The Interactive Fly
Zygotically transcribed genes
Regulation of Enhancer of split genes
Bearded family genes in the Enhancer of split Complex
Target specificities of Enhancer of split basic helix-loop-helix proteins
Introduction
Genes are often organized by function on the chromosome. The phenomenon of a group of genes with the same function and location on the chromosome signifies a so-called gene complex. The Enhancer of split complex (E[spl]-C) includes eight genes spread over 50 kilo bases on the Drosophila third chromosome. Other examples of Drosophila gene complexes include the Antennapedia complex (ANTP-C), the bithorax complex (BX-C) and the achaete-scute complex (AS-C). The hallmark of all these gene complexes, including the E(spl)-C is that within any complex the genes are evolutionarily related and jointly regulated.
All genes in the ANTP-C and BX-C code for homeodomain proteins. Together, they regulate the segment identity of the fly. Genes of the E(spl)-C and AS-C regulate neurogenesis and related differentiation pathways. They are structurally related as well. All are basic helix-loop-helix transcription factors. Each gene in any given cluster or complex of genes will have a similar function and structure to the others in its group. This means that the genes evolved by duplication, the more recently evolved genes emulating the structure and function of what earlier proved to be successful.
Fundamental differences exist between proteins when comparing one complex to another. For example, the AS-C transcription factors activate transcription of other genes, while the transcription factors of the E(spl)-C repress transcription. This difference is reflected in the activation domains of the respective proteins. Activation domains influence the way proteins interact with the trancription apparatus of the cell (Dawson, 1995).
Whereas AS-C coded proteins are proneural, meaning they initiate neurogenesis or nerve generation, E(spl)-C coded proteins are inhibitory for neurogenesis. It seems that the expansion of numbers of genes evolving by duplication to handle the ever increasing complexities of neurogenesis, has been matched by a similar expansion of genes, also by duplication, to suppress or regulate neurogenesis. In biological English, the term regulate is narrowed to refer only to the suppression of neurogenesis.
Many cell fate decisions in higher animals are based on intercellular communication governed by the Notch signaling pathway. Developmental signals received by the Notch receptor cause Suppressor of Hairless (Su[H]) to mediate transcription of target genes. In Drosophila, the majority of Notch target genes known so far is located in the Enhancer of split complex , encoding small basic helix-loop-helix (bHLH) proteins that presumably act as transcriptional repressors. The E(spl)-C contains three additional Notch responsive, non-bHLH genes: (E(spl) region transcript m4) m4 and malpha are structurally related, whilst m2 encodes a novel protein. All three genes depend on Su(H) for initiation and/or maintenance of transcription. The two other non-bHLH genes within the locus, m1 and m6, are unrelated to the Notch pathway: m1 might code for a protease inhibitor of the Kazal family, and m6 for a novel peptide. The five genes described in this paper are arrayed between mbeta and m7, both coding for bHLH proteins. Two other bHLH genes, m3 and m5 are intermingled with the five. Bearded and M4 are 16% identical. Furthermore, in transcripts of both Brd and m4 there are three common regulatory sequence motifs within the 3' UTR. These are known as the 'Brd box', the 'GY box' and the 'K box'. As in m4, the sequence motif of the Brd box is found twice in the 3'-UTR of malpha mRNA at similar positions but without a GY box. None of the other four non-bHLH E(spl)-C genes contains either Brd or GY box. The K box appears to be more common. It is found twice in the 3'-UTR of malpha and once each in the 3' UTRs of m2 and m6 (Wurmbach, 1999).
malpha and m4 embyonic expression patterns are nearly indistinguishable, and appear very similar to those of E(spl)-C bHLH genes, particularly m5, m7 and m8. The expression patterns suggest that both genes are under the same regulatory control as are the E(spl) bHLH genes and thus, might play a part in Notch mediated cell differentiation. Surprisingly, m2 transcripts also accumulate in a pattern reminiscent of the transcript distribution of E(spl) bHLH genes, although there are no structural similarites with either the bHLH or the m4/malpha genes. Therefore m2 might serve as a Notch target gene. Unlike the other E(spl)-C genes, the gene is expressed within neuronal cells in the embryo. m6 mRNA accumulates in the CNS, brain and PNS, and in imaginal tissues. m1 is expressed in the digestive tract. Su(H) is shown to be the transmitter of Notch signaling to malpha, m4 and m2. Thus there are three types of Notch responsive genes. The bHLH genes are represented by m8 and others. m4 and malpha share structural similarity with Bearded. These Bearded family proteins share a presumptive basic amphipatic alpha-helical domain but differ with regard to other conserved sequence elements. m2, coding for a novel protein, represents the third class of Notch responsive genes (Wurmbach, 1999).
Genes in the same biochemical pathway are more likely to interact. This genetic "truth" illustrates the power of genetic studies to unravel the mystery of how genes work. After decades of pure genetic studies of gene interaction, using mutation, crosses and examination of phenotypes, it has become clear that Notch and Enhancer of split are truely in the same genetic pathway.
Notch signals are carried from the cell surface to the nucleus by Suppressor of Hairless. Su(H) activates E(spl)-C genes, and these in turn set in motion a function called lateral inhibition. Lateral inhibition is the restriction of neural fate starting with a cluster of neurogenic cells to only a single cell.
Enhancer of split complex genes regulate Delta by acting through achaete-scute complex genes. Mutation of E(spl)-C genes or groucho cause an overproduction of sensory organs precursors at the expense of epidermis. Cells mutant for E(spl)-C genes or groucho inhibit neighouring wild-type cells destined for neural fate, thus causing them to adopt the epidermal fate. This inhibition requires the genes of the achaete-scute complex, which would be in a more active state in cells mutant for E(spl)-C genes or groucho. This active state would elevate Delta transcription, repressing the neural fate of neighboring cells. Thus there is a regulatory loop between Notch and Delta that is under the transcriptional control of the E(SPL)-C and AS-C genes (Heitzler, 1996).
E(spl)-C is distinct from AS-C in other ways. Each gene in the AS-C has its own identity and function in development; the genes of E(spl)-C are redundant, for the most part. Only two, the rogue gene groucho and the nominally defining gene of the complex, Enhancer of Split yield any noticeable phenotype when mutated. With the AS-C, there are outlying locus control regions known as enhancers, charged with fine tuning the regulation of genes to expression in specific developmental domains. Despite search efforts, enhancer type master regulators of the E(spl)-C have not been found to date. With E(spl)-C, each gene is regulated autonomously, and to a large degree, in an identical fashion. Thus E(spl)-C genes do not have enhancers that direct their expression to specific developmental fields. Instead, E(spl)-C genes are expressed wherever neurogenesis is taking place.
Although emphasis is placed on the lateral inhibition function of E(spl)-C genes, they also serve a positive function. Without them, cells that lose in the competition for neuroblast development do not remain healthy, and consequently undergo programmed cell death. Thus, E(spl)-C genes help maintain the integrity and function of the neuroepithelial cell layer from which neuroblasts arise.
Given the supposedly redundant bHLH genes of the E(spl)-C, one might suppose them to be under little evolutionary constraint and thus to evolve most rapidly. However a comparison of E(spl)-C between two species of Drosophila shows the entire E(spl)-C to have been highly conserved (Maier, 1993). This presents a paradox. How can the organization of a locus be maintained in evolution when the individual elements of that locus are largely dispensible?
In spite of their seeming redundency, the proteins coded for by E(spl)-C do have individual identities as far as their abilities to interact with themselves and with other basic HLH proteins.
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, but not with Lethal of scute. m-delta, m-beta m-gamma and m3, fail to interact with achaete-scute complex coded proteins. Of these four, only m-gamma interacts with m5. The others are non-interactive. It is thought that interactions that link products of the two complexes are essential for survival and correct determination of neural tissues. Thus 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).
The seven E(spl) basic HLH proteins can form homo- and heterodimers inter-se with distinct preferences. A subset of E(spl) proteins (MB 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 Daughterless. 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).
Enhancer of Split conplex genes manifest distinct patterns of expression in the wing imaginal disc. m8 and m7 mRNAs are detected in clusters of cells that correspond to the locations where sensory organ precursors (SOPs) develop. In addition m8 is also detected in cells at the dorsal/ventral boundary throughout the third instar. The expression of mgamma and mdelta is at times associated with the same SOPs, and at other times, with different SOPs. However mdelta and mgamma mRNAs are only detected in a subset of proneural clusters. Like m8, mgamma is also present at high levels at the dorsal/ventral boundary in early stages. The domain of mß is the most distinctive. It is expressed in the wing blade associated with developing veins, and is also present at the dorsal/ventral boundary and wing margin, and is expressed in a complex pattern elsewhere in the disc, with no simple association with developing sensory organs (de Celis, 1996). In the eye disc, m8 and m7 are expressed spanning the morphogenetic furrow, whereas mgamma and mdelta are expressed just posterior to the furrow. mgamma, mdelta and mß are expressed in the more posterior portions of the disc, where the recruitment of undifferentiated cells into ommatidial units occurs; there is little expression of m8 and m7 in this region (de Celis, 1996).
Achaete and Scute regulate E(spl)-C genes in a rather paradoxical fashion. Ac and Sc bind to the consensus E box sequence CANNTG, activating transcription in Enhancer of split, m7 and m8 (Singson, 1994). Achaete, Scute, and Lethal of scute, together with VND, act synergistically to specify the neuroectodermal expression of Enhancer of split complex genes. Autoregulatory interactions of E(spl)-C genes contribute to this regulation (Kramatschek, 1994).
It is now clear that E(spl)-C gene expression is totally dependent on lateral inhibition and the Notch pathway acting through Suppressor of Hairless. If this is true, then the role of E-boxes in the transcriptional activation of E(spl)-C genes is currently unclear. Perhaps VND activate proneural genes which in turn activate E(spl)-C genes through the Notch pathway. Achaete and Scute upregulate E(spl)m7 and Enhancer of split in a wing disc pattern very similar to that achaete and scute expression. This is surprising since the wild function of E(spl)-C genes is to antagonize the SOP cell fate within the proneural cluster. It is thought that other mechanisms (Notch signaling for example) normally operate to regulate the SOP expression or activity of E(spl)-C genes (Singson, 1994). In fact it has been shown that HLH-M5 and Enhancer of split are capable of binding as homo-and heterodimers to a sequence in the promoters of the Enhancer of split and achaete genes, called the N-box, which differs slightly from the consensus binding site (the E-box) for other bHLH proteins. In transient expression assays, both proteins were found to attenuate the transcriptional activation mediated by proneural bHLH proteins Lethal of Scute and Daughterless at the Enhancer of split promoter (Oellers, 1994).
Expression of the Drosophila Enhancer of split [E(spl)] genes, and their homologs in other species, is dependent on Notch activation. The seven E(spl) genes are clustered in a single complex and their functions overlap significantly; however, the individual genes have distinct patterns of expression. To investigate how this regulation is achieved and to find out whether there is shared or cross regulation between E(spl) genes, the enhancer activity of sequences from the adjacent E(spl)mbeta, E(spl)mgamma and E(spl)mdelta genes were analyzed and comparisons to E(spl)m8 were made. Although regulatory elements can be shared, most aspects of the expression of each individual gene are recapitulated by small (400-500 bp) evolutionarily conserved enhancers. Activated Notch or a Suppressor of Hairless-VP16 fusion are only sufficient to elicit transcription from the E(spl) enhancers in a subset of locations, indicating a requirement for other factors. In tissue culture cells, proneural proteins synergise with Suppressor of Hairless and Notch to promote expression from E(spl)mgamma and E(spl)m8, but this synergy is only observed in vivo with E(spl)m8. It is concluded that additional factors besides the proneural proteins limit the response of E(spl)mgamma in vivo. In contrast to the other genes, E(spl)mbeta exhibits little response to proneural proteins and its high level of activity in the wing imaginal disc suggests that wing-specific factors cooperate with Notch to activate the E(spl)mbeta enhancer. These results demonstrate that Notch activity must be integrated with other transcriptional regulators; since the activation of target genes is critical in determining the developmental consequences of Notch activity, these results provide a framework for understanding Notch function in different developmental contexts (Cooper, 2000).
E(spl)m8 is transcribed in all sensory organ clusters: E(spl)mdelta and E(spl)mgamma in a subset of sensory clusters but strongly in the developing eye, and E(spl)mbeta in the intervein regions of the wing primordium, at the dorsal/ventral boundaries of the wing and eye, and in the presumptive leg joints. To identify the regions responsible for conferring the specific expression patterns, 1- to 2-kb fragments from the region encompassing E(spl)mdelta, E(spl)mgamma, E(spl)mbeta were fused to a minimal promoter upstream of the lacZ gene to test for enhancer activity. For each of the three genes, the fragment adjacent to the promoter (mdelta1.9, mgamma1.1, and mbeta1.5) confers a pattern of expression that largely recapitulates the endogenous genes, although there are some notable exceptions: (1) neither mdelta1.9 nor mgamma1.1 generates the strong expression associated with the morphogenetic furrow that is observed with both genes; (2) the mdelta1.9 fragment fails to confer the tegula expression normally associated with E(spl)mdelta. Given the close proximity of the genes in the complex, it is possible that adjacent genes could share regulatory elements. Because mgamma1.1 confers strong expression in the tegula domain, it might account for the tegula expression of E(spl)mdelta as well as E(spl)mgamma. To test whether there is an insulator within mgamma1.1 that would prevent it acting on the adjacent E(spl)mdelta transcription unit, mgamma1.1 was inserted between the lacZ and CD2 coding sequences. Both proteins have similar patterns of expression, indicating that mgamma1.1 is able to regulate an upstream transcription unit and so could mediate the tegula expression of the upstream E(spl)mdelta. Further indirect support for the hypothesis that enhancers can act on neighboring genes comes from analysis of a P-element (K33) inserted at the E(spl)mgamma locus. When the sequences proximal to the P-element are deleted, as occurs in Df(3R)NF1P1, the inserted lacZ gene is now expressed in a pattern weakly resembling the distal E(spl)mbeta gene, even though none of the intervening sequences have been altered. These results indicate that the E(spl)mbeta enhancer has the potential to act on the E(spl)mgamma region, but in the wild-type chromosome it must be prevented by the sequences adjacent to the E(spl)mgamma promoter (Cooper, 2000).
Thus E(spl)mbeta, E(spl)mgamma, and E(spl)mdelta patterns can largely be recapitulated by DNA fragments of ~400-500 bp located close to the transcription start site. As expected, these fragments contain Su(H) binding sites, consistent with their responsiveness to Notch signaling. However, they are also sufficient to generate quite diverse patterns of expression. The fact that this activity resides in such localized enhancers contrasts with the organization of other genes expressed in similar complex patterns in the disc, such as proneural and intervein genes. These are regulated by an array of enhancers, each of which responds to a different combination of patterning genes. The comparative simplicity of the identified E(spl) enhancers suggests that they are unlikely to be regulated by a similar array, but are more likely to be responding to the next level in the hierarchy, i.e., to the factors that are themselves expressed in complex patterns (Cooper, 2000).
The suggestion that E(spl) genes are regulated by intermediates in the patterning hierarchy is consistent with the proneural proteins contributing to their regulation. However, this also presents an inconsistency, because the E(spl) products are not detected in the neural precursor cells where proneural proteins accumulate to highest levels. This study demonstrates that proneural proteins work synergistically with Su(H)/Nicd (the complex between Suppressor of hairless protein and the intracellular domain of Notch) to activate transcription from E(spl)m8 and E(spl)mgamma enhancers in cultured cells. For E(spl)m8, this synergy can also be demonstrated in vivo, as a combination of proneural proteins and Nicd leads to higher levels of m8-lacZ expression than either component alone. This combined regulation can explain why E(spl) genes are activated in the cells surrounding the sensory organ precursors, since these are cells where both proneural proteins and Notch activity would be present. In this respect the regulation of some E(spl) genes, in particular E(spl)m8, fits with a combinatorial model, which suggests that the activation of genes in response to signaling pathways involves the transcriptional response factor for the signaling pathway acting in combination with specific patterning genes (Cooper, 2000).
The combinatorial synergy between Notch and proneural proteins may be sufficient to explain E(spl)m8 regulation, but it is not sufficient to account for the expression of some other E(spl) genes. Two key points are highlighted by the different enhancers and tissues that have been analysed. The first is that there must be factors equivalent to the proneural proteins that synergise with Notch on the E(spl)mbeta enhancer. The second is that the competence of the E(spl) enhancers to respond to Su(H)/Nicd is spatially restricted by more than just the availability of an appropriate synergising activator. Unlike the other enhancers analysed, mbeta1.5 is highly sensitive to activated Notch and Su(H)VP16 throughout the wing pouch. Intriguingly, the E(spl)mbeta fragments confer much higher levels of expression than any of the other fragments tested, even though one of the two Su(H) sites in mbeta1.5 does not conform fully to a consensus binding site. The widespread activation of mbeta1.5 in the wing pouch and its poor response to proneural proteins suggest that the E(spl)mbeta enhancer responds to other activators. This explains why it is still possible for ectopic Nicd to promote increased levels of E(spl) proteins in scute10-1 discs. Under these conditions transcription of E(spl)mbeta [and possibly E(spl)m3] could still be increased in the wing pouch, even if E(spl)m8, E(spl)mgamma and E(spl)mdelta could not. These investigations have not yet identified specific activators that account for the activity of mbeta1.5, although there are binding sites for a variety of factors including two proteins expressed in the wing, Scalloped and Caupolican (Cooper, 2000).
The differences in the responses of mgamma1.1 and mdelta1.9 compared to E(spl)m8 argue that there is an additional level of regulation that limits the accessibility of mgamma1.1 and mdelta1.9 proneural proteins/Su(H). Thus, although mgamma1.1 and mdelta1.9 are targets for proneural proteins and Su(H)/Nicd, based on effects in tissue culture and/or in vivo, they cannot be activated very effectively within the wing pouch even when high levels of certain proneural proteins and/or Nicd are expressed ectopically. Likewise, mgamma1.1 and mdelta1.9 are largely resistant to activation by Su(H) VP16 in the wing pouch, although weak activation of mdelta1.9 is sometimes detected. Similar restrictions have been observed when an E(spl)m5 enhancer, whose Su(H) binding sites had been replaced with Gal4 UAS sites, was exposed to ubiquitous Gal4. This transgene could only be activated in a limited domain, indicating that Gal4 activity can also be influenced by E(spl) regulatory sequences (Cooper, 2000).
The factors that modulate the responsiveness of the enhancers to Su(H)/Nicd and activators such as proneural proteins also act through the small 180- to 500-bp enhancer fragments, and several different mechanisms can be envisioned that might account for this modulation. One is that there is a 'prefactor' that is necessary to initially modify the chromatin and allow entry of Su(H) and proneural proteins. Recent analyses of the mechanisms involved in gene activation demonstrate that there may be sequential stages in chromatin remodelling. If an earlier step of chromatin modification is needed before Su(H) and other activators can access the enhancers, the differential response of E(spl)m8 and E(spl)mgamma fragments to Su(H) VP16 in the wing pouch would arise from a requirement for different factors to implement this initial step. An alternative model is that the enhancer fragments are also targets for specific repressors, for example, mdelta1.9 and mgamma1.1 could be specifically repressed throughout most of the wing pouch. However, none of the truncations or site-specific mutations of the mgamma1.1 and mdelta1.9 fragments have ever led to ectopic activity, as would be indicative of loss of a repressor binding region (Cooper, 2000).
Su(H)VP16 mimics phenotypes produced by activated Notch both in Drosophila and in Xenopus consistent with the evidence that Su(H) is essential for activation of target genes, via its association with Nicd. Results from mammalian tissue culture cells, however, indicate that CBF/Su(H) also functions as a repressor, interacting with histone deacetylase (HDAC). There is as yet no evidence to support this model in Drosophila, but the low levels of residual expression from E(spl) enhancers in Su(H) mutant discs might be explained by this mechanism. If in wild-type discs, Su(H) is bound to E(spl) enhancers in association with HDAC, it could prevent any activation from proneural proteins until Nicd is present. In animals that lack Su(H), this repression would no longer occur, so that high levels of proneural proteins could activate the enhancers. In support of this reasoning it is found that in tissue culture cells some activation is elicited by proneural proteins alone, particularly of the E(spl)m8 reporter. Furthermore, the residual expression from mdelta1.9 and mgamma1.1 enhancers is greatest in the oldest discs, where the levels of proneural proteins are highest and residual maternal Su(H) protein would be lowest. The dual repressor/activator roles proposed for Su(H) are like those put forward for TCF/Pangolin, which becomes a transcriptional activator of Wnt/Wingless responsive genes upon binding to beta-catenin, but appears to act as a repressor in the absence of Wnt signalling (Cooper, 2000).
Previous studies of E(spl) regulation in the embryo suggested an element of autoregulation since expression of m8-lacZ is elevated in E(spl) mutant embryos. Similar effects are also seen with HES expression in tissue culture cells, where the levels of transcription decline after their initial activation. The data suggest that this is likely to be a general mechanism, since all four E(spl) enhancers are responsive to ectopic E(spl) proteins in vivo, especially mbeta1.5. Furthermore, in cells where the repressive function of E(spl) proteins is compromised, their expression levels increase. Both these results are compatible with autoregulatory negative feedback by E(spl) proteins, so that once a critical amount is produced these proteins inhibit their own expression. This negative feedback regulation could help to keep cells in a pliable state, for example, during neurogenesis, when the balance between proneural and E(spl) proteins is critical in determining whether a cell adopts the neural fate (Cooper, 2000).
Several results indicate that the individual enhancers are able to influence more than one E(spl) gene. (1) The fragment between E(spl)mdelta and E(spl)mgamma (mgamma1.1) confers strong tegula cluster expression and contains no insulator to prevent it from acting on the 5' E(spl)mdelta gene, suggesting that it normally acts on both transcription units and accounts for the tegula expression of both genes (although the possibility that there is an insulator within E(spl)mdelta itself has not been ruled out). (2) In the Df(3R)NF1P1 deletion, the E(spl)mbeta enhancers acts on the lacZ gene inserted at E(spl)mgamma, demonstrating that the regulatory elements have the potential to act on adjacent genes. Other evidence suggests that the complex E(spl) expression patterns involve a combination of shared and redundant elements. For example, although E(spl)mgamma and E(spl)mdelta are both expressed in the ommatidial field, only mdelta1.9 confers a high level of ommatidial expression: mgamma1.1 is much less robust. In the native E(spl) complex, these two elements could act in concert to give strong E(spl)mgamma expression in ommatidia (Cooper, 2000).
The sharing of regulatory elements means that there is significant overlap in the expression patterns of adjacent genes, which accounts for some of their redundancy. In addition the effects of deleting one gene could be rescued by residual elements influencing the expression of neighboring genes. The fact that there is some interdigitation of regulatory elements may also help to explain the conservation of the E(spl) complex, as has been argued for the paralogous Hox clusters in mammals where sharing of regulatory elements has been documented and is proposed to have helped constrain the organization of the clusters (Cooper, 2000).
During Drosophila development, transcriptional activation of genes of the Enhancer of split Complex (E(spl)-C) is a major response to cell-cell signaling via the Notch (N) receptor. Although the structure and function of the E(spl)-C have been studied intensively during the past decade, these efforts have focused heavily on seven transcription units that encode basic helix-loop-helix (bHLH) repressors; the non-bHLH members of the complex have received comparatively little attention. In this report, the structure, regulation and activity of the m1, m2 and m6 genes of the E(spl)-C are examined. E(spl)m2 and E(spl)m6 are found to encode divergent members of the Bearded (Brd) family of proteins, bringing to four (malpha, m2, m4 and m6) the number of Brd family genes in the E(spl)-C. The expression of both m2 and m6 is responsive to N receptor activity and both genes are apparently direct targets of regulation by the N-activated transcription factor Suppressor of Hairless. Consistent with this, both are expressed specifically in multiple settings where N signaling takes place. Particularly noteworthy is the finding that m6 transcripts accumulate both in adult muscle founder cells in the embryo and in a subset of adepithelial (muscle precursor) cells associated with the wing imaginal disc. Overexpression of either m2 or m6 interferes with N-dependent cell fate decisions in adult PNS development. Surprisingly, while misexpression of m6 impairs lateral inhibition, overexpression of m2 potentiates it, suggesting functional diversification within the Brd protein family. Reported here are initial studies of the structure, expression and regulation of the newest member of the Brd gene family, Ocho, which is located in the recently identified Bearded Complex (Lai, 2000).
The predicted protein products of both m2 and m6 contain highly basic domains with amphipathic character near their N termini. The basic domain of m2 is most similar to that of Tom, in that both contain a proline residue within this region. The basic domain in m6 is found at its extreme N terminus; it is likewise predicted to form a largely alpha-helical, strongly amphipathic structure. Thus, it is clear that a defining structural characteristic of Brd family proteins is present in both m2 and m6. Classification of m2 and m6 as Brd family proteins is further bolstered by the presence of two short sequence domains that are widely shared by members of the family. It is noted that the motif NXANE(K/R)(L/M) is common to m6, ma and m4, while Tom, Bob and Brd contain related sequences at comparable positions. A second motif, (I/L/V)P(L/V)X(F/Y)XXTXXGTFFW, is found near the C terminus of malpha, m2, m4 and Tom, while m6 contains the clearly related sequence VXXXXTXXGSFYW. The motif DRW(A/V)QA found at the extreme C-termini of ma, m4 and Tom is not present in m2 and m6. The 3' UTRs of both m2 and m6 contain single copies of the K box (TGTGAT), a negative post-transcriptional regulatory motif previously observed to be widely distributed among the 3' UTRs of genes in both the Brd-C and the E(spl)-C. The identification of E(spl)m2 and E(spl)m6 as members of the Brd family brings the number of Brd family genes in the E(spl)-C to four (Lai, 2000).
Uniquely among the known members of the Brd-C, Ocho has a strong concentration of predicted Su(H) binding sites in its proximal upstream region, a feature more typical of Brd family members in the E(spl)-C. Within the first 720 bp 5' to the presumed Ocho transcription start site, there are five sequences fitting the high-affinity Su(H) site consensus YGTGDGAA. In addition, a predicted high-affinity binding site (GCAGGTG) for proneural bHLH activators is found quite close to the start site at position -94. All five predicted Su(H) binding sites upstream of Ocho, as well as the single predicted proneural protein binding site, are indeed bound in vitro by the respective purified fusion proteins. These results suggest that Ocho is a direct target of regulation both by proneural bHLH activators and by Su(H) and the N pathway. By contrast to all other known Brd family genes, Ocho does not appear to include in its 3' UTR any of the known or putative post-transcriptional regulatory sequence elements (Brd box, K box, or GY box). Consistent with its possible regulation by proneural proteins and by N signaling, Ocho is expressed in external sensory organ and chordotonal organ proneural clusters in the wing, eye-antenna, haltere and leg discs. Ocho transcripts also appear in a very thin band in the vicinity of the morphogenetic furrow of the developing retina, evidently corresponding to a single column of cells. Strikingly, at most sites of its accumulation in imaginal disc epithelia, the majority of the Ocho transcript is apparently localized in very small apical 'dots', with markedly less signal in more basal positions. This same predominantly apical concentration of transcripts has not been observed for other Brd family genes, and its significance and control in the case of Ocho are under investigation (Lai, 2000).
It seems reasonable to postulate that an ancestral Brd family gene encoded a protein resembling the present-day E(spl)malpha, E(spl)m4, Tom and Ocho products, with their four shared domains. Though now significantly diverged in overall amino acid sequence, these paralogous proteins are very similar in size (138-158 aa) and have very similar domain organization. This proposal is supported by the existence of such an archetypal Brd family member (158 aa) in the silk moth Bombyx mori. The Lepidoptera and Diptera diverged approximately 200 million years ago, indicating that the Brd gene family is at least this old. The Brd and Bob proteins can be viewed as truncations of this archetypal Brd family protein, suggesting that a common ancestor of the Brd and Bob genes might have arisen by acquiring a premature termination codon. The E(spl)m2 and E(spl)m6 genes may have derived independently from an archetypal progenitor or progenitors; their predicted protein products can be seen to represent the loss of one [E(spl)m6] or two [E(spl)m2] of the four canonical domains, along with expansions or contractions in the length of non-conserved regions between the remaining domains. It is likely that these evolutionary changes in the domain composition of the Brd, Bob, m2 and m6 proteins contribute to functional diversity in this family (Lai, 2000).
The only structural element common to all eight Brd family proteins is the N-terminal basic amphipathic domain. These domains are themselves quite diversified and are classifiable into three groups: 'very strongly' amphipathic (Brd and Bob), 'less strongly' amphipathic (malpha, m4 and m6), and proline-containing (m2, Tom and Ocho). The observation that all Brd family proteins tested, with the exception of m2, induce qualitatively (but not quantitatively) similar phenotypes in GAL4-UAS misexpression experiments (i.e., interference with N pathway activity) suggests that they may interact with a common target, though the quality of the interaction may be influenced by the type of basic amphipathic domain present. The diversity of expression patterns among Brd family genes is no less striking. In both embryonic and imaginal tissue, these genes are deployed in a myriad of locations in which N signaling is used to elicit cellular responses and/or determine cell fates, and evidence is presented that all Brd family genes are direct targets of transcriptional regulation by Su(H). Nevertheless, the precise expression pattern of each Brd family member is unique, such that different combinations of Brd family genes are active at different sites of N pathway activity. Thus, the members of this family are differentially responsive to regulation by N receptor activity. The observation that promoter-reporter constructs for all Brd family genes tested to date recapitulate the expression pattern of the corresponding endogenous gene demonstrates that the selectivity of this response is mediated largely at the transcriptional level. Thus, it is suggested that evolution of transcriptional cis-regulatory sequences has been a major mechanism for diversification of Brd family gene expression and probably function as well (Lai, 2000).
Target specificities of Enhancer of split basic helix-loop-helix proteins
Related sites:
Alifragis, P., et al. (1997). A network of interacting transcriptional regulators involved in Drosophila neural fate specification revealed by the yeast two-hybrid system. Proc. Natl. Acad. Sci. 94(24): 13099-13104. Medline abstract: 98058775
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Dawson, S. R., et al. (1995). Specificity for the hairy/enhancer of split basic helix-loop-helix (bHLH) proteins maps outside the bHLH domain and suggests two separable modes of transcriptional repression. Mol Cell Biol 15: 6923-6931. Medline abstract: 96069402
de Celis, J. F., et al. (1996). Functional relationships between Notch, Su(H) and the bHLH genes of the E(spl) complex, the E(spl) genes mediate only a subset of Notch activities during imaginal development. Development 122: 2719-2728. Medline abstract: 96379733
Gigliani, F., et al. (1996). Interactions among the bHLH domains of the proteins encoded by the Enhancer of split and achaete-scute gene complexes in Drosophila. Mol. Gen. Genet. 251: 628-634. Medline abstract: 96335137
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Oellers, N., Dehio, M., and Knust, E. (1994). bHLH proteins encoded by the Enhancer of split complex of Drosophila negatively interfere with the transcriptional activation mediated by proneural genes. Mol. Gen. Genet. 244: 465-73. Medline abstract: 94359472
Schrons, H., Knust, E. and Campos-Ortega, J.A. (1992). The Enhancer of split complex and adjacent genes in the 96F region of Drosophila melanogaster are required for segregation of neural and epidermal progenitor cells. Genetics 132: 481-503. Medline abstract: 93051286
Singson, A., Leviten, M.W., Bang, A.G., Hua, X.H. and Posakony, J.W. (1994). Direct downstream targets of proneural activators in the imaginal disc include genes involved in lateral inhibitory signaling. Genes Dev. 8(17): 2058-2071. Medline abstract: 95047355
Tapanes-Castillo, A. and Baylies, M. K. (2004). Notch signaling patterns Drosophila mesodermal segments by regulating the bHLH transcription factor Twist. Development 131: 2359-2372. Medline abstract: 15128668
Wurmbach, E., Wech, I. and Preiss, A. (1999). The Enhancer of split complex of Drosophila melanogaster harbors three classes of Notch responsive genes. Mech. Dev. 80(2): 171-80. Medline abstract: 99173785
date revised: 30 August 2000
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