How conserved pathways are differentially regulated to produce diverse outcomes is a fundamental question of developmental and evolutionary biology. The conserved process of neural precursor cell (NPC) selection by basic helix-loop-helix (bHLH) proneural transcription factors in the peripheral nervous system (PNS) by atonal related proteins (ARPs) presents an excellent model in which to address this issue. Proneural ARPs belong to two highly related groups: the ATONAL (ATO) group and the NEUROGENIN (NGN) group. A cross-species approach was used to demonstrate that the genetic and molecular mechanisms by which ATO proteins and NGN proteins select NPCs are different. Specifically, ATO group genes efficiently induce neurogenesis in Drosophila but very weakly in Xenopus, while the reverse is true for NGN group proteins. This divergence in proneural activity is encoded by three residues in the basic domain of ATO proteins. In NGN proteins, proneural capacity is encoded by the equivalent three residues in the basic domain and a novel motif in the second Helix (H2) domain. Differential interactions with different types of zinc (Zn)-finger proteins mediate the divergence of ATO and NGN activities: Senseless is required for ATO group activity, whereas MyT1 is required for NGN group function. These data suggest an evolutionary divergence in the mechanisms of NPC selection between protostomes and deuterostomes (Quan, 2004).

ATO and NGN proteins share 47% identity in the bHLH domain including eight out of 12 amino acid residues in the DNA binding basic domain and are expressed in both the Drosophila and vertebrate PNS. However, differences in their usage for early NPC specification in the PNS between vertebrates and invertebrates have been noted. NGN proteins do not act early in NPC specification in invertebrates, whereas ATO proteins do not act early in NPC specification in vertebrates. Does this switch in the use of proneural proteins reflect a mechanistic difference or an inert change of expression pattern of otherwise functionally equivalent genes? To address this issue, a comparative analysis was initiated using Drosophila and Xenopus as model systems. To assay the proneural activity of mouse NGN1 and fly ATO in vertebrates, the mRNA of each was injected into a single blastomere of a two cell-stage Xenopus embryo. Neuronal induction was detected at stage 15 via whole-mount in situ hybridization for N-tubulin, an early marker of neuronal differentiation. Compared with uninjected embryos, injection of Ngn1 mRNA induces a large number of ectopic neuronal precursors on the injected side. By contrast, injection of Ato mRNA does not induce a significant increase in N-tubulin expression. Similarly, the injection of mRNA for Math1, a mouse ortholog of ato does not significantly increase N-tubulin expression at stage 15 but very few scattered N-tubulin positive cells can be seen at stage 19. Similar observations have been made for Xath1 which has been shown to be a much weaker inducer of neuronal precursors than NGN1. These data suggest that the Xenopus ectoderm responds robustly to NGN group proteins, but very weakly to ATO group proteins, to induce neurogenesis (Quan, 2004).

One explanation for the weakness of ATO and MATH1 activity in Xenopus is that NGN proteins are more potent neural inducers than ATO proteins and that, in parallel, stronger induction is needed in the vertebrate neuroectoderm than in the Drosophila neuroectoderm. To test this possibility, ATO proteins and NGN proteins were misexpressed in Drosophila using the UAS/Gal4 system and neural induction was assayed by counting the number of sensory bristles produced. Consistent with the fact that ato and Math1 completely rescue each other's loss of function, the two genes show very similar phenotypes and they were used interchangeably throughout the study. Expression of ngn2 with four different wing disc Gal4 drivers (C5-Gal4, 71B-Gal4, 32B-Gal4 and dpp-Gal4) showed no neural induction. Sixteen out of 23 ngn1 transgenic lines showed no neural induction. The other seven showed very weak induction with the strongest Gal4 driver, dppGal4. Therefore, the combination of dppGal4 and the strongest uas-ngn1 transgenic line were used in the rest of this study to determine the genetic and molecular basis of the difference in activity between ATO proteins and NGN proteins. The dppGal4 driver in Drosophila is used to induce genes of interest along the anteroposterior (AP) axis of the wing disc. Wild-type flies have no external sensory bristles or chordotonal organs (CHOs) on the AP axis of the wing blade. By contrast, a large number of sensory bristles is found along the AP axis of the wing with 100% penetrance when either MATH1 or ATO is expressed using dppGal4. In addition, both ATO and MATH1 induce CHOs. Expression of the strongest NGN1 transgenic line results in very few bristles in only 70% of the flies examined and no detectable CHOs. Quantitative analysis reveals that the number of sensory bristles induced by MATH1 is sixfold more than induced by NGN1. Identical observations were made in the few surviving flies under the same conditions using strong UAS-ATO lines. In the vertebrate PNS, NGN1 and NGN2 are sometimes co-expressed, and activate the expression of NeuroD group proteins. Therefore, it is possible that the weak neural induction of mouse NGN1 is due to the lack of homologs of NeuroD proteins in flies. However, co-expression of NGN1 and NGN2 or NGN1 and MATH3, a NeuroD group protein, failed to enhance the proneural activity of NGN1 in Drosophila (Quan, 2004).

One explanation for the very small number of bristles obtained after strong expression of NGN1 may be that the protein is able to induce NPCs, but most of these NPCs fail to differentiate properly and do not give rise to sensory organs. To test this possibility, NPC formation was examined directly upon expressing NGN1, ATO and MATH1 with dppGal4 in A101-lacZ flies. A101-lacZ is an NPC specific enhancer trap. The normal pattern of NPCs is revealed by anti-ß-GAL staining in third instar larval (L3) wing discs. Misexpression of ATO along the AP axis of the wing disc results in the induction of ectopic NPCs within the domain of ATO expression. By contrast, despite high levels of NGN1 expression, no detectable increase in NPCs is observed upon expression of NGN1. Similarly, ATO, but not NGN1, induced asense expression, another marker of NPC specification (Quan, 2004).

Is the weak activity of NGN1 specific to ectopic expression in the wing disc? Wide expression of NGN1 in embryos using da-Gal4 does not result in ectopic neurons. Finally, attempts were made to rescue the loss of ato in the eye imaginal disc using Gal4-7 and uas-ngn1. Gal4-7 induces expression anterior to the morphogenetic furrow and has been used to restore photoreceptors to ato mutant eye discs using scute and Math1. Expression of NGN1 in ato mutant discs did not result in any rescue nor did it induce ectopic R8 cells when expressed in control discs. For simplicity, the number of bristles was used as a quantitative assay for NPC formation for the remainder of the study (Quan, 2004).

To explore whether the differential activities of NGN proteins and ATO proteins can be understood at the level of the proteins themselves, a comparative analysis of the amino acid sequence of the basic domain was performed. Several studies have shown that important information is encoded by the basic domain, or specific residues therein. In addition, the 12 amino acids in the basic domain are sufficient to phylogenetically delineate ATO proteins and NGN proteins, arguing that sequence differences within the basic domain are of functional significance. However, previous studies did not investigate the genetic basis or address the evolutionary implications of the variation in basic domain sequence. ATO proteins and NGN proteins share eight residues out of 12 in the basic domain. One is variable, and the other three residues show almost absolute group specificity: they are highly conserved within each group but are essentially never the same between the two groups. To investigate whether this sequence specificity can explain the species-specific activities of ATO proteins and NGN proteins, a chimeric protein was created, exchanging the three group-specific amino acids in the basic domain of NGN1 for those present in ATO, named NGN^bATO. Expression of NGN^bATO induces the appearance of bristles along the AP axis of the wing in all transgenic lines examined. Strong UAS-NGN^bATO lines mimic strong UAS-ATO lines and result in significant lethality and more than 60 bristles per wing in the few surviving flies. Moderate UAS-NGN^bATO lines behave like moderate UAS-ATO lines and induce an average of 33 bristles per fly along the AP axis when compared with an average of seven for strongest UAS-NGN1 lines. Conversely, a chimeric protein was created exchanging the three group-specific amino acids in the basic domain of ATO to NGN1, named ATO^bNGN. Whereas the injection of Ato mRNA in Xenopus embryos has no significant effect on the N-tubulin expression pattern, the injection of Ato^bNGN mRNA induces N-tubulin expression, indistinguishable from that caused by the injection of NGN1. Therefore, the NGN^bATO mutant recovers the NPC inducing activity of ATO in Drosophila, and the ATO^bNGN mutant recovers the NPC inducing activity of NGN1 in Xenopus (Quan, 2004).

It is worthwhile to notice that only some of residues in the basic domain are directly contacting to DNA. The specific activities of ATO and NGN1 are unlikely to depend on differential DNA-binding activity, since ATO proteins and NGN proteins have identical DNA contact amino acids and can activate the NeuroD promoter via the same E-boxes in P19 cells. Interestingly, biophysical and DNA-binding studies comparing MASH1 and MyoD have shown that they display similar binding preferences leading to the conclusion that their different target specificities cannot be explained solely by differential DNA binding. Similar conclusions were made comparing ato and sc activities in neural subtype specification (Quan, 2004).

To investigate whether other functionally specific motifs exist in the bHLH domain of ARP proteins, the evolutionary trace (ET) analysis method was used. ET tracks residues whose mutations are associated with functional changes during evolution. This approach has been used to identify novel functional surfaces, and has recently been shown to be widely applicable to proteins. In practice, ET relies on the phylogenetic tree of a protein family and identifies residues of the alignment that are invariant within branches but variable between them. These positions are called 'class specific'. The smallest number of branches at which a position first becomes class specific defines its rank. The top ranked positions (1) do not vary. Very highly ranked positions (2-8) are such that they vary little and, whenever they do, there is also a major evolutionary divergence. By contrast, poorly ranked positions vary more often, and their variation does not seem to correlate with divergence. Thus, highly ranked positions tend to be functionally important, while poorly ranked ones tend not to be. When examining ARP bHLH domains, ET identified a number of positions that are jointly important in different bHLH domains, yet that undergo significant variation between them. These residues varied in rank from 2 to 7, suggesting that they can undergo non-conservative mutations that are likely to correspond to functional divergence events. These positions tend to be most conserved between NeuroDs and NGN proteins and then undergo variations in ATO proteins, suggesting that they are important for an activity shared by NGN proteins and NeuroDs, but absent in ATO proteins. The data above show that the ability to induce NPCs in vertebrates is precisely such an activity. To investigate further the role of these group-specific residues on functional specificity, a chimeric protein, named NGN^H2ATO (exchanging amino acids 37, 39, 43, 44 and 46 in Helix2 of NGN1 with those present in ATO), was created and tested in Drosophila. Expression of the strongest NGN^H2ATO transgenic line induces a maximum of two bristles along the AP axis of the wing per fly in 50% of the flies. Quantitative analysis shows that, unlike ATO, NGN^H2ATO induces an average of 0.8 bristles along AP axis per fly. These data indicate that the group-specific motif in Helix2 of ATO does not encode proneural activity in Drosophila. Conversely, a chimeric protein, named ATO^H2NGN, was generated exchanging the same five amino acids in Helix2 of ATO to those found in NGN1. Injection of ATO^H2NGN mRNA causes ectopic N-tubulin expression, indistinguishable from the injection of NGN1. Therefore, ATO^H2NGN recovers the activity of NGN1 in Xenopus. Taken together, the mutational analysis results agree with the predictions of the ET analysis indicating that the identified residues in Helix2 mediate the activity of NGN proteins but not of ATO proteins (Quan, 2004).

The data support the hypothesis that ATO proteins and NGN proteins act via different genetic pathways to specify NPCs in different species. What might those pathways be? One simple explanation may be that NGN1 is not able to form heterodimers with fly Daughterless (DA), a required partner protein for NPC specification. In order to test this possibility, co-IP experiments were performed, in which ³⁵S-labeled ATO, MATH1 or NGN1 were co-precipitated with DA-Myc using anti-Myc antibodies. In the presence of DA, mouse MATH1, fly ATO and mouse NGN1 are co-precipitated. Only background levels of NGN1 are detected in the absence of DA. These results suggest that mouse NGN1 can bind physically to fly DA in vitro. To test if DA and NGN1 can interact genetically in vivo, NGN1 was expressed in the absence of one copy of da. The number of sensory bristles produced by NGN1 along the AP axis is greatly decreased in a heterozygous da background. Therefore, mouse NGN1 can physically and genetically interact with fly da in Drosophila in a dose-sensitive manner (Quan, 2004).

Next, the possibility that mouse NGN1 does not respond to the Drosophila Notch signaling pathway was examined. To test this, neural induction by NGN1 was examined in the absence of one copy of Notch (N^+/–) or with the co-expression of Notch pathway genes. The proneural activity of NGN1 is enhanced in a N^+/– background. Conversely, NGN1 activity is completely inhibited by co-expression of a constitutively active form, N^intra or members of the E(Spl) complex, m8 and mdelta. These data demonstrate that mouse NGN1 can be regulated by the Notch signaling pathway in Drosophila. It should be noted that overexpression of ATO in a N heterozygous background results in almost complete lethality and in extremely deformed wings, owing (in part) to a very large number of bristles in the few surviving flies. Since both ATO and NGN proteins can respond to levels of Notch signaling but only ATO proteins can efficiently specify NPCs, it is possible that ATO proteins and NGN proteins use different mechanisms to interact with the Notch signaling pathway. One possibility is that NGN proteins are more sensitive than ATO proteins to levels of transcriptional inhibitors of proneural activity encoded by the E(spl) genes because NGN1 activity, like that of ATO, can be repressed by ectopic expression of E(spl) proteins. However, in contrast to what is observed with Notch, removing a copy of the E(spl) complex does not alter NGN1 activity, suggesting that NGN1 is not more sensitive to levels of transcriptional inhibitors of proneural activity (Quan, 2004).

NPC formation in Drosophila requires the Zn-finger protein Senseless (SENS). Fly proneural proteins first induce sens expression and then synergize with it in a positive feedback loop. This appears to enhance the ability of proneural genes to downregulate Notch signaling in the presumptive NPC. In vertebrates, Senseless-like proteins appear not to act in NPC formation, although they are expressed in the PNS. To test the possibility that SENS shows group specific interactions with bHLH proteins during NPC selection, the abilities of ATO and NGN1 to induce SENS were examined. SENS expression in wild-type L3 wing discs marks NPC formation. Ectopic SENS induction is detected along the AP axis of wing discs when ATO is misexpressed. However, SENS expression is not induced by NGN1. These data suggest that unlike ATO, NGN1 does not efficiently induce SENS expression. Whether lowering endogenous levels of Notch would allow NGN1 to induce SENS was examined. Expression of NGN1 in Notch heterozygous animals, although significantly increasing the number of induced bristles, fails to induce SENS expression when compared with N^+/– controls, arguing that NPCs induced by NGN proteins are specified via a different mechanism not normally used in Drosophila. Although NGN1 does not induce SENS, it is possible that synergy might occur if the requirement for SENS induction is bypassed. Therefore the ability of NGN1 and MATH1 to synergize with SENS in vivo was compared by co-expressing either NGN1 or MATH1 with SENS using a moderate scutellar Gal4 driver (C5-Gal4). Neural induction was examined by counting the ectopic bristles induced on the scutellum. Wild-type flies have four large bristles, or macrochaete, on their scutella. Expression of SENS or MATH1 alone with C5-Gal4 induces a number of ectopic microchaete, or small bristles, on the scutellum. No ectopic sensory bristles were found when NGN1 was expressed alone. Co-expression of NGN1 and SENS has the same effect on the scutellum as the misexpressing SENS alone. Co-expression of MATH1 and SENS, however, causes the appearance of a large number of both micro- and macrochaete. Finally, NGN1 or MATH1 were co-expressed in the absence of one copy of sens. No effect on NGN1 activity in a sens^+/– background was observed. By contrast, the average number of sensory bristles produced by MATH1 along the AP axis was reduced by 42% if a single copy of sens was removed suggesting dose-sensitive interactions. Thus, neither by loss nor gain of function criteria does NGN1 appear to interact with SENS, thus explaining its weak proneural activity and inability to efficiently antagonize Notch signaling in Drosophila. Therefore, SENS is a key extrinsic difference in how ATO proteins and NGN proteins regulate NPC selection (Quan, 2004).

In Xenopus, the C2HC-type Zn-finger protein X-MyT1 is expressed in primary neurons and can be induced by NGN proteins. In addition X-MyT1 has been suggested to play a role in NPC formation and to synergize with NGN proteins. In order to test if X-MyT1, like SENS, shows specificity in its interaction with ARP proteins, its ability to interact with NGN1 and ATO in Xenopus was compared. X-MyT1 mRNA was injected alone or co-injected with either Ngn1 or Ato mRNA. As expected, the injection of X-MyT1 increases the number of N-tubulin-expressing cells in the neural plate domains where neurons normally form, while the injection of Ngn1 mRNA alone leads to induction of N-tubulin expression. Co-injection of Ngn1 and X-MyT1 mRNAs results in very strong N-tubulin induction, pointing to a synergistic interaction between the two proteins. By contrast, co-injection of Ato and X-MyT1 mRNAs does not cause a detectable increase in N-tubulin expression compared with the injection of X-MyT1 mRNA alone. Similarly, the few ectopic N-tubulin-expressing cells observed when Math1 mRNA is injected are not increased by co-injection of Math1 and X-MyT1. Thus, X-MyT1 interacts specifically with NGN1 and not with ATO or MATH1. The data above demonstrate that the correct combination of ARP protein and Zn-finger protein is necessary for NPC induction (Quan, 2004).

Does the coding sequence difference mediate the divergence in the genetic interactions of ARPs? To test this, whether the chimeric proteins recover the ability to interact with the respective Zn-finger proteins was examined. Indeed, expression of NGN^bATO in Drosophila results in the induction of SENS, and the number of bristles induced by NGN^bATO in absence of one copy of sens (sens^+/–) is reduced by ~44%. In addition, strong synergy was observed by co-expression of NGN^bATO and SENS using the dppGal4 driver. Therefore, NGN^bATO is able to induce and interact with SENS in Drosophila. In Xenopus, just like Ngn1, co-injection of Ato^bNGN and X-MyT1 mRNAs results in synergy and very strong ectopic N-tubulin expression when compared with the injection of X-MyT1 or Ato^bNGN alone. Similarly, co-injection of ATO^H2NGN and X-MyT1 mRNAs results in synergy and very strong induction of N-tubulin expression suggesting that ATO^H2NGN and ATO^bNGN use the same mechanism of action as NGN1 (Quan, 2004).

At the developmental level, the data presented in this study can be explained by two possibilities. The first is that Drosophila and vertebrates use different bHLH proteins with divergent mechanisms for selecting similar cell types: the earliest born neural progenitors. Alternatively, NGN proteins may be involved in selecting neuronal (versus glial) rather than earliest born neural progenitors in vertebrates. This is certainly the case in the mammalian inner ear and it should be determined whether it is a more generally applicable rule, at least in the PNS. These two models for NGN function are not mutually exclusive. It is possible that in different lineages, NGN proteins select first neural, and then neuronal, precursors. This would be compatible with data from both flies and vertebrates showing that Notch signaling, in addition to having anti-neural effects, has also anti-neuronal and pro-glial effects during neural lineage development. Analysis of the fly NGN protein, TAP, may shed some light on this issue. At any rate, a comparative approach should provide a powerful tool for the systematic analysis of the pathways which program neural stem cells (Quan, 2004).

Regardless of the precise developmental step at which ATO proteins and NGN proteins act, it is clear that the genetic and molecular mechanisms by which they act are different, suggesting that the functions of ATO proteins and NGN proteins are regulated by different factors. Furthermore, it is clear that the group-specific amino acids underlie these molecular differences. At this point it is difficult to interpret the precise role of the group specific residues in molecular terms. Nonetheless, three possibilities seem reasonable. The first is that currently unknown proteins bind to these residues. The second is that these residues are sites of differential post-translational modifications which in turn influence the choice of target gene specificity. Finally, it is possible that while these residues do not bind to DNA themselves, they influence the three dimensional structure or the conformational changes which DNA binding residues assume upon contacting DNA. In this scenario, these residues do ultimately influence the choice of the binding site without themselves contacting it. The data illustrate the power of a comparative approach in identifying not only conserved, but also divergent, developmental mechanisms, and suggest a platform for screening for the genes mediating the divergence. It is noteworthy that NGN1, on the one hand, and XATH1 and MATH1, on the other, seem to have retained a type of proneural activity which is largely no longer needed in flies and vertebrates, respectively (Quan, 2004).

For a particular functional family of basic helix-loop-helix (bHLH) transcription factors, there is ample evidence that different factors regulate different target genes but little idea of how these different target genes are distinguished. The contribution was investigated of DNA binding site differences to the specificities of two functionally related proneural bHLH transcription factors required for the genesis of Drosophila sense organ precursors (Atonal and Scute). The proneural target gene, Bearded, is regulated by both Scute and Atonal via distinct E-box consensus binding sites. By comparing with other Ato-dependent enhancer sequences, an Ato-specific binding consensus that differs from the previously defined Scute-specific E-box consensus was defined, thereby defining distinct EAto and ESc sites. These E-box variants are crucial for function: (1) tandem repeats of 20-bp sequences containing EAto and ESc sites are sufficient to confer Atonal- and Scute-specific expression patterns, respectively, on a reporter gene in vivo; (2) interchanging EAto and ESc sites within enhancers almost abolishes enhancer activity. While the latter finding shows that enhancer context is also important in defining how proneural proteins interact with these sites, it is clear that differential utilization of DNA binding sites underlies proneural protein specificity (Powell, 2004).

Ato and Sc must interact with distinct DNA binding sites in vivo. In the target genes analyzed in this study, residues immediately flanking the 6-bp core E box allow the definition of distinct EAto and ESc consensus binding sites for Ato/Da and Sc/Da, respectively. It can be deduced that these variant E boxes consist of half sites, with Sc, Ato, and Da contacting GCAG, AWCAK, and STGK, respectively (proneural core sites underlined). Striking affirmation of binding site differences is provided by the common target gene Brd, which is regulated by Ato and Sc in a modular fashion via distinct E boxes in different enhancers. These E-box variations are crucial for function. They are sufficient to confer proneural protein-specific expression patterns on a GFP reporter gene in isolation from other enhancer sequences. Moreover, interchanging ESc and EAto sites within proneural enhancers almost abolishes enhancer activity and so is almost equivalent to destroying the E box. This shows that the correct proneural regulation of target genes requires the presence of a specific E-box binding site in combination with the selective ability to interact with factors bound to other sites within these enhancers (Powell, 2004).

In vivo DNA binding site differences underlie proneural specificity. Yet, paradoxically, evidence from misexpression and protein structure-function studies has strongly suggested that the target gene specificities of Ato and Sc (and their vertebrate homologues) result from specific interactions with protein cofactors and not from intrinsic differences in how proneural proteins contact DNA. For instance, DNA-contacting residues are shared between the proteins, and consequently, as show in this study, Ato/Da and Sc/Da have identical DNA binding properties in vitro. One way to reconcile these observations is that protein-protein interactions with cofactors may induce DNA binding specificity in proneural proteins by modifying the conformation of the DNA-contacting residues of the bHLH domain. It is also possible that DNA binding itself is not selective in vivo but that occupancy of different DNA binding sites triggers productive or unproductive conformational changes in the proneural proteins that influence their interaction with cofactor proteins. The favored view is that specific cofactor interactions will induce distinctive DNA binding affinities; the conformational changes induced by each may be subtle individually but may be interdependent and mutually reinforcing (Powell, 2004).

Significantly, ato can rescue mutations of its mouse orthologue, Math1, and vice versa, suggesting that DNA site preferences will be conserved among vertebrate orthologues. A number of functional E boxes have been characterized in vertebrate neural-specific genes, and in two cases the interacting bHLH protein is likely to be an Ato orthologue: an autoregulatory site in the Math1 promoter (TCAGCTGG) and a proposed Xath5 site in the 3 nAChr promoter (ACAGCTGG). Thus, in these cases the E boxes match EAto in the 5 flanking base (Powell, 2004).

For correct enhancer function, proneural proteins must interact differentially with other DNA binding factors. Although E-box consensus differences underlie specificity, enhancer context is usually also crucial for this specificity to be manifest. In swapping EAto and ESc sequences between the sc and ato autoregulatory enhancers, in only one case was a corresponding 'swap' in enhancer specificity observed: Ato could be made to regulate the sc-SMC-E enhancer via an EAto site. Otherwise, alteration of E-box flanking bases resulted in a severe loss of enhancer activity. This suggests that recruiting a different proneural protein cannot alone change the function of an enhancer. Correct proneural target enhancer function requires a combination of the correct E-box sequence and the ability to interact with other factors bound to the enhancer. This is reminiscent of the cooperative interaction between MyoD and MEF2 in myogenesis and of interaction between Sc/Da and Pannier/Chip to activate ac in a specific part of the thorax. For the Ato enhancer a requirement for cooperative interaction between Ato/Da and the ETS protein Pointed, bound to a site adjacent to the EAto site has been shown (zur Lage, submitted, cited in Powell, 2004). Similarly, neurogenin 2 interacts with LIM factors during the activation of subtype-specific target genes. The finding that EAto and ESc sites encode much specificity in artificial enhancers suggests that tandem E boxes remove the requirement for interaction with factors bound to other DNA sites, perhaps because cooperative binding between proneural proteins themselves is then sufficient and may even allow the recruitment of cofactors by protein interactions alone. Interestingly, the converse situation may also occur: for both the ato and sc enhancers, there is a low level of expression remaining after swapping of E boxes. This suggests that the original bHLH protein can be recruited to the 'wrong' E-box sequence, inefficiently, by interaction with cofactors. A basis for this can be found with MyoD, where interaction with Sp1 allows MyoD to bind to a nonideal site in the human cardiac alpha-actin promoter (Powell, 2004 and references therein).

Another indication of the importance of enhancer context is that parent enhancers support patterns different from those of the isolated E boxes, at least in the case of Ato. [ato-E1]7-GFP is widely expressed in Ato-specific regions in the embryo, whereas the parent ato-FCO-E enhancer is limited to a small subset of chordotonal SOPs (zur Lage, submitted cited in Powell, 2005). In discs, ato-E1 drives expression relatively poorly in FCO precursors compared with ato-FCO-E. TAKR86C is even more extreme: the TAKR86C enhancer is normally active only in a single embryonic chordotonal precursor (the P cell), but the TAKR86C-E2 site drives Ato-dependent expression in the larval and adult eye and not in the P cell. Clearly, the parent enhancers must have other regulatory inputs that restrict expression (Powell, 2004).

Despite the importance of enhancer context and interaction with other factors, the ESc and EAto sequences support strikingly specific expression patterns when taken out of their enhancers. All tandem repeat E-box constructs tested were activated almost exclusively during PNS neurogenesis, despite the presence of some 24 class A factors in Drosophila. None were activated during CNS neurogenesis, myogenesis, or mesoderm formation, even though AS-C proteins function during the former two processes. In the case of two sites, sc-E1 and ato-E1, expression is remarkably consistent, with regulation solely by Ato or Sc, respectively, in PNS neurogenesis; the sites alone must contain all of the information necessary for specific recognition. It is remarkable that ato-E1 does not respond in vivo to the Ato-related protein Cato or Amos, even though the latter has a basic region almost identical to that of Ato and might be expected to have the same DNA binding properties. The main exception to this specificity is the presence of expression in embryonic ectodermal stripes. These resemble muscle attachment sites, suggesting recognition by the Ato superfamily member Delilah. The conclusion is that tandem duplications can overcome the need for DNA binding sites for other factors. Cooperative binding of proneural proteins may negate the need for cofactor interactions, or, as suggested above, cooperative binding may allow the recruitment of cofactors directly (Powell, 2004).

There are dramatic differences between the two Ato sites tested. Unlike ato-E1, the TAKR86C-E2 site drives expression in only a subset of Ato locations; it appears to be photoreceptor specific despite containing a good class A core E-box match (CAGGTG). This opens up the possibility that there may be different subtypes of Ato binding sites. The spatially restricted recognition of TAKR86C-E2 also implies that cellular context is important in how different sites are recognized. One may speculate, for instance, that eye-specific DNA binding properties of Ato may be conferred by interaction with PAX6 proteins. Interestingly, diversity of E-box expression patterns correlates with variability in the consensus sequences. The ESc consensus sequence (based on some 23 sites) is less variable than the Ato/Da consensus, even though the latter is based on only three sites. It is suggested that regulatory fine-tuning by E-box variation is more important for Ato target genes than for Sc target genes (Powell, 2004).

In summary, the E-box sequences and their flanking bases contain impressively sufficient information for regulation by specific proneural proteins. However, there is further complexity: at least the two Ato sites tested support different patterns and have a different relationship with their parent enhancers. Subtle variations in regulation by proneural proteins may therefore contribute to variations in target gene expression; indeed, there may be no such thing as a typical target site or target gene. This may also be true for common target genes: despite the modular regulation of Brd, the possibility is not ruled out that within the spectrum of proneural E boxes there are some sites that are jointly recognized by Sc and Ato in vivo and that this would be another mechanism for regulating common target genes (Powell, 2004).