sites, respectively (Cave, 2005).
The transcriptional synergy between NICD and proneural proteins mediated by the SPS element is crucial for the coactivation by the Mastermind (Mam) protein. Coexpression of Mam with both dNICD and proneural proteins provides a strong coactivation of transcription of the wild-type m8 promoter. However, this strong coactivation is not observed with any of the non-wild-type m8 SPS variants, which also cannot mediate Notch-proneural synergy. Thus, coactivation by both the NICD and Mam cofactors is strongly dependent on synergistic interactions with proneural combinatorial cofactors, and the specific SPS architecture is critical for mediating this synergy (Cave, 2005).
The native m8 promoter studies tested whether the organization of the S binding sites in the SPS are necessary to mediate the Notch-proneural synergy. In order to test which of these architectural features are sufficient to mediate that synergy, a set of synthetic promoters was created carrying the same SPS variants mentioned above in combination with A sites (SPS-4A reporter). These synthetic promoters thus contain the sites predicted to mediate the synergy but lack the other sites present in the native m8 promoter, which might also be necessary. This reductionist approach allows for the identification of a minimal promoter that contains only those sites that are necessary and sufficient to mediate the Notch-proneural synergy. All of these synthetic reporters are modestly activated by expression of proneural proteins alone, but expression of dNICD alone gives no activation. By contrast, only the SPS-4A reporter containing the wild-type SPS (SF-SR) mediates clear synergistic activation when dNICD and proneural proteins are coexpressed, and none of the SPS variants do so (Cave, 2005).
Given that functional CSL/NICD/Mam ternary complexes have been shown to assemble on single S sites and activate transcription, it was expected that promoters with single S sites could be activated at low levels by expression of dNICD in the absence of the proneural proteins and that promoters with two S sites might have more activity than single S sites. However, it was surprising to observe that all of the m8 and synthetic promoters, even with the wild-type SPS element, have very low or no activity when dNICD is expressed alone. Thus, the SPS binding site architecture does not appear to facilitate recruitment of functional NICD coactivator. This argues against previous proposals that suggested that the SPS architecture might function to recruit other proteins to the promoter. Thus, given that the wild-type SPS architecture is necessary and sufficient for Notch-proneural synergy, these results indicate that the function of the SPS element is to enable synergistic interactions with proneural proteins (Cave, 2005).
The synthetic promoters do not carry bHLH R sites, which are present in all E(spl)-C gene promoters. Thus, these sites clearly are not necessary for Notch-proneural synergy, although they may modulate it in vivo. It has been proposed that other repressor proteins bind the 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 (SF-SR) SPS-4A promoter, and mNICD alone does not produce any significant activation of the promoter. However, clear transcriptional synergy is observed with the wild-type SPS promoter when both mNICD and neural bHLH A proteins are coexpressed. Moreover, SPS-mediated synergy requires nearly the same organizational features of S binding sites as observed in Drosophila. Neither of the single S site promoters can mediate that synergy, nor can most of the orientation variants. Although the SR-SR promoter is activated following coexpression of both the mNICD and bHLH A proteins, it is not activated by mNICD alone (Cave, 2005).
These results indicate that the potential for transcriptional synergy between NICD and neural bHLH A proteins has been conserved in a mammalian cell system and that the SPS+A code is sufficient and critical for mediating that transcriptional synergy. This raises the possibility that there may be mammalian genes that are regulated by neural bHLH A proteins and Notch signaling via this code. Although there is an SPS element conserved in the HES-1 promoter, HES-1 does not have an A site in its proximal promoter region, and HES-1 is not activated by expression of bHLH A genes. Thus, HES-1 appears to be similar to the Drosophila E(spl)-C m3 bHLH R gene, which also has an SPS but no obvious nearby A site. Whole-genome searches are being performed for genes in mammalian systems that may be regulated by the SPS+A code (Cave, 2005).
It has been proposed that the architecture of the SPS element may mediate cooperative binding of a second CSL protein once an initial CSL protein binds the DNA. Using electromobility gel shift assays to test for cooperative binding, the ability was compared of bacterially expressed and partially purified Drosophila Su(H) protein to bind DNA probes containing either the wild-type m8 SPS or an m8 SPS with one S site mutated. If there is cooperativity, one would expect to observe the band corresponding to two DNA bound CSL proteins to be as strong or stronger than the band corresponding to a single CSL protein bound to DNA. The single S site probe serves as a control because it cannot be cooperatively bound by two Su(H) proteins, and it also serves to identify the band corresponding to a single Su(H) protein bound to the wild-type SPS probe. Similar amounts of Su(H) protein bind strongly to the wild-type probe and to the single-site probe. In particular, because single protein binding to the wild-type DNA probe did not facilitate or stabilize simultaneous binding of two S proteins, Su(H) does not appear to bind cooperatively to the two S sites in the wild-type probe. These results suggest that CSL proteins do not bind cooperatively to the SPS in vivo, although posttranslational modifications in vivo could affect these binding properties Cave, 2005).
In addition, the protein binding affinity for the SF-SR and SR-SF probes appears to be comparable, although the reversed orientation of the two S sites would have likely disrupted cooperative binding if it were present. This result strongly suggests that the complete lack of activation by SR-SF sites in all of the promoters tested is not due simply to decreased ability of Su(H) protein to bind to the SR-SF orientation variant Cave, 2005).
To test the in vivo relevance of the conserved S binding site orientation in SPS elements, transgenic flies were created carrying β-galactosidase reporter genes driven by native m8 promoters containing either the wild-type (SF-SR) or SR-SF variant SPS elements. Wing and eye imaginal discs containing m8 promoters with the wild-type SPS element produced strong expression in proneural cluster regions, similar to the pattern described for endogenous m8. By contrast, comparably stained wing and eye discs carrying the m8 promoter reporters with the SR-SF SPS variant showed no expression or very low levels of expression, respectively. Extended staining of discs containing the SR-SF element revealed clear but weak expression in a pattern of single cells that resembles the distribution of neural precursors in the wing discs and eye discs. This is likely due to activation via the A site by proneural proteins because proneural levels are highest in the precursor cells. However, there was no expression in the surrounding nonprecursor cells within the proneural clusters even though Notch signaling is activated in these cells. Similar neural precursor-specific m8 reporter expression patterns have been observed when the S binding sites are eliminated, indicating that reversal of the S binding site orientations is functionally equivalent to eliminating them for this aspect of Notch target gene expression. These in vivo results confirm that the conserved orientation of the S binding sites in the wild-type SPS element is essential for nonprecursor cell specific upregulation of the SPS+A bHLH R m8 genes in response to Notch signaling in proneural clusters (Cave, 2005).
To gain an insight into the molecular mechanism underlying the strong transcriptional synergy between Notch signaling and bHLH A proteins on the m8 and SPS-4A promoters, whether this synergy involves a direct physical interaction was tested by using yeast two-hybrid assays with the Drosophila proteins. These experiments revealed that the Daughterless N-terminal domain directly and specifically interacts with the Su(H) protein in the absence of the bHLH domain and C terminus (Cave, 2005).
Using transcription assays in Drosophila cells, whether the Da N terminus (DaN construct), which contains a transcription activation domain, can synergistically activate the m8 promoter was tested in the absence of both its bHLH DNA binding domain and a heterodimerization partner, like Ac. The Da N-terminal protein synergistically activates the m8 promoter when dNICD is coexpressed, apparently by direct binding of the DaN protein to endogenous CSL bound to the SPS element. These results indicate that the Notch-proneural transcriptional synergy is not mediated by cooperative DNA binding interactions between the Su(H) and proneural proteins, although such cooperative binding may mediate transcriptional synergy between some combinatorial cofactors. These results suggest that a direct interaction between Su(H) and the Da N-terminal fragment, which can occur independent of NICD, facilitates the formation of an active transcription complex when NICD is also present during Notch signaling (Cave, 2005).
These results suggest that the SPS architecture functions to enable a direct physical interaction between Su(H) and Da proteins, thus providing a molecular mechanism for the observed Notch-proneural synergy that is mediated by the SPS element. This interaction could stabilize the recruitment or functional activity of NICD, which then recruits Mam, and could explain the strong dependence of both NICD and Mam coactivation functions on the presence of proneural proteins (Cave, 2005).
In previous studies, it has been proposed that neither the synergistic activation nor the transcriptional repression mediated by CSL protein complexes imply direct interactions between CSL and DNA bound combinatorial cofactors; rather, it is likely that CSL proteins exert their effects through the recruitment of non-DNA binding cofactors, such as chromatin modifying enzymes. While this might be the case for some Notch target gene promoters, in the case of m8, the results indicate that the mechanism underlying the synergistic interactions between CSL/NICD and bHLH A proteins does involve direct physical interactions (Cave, 2005).
A mechanistic model is proposed for programming Notch-proneural synergy with the SPS+A transcription code. These studies demonstrate that there are important functional differences between SPS and non-SPS organizations of S binding sites. The critical role of the SPS binding site architecture is not predicted or explained by the previous models for Notch target gene transcription. Previous models suggest that transcription is promoted by the binding of NICD to CSL, which displaces CSL bound corepressors, thus allowing transcriptional synergy with other DNA bound combinatorial cofactors. These models have not distinguished between Notch target genes with regulatory modules that contain SPS or non-SPS configurations of S binding sites, nor do they explain or predict the critical function of the SPS binding site architecture in mediating Notch-proneural transcriptional synergy (Cave, 2005).
A revised model is proposed that incorporates the essential requirement for the specific SPS binding site architecture in combination with the proneural A binding sites for transcriptional activation of m8 and the other SPS+A bHLH R genes. These genes each contain an SPS+A module and exhibit similar cell-specific upregulation in nonprecursor cells in proneural clusters. In this new model, the specific architecture of the S sites in the SPS element directs the oriented binding of Su(H) so that it is in the proper orientation and/or conformation to enable a direct interaction with Da. This interaction is an essential prerequisite for subsequent recruitment and/or functional coactivation by NICD during Notch signaling. This Notch-proneural complex is then further activated by subsequent recruitment of Mam (Cave, 2005).
It is interesting to note that the mammalian homologs of each of the Su(H), NICD, and Da proteins have been shown to interact with the p300 coactivator; thus, when complexed together, these proteins could potentially function combinatorially to recruit p300 or a related coactivator (Cave, 2005).
In Drosophila and mammals, Notch signaling is used throughout development to activate many different target genes, and in multiple developmental pathways. Thus, it is of paramount importance that the proper target genes are selectively activated in the proper cell-specific patterns. It is known that Notch signaling can activate genes through non-SPS configurations of S sites in certain other target genes. For example, expression of the Drosophila genes single minded, Su(H), and vestigal have all been shown to be regulated by Notch signaling, and all have single S sites or multiple unpaired S sites but no SPS elements in their promoter and/or enhancer regions (Cave, 2005).
The results show that for essentially every promoter tested, NICD cannot activate in the absence of neural bHLH A combinatorial cofactors, suggesting that NICD may always require a combinatorial cofactor to activate target genes. If so, the non-SPS Notch target genes are likely also to have specific combinatorial cofactors. The results also clearly show that the Notch-proneural combinatorial synergy requires a specific configuration of S sites, the SPS. There may be other specific configurations of S binding sites that mediate synergy for different classes of combinatorial cofactors for Notch signaling (Cave, 2005).
Together, these observations suggest that specific, but unknown, non-SPS configurations of sites may program the interactions between Notch complexes and the proper combinatorial cofactors. It is speculated that these non-SPS configurations might be unique to each target gene, or it is possible that there are specific patterns or classes of S binding site configurations -- an 'S binding site subcode' -- that determine cofactor specificity. Thus, the results suggest that selective Notch target gene activation may be programmed by distinct Notch transcription codes in which specific configurations of S binding sites mediate selective interactions with specific combinatorial cofactors (Cave, 2005).
Elucidating the various transcription codes controlling target gene activation during Notch signaling will be an important goal for future studies. The results have clearly shown that the architecture of transcription factor binding sites can be crucial for control of cell-specific Notch target gene activation. The studies presented here give a glimpse into the molecular mechanisms by which a one dimensional pattern of DNA binding sites can program cell-specific patterns of gene expression (Cave, 2005).
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).
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 M7ACT and MbetaACT and that induction of ectopic bristles in the Drosophila wing and notum by M7ACT 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 M7ACT can induce certain ectopic leg bristles in the absence of the achaete and scute genes. In the latter context, M7ACT 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 lzr15 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).
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 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 km values for the protein substrate, the Kcat 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 Kcat with marginal effects on the km. 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).
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 (m7ACT), 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 m7ACT is able to activate ac and sc transcription. Therefore, it is formally possible that m7ACT 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 m7ACT 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 m7ACT able to induce sens expression in the absence of ac and sc? To address this question, it was confirmed that overexpression of m7ACT can produce several extra bristles in a sc10-1 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).
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. sc10-1 is a null allele for both ac and sc, the only proneural proteins expressed in the wing disk. In sc10-1 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 EB 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 EB 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 EB/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 sc10-1 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 sc10-1 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 sc10-1 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 groE48 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:
groE48 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 groE48 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).
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
sc1-260 and sc1-290 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-sc1-260 or
UAS-sc1-290. 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).
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 46th (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).
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 a