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).

How does the Enhancer of split complex function?

split is an alternative name for the gene Notch. When Notch is mutated, the results are often not lethal. Enhancer of split exaggerates for the worse the effects of Notch mutation. In genetic terms, Notch and Enhancer of split are said to interact.

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).

Regulation of Enhancer of split genes

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

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

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

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

The combinatorial synergy between Notch and proneural proteins may be sufficient to explain E(spl)m8 regulation, but it is not sufficient to account for the expression of some other E(spl) genes. Two key points are highlighted by the different enhancers and tissues that have been analysed. The first is that there must be factors equivalent to the proneural proteins that synergise with Notch on the E(spl)mbeta enhancer. The second is that the competence of the E(spl) enhancers to respond to Su(H)/Nicd is spatially restricted by more than just the availability of an appropriate synergising activator. Unlike the other enhancers analysed, mbeta1.5 is highly sensitive to activated Notch and Su(H)VP16 throughout the wing pouch. Intriguingly, the E(spl)mbeta fragments confer much higher levels of expression than any of the other fragments tested, even though one of the two Su(H) sites in mbeta1.5 does not conform fully to a consensus binding site. The widespread activation of mbeta1.5 in the wing pouch and its poor response to proneural proteins suggest that the E(spl)mbeta enhancer responds to other activators. This explains why it is still possible for ectopic Nicd to promote increased levels of E(spl) proteins in scute^10-1 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).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Bearded family genes in the Enhancer of split Complex

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).

A novel interaction between hedgehog and Notch promotes proliferation at the anterior-posterior organizer of the Drosophila wing

Notch has multiple roles in the development of the Drosophila melanogaster wing imaginal disc. It helps specify the dorsal-ventral compartment border, and it is needed for the wing margin, veins, and sensory organs. Evidence is presented for a new role: stimulating growth in response to Hedgehog. This study shows that Notch signaling is activated in the cells of the anterior-posterior organizer that produce the region between wing veins 3 and 4, and strong genetic interactions are described between the gene that encodes the Hedgehog pathway activator Smoothened and the Notch pathway genes Notch, presenilin, and Suppressor of Hairless and the Enhancer of split complex. This work thus reveals a novel collaboration by the Hedgehog and Notch pathways that regulates proliferation in the 3-4 intervein region independently of Decapentaplegic (Casso, 2011).

This article shows activation of N signaling at the wing AP organizer by defining with cellular resolution the expression patterns of N protein and N pathway reporters in relation to the AP organizer, and dependence on Hh signaling is shown. Strong interactions are also shown between hh- and N-signaling pathways, and it is confirmed that the activation of N signaling is necessary for the normal growth of the AP organizer. This work uncovers a previously unknown activity of the Hh pathway in mitogenesis at the AP organizer: the activation of N signaling. These results are surprising in that they show that the roles of N signaling in the growth of the wing are not limited to the function of the DV organizer and a general growth-promoting function in the wing: N signaling also induces growth downstream of hh at the AP organizer (Casso, 2011).

N is essential for the cells that give rise to the DV margin, veins, and sensory organs of the wing, and its expression is elevated in the progenitors that produce these structures. The DV margin progenitors, which transect the wing disc in a band that is orthogonal to the Hh-dependent AP organizer, express wg in response to N. These wg-expressing cells function as a DV organizer, and several lines of evidence suggest that the AP and DV organizers function independently: Hh signaling along the AP axis is not N-dependent, N signaling along the DV axis is not hh-dependent, and targets regulated by the AP and DV organizers are not the same. The findings reported in this study show that, separately from its roles elsewhere in the wing disc, N signaling has an essential mitogenic role in the cells of the AP organizer region (Casso, 2011).

While N can stimulate growth by inducing expression of wg (as it does in the DV organizer), hyper-activation of N signaling near the AP border of the wing pouch causes overgrowth that is independent of wg. wg is not normally expressed along the AP axis, but this study found that N signaling is activated at the AP compartment border in late third instar discs, pupal discs, and pupal wings. Through vn expression, Hh signaling at the AP compartment border increases expression of Dl flanking the organizer, and Hh signaling activates N in the 3-4 intervein region. While a role for Ser at the AP organizer has not been directly investigated, Ser expression in the wing disc is very similar to that of Dl, with high levels of Ser in the vein 3 and 4 primordia as well as along the DV border. The results show that growth of the 3-4 intervein region, long known to be dependent on Hh, is also dependent on Hh-induced activation of N (Casso, 2011).

Expression of N pathway reporters and components and genetic interactions support this model of regulation of the intervein region. The reporters Su(H)lacZ and E(spl)m-α-GFP express at the AP border in a Hh-dependent manner. Elevated levels of N protein expression on the anterior side of the AP border require Vn signaling. This N region is flanked by Dl expression in the vein 3 and vein 4 primordia; Dl expression is known to be dependent upon expression of the Hh target vn. Genetic interactions between smo RNAi and N and between smo RNAi and N pathway components [e.g., the Psn intramembrane protease, which activates N; the Su(H) transcriptional co-activator; the Su(dx) E3 ubiquitin ligase, which monitors levels of N protein; and the E(spl) complex of N transcriptional targets] also indicate a functional link between the Hh and N systems (Casso, 2011).

The model for the role of N in the 3–4 intervein region is consistent with previous reports of expression patterns of the E(spl) genes E(spl)m8, M-β, and M-α. Ectopic expression of HLH-mδ and m8 rescues smo RNAi. Although HLH-mδ does not appear to be expressed in the AP organizer in a wild-type wing because the E(spl) genes are thought to have partially overlapping functions, the fact that mδ phenocopies the rescue by m8 reinforces the conclusion that the function of the E(spl) genes is critical to inducing growth at the AP organizer. Importantly, these findings show that the cells that activate N are the anterior cells of the AP organizer and are not associated with development of veins in pupal wings. Vein 4 develops within the posterior compartment and in many cases has posterior cells between it and the AP border. Since activation of these reporters was never observed extending into posterior territory, their expression correlates better with the position of the AP organizer than with vein/intervein territories at the stages that were examined. It should be noted that no single readout currently available marks all tissues in which N is activated. The E(spl) genes, for example, express in a variety of spatial and temporal patterns in response to N, and these patterns are only partially overlapping. The possibility cannot be excluded that N signaling is also activated along the stripe of Dl expression in the vein 3 primordium or that signaling could be occurring in the entire broad stripe of elevated N expression in the AP organizer. No changes were seen in proliferation using a direct readout such as phosphohistone staining of mitotic cells to visualize increases or decreases in growth at the AP organizer. These proliferation assays mark cell cycle progression at a single time point in fixed tissues, and the changes that were seen in the adult wing could be due to one or two fewer cell division cycles occurring over the course of days of development (Casso, 2011).

The findings indicate a link between the Hh and N pathways and suggest a model in which the domain of N activation at the AP border [manifested by Su(H)lacZ expression] is a consequence both of flanking cells that express high levels of Dl and of Hh signaling. The proposed role for Hh signaling is multifaceted: Hh is required for vn expression, which is itself required for high levels of Dl expression in the vein 3 stripe and the vein 4 stripe and for N expression at the AP organizer. Although whether Dl expression in veins 3 and 4 activates N signaling has not been directly tested, vn function is necessary for N activation, and the reciprocal relationship between cells expressing high levels of Dl and neighboring cells expressing high levels of N is well established (Casso, 2011).

Interactions between the Sonic hedgehog (SHH) and N signaling pathways have been identified previously in vertebrates. Particularly noteworthy for their relevance to the interactions that were found in the Drosophila wing disc are the increased expression of the Serrate-related N ligand, Jagged 1, in the mouse Gli3^Xt mutant; reduced expression of Jagged1 and Notch2 in the cerebella of mice with reduced SHH signaling; regulation of the Delta-related ligand, DNER, by SHH in Purkinje neurons and fetal prostate; activation of N signaling in neuroblastomas in Ptch^+/– mice with elevated SHH signaling; and Notch2 overexpression in mice carrying an activated allele of smo. These studies establish a positive effect of SHH signaling on the N pathway, consistent with the current data (Casso, 2011).

In Drosophila, there have been several reports of interactions between the N and Hh pathways. In the wing pouch, for example, expression levels of the Hh targets ptc, ci, col, and en are markedly lower at the intersection of the AP and DV borders than elsewhere in the AP organizer. This repression is mediated by wg. In addition, N and col function together to determine the position of wing veins 3 and 4. However, loss of function of either col or vn did not show interactions with smo RNAi (Casso, 2011).

N functions in two types of settings. One is associated with binary fate choices; it involves adjacent cells that adopt either of two fates on the basis of the activation of N signaling in one cell and inactivation in the other. In these settings, activation of N not only induces differentiation in a designated cell, but also blocks activation of N in the neighbors. The second type of setting does not induce a binary fate choice, but instead activates the pathway at the junction of two distinct cell types. N pathway activation at the DV border in the wing is one example; in this setting, N is activated in a band that straddles the DV border and the N ligands Dl and Ser signal from adjacent domains from either the dorsal (i.e., Dl) or the ventral (i.e., Ser) side. Activation of N in the 3-4 intervein region at the AP border appears to be of this second type: it occurs adjacent to regions of elevated Dl expression at the apposition of anterior and posterior cell types. There is no apparent binary fate choice in this region of the wing (Casso, 2011).

In ways that are not understood well, development of the 3-4 intervein region is controlled differently from other regions of the wing pouch. Whereas Hh induces expression of Dpp, and Dpp orchestrates proliferation and patterning of wing pouch cells generally, Dpp does not have the same role in the 3-4 intervein cells. For these cells, Hh appears to control proliferation and patterning directly. For example, the lateral regions of wings that develop from discs with compromised Dpp function are reduced, but their central regions, between veins 3 and 4, are essentially normal. Downregulation of Dpp activity and repression of expression of the Dpp receptor appears to be the basis for this insensitivity. In contrast, partial impairment of Hh signal transduction that is insufficient to reduce Dpp function (such as in fu mutants or in the smo RNAi genotypes that were characterized) results in wings that are normal in size and pattern except for a small or absent 3-4 intervein region. Since the 3-4 intervein cells divide one to two times in the early pupa during disc eversion and wing formation, the direct role of Hh in regulating these cells may be specific to this post-larval period. N signaling has a well-described mitogenic function in the wing. Ectopic signaling causes hyper-proliferation, while clones that impair the activation of the pathway reduce growth. The current findings indicate that Hh regulates proliferation of cells in the 3-4 intervein region at least in part by activating N signal transduction (Casso, 2011).

The idea that this model promotes is that Hh-dependent activation of N at the AP organizer is stage- and position-specific. This model is consistent with the complex pattern of N expression and activation in the wing, since different pathways may regulate N in different locations. It is also consistent with the proposed role of N regulating the width and position of veins 3 and 4, since the processes that establish the veins and control proliferation of the intervein cells need not be the same, even if they are interdependent. The temporal specificity that this study describes represents an example of how complex patterns are generated with a limited number of signaling pathways -- in this case by using N signaling for different outcomes at different times and in different places. Throughout larval development, Dpp regulates proliferation and patterning in the wing disc. In the pupal wing, Dpp takes on a new instructive vein-positioning function. There is no evidence that Hh regulates Dpp in the pupal wing, and moreover, the cells that had produced Dpp at the AP organizer no longer do so and no longer function as a AP organizers. These data show that N also takes on a new role during late larval and pupal stages: functioning at the AP organizer to regulate growth in response to Hh signaling (Casso, 2011).

JAK/Stat dependent m5 regulates heart precursor diversification in Drosophila

Intercellular signal transduction pathways regulate the NK-2 family of transcription factors in a conserved gene regulatory network that directs cardiogenesis in both flies and mammals. The Drosophila NK-2 protein Tinman (Tin) was recently shown to regulate Stat92E, the JAK/Stat pathway effector, in the developing mesoderm. To understand whether the JAK/Stat pathway also regulates cardiogenesis, a systematic characterization was performed of JAK/Stat signaling during mesoderm development. Drosophila embryos with mutations in the JAK/Stat ligand upd or in Stat92E have non-functional hearts with luminal defects and inappropriate cell aggregations. Using strong Stat92E loss-of-function alleles, this study shows that the JAK/Stat pathway regulates tin expression prior to heart precursor cell diversification. tin expression can be subdivided into four phases and, in Stat92E mutant embryos, the broad phase 2 expression pattern in the dorsal mesoderm does not restrict to the constrained phase 3 pattern. These embryos also have an expanded pericardial cell domain. The E(spl)-C gene HLHm5 is shown to be expressed in a pattern complementary to tin during phase 3, and this expression is JAK/Stat dependent. In addition, E(spl)-C mutant embryos phenocopy the cardiac defects of Stat92E embryos. Mechanistically, JAK/Stat signals activate E(spl)-C genes to restrict Tin expression and the subsequent expression of the T-box transcription factor H15 to direct heart precursor diversification. This study is the first to characterize a role for the JAK/Stat pathway during cardiogenesis and identifies an autoregulatory circuit in which tin limits its own expression domain (Johnson, 2011).

tin expression can be divided into four distinct spatial-temporal phases. Phase 1 tin expression initiates after gastrulation during which Twist (Twi) activates pan-mesodermal tin expression via the enhancer tinB. Phase 2 begins after FGF-mediated mesoderm spreading in which Dpp signals produced by the dorsal ectoderm maintain tin expression throughout the dorsal mesoderm via a second enhancer, tinD. It is during phase 2 that Tin specifies the major dorsal mesoderm derivatives. Phase 3 initiates after dorsal mesoderm progenitor specification and is characterized by a pronounced restriction of tin to the cardiac and visceral muscle progenitors. Upd and Upd2 are expressed in the ventral ectoderm during the transition from phase 2 to phase 3 expression. Phase 4 initiates after precursor specification and is characterized by further restriction of tin to the cardiac precursors that give rise to the contractile cardiomyocytes and the noncontractile pericardial nephrocytes. Phase 4 expression further directs heart cell diversification and maturation and is dependent on a third enhancer element, tinC (Johnson, 2011 and references therein).

To test the hypothesis that the JAK/Stat pathway functions in the cardiac-specific gene regulatory network, a systematic characterization was performed of JAK/Stat signaling during mesoderm development. The JAK/Stat pathway regulates final cardiac morphology as well as heart precursor diversification. Stat92E loss-of-function analysis identified a discrete function for the JAK/Stat pathway in restricting tin during the transition from phase 2 to phase 3 expression. In addition, Stat92E embryos have an expanded pericardial cell domain arguing that the JAK/Stat pathway regulates tin to ensure proper heart precursor diversification. Mechanistically, it was found that the E(spl)-C gene HLHm5 is expressed in a complementary pattern to tin during phase 3 expression and that the JAK/Stat pathway activates HLHm5 expression in the dorsal mesoderm. The E(spl)-C genes in turn repress twi expression to preserve cardiac morphology and restrict tin and H15 expression to direct heart precursor diversification. These findings provide the first evidence of a role for the JAK/Stat pathway in cardiogenesis and identify a novel tin autoinhibitory circuit involving Stat92E and E(spl)-C (Johnson, 2011).

Stat92E is a direct Tin target gene during phase 2 expression; however, Stat92E is expressed in segmented stripes at this stage whereas tin is expressed throughout the dorsal mesoderm. In addition, embryos lacking only the maternal contribution of Stat92E have mesoderm patterning defects. Tin-regulated Stat92E zygotic transcription is therefore insufficient to coordinate mesoderm development. These data suggest that maternally contributed Stat92E is activated in response to segmented Upd and Upd2 activity, binds the Stat92E locus and co-activates Stat92E zygotic transcription with Tin. In addition, ChIP-chip experiments identified Stat92E binding activity and a conserved Stat92E consensus binding sites (SCBS) within the Tin-responsive Stat92E mesoderm enhancer. It is concluded that Stat92E and tin co-regulate a brief, spatially restricted JAK/Stat signaling event that patterns the mesoderm (Johnson, 2011).

Phase 3 tin expression promotes cell-type diversification and differentiation within the dorsal mesoderm and is indirectly activated by Wg via the T-box transcription factors in the Dorsocross complex and the GATA factor Pannier. A key finding of this study is that the JAK/Stat pathway activates the transcriptional repressor HLHm5 in the dorsal mesoderm to establish phase 3 tin expression. Because the HLHm5 cis-regulatory region lacks a conserved SCBS, it is predicted that Stat92E regulates HLHm5 expression through a non-consensus binding site. Alternatively, Stat92E acts at long distances to regulate gene expression. The SCBSs in E(spl)-C could be a platform from which Stat92E regulates multiple E(spl)-C genes that, in turn, regulate HLHm5 expression. In either event, Stat92E-mediated activation of E(spl)-C genes restricts tin in the dorsal mesoderm to establish phase 3 expression. Tin, therefore, establishes an autoinhibitory circuit by activating its own repressors in the JAK/Stat pathway and in E(spl)-C (Johnson, 2011).

Both Stat92E and Df(3R)Espl^δmδ-m6 embryos show an increased number of Tin+ pericardial cells and an expanded H15 expression domain. Misexpressing mid or H15 in the mesoderm expands the number of Tin+ cells in the dorsal vessel and embryos misexpressing mid show a phenotype strikingly similar to Stat92E and E(spl) embryos. As mid, and presumably H15, are positively regulated by Tin during St11/12, unrestricted tin expression in Stat92E or Df(3R)Espl^δmδ-m6 embryos expands the H15 expression domain. Ectopic H15 then specifies supernumerary Tin+ pericardial cells. Because mid expression is not affected in Stat92E embryos, the expression of mid and H15 must be controlled by distinct mechanisms and might have non-redundant functions (Johnson, 2011).

Although the Twi target genes directing mesoderm development and subdivision have been studied in detail, the molecular mechanism that restricts twi expression after gastrulation remains unclear. One regulator of twi is the Notch signaling pathway, which acts through E(spl)-C genes to restrict twi expression. However, Notch signaling appears to be active throughout the mesoderm after gastrulation. This study suggests that segmented JAK/Stat signaling activity differentially upregulates E(spl)-C gene expression in concert with Notch to produce periodic twi expression in the mesoderm. In addition, pan-mesodermal twi expression causes cardiac defects independently of cell fate specification, suggesting that the cardiac morphology defects in Stat92E embryos are due to dysregulated twi expression. These results highlight a previously unrecognized role for the JAK/Stat pathway and Twi in regulating cardiogenesis (Johnson, 2011).

Pericardial cell hyperplasia without a concomitant loss of cardioblasts has been reported for dpp hypomorphic embryos and lame duck (lmd) embryos. A late Dpp signal, which occurs after the Dpp signal that regulates phase 2 tin expression, instructs the Gli-like transcription factor Lmd to repress Tin expression in fusion competent myoblasts (FCMs). Tin expression appears to expand into the FCM domain in Stat92E embryos; however, the closest Stat92E chromatin binding domain is over 120 kb distal to the lmd transcriptional start site. This study highlights the possibility that sequential JAK/Stat and then Dpp signals regulate Lmd function to direct heart precursor diversification (Johnson, 2011).

In vertebrates, skeletal myogenesis initiates with the periodic specification of somites in the presomitic mesoderm. Cyclical expression of hairy1 in the chick, the hairy- and E(spl)-related family (Her) in zebrafish, and the orthologous Hes family in mice are under the control of Notch-Delta signaling. Loss of her1 and her7 alters the periodicity with which somite boundaries are specified in fish, and artificially stabilizing Hes7 causes somites to fuse in the mouse. Thus, mesoderm segmentation is governed by Notch-Delta regulation of the E(spl)-C genes in both insects and vertebrates indicating that the two processes share molecular homology. A cell culture model of somitogenesis shows that oscillating Hes1 expression is dependent on Stat activity. This study supports the exciting possibility that JAK/Stat signaling and E(spl)-C form a conserved developmental cassette directing mesoderm segmentation throughout Metazoa (Johnson, 2011).

Notch signaling downstream target E(spl)mβ is dispensable for adult midgut homeostasis in Drosophila

Adult tissue homeostasis is maintained by residential stem cells through the proper balance of stem cell self-renewal and differentiation. The adult midgut of Drosophila contains multipotent intestinal stem cells (ISCs), and Notch signaling plays critical roles in the proliferation and differentiation of these ISCs. However, how Notch signaling downstream targets regulate ISC proliferation and differentiation still remains unclear. This study found that Notch signaling downstream targets E(spl)mβ and E(spl)mα are differentially expressed in ISCs and their progeny. Interestingly, midgut homeostasis was not affected in E(spl)mβ null mutant. No obvious defects are observed in the intestines ectopically expressing E(spl)mβ or E(spl)mα. Importantly, the defects in ISC proliferation and differentiation observed in Notch mutant cannot be rescued by ectopic expression of E(spl)mβ or E(spl)mα. Together, these data indicate that the proliferation and differentiation of ISCs are not regulated by individual Notch downstream target, but by different downstream targets collectively (Lu, 2015).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Enhancer of Split Gene Complex: architecture and coordinate regulation by Notch, Cohesin and Polycomb group proteins

The cohesin protein complex functionally interacts with Polycomb group (PcG) silencing proteins to control expression of several key developmental genes, such as the Drosophila Enhancer of split gene complex [E(spl)-C]. The E(spl)-C contains twelve genes that inhibit neural development. In a cell line derived from central nervous system, cohesin and the PRC1 PcG protein complex bind and repress E(spl)-C transcription, but the repression mechanisms are unknown. The genes in the E(spl)-C are directly activated by the Notch receptor. This study shows that depletion of cohesin or PRC1 increases binding of the Notch intracellular fragment (NICD) to genes in the E(spl)-C, correlating with increased transcription. The increased transcription likely reflects both direct effects of cohesin and PRC1 on RNA polymerase activity at the E(spl)-C, and increased expression of Notch ligands. By chromosome conformation capture this study found that the E(spl) C is organized into a self-interactive architectural domain that is co-extensive with the region that binds cohesin and PcG complexes. The self-interactive architecture is formed independently of cohesin or PcG proteins. It is posited that the E(spl)-C architecture dictates where cohesin and PcG complexes bind and act when they are recruited by as yet unidentified factors, thereby controlling the E(spl)-C as a coordinated domain (Schaaf, 2013).

These studies investigated the regulation of the E(spl)-C complex by cohesin, PRC1, and the Putzig (Chro-Z4/Pzg) protein complex in CNS derived BG3 cells, in which the E(spl)-C has a rare restrained state with a cohesin-H3K27me3 overlap. The E(spl)-C has a highly self-interactive structure that is unexpectedly independent of these protein complexes and the level of gene expression. Depletion of any of these three protein complexes, however, significantly increases E(spl)-C transcription. The effects of these three protein complexes on E(spl)-C expression likely reflect changes in expression of Notch ligands, and in the cases of cohesin and PRC1, potentially direct effects on activator and Pol II activity at the E(spl)-C genes (Schaaf, 2013).

Chromosome conformation capture (3C) analysis revealed that the E(spl)-C has a structure in which all positions within the complex interact with each other at a high frequency, but not with flanking regions. Surprisingly, this study found that this architecture is independent of cohesin, the PcG complexes, the Chro-Pzg/Z4 complex, transcription, and stage of the cell cycle. Thus it is not known which factors establish this striking architecture, which defines the E(spl)-C as a structurally independent domain. It is also not yet known which factors control recruitment of cohesin and PcG complexes to the locus. It is speculated, however, that this architecture coordinates transcriptional control of the entire E(spl)-C, based on the finding that in BG3 cells, cohesin, PRC1, and the ubiquityl-Histone H2 (H2Aub) and H3K27me3 histone modifications made by the PRC1 and PRC2 complexes are co-extensive within this architectural domain. Although no known insulators or boundary elements flank the E(spl)-C, and depletion of the CP190 protein required for activity of all known Drosophila insulators does not alter E(spl)-C expression, it is likely that the unknown factors that form this structure limit the spread of these protein complexes and histone marks. The E(spl)-C architectural domain may be evolutionarily significant, because Notch-regulated Enhancer of split complexes with similar structures are conserved in insects and crustaceans over 420 million years (Schaaf, 2013).

Possible clues to the identities of the factors that control the E(spl)-C architecture and/or the recruitment of cohesin and PcG complexes may arise in genetic screens for factors that alter E(spl)-C sensitive phenotypes, such as the Nspl-1 rough eye and bristle phenotypes. These phenotypes are sensitive to mutations in the E(spl)-C and cohesin genes in a highly dosage-sensitive manner, and modest changes in the E(spl)-C architecture or recruitment of cohesin or PcG proteins may have similar effects (Schaaf, 2013).

There is coordinate regulation of gene complexes by cohesin in mammalian cells. The Protocadherin beta (Pchdb) gene complex is downregulated in the embryonic fibroblasts and brains of mice heterozygous mutant for the Nipbl cohesin loading factor, and brains of mice that are homozygous mutant for the SA1 cohesin subunit, and cohesin is involved in enhancer-promoter looping in the Protocadherin alpha (Pchda) complex, helping determine which genes in the complex are active. While this is a positive role for cohesin, as opposed to the repressive role that occurs in the E(spl)-C, it is possible that the protocadherin gene clusters also have a higher order architecture that dictates how cohesin functions within the gene complex. Recent genome-wide analysis also indicates that there are constitutive higher order looping architectures that may organize cell-type specific interactions on a shorter scale, and that cohesin contributes to both types of structures (Schaaf, 2013).

Prior studies showed that depletion of cohesin or PRC1 increases expression of the Serrate Notch ligand gene. This likely explains part of the increase in E(spl)-C transcription upon cohesin and PRC1 depletion, because the E(spl)-C genes are directly activated by Notch. Consistent with this idea, this study detected increases in NICD association with the HLHmβ and HLHm3 genes upon cohesin or PRC1 depletion. EDTA treatment confirms that increasing Notch activation increases NICD binding to the E(spl)-C genes (Schaaf, 2013).

Because cohesin and PRC1, unlike the Chro-Pzg/Z4 complex, bind directly to the E(spl)-C, it is also possible that they also directly control association of NICD with the Su(H) protein bound upstream of the active genes. For example, they could potentially interact with NICD or the Su(H) complex, and interfere with NICD association, or somehow facilitate ubiquitination and degradation of NICD. The lack of an effect of cohesin or PRC1 depletion on NICD association with E(spl)-C genes after EDTA treatment does not rule out this possibility, because under these conditions, the amount of NICD is no longer limiting (Schaaf, 2013).

It remains to be determined if the multiple effects of cohesin on Notch function seen in Drosophila, including regulation of Notch ligand and target genes, also occur in mammals. If so, this could underlie many of the development deficits seen in Cornelia de Lange syndrome, caused by mutations in NIPBL and cohesin subunit genes. Mutations in Notch receptor and ligand genes cause Alagille and other syndromes that affect many of the same tissues as CdLS (Schaaf, 2013).

The possibility cannot be ruled out that cohesin and PRC1 directly repress E(spl)-C transcription independently of any effects on Notch ligand expression or NICD association with the E(spl)-C genes. This is because both bind throughout the complex, and the PRC1-generated H2Aub repressive histone mark is co-extensive with the E(spl)-C architectural domain. Importantly, all genes in BG3 cells that show rare overlap of cohesin and the PRC2-generated H3K27me3 modification, such as the invected and engrailed gene complex, show substantial increases in transcription upon cohesin or PRC1 depletion, even though they are not Notch activated. It is highly unlikely that cohesin or PRC1 depletion increases the expression of all the diverse activators that control these genes, and more likely that cohesin and PRC1 directly repress their transcription (Schaaf, 2013).

At all genes examined that are strongly repressed by cohesin, cohesin restricts the transition of paused RNA Pol II into elongation, irrespective of whether or not they have the H3K27me3 mark (Fay, 2011). PRC1 restricts entry of paused Pol II into elongation at active genes that bind cohesin and PRC1, but lack PRC2 and the H3K27me3 modification (Schaaf, 2013b). It is thus posited that cohesin and PRC1 together restrict transition of the paused Pol II present at the active E(spl)-C genes into elongation. Because co-depletion of cohesin and PRC1 does not synergistically increase transcription, it is thought likely that they function together at the same step. Cohesin and PRC1 directly interact with each other, and cohesin facilitates binding of PRC1 to active genes that lack the H3K27me3 mark. Cohesin depletion, however, does not significantly alter PRC1 association with the E(spl)-C, likely because PRC1 binding is stabilized by the known interaction of PRC1 with H2K27me3. PRC1 is thus not sufficient to repress E(spl)-C transcription in the absence of cohesin, indicating that cohesin has roles that extend beyond its interaction with PRC1 (Schaaf, 2013).

The super elongation complex drives neural stem cell fate commitment

Asymmetric stem cell division establishes an initial difference between a stem cell and its differentiating sibling, critical for maintaining homeostasis and preventing carcinogenesis. Yet the mechanisms that consolidate and lock in such initial fate bias remain obscure. This study used Drosophila neuroblasts to demonstrate that the super elongation complex (SEC) acts as an intrinsic amplifier to drive cell fate commitment. SEC is highly expressed in neuroblasts, where it promotes self-renewal by physically associating with Notch transcription activation complex and enhancing HES (hairy and E(spl)) transcription. HES in turn upregulates SEC activity, forming an unexpected self-reinforcing feedback loop with SEC. SEC inactivation leads to neuroblast loss, whereas its forced activation results in neural progenitor dedifferentiation and tumorigenesis. These studies unveil an SEC-mediated intracellular amplifier mechanism in ensuring robustness and precision in stem cell fate commitment and provide mechanistic explanation for the highly frequent association of SEC overactivation with human cancers (Liu, 2017).

Both normal development and tissue homeostasis rely on the remarkable capacity of stem cells to divide asymmetrically, simultaneously generating one identical stem cell and one differentiating progeny. Extensive studies have unveiled how extrinsic niche signals and intrinsic cell polarity cues ensure proper orientation of mitotic spindle and, hence, asymmetric division of stem cells. However, it remains unclear whether the initial fate bias, established by unequal exposure to niche signals or differential partitioning of cell fate determinants, can be immediately and automatically consolidated and stabilized into distinct and irreversible cell fate outcomes. In fact, in vivo timelapse imaging of the developing zebrafish hindbrain using the Notch activity reporter showed that, immediately after the asymmetric division of a radial glia progenitor, Notch activity is not noticeably biased in the paired daughter cells. Instead, the differential Notch activity in the pair of daughter cells only gradually increases afterward, over a time span of 3-8 hr, indicating the existence of a progressive and tightly regulated transition phase between the initial cell fate decision and the ultimate cell fate commitment. Stem cells and progenitors, especially the fast-cycling ones, face the daunting challenges of ensuring timely, precise, and robust cell fate determination in every cell cycle and are likely to achieve so through rapid amplification of the initial small fate bias upon their asymmetric division. In electronics, a device called an amplifier magnifies a small input signal to a large output signal until it reaches a desired level. Conceivably, a similar 'amplifier' mechanism could be employed in the stem cells or progenitors to accelerate the transition phase and drive cell fate commitment. Dysregulation of such an amplifier could cause an imbalance between self-renewal and differentiation, resulting in impaired tissue homeostasis. However, the regulatory modules governing the transition phase from stem cell fate decision to fate commitment, especially the identity and control of this putative 'amplifier,' remain largely unexplored (Liu, 2017).

Drosophila type II neural stem cells (NSCs), known as neuroblasts (NBs), provide an excellent model system for studying stem cell fate commitment. Firstly, distinct from type I NB lineages, type II NB lineages contain transit-amplifying cells called intermediate neural progenitors (INPs), similar to mammalian NSC lineages in both functional and molecular criteria, yet with much simpler anatomy and lineage composition. Each type II NB undergoes stereotypic, self-renewing divisions to produce immature INPs, which, upon maturation, undergo a few rounds of asymmetric, self-renewing divisions to give rise to ganglion mother cells (GMCs) that subsequently generate post-mitotic neurons or glia. Secondly, the identity of each cell type in the NB lineages can be unambiguously determined by a combination of cell fate makers as well as by their geological positions within the lineages. Thirdly, the molecular mechanisms underlying initial NB versus INP fate decision are well understood. Unidirectional Notch signaling is both necessary and sufficient to promote type II NB self-renewal. At each division, type II NBs asymmetrically segregate differentiation-promoting determinants, such as Notch antagonist Numb, into immature INPs. As a consequence, Notch pathway effector HES (hairy and E(spl)) genes, such as E(spl)mγ, are highly expressed in NBs but not in immature INPs. HES genes, encoding basic helix-loop-helix (bHLH) transcription factors, are crucial for promoting NB self-renewal. Importantly, numb mutant immature INPs fail to complete maturation but instead revert fate back into NBs and result in tumorigenesis, indicating that the asymmetric segregation of Numb protein is critical for establishing the initial fate bias between a type II NB and its sibling INP. However, whether such initial bias is sufficient to confer differential Notch activity and achieve definitive fate commitment is currently unclear. Lastly, type II NBs undergo fast cell divisions, dividing every 2 hr, placing them under huge pressure to timely yet precisely achieve differential fate outcomes. Therefore, within type II NB lineages, a regulatory module that drives robust cell fate commitment is likely to exist, plausibly with high activity (Liu, 2017).

Overactivation of Notch signaling leads to immature INP dedifferentiation and tumorigenesis, providing a sensitized background for identifying factors pivotal for NB or INP fate commitment. In such a genetic background a genome-wide RNAi-based screen was carried out for genes whose downregulation specifically suppresses the supernumerary NB phenotype induced by Notch overactivation, and subunits of the super elongation complex (SEC) were identified. The SEC is composed of the elongation factor ELL (eleven-nineteen lysine-rich leukemia) 1/2/3, the flexible scaffolding protein AFF (AF4/FMR2 family) 1/2/3/4, the ELL-associated factor EAF1/2, eleven-nineteen leukemia (ENL)/AF9, as well as the Pol II elongation factor P-TEFb consisting of cyclin T (CycT) and cyclin-dependent kinase 9 (CDK9). The screen identified all subunits of SEC except EAF and ENL/AF9, suggesting that SEC interplays with Notch signaling in promoting NB self renewal. The SEC subunits were originally identified as frequent translocation partners of MLL (mixed-lineage leukemia) in inducing leukemogenesis, and play key roles in c-Myc-dependent carcinogenesis and HIV viral DNA transcription. Previous studies demonstrated that SEC executes its functions by inducing rapid gene transcription, mainly through phosphorylating RNA polymerase II (Pol II) C-terminal domain and releasing it from promoter-proximal pausing (Liu, 2017).

This study shows that the SEC is specifically expressed in Drosophila NBs, where it acts as an amplifier to drive type II NB fate commitment. SEC exerts its function by physically associating with Notch transcription activation complex to stimulate dHES (Drosophila HES) transcription. dHES in turn promotes SEC expression/activity. Thus, driven by a self-reinforcing feedback loop between SEC and Notch signaling, an initial small bias of Notch activity between an NB and its sibling INP is rapidly amplified and consolidated into robust and irreversible fate commitment (Liu, 2017).

Is the establishment of an initial fate bias at the end of stem cell asymmetric division truly the end, or just the beginning of the end? The current findings revealed that a progressive and tightly controlled transition phase exists between the initial fate decision and the final definitive fate commitment. The results identified the evolutionarily conserved SEC as a crucial intrinsic amplifier, accelerating this previously overlooked fate transition phase and ensuring NSC fate commitment in Drosophila type II NB lineages. Inactivation of SEC prevents the self-reinforcing feedback loop between SEC and Notch signaling from running, resulting in NBs with ambiguous stem cell identity and ultimate fate loss. Conversely, ectopic overactivation of SEC initiates and sustains this positive feedback loop within progenitors, driving dedifferentiation and tumorigenesis. It is interesting to note that, as one of the most active P-TEFb-containing complexes in controlling rapid transcriptional induction in response to dynamic developmental or environmental cues, SEC is particularly suitable for being an amplifier in driving timely cell fate commitment. Since fast-cycling stem cells are under huge pressure to achieve robust fate determination in every cell cycle, it is not surprising that they employ SEC as a regulatory component to induce immediate activation of master fate-specifying genes that in turn form a self-amplifying loop with SEC to rapidly magnify the initial fate bias and ensure prompt fate commitment (Liu, 2017).

Such an intracellular amplifier mechanism revealed by these studies might complement the well-established intercellular lateral inhibition mechanism and represent a general, cell-autonomous paradigm to ensure robustness and precision in binary cell fate commitment. Lateral inhibition is a widely used mechanism underlying cell fate diversification, whereby unidirectional Notch signaling utilizes intercellular feedback loops to amplify an initial small difference between adjacent daughter cells, and eventually confers distinct cell fates. Lateral inhibition relies on intercellular interactions between adjacent cells. This study proposes a model whereby an intracellular amplifier mechanism may also diversify cell fates (Liu, 2017).

The intracellular amplifier and intercellular lateral inhibition mechanisms, both acting through feedback loops, are not mutually exclusive. Instead, they are complementary to each other and can be used concomitantly or sequentially to achieve differential fate outcomes in a timely, precise, and robust manner. An amplifier design often employs negative feedback to prevent excessive amplification. In this study the dHES-Earmuff/Brahma-SEC double-negative regulatory mechanism that this study has revealed in NBs might also operate in neural progenitors, where the Erm/Brm complex could serve as a crucial 'brake' to prevent the Notch-SEC-Notch self-reinforcing positive feedback loop from starting (Liu, 2017).

Notch signaling plays a conserved role during vertebrate embryonic neurogenesis in maintaining the undifferentiated status of NSCs. Intriguingly, expression of HES-1, a primary target of Notch pathway in mammalian neural development, oscillates every 2 hr. It has been proposed that oscillations in HES-1 expression drive fluctuations in gene expression, resulting in differential expressions between neighboring cells, which needs be further amplified to confer distinct cell fates. How such an amplification step is triggered and modulated remains elusive. Given that SEC is highly conserved in mammals, it is interesting to speculate that a similar amplifier mechanism is employed to ensure mammalian NSC fate commitment. Whether SEC interplays with Notch signaling to drive cell fate commitment in other stem cell lineages also warrants future investigation (Liu, 2017).

Despite extensive studies elucidating how SEC regulates transcription elongation, the in vivo function of SEC in normal development and physiology remains enigmatic. The current results indicate that SEC is highly expressed in Drosophila NSCs, where it is recruited by the Notch transcription activation complex to stimulate the transcription of dHES genes and promote self-renewing fate. Interestingly, the dHES genes in fly larval brain NB lineages are non-pausing genes, raising the possibility that SEC promotes the transcriptional activation of dHES in the absence of paused Pol II. Consistent with this view, recent studies have demonstrated that the rapid transcriptional induction of some nonpausing genes, such as Cyp26a1 in human embryonic stem cells and a subset of pre-cellular genes in early Drosophila embryos, depends on SEC activity and Pol II occupancy. The current findings that SEC physically and genetically interplays with the dCSL-NICD-MAM transcription activation complex to activate dHES transcription thus provide a unique physiological context for elucidating the detailed molecular mechanisms underlying transcriptional induction of non-pausing genes by SEC (Liu, 2017).

The upstream signals and molecular mechanisms controlling SEC activity in normal development or physiology are just unfolding. It has been previously shown that the activity of SEC could be regulated by modulating the kinase activity of CDK9, the catalytic subunit of SEC. The results unveil a new and unexpected mechanism underlying the control of SEC: the Notch-HES axis spatially restricts SEC activity within NSCs by cell-autonomously promoting the protein abundance of dAFF and dELL, two regulatory subunits of SEC. Consistently, overactivation of Notch signaling led to dedifferentiation of immature INPs, in which the expression levels of dAFF/dELL and, hence, the activity of SEC evidently increase (Liu, 2017).

Dysregulation of the SEC subunits is frequently associated with various human cancers including leukemia and glioblastoma. However, in most cases, whether SEC acts as a cancer driver or passenger is unclear. Furthermore, whether SEC subunits exert their oncogenic or tumor suppressive roles as a component of SEC or independent of SEC remains poorly understood. Intriguingly, the results show that overexpression of dELL and dAFF but not either alone induces a dramatic surge of dHES expression in immature INPs and causes progenitorderived tumor. These findings strongly suggest that, in NSC lineages, SEC drives tumorigenesis as an integral complex and exerts its oncogenic function in a dose-dependent manner. Supporting this view, the kinase activity of CDK9 is essential for dELL/dAFF-induced tumorigenesis. It will be interesting to investigate whether upregulation of dELL/dAFF abundance is sufficient to induce carcinogenesis in other biological contexts. Tne findings highlighting the self-reinforcing feedback loop between SEC and Notch signaling in driving tumorigenesis further suggest that CDK9 inhibitors could be pursued as an effective therapy for Notch overactivation-induced tumors (Liu, 2017).

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

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

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

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

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

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

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

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

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

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

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

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

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

Repression of differentiation genes by Hes transcription factors fuels neural tumour growth in Drosophila

Neural stem cells (NSC) divide asymmetrically to generate a cell that retains stem cell identity and another that is routed to differentiation. Prolonged mitotic activity of the NSCs gives rise to the plethora of neurons and glial cells that wire the brain and nerve cord. Genetic insults, such as excess of Notch signaling, perturb the normal NSC proliferation programs and trigger the formation of NSC hyperplasias, that can later progress to malignancies. Hes proteins are crucial mediators of Notch signaling and in the NSC context they act by repressing a cohort of early pro-differentiation transcription factors. Downregulation of these pro-differentiation factors makes NSC progeny cells susceptible to adopting an aberrant stem cell program. It has been recently shown that Hes overexpression in Drosophila leads to NSC hyperplasias that progress to malignant tumours after allografting to adult hosts. This study combined genetic analysis, tissue allografting and transcriptomic approaches to address the role of Hes genes in NSC malignant transformation. The E(spl) genes are important mediators in the progression of Notch hyperplasias to malignancy, since allografts lacking the E(spl) genes grow much slower. RNA profiling is presented of Hes-induced tumours at two different stages after allografting. The same cohort of differentiation-promoting transcription factors that are repressed in the primary hyperplasias continue to be downregulated after transplantation. This is accompanied by an upregulation of stress-response genes and metabolic reprogramming. It is concluded that the combination of dedifferentiation and cell physiology changes most likely drive tumour growth (Voutyraki, 2021).

Target specificities of Enhancer of split basic helix-loop-helix proteins

Continued: see Enhancer of split complex part 2/2

Related sites:

The function and regulation of groucho and Enhancer of split

Notch and Delta and the dual function signaling protein and transcription factor, Suppressor of Hairless

VND ( also known as NK2) protein: another regulator of E(spl)-C

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date revised: 15 April 2015

Zygotically transcribed genes

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