BIOLOGICAL OVERVIEW

The decision of ectodermal cells to adopt the sensory organ precursor fate in Drosophila is controlled by two classes of basic-helix-loop-helix transcription factors: the proneural Achaete (Ac) and Scute (Sc) activators promote neural fate, whereas the E(spl) repressors suppress it. E(spl) proteins m7 and mgamma are potent inhibitors of neural fate, even in the presence of excess Sc activity and even when their DNA-binding basic domain has been inactivated. Furthermore, these E(spl) proteins can efficiently repress target genes that lack cognate DNA binding sites, as long as these genes are bound by Ac/Sc activators. This activity of E(spl)m7 and mgamma correlates with their ability to interact with proneural activators, through which they are probably tethered on target enhancers. Analysis of reporter genes and sensory organ (bristle) patterns reveals that, in addition to this indirect recruitment of E(spl) onto enhancers via protein-protein interaction with bound Ac/Sc factors, direct DNA binding of target genes by E(spl) also takes place. Irrespective of whether E(spl) are recruited via direct DNA binding or interaction with proneural proteins, the co-repressor Groucho is always needed for target gene repression (Giagtzoglou, 2003).

Basic helix-loop-helix (bHLH) proteins constitute a large family of transcriptional regulators that are characterized by a basic DNA-binding domain contiguous with a dimerization domain consisting of two amphipathic alpha-helices separated by a loop. Members of this family are implicated in a multitude of biological functions, from proliferation to response to toxic stress. Most notable are members of a class of bHLH proteins, termed Class II, that are capable of directing cells towards specific fates; well-studied examples are the myogenic and the proneural factors. These bHLH proteins dimerize (via their HLH domains) with ubiquitous bHLH Class I co-factors, also known as E-proteins, as a prerequisite to DNA binding. The heterodimer acts as a transcriptional activator of multiple target genes, some of which encode transcription factors, thus setting off a cascade of gene regulation that implements the particular developmental program. Ac, Sc and L'sc are among the proneural bHLH proteins in Drosophila and together with the E-protein Daughterless (Da) are responsible for specifying most CNS and external sensory neural precursors (Giagtzoglou, 2003 and references therein).

Within the anlagen of the CNS and PNS, proneural genes are initially expressed in groups of cells termed proneural clusters. From these broad domains, only a subset of cells will commit to the neural fate. These neural precursors transiently upregulate proneural gene expression and activate a number of neural differentiation genes, such as asense, senseless/Lyra, deadpan and others, which are direct transcriptional targets of proneural bHLH activators. The remaining cells of the proneural cluster are inhibited from embarking into a neural pathway and will either continue proliferation or differentiate to alternative cell types, such as epidermis. This is the outcome of intercellular signaling within the proneural cluster, which is mediated by the Notch pathway and is termed lateral inhibition. Cells that receive a high level of Notch signal cannot turn on the proneural target genes (such as ase, dpn, etc.); this block requires the activity of members of yet another class of bHLH proteins, named Class VI or HES proteins. The seven clustered E(spl) genes in Drosophila, m8, m7, m5, m3, mß, mgamma and mdelta, encode Class VI bHLH proteins and are directly turned on (transcriptionally) by Notch signaling. Their products accumulate in all cells of the proneural cluster, but are minimal within the neural precursors; they can be therefore considered 'anti-neural' proteins. Indeed, deletion of the entire E(spl) locus results in severe overcommitment of neural precursors. However, mutations in individual E(spl) genes display no phenotypic defects, as a result of partial functional redundancy, a fact that prohibits forward genetic dissection of E(spl) protein function (Giagtzoglou, 2003 and references therein).

The link between proneural bHLH proteins, Notch signaling and HES proteins is evolutionarily conserved, since it is also encountered in vertebrates, where the cellular events of neurogenesis are very distinct from those in insects. In both phylogenetic groups, allocation of neural versus non-neural fates is the outcome of two antagonistic bHLH activities: proneural proteins that promote neurogenesis and HES proteins that inhibit it. As in Drosophila, in vertebrates some HES genes are direct transcriptional targets of Notch. Despite the central importance of these bHLH transcription factors in early neural commitment, there are many gaps in knowledge of the regulatory circuits underlying neurogenesis, both in terms of the target genes of proneural and HES genes, and in terms of the mechanisms of gene activation and repression by these bHLH proteins. It was originally proposed that E(spl) proteins might block neurogenesis in Drosophila by repressing proneural genes. More recent data suggest that this is true only for specific enhancers of the proneural genes that are autostimulatory and sensory organ precursor (SOP) specific, while the major function of E(spl) proteins is to repress downstream target genes of the proneural proteins. HES proteins are indeed transcriptional repressors. Key amino acid differences between the basic domains of HES and Ac/Sc proteins endow these different bHLH factors with distinct target site specificities: Da-Ac/Sc heterodimers bind the E_A box GCAGSTG (Singson, 1994), whereas E(spl) homodimers preferentially bind to E_B-boxes (CACGTG) and variants thereof, the C and N boxes (CACGCG and CACNAG, respectively) (Jennings, 1999; Oellers, 1994; Ohsako, 1994; Tietze, 1992; Van Doren, 1994). E_A, E_B, C and N boxes are encountered clustered in enhancers of proneural target genes, such as ase and dpn, which are expressed strongly in the neural precursor and repressed in the remaining proneural cluster cells. The importance of E_A sites in such enhancers has been confirmed by mutagenesis; ablation of E_A boxes leads to loss of transcriptional activity (Culi, 1998; Jarman, 1993). The same does not hold true, however, for E_B/C/N boxes; mutation of these does not lead to derepression of reporter genes -- mutant versions of the sc SMC enhancer lacking all E(spl) binding sites are still expressed only in the SOPs (Culi, 1998). Furthermore, E(spl) proteins retain residual activity after disruption of their DNA-binding basic domain, although this is still somewhat controversial. As a result, alternative models regarding the mechanism of target gene repression by E(spl) have been suggested. One proposes that E(spl) can sequester activator complexes away from DNA. A second model proposes that E(spl) proteins may be recruited to target enhancers indirectly, via interactions with other uncharacterized DNA bound factors (Giagtzoglou, 2003 and references therein).

The simplest explanation for the fact that proneural target enhancers can be repressed by E(spl) in the absence of cognate DNA-binding sites is that E(spl) proteins use a DNA-binding-independent mechanism for proneural target gene repression, instead of, or in addition to, a DNA-binding-dependent one. In the present work, it was asked if this is indeed the case. In vivo data is presented that strongly support protein-tether-mediated recruitment of some E(spl) repressors onto DNA -- interestingly, this is achieved via protein-protein interactions with proneural activators. Direct DNA binding also contributes significantly to E(spl) activity, while activator sequestering is unlikely to be used by E(spl) proteins to counteract proneural function (Giagtzoglou, 2003).

The first indication that E_B/C sites are dispensable for E(spl)-mediated repression came from reporter gene analysis in transfected Schneider S2 cells. T5-0.9wt/luc, a luciferase reporter driven by the proximal 5' regulatory region of the ac gene was used. This fragment probably constitutes an autoregulatory element, since it contains three E_A boxes and can be activated by Da/Sc or Da/Ac; it also contains one C-box needed for repression by the E(spl)-related protein Hairy. When an E(spl) expression plasmid (expressing either E(spl)m7, mgamma or mdelta) is included in addition to those expressing da and sc in a transient transfection experiment, repression of T5-0.9wt/luc was observed. A mutant version of the same reporter, T5-0.9mut/luc, was also used -- in this reporter the C box had been mutated, disabling repression by Hairy. E(spl)m7 and mgamma were still capable of repressing the mutant reporter, whereas E(spl)mdelta had lost the ability to repress, in fact it somewhat activated transcription [an unexplained result, also observed with Hairy. It thus appears that different members of the HES family of repressors may use different mechanisms of repression, with Hairy and E(spl)mdelta being strictly dependent on a DNA target site, versus E(spl)m7 and mgamma retaining activity in the absence of direct DNA binding (Giagtzoglou, 2003).

To gain more insight into this novel repression mechanism of E(spl)m7 and mgamma, an in vivo system was used. An artificial reporter gene in the fly driven solely by E_A boxes was used to avoid the possibility of E(spl) proteins binding to atypical sites, a behavior for which there is ample precedent (Chen, 1997; Culi, 1998; Yang, 2001), and may have been the cause of repression of T5m-luc in transfection experiments. The EE4-lacZ reporter, consisting of eight tandem E_A boxes in front of a minimal promoter was shown (Culi, 1998) to respond to proneural proteins by turning on in all proneural cluster cells in the wing disk. The response of EE4-lacZ was assayed in larval imaginal disks in response to E(spl) proteins expressed using the Gal4/UAS system. Overexpression of E(spl)m7 abolishes EE4-lacZ activity, whereas E(spl)mdelta only moderately reduces expression. This was somewhat surprising, given that E(spl) proteins do not recognize the E_A target site (Culi, 1998; Jennings, 1999; Oellers, 1994). Thus, the possibility was entertained that the repression by E(spl) was not a direct effect on the EE4 enhancer, rather it could have arisen from the fact that overexpression of E(spl) represses endogenous proneural genes, which in turn are needed to activate EE4. Therefore Ac protein was visualized in wing disks overexpressing E(spl)m7. The overall proneural pattern of Ac was not altered, but expression levels were variably reduced within the overexpression domain. Strongly expressing SOP cells within proneural clusters were never seen, in agreement with the well-established sensory-organ suppressive activity of E(spl) proteins (Giagtzoglou, 2003).

In order to test more rigorously the mechanism of E(spl)-mediated repression of EE4-lacZ and to avoid the fluctuation of endogenous proneural protein levels caused by E(spl) overexpression, the need for endogenous proneural proteins was bypassed altogether by providing excess Sc exogenously. A UAS-sc transgene was expressed alone or together with UAS-E(spl) transgenes. Ectopic Sc gave the expected broad, yet patchy, ectopic activation of EE4-lacZ. Patchy activation of proneural target genes has been observed before and apparently reflects stochastic damping of Sc activity, at least partly because of induction of endogenous E(spl) genes. Co-expression of E(spl)m7 results in strong repression of the EE4 enhancer, whereas E(spl)mdelta did not affect activation by UAS-sc. The same effects were observed using two different GAL4 lines, pnr-GAL4 and omb-GAL4, which drive expression in a central wing pouch region. It thus appears that E(spl)m7, but not mdelta, can repress transcription of EE4-lacZ without directly binding to DNA, consistent with the different behavior of these proteins in transfection assays. mdelta still weakly represses EE4-lacZ transcription, most probably through repression of activators, such as sc. Another UAS-E(spl) transgene, E(spl)mgamma, was able to repress UAS-sc-driven activation of EE4-lacZ, similar to E(spl)m7 (Giagtzoglou, 2003).

If direct DNA binding is dispensable for the repression by E(spl)m7 and mgamma of EE4-lacZ, mutant versions that lack the DNA-binding basic domain should be functional. E(spl)m7KNEQ, a double point mutation in the basic domain, which abolishes DNA binding, was generated, and it was tested in transgenic flies. UAS-E(spl)m7KNEQ has strong repressive activity on EE4-lacZ when expressed either alone or together with UAS-sc, confirming the dispensability of the basic domain in this assay. UAS-mgammaKNEQ, which bears the same basic domain inactivating mutations as m7KNEQ, was also capable of repressing EE4-lacZ, even in the presence of exogenous UAS-sc (Giagtzoglou, 2003).

In a converse experiment, the activity of the EE4-lacZ reporter was examined in loss-of-function backgrounds for E(spl). EE4-lacZ was consistently more active in a mutant background lacking E(spl)m7 and m8, as compared with wild type. This happens even though the number and pattern of SOPs in this mutant background is identical to wild type, presumably owing to the activity of the remaining E(spl) genes. It is concluded that activity of E(spl)m7 and m8, the two most highly expressed E(spl) genes in wing disk proneural clusters, attenuates EE4-lacZ expression. Since E(spl) genes other than m7 and m8 were still present in the above genetic background, the response of EE4-lacZ was tested in homozygous clones of a deficiency removing the entire E(spl) locus. Increased levels of ß-galactosidase expression were again observed within mutant patches, confirming the response of EE4-lacZ to E(spl) activity, despite the absence of E(spl) binding sites on this reporter. A caveat in interpreting these experiments is that E(spl) loss-of-function may increase sc expression, which would then act on the EE4-lacZ reporter (Giagtzoglou, 2003).

Ectopic expression of sc in flies is known to induce formation of supernumerary chetae, reflecting induction of endogenous Sc target genes. Individual UAS-E(spl) transgenes were tested for their ability to block ectopic cheta production by sc. When expressed alone by pnr-Gal4, all UAS-E(spl) genes inhibited formation of both macro- and mirco-chetae, resulting in a bald stripe in the center of the thorax. This was even true for UAS-E(spl)m7KNEQ and E(spl)mgammaKNEQ, suggesting that, under the conditions of this assay, direct DNA binding (to presumably natural target genes controlling SOP fate) is dispensable. When co-expressed with UAS-sc, UAS-E(spl)m7 and mgamma, as well as E(spl)m7KNEQ and mgammaKNEQ, still produced completely bald thoracic stripes, indicating that these proteins can inhibit the activity of both endogenous and overexpressed Sc on (endogenous) target genes very effectively. By contrast, UAS-E(spl)mdelta only partially suppresses the ectopic bristle phenotype of UAS-sc. This behavior was essentially the same as that documented above using EE4-lacZ and was further confirmed by assaying the expression of two target genes, SMC-lacZ and ase. The sole difference was that E(spl)mdelta could partially decrease the number of ectopic bristles, while having no effect on EE4-lacZ activation. The bristle/SOP suppressive activity of E(spl)mdelta is attributed to DNA-binding-dependent repression of proneural target genes. Taken together, reporter and bristle repression assays demonstrate that E(spl)m7 and mgamma, but not mdelta, can repress an E_A-driven artificial reporter gene, as well as endogenous target genes, despite the overexpression of sc. Based on the fact that basic domain mutated versions of E(spl)m7 and mgamma are much more potent repressors than mdelta, it is concluded that in this assay some activity of E(spl) proteins other than their direct DNA binding ability is most important in target gene repression (Giagtzoglou, 2003).

The most important conclusions from this study are the following: (1) targets of E(spl) repression are the target genes of the proneurals, and to a lesser extent the proneural genes themselves; (2) E(spl) recruitment onto target genes can occur via direct DNA binding (to E_B/C/N boxes), but also via interactions with E_A-box-bound proneural activators; (3) sequestration of the proneural activators off DNA, a mechanism employed by the Emc/Id family of HLH proteins, does not seem to operate in the case of E(spl), and (4) in both DNA-mediated and activator-mediated modes of E(spl) tethering to target genes Groucho recruitment is required for repression (Giagtzoglou, 2003).

It is sometimes assumed that E(spl) proteins suppress neurogenesis solely by repressing proneural gene transcription. This is not the case, since E(spl)m7 and mgamma can completely block sensory organ commitment in a background of exogenously (transgenically) provided high levels of Sc. Target genes (genuine and artificial) that are activated by Da/Sc are still repressed by E(spl)m7 and mgamma in the above genetic background. This is consistent with the earlier observation that E(spl) overexpression has only a moderate effect on ac expression, whereas it completely represses downstream targets, such as SMC-lacZ (Culi, 1998), ase or EE4-lacZ. Even though ac and sc are not the main targets of E(spl), some of their enhancers are repressible by E(spl). ac and sc genes elaborate expression pattern is dependent on a number of prepattern enhancers, which are controlled by patterning systems and are weakly, if at all, repressible by E(spl) (Gomez-Skarmeta, 1995). One enhancer each of ac [the proximal 900bp (Martinez, 1993) and sc (the SMC enhancer) (Culi, 1998) has been described that is repressible by E(spl). Both of these are autoregulatory inasmuch as they contain E_A boxes and are activated by Da/Sc or Ac, hence they act to boost ac/sc levels after transcription has been initiated via the prepattern enhancers; in this context the SMC and ac-proximal enhancers can be viewed as 'target genes' of the proneural proteins (Giagtzoglou, 2003).

Another piece of evidence in favor of regulation of proneural target genes (rather than proneural genes themselves) by E(spl) is that E(spl)m7VP16 can activate the neural pathway in genetic backgrounds mutant for ac and sc. Other than displaying aberrant spacing, bristles produced in such a background are normal, at least in external appearance. This is consistent with E(spl)m7VP16 binding and activating many, perhaps all, target genes of Ac/Sc (not just the autoregulatory ac/sc enhancers), bypassing the need for proneural proteins. One should be aware, however, that there are other bHLH proneural genes, besides ac and sc, in the fly genome; e.g. l'sc is not affected by the sc^10-1 allele used in this study. Although l'sc is not normally expressed in the larval wing disk, it is conceivable that it is turned on by E(spl)VP16 activators and then takes over the task of activating the panel of downstream genes. Another potential candidate that might single-handedly mediate the sensory-organ promoting activity of E(spl)VP16 is ase, a SOP-specific gene that bears homology to the proneural genes of the ac/sc family and can act as a proneural gene itself. Thus, it is a matter of further research whether the bristle-induction ability of E(spl)VP16 in a sc^10-1 background is channeled through activation of a single E(spl) target gene or of a number of target genes (Giagtzoglou, 2003).

All proneural target genes contain E_A boxes, via which the Da/proneural activators exert their effect. Analysis of the EE4-lacZ enhancer has revealed that the same E_A boxes are sufficient for E(spl)-mediated repression, even though the latter bind a different class of target sites, the E_B/C/N-boxes. Based on the data presented in this work, it is proposed that this is achieved by enhancer recruitment of E(spl) proteins via protein-protein interactions with proneural activators. This study has focused on three E(spl) proteins. Two, m7 and mgamma, have been shown to interact with both Da and Ac/Sc (Alifragis, 1997) and in the present study display an equivalent ability to be indirectly recruited onto DNA by Da/Sc. The third, E(spl)mdelta, shows no proneural-mediated recruitment activity, apparently because of its inability to interact with either Da or Sc. Perhaps this Da/Sc-binding activity of some of the E(spl) proteins has evolved to enable them to repress all proneural target genes effectively without the need for direct DNA binding. Ac and Sc seem to play a central role in this repression mechanism, since the ubiquitous Da is not sufficient to recruit E(spl)-VP16 proteins to EE4-lacZ and other proneural target enhancers (Giagtzoglou, 2003).

Even though E(spl) proteins can be recruited onto their target genes via proneural complexes, all characterized proneural target enhancers (e.g. SMC, ac-proximal, ase, dpn, neur) do bear E_B/C/N-boxes in addition to E_A-sites. Likewise, all E(spl) proteins possess well-conserved DNA-contacting basic domains. Two observations from this work strongly suggest that direct DNA binding is also used in the repression of target genes by E(spl). (1) A significant suppression of bristle formation by E(spl)mdelta is observed upon co-expression with Sc. This can only be interpreted as repression of Sc targets by E(spl)mdelta by direct binding to their E_B/C/N-boxes, since it has been established that E(spl)mdelta is incapable of proneural-mediated enhancer binding. (2) E(spl)m7VP16, but not a basic region mutant version, turns on bristle commitment in the absence of proneural genes, pointing towards DNA-binding-dependent recruitment onto proneural target genes (Giagtzoglou, 2003).

The realization that some E(spl) proteins can act as both repressors and co-repressors of the proneurals prompts reconsideration of the proneural proteins as dedicated transcriptional activators; they seem to be equally important in effecting repression of their target genes. Other transcriptional activators, such as Dorsal and HNF4 can act as repressors in certain contexts, suggesting that this may be quite a widespread mechanism (Giagtzoglou, 2003).

This study has used a transgenic approach to establish the ability of E(spl) proteins to be recruited onto target genes by the two mechanisms discussed above. It cannot be predicted whether in a wild-type background the two mechanisms are used exclusively of one another or simultaneously. The presence of E_B/C/N-boxes in close proximity to E_A-boxes in enhancers of proneural target genes favors the latter possibility, namely that proneural and E(spl) proteins each bind their cognate target sites and subsequently also interact at the protein level. Protein-protein interaction concomitant with DNA binding may enable cooperative enhancer binding, which would ensure a rapid response of target genes to changes in concentration of proneural and E(spl) proteins (Giagtzoglou, 2003).

Having realized the plausibility for two (alternative or simultaneous) modes of E(spl) recruitment onto target enhancers, a complete picture still does not exist of what it takes (in terms of transcriptional regulation) to achieve a robustly laterally inhibited response to proneural activity; in other words, to turn on a proneural target gene solely in the neural precursor. The artificial EE4-lacZ enhancer, though responsive to wild-type levels of E(spl) is still not fully repressed, and is expressed in most cells of a wild-type proneural cluster. By contrast, another enhancer that also lacks E_B/C/N boxes has been reported to be fully repressible by wild-type levels of E(spl): SMCN-delta147-181 is a mutant version of the SMC enhancer lacking all E(spl)-binding sites, but containing two E_A-boxes -- this enhancer expresses solely in the neural precursor (SOP) and not in surrounding proneural cluster cells (Culi, 1998). One can hypothesize that additional factors binding SMCN-delta-147-181 favor the formation of a repressive DNA-protein complex in the E(spl)-containing non-SOPs. Indeed this enhancer contains two copies each of conserved alpha and ß boxes (bound by unknown factors) interspersed with the E_A boxes (Culi, 1998). One or both of these factors may cooperate with low (wild-type) levels of E(spl) (bound to E_A via interaction with the proneural complex) to stabilize Gro binding to this enhancer; indeed Gro often has to simultaneously interact with more than one DNA bound factor to gain access to an enhancer (Giagtzoglou, 2003).

Natural proneural target enhancers contain E_A, E_B, C, N, alpha and ß boxes, in addition to binding sites for other factors, such as the Zn-finger protein Senseless. Some of these enhancers (e.g. SMC, ase, dpn, neur) are expressed solely in the neural precursor, whereas others [ac proximal, sca, various E(spl) enhancers] are expressed more widely within the proneural cluster, apparently not responding (or less responsive) to lateral inhibition. Yet, the two types of enhancers are not obviously different with respect to types of target sites contained. Perhaps it is the exact number and arrangement of the various target sites and DNA-bound factors that defines the threshold level of lateral inhibition that each enhancer is responsive to. Seen in this light, it is conceivable that interaction of E(spl) with proneural factors (and perhaps other factors within a large protein-DNA complex) may bring about conformational changes, which are needed to fine-tune crosstalk of these transcription factors with co-activators, co-repressors and other components of the transcriptional machinery. Characterizing these regulatory interactions will improve insight on the transcriptional mechanisms that mediate neural fate acquisition and will be a major challenge for the future (Giagtzoglou, 2003).

GENE STRUCTURE

cDNA clone length- 723

Bases in 5' UTR - 125

Exons- 1

Bases in 3' UTR- 47

PROTEIN STRUCTURE

Amino Acids- 186

Structural Domains

Genetic evidence suggests that E(spl), one of the neurogenic loci of Drosophila, is a gene complex comprising an as yet incompletely established number of transcription units. In order to correlate the various transcription units with E(spl) functions, wild-type flies were transformed with genomic DNA encoding the transcription unit m8 from the mutant E(spl)D, which was known to be altered in embryos carrying this mutant allele. Transformants show the same dominant enhancement of the spl phenotype as E(spl)D itself. Since m8 has a virtually identical pattern of expression as m4, m5 and m7, the sequence of each of these four transcripts was determined. The deduced protein products of m5, m7 and m8 exhibit extensive sequence homology with each other. All three encode a sequence similar to one of the conserved domains of representatives of the vertebrate myc gene family which is also present in the deduced protein sequences of the Drosophila achaete-scute gene complex (Klambt, 1989).

date revised: 15 April 2003

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