The Interactive Fly
Zygotically transcribed genes
Seven Enhancer of split genes in Drosophila melanogaster encode basic-helix-loop-helix transcription factors that are components of the Notch signaling pathway. They are expressed in response to Notch activation and mediate some effects of the pathway by regulating the expression of target genes. Using random oligonucleotide selection, the optimal DNA binding site for the Enhancer of split proteins has been determined to be a palindromic 12-bp sequence, 5'-TGGCACGTG(C/T)(C/T)A-3', which contains an E-box core (CACGTG). This site is recognized by all of the individual Enhancer of split basic helix-loop-helix proteins, consistent with their ability to regulate similar target genes in vivo. The 3 base pairs flanking the E-box core are intrinsic to DNA recognition by these proteins and the Enhancer of split and proneural proteins can compete for binding on specific DNA sequences. Furthermore, the regulation conferred on a reporter gene in Drosophila by three closely related sequences demonstrates that even subtle sequence changes within an E box or flanking bases have dramatic consequences on the overall repertoire of proteins that can bind in vivo (Jennings, 1999).
The three related sequences studied were the B1, A1 and A2 sites. The B1 and A1 sites contain optimal flanking bases and differ by a single-base substitution that switches the E box from a class B site (B1) to a class A site (A1). The A2 site has the class A E-box core but suboptimal flanking sequences. The consensus binding site for the E(spl)bHLH proteins contains a class B canonical E-box (CACGTG). This is compatible with the presence of the key arginine residue in the basic region that is characteristic of all other bHLH proteins that recognize class B sites and which contacts the central G. The selected site differs from the previously identified N box (CANNAG), indicating that the latter may not be generally representative of E(spl) target sites -- in vitro, the N box is a much lower affinity target that the class B site. The 12-bp palindrome (the optimal site) is termed the ESE box. Flanking bases have been implicated in DNA binding by other bHLH proteins, including c-Myc and Hairy, based on in vitro assays and X-ray crystallography studies that reveal interactions between bHLH proteins and bases outside the E-box core. However, the flanking bases preferred by c-Myc and Hairy differ from those selected by E(spl)bHLH proteins, indicating that in vivo, the sequences immediately surrounding an E box are important for determining exactly which bHLH proteins bind there to regulate transcription (Jennings, 1999 and references).
The interactions with flanking bases helps to explain the specificity in vivo of different bHLH proteins, an important factor given the large number of bHLH proteins identified to date. The in vivo expression patterns produced by E boxes with different flanking bases in these experiments emphasizes the significance of the flanking sequences. For example, a comparison between the A2 and A1 sites demonstrates that the former is a target for many more transcriptional activators. These experiments also illustrate the relevance of different E-box core sequences, since a single-base difference within the E-box core (A1 to B1) is sufficient to prevent binding of proneural proteins and other activators. This is in agreement with earlier studies that argued that proneural proteins and E(spl)bHLH repressors recognize sites with distinct types of E-box cores. However, these results show that E(spl)bHLH repressors prefer the class B core, which is recognized by many different bHLH activators and repressors, over the class C core. Class C has been designated the target for repressor bHLH proteins that contain a proline residue in the basic domain. The class C site (CACGCG) is the optimal binding site for the Drosophila Hairy protein, whose basic domain contains a proline residue but differs from E(spl)bHLH proteins in 7 of the 11 remaining residues, which could account for the different profile of DNA binding specificities. The distinctions in the DNA binding specificities could be significant for studies of the vertebrate homologs of the E(spl)bHLHs and Hairy. Overall, the in vitro binding experiments and the activity of different sites in vivo demonstrate that the bHLH proteins that were tested can recognize a specific range of target sequences and that both core and flanking bases are important for determining the binding specificity (Jennings, 1999 and references).
Although flanking bases may distinguish sites for different types of E-box binding proteins, there are no significant differences in the bases recognized by individual E(spl) proteins; the same consensus binding site was derived for each of three proteins tested. There were subtle differences in the ranges of oligonucleotides, with Mdelta selecting a broader range of variants at the flanking sites than Mgamma and M3 and the latter two proteins exhibiting more tolerance for variants in the core E box, but experiments comparing the affinity of the proteins for these variant sites reveal no detectable bias (Jennings, 1999).
The binding specificities observed are all for homodimers of individual E(spl) proteins. In places where more than one E(spl)bHLH protein is expressed (e.g., proneural clusters), it is possible that the proteins form heterodimers among themselves to bind DNA and repress transcription. However, given that the amino acid sequences of the DNA binding domains and the DNA binding preferences of the individual E(spl)bHLH proteins are so similar, it seems unlikely that heterodimers between E(spl)bHLH proteins would differ greatly from homodimers in their DNA binding sequence preferences. In addition, during several developmental processes, a single E(spl)bHLH protein predominates (e.g., Mbeta in the presumptive intervein region of the wing), indicating that E(spl)bHLH proteins are likely to function as do homodimers. There is also no evidence to suggest that the E(spl)bHLH proteins are required to form heterodimers with other bHLH family members to bind DNA and repress gene transcription in response to Notch signaling. Thus, the homodimers analyzed in these experiments likely represent complexes that are functional in vivo (Jennings, 1999 and references).
The overall similarity in the binding of different E(spl) proteins in vitro suggests that they are capable of recognizing the same targets in vivo and is consistent with the phenotypes observed when the individual proteins are expressed ectopically. Ectopic expression of M8, M5, Mbeta, Mdelta, and M7 all produce phenotypes of vein and bristle loss. Both Mbeta and M7 are able to interact with DNA sequences regulating achaete. The ability to recognize the same DNA target sequences could explain the apparent redundancy between the E(spl) genes, as they would all have the potential to act in the same processes. The observation that specific E(spl)bHLH proteins are more or less efficient in regulating different processes (e.g., Mbeta more effective at suppressing veins and M8 more effective at suppressing bristles) is thus more likely to be consequence of differences in protein:protein interactions than of differences in target recognition (Jennings, 1999 and references).
In the absence of E(spl)bHLH proteins, proneural protein expression persists at high levels in all cells of a proneural cluster. Thus, one action of E(spl)bHLH proteins is to antagonize the proneural proteins, with the ultimate consequence that proneural gene expression is repressed. It has been proposed that E(spl)bHLH proteins exert their influence by binding to regulatory regions within the AS-C and repressing transcription of the proneural genes. This hypothesis is supported by the observations that expression of Achaete is induced by M7ACT and MbetaACT and that induction of ectopic bristles in the Drosophila wing and notum by M7ACT is abolished in the absence of proneural proteins. One putative binding site for the E(spl)bHLH proteins, that upstream of the achaete gene, has the sequence 5'-CGGCACGCGACA-3' (Hairy site). Mgamma will bind this site in vitro, and M7 can bind this sequence and repress transcription in a cotransfection assay in Drosophila S2 cultured cells. However, mutation of this site in vivo results in a phenotype resembling that caused by mutations in hairy rather than in the E(spl)-C. This fits with the observation that this sequence conforms to an optimal Hairy DNA binding site but is a suboptimal site for the E(spl) proteins and indicates that the E(spl) proteins do not recognize this sequence in vivo. Thus, if E(spl) proteins are directly repressing achaete expression, there should be more optimal target sites elsewhere within the AS-C. Indeed, a search of recently available AS-C genomic sequence identifies >10 sequences with good matches to ESE boxes, in addition to the sites that have been identified by in vitro binding assays (Jennings, 1999 and references).
An alternative hypothesis is that the primary function of the E(spl)bHLH proteins is to antagonize the actions of proneural proteins posttranscriptionally. Evidence in support of this comes from experiments in which L'sc is ectopically expressed using a heterologous promoter that is not subject to direct regulation by E(spl)bHLH proteins. Under these conditions L'sc expression results in isolated ectopic bristles, rather than clusters of bristles, demonstrating that lateral inhibition is still able to restrict neural fate to a single cell even though l'sc transcription is insensitive to Notch signaling. This implies that E(spl)bHLH proteins are able to antagonize proneural genes in ways other than by repressing their transcription. One possibility is that the E(spl) proteins can interact with the same targets as proneural proteins, but that they repress rather than activate transcription. The ability of E(spl) proteins to bind to the B1 and A1 sequences and repress transcription from a heterologous promoter is consistent with this model, as is the observation that M7ACT can induce certain ectopic leg bristles in the absence of the achaete and scute genes. In the latter context, M7ACT is likely to be acting on genes with functions downstream of the proneural proteins to cause neural differentiation. In addition, the E(spl)bHLH proteins are involved with developmental processes that do not involve the proneural proteins, e.g. wing vein development; thus, they cannot act solely to repress proneural gene transcription during development (Jennings, 1999 and references).
How might E(spl)bHLH repress transcription of target genes? The
closely related protein Hairy has been shown to repress transcription in a dominant manner even when its binding sites are located at some
distance from the promoter, leading to the hypothesis that Hairy is able to mediate stable, inheritable repression of the
target genes. It is anticipated that E(spl)bHLH repression will be
transitory, so that if Notch signaling were terminated, the E(spl)
proteins would decay and the target genes would be susceptible to
reactivation. Although proneural and E(spl)bHLH proteins optimally prefer different core E-box binding sites, so that independent binding
to target genes appears likely, the importance of the bases flanking
the E box in target recognition means that there is potential for
overlap in the binding sites of the two groups of proteins. Thus, in
cells where expression of E(spl)bHLH proteins is induced by Notch
signaling, the proteins accumulate to high levels and could compete
for binding to proneural protein target sites of the A1 type described
here. Among the E-box sequences recognized by proneural proteins in
vitro that have been described, at least a subset have good matches
with the ESE consensus and thus could be recognized by both classes of
proteins. Now that
the sequence preferences of the E(spl)bHLH proteins have been identified, when target
genes of proneural and E(spl)bHLH proteins have also been identified and
their regulatory regions analyzed, it will be possible to determine
whether the sites present offer the potential for competition (e.g., by
resembling A1 sites) or whether they have the features of
completely distinct binding sites for E(spl)bHLH, Hairy, proneural,
and other bHLH proteins (Jennings, 1999 and references).
One of the first steps in embryonic mesodermal differentiation is allocation of cells to particular tissue fates. In Drosophila, this process of mesodermal subdivision requires regulation of the bHLH transcription factor Twist. During subdivision, Twist expression is modulated into stripes of low and high levels within each mesodermal segment. High Twist levels direct cells to the body wall muscle fate, whereas low levels are permissive for gut muscle and fat body fate. Su(H)-mediated Notch signaling represses Twist expression during subdivision and thus plays a critical role in patterning mesodermal segments. This work demonstrates that Notch acts as a transcriptional switch on mesodermal target genes, and it suggests that Notch/Su(H) directly regulates twist, as well as indirectly regulating twist by activating proteins that repress Twist. It is proposed that Notch signaling targets two distinct 'Repressors of twist' - the proteins encoded by the Enhancer of split complex [E(spl)C] and the HLH gene extra machrochaetae (emc). Hence, the patterning of Drosophila mesodermal segments relies on Notch signaling changing the activities of a network of bHLH transcriptional regulators, which, in turn, control mesodermal cell fate. Since this same cassette of Notch, Su(H) and bHLH regulators is active during vertebrate mesodermal segmentation and/or subdivision, this work suggests a conserved mechanism for Notch in early mesodermal patterning (Tapanes-Castillo, 2004).
Analysis of Notch mutant embryos revealed that Notch signaling is essential for Twist regulation at mesodermal subdivision. However, comparison of Notch and Su(H) mutant embryos indicated that Notch regulates Twist differently from Su(H). At stage 10, uniform high Twist expression was maintained in Nnull mutants; by contrast, Su(H)null mutants have a wild-type-like Twist pattern. Furthermore, while constitutive activation of Notch represses Twist expression at stage 10, constitutive expression of a transactivating form of Su(H) [Su(H)-VP16] increases Twist expression. Despite these differences, double mutant analysis and rescue experiments demonstrate that Notch requires Su(H) to repress Twist. Moreover, further rescue experiments show that Notch signaling acts as a transcriptional switch, which alleviates Su(H)-mediated repression and promotes transcription. In addition, genetics, combined with promoter analysis, suggest that Notch and Su(H) have multiple inputs into twist. Notch/Su(H) signaling both directly activates twist and indirectly represses twist expression by activating proteins that repress Twist. Finally, the data indicate that Notch targets two distinct 'Repressors of twist' - E(spl)-C genes and Emc. It is proposed that Notch signaling activates expression of E(spl)-C genes, which then act directly on the twist promoter to repress transcription. Since removing groucho enhances the phenotype of the E(spl)-C
mutant embryos, it is suggested that the corepressor, Groucho, acts with E(spl)-C proteins and the Hairless/Su(H) repressive complex to mediate direct repression of twist. The second 'Repressor of twist', Emc, mediates repression of Twist in an alternative fashion. It is hypothesized Emc activity inhibits dimerization of Da with itself or another bHLH protein. This, in turn, prevents Da from binding DNA and activating twist transcription. Since Emc is expressed in the embryo prior to stage 10, it is likely that the transition from uniform high Twist expression to a modulated Twist pattern involves Emc inhibition of Da activity at stage 9. In conclusion, this work uncovers how Notch signaling impacts a network of mesodermal genes, and specifically Twist expression. Given that Notch signaling directs cell fate decisions in many Drosophila embryonic and adult tissues and that Notch regulates Twist in adult flight muscles, these data may suggest a more universal mode of Notch regulation (Tapanes-Castillo, 2004).
The distinct mesodermal phenotypes of Notch and Su(H)
mutants can be explained by Notch acting as a transcriptional switch. This
aspect of Notch signaling has been described in other systems, and the
early Drosophila mesoderm appears no different in this regard.
However, these data suggest that there is more to the phenotypes; that is, additional layers of Notch regulation in the transcriptional control of twist (Tapanes-Castillo, 2004).
Genetic experiments, as well as promoter analysis, raised the hypothesis
that Notch signaling regulates twist directly, as well as indirectly
by activating expression of a 'repressor of twist.' This indirect
repression of twist concurs with the role of Notch in activating
E(spl) transcriptional repressors. Moreover, a mechanism involving
direct and indirect regulation is consistent with Su(H) mutant
phenotypes. In Su(H)null embryos, neither twist
nor repressor of twist (for example, emc) are repressed. The
de-repression of both genes at the same time results in Twist expression
appearing 'wild-type-like'. When a constitutively activating form of Su(H) is expressed, both twist and repressor of twist are activated.
In these embryos, high Twist domains are expanded, but uniform high Twist
expression is not observed because repressor of twist is
expressed (Tapanes-Castillo, 2004).
However, simple direct and indirect regulation [through emc and
E(spl)-C genes] by Notch still does not fully explain the phenotypes of Notch mutants. Both twist
and repressor of twist should be repressed in
Nnull embryos because Su(H) will remain in its repressor
state. While the Nnull phenotype was consistent with
repressor of twist being repressed, twist was still strongly
expressed. Additionally, constitutive Notch activation should cause both twist and repressor of twist to be expressed. Consequently,
Nintra was expected to cause a phenotype similar to that caused by Su(H)-VP16. Contrary to these predictions, panmesodermal expression of
Nintra represses Twist, consistent with only repressor of
twist being strongly expressed. Taken together, these results suggested that at stage 10, the twist promoter is less receptive to Notch/Su(H) activation than to Notch/Su(H) repression. As a result, constitutive activation of Notch represses twist, while loss of Notch activates twist ectopically (Tapanes-Castillo, 2004).
While Notch signaling has the ability to activate twist,
Notch/Su(H) signaling ultimately leads to repression of twist at
stage 10. This predominance of repression can be explained in two ways: (1) direct Notch activation of the twist promoter is overpowered by Notch activated repressors of twist; and (2) a repressor of twist gene, such as E(spl), is more responsive to Notch/Su(H) activation than twist. These ideas are discussed below in light of the results (Tapanes-Castillo, 2004).
The first model proposes that while Notch signaling might directly promote both twist and repressor of twist activation, repressors of twist might suppress an increase in twist transcription. The data suggest that Notch regulates multiple repressors of twist, including E(spl)-C genes and Emc. On the twist promoter, these multiple repressors could overwhelm Su(H) activation. Hence, twist would be transcriptionally repressed rather than activated. In Su(H)-VP16 embryos, the constitutive activating ability of Su(H) on the twist promoter might inhibit some of this repression. Consequently, Twist is ectopically expressed at high levels (Tapanes-Castillo, 2004).
Jennings, B. H., Tyler, D. M. and Bray, S. J. (1999). Target specificities of Drosophila Enhancer of split basic helix-loop-helix proteins. Mol. Cell. Biol. 19: 4600-4610
Schlatter, R. and Maier, D. (2005). The Enhancer of split and Achaete-Scute complexes of Drosophilids derived from simple ur-complexes preserved in mosquito and honeybee. BMC Evol. Biol. 5: 67. 16293187
Tapanes-Castillo, A. and Baylies, M. K. (2004). Notch signaling patterns Drosophila mesodermal segments by regulating the bHLH transcription factor Twist. Development 131: 2359-2372. 15128668
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