atonal: Biological Overview | Evolutionary Homologs | Regulation | Targets of Activity and Protein Interactions | Developmental Biology | Effects of Mutation | References

Gene name - atonal

Synonyms -

Cytological map position - 84F

Function - transcription factor

Keywords - proneural - eye and peripheral nervous system

Symbol - ato

FlyBase ID:FBgn0010433

Genetic map position - 3-[48]

Classification - bHLH

Cellular location - nuclear



NCBI links: Precomputed BLAST | EntrezGene | UniGene
Recent literature
Okumura, M., Kato, T., Miura, M. and Chihara, T. (2015). Hierarchical axon targeting of Drosophila olfactory receptor neurons specified by the proneural transcription factors Atonal and Amos. Genes Cells [Epub ahead of print]. PubMed ID: 26663477
Summary:
Sensory information is spatially represented in the brain to form a neural map. It has been suggested that axon-axon interactions are important for neural map formation; however, the underlying mechanisms are not fully understood. This study used the Drosophila antennal lobe, the first olfactory center in the brain, as a model for studying neural map formation. Olfactory receptor neurons (ORNs) expressing the same odorant receptor target their axons to a single glomerulus out of approximately 50 glomeruli in the antennal lobe. Previous studies have shown that the axons of Atonal ORNs, specified by Atonal, a basic helix-loop-helix (bHLH) transcription factor, pioneer antennal lobe formation; however, the details remain to be elucidated. This study shows that genetic ablation of Atonal ORNs affects antennal lobe structure and axon targeting of Amos ORNs, another type of ORN specified by the bHLH transcription factor Amos. During development, Atonal ORNs reach the antennal lobe and form the axon commissure before Amos ORNs. It was also found that N-cadherin knockdown specifically in Atonal ORNs disrupts the glomerular boundary in the whole antennal lobe. These results suggest that Atonal ORNs function as pioneer axons. Thus, correct axon targeting of Atonal ORNs is essential for formation of the whole antennal lobe.

Quan, X. J., Yuan, L., Tiberi, L., Claeys, A., De Geest, N., Yan, J., van der Kant, R., Xie, W. R., Klisch, T. J., Shymkowitz, J., Rousseau, F., Bollen, M., Beullens, M., Zoghbi, H. Y., Vanderhaeghen, P. and Hassan, B. A. (2016). Post-translational control of the temporal dynamics of transcription factor activity regulates neurogenesis. Cell 164: 460-475. PubMed ID: 26824657
Summary:
Neurogenesis is initiated by the transient expression of the highly conserved proneural proteins, bHLH transcriptional regulators. This study discovered a conserved post-translational switch governing the duration of proneural protein activity that is required for proper neuronal development. Phosphorylation of a single Serine at the same position in Scute and Atonal proneural proteins governs the transition from active to inactive forms by regulating DNA binding. The equivalent Neurogenin2 Threonine also regulates DNA binding and proneural activity in the developing mammalian neocortex. Using genome editing in Drosophila, this study showed that Atonal outlives its mRNA but is inactivated by phosphorylation. Inhibiting the phosphorylation of the conserved proneural Serine causes quantitative changes in expression dynamics and target gene expression resulting in neuronal number and fate defects. Strikingly, even a subtle change from Serine to Threonine appears to shift the duration of Atonal activity in vivo, resulting in neuronal fate defects.
Zhou, Q., DeSantis, D. F., Friedrich, M. and Pignoni, F. (2016). Shared and distinct mechanisms of atonal regulation in Drosophila ocelli and compound eyes. Dev Biol 418: 10-16. PubMed ID: 27565023
Summary:
The fruit fly Drosophila melanogaster has two types of external visual organs, a pair of compound eyes and a group of three ocelli. At the time of neurogenesis, the proneural transcription factor Atonal mediates the transition from progenitor cells to differentiating photoreceptor neurons in both organs. In the developing compound eye, atonal (ato) expression is directly induced by transcriptional regulators that confer retinal identity, the Retinal Determination (RD) factors. Little is known, however, about control of ato transcription in the ocelli. Here we show that a 2kb genomic DNA fragment contains distinct and common regulatory elements necessary for ato induction in compound eyes and ocelli. The three binding sites that mediate direct regulation by the RD factors Sine oculis and Eyeless in the compound eye are also required in the ocelli. However, in the latter, these sites mediate control by Sine oculis and the other Pax6 factor of Drosophila, Twin of eyeless, which can bind the Pax6 sites in vitro. Moreover, the three sites are differentially utilized in the ocelli: all three are similarly essential for atonal induction in the posterior ocelli, but show considerable redundancy in the anterior ocellus. Strikingly, this difference parallels the distinct control of ato transcription in the posterior and anterior progenitors of the developing compound eyes. From a comparative perspective, these findings suggest that the ocelli of arthropods may have originated through spatial partitioning from the dorsal edge of an ancestral compound eye.
Zhou, Q., Yu, L., Friedrich, M. and Pignoni, F. (2016). Distinct regulation of atonal in a visual organ of Drosophila: organ-specific enhancer and lack of autoregulation in the larval eye. Dev Biol [Epub ahead of print]. PubMed ID: 27693434
Summary:
Drosophila has three types of visual organs, the larval eyes or Bolwig's organs (BO), the ocelli (OC) and the compound eyes (CE). In all, the bHLH protein Atonal (Ato) functions as the proneural factor for photoreceptors and effects the transition from progenitor cells to differentiating neurons. This work investigates the regulation of ato expression in the BO primordium (BOP). Surprisingly, atotranscription in the BOP was found to be is entirely independent of the shared regulatory DNA for the developing CE and OC. The core enhancer for BOP expression, atoBO, lies ~6kb upstream of the ato gene, in contrast to the downstream location of CE and OC regulatory elements. Moreover, maintenance of ato expression in the neuronal precursors through autoregulation-a common and ancient feature of ato expression that is well-documented in eyes, ocelli and chordotonal organs-does not occur in the BO. The atoBO enhancer contains two binding sites for the transcription factor Sine oculis (So), a core component of the progenitor specification network in all three visual organs. These binding sites function in vivo and are specifically bound by So in vitro. Taken together, these findings reveal that the control of ato transcription in the evolutionarily derived BO has diverged considerably from ato regulation in the more ancestral compound eyes and ocelli, to the extent of acquiring what appears to be a distinct and evolutionarily novel cis-regulatory module.
Weinberger, S., Topping, M. P., Yan, J., Claeys, A., De Geest, N., Ozbay, D., Hassan, T., He, X., Albert, J. T., Hassan, B. A. and Ramaekers, A. (2017). Evolutionary changes in transcription factor coding sequence quantitatively alter sensory organ development and function. Elife 6. PubMed ID: 28406397
Summary:
'Toolkit' genes are highly conserved developmental regulators. While changes in their regulatory elements contribute to morphological evolution, the role of coding sequence (CDS) evolution remains unresolved. This study used CDS-specific knock-ins of the proneural transcription factor Atonal homologs (ATHs) to address this question. Drosophila Atonal CDS was endogenously replaced with that of distant ATHs at key phylogenetic positions, non-ATH proneural genes, and the closest CDS to ancestral proneural genes. ATHs and the ancestral-like gene rescued sensory organ fate in atonal mutants, in contrast to non-ATHs. Surprisingly, different ATHs displayed a gradient of quantitative variation in proneural activity and the number and functionality of sense organs. This proneural potency gradient correlated directly with ATH protein stability, including in response to Notch signaling, independently of mRNA levels or codon usage. This establishes a distinct and ancient function for ATHs and demonstrates that CDS evolution can underlie quantitative variation in sensory development and function.
BIOLOGICAL OVERVIEW

Drosophila has four types of sensory elements: external sense organs, multiple dendritic neurons, chordotonal neurons, and photoreceptors. The latter two do not require the proneural genes of the achaete-scute complex (AS-C) in order to develop and function properly, but they do need atonal, a neurogenic gene that behaves in many ways like the genes of AS-C. atonal gets its name from the disruptive effects the gene's mutation has on chordotonal neuron differentiation. Mutants are completely difficient in this lateral sense organ (Jarman, 1995).

Early in embryonic development, atonal is expressed in all chordotonal organ progenitor cells, but its later expression is restricted to only a particular set of precursors, through a process of lateral inhibition. Notch does not seem to be required for this process in chordotonal organs, and the mechanism is not well understood. Mutation in atonal also disrupts eye development (Jarman, 1995).

Atonal protein is produced in all cells just anterior to the morphogenic furrow. Expression is downstream of hedgehog, an important gene involved in regulating the progression of the furrow. As the furrow progresses, most cells that express atonal in front of the furrow lose that capacity. atonal expression behind the furrow is confined to the R8 progenitors, whose fate atonal determines. atonal is produced within the context of the furrow, which is only rudimentary in atonal mutants. atonal is a neurogenic gene, functioning in the place of achaete-scute complex bHLH genes, both in the eye and in chordotonal organs.

The function of Atonal is best illustrated by its role in chordotonal organ development. A scolopidium, the basic unit of chordotonal organs, consists of four cells: a neuron with a single dendrite, the scolopale cell, cap cell and ligament cell. The scolopale cell (a glial cell) forms a sheath around the dendrite, while the cap cell and ligament cell mediate the attachment of the chordotonal organ to the body cell. Expression of atonal is restricted to a subset of atonal-requiring chordotonal precursors, called founder precursors. In atonal mutants, all chordotonal organs are absent except for one scolopidium of Ich5, the abdominal pentascolopidial organ (Ich5 consists of five scolopidia organized in a linear array). This one scolopidium formed in atonal mutants is atonal independent. The atonal independent precursor corresponds to the earliest chordotonal precursor (precursor C1) and corresponds to the P cell which gives rise to the anterior-most scolopidium of Ich5 (zur Lage, 1997).

EGF receptor signaling is required in neural recruitment during formation of Drosophila chordotonal sense organ clusters. A total of five neural precursors express atonal in abdominal segments, and this number is too few to explain the formation of the eight scolopidia in each abdominal segment. Of the five precursors, C-1, C2 and C3 contribute to Ich5, C4 migrates slightly anterodorsally and gives rise to v'ch1, the dorsal most scolopidium, which is solitary. C5 contributes to vchAB, a more ventral pair of scolopidia. The remaining precursors require Egf-R signaling for their selection. Signaling by the founder precursors is initiated by atonal activating (directly or indirectly) rhomboid expression in the founder cells. It should be noted that in some developmental processes, rhomboid appears to function in the signal-receiving cells, such as in the patterning of ovarian follicle cells. It is not believed that this is the case in chordotonal-precursor formation, because rho is expressed in precursors that do not require rhomboid function (C1-C5 are formed even in rhomboid mutants). Signaling by these founder precursors, presumably through the EGF receptor ligand Spitz, then provokes a response in the surrounding ectodermal cells, as shown by the activation of expression of the Egf-R target genes pointed and argos. The signal and response then leads to recruitment of some of the ectodermal cells to the chordotonal precursor cell fate. Egf-R hyperactivation by misexpression of rhomboid results in excessive chordotonal precursor recruitment. Argos functions in a feedback mechanism to prevent the excess recruitment of additional ectodermal cells. The increase in the number of scolopidia caused by Egf-R hyperactivation is confined to an enlargement of existing cluster sizes: no new chordotonal clusters are formed. A two step mechanism is postulated for the formation of clusters of chordotonal precursors. In the first step, precursors C1-C5 are selected as founder precursors by the conventional route of proneural gene expression and lateral inhibition. In a distinct second phase, these precursors then signal to adjacent ectodermal cells via the Egf-r pathway, inducing some of them to become chordotonal precursors (secondary or recruited precursors). This two-step process is strongly reminiscent of the way atonal acts in neurogenesis in compound eyes. Here, atonal expression is initially refined by lateral inhibition, until atonal is expressed in only the founding R8 precursor, which then recruits R1-R7 in a mechanism that does not require the activation of atonal in these cells (zur Lage, 1997).

The selection of Drosophila sense organ precursors (SOPs) for sensory bristles is a progressive process: each neural equivalence group is transiently defined by the expression of proneural genes (proneural cluster), and neural fate is refined to single cells by Notch-Delta lateral inhibitory signalling between the cells. Unlike sensory bristles, SOPs of chordotonal (stretch receptor) sense organs are tightly clustered. It has been shown that for one large adult chordotonal SOP array (the adult femoral chordotonal sense organ), clustering results from the progressive accumulation of a large number of SOPs from a persistent proneural cluster. This is achieved by a novel interplay of inductive epidermal growth factor- receptor (EGFR) and competitive Notch signals. EGFR acts in opposition to Notch signaling in two ways: it promotes continuous SOP recruitment despite lateral inhibition, and it attenuates the effect of lateral inhibition on the proneural cluster equivalence group, thus maintaining the persistent proneural cluster. SOP recruitment is reiterative because the inductive signal comes from previously recruited SOPs (zur Lage, 1999).

The adult femoral chordotonal sense organ arises from a group of some 70-80 SOPs. A developmental analysis of Ato expression has revealed that these SOPs accumulate over an extended period of time in the dorsal region of each leg imaginal disc during the third larval instar and early pupa. The continued expression of Ato implies a sustained requirement for proneural function throughout the process of SOP accumulation. Unusually, Ato is persistently expressed in a group of ectodermal cells identified as the proneural cluster (PNC). From this PNC, cells are funnelled inward into a cavity formed by the folding of the disc. This invagination later becomes visible as a distinctive 2-cell wide intrusion, which is referred to as the 'stalk'. Cells at the deepest end of the stalk undergo shape changes to form an amorphous inner SOP mass. Invaginating cells are characterised by upregulation of Ato expression, a characteristic of SOP commitment. Surprisingly, SOP markers (Ase protein and the A101 enhancer trap line) are not expressed in all the stalk SOPs. Instead, these markers are only apparent in older cells, particularly at the time when they become part of the inner mass (which is therefore referred to as mature SOPs). Despite this, entry into the stalk seems to mark SOP commitment, since both the stalk and the mature SOPs are absent in discs from ato mutant larvae. This apparent intermediate stage may not have a counterpart in external sense organ precursor formation, although there is some evidence for multiple steps between the uncommitted cell and the SOP (the so-called pre-sensory mother cell state). Initially, Ato remains activated in all invaginated SOPs. This extended period of proneural gene expression is unusual since AS-C proneural expression is typically switched off in SOPs shortly after commitment. Later, at approximately 6 hours before puparium formation (BPF), Ato expression is switched off synchronously in the mature SOPs, although expression remains in the stalk SOPs and the PNC. At this point there is very little overlap between Ato and Ase or A101 (zur Lage, 1999).

The process of chordotonal SOP formation described above is at odds in several respects with the well-known paradigm of SOP selection for sensory bristles. In the latter, the solitary SOP expresses Delta, which triggers expression in the PNC of genes of the E(spl)-C, thereby preventing further SOP commitment and forcing loss of AS-C expression and neural competence. In the case of the femoral chordotonal organ, newly committed cells from the PNC are in contact with previously committed SOPs in the stalk, but are apparently not receiving (or not responding to) lateral inhibition signals from these to prevent their commitment. Likewise, the presence of committed SOPs does not switch off ato expression in the PNC. Nevertheless, components of the N-Dl pathway are expressed in patterns consistent with lateral inhibition. The newly formed SOPs express Dl, suggesting that they send inhibitory signals, while the PNC expresses mgamma, a member of the E(spl)-C, suggesting that these cells are responding to the Notch-Delta signal. Indeed, mgamma is coexpressed with ato in the PNC throughout the development of the SOP cluster. Chordotonal SOP formation is shown to be sensitive to N inhibitory signaling. Strong activation of N signaling or its effectors can inhibit chordotonal SOP formation. Thus, N signaling has an important role to play: it acts to limit the process of SOP selection from the PNC. Some mechanism, however, must prevent N signaling from completely inhibiting multiple SOP formation (zur Lage, 1999).

The progressive accumulation of chordotonal SOPs suggests that a recruitment mechanism could explain the clustering of SOPs. The Drosophila Egfr signaling pathway is involved in a number of recruitment processes in development, and a role for Egfr signaling has been demonstrated in the induction of embryonic chordotonal precursors (zur Lage, 1997). Although there appear to be significant differences in the process of SOP formation in imaginal discs, as compared with the embryo, it was asked whether Egfr signaling is also involved in forming the femoral chordotonal cluster. To address this question, the pathway was conditionally disrupted by expressing a dominant negative form of Egfr protein. Expression of UAS-Egfr DN results in a dramatic loss of chordotonal SOPs in late third instar imaginal leg discs (as judged by Ase protein expression or the A101 enhancer trap line). This demonstrates that Egfr signaling is required for the process of femoral chordotonal SOP formation. In contrast, the appearance of bristle SOPs is unaffected, arguing against the possibility of a nonspecific effect on SOPs in general (zur Lage, 1999).

To determine whether Egfr signaling controls SOP number, expression of components of the Egfr pathway that determine the level of signaling was forced, thus resulting in hyperactivation of the pathway. pointed (pnt) is an effector gene that encodes a transcription factor and is activated in cells responding to Egfr signaling. Both rho and pnt are expressed during chordotonal SOP formation. Indeed, forced expression of rho or pnt increases chordotonal SOP formation. Egfr could promote SOP formation by stimulating the commitment of PNC cells or by stimulating proliferation of SOPs. Both functions would be consistent with known Egfr roles, but the current investigations favour the former. Analysis of Ato expression in leg discs in which rho has been misexpressed reveals a large invagination of cells and a smaller PNC. Shrinking of the PNC was confirmed by the reduced extent of mgamma expression. These observations are consistent with an increased rate of SOP commitment upon Egfr hyperactivation. Moreover, this effect is reminiscent of the effect of N loss of function on Ato expression, suggesting that Egfr signaling supplies the mechanism that interferes with lateral inhibition of SOP commitment (zur Lage, 1999).

Although it seems that cells of the PNC and stalk are held in a state of mitotic quiescence throughout the time that SOP fate decisions are being made, BrdU is incorporated in the older (mature) SOPs. The experiments so far have indicated that Egfr signaling affects SOP commitment from the PNC. To determine more precisely the spatial patterning of Egfr activity required for SOP clustering and N antagonism, the expression patterns of key components of the pathway were characterized. Localized expression of rho appears to play a central role in spatial restriction of Egfr activity in cases where Spi is the ligand; in these cases it appears to mark the cells that are a source of signaling. During development of the femoral chordotonal organ, rho is expressed in a very restricted pattern: RHO mRNA is only detected in the SOPs, becoming confined in the late third instar larva to the youngest SOPs at the top of the stalk. To identify the cells responding to rho-effected signaling, an antibody that detects the dual-phosphorylated (activated) form of the ERK MAP kinase (dp-ERK) was used. In leg imaginal discs, dp-ERK is detected in a confined area corresponding to the uppermost (youngest) stalk SOPs. Thus, like rho, dp-ERK is expressed in the newly formed stalk SOPs. Double labelling for RHO RNA and dp-ERK confirms this, but also suggests that the overlap in expression is not complete: dp-ERK is detected above the uppermost rho-expressing cells of the stalk, probably in one or a few cells of the proneural cluster as they funnel into the stalk. This suggests that Egfr promotes SOP commitment as a consequence of direct signaling from previous SOPs to overlying PNC cells. Since rho expression is itself activated upon SOP commitment, this process occurs cyclically: the newly recruited SOPs are in turn able to signal to further overlying PNC cells. That is, recruitment is reiterative. Egfr signaling via Spitz has been shown to help to maintain neural competence by attenuation of Notch directed lateral inhibition. The opposing forces of Notch and Egfr signaling are thought to be played out through direct Notch and Egfr signaling between the epidermal proneural cells, which bear Notch, and the SOP, which sends inhibitory signals through the Delta ligand, and stimulatory signals through the Spitz ligand (zur Lage, 1999).

Reiterative recruitment alone cannot entirely explain the accumulation of SOPs. Such an accumulation also relies on the persistence of the competent pool of PNC cells from which SOPs can be recruited. For AS-C PNCs, this does not occur, because the mutual inhibition required for continued competence is unstable and resolves quickly to a state of lateral inhibition once the SOP emerges from the PNC. This results in rapid shutdown of AS-C expression and hence competence within the PNC. It is possible that the members of E(spl)-C that are expressed in the PNC (notably mgamma and mdelta) are less aggressive inhibitors of proneural gene expression than the E(spl)-C members expressed in AS-C PNCs (m5 and m8). The results obtained in the femoral SOP suggest, however, that Egfr has a role to play in maintaining the PNC by partially attenuating lateral inhibition on a PNC-wide scale. Thus, the PNC is not completely shut off by inhibition from SOPs, but instead kept in check, allowing continued mutual inhibition and maintenance of competence but not allowing general SOP commitment. Since neither rho nor dp-ERK are detected in the PNC as a whole, this function of Egfr could be indirect and achieved through partial attenuation of Dl signaling from the stalk SOPs themselves. The trans- or auto-activation of EGFR signaling between the stalk SOPs (as suggested by the co-expression of dp-ERK and rho) might be an indicator of this function. It is also possible, however, that Egfr signaling is direct and that the dp-ERK antibody is not sensitive enough to detect expression in the PNC cells (zur Lage, 1999).

Specificity of Atonal and Scute bHLH factors: analysis of cognate E box binding sites and the influence of Senseless

The question of how proneural bHLH transcription factors recognize and regulate their target genes is still relatively poorly understood. It has been shown that Scute (Sc) and Atonal (Ato) target genes have different cognate E box motifs, suggesting that specific DNA interactions contribute to differences in their target gene specificity. This study shows that Sc and Ato proteins (in combination with Daughterless) can activate reporter gene expression via their cognate E boxes in a non-neuronal cell culture system, suggesting that the proteins have strong intrinsic abilities to recognize different E box motifs in the absence of specialized cofactors. Functional comparison of E boxes from several target genes and site-directed mutagenesis of E box motifs suggests that specificity and activity require further sequence elements flanking both sides of the previously identified E box motifs. Moreover, the proneural cofactor, Senseless, can augment the function of Sc and Ato on their cognate E boxes and therefore may contribute to proneural specificity (Powell, 2008).

The proneural proteins exhibit very precise specificity in activation of different neurogenesis programmes. It has been suggested that utilization of different E-box motifs as binding sites may partly underlie this specificity. This was based on the finding that E boxes from Sc- and Ato-specific target genes conform to different consensus motifs. This study found further support for observation. In a cell culture assay, artificial enhancers of concatemers of EAto or ESc sequences generally show specific activation by Ato or Sc proteins, respectively. Nevertheless, the results also show that E box activity and specificity depends on complex features of the DNA surrounding the proneural-specific motifs both in cell culture and in vivo. The task of predicting by sequence analysis how proneural proteins regulate targets remains formidable (Powell, 2008).

Transcription factor activity depends on a complex interplay of interactions with DNA and with other protein factors, including those bound to other sites within the enhancer. To concentrate on the role that proneural protein interaction with E-box binding sites plays in specificity, synthetic enhancers of concatemers of E-box-containing sequences were studied in a cell culture reporter gene assay. A previous study of Ato or Sc-specific enhancers relied on the analysis of expression patterns produced in transgenic flies carrying GFP reporter gene constructs. In that study, specific regulation by Sc or Ato was inferred indirectly from patterns of GFP expression. This study showed that much of this inferred specificity is also seen in a cell culture reporter gene assay, strongly supporting the conclusion that Ato and Sc directly use different E box motifs. Thus, in general, the specificity of E box response (ratio of response to Sc and Ato) could be predicted from matches to ESc or EAto motifs identified previously. In most cases, this specificity also corresponded to the specificity of the native enhancer from which the E box was taken. An interesting exception is sens-E1: while this E box is proposed to respond to both Ato and Sc in vivo, it responds slightly better to Sc than to Ato in culture, which is more consistent with its ESc motif. It will be important to determine what other enhancer features allow such an E box to function as a common target of Ato and Sc in vivo (Powell, 2008).

Importantly, E box specificity is achieved without the appropriate cellular and developmental context of neurogenesis: S2 cells are embryonic, non-neural cells of likely hematopoietic origin and are not expected to contain neural-specific factors. The results therefore indicate that proneural factors have intrinsic ability to use different E box motifs without the need for interactions with neural specific cofactors. The ESc and EAto motifs differ most notably in the bases immediately flanking the 5' end of the 6-bp core sequence (NG vs. AW). There is evidence from the crystal structure of the MyoD bHLH domain–DNA complex that protein contacts are made with bases in this position, suggesting that similar direct contacts may influence E box utilization by proneural proteins. The basic region amino acids making these contacts (R110, R117 and E118) are conserved in the proneural proteins, but in Ato the arginines are separated by three amino acids (LAA, equivalent to MyoD KAA) that are absent in Sc. Thus despite the apparent conservation of DNA-contacting residues, one might predict strong differences in how the proneural proteins interact with the flanking nucleotides. SPR analysis shows Ato/Da to bind to ato-E1 and sc-E1 with similar affinity. Rather than affecting E box affinity, it is possible that subtle differences in binding contacts may cause conformational effects that affect the transactivation ability of the proneural protein (Powell, 2008).

The above results point to the importance of distinct Ato and Sc E box motifs for proneural specificity. Several findings, however, demonstrate that these motifs are heavily dependent on the wider DNA context. For instance, the E(spl)mγ-E2(C4 > G), sens-E1 and sc-E1 binding sites show very large differences in activity in cell culture, even though they have identical perfect ESc motifs at their core (gCAGGTGt). The effect of DNA context is also seen in the general inability, in the cell culture assay, to swap the proneural specificities of sc-E1 and ato-E1 by mutating the immediate 5' flanking bases of the core E box. Such changes generally result in loss of E box activity rather than a clear change in specificity. These results indicate that the ESc and EAto motifs are generally not sufficient for activity or specificity in the cell culture assay and that the surrounding DNA context is important (even within the short 20-bp sequences used) (Powell, 2008).

Interestingly, in some circumstances specificity could be manipulated more successfully in vivo: (sc-E1 GG > AA)6-GFP transgenic flies showed GFP expression consistent with strongly reduced activation by Sc and a gain of activation in some specific locations by Ato. However, this mutated motif did not respond to ectopically expressed Ato, perhaps suggesting that improved specificity in vivo results from cofactors expressed in locations of endogenous Ato expression and function (Powell, 2008).

Overall, the results above show that further sequences on both flanks of the ESc and EAto box motifs are also important for specificity and activity. One possibility is that the 20-bp DNA sequences used to construct the concatemers may include flanking sequences that interact with other protein factors to influence proneural specificity. Such adjacent sites have been identified for some mouse proneural E box binding sites. Moreover, in its native enhancer, ato-E1 is adjacent to an Ets-domain transcription factor binding site (although this site is mutated in the constructs used in this study). However, such cofactors would need to be expressed in S2 cells. Moreover, although the flanking sequences of the ato-E1 and sc-E1 sites are strongly conserved among Drosophila species, no obvious shared sequence motifs were found in the 5' and 3' flanks of known Drosophila E boxes that might be cofactor binding sites. Whilst there is a potential POU factor binding sequence 5' of the ato-E1 site, no members of the Drosophila POU family appear to be expressed during early neurogenesis. Alternatively, the further flanking bases may influence bHLH heterodimer interaction either through direct contacts or through indirect conformational effects. It is interesting that 3' bases appear important as these may be predicted to affect Da interaction. It is notable that the Da homologue, E2A, has different half-site preferences when bound to Twi or MyoD (Powell, 2008).

The specificity of E-box concatemer constructs is generally more complete in vivo than in the S2 luciferase assay -- notably proneural proteins can generally activate non-cognate E boxes to some extent in cell culture but not in vivo. One possibility is that the intrinsic specificity of proneural proteins must normally be enhanced by interaction with cofactors that are not present in S2 cells. In the cell culture assay, at high proneural levels it was found that Sens enhanced proneural activity in a general manner. None of the constructs tested contain Sens binding motifs, so it is likely that enhancement occurs in a DNA-binding independent manner via protein–protein interactions. At low proneural concentrations, however, the effect of Sens enhancement becomes selective. For many of the constructs tested, Sens only enhanced the activity of proneural proteins for concatemers consisting of their cognate E box. It is suggested that proneural–Sens interaction may enhance the specificity of proneural–E box interaction. Thus, this is an interesting case in which proneural specificity can be influenced by a common cofactor, rather than requiring interaction with different subtype-specific cofactors. It remains to be determined whether Sens would enhance specificity on native enhancers as well as concatemer constructs. Moreover, it seems unlikely that Sens is a specificity factor for all proneural target genes. However, the results are consistent with Sens acting as a specificity cofactor in certain contexts -- such as the proneural autoregulatory enhancers active in SOPs where there are high levels of Sens and proneural proteins present. Other non-DNA binding proneural protein interactors, such as Chip may have a similar effect in other contexts (Powell, 2008).

The effect of Sens could be explained by two models. First, interaction of a proneural protein with a specific E-box motif may give rise to a specific conformation which results in an increased affinity for Sens protein. Alternatively, the Sens–proneural protein interaction may alter the proneural bHLH domain conformation thereby increasing its affinity for its cognate binding site (i.e., an induced fit model). Indeed, variation in MyoD bHLH protein DNA sequence preferences have been previously observed to be the result of effects on basic region conformation arising because of binding partner differences or amino acid composition of the basic region. In this view, proneural specificity relies on a combination of cognate DNA sequence recognition and protein–protein interactions. Important future work will be the identification of the amino acid residues of Ato and Sc necessary for their interaction with Sens and the determination of whether these influence DNA recognition (Powell, 2008).


GENE STRUCTURE

cDNA clone length - 1492

Bases in 5' UTR - 334

Exons - two

Bases in 3' UTR - 222


PROTEIN STRUCTURE

Amino Acids - 312

Structural Domains

The bHLH domain is at the C-terminus. Preceding this is an acidic sequence that also contains a PEST sequence which aids in protein degradation (Jarman, 1993).


atonal: Evolutionary Homologs | Regulation | Targets of Activity and Protein Interactions | Developmental Biology | Effects of Mutation | References

date revised: 3 MAR 97 

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.