Protein Interactions and Post-transcriptional Regulation

Achaete and Scute form heterodimers with Daughterless, which is expressed ubiquitously (van Doren, 1994 and Cabrera, 1994).

Extra machrochaetae forms heterodimers with Daughterless, Achaete and Scute, and negatively antagonizes them (van Doren, 1994 and Cabrera, 1994), by preventing their binding to DNA.

The simultaneous reduction in the levels of Dorsal and any one of several helix-loop-helix (HLH) proteins results in severe disruptions in the formation of mesoderm and neuroectoderm. The area of twist and snail expression in the presumptive mesoderm is severely reduced in dl-da double heterozygotes. The same is true in dorsal ac-sc double heterozygotes. Certain triple heterozygous combinations essentially lack mesoderm as a result of a block in ventral furrow formation during gastrulation. HLH proteins that have been implicated previously in sex determination and neurogenesis (daughterless, achaete, and scute) are required for the formation of these embryonic tissues. Evidence suggests that DL-HLH interactions involve the direct physical association of these proteins in solution mediated by the rel and HLH domains (Gonzalez-Crespo, 1993).

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).

The GATA factor Pannier activates the achaete-scute (ASC) proneural complex through enhancer binding and provides positional information for sensory bristle patterning in Drosophila. Chip acts as a cofactor of the dorsal selector Apterous, and both Apterous and Chip also regulate ASC expression. Chip cooperates with Pannier in bridging the GATA factor with the HLH Ac/Sc and Daughterless proteins to allow enhancer-promoter interactions, leading to activation of the proneural genes, whereas Apterous antagonizes Pannier function. Within the Pannier domain of expression, Pannier and Apterous may compete for binding to their common Chip cofactor, and the accurate stoichiometry between these three proteins is essential for both proneural prepattern and compartmentalization of the thorax (Ramain, 2000).

Pnr is a member of the GATA-1 family of transcription factors and activates proneural function by binding to the dorsocentral (DC) enhancer located 4 kb and 30 kb upstream of ac and sc, respectively. Reported in this study is the characterization of ChipE, a viable allele of Chip, that interacts with pnr genetically. ChipE mutants show reduced ac-sc expression in the DC, associated with loss of DC bristles, and produce a phenotype similar to that of loss-of-function pnr alleles. This genetic interaction correlates with a physical interaction between Chip, Pnr, and the bHLH heterodimers (Ac/Sc-Da). Pnr interacts with the N terminus of Chip through its COOH terminus encompassing two helices that are conserved between D. melanogaster and D. virilis and that are probably both involved in protein-protein interactions. Chip dimerizes with the bHLH heterodimers through its C-terminal LID, known to mediate heterodimerization with LIM-containing proteins (Ramain, 2000).

In vertebrates, the Ldb1/NLI protein associates with GATA-1, Lmo2, and the bHLH E47, Tal1/SCL in an erythroid complex whose function is poorly understood. A similar Drosophila complex functions in vivo to regulate the ac/sc genes directly during establishment of the proneural prepattern.

Accurate coexpression of ac/sc is mediated by Pnr binding to the DC. The ac and sc promoters include E boxes that are targets for the Ac/Sc-Da heterodimers and support autoregulation during development. In cultured chicken embryonic fibroblast (CEF) cells, Pnr and the Ac/Sc-Da heterodimer activate expression of a CAT reporter linked respectively to the DC enhancer and to the ac promoter. Physical interactions between Pnr and the bHLHs lead to synergistic activation of the reporter when the regulatory sequences are associated. Pnr and the Ac/Sc-Da heterodimers are both required in flies for expression of a LacZ reporter linked to the promoter associated with the enhancer, but the analysis of ChipE shows that Chip is also required for full activation in vivo. The interactions between Pnr and the bHLH mediated by Chip suggest that Pnr might also be involved in autoregulation. Interestingly, Chip interacts with Ac/Sc through the Ac/Sc bHLH domains, and it has been shown that the overexpression of a homologous bHLH domain is sufficient to mediate the proneural function of Ac/Sc (Ramain, 2000).

Chip has been identified in a genetic screen for mutations that reduce activity of the wing margin enhancer of the cut locus, and it has been proposed that Chip may act as a bridge allowing enhancer-promoter communications. Thus, if the flies have a unique functional Chip allele, they display a cut margin phenotype, and this effect is observed exclusively when they carry a gypsy insertion between the enhancer and the promoter on one chromosome. It has been proposed that binding of the Su(Hw) insulator protein to the gypsy insertion blocks communication on the mutant chromosome, thereby interfering with the functioning of the wild-type homolog. The interchromosomal insulation is detectable when Chip activity is reduced, and Chip and Su(Hw) are antagonistic to each other, suggesting that Chip may be a facilitator target of Su(Hw) (Ramain, 2000).

The ChipE mutation specifically disrupts interactions with the bHLH and strongly affects the expression of a LacZ reporter linked to the ac promoter/DC enhancer in flies, suggesting that Chip also mediates enhancer-promoter communication in the ASC. Further evidence is provided by the Hw1 mutant. Hw1 carries a gypsy insertion within ac such that sc, which is located further downstream from the DC enhancer, is no longer expressed. In addition, the removal of the gypsy insulator largely restores sc expression in the DC proneural cluster (Ramain, 2000).

Thus, a complex containing Pnr, Chip, and the Ac/Sc-Da heterodimer activates ac-sc expression, and its function is antagonized by Ush, Ap, and Emc. Ush and Emc dimerize respectively with Pnr and the HLH Ac/Sc. The repressing effect of Ap may reflect its ability to interact with Chip, thereby depriving Pnr of its essential cofactor. Alternatively, Ap may weaken the enhancer activity of Pnr. Thus, Ap may compete directly with Chip for binding to Pnr (Ramain, 2000).

Within the domain of Pnr expression, Ap and Pnr compete for binding to their common Chip cofactor. Ap activity is mediated by a Chip dimer, whereas activation of ac-sc by Pnr requires a Chip monomer. Chip acts as a bridge between the Ac-Da heterodimer bound to the E boxes of the ac promoter and Pnr bound to the GATA sites of the DC enhancer. The activity of the resulting complex is antagonized by Ush and Emc, which negatively regulate Pnr and Ac/Sc functions during development. The repressing effect of Ap is mediated either by dimerization of Ap with Chip and/or Pnr or by Chip-assisted binding of Ap to sites located between the DC enhancer and the ac promoter. Additional cofactors, such as dLMO, may participate in this complex (Ramain, 2000).

Chip is required in flies for ASC activation, whereas it appears dispensable in CEF cells. This observation may reflect the nature of the reporter used in the transfection experiments where the DC enhancer is close to the ac promoter and poorly mimics the genomic organization of the ASC, where the DC enhancer has to regulate ac and sc simultaneously. Furthermore, the chromatin structure and its modifications associated with gene expression are probably not reproduced in the transient expression assay. Thus, ASC expression in flies may require additional coactivators recruited by Chip, including chromatin remodeling factors. Moreover, the activation of ac/sc probably requires the assembly of a higher-order nucleoprotein complex containing multiple transcription factors (enhanceosome), and Chip may allow the correct positioning of Pnr and the Ac/Sc-Da heterodimer in this structure (Ramain, 2000).

ChipE mutants affect the scutellar and dorsocentral bristles in opposite fashions. It will be of interest to compare the regulation of the activity of the corresponding enhancers by Chip and Pnr (Ramain, 2000).

It has been proposed that appropriate combinations of proteins represent the positional cues that activate a given enhancer of the ASC complex. The disc is divided in large territories, but almost nothing is known concerning how these territories are further subdivided or how the positional information revealed by the accurate ac-sc expression is created. The present study provides a link between the spatial regulation of the proneural genes and the compartmentalization of the disc. ac-sc expression is stimulated by a complex containing the prepattern factor Pnr, Chip, and the bHLH proteins Ac/Sc and Da. Chip is an essential cofactor of the dorsal selector Ap, and these interactions coordinate the spatial transcription of the proneural genes. Ap is expressed specifically in the dorsal compartment of the wing pouch, and the juxtaposition of Ap-expressing with Ap-nonexpressing cells defines the dorsal/ventral organizing boundary where wingless (wg) expression is induced. On the thorax, ap and Chip are ubiquitously expressed, whereas wg expression occurs in a stripe straddling the lateral border of the domain of pnr expression. Moreover, Pnr activates wg. Pnr associates with Chip, and the domain of pnr expression appears devoid of Ap activity. As a consequence, this domain may define a boundary between a region devoid of Ap activity and a region where Ap is active. Alternatively, Pnr may associate with Ap, and the resulting heterodimer may regulate wg. Further studies will help to resolve this issue (Ramain, 2000).

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).

E(spl) proteins interact selectively with proneural ones in a yeast two-hybrid assay; E(spl)m7 and mgamma interact with Ac, Sc and Da, whereas mdelta interacts with none. Tests with mutant E(spl) proteins indicate that some activity of E(spl) proteins other than their direct DNA binding ability is most important in target gene repression. In the light of these results, it is possible that the ability of E(spl) proteins to interact with activator bHLH proteins might underlie the ability of the former to repress target genes in the absence of direct DNA binding and enhance their potency in neural fate suppression. The question arises as to how interaction with proneural proteins might help realize this potent repressive activity: do E(spl) proteins sequester proneural activators off the target DNA or do they use the proneural complexes as tethers to bind to DNA? A way to approach the question of whether a repressor works on or off DNA uses a fusion of a strong transcriptional activation domain (VP16) to a repressor; this is tested for its ability to activate transcription, which can only happen if the VP16 domain is tethered to the DNA. If, however, the repressor works by sequestering activators off DNA, the VP16-tagged repressor should still be able to repress (rather than activate) target genes (Giagtzoglou, 2003).

A hybrid E(spl)m7VP16 protein was expressed in wing disks and its effect on EE4-lacZ was assayed. In both pnr-Gal4 and omb-Gal4 expression domains, strong activation of EE4-lacZ was observed, suggesting that E(spl)m7VP16 is somehow tethered to this artificial enhancer. Rather than being ubiquitous, activation by E(spl)m7VP16 was patterned in a way that strongly resembles the proneural pattern, suggesting that E(spl)m7VP16 is tethered to EE4-lacZ via proneural complexes. To demonstrate this, the same effector-reporter combination was assayed in both loss-of-function and gain-of-function backgrounds for proneural genes. sc10-1 is a null allele for both ac and sc, the only proneural proteins expressed in the wing disk. In sc10-1 wing disks, EE4-lacZ was not expressed and could not be activated by E(spl)m7VP16. In the converse experiment, ectopic Sc was supplied by co-expressing UAS-sc with UAS-m7VP16; in this case, the pattern of EE4-lacZ activation was broadened to encompass the whole expression domain and was not restricted to proneural clusters. It therefore appears that it is the availability and spatial distribution of proneural proteins that determines the pattern of activation of EE4-lacZ by E(spl)m7VP16. The simplest way to account for this finding is to propose that E(spl)m7VP16 is recruited onto DNA using the proneural complexes (and not some other DNA-bound factor) as tethers. This was confirmed by testing the ability of two other E(spl)VP16 variants: E(spl)mgammaVP16 and mdeltaVP16. Whereas the former behaves identically to E(spl)m7VP16, E(spl)mdeltaVP16 has no effect on EE4-lacZ expression. The inability of E(spl)mdeltaVP16 to become recruited onto EE4-lacZ is attributed to its inability to interact with the proneural protein-tethering factors (Giagtzoglou, 2003).

Enhancer-promoter communication mediated by Chip during Pannier-driven proneural patterning is regulated by Osa

The GATA factor Pannier activates proneural achaete/scute (ac/sc) expression during development of the sensory organs of Drosophila through enhancer binding. Chip bridges Pannier with the (Ac/Sc)-Daughterless heterodimers bound to the promoter and facilitates the enhancer-promoter communication required for proneural development. This communication is regulated by Osa, which is recruited by Pannier and Chip. Osa belongs to Brahma chromatin remodeling complexes, and this study shows that Osa negatively regulates ac/sc. Consequently, Pannier and Chip also play an essential role during repression of proneural gene expression. This study suggests that altering chromatin structure is essential for regulation of enhancer-promoter communication (Heitzler, 2003).

ChipE is a viable allele of Chip that is associated with a point mutation in the LIM-interacting domain (LID), which specifically reduces interaction with the bHLH proteins Ac, Sc, and Da. As a consequence, the ChipE mutation disrupts the functioning of the proneural complex encompassing Chip, Pnr, Ac/Sc, and Da. A homozygous ChipE mutant shows thoracic cleft and loss of the DC bristles, similar to loss of function pnr alleles (Heitzler, 2003).

To identify new factors that regulate this proneural complex, a screen was performed for second-site modifiers of the ChipE phenotypes. One allele of osa (osaE17) was found among the putative mutants. OsaE17 corresponds to a loss-of-function allele, and homozygous embryos die with normal cuticle patterning. Both osaE17 and null alleles of osa (osa616 or osa14060) enhance the cleft but suppress the loss of DC bristle phenotypes of ChipE flies. Indeed, ChipE flies with only one copy of osa+ (ChipE;osa616/+) are weak and sterile but show wild-type DC bristle pattern (Heitzler, 2003).

These genetic interactions suggest that Osa can antagonize the function of Pnr. Moreover, overexpressed Osa (+/UAS-osa;Gal4-pnrMD237/+) induces a thoracic cleft and the loss of DC bristles similar to the loss-of-function pnr alleles. In contrast, loss-of-function osa alleles display an excess of DC bristles similar to overexpressed Pnr. For example, (osa14060/+), (osa616/+), and (osaE17/+) flies exhibit respectively 2.35 ± 0.12, 2.38 ± 0.12, and 2.43 ± 0.17 DC bristles per heminotum (Oregon wild-type flies have 2.00 DC bristles/heminotum). Furthermore, transallelic combination of osa14060 with the hypomorphic osa4H (osa4H/osa14060) accentuates the excess of DC bristles compared with (osa14060/+). (osa4H/osa14060) flies display 4.17 ± 0.19 DC bristles per heminotum. In contrast, (osa4H/osa4H) flies display 2.50 ± 0.11 DC bristles per hemithorax. The development of the extra DC bristles revealed by phenotypic analysis was compared with the positions of the DC bristle precursors detected with a LacZ insert, A101, in the neuralized gene that exhibits staining in all sensory organs. In (osa14060/osa4H) discs, additional DC precursors are observed that lead to the excess of DC bristles. The pnrD alleles encode Pnr proteins carrying a single amino acid substitution in the DNA binding domain that disrupts interaction with the U-shaped (Ush) antagonist. Consequently, PnrD constitutively activates ac/sc, leading to an excess of DC bristles. This excess is accentuated when osa function is simultaneously reduced (pnrD1/osa616) (Heitzler, 2003).

Since osa shows genetic interactions with trithorax group genes encoding components of the Brm complex like moira (mor) and brm, whether mutations in mor and brm suppress the ChipE phenotype was investigated. Loss of one copy of brm+ in (ChipE; brm2/+) flies suppresses the lack of DC bristles observed in ChipE flies, similar to loss of one copy of osa+. This shows that brm and osa both act during Pnr-dependent patterning, in agreement with the fact that they have been shown to be associated in the Brm complex. In contrast, reducing the amount of Mor by half [(ChipE;mor1/+) flies] is not sufficient to modify the ChipE phenotype. This does not definitely exclude the possibility that mor is directly or indirectly involved, via the Brm complex, in Pnr-dependent patterning (Heitzler, 2003).

The complete osa open reading frame of 2715 amino acids and the intronic splicing signals were PCR amplified from genomic DNA prepared from homozygous embryos (osaE17 and osa14060) and homozygous first instar larvae (osa4H). For osa14060 and osa4H, the sequence analysis revealed a single mutation in the N terminus that causes a glutamine to stop codon substitution. The conceptual translation of osa14060 leads to a truncated Osa protein lacking both functional domains, whereas Osa4H retains the ARID domain but lacks the C-terminal EHD. Wild-type osa function is necessary for patterning of the DC bristles. Although osaE17 behaves as a stronger allele than osa14060 and osa4H, molecular identity of the mutation is unknown. Hence, the osaE17 phenotype may result from a mutation in regulatory sequences that affects osa expression (Heitzler, 2003).

It has been shown that a complex containing Pnr, Chip, and the (Ac/Sc)-Da heterodimer activates proneural expression in the DC proneural cluster and promotes development of the DC macrochaetae. Osa and Pnr/Chip have antagonistic activities during development because loss of osa function (osa4H and osa14060) displays additional DC bristles. However, since the current study reveals that osa genetically interacts with pnr and Chip, it was asked whether Osa physically interacts with the Pnr and Chip proteins. Immunoprecipitations of protein extracts made from Cos cells cotransfected with expression vectors for tagged Osa and either Pnr or tagged Chip were immunoprecipitated. Because Osa is a large protein, several expression vectors encoding contiguous domains of Osa were used. Osa coimmunoprecipitates with Pnr and Chip and can be detected on Western blots with appropriate antibodies. The interactions appear to require the overlapping domains Osa E (His1733/Glu2550) and Osa F (Ala2339/Ala2715) corresponding to the EHD. Enhancer-promoter communication during proneural activation and development of the DC bristles requires regulatory sequences scattered over large distances and appears to be negatively regulated by interaction of Pnr and Chip with Osa through the EHD. Interestingly, the EHD is not conserved in yeast. In yeast, the UAS sequences are generally close to the promoter and there is no requirement for long-distance interactions. This observation could support the idea that the EHD is essential for long-distance enhancer-promoter communication. Alternatively, yeast may just lack proteins like Chip or Pnr (Heitzler, 2003).

The DNA-binding domain and the C-terminal region are essential for the function of Pnr during development of the DC sensory organs. The pnrVX1 and pnrVX4 alleles (collectively pnrVX1/4) are characterized by frameshift deletions that remove two C-terminal alpha-helices and result in reduced proneural expression and loss of DC bristles (Heitzler, 2003).

The molecular interactions between Osa and PnrD1 and between Osa and PnrVX1 were investigated. PnrD1 protein interacts with the EHD as efficiently as wild-type Pnr. In contrast, the physical interaction is disrupted when the C terminus of Pnr encompassing the alpha-helices is removed. Because the C terminus of Pnr is required for the Pnr-Osa interaction in transfected cells extracts, the abilities of in vitro translated 35S-labeled Osa domains to bind to GST-CTPnr attached to glutathione-bearing beads were investigated. Only Osa E and Osa F interact with the C terminus of Pnr. The interaction between Chip and Osa was examined and it was found that Osa associates with the N-terminal homodimerization domain of Chip, and Osa was found to be required for the interaction between Chip and Pnr. Furthermore, Osa E and Osa F also bind to immobilized GST-Chip. Deletion of the alpha helix H1 disrupts the interactions between Pnr and Osa. Interestingly, the same deletion also disrupts the interaction with Chip. Therefore, the functional antagonism between Chip and Osa during neural development may result from a competition between these proteins for association with Pnr. Alternatively, the deletion of H1 may affect the overall structure of the C terminus of Pnr and disrupt the physical interactions with Chip and Osa. To discriminate between these hypotheses, immunoprecipitations of protein extracts containing a constant amount of Pnr, a constant amount of the tagged Osa E domain, and increasing concentrations of Chip were performed. Pnr immunoprecipitates with immunoprecipitated tagged Osa E and the amount of Pnr immunoprecipitated increases in the presence of increasing concentrations of Chip. The presence of increasing amounts of Chip does not inhibit the Osa-Pnr interaction as would be expected if Osa and Chip were to compete for binding to Pnr. In contrast, it suggests that Chip and Pnr act together to recruit Osa and to target its activity and possibly the activity of the Brm complex to the ac/sc promoter sequences (Heitzler, 2003).

Using expression vectors encoding contiguous domains of Osa, it was shown that the EHD of Osa mediates interactions with Pnr and Chip. Because the EHD is lacking in the truncated Osa14060 and Osa4H, it is hypothesized that the loss of interaction with Pnr and Chip are responsible for the excess of DC bristles observed in osa4H and osa14060 (Heitzler, 2003).

To investigate whether these interactions between Osa, Pnr, and Chip function in vivo during DC bristle development, the effects of both loss of function and overexpression of osa were examined on the activity of a LacZ reporter whose expression is driven by a minimal promoter sequence of ac fused to the DC enhancer (transgenic line DC:ac-LacZ). It was found that expression of the LacZ transgene is increased in osa14060/osa4H wing discs when compared with the wild-type control. For overexpression experiments, the UAS/GAL4 system was used, using as a driver the pnrMD237 strain that carries a GAL4-containing transposon inserted in the pnr locus (driver: pnr-Gal4). This insert gives an expression pattern of Gal4 indistinguishable from that of pnr. It was found that overexpressed Osa leads to a strong reduction of LacZ staining in the DC area, consistent with the lack of DC bristles. Thus, overexpressed Osa represses activity of the ac promoter sequences required for DC ac/sc expression and development of the DC bristles. It has been previously reported that wingless expression is also required for patterning of the DC bristles. However, the repressing effect of Osa on development of the DC bristles is unlikely to be the result of an effect of Osa on wingless expression because overexpressed Osa driven by pnrMD237 has no effect on the expression of a LacZ reporter inserted into the wingless locus. Thus, Osa acts through the DC enhancer of the ac/sc promoter sequences to repress ac/sc and neural development (Heitzler, 2003).

ChipE disrupts the enhancer-promoter communication and strongly affects expression of the LacZ reporter driven by the ac promoter linked to the DC enhancer. Because null alleles of osa suppress the loss of DC bristles displayed by ChipE, the consequences of reducing the dosage of osa was examined in ChipE flies. The expression of the LacZ reporter is not affected in ChipE flies when Osa concentration is simultaneously reduced (Heitzler, 2003).

In conclusion, Pnr function during proneural patterning is regulated by interaction with several transcription factors. Pnr function is negatively regulated by Ush, which interacts with its DNA-binding domain. Chip associates with the C terminus of Pnr, bridging Pnr at the DC enhancer with the AC/Sc-Da heterodimers bound at the proneural promoters, thus activating proneural gene expression. The current study reveals that Pnr function can also be regulated by interaction with Osa. Thus, Osa activity is specifically targeted to ac/sc promoter sequences and the binding of Osa therefore has a negative effect on Pnr function, leading to reduced expression of the proneural ac/sc genes. Osa belongs to Brm complexes, which are believed to play an essential role during chromatin remodeling necessary for gene expression. For example, in vitro transcription experiments with nucleosome assembled human beta-globin promoters have shown that the BRG1 and BAF155 subunits of the mammalian SWI/SNF homolog are essential to target chromatin remodeling and promote transcription initiation mediated by GATA-1. In contrast to what was observed in vitro, the current results suggest that in vivo the SWI/SNF complexes can also act to remodel chromatin in a way that represses transcription. Alternatively, the observed repression of proneural genes may simply define a novel function of Osa, independent of chromatin remodeling (Heitzler, 2003).

Drosophila LIM-only is a positive regulator of transcription during thoracic bristle development

The Drosophila LIM-only (Lmo) protein DLMO functions as a negative regulator of transcription during development of the fly wing. This study reports a novel role of Dlmo as a positive regulator of transcription during the development of thoracic sensory bristles. New dlmo mutants, which lack some thoracic dorsocentral (DC) bristles, were isolated. This phenotype is typical of malfunction of a thoracic multiprotein transcription complex, composed of Chip, Pannier (Pnr), Achaete (Ac), and Daughterless (Da). Genetic interactions reveal that dlmo synergizes with pnr and ac to promote the development of thoracic DC bristles. Moreover, loss-of-function of dlmo reduces the expression of a reporter target gene of this complex in vivo. Using the GAL4-UAS system it was also shown that dlmo is spatially expressed where this complex is known to be active. Glutathione-S-transferase (GST)-pulldown assays showed that Dlmo can physically bind Chip and Pnr through either of the two LIM domains of Dlmo, suggesting that Dlmo might function as part of this transcription complex in vivo. It is proposed that Dlmo exerts its positive effect on DC bristle development by serving as a bridging molecule between components of the thoracic transcription complex (Zenvirt, 2008).

The results presented in this study uncover a novel role of Dlmo in regulation of the development of the thoracic DC bristles. Homozygous, or hemizygous, loss-of-function (dlmohdp) mutants lack the anterior pair of the DC bristles. Moreover, these dlmo mutants displayed genetic interactions with mutants in genes known to regulate DC bristle development, such as pnr and ac, to reduce the number of DC bristles. Consistently, overexpression alleles of dlmo (dlmoBx) also exhibited genetic interactions with these pnr and ac mutants, resulting in an increased number of bristles. In addition, the finding that overexpression of pnr under the regulation of dlmo-GAL4 affects DC bristle development suggests that dlmo is expressed in the region of the wing disc that gives rise to these bristles (Zenvirt, 2008).

These results suggest a role of Dlmo in positive regulation of transcription. The negative role of Dlmo in modulation of transcription during Drosophila wing development has been well documented. The findings indicate that in another context, namely in regulation of DC bristle development by the Chip, Pnr, Ac and Da (CPAD) complex, Dlmo has another role, as a positive regulator of transcription. Lowering the level of Dlmo (in dlmohdp mutants) results in a reduction in the expression of a reporter driven by regulatory sequences of a bona fide target gene of the CPAD transcription complex, suggesting that Dlmo is a positive regulator of CPAD-dependent transcription. While the mechanism by which Dlmo positively regulates transcription in the context of the CPAD complex remains to be elucidated, a first clue to this mechanism may lie in the finding that Dlmo can bind constituent proteins of this complex, including Pnr and Chip, in vitro. Should these interactions also take place in vivo, Dlmo may exert its positive role in transcriptional regulation as a component of the CPAD complex (Zenvirt, 2008).

Insights into the mechanism of positive transcriptional regulation by Dlmo can be gleaned from LMO2, one of the mammalian homologs of Dlmo. LMO2 was demonstrated to participate in a multiprotein transcription complex that contains Ldb1, a GATA factor (GATA-1 or GATA-2), and the bHLH transcription factors TAL1 and E2A, which are homologous, respectively, to the fly components of the CPAD complex, Chip, Pannier, Achaete, and Daughterless. Various lines of evidence indicate that in mammals LMO2 serves as a bridge between components of the complex, and silencing of LMO2 causes disruption of the complex and decreases in the activation of transcription of its target genes, just as does silencing of Ldb1 or Tal1. Similarly to LMO2, Dlmo might serve as a bridge between components of the CPAD complex. LIM domains are protein-interaction modules and could serve Dlmo to bind components of the CPAD complex. This suggestion is supported by the finding that each single LIM domain of Dlmo is capable of binding components of the CPAD complex in vitro, and it agrees with similar reports on other LIM-containing proteins. Notably, a single LIM domain from LMO2 and LMO4 is sufficient to interact with Ldb1 or the related protein CLIM-1a. However, both LIM domains are required for the highest-affinity interactions (Zenvirt, 2008).

This proposed mode of action of Dlmo, as a bridging molecule, which binds a different protein through each one of its LIM domains, predicts that a Dlmo molecule with one defective LIM domain and one intact LIM domain would bind only one protein at a time and not be able to bridge between molecules. Indeed, in new dlmo mutants it was found that deletions that span the second zinc finger of the second LIM domain of Dlmo, namely dlmohdp48-1 and dlmohdp185-1, resulted in dlmo loss-of-function mutations. These mutants display partial loss of thoracic DC bristles along with reduced expression of a target gene of the thoracic transcription complex. Interestingly, the wing size of these mutants is normal, unlike the small wings of mutants with lesions in the 5'-UTR of Dlmo, such as dlmohdp58-1, dlmohdp67-2, and dlmohdpR590. This may suggest that the defective Dlmo protein, which has only a single intact LIM domain, is sufficient for its function in the context of the wing, where Dlmo acts as a negative regulator that binds only one protein (CHIP), but is not sufficient when Dlmo acts as a bridging molecule in the thoracic CPAD transcription complex. Finally, the finding that Dlmo can bind other Dlmo molecules to generate homodimers or multimers might provide Dlmo with a greater flexibility of bridging between distant components of the complex. This possibility remains to be examined (Zenvirt, 2008).

In conclusion, Dlmo appears to have a dual role in regulation of transcription, depending on the context. Such a phenomenon has been documented for other transcription cofactors, whose dual function in transcription regulation varies according to their binding partners, the specific tissue, or the developmental stage. Likewise, these results indicate Dlmo has such a dual role, being a negative regulator with respect to the Ap-Chip complex and a positive regulator in the context of the CPAD complex (Zenvirt, 2008).

Proneural proteins Achaete and Scute associate with nuclear actin to promote external sensory organ formation

Basic helix-loop-helix (bHLH) proneural proteins promote neurogenesis through transcriptional regulation. Although much is known about the tissue-specific regulation of proneural gene expression, how proneural proteins interact with transcriptional machinery to activate downstream target genes is less clear. Drosophila proneural proteins Achaete (Ac) and Scute (Sc) induce external sensory organ formation by activating neural precursor gene expression. Through co-immunoprecipitation and mass spectrometric analyses, this study found that nuclear but not cytoplasmic actin associates with the Ac and Sc proteins in Drosophila S2 cells. Daughterless (Da), the common heterodimeric partner of Drosophila bHLH proteins, was observed to associate with nuclear actin via proneural proteins. A yeast two-hybrid assay revealed that the binding specificity between actin and Ac or Sc is conserved in yeast nuclei without the presence of additional Drosophila factors. It was further shown that actin is required in external sensory organ formation. Reduction in actin gene activity impaired proneural protein-dependent neural precursor gene expression as well as neural precursor formation. Furthermore, increased nuclear actin levels, by expression of nucleus-localized actin, elevated Ac/Da-dependent gene transcription as well as Ac-mediated external sensory organ formation. Taken together, these in vivo and in vitro observations suggest a novel link for actin in proneural protein-mediated transcriptional activation and neural precursor differentiation (Hsiao, 2013).

Post-transcriptional regulation

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, 1998).

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, 1998).

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, 1998 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, 1998).

achaete: Biological Overview | Evolutionary Homologs | Transcriptional regulation | Targets of activity | Developmental Biology | Effects of Mutation | References

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