Enhancer of split


Protein Interactions

Hairy, Deadpan and E(SPL) proteins have three evolutionarily conserved domains required for their function: the bHLH, Orange, and WRPW domains. However, the suppression of Scute activity by Hairy does not require the WRPW domain. The Orange domain is an important functional domain that confers specificity among members of the Hairy/E(SPL) family. A Xenopus Hairy homology conserves not only Hairy's structure but also its biological activity. Transcriptional repression by the Hairy/E(SPL) family of bHLH proteins involves two separable mechanisms: repression of specific transcriptional activators, such as Scute, through the bHLH and Orange domains, and repression of other activators via interaction of the C-terminal WRPW motif with corepressors, such as the Groucho protein (Dawson, 1995).

The basic HLH domain of the proteins coded for by the Enhancer of split and achaete-scute complexes differ in their ability to form homo- and heterodimers. The bHLH domains of E(spl)C proteins m5, m7 and m8 interact with bHLH domains of the Achaete and Scute proteins. These bHLH domains form an interaction network which may represent the molecular mechanism whereby the competent state of proneural genes is maintained until the terminal determination to neuroblast identity occurs (Gigliani, 1996).

Neural fate specification in Drosophila is promoted by the products of the proneural genes, such as those of the achaete-scute complex, and is antagonized by the products of the Enhancer of split [E(spl)] complex, hairy, and extramacrochaetae. Since all these proteins bear a helix-loop-helix (HLH) dimerization domain, their potential pairwise interactions were investigated using the yeast two-hybrid system. The fidelity of the system was established by its ability to closely reproduce the already documented interactions among Daughterless, Achaete, Scute, and Extramacrochaetae. The seven E(spl) basic HLH proteins can form homo- and heterodimers inter-se with distinct preferences. A subset of E(spl) proteins (MB and M5) can heterodimerize with Da, another subset (M3) can heterodimerize with proneural proteins, and yet another (Mbeta, Mgamma and M7) with both, indicating specialization within the E(spl) family. Hairy displays no interactions with any of the HLH proteins tested. It does interact with the non-HLH protein Groucho, which itself interacts with all E(spl) basic HLH proteins, but with none of the proneural proteins or Da. An investigation was carried out of the structural requirements for some of these interactions, using site-specific and deletion mutagenesis. Deletion analysis of M3 and Scute is consistent with their interaction being mediated by their respective bHLH domains. The dependence of the E(spl)-activator HLH interactions on the HLH domain is nicely reflected in the fact that the functional grouping of the E(spl) proteins correlates well with the amino acid sequences of their bHLH domains, e.g., M5 and M8 have highly similar bHLH regions, different from those of the M7/Mbeta/ and Mgamma group, which also display high intragroup similarity. The strong interactions observed between E(spl) proteins and proneural proteins might lead one to hypothesize the E(spl) proteins act like Extramachrochaetae, i.e., by sequestering HLH activators. This is unlikely, since residual activities of E(spl) proteins with mutated basic domains have only weak residual activities (Alifragis, 1997).

Drosophila neurogenesis requires the opposing activities of two sets of basic helix-loop-helix (bHLH) proteins: proneural proteins, which confer on cells the ability to become neural precursors, and the Enhancer-of-split [E(spl)] proteins, which restrict such potential as part of the lateral inhibition process. Do E(spl) proteins function as promoter-bound repressors? The answer was sought by examining the effects on neurogenesis of an E(spl) derivative containing a heterologous transcriptional activation domain [E(spl) m7Act (m7Act)]. The activator domain is derived from VP16. m7Act contains the bHLH and adjacent putative helical domains from m7 but lacks the last 43 amino acids of the protein, including the C-terminal WRPW tetrapeptide required for repressor activity of the related Hairy protein. In contrast to the wild-type E(spl) proteins, m7Act efficiently induces neural development, indicating that it binds to and activates target genes normally repressed by E(spl). Persistent expression of wild-type E(spl) proteins causes loss of neural precursors and sensory bristles and also suppresses wing vein formation. By contrast, m7Act efficiently induces supernumerary external sense organs, as predicted if E(spl) proteins function as direct repressors. An equivalent m7 truncation lacking the VP16 activation domain has no phenotypic effects, indicating that m7Act does not function by passively interfering with endogenous E(spl) activity, but instead acts as a transcriptional activatior. Mutations in the basic domain disrupt m7Act activity, suggesting that its effects are mediated through direct DNA binding. m7Act causes ectopic transcription of the proneural achaete and scute genes. These results support a model in which E(spl) proteins normally regulate neurogenesis by direct repression of genes at the top of the neural determination pathway (Jimenez, 1997).

E(spl) antagonizes Senseless, a binary switch, during sensory organ precursor selection

During sensory organ precursor (SOP) specification, a single cell is selected from a proneural cluster of cells. Evidence is presented that Senseless (Sens), a zinc-finger transcription factor, plays an important role in this process. Sens is directly activated by proneural proteins in the presumptive SOPs and a few cells surrounding the SOP in most tissues. In the cells that express Sens low levels, Sens acts in a DNA-binding-dependent manner to repress transcription of proneural genes. In the presumptive SOPs that express Sens at high levels, Sens acts as a transcriptional activator and synergizes with proneural proteins. It is therefore proposed that Sens acts as a binary switch that is fundamental to SOP selection (Jafar-Nejad, 2003).

Proneural genes have been shown to be required for sens expression. To determine whether proneurals directly activate sens expression, the putative enhancers of sens were identified and were scanned for proneural protein-binding sites (E boxes). An 11-kb genomic fragment containing the sens locus is able to rescue the sens mutant phenotype. To identify the embryonic and imaginal disc enhancers, three genomic DNA fragments were used to create lacZ reporter transgenes. Both 5.9-kb and 3.4-kb fragments are sufficient to drive expression in the embryonic PNS in a pattern similar to endogenous sens. To refine sens enhancers, the 3.4-kb enhancer was divided into nine overlapping fragments. Fragments 8 and 9 induced lacZ expression in a pattern similar to the original 3.4-lacZ line, indicating that both contain regulatory elements sufficient for sens expression in the embryonic PNS. Fragments 8 and 9 were further divided into overlapping fragments. Only 9-1-lacZ expresses the reporter in a pattern similar to the 3.4-lacZ. Inspection of the 9-1 sequence showed that it contains a single E box. The recently sequenced genome of Drosophila pseudoobscura, a species 25-30 myr divergent from Drosophila melanogaster was used to align the genomic regions. The alignment showed that the E box, as well as several other elements in the 9-1 enhancer, is fully conserved. Upon mutation of this E box from CAGGTG to CCGGTG, most of the PNS cells failed to express lacZ, and staining in other cells was much weaker than for the wild-type transgene. These data indicate that proneural genes directly regulate the transcription of sens (Jafar-Nejad, 2003).

It is thought that the two core nucleotides of the E box as well as its flanking sequences are involved in the specificity of each E box for its cognate bHLH transcription factor. It was intriguing that expression of the lacZ marker is almost abolished in chordotonal organs that are dependent on atonal (ato) as well as in external organs and multiple dendritic organs that are dependent on ac, sc, and amos. Because the 9-1 fragment contains only a single E box, the data suggest that different proneural proteins can bind the same E box in vivo. Therefore EMSA was performed to determine whether a variety of Da-proneural heterodimers can shift a wild-type or an E box-mutated probe taken from the 9-1 sequence. Da homodimer, Ato/Da heterodimer, Ac/Da heterodimer, and Sc/Da heterodimer were all able to bind to this E box. Mutation from A to C in the second position of the E box abolished binding for all protein combinations tested, suggesting that these interactions are sequence specific. It is concluded that at least three proneural proteins (Ac, Sc, and Ato) directly regulate sens expression in the embryonic PNS, and that they may bind the same site in vivo (Jafar-Nejad, 2003).

To examine whether sens regulation in the precursors of the adult PNS is also under direct proneural regulation, the 9-1-lacZ and 9-1-mut-lacZ expression patterns were compared in the SOPs of the thoracic microchaetae. Similar to what was observed in embryos, a single-nucleotide change in the 9-1 E box abolishes most of the lacZ expression in pupae of the same age, again suggesting direct regulation of sens by proneurals (Jafar-Nejad, 2003).

The effects of loss- and gain-of-function of proneural genes on sens expression were assessed in the imaginal discs of third instar larvae. Because fragments 9 and 9-1 do not drive lacZ at this stage, enhancer 8 was used. The 8-lacZ transgene drives lacZ expression in several wing SOPs in late third instar larvae. To determine whether proneural genes are able to control 8-lacZ expression, Sc was overexpressed in the wing pouch using the C5-GAL4 driver. Many more cells express lacZ in the wing pouch than in wild type, indicating that the Sc protein is able to induce lacZ expression ectopically. However, removal of the activity of both ac and sc genes results in loss of lacZ expression in all of the ac/sc-dependent SOPs. The precursors of the ventral radius and the femoral chordotonal organs still express lacZ, since these cells are dependent on Ato expression. Moreover, upon Ato overexpression driven by dpp-GAL4, 8-lacZ is strongly induced at the A/P boundary. Together, these data indicate that proneural proteins regulate sens expression in the precursors of the adult PNS. Fragment 8 contains two E boxes, one of which is fully conserved between D. pseudoobscura and D. melanogaster. Band-shift experiments show that the Ac/Da heterodimer can bind to a radioactive probe that contains the conserved E box of fragment 8, further suggesting that proneurals directly regulate sens expression (Jafar-Nejad, 2003).

E(spl) proteins are known to prevent SOP formation through transcriptional repression of proneural gene expression. Whether they affect sens expression was examined. scabrous (sca)-GAL4 was used to express E(spl)m8 in the SOPs and a few cells around the SOPs in third instar imaginal discs. lacZ expression is abolished in most or all cells. Moreover, misexpression of an 'activator' version of E(spl)m7 (m7ACT), in which the Gro-binding motif is replaced with the VP16 transactivator domain, caused numerous extra lacZ-positive cells when driven in the wing pouch. These observations suggest that E(spl)m7 and E(spl)m8 proteins are also involved in the transcriptional regulation of sens and that proneural proteins and E(spl) proteins have an antagonistic relationship in transcriptional control of sens. E(spl) proteins are known to bind to proneural gene enhancers and m7ACT is able to activate ac and sc transcription. Therefore, it is formally possible that m7ACT is indirectly activating the sens enhancer through its up-regulation of proneural proteins. However, it has been shown that even in the absence of endogenous ac and sc, overexpression of m7ACT causes extra bristle formation, suggesting that the E(spl) proteins not only regulate proneural gene expression, but also regulate the expression of one or more of proneural target genes. Is m7ACT able to induce sens expression in the absence of ac and sc? To address this question, it was confirmed that overexpression of m7ACT can produce several extra bristles in a sc10-1 background. Staining of the imaginal wing discs of these flies shows that there are many Sens-positive cells in the anterior part of the presumptive notum, where the Eq-GAL4 driver used in this experiment is expressed. The data suggest that sens is one of the targets of the E(spl) proteins. Altogether, sens enhancers seem to be able to integrate the positive and negative inputs from proneural and E(spl) proteins, respectively (Jafar-Nejad, 2003).

Protein-protein interactions play a significant role in determining how a transcription factor regulates its target genes. To identify proteins that bind Sens, a yeast two-hybrid (YTH) screen was performed. Of 38 positives sequenced from the screen, seven correspond to members of the E(spl) complex. To confirm the interactions identified in yeast, coimmunoprecipitation (co-IP) assays were performed using in vitro-translated E(spl) proteins and myc-tagged Sens. A monoclonal anti-myc antibody can precipitate E(spl)m7, E(spl)m8, and E(spl)m5 only in the presence of myc-Sens. The yeast two-hybrid assay was used to identify the interaction motif in each partner. Testing a series of Sens deletion constructs showed that a 25-amino acid fragment of Sens (amino acids 276-300) is necessary and sufficient for Sens/E(spl) interaction. To further delineate the interaction motif, the 25 amino acids were mutated to alanines five at a time and five mutant sens constructs were generated. The YTH assays suggested that a 15-amino acid deletion (Sens-del) would abrogate the interaction for all three members of the E(spl) complex. This was indeed observed (Jafar-Nejad, 2003).

Proteins of the E(spl) complex have several conserved motifs, for example, a basic domain, a Helix Loop Helix (HLH) domain, an Orange domain, a caseine kinase-binding motif (CK), and a WRPW or Gro interaction domain (W). To find the interaction motif in the E(spl) proteins, deletion constructs of E(spl)m8 were created and their ability to bind Sens was tested in yeast. The Orange domain was necessary and sufficient for the Sens/E(spl)m8 interaction. The 25-amino acid motif of Sens in isolation interacts with the Orange domain of E(spl)m8 in isolation in the yeast two-hybrid assay. The Orange domain is conserved in all members of the Hairy-E(spl) family of proteins and there is evidence that this domain is functionally important. Alignment of the Orange domains of E(spl)m5, E(spl)m7, and E(spl)m8 prompted mutation of three amino acids in each of the two conserved motifs to alanine and the ability of mutant m8 proteins to interact with Sens was tested. Replacement of EVS with AAA or THL with AAA is sufficient to abolish the interaction of E(spl)m8 with Sens in the yeast assay. In summary, the data indicate that Sens and E(spl) proteins interact in yeast and in vitro (Jafar-Nejad, 2003).

To explore the mechanism by which Sens promotes SOP specification, how Sens regulates proneural gene expression was studied. In sens mutant clones, proneural proteins fail to accumulate in the SOPs. A strong synergism exists in the ability of Sens and Sc to promote extra bristle formation. Whether there is an in vivo synergy between Sens and Ac was examined. acHw-1 exhibits an occasional extra macrochaetae on the notum because of an increase in ac transcript level. Overexpression of Sens with sca-GAL4 also causes a number of extra micro- and macrochaetae on the notum, and overexpression of Sens in an acHw-1 background causes many more extra macrochaetae than the sum of the two genotypes alone. Therefore, there is a synergy between the bristle-promoting effects of the two proteins in vivo (Jafar-Nejad, 2003).

An assay to determine whether Sens can affect ac gene transcription was established in Drosophila S2 cells. The reporter construct used in this assay was a 470-nucleotide fragment of the ac gene containing the ac promoter region fused to the firefly luciferase. This fragment contains the Hairy-E(spl)-binding site and the three E boxes that are involved in ac regulation by proneural and E(spl) proteins. Moreover, during the course of microchaetae SOP specification, expression driven by the ac proximal enhancer/promoter refines from the proneural cluster to a single cell, supporting the notion that it can serve as an SOP-specific regulatory region. The constitutively active actin5 promoter was used to drive the expression of Da, Ac, Sens, or E(spl)m8. Sens alone does not activate the ac470-luc construct. Cotransfection of minimal amounts of actin5-da and -ac activates the luciferase expression about 10-fold. However, adding an additional 20 ng of the actin5-sens leads to a dramatic activation of the ac promoter (>500-fold). It is concluded that Sens can activate ac transcription through synergism with Ac/Da heterodimer in Drosophila S2 cells. These findings suggest that there is a parallel between the in vivo and transcriptional synergy observed between Sens and Ac (Jafar-Nejad, 2003).

Because E(spl)m8 strongly antagonizes SOP specification, it was postulated that E(spl)m8 may decrease the synergistic activation of ac by Sens. Cotransfection of 100 ng of actin5-E(spl)m8 with 1 ng actin5-da and -ac does not significantly repress the luciferase activity induced by these proneural proteins. However, cotransfection of actin5-E(spl)m8 together with sens, da, and ac constructs inhibits the synergy in a dose-dependent manner. In summary, Sens is able to strongly synergize with the proneural proteins in vivo and in vitro, and this synergism is antagonized by E(spl) proteins in a dose-dependent manner (Jafar-Nejad, 2003).

To determine whether the E(spl) antagonism of the Sens synergism operates in vivo, it was first documented that overexpression of Sens at high levels in the wing pouch produces a vast excess of bristles in the wing. In addition, extra vein tissue and thickening of the wing veins is observed. E(spl)m8 overexpression with the C5-GAL4 driver causes loss of wing vein tissue, as well as loss of some of the dorsal wing margin bristles. When Sens and E(spl) are coexpressed, the two proteins suppress each other's phenotypes; the number of extra bristles is decreased significantly, and many wing veins are restored. A higher magnification shows that there are still extra bristles as well as some aberrant vein tissue. Taken together, these data support the notion that Sens and E(spl)m8 have antagonistic effects at the level of proneural gene expression, in agreement with their in vivo effects on bristle formation. Finally, whether the physical interaction between Sens and E(spl) plays a role in their antagonism on ac enhancer was examined in S2 cells. The Sens-del, which lacks the 15-amino acid E(spl)-interacting motif, can synergize with Ac and Da similar to wild-type Sens. However, the ability of E(spl)-m8 to antagonize the synergy between Sens-del and proneural proteins is impaired when compared with its effect on the wild-type Sens/Ac/Da synergy, suggesting that the physical interaction between Sens and E(spl) plays a role in their antagonistic effect (Jafar-Nejad, 2003).

A model for selection of an SOP from a proneural cluster is proposed in which an intricate set of feedback loops between various transcription factors determines, through the action of Sens and E(spl), the selection of the adult SOP. Most cells of a proneural cluster first express relatively low levels of proneural proteins. This leads to transcriptional activation of E(spl) genes in the cluster. E(spl) proteins, together with the corepressor Gro, then prevent the up-regulation of proneural gene expression in the cluster. It is thought that prepattern factors then lead to a higher level of proneural protein expression in a smaller group of cells of the proneural cluster, the proneural field. It is proposed that this higher level of proneural expression, probably together with the prepattern factors, induces low levels of Sens expression in the proneural field or an area that is even smaller. Consistent low levels of Sens staining are observed in groups of cells in the pupal microchaetae field, embryos, wing, and eye discs. These domains that are part of the proneural cluster colabel with proneural proteins, and a single or a few cells are typically selected from these domains to induce higher levels of Sens. It is proposed that Sens plays a critical role in the SOP through transcriptional synergy with proneural proteins. In addition, the data suggest that Sens plays a role in repressing proneural expression in non-SOP cells. Hence, it is proposed that Sens acts as a binary switch in the refinement of the proneural field that will lead to SOP selection (Jafar-Nejad, 2003).

The data also suggest that sens transcription is mediated directly through proneural binding to E boxes in the sens enhancers. In addition, sens enhancers integrate two opposing forces, the positive regulation by proneural and the negative regulation mediated by E(spl) proteins, similar to SOP-specific enhancers of the proneural genes (Jafar-Nejad, 2003).

Because E(spl) prevents the up-regulation of the proneural gene and sens expression, this repressive effect must be overcome if some cells of the proneural field are to be selected as SOPs. In fact, it has been shown that by repressing E(spl)m8 and other repressors of sens, Su(H) plays a positive role in the SOP fate promotion. It is also known that proneural proteins positively regulate E(spl) gene expression, which will prevent further up-regulation of proneural proteins. This negative feedback has prompted the idea that to accumulate large amounts of proneural proteins in the SOP, the equilibrium between the proneural and E(spl) proteins should be displaced in favor of proneurals. It is proposed that the synergy between Sens and Da/Ac on the ac regulatory region is a key mechanism for the up-regulation of ac transcription. In this model, Sens accelerates proneural gene expression and proneural protein accumulation, overruling the negative feedback conferred by E(spl). This hypothesis is supported by the observation that the synergy between Sens and proneurals is highly sensitive to the levels of E(spl) protein in the transcription assay, as well as in vivo. Ac up-regulation will lead to further Sens production and increased synergistic activation of ac transcription. In the absence of Sens, the presumptive SOPs fail to up-regulate proneural gene expression. Hence, Sens will render the presumptive SOP less sensitive to N signaling. This is also supported by the observation that coexpression of Sens and proneurals is able to produce closely spaced bristles, indicating highly inefficient N signaling. In summary, it is proposed that the balance between the levels of the Sens and E(spl) proteins determines the SOP selection (Jafar-Nejad, 2003).

The synergistic model of proneural gene activation predicts that low levels of Sens and proneural proteins may suffice to override the E(spl) inhibition. However, many cells that express sens and proneural genes fail to up-regulate proneural gene expression. At low levels, Sens acts as a repressor of ac transcription, suggesting that in addition to the relative levels of E(spl), the relative levels of proneural proteins and Sens also play a critical role in SOP selection. In those areas of the proneural field in which Sens and proneural protein levels are low, not only is the transcriptional synergy absent, but there is also a weak repression of proneural gene expression. This should lead to a rapid loss of Sens expression and a failure to adopt the SOP fate. Analysis of the Sc expression pattern in sens clones that include the wing margin confirms that in the absence of Sens function, the broad Sc expression in the wing margin persists, and at the same time, the presumptive SOPs fail to up-regulate Sc protein. This is further supported by the observation that overexpression of low levels of Sens causes bristle loss in the wing margin (Jafar-Nejad, 2003).

The mechanism by which Sens represses transcription of proneural genes is probably through DNA binding. When the S box is mutated, Sens is unable to repress ac transcription. This finding is corroborated with in vivo observations that the ac minigene with the mutated Sens-binding site is a more potent inducer of bristle formation than the wild-type minigene. It is therefore concluded that the transcriptional repression of the ac promoter by Sens is mediated through DNA binding (Jafar-Nejad, 2003).

Altogether, these data support a model in which Sens promotes the SOP fate in one cell by activating ac transcription, whereas it prevents SOP fate in the neighboring cells by repressing ac transcription. The relative levels of Sens, proneural, and E(spl) proteins seem to be the major determinants of these fate decisions. Therefore, it is proposed that Sens acts as a binary switch in SOP determination by affecting a series of interconnected positive and negative regulatory loops to refine the potential for a specific fate from a group of cells to a single cell, the SOP (Jafar-Nejad, 2003).

Post-transcriptional Regulation

Cell-cell interactions mediated by the Notch receptor play an essential role in the development of the Drosophila adult peripheral nervous system (PNS). Transcriptional activation of multiple genes of the Enhancer of split Complex [E(spl)-C] is a key intracellular response to Notch receptor activity. Most E(spl)-C genes contain a novel sequence motif, the K box (TGTGAT), in their 3' untranslated regions (3' UTRs). Three lines of evidence are presented that demonstrate the importance of this element in the post-transcriptional regulation of E(spl)-C genes: (1) K box sequences are specifically conserved in the orthologs of two structurally distinct E(spl)-C genes (m4 and m8) from a distantly related Drosophila species; (2) the wild-type m8 3' UTR strongly reduces accumulation of heterologous transcripts in vivo, an activity that requires its K box sequences, and (3) m8 genomic DNA transgenes lacking these motifs cause mild gain-of-function PNS defects and can partially phenocopy the genetic interaction of E(spl)D with Notchspl. Although E(spl)-C genes are expressed in temporally and spatially specific patterns, K box-mediated regulation is ubiquitous, implying that other targets of this activity may exist. In support of this, sequence analyses are presented that implicate genes of the iroquois Complex (Iro-C) and the gene engrailed as additional targets of K box-mediated regulation (Lai, 1998a).

The 3' untranslated regions (3' UTRs) of Bearded, hairy, and many genes of the E(spl)-C contain a novel class of sequence motif, the GY box (GYB, GUCUUCC); extra macrochaetae contains the variant sequence GUUUUCC. The 3' UTRs of three proneural genes include a second type of sequence element, the proneural box (PB, AAUGGAAGACAAU). The full 13 nt PB is found once each in ac, l'sc, and ato, along with a second, variant version in both l'sc and ato. The presence of these motifs in such distantly related paralogs as hairy and certain bHLH genes of the E(spl)-C (for the GYB), and ato and two genes of the AS-C (for the PB), indicates that both classes of sequence element are subject to strong selection. Furthermore, both the PB and the GYB are conserved in the orthologs of ac and E(spl)m4 from the distantly related Drosophilids D. virilis and D. hydei, respectively, though these 3' UTRs are otherwise quite divergent from their D. melanogaster counterparts. These findings strongly suggest functional roles for both of these sequence elements (Lai, 1998b).

Intriguingly, the central 7 nt of the PB and the GYB are exactly complementary, and are often located within extensive regions of RNA:RNA duplex predicted to form between PB- and GYB-containing 3' UTRs. Indeed, using in vitro assays, RNA duplex formation has been observed between the ato/Brd and ato/m4 3' UTR pairs that is PB- and GYB-dependent. It is noteworthy that the predicted duplex interactions involving the GYB of Brd are significantly stronger than those involving the GYBs of the other transcripts. For example, Brd and ato are perfectly complementary over 18 contiguous nucleotides. This difference in the degree of PB:GYB-associated complementarity is likely to have functional consequences (Lai, 1998b).

In C. elegans, small antisense RNAs encoded by lin-4 mediate translational repression of lin-14 and lin-28 transcripts by binding to complementary sequences in their 3' UTRs. In Drosophila, PB- and GYB-bearing transcripts may likewise participate in a regulatory mechanism mediated by RNA:RNA duplexes, but with the feature that both partners are mRNAs that also direct the synthesis of functionally interacting proteins. The opportunity to form such duplexes clearly exists, since transcripts from proneural genes and their regulators very frequently accumulate in coincident or overlapping patterns. Moreover, while 7 nt is the minimum length of complementarity between any PB and any GYB, the longest possible uninterrupted duplex between a given GYB-bearing transcript and a given proneural partner is almost always considerably longer (8-12 nt). It is worth noting that in a lin-4/lin-14 duplex that has been shown to be sufficient for proper regulation in vivo, the longest region of uninterrupted complementarity is only 7 nt (Lai, 1998b and references therein).

The formation of the postulated RNA duplexes may serve to regulate proneural gene function, consistent with the known roles of hairy, emc, and the bHLH genes of the E(spl)-C. This might explain occasional C-to-U transitions in the GYB sequence (in emc and D. hydei m4); these variants retain complementarity with the PB due to G:U base-pairing. It is equally plausible that GYB-containing transcripts are regulated by duplex formation. A third very interesting possibility is that RNA:RNA duplexes formed between PB- and GYB-containing transcripts function to initiate a downstream regulatory activity affecting as-yet-unknown targets. Ample precedent exists establishing the trans-regulatory potency of double-stranded RNA. In any case, the apparent capacity of transcripts from the proneural genes and their regulators to form duplexes in their 3' UTRs suggests further complexity in the already complex regulatory interactions that control Drosophila neurogenesis (Lai, 1998b).

Micro RNAs are a large family of noncoding RNAs of 21-22 nucleotides whose functions are generally unknown. A large subset of Drosophila RNAs has been shown to be perfectly complementary to several classes of sequence motif previously demonstrated to mediate negative post-transcriptional regulation. These findings suggest a more general role for micro RNAs in gene regulation through the formation of RNA duplexes (Lai, 2002).

A new strategy of gene regulation was defined by the activities of Caenorhabditis elegans let-7 and lin-4. These RNA molecules of 21-22 nt are complementary to the 3' untranslated regions (UTRs) of target transcripts and mediate negative post-transcriptional regulation through RNA duplex formation. Several recent reports now reveal that a large family of RNAs of 21-22 nt, collectively termed micro RNAs (miRNAs), exists in organisms as diverse as worms, flies and humans. Although it was presumed that many of these new miRNAs would also act in post-transcriptional gene regulation, initial searches did not reveal obvious targets based on sequence complementarity (Lai, 2002).

In Drosophila, two 3'-UTR sequence motifs, the K box (cUGUGAUa) and the Brd box (AGCUUUA) mediate negative post-transcriptional regulation. Although originally identified in the 3' UTRs of Notch pathway target genes encoding basic helix-loop-helix (bHLH) repressors and Bearded family proteins, modes of regulation mediated by both motifs are spatially and temporally ubiquitous. This suggests that at least some of the many other Drosophila transcripts that contain K boxes or Brd boxes in their 3' UTRs are also actively regulated by these motifs. Since RNA-binding proteins typically show relatively relaxed binding specificities, it was hypothesized that an RNA component might be involved in recognition of these highly constrained motifs. This was bolstered by the finding that another motif common to the 3' UTRs of many of the same Notch pathway target genes, the GY box (uGUCUUCC), is complementary to and mediates RNA duplex formation through the proneural box (AUGGAAGACAAU), a motif located in the 3' UTRs of transcripts encoding proneural bHLH activators, (Lai, 2002).

Drosophila miRNAs encoded by 11 of 21 distinct genomic miRNA loci are complementary to the K box at their 5' end, with all but miR-11 having a perfect (8/8) antisense match to the extended K box consensus (UAUCACAG). Notably, the most 5' nucleotide of miR-11 is a cytosine residue, making it complementary to the second most common nucleotide at this position in identified K boxes. In addition, perfect antisense matches to the Brd box and GY box were found at the 5' ends of fly miR-4 and fly miR-7, respectively. The precise complementarity of these miRNAs to K box, Brd box and GY box motifs suggests that they bind these sequences in 3' UTRs and, in the case of the former two motifs, mediate negative post-transcriptional regulation. Complementarity between miRNAs and 3' UTRs extends beyond core sequence motifs in many cases, providing additional support for the existence of the proposed RNA duplexes. Examples exist of extended miRNA complementarity to 3' UTRs containing K boxes, Brd boxes and GY boxes. Complements to all three sequence motifs are located exclusively at the 5' ends of miRNA, suggesting that some aspect of regulation may be shared by these different miRNAs. For example, a common factor might be involved in the recognition or stabilization of these short miRNA-3' UTR duplexes (Lai, 2002).

Several miRNAs complementary to K boxes (miR-11 and the miR-2b and miR-13 subfamilies) are broadly expressed throughout Drosophila development, consistent with their proposed involvement in temporally ubiquitous regulation mediated by K boxes; the GY box-complementary miRNA miR-7 is similarly broadly expressed during development. The expression of the single identified Brd box-complementary miRNA miR-4 is restricted to embryogenesis. However, since the search for miRNAs has not yet been saturating, other miRNAs complementary to Brd boxes that are expressed later in development might yet be found (Lai, 2002).

The regulatory role of the K box and Brd box in other organisms has not yet been tested. Nevertheless, the presence of their complements in worm and human miRNAs suggests that these modes of regulation have potentially been conserved. Notably, the complements to these motifs are also located specifically at the 5' ends of miRNA. The restricted location of complements in these different species further suggests that the regulatory targets of many other miRNAs will be determined by the sequence of their 5' ends. In agreement with this idea, most of the known lin-4 and let-7 target sequences also involve perfect complements with the 5' ends of these miRNAs. Systematic searches for the complements of other 5' miRNA ends in 3' UTRs may therefore identify new post-transcriptional regulatory sequence elements. It should be noted, however, that despite the existence of three conserved sites in the lin-14 3' UTR that include perfect complements to lin-4, normal regulation of lin-14 actually depends on variant lin-4 binding sites containing a bulged nucleotide in the 5' complementary region. Thus, this rule is probably not absolute (Lai, 2002).

Initially, miRNAs are transcribed as RNAs of approximately 70 nt containing a stem-loop structure; these are cleaved by the RNAse III enzyme Dicer to generate the mature miRNA. Curiously, only a single strand of the duplex precursor stem structure is generally stable and is recovered as miRNA. The model proposed here may help to explain this phenomenon, since the strand that is complementary to these identified 3' UTR motifs is nearly exclusively the one that is isolated as miRNA. The single exception is miR-5, whose sequence contains a K box. Notably, miR-5 and the K box-complementary miRNAs miR-6-1,2,3 (whose loci are incidentally located next to each other in the genome) are complementary at 20 of 21 continuous nucleotide positions. This suggests that miR-5 might influence or possibly interfere with the ability of miR-6-1,2,3 to interact with 3' UTRs that contain K boxes (Lai, 2002).

Negative regulation by K box- and Brd box-complementary miRNA must differ from lin-4-mediated regulation, because K boxes and Brd boxes have significant, though distinct, effects on both transcript stability and translational efficiency, whereas lin-4 is thought to act at a step following translational initiation. The GY box does not seem to have a strong effect at the cis-regulatory level. Other miRNAs may show additional regulatory capacities; efforts are underway to understand the different molecular mechanisms of regulation mediated by miRNA-3' UTR RNA duplexes (Lai, 2002).

Drosophila CK2 regulates lateral-inhibition during eye and bristle development by targeting E(spl) repressors

Lateral inhibition is critical for cell fate determination and involves the functions of Notch (N) and its effectors, the Enhancer of Split Complex, E(spl)C repressors. Although E(spl) proteins mediate the repressive effects of N in diverse contexts, the role of phosphorylation has been unclear. This study implicates a common role for the highly conserved Ser/Thr protein kinase CK2 during eye and bristle development. Compromising the functions of the catalytic (α) subunit of CK2 elicits a rough eye and defects in the interommatidial bristles (IOBs). These phenotypes are exacerbated by mutations in CK2 and suppressed by an increase in the dosage of this protein kinase. The appearance of the rough eye correlates, in time and space, to the specification and refinement of the ‘founding’ R8 photoreceptor. Consistent with this observation, compromising CK2 elicits supernumerary R8’s at the posterior margin of the morphogenetic furrow (MF), a phenotype characteristic of loss of E(spl)C and impaired lateral inhibition. Compromising CK2 elicits ectopic and split bristles. The former reflects the specification of excess bristle SOPs, while the latter suggests roles during asymmetric divisions that drive morphogenesis of this sensory organ. In addition, these phenotypes are exacerbated by mutations in CK2 or E(spl), indicating genetic interactions between these two loci. Given the centrality of E(spl) to the repressive effects of N, these studies suggest conserved roles for this protein kinase during lateral inhibition. Candidates for this regulation are the E(spl) repressors, the terminal effectors of this pathway (Bose, 2006).

Neurogenesis reflects the outcome of a complex balance between the activities of transcription factors that favor this cell fate (ASC/Ato) and those that oppose it (E(spl)). It is increasingly apparent that formation of the eye and bristle are predicated on a similar mechanistic framework, even though the proneurals that participate in these two developmental programs are distinct. For example, the PNCs in the eye (the R8 cell) require ato, while those in the bristle (macrochaetes and IOBs) require ASC. Nevertheless, one common feature of the resolution of PNCs in the eye and the bristle is the centrality of E(spl)C, since loss of E(spl)C leads to exaggerated neurogenesis in both contexts. In the eye it leads to excess R8’s, rough eyes, and duplicated IOBs, while in the bristles this manifests as ectopic, split and missing bristles. Extensive analyses have identified the genes involved with these developmental programs, the feedback loops that reinforce proneural expression in R8’s/SOPs, and the role of E(spl)C for lateral inhibition. In contrast, it has remained unclear how phosphorylation contributes to the dynamics of this process (Bose, 2006).

It has been thought that transcription of E(spl) is, by itself, necessary and sufficient for lateral inhibition. This model emerged from studies on bristle development, where ectopic E(spl) proteins extinguished the SOPs, whereas loss of E(spl) favored this cell fate. This model needs qualification because similar outcomes have not been recapitulated in the eye. In this context, loss of E(spl) demonstrably compromises lateral inhibition and elicits excess R8’s. However, ectopic expression of E(spl) members does not block the R8 fate, and consequently the eye displays the normal hexagonal packing of the ommatidia; the only defect is loss of the IOBs whose developmental program bears similarities to that of the macrochaetes. In contrast, R8 formation is blocked by the truncated M8* protein encoded by the E(spl)D allele, or by the CK2 phophomimetic variant M8SD. It is important to note that the eye defect of E(spl)D requires Nspl, a recessive allele that attenuates ato, but not E(spl), expression. The inability of M8* to recruit Gro, which compromises repression, thus necessitates a sensitized background, one conferred by Nspl. Accordingly, M8SD (which binds Gro) elicits eye defects independent of Nspl (Karandikar, 2004). Based on the observation that both M8* and M8SD display exacerbated and equivalent interactions with Ato, it was proposed that CK2 phosphorylation switches M8 into an active repressor by uncovering the Orange domain, and it is this regulation that is bypassed by the E(spl)D mutation (Karandikar, 2004). Given that the Orange domain mediates binding to other proneurals as well, this regulation by CK2 should have been more general to lateral inhibition. These studies suggest just such a role in the eye and the bristle (Bose, 2006).

This work supports the notion that CK2 is a participant in lateral inhibition. Compromising CK2 by a number of independent routes, i.e., in wild type and backgrounds mutant for CK2 and E(spl), elicits neural defects in the eye and bristle. These include rough eyes due to the specification of excess 'founding' R8 cells, and ectopic bristles (macrochaetes and IOBs) due to the specification of excess SOP’s. These phenotypes are hallmarks of impaired lateral inhibition, and have been previously described for loss of function of the E(spl)C. Evidence is provided for genetic interactions between CK2 and E(spl). While these studies provide multiple lines of evidence, the absence of suitable antibodies have precluded a formal demonstration that E(spl) repressors are, in fact, phosphorylated in cells undergoing lateral inhibition. Nevertheless, the congruence of the results utilizing CK2-RNAi or CK2-DN in conjunction with extant mutants and cell fate in imaginal discs, together, constitute a plausible argument supporting a role for this protein kinase (Bose, 2006).

These studies also suggest secondary roles for CK2 in the bristle lineage. In contrast to R8 patterning, the roles of N and E(spl) are different during bristle morphogenesis. In the case of the macrochaete or the IOB, N and E(spl) are re-deployed following SOP selection. Specifically, the SOP gives rise to the pI neuroblast that undergoes two asymmetric divisions to generate four cell types characteristic of the sensillum; socket, shaft, sheath and neuron, and these divisions are dependent on N- and E(spl)-inhibitory signaling. Thus loss of E(spl) following SOP selection manifests as split bristles (aberrant division of the pIIa cell) or missing bristles (aberrant division of the pI cell). The split bristles described thus suggest a role for CK2 during the socket-to-shaft sister cell fate. In contrast, while the missing bristles suggest a role for CK2 during the pIIa-vs-pIIb fates, this phenotype could result from loss of the SOP itself, a possibility if CK2 levels become rate limiting for cell division. Despite the fact that the timing of the asymmetric divisions of pI, pIIa and pIIb are well known, CK2-RNAi or CK2-DN are not suitable for dissecting the roles of CK2 at these later steps of bristle development. Conditional alleles of CK2, e.g., temperature-sensitives, will be necessary to better define its roles during specification of these sister-cell fates. One major question that emerges from these studies is why is phosphorylation necessary, given that not all members of the E(spl)C are targets of CK2. It is thought that evolutionary principles, the diversities and/or affinities of interactions between E(spl)C and ASC/ato, and their spatial expression patterns, perhaps, offer insights (Bose, 2006).

Of the seven E(spl) proteins, three (M8, M5, and M7) are targeted by CK2, and these are also the most closely related. Among all E(spl) members, two regions largely account for length heterogeneity and divergence. These are sequences between HLH and Orange and those between Orange and WRPW, the CtD. However, within the CtD of M8 (and M5 and M7 as well) is a highly invariant sequence, the phosphorylation domain (P-domain) that harbors the CK2 site. Given the phylogenetic relationships of these species, it is noteworthy that over a period of ~50 million years the P-domain and the CK2 site have been remarkably conserved. For example, of all M8 homologs, only D. pseudoobscura, D. grimshawii and D. hydei harbor a Glu residue, in place of Asp, at the n + 3 position of the CK2 phosphoacceptor. While it has not been experimentally confirmed that these homologs are phosphorylated, the possibility is high because this change still conforms to the consensus (S/T-D/E-x-D/E) for recognition by CK2. The virtually identical consensus site that is present in mammalian Hes6 is targeted by CK2 (Gratton, 2003) in vivo (Bose, 2006).

The mechanisms by which E(spl) proteins mediate repression have been intensely studied. In essence, E(spl) proteins repress ASC/Ato. Repression was initially thought to involve binding to a DNA sequence, the N-box. This, however, is not the case, because E(spl) proteins neutralized for DNA-binding still function as potent repressors. Furthermore, no N-box has been found in the regulatory region of ato, while that in sc is dispensable for repression in non-SOPs. It is now thought that direct (protein–protein) interactions between E(spl) and proneurals are more critical for repression, the protein–tether model (Giagtzoglou, 2003). In this model, repression by E(spl) occurs via direct interactions with enhancer bound proneurals, rather than by activator sequestration. This model is consistent with direct interactions between E(spl) and ASC/Ato proteins. It was, in fact, the analyses of various binary combinations that were the first to suggest that these interactions are regulated and non-redundant, two aspects that appear relevant to the current findings (Bose, 2006).

Analysis of M8 and its E(spl)D encoded variant, M8* provided the first hint that these antagonistic interactions are regulated. For example, it has been reported that, in addition to Ato, M8* interacts with a much higher affinity with Ac, Sc, and Ase. A similar case is described for M8SD, which interacts with Ato or L’sc with affinities significantly higher than M8 or its non-phosphorylatable variant M8SA (Karandikar, 2004). It is noteworthy that phosphorylation of mammalian Hes6 by CK2 is also a pre-requisite for its interactions with Hes1 (Gratton, 2003). Thus CK2 phosphorylation influences antagonistic interactions between the E(spl) and the ASC/Ato. Because these studies have employed two hybrid, instead of direct protein, approaches the possibility that these are kinetic effects remains open. This interpretation is consistent with the observations that a 2× dosage of a UAS-mδ construct interferes with Ato and blocks eye development in the wild type, whereas that of m7, m5 or m8 requires Nspl. Together, these findings argue that E(spl)-ASC interactions are of variable strengths and are isoform-specific. Given that only a subset of E(spl) and ASC members are expressed in the eye and wing disc, the possibility thus arises that distinct domains of ASC define sub-regions of the proneural field. In this context, E(spl) members might have been selected based on their affinities and/or specificities for these proneural factors. Thus the type of E(spl) repressors that are deployed might reflect the combinations and levels of proneurals, with CK2 playing an integrative role. The currently available techniques preclude a distinction between these possibilities (Bose, 2006).

It is presently unclear if/how CK2 activity is modulated during neurogenesis. Expression of this enzyme appears to be constitutive in the eye and wing disc (Karandikar, 2004). Holoenzyme formation, proposed to be a dynamic process in vivo, represents an attractive regulatory mechanism, given that CK2β modulates substrate recognition and that the fly CK2β gene encodes for non-redundant isoforms of this regulatory subunit. Alternatively, CK2 might be regulated by assembly into multiprotein complexes and/or via interactions with protein phosphatases. Such a coordinated function has been described for regulation of Period, the central component of the circadian clock, by CK2 and the phosphatase PP2A. Future studies aimed at the identification of protein phosphatase(s) that counteract the phosphorylation of E(spl)m8/5/7 by CK2, or multiprotein complexes containing E(spl) and/or CK2 will be required to better define the regulatory dynamics of this process during eye and bristle development (Bose, 2006).

Enhancer of split: Biological Overview | Evolutionary Homologs | Regulation | Targets of Activity | Developmental Biology | Effects of Mutation | References

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