Myocyte enhancer factor 2


REGULATION

Targets of Activity

Since nautilus is expressed later than Mef2, it does not appear to take a role in the activation of Mef2. Likewise, the initiation of nau expression does not require Mef2 (Bour, 1995). Mutants exhibit an absence of alpha PS2 integrin gene expression. It appears that Mef2 regulates inflated, the alpha PS2 integrin gene (see myospheroid) through a visceral muscle enhancer of that gene (Rangarayakulu, 1995). Despite the appearance of an apparently normal dorsal vessel in Mef2 mutants, including the expression of Beta 3 tubulin, individual myosin subunit genes (myosin heavy chain, myosin alkaline light chain, and myosin light chain-2) are not expressed in the mutant dorsal vessel (Ranganayakulu, 1995).

Drosophila tropomyosin I (TmI) gene is a target gene for mef2 regulation. The TmI gene contains two different muscle enhancers within the first intron of the gene. Both enhancers contain a Mef2 binding site, and mutation in either site reduces expression in somatic body wall muscles. For one enhancer, a high level of gene expression in somatic muscles requires the cooperative activity of Mef2 and a cis-acting muscle activator region located within the enhancer. There is little effect in these mutants on TmI expression in developing visceral muscles and dorsal vessel (heart), despite the fact that Mef2 is expressed in these muscles, indicating that TmI is regulated differently in these muscles (Lin, 1996).

Mef2 is a positive regulator of Drosophila Tromomyosin I in the body-wall muscles of embryos, larvae, and adults. Ectopic expression of Mef2 in the epidermis and ventral midline cell in embryos activates the expression of TmI and other muscle genes in these tissues; this activation is stage-dependent, suggesting a requirement for additional factors. Among the genes activated by Mef2 are ß3-Tubulin, a cytoskeletal protein found in myoblasts, muscle cells and some nonmuscle cells and Muscle LIM protein at 60A, a Drosophila homolog of the vertebrate muscle LIM protein, which is a positive regulator of myogenic differentiation. Mesodermal nautilus expression is diminished, but there is no indication of ectopic Mef2 activating nautilus in the ectoderm or ventral midline. Ectopic expression of Mlp60A is observed only in the ventral midline cells but not in the epidermis of embryos that ectopically express Mef2 in the epidermis and ventral midline. Furthermore, ectopic expression of Mef2 in the epidermis results in a decrease in the expression of signaling molecules in the epidermis and a failure of the embryo to properly form body-wall muscles. Muscle VA1 of the ventral group body-wall muscles is missing and muscle VA2 is much shorter than that found in wild type embryos. Muscles VO 4 to 6 are also much shorter than those found in wild-type embryos and often fuse to one another. LT1 to LT4 of the lateral group body-wall muscles are disorganized. These results indicate that Mef2 can function out of context in the epidermis to induce the expression of muscle genes and interfere with a requirement for the epidermis in muscle development. The level of Mef2 in the mesoderm and/or muscles in embryos is critical to body-wall muscle formation; however, no effect is observed on the development of the visceral muscle or dorsal vessel (M.-H. Lin, 1997)

A genetic hierarchy of interactions, involving myogenic regulatory factors of the MyoD and myocyte enhancer-binding 2 (MEF2) families, serves to elaborate and maintain the differentiated muscle phenotype through transcriptional regulation of muscle-specific target genes. Much work suggests that members of the cysteine-rich protein (CRP) family of LIM domain proteins also play a role in muscle differentiation; however, the specific functions of CRPs in this process remain undefined. Two members of the Drosophila CRP family, the muscle LIM proteins Mlp60A and Mlp84B, have been characterized that show restricted expression in differentiating muscle lineages. To extend this analysis of Drosophila Mlps, the expression of Mlps have been characterized in mutant backgrounds that disrupt specific aspects of muscle development. A genetic requirement is found for the transcription factor dMEF2 in regulating Mlp expression, and dMEF2 can bind, in vitro, to consensus MEF2 sites derived from those present in Mlp genomic sequences. These data suggest that the Mlp genes may be direct targets of dMEF2 within the genetic hierarchy controlling muscle differentiation. Mutations that disrupt myoblast fusion fail to affect Mlp expression. In later stages of myogenic differentiation, which are dedicated primarily to assembly of the contractile apparatus, the subcellular distribution of Mlp84B has been analyzed in detail. Immunofluorescent studies reveal the localization of Mlp84B to muscle attachment sites and the periphery of Z-bands of striated muscle. Analysis of mutations that affect expression of integrins and alpha-actinin, key components of these structures, also fail to perturb Mlp84B distribution. In conclusion, molecular epistasis analysis has been used to position Mlp function downstream of events involving mesoderm specification and patterning, concomitant with terminal muscle differentiation. Furthermore, these results are consistent with a structural role for Mlps as components of muscle cytoarchitecture (Stronach, 1999).

The Mlp60A gene exhibits three exons interrupted by two small introns, one in the 5' untranslated region and another in the coding region. The Mlp84B gene contains one noncoding and one coding exon separated by a single large intron. Analysis of noncoding DNA within and surrounding the Mlp genes reveals the presence of multiple A/T-rich sequences matching exactly the reported MEF2 target binding consensus sequence. The Mlp60A gene contains three potential dMEF2 binding sites; two of these sites are located in the region 5' to the start of gene, whereas the third is found 3' to the coding sequence. Six putative dMEF2 binding sites are found in the Mlp84B gene. Four of the six are clustered in the intron, another is located 3' to the coding region of the gene, and another is contained completely within the first exon (Stronach, 1999).

Knowledge of the subcellular distribution of a protein often contributes substantially to an understanding of its function. In embryonic somatic muscles, Mlp60A and Mlp84B are found in both the nuclear and cytoplasmic compartments, consistent with either a regulatory or structural role in differentiating muscle. When expressed in rat embryo fibroblast cells, Drosophila Mlps showed a specific association with the actin cytoskeleton. To determine the precise localization of Mlps within mature myofibrils at higher resolution, whole, third instar larval midguts were double labelled using antibodies directed against the Mlps and alpha-actinin, which marks Z-bands. Surrounding the midgut, elongated visceral mesodermal cells form a lattice of transverse and longitudinal fibers. Although these cells do not undergo myoblast fusion, they appear striated and display sarcomeric repeats. Within the midgut visceral mesoderm, alpha-actinin prominently localizes to Z-bands. Z-bands demarcate the ends of individual sarcomeres, where the barbed ends of actin thin filaments terminate. In the same tissue, Mlp84B distributes as a doublet that flanks each Z-band. As seen in merged images, Alpha-actinin and Mlp84B are localized in adjacent regions. Mlp84B extends away from the periphery of the Z-band, whereas alpha-actinin is clearly more restricted. The localization of Mlp84B to discrete sites within the muscle sarcomere provides evidence for a specific association of Mlp84B with the microfilament cytoskeleton in vivo. Although Western immunoblot analysis reveals that Mlp60A is present in isolated midgut preparations, the protein was not detected using immunofluorescent methods. It is unclear why Mlp60A protein was not observed in situ, but perhaps within the mature myofibril, Mlp60A is complexed with protein partners such that the epitopes recognized by the antibodies were masked (Stronach, 1999).

The beta 3 tubulin gene is a structural gene expressed during mesoderm development from the extended germ band stage onward. Expression within the individual mesodermal derivatives is guided by different control elements. The upstream regions allow expression in the dorsal vessel and the somatic mesoderm, while enhancers localized in the first intron guide expression in the visceral mesoderm. In addition to this tissue-specific mode of transcriptional control, beta 3 tubulin is regulated in a distinct stage-specific manner. From stage 10 to stage 12, expression in the visceral mesoderm is regulated along the anterior-posterior axis by a first intron localized enhancer; this regulation is mediated by homeotic selectors genes like Ubx. After stage 12, an additional farther 3'-located enhancer mediates expression in the visceral and somatic mesoderm. In the epidermis, transcription of the beta 3 tubulin gene is repressed by Engrailed (Damm, 1998).

Deletion analysis carried out in transgenic flies reveals independent regulatory elements for the dorsal vessel and the somatic mesoderm. These elements are located upstream of the transcriptional start site. For expression in the somatic mesoderm, a 279-bp region is absolutely essential. This region contains a binding site for the Drosophila myocyte-specific enhancer binding factor 2 (D-MEF2). Deletion or mutation of this D-MEF2 binding site strongly reduces transcription. This pattern is consistent with the strongly reduced expression of beta 3 tubulin in D-mef2 mutant embryos. This analysis furthermore reveals that the D-MEF2 binding site acts in concert with nearby cis regulatory elements; D-MEF2 cannot, on its own, direct beta 3 tubulin expression. These data show that the upstream control region of the beta 3 tubulin gene is an early target of the D-MEF2 transcriptional activator (Damm, 1998).

muscleblind (mbl) is a recently described Drosophila gene involved in terminal differentiation of adult ommatidia. mbl is a nuclear protein expressed late in the embryo in pharyngeal, visceral, and somatic muscles, the ventral nerve cord, and the larval photoreceptor system. All three mbl alleles studied exhibit a lethal phenotype and die as stage 17 embryos or first instar larvae. These larvae are partially paralyzed, show a characteristically contracted abdomen, and lack striation of muscles. An analysis of the somatic musculature shows that the pattern of muscles is established correctly, and they form morphologically normal synapses. Ultrastructural analysis, however, reveals two defects in the terminal differentiation of the muscles: inability to differentiate Z-bands in the sarcomeric apparatus and reduction of extracellular tendon matrix at attachment sites to the epidermis. Failure to differentiate both structures could explain the partial paralysis and contracted abdomen phenotype. Analysis of mbl expression in embryos that are either mutant for Dmef2 or ectopically express Dmef2 places mbl downstream of Dmef2 function in the myogenic differentiation program. mbl, therefore, may act as a critical element in the execution of two Dmef2-dependent processes in the terminal differentiation of muscles (Artero, 1998).

It is unknown how the general patterning mechanisms that subdivide the mesoderm initiate different pathways of cell differentiation. One route to understanding these events is to isolate and analyse genes specifically expressed early in this differentiation process. A novel molecular screen has been undertaken in Drosophila in a systematic search for such genes. The approach utilizes subtractive hybridization coupled to directional cDNA library construction. Libraries were made from as little as 20 mg total RNA isolated from hand-picked embryos of defined stage of development and genotype. In a one-step procedure, the subtraction was 6.5- to 7.25-h wild-type embryos minus 6.5- to 7.25-h twist (twi) zygotic mutant embryos. A two-step procedure in which maternally expressed sequences were subtracted from each of these cDNA libraries, before subtracting twi from wild-type, increases the subtraction efficiency. Subtraction results in a cDNA population enriched more than 100-fold for mesodermal cDNAs. This was screened by determination of the embryonic expression pattern of each clone in a high throughput procedure followed by DNA sequencing. The method, which is comprehensive and does not discriminate against rarer cDNAs, is generally applicable and calculations show that it would work for just 10 embryos. Analysis of one clone, Dmeso18E, reveals a gene encoding a putative nuclear protein of 553 amino acids. The protein is novel; its expression is mesoderm-specific, twi-dependent, and occurs early during somatic, visceral, and heart muscle differentiation. Two pivotal regulators of mesoderm development and gene expression are Dmef2 and tinman (tin). Analysis of Dmeso18E expression reveals new aspects to their roles: there are effects of Dmef2 on developing muscle much earlier than hitherto believed, and there is tin-independent gene expression in, and invagination of, prospective midgut visceral muscle cells. Dmeso18E expression is regulated by Dmef2, although some expression is Dmef2-independent. The tin-independent and Dmef2-independent expression of Dmeso18E indicates that it either occupies a link between twi and genes like tin and Dmef2 or it lies in a parallel pathway of gene activation (Taylor, 2000).

Thus, there is tin-independent expression of Dmeso18E in the cells that would go on to form the visceral muscle. Dmeso18E expression can be compared to that of bagpipe, which is expressed in the mesoderm from stage 8, like Dmeso18E, and then in segmentally repeated patches corresponding to the visceral muscle primordia as they form during stages 10 and 11. However, in contrast to Dmeso18E, expression of the bap gene is not activated in the germ band in tin mutants and is a candidate target gene for Tinman. Two possible explanations follow for the presence of Dmeso18E mRNA in tin mutants in cells that would go on to form visceral muscle: (1) there may be a tin-independent route for activation of transcription in prospective visceral muscle cells; (2) it could be mRNA remaining from an earlier transcription activation event before the visceral muscle progenitors form. However, this would require that Dmeso18E RNA be quite stable -- however, the dynamic nature of its embryonic expression pattern suggests a relatively short half-life. Whatever the mechanism that lies behind this expression, the main point is that a muscle-specific gene that does not require tin is expressed in the visceral muscle primordia. Together with the tin-independent invagination of these cells, these results show that some features of the cells that will form the visceral muscle do not require tin (Taylor, 2000).

To identify regulatory events occurring during myogenesis, the transcriptional regulation of a Drosophila melanogaster actin gene, Actin 57B, was characterized. Act57B transcription is first detected in visceral muscle precursors and is detectable in all embryonic muscles by the end of embryogenesis. Through deletion analysis a 595 bp promoter element has been identified that is sufficient for high levels of expression in all three muscle lineages. This fragment contains a Mef2 binding site conserved between D. melanogaster and Drosophila virilis that binds Mef2 protein in embryo nuclear extracts. Mutation of the MEF2 site severely reduces promoter activity in embryos, and in Mef2 mutants Act57B expression is severely decreased, demonstrating that Mef2 is an essential regulator of Act57B. Mef2 likely acts synergistically with factors bound to additional sequences within the 595 bp element. These findings underline the importance of Mef2 in controlling differentiation in all muscle lineages. These experiments reveal a novel regulatory mechanism for a structural gene where high levels of expression in all embryonic muscles is regulated through a single transcription factor binding site (Kelly, 2002).

Mef2 function alone is not sufficient to induce Act57B expression at high levels in muscle cells. LacZ reporter constructs with the Mef2 site and flanking sequences, but lacking region b (consisting of about half of the 595 bp promoter element), had reduced reporter activity. In addition, ectopic expression of Mef2 under control of a heat shock promoter does not induce expression of Act57B. Low levels of Act57B expression occur in the absence of Mef2 function, either when the Mef2 site is mutated in the minimal reporter construct or in a Mef2 mutant background. The simplest explanation for these results is that Mef2 functions with additional factor(s) to regulate muscle gene expression and that the presumptive cofactor is also restricted in its activity to the musculature (Kelly, 2002).

However, the identity of this putative cofactor is far from clear. PAR-domain protein 1 has been shown to be a Mef2 cofactor in Drosophila, regulating TmI gene expression in the somatic mesoderm, however there are no PDP1 binding sites contained within the minimal Act57B enhancer fragment. In vertebrate systems the myogenic bHLHs (MyoD, myogenin, Myf5, MRF4) have been shown to regulate gene expression with Mef2 through E-boxes. Again, there are no E-boxes contained within the minimal Act57B enhancer. Since the upstream region required for high promoter activity contains areas of strong sequence conservation between D. melanogaster and D. virilis, it is likely that these might represent binding sites for the putative cofactors. It is unclear at this time exactly what factors may bind to this region (Kelly, 2002).

All of the data presented in this paper indicate that the Act57B locus is under the positive control of Mef2 and potential cofactors. Moreover, none of the deletion constructs show inappropriate expression, suggesting the absence of cis-acting negative regulatory sequence within the enhancer. However, there is a time during early embryogenesis when Mef2 is present, but Act57B is not expressed. This could simply be due to the absence of positively acting cofactors at this stage in development. Alternatively, this could be the result of negative regulators interacting directly with factors bound to the Act57B promoter (Kelly, 2002).

The Drosophila C2-H2-type zinc-finger transcription factor CF2 has been shown to regulate follicular cell fate determination during oogenesis. CF2 is also expressed in the developing muscles of the embryo where it first appears at stage 12 at the time of skeletal myoblast fusion. Later it is expressed in all muscle lineages including skeletal, visceral and cardiac. Epistatic analysis has shown that CF2 expression is dependent on the myogenic factor MEF2 (Bagni, 2002).

The distinct muscles of an organism accumulate different quantities of structural proteins, but always maintaining their stoichiometry. However, the mechanisms that control the levels of these proteins and that co-ordinate muscle gene expression remain to be defined. The paramyosin/miniparamyosin gene encodes two thick filament proteins transcribed from two different promoters. The regulatory regions were analyzed that control expression of this gene and that are situated in the two promoters, the 5' and the internal promoters, both in vivo and in silico. A distal muscle enhancer containing three conserved MEF2 motifs is essential to drive high levels of paramyosin expression in all the major embryonic, larval and adult muscles. This enhancer shares sequence motifs, as well as its structure and organisation, with at least four co-regulated muscle enhancers that direct similar patterns of expression. However, other elements located downstream of the enhancer are also required for correct gene expression. Other muscle genes with different patterns of expression, such as miniparamyosin, are regulated by other basic mechanisms. The expression of miniparamyosin is controlled by two enhancers, AB and TX, but a BF modulator is required to ensure the correct levels of expression in each particular muscle. A mechanism of transcriptional regulation is proposed in which similar enhancers are responsible for the spatio-temporal expression of co-regulated genes. However, it is the interaction between enhancers that ensures that the correct amounts of protein are expressed at any particular time in a cell, adapting these levels to their specific needs. These mechanisms may not be exclusive to neural or muscle tissue and might represent a general mechanism for genes that are spatially and temporally co-regulated (Marco-Ferreres, 2005).

Marin, M. C., Rodriguez, J. R. and Ferrus, A. (2004). Transcription of Drosophila troponin I gene is regulated by two conserved, functionally identical, synergistic elements. Mol. Biol. Cell 15, 1185-1196. PubMed Citation: 14718563

Transcription of Drosophila troponin I gene is regulated by two conserved, functionally identical, synergistic elements

The Drosophila wings-up A gene encodes Troponin I. Two regions, located upstream of the transcription initiation site (upstream regulatory element) and in the first intron (intron regulatory element), regulate gene expression in specific developmental and muscle type domains. Based on LacZ reporter expression in transgenic lines, upstream regulatory element and intron regulatory element yield identical expression patterns. Both elements are required for full expression levels in vivo as indicated by quantitative reverse transcription-polymerase chain reaction assays. Three myocyte enhancer factor-2 binding sites have been functionally characterized in each regulatory element. Using exon specific probes, it was shown that transvection is based on transcriptional changes in the homologous chromosome and that Zeste and Suppressor of Zeste 3 gene products act as repressors for wings-up A. Critical regions for transvection and for Zeste effects are defined near the transcription initiation site. After in silico analysis in insects (Anopheles and Drosophila pseudoobscura) and vertebrates (Ratus and Coturnix), the regulatory organization of Drosophila seems to be conserved. Troponin I (TnI) is expressed before muscle progenitors begin to fuse, and sarcomere morphogenesis is affected by TnI depletion as Z discs fail to form, revealing a novel developmental role for the protein or its transcripts. Also, abnormal stoichiometry among TnI isoforms, rather than their absolute levels, seems to cause the functional muscle defects (Marin, 2004).

This in vivo study reveals two regulatory regions, URE and IRE, located immediately upstream and downstream to the transcription initiation site and defined by a characteristic array of binding sites for the transcription factors Mef2, Biniou, and Tinman. The regions are qualitatively identical in their effects, but both are required for proper levels of transcription. Given the span of the genomic fragments tested for the reporter expression, it seems that the full set of positive regulatory elements has been identified. Putative repressor sites, however, remain to be identified. Finally, the transvection experiments suggest that, in addition to the cis-requirements, transcription is also dependent on trans-effects occurring probably at a small critical region close to the putative promoter (Marin, 2004).

The genomic fragments analyzed in LacZ reported transgenes allow identifying regions that contain positive regulatory elements that direct expression to specific tissues and developmental stages. These regulatory modules are revealed as overlapping stretches of DNA rather than separate and mutually exclusive units. For example, the modules for somatic and visceral muscles share ∼1 kb of sequences. None of the smaller fragments tested that subdivide this 1 kb, however, could reproduce the original somatic or visceral expression patterns. The case has precedents in other genes such as mef2, tubulin, or tropomyosin 2, and illustrate the intimate relationship between specific sequences (i.e., enhancers or repressors) and the topology of the surrounding chromatin in the context of proper gene expression (Marin, 2004 and references therein).

The location of the two regulatory regions could sustain a particular chromatin structure, perhaps of a hair-pin type, for normal transcription. The spacing requirement and the linear order of interference effects shown by the three rearrangements support this speculation. In females, normal transcription requires correct pairing between both IRE + URE complements, and the spacing becomes less critical as long as it is the same in both chromosomes. The transvection effect that takes place when two homologous copies of the gene are present clearly implies enhanced transcription from the trans-homologue, as demonstrated by QRT-PCR assays in genotypes that allow to discriminate the chromosomal origin of some transcripts (Marin, 2004).

The initiation of transcription is clearly dependent on Mef2. Maintenance, however, does not seem to rely exclusively on this transcription factor. The difference between these two types of transcription has been recently documented, and the present case may indicate that it is a general phenomenon. Because the analyzed regions contain canonical binding sites for Tinman and Biniou, it seems puzzling why these factors are not able to drive gene expression in a mef2 null background. One possibility is that Mef2, in addition to its direct DNA binding activity on wupA and its role as a transcription factor, acts also as a trancriptional cofactor for Tinman and Biniou. Also, the role of other transcription factors such as the one encoded in Dmeso18E remains to be integrated into this scenario. Purification and analysis of the corresponding protein complexes would be required to test these speculations (Marin, 2004).

Concerning the Trithorax group of transcriptional cofactors assayed, the data demonstrate that zeste acts as a repressor for wupA. In addition, Su(z)3 is also a repressor and it behaves similarly to zeste with respect to transvection effects. Additional data on a third gene, Trithorax-like, which encodes the GAGA factor yielded similar effects. They represent cis- and trans-requirements for normal transcription. There seems to be, however, a critical domain near the promoter where the effects of these repressors become evident. Based on the immunity of PG31 heterozygotes to Trithorax group mutant backgrounds, this critical region could be defined by the-249 position as the upstream limit. In addition, the perfect transvection in PL87/PG31 heterozygotes suggest also a critical region of pairing for transcription. It is plausible that both critical regions are coincident. Presumably, pairing of these two rearrangements will be facilitated by the common sequences and size of the inserts. Their different site of insertion and the different transgenes, however, most likely will distort pairing to some extent. Thus, the critical region for transvection might be as small as 30 base pairs upstream of the initiation site. Extensive studies in the gene yellow have reached the same conclusion where the critical region for transvection seems to be the TATA box and an initiator element located in cis. The case of wupA, which does not contain TATA box, suggests that the critical region is the promoter per se, independently of its type. These observations should help to direct future in vitro studies with chromatin fragments (Marin, 2004).

It may seem counterintuitive the observation that z and Su(z)3 mutant backgrounds result in an increase of transcription at wupA, whereas the phenotypic effect shows a loss of transvection. Because the transcriptional change has been consistently observed in all genotypes assayed, including the flies sorted by wing position, it is evident that the phenotype does not correlate with the absolute levels of TnI transcripts. Furthermore, in spite of the very low levels of transcription in 23437/hdp3 females, they are viable and muscles are functional, except those involved in flight. The most plausible interpretation of these observations is that the deleterious effect on muscle structure results from changes in the stoichiometry of certain TnI isoforms rather than in their absolute levels. It is likely that, as in TnI isoform replacement experiments with the vertebrate homologues, the unbalance of certain TnI isoforms lead to unsuitable thin filaments in vivo. If this is also the case in humans, certain muscular diseases, particularly those revealed under intense exercise, may result from mutations in regulatory regions, and thus may have escaped detection under standard sequencing procedures. In this context, the conserved gene regulatory array should be useful to guide future mutant screenings in humans (Marin, 2004).

Z discs are thought to be the anchoring points where thin filaments exert force and contract the sarcomere during muscle activity. It is somewhat unexpected that reduced levels or structural modifications of TnI can result in defective Z discs. The aspect and spacing of these Z disk-like structures suggest that a Z disk results from the lining of independent substructures deposited on the thin filaments and latter organized in register. It is worth noting, however, that a thin filament does not necessarily anchor at a Z disk. The quasi-normal sarcomeres from double mutants in troponin I, hdp2, and myosin or tropomyosin, Su(hdp2), show frequent cases of thin filaments extending more than one sarcomere in length. All these abnormal features of Z discs observed in mutants that involve TnI may indicate additional developmental functions of this protein beyond the well known regulatory role in sarcomere mechanics. Alternatively, these features may result from depletion of bona fide Z disk components whose expression is downregulated because of TnI mutations. This possibility would require a coordinated regulation of gene expression among thin filament components (Marin, 2004).

Vertebrate TnI encoding genes are not yet amenable to the in vivo analysis that Drosophila allows. Nevertheless, previous studies on the quail fast and slow TnI genes show functional evidences of a very similar regulatory structure to that described here for wupA. Equivalent regions to IRE and URE can be revealed by sequence analysis in other TnI members with the exception of the cardiac gene. The array of regulatory regions and their characteristic features seem fairly well conserved in TnI encoding genes of insects and vertebrates. This observation will be relevant toward the design of tools that aim to mimic the native gene expression in otherwise pathological conditions. Beyond this utilitarian use, the conservation of the TnI regulatory landscape in other genes that encode thin filament components might be indicative of common trends that would ensure proper quantitative expression of these components and, eventually, could help to translate gene regulation into physiology (Marin, 2004).

Mapping Dmef2-binding regulatory modules by using a ChIP-enriched in silico targets approach

Mapping the regulatory modules to which transcription factors bind in vivo is a key step toward understanding of global gene expression programs. A chromatin immunoprecipitation (ChIP)-chip strategy has been developed for identifying factor-specific regulatory regions acting in vivo. This method, called the ChIP-enriched in silico targets (ChEST) approach, combines immunoprecipitation of cross-linked protein-DNA complexes (X-ChIP) with in silico prediction of targets and generation of computed DNA microarrays. Use of ChEST in Drosophila is described to identify several previously unknown targets of myocyte enhancer factor 2 (MEF2), a key regulator of myogenic differentiation. The approach was validated by demonstrating that the identified sequences act as enhancers in vivo and are able to drive reporter gene expression specifically in MEF2-positive muscle cells. Presented here, the ChEST strategy was originally designed to identify regulatory modules in Drosophila, but it can be adapted for any sequenced and annotated genome (Junion, 2005).

To predict Dmef2-dependent CRMs, the Drosophila genome was scanned for modules containing one of the three previously described in vivo-acting Dmef2 binding sites. Because muscle differentiation events are controlled by the synergistic action of MADS-box (Mef2 family) and E-box (Twist and MyoD bHLH family) factors modules were sought containing Mef2- and E-box-binding sites. This scanning procedure led to the identification of ~1,243 potential Dmef2-binding CRMs, from which 99 modules were selected, amplified, and spotted to produce a computed Dmef2-CRM array. Three of four previously described Dmef2-CRMs, located in the vicinity of Paramyosin, Act57B, and Dmef2, were identified during genome scanning and selected for spotting as positive control. The fourth in vivo-acting Dmef2-CRM located close to b3-tubulin did not come out from the screen because of the high number of E-box sites used as the scanning criterion. The CRMs located in the vicinity of genes that are not expressed in the mesoderm or genes of unknown expression and function (some of CGs) were rejected. In parallel, X-ChIP was used to isolate DNA fragments to which Dmef2 binds in vivo. ChIP-DNA immunoprecipitated either with Dmef2 antibody or with nonimmune serum was then labeled and used to probe the Dmef2-CRM array. The three previously described in vivo-acting CRMs were found enriched in ChIP-DNA immunoprecipitated with Dmef2 antibody. Importantly, numerous other in silico-identified Dmef2-CRMs were enriched in ChIP-DNA, thus demonstrating the efficacy of the ChEST method (Junion, 2005).

Of the 99 in silico-predicted Dmef2-CRMs, 62 were enriched in the DNA immunoprecipitated with anti-Dmef2 antibody. The CRM-associated Dmef2 targets included genes expressed in all muscle cell types, in which Dmef2 has previously been reported to function. In addition to expected candidates encoding fusion (Lmd, Hibris) and structural (Ket, Pod1) muscle proteins, a large number of CRM-associated Dmef2 target genes coded for TFs and signal transduction proteins. For example, CRMs upstream of Fz2 and within the introns of Ci and Pan indicate a potential role of Dmef2 in transcriptional regulation of genes transducing to the mesodermal cells ectodermal Wg and Hh signals, whereas CRMs within the introns of If and Pka-C3 suggest that by regulating transcription of these genes Dmef2 is involved in the attachment of muscle fibers and in fiber contraction, respectively. In some cases (e.g., Kettin, NetB, N-cad), several Dmef2-dependent CRMs were mapped in the vicinity of the same gene, highlighting the complexity of transcriptional regulation in which Dmef2 is involved. Analysis of the position of CRMs in relation to adjacent genes revealed that the majority of ChIP-enriched modules are located upstream (42%) or within the introns (39%) of target genes. In these two categories, the most frequent positions of Dmef2-CRMs appear between 1 and 5 kb upstream of the gene and within the first intron (Junion, 2005).

To determine whether the ChEST-identified DNA fragments are able to act as regulatory modules in vivo, ten Dmef2-CRMs by reporter gene transgenesis were tested. Nine of 10 CRMs were found to drive reporter gene expression in Dmef2-positive muscle cells. In all transgenic Drosophila lines, lacZ reporter expression at least partially reproduced endogenous gene expression, indicating that the identified CRMs were bona fide enhancers of adjacent genes. For example, the Ket-1, Ket-2, and Ket-3 CRMs laying in the vicinity of the kettin gene, which encodes a giant muscle protein required for the formation and maintenance of normal sarcomere structure, were found to drive lacZ expression in distinct subsets of differentiating body wall muscles. These data indicate that the pan-muscular expression of kettin is regulated in a muscle-type-specific manner, and by multiple Dmef2-binding enhancers. Interestingly, four other analyzed CRMs located within the introns of N-cad and acon and upstream of fz2, sfl, and Meso18E also drive lacZ expression in discrete subsets of somatic muscle precursors. The muscle-type-restricted activity of these modules suggests that both CRM regulators (Dmef2, E-box factors) and their target genes are involved in different aspects of muscle precursors diversification, including muscle fiber shape and axial positioning. Alternatively, the observed muscle-type-specific expression of lacZ may result from the limited size (250-300 bp) of genomic sequences tested. In embryos carrying a Dmef2-dependent CRM found in the intron of If, lacZ expression is detected in a group of ventrolateral muscles. This lacZ pattern correlates with distribution of endogenous If, which accumulates at the extremities of ventrolateral muscle fibers and is required for their correct epidermal insertion. The reduced level of target gene expression in Dmef2 mutant embryos provides an additional support for Dmef2-dependent in vivo regulation of ChEST-identified CRMs (Junion, 2005).

Compared with other genomewide ChIP-chip approaches, ChEST does not require access to the whole-genome tiling arrays. It is dedicated to the identification of a subset of factor-specific CRMs that can be predicted by using computer-assisted methods. Depending on the TF and the organism concerned, the predictability of CRMs may represent a limiting factor. This study used ChEST to identify Dmef2-regulated CRMs in Drosophila. Because Dmef2 binds to long, well characterized DNA sequences and the Drosophila genome is well adapted to CRM prediction (rich in annotations), this approach created an attractive framework for analysis and contributed to a high percentage (63%) of ChIP-enriched CRMs. The fact that 37% of in silico-selected Dmef2-CRMs have not been found as significantly enriched in ChIP material results from one of the following reasons or from a combination of these reasons: (1) inefficient ChIP due to restricted expression of target genes (13 CRMs listed lay close to genes expressed in a short time window or in a subset of mesodermal cells); (2) inefficient ChIP due to chromatin conformation, and/or (3) false positives generated during the computer-assisted genome scanning (Junion, 2005).

To test ChEST in more challenging conditions, it was applied for mapping CRMs to which bind two other myogenic factors, Lame duck (Lmd) and Ladybird early (Lbe). Compared with Dmef2, Lmd and Lbe are expressed in a progressively more restricted subset of cells in the embryo, and their in vivo binding sites have not yet been identified, meaning only the in vitro consensus binding sequences can be used for CRM prediction. In such suboptimal conditions, the efficacy of ChEST dropped from 63% (Dmef2) to 34% of ChIP-enriched CRMs for Lmd and <20% for Lbe. Thus, in Drosophila, ChEST can also be used (with a lower efficacy) for mapping CRMs to which bind transcription factors recognizing short and/or in vitro-only characterized DNA sequences (Junion, 2005).

Furthermore, because the genome annotations for several invertebrate and vertebrate organisms are rapidly progressing, ChEST is expected to be easily adapted to other systems. Otherwise, targets for the evolutionarily conserved TFs can be identified in Drosophila and then validated in other organisms. One possible application of ChEST in the future is to use it in cell- or tissue-culture systems to follow the repertoire of target CRMs for TFs involved in important biological processes, including human pathologies (Junion, 2005).

Drosophila Dmef2 is required for the terminal differentiation of skeletal, visceral, and cardiac musculature but seems not to be involved in early myogenic events such as muscle specification and diversification. It directs mesodermal cell differentiation programs by regulating transcriptional activity of genes associated with these processes. Here, among the identified Dmef2-binding CRMs, those were found that map in the vicinity of ket, Tm1, Act87E, and Pod1 genes encoding muscle structural proteins and a few others located close to Lmd, N-cad, NetB, Hibris, Sema-5c, If, dei, and Flw genes implicated in cell adhesion/fusion or muscle attachment, thus supporting previously described muscle differentiation functions of Dmef2. The identification of Lmd, Hibris, and N-cad CRMs is consistent with the involvement of Dmef2 in myoblast fusion processes, which are affected in Dmef2 mutant embryos. Furthermore, the presence of Dmef2-dependent CRMs close to NetB, which is involved in the attraction of motoneurons, suggests that the Dmef2-dependent formation of presynaptic active zones may involve NetB. However, in addition to these novel but anticipated Dmef2 targets, the data indicate that Dmef2 also contributes to the transcriptional regulation of genes implicated in muscle specification and diversification. Candidates that indicate an early myogenic role of Dmef2 include components of the Wg, Hh, and RTK signaling pathways (Ci, Pan, GATAe, AbdA, Jeb, Sfl, Fz2, ttk, argos, stumps, Concertina, and Src42A) that play key roles in the specification of myogenic lineages and their subsequent diversification (Junion, 2005).

The role of Dmef2 in the initial steps of myogenesis is in agreement with the early, twist-dependent phase of Dmef2 expression, and is further supported by the previously described regulation of early expression of Dmeso18E. Another unexpected group of potential transcriptional targets of Dmef2 corresponds to genes encoding proteins involved in ion transport, channel activity, and metabolism (e.g., Slo, Itp-r83A, NheII, and Acon). The regulation of this class of genes may reflect a role for Dmef2 in the transmission of neural stimulation and muscle contraction. The maintenance of Dmef2 expression in fully differentiated adult muscles is in agreement with such a function. In conclusion, the ChEST approach presented here has led to the identification of a set of previously unknown Dmef2-dependent regulatory modules whose activity and adjacent genes indicate novel myogenic functions for Dmef2 (Junion, 2005).

Single-minded, Dmef2, Pointed, and Su(H) act on identified regulatory sequences of the roughest

Roughest (Rst) is a cell adhesion molecule of the immunoglobulin superfamily that has multiple and diverse functions during the development of Drosophila melanogaster. The pleiotropic action of Rst is reflected by its complex and dynamic expression during the development of Drosophila. By an enhancer detection screen, several cis-regulatory modules have been identified that mediate specific expression of the roughest gene in Drosophila developmental processes. To identify trans-regulators of rst expression, the Gal4/UAS system was used to screen for factors that were sufficient to activate Rst expression when ectopically expressed. By this method the transcription factors Single-minded, Pointed.P1, and Su(H)-VP16 were identified. Furthermore, these factors and, in addition, Dmef2 are able to ectopically activate rst expression via the previously described rst cis-regulatory modules. This fact and the use of mutant analysis allocates the action of the transcription factors to specific developmental contexts. In the case of Sim, it could be shown to regulate rst expression in the embryonic midline, but not in the optic lobes. Mutagenesis of Sim consensus binding sites in the regulatory module required for rst expression in the embryonic midline, abolishes rst expression; indicating that the regulation of rst by Sim is direct (Apitz, 2005).

Rst has complex and multifaceted functions throughout the development of the fly, which include myogenesis, eye development, as well as axonal pathfinding in the optic lobes. To gain a better understanding of these functions at the levels of gene regulation and signal transduction, a number of tests were designed to identify both the transcriptional activators and their respective targets surrounding the rst locus. In a preceding study (Apitz, 2004), a number of DNA segments upstream of rst were characterized and regulatory regions were discovered that mediate gene expression in myoblasts, midline, and eyes, respectively. In the present study these results were supplemented with an in vivo screen to identify regulators of rst expression using the Gal4/UAS system. Several factors were discovered that are able to induce ectopic Rst expression and to activate reporter gene expression via rst cis-regulatory sequences (Apitz, 2005).

The experimental route taken to identify protein factors involved in the regulation of the rst gene is based on the detection of their potential to induce ectopic Rst expression in vivo. The use of sca-Gal4 as a driver line in this experimental approach is based on the following criteria. sca-Gal4 mediates expression in neuroectodermal cells of the embryo. At embryonic stage 10, these cells can be examined for ectopic Rst expression because no endogenous Rst expression is found at this time in these cells; this allows operators to obtain clear-cut and unequivocal results. The use of alternative Gal4 driver lines did not prove suitable because of the dynamic expression of Rst during all developmental stages, and due to its subcellular localization. For example, when dll-Gal4 is used as a driver, it is difficult to distinguish between ectopic Rst expression induced in the apical tips of cells of the leg discs, and endogenous Rst expression in the overlaying ectodermal cells. Furthermore, the use of sca-Gal4 has the advantage that neuroectodermal cells are not fully differentiated cells. This may more closely resemble the developmental state of the cells in which Rst expression is normally induced endogenously, e.g., in undifferentiated cells of the developing eye disc. However, this approach generally fails to reveal transcription factors that need a coactivator for induction of rst expression, which is not present in the cells of the neuroectoderm at embryonic stage 10. This may explain the failure of Dmef2 to induce rst expression at this stage. It was shown, however, that Dmef2 is able to induce rst expression at later stages by the use of rst-lacZ constructs. The function of the different rst-lacZ constructs has been linked to specific developmental circumstances (Apitz, 2004) and their activation by corresponding factors is consistent with the known roles of these proteins in development (Apitz, 2005).

Ectopic expression of a constitutively active Pnt variant (Pnt.P1) mediates strong activation of Rst expression in neuroectodermal cells. Since Pnt.P1 recognizes the same target sequences as its splice variant Pnt.P2, the nuclear effector of the Ras-MAPK pathway, ectopic activation of Rst expression by Pnt.P1 is consistent with a regulation by the Ras-MAPK pathway. Similarly, the ectoptic activation of Rst expression in neuroectodermal cells by Su(H)-VP16 points to a regulation of rst by the Notch pathway (Apitz, 2005).

The Ras-MAPK and the Notch pathways display significant crosstalk during developmental processes in Drosophila, e.g., in cell fate specification of the eye disc. It is difficult to elucidate a possible regulation of a candidate gene by mutant analysis if it is activated by both pathways. In mutants, for one of the pathways, the activity of the other pathway will ensure residual expression of the candidate gene under scrutiny. rst expression is activated by both pathways and single mutant analysis did not reveal a significant loss of expression. Both pathways converge on regulatory elements contained within F6 and not in F5. Furthermore, a regulatory module is present in the nonoverlapping part of F6 that is activated in IOC before apoptotic decisions are made in these cells (Apitz, 2004). This module is located within an approximately 600-bp sequence and is active during several apoptotic decisions (Apitz, 2004). Consensus binding sites for Su(H) and Pnt in this module are conserved between D. melanogaster and D. pseudoobscura. Both the Ras-MAPK and Notch pathways are involved in apoptotic processes of IOC cells. Together, these data suggest that rst transcription is regulated by these pathways in the context of apoptotic decisions (Apitz, 2005).

The transactivation screen shows that Dmef2 acts on rst cis-regulatory sequences: the overlapping reporter gene constructs rstF5 -lacZ and rstF6 -lacZ are both ectopically activated by Dmef2 in the scabrous expression domain. Regions F5 and F6 contain partially overlapping sequences and it is likely that Dmef2 acts on a sequence interval bracketed by this overlap. This region contains a regulatory module for expression in the mesoderm (Apitz, 2004). This sequence contains a putative Dmef2 binding site that is also conserved between D. melanogaster and D. pseudoobscura. The sequence of this putative element exactly matches a Dmef2 binding site found in the enhancer of β3 tubulin. Therefore, it is argued that rst may be a direct target of Dmef2. This is consistent with similar mesodermal expression patterns and mutant phenotypes of Dmef2 and rst. In contrast, Rst is expressed in the mesoderm of Dmef2 loss-of-function mutants. An explanation for this result is provided by analysis of rst cis-regulatory sequences. rst is regulated in the mesoderm by at least two independent regulatory modules that mediate a differential expression pattern (Apitz, 2004). One is contained in F6p, the other in F5p. This points to a mesodermal regulation of rst by at least two factors, one of which is still active in Dmef2 mutants (Apitz, 2005).

MicroRNA1, regulated by Mef2, influences cardiac differentiation in Drosophila and regulates Notch signaling

MicroRNAs (miRNAs) are genomically encoded small RNAs that hybridize with messenger RNAs, resulting in degradation or translational inhibition of targeted transcripts. The potential for miRNAs to regulate cell-lineage determination or differentiation from pluripotent progenitor or stem cells is unknown. MicroRNA1 (miR-1) is an ancient muscle-specific gene conserved in sequence and expression in Drosophila. Drosophila miR-1 (dmiR-1) is regulated through a serum response factor-like binding site in cardiac progenitor cells. Loss- and gain-of-function studies demonstrated a role for dmiR-1 in modulating cardiogenesis and in maintenance of muscle-gene expression. In vivo evidence is provided that dmiR-1 targets transcripts encoding the Notch ligand Delta, providing a potential mechanism for the expansion of cardiac and muscle progenitor cells and failure of progenitor cell differentiation in some dmiR-1 mutants. These findings demonstrate that dmiR-1 may 'fine-tune' critical steps involved in differentiation of cardiac and somatic muscle progenitors and targets a pathway required for progenitor cell specification and asymmetric cell division (Kwon, 2005).

The single orthologue of miR-1 in Drosophila, dmiR-1, is nearly identical in sequence to mouse and human miR-1. In situ hybridization revealed dmiR-1 transcripts in presumptive mesodermal cells as early as stage 5 (2.2-2.8 h) of Drosophila development. This pattern changed dynamically throughout gastrulation, but dmiR-1 consistently marked mesodermal cells. Transcripts persisted in later stages of cardiac and somatic (body wall) muscle differentiation, as in mice, and were also found in visceral muscles of the gut. dmiR-1 expression overlaps, but precedes, that of dmef2, a transcriptional regulator of muscle precursors (Kwon, 2005).

To determine whether transcriptional regulation of miR-1 is evolutionarily conserved, ten kb of genomic DNA surrounding Drosophila melanogaster and Drosophila pseudoobscura miR-1 genes were aligned to find regions of sequence conservation. Transgenic flies containing conserved islands 4.6 kb upstream of dmiR-1 adjacent to the gene encoding nuclear GFP (nGFP) recapitulates the endogenous dmiR-1 expression in all muscle types. Cardiac nGFP expression coincided with dmef2 expression in cardial cells and was present in pericardial cells, which do not express dmef2. The basic helix–loop–helix transcription factor Twist is essential for mesoderm specification and regulates dmiR-1 in certain domains, so the expression of nGFP driven by the dmiR-1 enhancer was directly compared with Twist expression. Whereas there was considerable overlap, the dmiR-1 enhancer directs expression in many areas of low Twist expression, including cardiac and visceral muscle progenitors, suggesting twist-independent regulation in these domains (Kwon, 2005).

Deletion analyses indicated that a 2.5-kb region was sufficient for expression in all domains of dmiR-1 expression except pericardial cells. Within this domain, a 720-bp genomic region containing a highly conserved SRF-like binding site recapitulates the expression directed by the 2.5-kb fragment. SRF, which is closely related to MEF2, controls the expression of genes involved in muscle differentiation, cell migration and proliferation. Prior studies showed that SRF is an obligate activator of miR-1 expression during cardiac development in the mouse. Mutation of the SRF-like site in flies abolishes nuclear GFP expression in cardiac and visceral muscle cells but not somatic muscle. In vitro, Drosophila SRF (DSRF) weakly activates transcription of a luciferase reporter through the SRF-like binding site. Addition of the potent cardiac and smooth muscle-specific SRF cofactor myocardin-related transcription factor robustly activated luciferase activity dependent on an intact SRF-like binding site. This observation is consistent with regulation of miR-1 in mice, but the possibility cannot be ruled out that Dmef2 also regulates cardial expression of dmiR-1 through this site independently or cooperatively with SRF (Kwon, 2005).

To begin to define the functions of dmiR-1 in vivo, two Exelixis lines of Drosophila containing FRT sites surrounding the dmiR-1 gene were used to generate a FRT-FLP-based deletion of the dmiR-1 locus. Successful excision of dmiR-1, the only known or predicted gene in the 31-kb deleted interval, was confirmed by sequence analysis and RT-PCR. Homozygous dmiR-1 deletion was 100% lethal, but a spectrum of severity was observed, with approximately one-third dying at embryonic stages, one-third around hatching, and the remaining at larval stages. Homozygous mutant larvae were abnormally lethargic compared with their heterozygous siblings before death. The embryonic and larval lethality was fully rescued by overexpression of dmiR-1 by using a mesoderm specific twi-Gal4 driver or by a 5.1-kb transgene encompassing the dmiR-1 genomic locus including the 4.6-kb enhancer and the sequence encoding dmiR-1, consistent with dmiR-1 being the sole gene within the deleted region responsible for the lethal phenotype. The variability in phenotype may be related to previously described maternal dmiR-1 transcripts, redundancy with other miRNAs or may simply reflect the role of dmiR-1 in 'fine-tuning' whether cells achieve the thresholds of critical proteins to initiate critical developmental events (Kwon, 2005).

Because one-third of all dmiR-1 mutants died around the time of hatching and another one-third at larval stages with poor mobility, whether there might be a discernable muscle defect was investigated. Nearly half of all dmiR-1 mutant embryos displayed severe defects in muscle gene expression with down-regulation of sarcomeric genes such as myosin heavy chain (MHC), indicating a late requirement for miR-1 to maintain muscle differentiation. This phenotype was also uniformly rescued by dmiR-1 under the control of the twist driver, indicating that the muscle differentiation defect was due to loss of dmiR-1 and not other sequences within the deleted region (Kwon, 2005).

Because miRNAs typically target numerous mRNAs, the phenotype of dmiR-1 mutants is likely due to down-regulation of multiple critical proteins. Despite the likely complexity of targets, attempts were made to identify mRNA targets of miR-1 in flies that might be involved in dmiR-1-dependent lineage determination and differentiation decisions. Although mouse miR-1 targets transcripts encoding the cardiac-enriched basic helix-loop-helix transcription factor Hand2, no miR-1-binding sites were identified in the 3'-UTR of Drosophila hand, suggesting alternative targets in flies. Because the more severe dmiR-1 gain- and loss-of-function phenotypes were reminiscent of progenitor defects induced by altering Notch signaling, the 3'-UTRs of genes involved in the Notch pathway were examined for potential sequence matching and accessibility to dmiR-1 (Kwon, 2005).

Several conserved putative miR-1-binding sites were found in the 3'-UTR of the gene encoding Delta, a membrane-bound ligand for Notch. Upon interaction with Delta, Notch is cleaved, allowing the Notch intracellular domain to translocate into the nucleus and regulate gene expression. Signaling between neighboring Delta- and Notch-expressing cells is necessary for lateral inhibition and asymmetric cell fates during lineage determination and involves repression of Delta in Notch-expressing cells and similar repression of Notch in adjacent Delta-expressing cells. Notch signaling also later regulates differentiation of numerous cell types, including cardial cells (Kwon, 2005).

Introduction of one of the putative dmiR-1-binding sites from the Delta 3'-UTR into the 3'-UTR of luciferase resulted in dose-dependent and specific down-regulation of luciferase activity in the presence of dmiR-1 in cultured fly S2 cells. Although the in vitro data supported Delta as a dmiR-1 target, attempts were made to determine whether dmiR-1 affected Delta protein levels in vivo. Available Delta antibodies are not sensitive enough to distinguish levels of Delta protein in embryonic muscle precursors. Therefore, an in vivo assay was used involving the well described role of Delta-Notch signaling in the developing wing disc, where disruption of Delta results in thickening of fly wing veins. Delta protein is normally detectable and expressed in two perpendicular stripes in the wing pouch. dmiR-1 was overexpressed along one of the two stripes using a dpp-Gal4 driver and the effects on Delta protein expression were assayed. Delta protein was markedly reduced exclusively in the domain of dmiR-1 expression, providing in vivo support for Delta as a target of miR-1 in flies. dmiR-1-induced loss of Delta in this specific subdomain of the wing resulted in thickening of wing veins, recapitulating the loss-of-Delta phenotype. The shortened-leg phenotype upon dmiR-1 overexpression provided further evidence of dmiR-1's effects on the Notch pathway, because this, too, was similar to the phenotype of flies lacking Delta. Together, the in vivo experiments provided compelling evidence that dmiR-1 can regulate Delta protein levels, providing a potential means to fine-tune cellular responses to Notch signaling. Given the recognized role of Notch signaling in asymmetric cell division of muscle progenitors, dmiR-1 regulation of Delta, along with other dmiR-1 targets, may be important in cardiac lineage determination events (Kwon, 2005).

A core transcriptional network for early mesoderm development in Drosophila consists of Twist, Mef2, Tinman and Dorsal

Embryogenesis is controlled by large gene-regulatory networks, which generate spatially and temporally refined patterns of gene expression. This study reports the characteristics of the regulatory network orchestrating early mesodermal development in the fruitfly, where the transcription factor Twist is both necessary and sufficient to drive development. Through the integration of chromatin immunoprecipitation followed by microarray analysis (ChIP-on-chip) experiments during discrete time periods with computational approaches, >2000 Twist-bound cis-regulatory modules (CRMs) were identified and almost 500 direct target genes. Unexpectedly, Twist regulates an almost complete cassette of genes required for cell proliferation in addition to genes essential for morophogenesis and cell migration. Twist targets almost 25% of all annotated Drosophila transcription factors, which may represent the entire set of regulators necessary for the early development of this system. By combining in vivo binding data from Twist, Mef2, Tinman, and Dorsal an initial transcriptional network was constructed of early mesoderm development. The network topology reveals extensive combinatorial binding, feed-forward regulation, and complex logical outputs as prevalent features. In addition to binary activation and repression, it is suggested that Twist binds to almost all mesodermal CRMs to provide the competence to integrate inputs from more specialized transcription factors (Sandmann, 2007).

Twist-bound enhancers and direct Twist target genes

ChIP-on-chip was performed at two consecutive developmental time periods: 2-4 h (stages 5-7) and 4-6 h (stages 8-9), covering the stages of gastrulation, mesoderm expansion, migration, and early subdivision into different primordia. For each time period, four independent ChIPs were performed using two different anti-Twist antibodies to reduce possible off-target effects (Sandmann, 2007).

To systematically identify Twist-bound regions in an unbiased, global manner, a high-density microarray tiling across the Drosophila melanogaster genome was designed with ~380,000 60mer oligonucleotide probes. Twist binds to E-box motifs: As a degenerate E-box (CANNTG) is expected to occur every ~256 base pairs (bp) in the Drosophila genome, a 60mer oligonucleotide was designed for each E-box motif within the nonrepetitive, noncoding regions of the genome. This design made no assumptions about the specificity of the E-box bound by Twist, yet ensured all putative E-boxes were covered and that each Twist-bound sequence was detected by at least two neighboring 60mers (Sandmann, 2007).

These experiments identified 2096 nonoverlapping genomic regions significantly bound by Twist within one or both developmental time periods. This set includes all known Twist-bound enhancers tested, except the eve-cardiac enhancer that is regulated outside the period of development assayed. The majority of Twist-bound regions are found within introns of gene loci, rather than noncoding 5' and 3' regions. A similar positional bias was also observed for p53 and Krüppel, suggesting that introns close to the transcriptional start site represent hotspots for active CRMs. Intronic binding of Twist correlates significantly with the misregulation of these genes' expression in twist loss-of-function mutant embryos and their expression within the ventral blastoderm and mesoderm (Sandmann, 2007).

One of the major challenges for ChIP-on-chip studies is to accurately link the TF-bound enhancers to their appropriate target gene. Rather than simply taking the closest 5' or 3' gene, a more stringent approach was taken and a Twist-bound region was not assigned to a gene based on proximity alone. The results demonstrate that Twist binds more frequently to gene loci genetically downstream from the TF and/or expressed in the same cells as the TF. These criteria to systematically match all 2096 Twist-bound regions (intronic or intergenic) to their likely targets, leading to a high-confidence gene assignment for 854 Twist-bound sequences. This increased the number of Twist direct targets from the previously known 11 to 494 genes. All Twist-bound regions and surrounding genes can be visualized and searched at http://furlonglab.embl.de (Sandmann, 2007).

The RedFly database contains a comprehensive collection of previously described Drosophila enhancers, mainly characterized through single gene studies. Of the 2096 Twist-bound regions, 143 overlap with known enhancers for 62 genes, confirming that these regions have regulatory potential in vivo. Twist was not known to bind to many of these enhancers; this overlap therefore provides strong evidence for a regulatory link between Twist and the 62 target genes (e.g., Abd-A, Abd-B, aop, Brd, slp1, and bap). To further examine the regulatory potential of Twist-bound regions, reporter constructs of new putative enhancer sequences were tested in transgenic animals. Six Twist-bound regions within or close to the following gene loci were assayed: T48, trbl, retn, CG4221, CG8788, and CG32372. All regions proved sufficient to function as enhancers in vivo and could reproduce all or part of the endogenous spatio-temporal gene-expression pattern (Sandmann, 2007).

The T48, tribbles, retained, CG4221, and CG8788 enhancers initiate expression within the early blastoderm. The T48 module mirrors the expression of the endogenous gene within the presumptive mesoderm. The zygotic expression of tribbles is highly dynamic, which is reflected by the assayed CRM. This enhancer drives expression very transiently in the ventral blastoderm and quickly becomes ubiquitously expressed. The relatively small enhancer region for retained is activated in the anterior and posterior ventral blastoderm, where it is coexpressed with Twist, and its expression extends into the dorsal blastoderm. The CRMs for CG4221 and CG8788 initiate expression in the presumptive mesoderm, and continue to drive expression throughout the trunk mesoderm at later stages. The expression of the CG32372 module initiates after gastrulation in the head mesoderm, a domain that overlaps with twist expression. It is interesting to note that Twist binds to multiple enhancer regions for many of these genes. This feature is also evident more globally: Almost 50% of Twist target genes have two or more Twist-bound enhancers, reflecting the complexity of their regulation (Sandmann, 2007).

In summary, these results demonstrate that ChIP-on-chip experiments provide a sensitive and accurate global map of Twist-bound regulatory regions during key stages of early mesoderm development (Sandmann, 2007).

Twist activity is essential for target gene expression

To assay the requirement of Twist function for target gene expression, the expression was examined of six novel direct targets in twist mutant embryos. These genes are expressed in the presumptive mesoderm prior to gastrultion, and therefore at stages when the role of twist function can be assessed. Mesodermal cells are absent in twist mutant embryos later in development due to a block in gastrulation. Triple-fluorescent in situ hybridization was performed using probes directed against twist (blue channel; while twist1 is a protein-null allele, twist RNA is still expressed), inflated (red channel; this gene is dependent on twist for its expression and was used as a marker to distinguish homozygous mutant embryos from their siblings), and a probe directed against one of the six direct target genes (green channel). The spatial expression of all six targets overlaps with twist within the presumptive mesoderm (Sandmann, 2007).

Importantly, twist activity is essential for the expression of five out of six genes examined. Note, for CG32982 and CG9005, residual expression remains outside the twist expression domain in the dorsal and posterior blastoderm, respectively. These results, in combination with in vivo binding data, indicate that Twist binding to a CRM is a prerequisite to activate target gene expression for a large percentage of its targets. The role of Twist binding to the NetA enhancer remains unclear. Twist may act redundantly with other TFs, or alternatively may function in a more subtle manner to modulate the levels of expression (Sandmann, 2007).

Twist and Dorsal collaborate much more extensively than previously predicted

One of the earliest functions of Twist within the pregastrula embryo is the coregulation of D-V patterning with the NFkappaB ortholog Dorsal. Dorsal acts as a morphogen by regulating its target genes at (at least) three threshold concentrations along the D-V axis. Type I-regulated Dorsal enhancers receive high levels of Dorsal, contain low-affinity Dorsal sites and drive expression in ventral mesodermal domains (e.g., sna, htl, twi). Type II enhancers receive intermediate levels of Dorsal and drive expression in mediolateral domains of different sizes (e.g., sim, brk, vn), while Type III enhancers receive low levels of Dorsal, contain high-affinity Dorsal sites, and can be either activated (sog, ths) or repressed (dpp, tld, zen) by Dorsal. This system has been studied so intensively that the level of knowledge is sufficient for quantitative modeling of cis-regulatory interactions. It was therefore of interest to determine whether global analysis could reveal new insights into this process. The data identified in vivo binding of Twist to both Type I and II Dorsal enhancers, as expected. The boundaries of Twist binding are in remarkable agreement with the limits of characterized minimal enhancers (e.g., htl, rho, and ths). More importantly, new CRMs were identified for several of these well-characterized genes (Sandmann, 2007).

Seven novel enhancers for D-V patterning genes reveal the regulatory complexity of Twist-bound CRMs: The cactus, stumps, and wntD enhancers drive expression in a domain overlapping Twist within the ventral blastoderm and likely represent Type I enhancers. Cactus, an IkappaB ortholog, is expressed both maternally and zygotically and sequesters Dorsal within the cytoplasm. While the regulation of zygotic cactus expression was previously not understood, these data reveal a Twist-bound CRM that is sufficient to drive expression in the presumptive mesoderm. Twist also binds to a CRM of Toll. Although the function of cactus'and Toll's zygotic regulation remains unclear in Drosophila, positive feedback regulation of zygotic Toll-receptor expression is required to refine the Dorsal nuclear gradient in the flour beetle Tribolium castaneum (Sandmann, 2007).

The stumps CRM is expressed in a subset of Twist-expressing cells, yielding a salt and pepper pattern that may reflect the requirement for a second, partially redundant enhancer (e.g., the 'stumps_early' enhancer) to give robust expression. The wntD CRM is highly expressed at the anterior and posterior poles of the ventral blastoderm, but is very weakly expressed within the central region. This mirrors the transient expression of the endogenous gene at this stage of development. This single enhancer reflects the regulatory logic deduced from genetic studies: The inputs from Twist and Dorsal activate WntD, while Snail represses its transcription within the presumptive mesoderm. The CRM for crumbs reproduces the endogenous genes expression. This 480-bp region can function as an enhancer in the ectoderm while acting as a silencer within the ventral blastoderm. This ventral repression is most likely due to direct input from Snail on this CRM. Therefore, even at the same stage of development, these four Twist-bound CRMs drive expression in different spatial patterns within a small population of cells. This complexity is clearly mediated by context-dependent integration of additional inputs. Three additional CRMs for mir-1 (Type I), vn, and sim (Type II Dorsal targets) drive expression later in development, reproducing part of the endogenous gene’s expression (Sandmann, 2007).

Unexpectedly, Twist also binds to characterized Dorsal Type III enhancers known to regulate dpp, ind, and ths. Dorsal and its associated corepressors Cut, Retained, and Capicua recruit Groucho to repress dpp, confining its expression to the dorsal blastoderm. The cobinding of Twist and Dorsal to Type III CRMs suggests that these factors may also collaborate in transcriptional repression. Interestingly, Twist binds to regulatory regions of all three Dorsal corepressors, providing another level at which Twist may modulate Dorsal-mediated repression. Overall, this exhaustive map of new CRMs for D-V patterning genes greatly extends previous knowledge and will likely improve predictive models for this system (Sandmann, 2007).

Twist targets functional modules required for diverse aspects of mesoderm development

Twist is not only required for D-V patterning. The 494 direct target genes are significantly enriched in functional groups of genes involved in cell communication, signal transduction, cell motility, and cell adhesion. Genes in these categories are essential for multiple aspects of development, including gastrulation and directed migration of mesodermal cells. Genetic studies have demonstrated a requirement for twist in these processes; however, the molecular mechanism remained ill-defined. These data reveals Twist binding to CRMs for entire functional modules necessary for both gastrulation and migration (the FGF pathway) (Sandmann, 2007).

The present study highlights a new direct connection between Twist and many key components involved in cell cycle progression and cell growth. Members of both the Cdk2/CyclinA/B and Cdk2/CyclinE complexes are targeted, as well as modifiers of their activity and genes involved in cytokinesis and replication. In many cases, Twist binds to several CRMs of these genes (e.g., cyclinE and E2f), revealing the complexity of their regulation. This surprising link between Twist and the cell cycle is highly likely to be of regulatory significance; twist mutant embryos have proliferative defects that can be genetically separated from the block in mesoderm gastrulation (Sandmann, 2007).

These three functional groups of target genes (involved in morphogenesis, migration, and cell proliferation) have been defined as essential developmental network 'plug-ins.' Twist orchestrates early mesoderm development by binding to CRMs of virtually all genes within functional groups essential for gastrulation, mesoderm proliferation, migration, and specification. In contrast, few CRMs for genes involved in terminal differentiation (e.g., sarcomere structure) are targeted by Twist (Sandmann, 2007).

Twist is a highly connected hub targeting a large repertoire of TFs

This global map of Twist-bound CRMs provides a first glimpse of Twist’s connectivity to the rest of the regulatory genome. Remarkably, TFs represent the largest group of Twist targets: Twist binds to CRMs of a striking 25% (113/454) of all sequence-specific Drosophila TFs. Among these are TFs essential for mesoderm development, including gap (hb, hkb, kr, kni), pair rule (eve, slp, opa, odd, prd, run), and segmentation genes (en, hh, ptc, wg), as well as homeotic genes (pb, Scr, Antp, Abd-A, Abd-B, Ubx). These classes of target genes implicate a new role for Twist in the establishment or maintenance of anterior-posterior patterning within the mesoderm in addition to its known role in D-V axis formation. Although the function of many of the remaining TFs is unknown, this data links these regulators to mesoderm development. The sheer number of TFs regulated by Twist does not support a simple hierarchical network, where Twist regulates a small set of TFs, which in turn control another layer of regulators, and so forth. Rather, the data suggests a model for Twist contributing to the regulation of the majority of TFs involved in every aspect of early mesoderm development (Sandmann, 2007).

Temporal enhancer occupancy by Twist reveals stage-specific coregulators

Although Twist is expressed during both developmental time periods assayed, it binds to CRMs in a temporally regulated manner. Approximately half of the enhancer regions are detected at both time periods, indicating continuous binding of Twist throughout these developmental stages. In contrast, 23% of Twist-CRMs are only bound in early development (2-4 h), while 26% are specific to later time periods (4-6 h). This dynamic occupancy reveals that the ability of Twist to bind to CRMs is tightly controlled beyond the mere presence of a suitable binding site, and is likely regulated by other TFs that aid or inhibit binding. To identify additional regulators that could differentiate between temporally bound CRMs, a search was performed for overrepresented sequence motifs, using two complementary computational approaches: statistical enrichment of position weight matrices (PWMs) for characterized TFs, and the de novo detection of overrepresented motifs (Sandmann, 2007).

Twist and Snail consensus motifs are significantly overrepresented in all three groups of CRMs, indicating a potential for extensive cobinding between these two TFs. In contrast, Dorsal motifs are exclusively enriched in the early-bound CRMs, and not in the late group. While Tinman motifs are specifically overrepresented in the continuous and late-bound CRMs. A number of other motifs were also uncovered, including sites for potential Twist/Daughterless heterodimers, suggesting additional mechanisms to generate diverse outputs from Twist-CRMs (Sandmann, 2007).

These data reveals Twist binding to almost all previously characterized Dorsal enhancers. Twist and Dorsal are known to interact physically and to coregulate enhancers in the early, but not the late, time window of this experiment. It is therefore hypothesized that Dorsal may be coregulating many of the newly discovered Twist CRMs, in keeping with the specific enrichment of Dorsal consensus motifs within these enhancers. To experimentally test Dorsal's presence on predicted sites in vivo, ChIP experiments were performed at 2-4 h of development. Significant binding of Dorsal was detected by quantitative real-time PCR to all seven predicted sites tested. Similarly, since Tinman consensus sites were significantly enriched in 4-6-h CRMs, the in vivo occupancy of predicted sites by Tinman was tested at this stage of development. ChIP experiments detected significant binding of Tinman to 10 of 11 sites tested. Given the large number of early and late CRMs, the enrichment of these motifs highlights extensive combinatorial binding of Dorsal and Twist at 2-4 h, and Tinman and Twist at 4-6 h. A substantial part of Twist's temporal specificity likely stems from its association with these upstream and downstream coregulators (Sandmann, 2007).

A core transcriptional network for early mesoderm development

To delineate the combinatorial relationships between Twist and other TFs, an initial transcriptional network was generated for early mesoderm development. The temporal binding map for Twist was integrated with in vivo binding data for Mef2, Dorsal, and Tinman. A previous study of Mef2-bound enhancers offers the largest collection of regulatory regions bound at this stage of development to date. As it is difficult to visualize all 494 Twist target genes, focus was placed on TFs whose CRMs are cobound by two or more regulators during these stages of development. Therefore, all links in this network represent direct connections to the same CRM at the same stages of development (Sandmann, 2007).

The resulting core network of 51 TFs is already relatively complex, with nine genes [nau, E(spl), eve, bap, Ubx, lbe, odd, hth, and Ptx1] being targeted by three out of the four examined regulators. The topology of the network provides several insights into how Twist functions to regulate multiple aspects of early mesoderm development. Extensive combinatorial binding and feed-forward regulation are abundant features. Dorsal activates twist, which in turn coregulates the majority of known direct Dorsal targets. This network motif is even more prominent within the mesoderm: Twist regulates the expression of Mef2 and tinman, and cobinds with these TFs to many of their targets' enhancers. In fact, Twist co-occupies 42% of all Mef2-bound enhancers during early mesoderm development. Depending on the logical inputs from the two upstream regulators (transcriptional repression or activation), feed-forward loops can aid in cellular decision making by filtering out noisy regulatory inputs or control the timing of a transcriptional response. For example, early gene expression in the mesoderm (e.g., activation of tin) depends on Twist alone, while transcription of other genes initiated at a later stage may require the input from both Twist and Tinman proteins (Sandmann, 2007).

Twist-bound CRMs correspond to silencers as well as enhancers of transcription

Through the integration of ChIP-on-chip analysis with expression profiling data during early stages of Drosophila development, this study has identified >2000 Twist-bound regulatory regions and almost 500 direct target genes. This data, in combination with in vivo binding data for other TFs, lays the foundation of a transcriptional network describing early mesoderm development. The resulting network view reveals regulatory features that form the basis of Twist's functional versatility (Sandmann, 2007).

The data revealed extensive Twist binding to characterized Dorsal enhancers and also, surprisingly, to Dorsal-regulated silencers (e.g., dpp). Moreover, many of the new regulatory regions identified for D-V patterning genes can function either as enhancers or integrated enhancer-silencer modules (e.g., WntD and crumbs). This ability of Twist to act within the context of silencers, as well as enhancers, may partially explain the widespread recruitment of Twist to many regulatory regions and its ability to regulate diverse developmental processes (Sandmann, 2007).

An attractive molecular explanation for this bifunctionality is the potential of Twist to form both homodimers and heterodimers. Twist homodimers drive gene activation in Drosophila, while Twist-Daughterless heterodimers are associated with transcriptional repression. This model is supported by the significant overrepresentation of the Twist/Daughterless heterodimer consensus motif in both 2-4-h and 4-6-h CRMs. Direct protein-protein interactions between Twist and Dorsal is an alternative mechanism for Twist's incorporation into repressive complexes (Sandmann, 2007).

A network with unexpected topology governs early mesoderm development

Although the network generated in this study is far from complete, it represents the largest set of combinatorial-bound CRMs during these stages of development described to date, and therefore provides a comprehensive resource to decipher general regulatory principles. The resulting network topology was surprising. Instead of Twist regulating a restricted group of TFs, which in turn regulate a successive wave of transcription in a relay model, Twist directly impinges on CRMs for the vast majority of genes expressed in the early mesoderm (Sandmann, 2007).

The extent of combinatorial binding was also unanticipated. There is extensive cobinding of Twist and Dorsal to early 2-4-h CRMs. In fact, the presence of Dorsal binding may be a general prerequisite for Twist binding to enhancers specific for early development. The cooperative binding of Dorsal and Twist to the rho and sim CRMs supports this model. At 4-6 h of development, the composition of TFs impinging on Twist-bound CRMs changes. Although genome wide ChIP-on-chip data is currently not available for Tinman, the significant overrepresentation of Tinman motifs in Twist-bound CRMs and the ability of Tinman to bind to the majority of sites tested indicates prevalent combinatorial binding between these two TFs during 4-6 h of development. Comparing Twist-bound CRMs with a previously generated data set for Mef2 revealed extensive cobinding to enhancers early in development. Converging regulatory connections through combinatorial binding can produce diverse logical outputs, depending on the nature of the TFs. The cobinding of several pan-mesodermal TFs (Twist, Tinman, and Mef2) may ensure robust gene expression. While in other contexts (for example, the WntD-enhancer) the combined inputs of Twist and Snail allow for spatial fine-tuning of gene expression (Sandmann, 2007).

The core network also revealed an abundance of feed-forward loops, providing directionality during early mesoderm development. This is prevalent with both upstream regulators of Twist (Dorsal and Twist) and downstream regulators (Tinman and Twist and Mef2 and Twist). This network motif will likely become even more widespread as additional ChIP-on-chip data becomes available. Twist targets an astounding number of TFs, which may represent an almost complete repertoire of TFs required for early mesoderm development. It is tempting to speculate that Twist participates in feed-forward regulation, with many of these factors through combinatorial binding to different subsets of the ~2000 Twist-bound CRMs (Sandmann, 2007).

Temporal network dynamics reflect developmental progression

Both the composition and connectivity of regulatory networks describing developmental progression will naturally change over time. To capture dynamic changes within the early mesodermal network, these experiments were performed at consecutive time periods. The data reveals temporally regulated binding of Twist to three classes of CRMs: early, continuous, and late. Similar temporally restricted enhancer occupancy has also been observed for other regulators with broad temporal expression, suggesting that this may be a general feature of developmental networks—e.g., MyoD, PHA-4, and Mef2 (Sandmann, 2007).

The temporal occupancy of specific CRMs by Twist reflects the development of this tissue. At 2-4 h of development, Twist and Dorsal coregulate genes essential for D-V patterning. Twist also targets an almost complete set of genes essential for gastrulation and is required to progress to the next phase of development, mesoderm maturation. During this developmental window, the predominant target genes are part of functional modules essential for the cell migration, proliferation, patterning, and specification events occurring within the mesoderm at these stages. As expected for a TF essential for early aspects of mesoderm development, Twist does not bind to significant numbers of CRMs for genes involved in terminal differentiation. This is in sharp contrast to Mef2, which first co-occupies CRMs involved in early mesoderm development with Twist, and later selectively regulates an alternative group of CRMs driving genes involved in later aspects of differentiation; e.g., sarcomere structure or muscle attachment (Sandmann, 2007).

Conserved regulation of functional classes of genes by Twist

Integrating these data with genetic evidence from other species suggests that the regulation of several functional gene cassettes by Twist is conserved throughout evolution, from flies to man. These include (1) the FGF signaling pathway: Mutations in human FGF receptors phenocopy mutants in human twist (Htwist). (2) Genes implicated in epithelial-mesenchymal transitions (EMTs): In mice and humans, Twist facilitates tumor metastasis through the promotion of EMTs. (3) Cell proliferation and apoptosis: Htwist has been classified as a potential oncogene, since it maintains tissue culture cells in a proliferative state. Interestingly, ectopic expression of Htwist in Drosophila also induces proliferation and inhibits p53-dependent apoptosis, indicating that the ability to regulate these processes is conserved. However, for each process, only a few Twist-regulated genes have been known. Extrapolating from the current findings in flies points toward a role for Twist in the direct regulation of entire gene modules required for each process in vertebrates (Sandmann, 2007).

An emerging model for Twist as a global competence factor for mesoderm development

The results provide an initial global view of the transcriptional network describing early mesoderm development within the metazoan Drosophila. Twist resides at the top of this network and binds to CRMs for the vast majority of genes that need to be expressed during these stages. In many cases, Twist is essential and sufficient to drive expression of the target gene. In other cases, however, the contribution of Twist remains unclear (e.g., crumbs and NetA) . Rather than acting as a binary switch, Twist may act redundantly with other TFs. Alternatively, Twist may provide the competence for more specific TFs to bind to these CRMs; for example, by acting as a pioneer TF to facilitate chromatin remodeling (Sandmann, 2007).

In species as diverse as flies, jellyfish, and mice, Twist is only expressed in mesodermal cells when they are in an immature state, and loss of twist expression correlates with the initiation of differentiation. Moreover, overexpression of Twist-1 in mice is sufficient to block osteoblast differentiation. It is suggested that Twist provides the mesoderm with the competence to be pluripotent: first, by providing these cells with the components necessary to respond to inductive cues directing further specification; and second, by providing an almost universal repertoire of mesodermal CRMs with the competence to respond to other TFs. Once bound by Twist, these regulatory regions may be primed for activation by more specialized TFs, and thereby allow rapid developmental progression at the appropriate time (Sandmann, 2007).

Myocyte enhancer factor 2 and chorion factor 2 collaborate in activation of the myogenic program in Drosophila

The process of myogenesis requires the coordinated activation of many structural genes whose products are required for myofibril assembly, function, and regulation. Although numerous reports have documented the importance of the myogenic regulator myocyte enhancer factor 2 (MEF2) in muscle differentiation, the interaction of MEF2 with cofactors is critical to the realization of muscle fate. This study identified a genomic region required for full MEF2-mediated activation of actin gene expression in Drosophila, and the zinc finger transcriptional regulator Chorion factor 2 (CF2) was identified as a factor functioning alongside MEF2 via this region. Furthermore, although both MEF2 and CF2 can individually activate actin gene expression, these two factors collaborate in regulating the Actin57B target gene in vitro and in vivo. More globally, MEF2 and CF2 synergistically activate the enhancers of a number of muscle-specific genes, and loss of CF2 function in vivo results in reductions in the levels of several muscle structural gene transcripts. These findings validate a general importance of CF2 alongside MEF2 as a critical regulator of the myogenic program, identify a new regulator functioning with MEF2 to control cell fate, and provide insight into the network of regulatory events that shape the developing musculature (Tanaka, 2008).

A major function of the mesodermal germ layer during embryogenesis is the generation of myogenic tissue and the subdivision of cells into skeletal, cardiac, and visceral muscle precursors. This process has been broadly conserved during evolution. Specification of the three major muscle lineages is conserved from Drosophila to mammals, and conserved structural genes are activated in each lineage to provide power for locomotion, circulation, and digestion (Tanaka, 2008).

The transcription factors that mediate muscle cell differentiation are also broadly conserved. Foremost among these proteins are members of the myocyte enhancer factor 2 (MEF2) family of transcriptional regulators. MEF2 proteins dimerize and bind to DNA via conserved MADS and MEF2 protein domains, and MEF2 proteins recognize an AT-rich sequence, which is found in the promoters and enhancers of numerous muscle-specific genes. MEF2-encoding genes are found in all animal genomes, from single-gene copies in Caenorhabditis elegans (named CeMef2) and Drosophila melanogaster (named Mef2), to four genes in mammals (named mef2a to -d) (Tanaka, 2008).

Consistent with the pervasiveness of MEF2 sites in muscle-specific genes and the conservation of this gene family during evolution, mutational studies in several animal models have established important roles for MEF2 factors in muscle differentiation. In Drosophila, inactivation of the single Mef2 gene resulted in a complete failure of differentiation for all muscle lineages. In mice, inactivation of mef2c resulted in a failure of normal cardiac and visceral muscle development. Furthermore, inactivation of murine mef2a caused cardiac failure shortly after birth. Also, expression of dominant-negative isoforms of MEF2 predicted to inhibit all MEF2 function resulted in the inhibition of skeletal myogenesis in mammalian cells in vitro and in interference in cardiac development in vivo. Finally, a point mutation in the human gene encoding MEF2A is associated with susceptibility to cardiac disease. These studies present a strong argument for the importance of MEF2 proteins in muscle development and disease (Tanaka, 2008).

Despite the critical requirement of MEF2 for muscle differentiation, MEF2 proteins are not themselves capable of activating the myogenic program in naive cells in tissue culture. Similarly in Drosophila, ectopic expression of Mef2 in the ectoderm caused the activation of some muscle-specific markers, and yet failed to induce myogenesis at high levels. These findings suggested that MEF2 must act with specific cofactors in order to control myogenesis. Much progress has subsequently been made in defining both positive and negative cofactors for MEF2, in a variety of muscle tissues. MEF2 collaborates positively with skeletal muscle-specific members of the basic helix-loop-helix family of transcriptional regulators, including MyoD and myogenin, to control skeletal myogenesis. In addition, the SAP domain proteins Myocardin and MASTR also stimulate the transcriptional activity of MEF2 in cardiac and skeletal muscle tissue, respectively. MEF2 proteins also interact with ubiquitous factors, such as the p300 coactivator and members of the histone deacetylase family of transcriptional repressors (Tanaka, 2008).

In Drosophila, there is relatively little direct data concerning the identification of cofactors that might collaborate with MEF2 to control myogenesis. Nevertheless, a number of enhancers for muscle-specific genes have been described, many of which contain MEF2 binding sites that are critical for full gene activation. Among these, the promoter has been described for the Act57B actin gene in Drosophila (Kelly, 2002). Act57B is the predominant embryonic muscle actin, being expressed at high levels in all of the major muscle lineages and also being one of the earliest markers of muscle differentiation in the embryo. Activation of Act57B expression in all embryonic muscle lineages is controlled by a proximal 595-bp promoter element. Within this region, full transcriptional activation required the integrity of a single MEF2 binding site at position -209 relative to the transcriptional start site, validating the importance of MEF2 as a direct regulator of myogenesis in this system. This finding was also supported by the study of Sandmann (2006), who showed, using microarray analysis, that Act57B expression was strongly downregulated in Mef2 mutants. Furthermore, the Sandmann study utilized chromatin immunoprecipitation-microarray analyses to identify the MEF2 site at position -209 as an in vivo target of MEF2 (Tanaka, 2008).

An ~300-bp region distal to the MEF2 site is critical for full Act57B activation in embryonic muscle tissue, suggesting the existence of a MEF2 cofactor that interacted with this distal sequence. This analysis permitted a directed approach to be carried out to identify and functional characterize a MEF2 collaborating factor in Drosophila. This study delineates sequences within the 300-bp distal region that are required for full gene activation in vivo; a nuclear factor binds to this region with the characteristics identical to that of the C2H2 zinc finger transcriptional regulator chorion factor 2 (CF2). This study further shows that MEF2 and CF2 can each activate Act57B expression in vitro and in vivo and that these factors function synergistically to maintain high levels of actin expression in the Drosophila embryo. More globally, MEF2 and CF2 synergistically activate other muscle structural genes, and loss of CF2 function results in reductions in the expression levels of several muscle structural genes, including Act57B. These findings describe the first collaborating factor for MEF2 in the Drosophila system and further delineate the network of transcriptional events required for normal muscle development (Tanaka, 2008).

A region of the Act57B promoter from positions -593 to +2 can provide robust levels of muscle-specific expression of a lacZ reporter gene in transgenic animals. Within this fragment, at position -209, is a conserved MEF2 binding site that was critical for full promoter activity. Furthermore, 5' deletion of 315 nucleotides to generate a -278/+2 Act57B-lacZ reduced reporter gene expression. These findings established the importance of the MEF2 binding site for full activation of the Act57B gene (Kelly, 2002). However, since the -278/+2 construct showed reduced reporter activity despite retaining the MEF2 binding site at position -209, the data also indicated that a genomic region separate from the MEF2 site was critical for normal levels of gene expression, which might therefore interact with a cofactor for MEF2 (Tanaka, 2008).

To more precisely delineate regions critical to Act57B expression, a further 5' deletion analysis of the -593/+2 fragment was performed. At least three transgenic lines were assessed for each construct, and representative images are presented. Expression of the full-length construct was first detected at stage 11 of embryogenesis in visceral muscle precursors, and by stage 12 in skeletal muscle and visceral muscle precursors; high levels of reporter gene expression were still observed at stage 16 in all muscle lineages. 5' deletion of ~70 bp to position -521 did not affect the initiation of Act57B expression at stage 11, although reporter gene expression levels were consistently reduced at stage 16. This was apparent by the lesser ease with which individual muscle fibers could be distinguished in stained embryos at higher magnifications. A further 5' deletion of 130 bp to position -390/+2 showed no additional strong reduction in reporter gene expression. In contrast, the smallest -278/+2 Act57B-lacZ showed reduced reporter activity at stage 12, and reporter levels also appeared to be further reduced at stage 16 compared to larger genomic fragments that were tested (Tanaka, 2008).

These studies demonstrated that more than one region of the Act57B upstream promoter is required for normal reporter gene expression in vivo. Although it is difficult to assign relative levels of expression in these immunohistochemical stains, two regions seem most critical to normal expression: the region between positions -593 and -521 is a genomic element required for full gene activation at later stages of development, and a more proximal region from positions -390 to -278 is important for the initiation of Act57B expression at stages 11 and 12, as well as for later sustained Act57B expression. Subsequent studies therefore focused initially upon the identification of factors interacting with this more proximal region (Tanaka, 2008).

In order to determine whether factors existed that were capable of interacting with the Act57B promoter region, electrophoretic mobility shift assays were carried out using as a probe a -390/-245 region of the Act57B gene. When this fragment was mixed with nuclear extracts prepared from 12- to 24-h-old Drosophila embryos, the formation was observed of a slowly migrating complex, which is hypothesized to corresponded to a transcription factor capable of regulating Act57B expression (Tanaka, 2008).

To more precisely locate the region of the DNA probe with which the nuclear factor was interacting, binding reactions were compated with sets of double-stranded oligonucleotides spanning the entire probe sequence. It was found that, whereas most of the oligonucleotide competitors failed to reduce the intensity of the bound complex, double-stranded DNA probes of length approximately 30 bp, termed here oligonucleotides 11 and 12, effectively competed for the formation of the complex (Tanaka, 2008).

These findings were important for two reasons. First, the observation that some oligonucleotides were capable of competition, whereas others were not, indicated that the interaction of the nuclear factor with the Act57B probe was sequence specific. Second, competition assays identified more precisely the region of the Act57B gene that was interacting with the nuclear factor (Tanaka, 2008).

Observation of the DNA sequence corresponding to the locations of oligonucleotides 11 and 12 revealed several repeats of a 5'-TATA-3' motif, which lies at the core of the recognition sequence of the transcription factor CF2. Since CF2 is a C2H2 zinc finger protein that regulates gene expression in the chorion cells of the maturing egg in Drosophila, and since embryonic expression of CF2 is exclusive to the three muscle lineages of the mesoderm (Bagni, 2002), it was reasoned that CF2 might be interacting specifically with the Act57B enhancer (Tanaka, 2008).

To determine whether CF2 could interact with the Act57B gene enhancer, an electrophoretic mobility shift assay was carried out using as a probe the -390/-245 region and using the CF2 protein generated in vitro for binding. A robust interaction was found between CF2 and the -390/-245 region, and this interaction was competed for with excess nonradioactive -390/-245 sequence. More importantly, when the interaction of CF2 and -390/-245 probe was competed using the oligonucleotides spanning the region, CF2 binding was competed for only by oligonucleotides 11 and 12 (Tanaka, 2008).

These findings firstly demonstrated that CF2 is capable of interacting with the Act57B promoter. This is likely to be a relevant interaction given the broad expression of CF2 in the embryonic mesoderm. Second, these results showing identical responses to binding competition by a nuclear factor and by CF2 protein strongly suggested that CF2 is the interacting factor identified in nuclear extracts. It is noted that when CF2 protein is synthesized in vitro the electrophoretic mobility shift assays show two shifted bands of very similar mobilities. This probably arises from the synthesis of two CF2 isoforms in the in vitro expression system. Whether this arises from posttranslational modification of the CF2 protein or from internal translation initiation is not clear. Nevertheless, there is a strong and specific interaction between CF2 protein and the Act57B promoter region (Tanaka, 2008).

Clearly, the C2H2 zinc finger transcription factor CF2 is able to bind potently to regions of the Act57B promoter that were critical for full levels of gene expression, suggesting that it might be a positive regulator of Act57B transcription. To test the hypothesis that CF2 is an Act57B activator, a series of cell culture experiments was initiated aimed at determining whether MEF2 or CF2 can activate Act57B promoter-lacZ constructs. First, Drosophila S2 cells were cotransfected with the full-length -593/+2 promoter-lacZ, along with a MEF2 expression plasmid. This resulted in strong and reproducible activation of the reporter. In contrast, a point mutation of the putative MEF2-binding site within the -593/+2 promoter-lacZ construct dramatically impaired MEF2-dependent reporter responsiveness. These results confirmed the importance of the sole MEF2 site in this construct for Act57B gene activation (Tanaka, 2008). When the ability of CF2 to activate the wild-type Act57B-lacZ construct was tested, significant activation of the reporter occured, suggesting that CF2 was indeed an activator of this gene. To determine whether CF2 activates Act57B via the sites identified in vitro, the ability of CF2 to activate the 5'-deleted reporter constructs was tested. Indeed, deletion of 5' nucleotides to -390/+2, predicted to remove the most distal CF2 binding sites, reduced reporter activation by CF2 by ca. 50% compared to the full-length promoter fragment. The shortest version of the Act57B promoter, -278/+2, lacking both of the predicted CF2-binding regions, was completely unable to respond to CF2. Similarly, tests were performed to see whether the CF2-binding regions alone are sufficient for reporter activation, in the proximally truncated construct -593/-245. Here, there was strong CF2-dependent activation, although it did not reach full strength compared to the -593/+2 promoter construct. Apparently, some unknown regulatory elements in the proximal part of the Act57B promoter, perhaps its basal promoter, may additionally boost transcriptional activation initially conferred by the CF2-binding regions. Also, comparative analysis of this construct might be complicated by the observation that its basic activity was more than two times higher than that of the other tested constructs (Tanaka, 2008).

Taken together, these provide strong support for the identification of CF2 as an activator of Act57B: CF2 is capable of binding to two regions of the promoter, which were shown to be important for full reporter gene expression in vivo; furthermore, CF2 is identified as a strong transcriptional activator of the Act57B promoter via these same sequences in tissue culture (Tanaka, 2008).

Analyses performed on the promoter of Act57B provided direct evidence that CF2 can independently initiate and synergistically collaborate with MEF2 to maximize expression of this muscle-specific gene. However, it was of particular interest to investigate whether CF2 is more broadly involved in the regulation of the transcriptional activities of other structural muscle genes. In order to address this question, promoter reporters were analyzed of two genes for which the muscle enhancers had been identified: Troponin I (TnI) and Myosin heavy chain (Mhc) (Tanaka, 2008).

Transfection studies in cell culture indicated that CF2, as well as MEF2, could individually be effective transcriptional activators for both TnI and Mhc reporters. This is the first direct evidence in support of the roles of MEF2 and CF2 upon these structural genes. More importantly, it was found that CF2 and MEF2 also synergistically act upon the promoter-lacZ constructs for both TnI and Mhc. These findings supported a general role for the collaboration of CF2 and MEF2 in the control of embryonic muscle development in Drosophila. It was interesting that the titration analyses of the TnI and Mhc reporters showed different degrees of synergism between MEF2 and CF2: whereas dual activation of the TnI reporter with MEF2 and CF2 just slightly exceeded the predicted additive limit, the Mhc reporter showed a remarkable boost of activation conferred by the combination of these two factors (Tanaka, 2008).

Given the demonstrations that CF2 could activate Act57B gene expression in vivo and in tissue culture, it was shown that tests of loss of CF2 function resulted in a reduction or loss of expression of muscle structural genes. A Cf2 mutant named Df(2L)gamma, described by Bagni (2002), comprises a deletion of the Cf2 transcribed region and is homozygous larval lethal. In order to determine whether the loss of Cf2 function affects muscle structural gene expression, homozygous mutant embryos (at stage 16-17) or first-instar larvae were collected and assayed for the accumulation of the muscle-specific Act57B, TnI, and Mhc transcripts relative to the expression of the housekeeping Act5C cytoplasmic actin gene by using quantitative RT-PCR. As controls, heterozygous siblings were collected at embryonic or larval stages; these were assayed for expression of the same genes (Tanaka, 2008).

In embryos, there was an ~10% reduction in the levels of expression of all three muscle structural genes assayed relative to the Act5C control expression levels. While this change in expression is relatively small, it must be borne in mind that removal of the CF2 sites from the Act57B-lacZ reporter construct results in similarly moderate effects upon reporter gene expression, even though these two separate experiments cannot be directly compared. More strikingly, at the larval stage the reductions in the levels of muscle gene expression were much more severe, suggesting that a major function of CF2 might be to maintain high levels of expression in fully differentiated tissues. This reduction in the expression of the three genes tested is highly reproducible (Tanaka, 2008).

Overall, these studies identify CF2 as a potent activator of muscle gene expression both in vitro and in vivo and show that CF2 functions alongside MEF2 in controlling high levels of expression of muscle-specific genes (Tanaka, 2008).

This study has identified the first known collaborative factor for MEF2 function in Drosophila. CF2, a transcriptional regulator first identified as an activator of chorion protein genes in the female ovary, is expressed in all three muscle lineages in the embryonic mesoderm. The data indicate that CF2 interacts with the Act57B promoter both in the context of embryonic nuclear extracts and when expressed in vitro, and CF2 binding sites correspond precisely to the genomic regions required for full Act57B-lacZ activation. Furthermore, overexpression studies in vitro and in vivo establish a synergistic relationship between MEF2 and CF2 in the activation of Act57B. These studies define the first known embryonic function for this zinc finger transcriptional regulator. Nevertheless, it is also noted that even in the absence of CF2 sites, in the -270/+2 Act57B-lacZ construct there is significant muscle-specific expression. This indicates that CF2 contributes to high levels of structural gene expression but that it is not essential for the activation of Act57B (Tanaka, 2008).

Removing the genomic regions containing CF2 binding sites had clear effects upon Act57B-lacZ expression at stage 16. In contrast, removal of CF2 function from the embryos in the mutant analysis resulted in a more modest reduction in Act57B transcription. Although these two experiments utilize different approaches and different readouts, what might be the cause of this apparent discrepancy? The most reasonable explanation is that there are probably additional regulators of Act57B that bind to the genomic regions affected in the deletion analyses; thus, the deletions remove the influence not only of CF2 but also of other muscle-specific activators. In contrast, deletion of the CF2 gene might remove only the influence of CF2 from actin gene expression. Future deletion and point mutant analyses, carried out in the manner applied in this study, would test this hypothesis and could ultimately shed light upon the identity of such additional factors (Tanaka, 2008).

More broadly, MEF2 and CF2 can activate other muscle structural genes in Drosophila. Both of the enhancers that tested have predicted binding sites for MEF2 and CF2 proteins (TnI and Mhc); however, the current study privides the first direct functional tests of whether MEF2 and CF2 can transcriptionally activate these enhancers. The findings suggest strongly that the collaboration of MEF2 and CF2 during fly embryonic muscle development is a general and important phenomenon (Tanaka, 2008).

A simple tissue culture assay was used to demonstrate that MEF2 and CF2 collaborate to activate endogenous genes in the context of the intact genome, in addition to transiently transfected reporter constructs. The assay closely supports the findings from other experiments presented in this study and is somewhat more quantifiable than ectopic expression assays in embryos. This is because embryos already express significant levels of the targets genes in the mesoderm, whereas in vitro the baseline from nontransfected cells is essentially zero. Similarly, the lacZ reporter vectors that were use frequently show leaky promoter activity, which might ultimately result in an underestimation of the true activation of target promoter-lacZ constructs. In this sense, the activation of endogenous genes in S2 cells is highly informative (Tanaka, 2008).

Presumably, a more complete set of MEF2/CF2 targets could be generated by cotransfecting S2 cells with expression plasmids for CF2 and MEF2 and then performing an array analysis of transcripts upregulated after this treatment. Such a data set would significantly overlap with the analysis of mesoderm differentiation described by Sandmann (2006), although it might have the advantage of indicating which genes are direct targets of combinatorial activation by MEF2/CF2 (Tanaka, 2008).

Along these lines, the current studies also indicate that there are subtle, yet potentially important, differences between the responsiveness of different target genes to activation by MEF2 and CF2 alone. In tissue culture, Act57B is readily activated by MEF2 and CF2 individually, and yet TnI and Mhc induction are detected far less readily. This might be a simple result of different ease of detection for different targets. Alternatively, there might be a more mechanistic basis for these differences. One explanation might be related to the proximity of binding sites of the respective factors to the target promoter. For Act57B, both MEF2 and CF2 sites that were mapped are within 600 bp of the transcription start site. In contrast, for Mhc most of the candidate sites are located in the first intron, at least 800 bp away from the transcription start site. In contrast the other hand, putative binding sites for MEF2 and CF2 are in relatively close proximity to the TnI transcription start site. Thus, it is not clear whether binding site proximity plays a role in the activation of genes in vitro, although it should be noted that for TnI and Mhc many of the sites have yet to be validated by DNA-binding assays (Tanaka, 2008).

The data of Bagni (2002) indicate that Cf2 expression is dependent upon the function of the Mef2 gene in Drosophila embryos; these authors also show that MEF2 levels are unaffected in Cf2 mutants, at least at the embryonic stage. It is therefore most likely that the reduction in muscle structural gene transcripts in Cf2 mutants arises directly from a loss of CF2 protein rather than from indirect effects upon MEF2 levels. Clearly, one of the means by which MEF2 activates myogenesis at high levels is via the activation of its own collaborative factors. There is some precedent for this mechanism, both in muscle and other systems. In Drosophila cardiac development, the homeodomain transcription factor Tinman is a direct activator of the GATA factor pannier, after which Tin and Pnr can collaborate to activate structural genes such as Sulfonylurea receptor. The activation of, and then collaboration with, a cofactor appears to be a commonly used genomic regulatory mechanism (Tanaka, 2008).

CF2 activity and enhancer integration are required for proper muscle gene expression in Drosophila

The creation of the contractile apparatus in muscle involves the co-activation of a group of genes encoding muscle-specific proteins and the production of high levels of protein in a short period of time. The transcriptional control of six Drosophila muscle genes that have similar expression profiles were analyzed, and these mechanisms were compared with those employed to control the distinct expression profiles of other Drosophila genes. The regulatory elements controlling the transcription of co-expressed muscle genes share an Upstream Regulatory Element and an Intronic Regulatory Element. Moreover, similar clusters of MEF2 and CF2 binding sites are present in these elements. CF2 depletion alters the relative expression of thin and thick filament components. It is proposed that the appropriate rapid gene expression responses during muscle formation and the maintenance of each muscle type is guaranteed in Drosophila by equivalent duplicate enhancer-like elements. This mechanism may be exceptional and restricted to muscle genes, reflecting the specific requirement to mediate rapid muscle responses. However, it may also be a more general mechanism to control the correct levels of gene expression during development in each cell type (García-Zaragoza, 2008).

This study demonstrates that a large group of Drosophila muscle genes that present the same expression profile during development share a basic regulatory organization. This occurs both in genes encoding thick filament components namely, myosin heavy chain (Mhc), paramyosin (PM), tropomyosin 1 (Tm1) and tropomyosin 2 (Tm2), troponin T (TnT) and troponin I (TnI). genes encoding components of the thin filament. The elements involved are an Upstream Regulatory Element (URE) situated in the 5' upstream regions and an Intronic Regulatory Element (IRE) situated in introns 1 and 2 of each gene. Each of these regulatory elements, the URE and IRE, is sufficient to independently activate transcription in all muscle types and hence, they are inferred to be functionally equivalent. Moreover, these regulatory elements are mostly conserved among Drosophilidae. However, in each particular muscle, LacZ reporter activity varies depending on whether the URE or the IRE element is directing transgene expression. Thus, PM expression is very high in the IFM where the IRE element governs reporter expression, whereas it is low when directed by the URE element. In summary, either the URE or IRE elements independently govern transgene expression in all muscle types at all developmental stages. While the URE or IRE elements can dictate when and where these co-regulated genes are expressed during development, on their own they are unable to dictate the correct levels of protein expression in each muscle type (García-Zaragoza, 2008).

It has been demonstrated that the regulation of TnT transcription depends on the synergistic interaction between these two elements (Mas, 2004). Moreover, this synergism varies in each particular muscle type in order to produce the different specific levels of protein required. In conjunction with the data presented in this study and with that from similar studies performed on the TnI gene (Marin, 2004), it is concluded that the correct levels of expression of co-activated muscle genes are established in each muscle type through a direct or indirect interaction between the URE and IRE elements. Thus, this is considered to be a general mechanism that co-ordinates the expression of co-regulated muscle genes, establishing the proper levels of protein required by each muscle type and mediating the rapid responses required for muscle formation (Garcí-Zaragoza, 2008).

In mammals, duplicate enhancer-like elements capable of independently activating transcription in all muscle types and of directing the correct spatio-temporal expression of the same gene have yet to be identified. However, a number of modules or partial regulatory elements, located either upstream (USE/SURE) or downstream (IRE/FIRE) of the transcription initiation site confer transcriptional specificity to slow (TnIs) and fast (TnIf) genes in mouse muscle C2C12 cells. The importance of upstream and downstream elements in driving fibre specific expression of fast and slow TnI genes has been highlighted in studies of transgenic mice. As a result, additional unidentified enhancers were claimed to be necessary to recapitulate the quantitative endogenous expression of the TnI genes. In the context of the current data, further studies will be necessary to determine if, as in Drosophila, mammals use duplicate regulatory elements to establish the proper levels of gene expression in each muscle type. Indeed, preliminary data is available suggesting that these mechanisms might be totally or partially conserved (García-Zaragoza, 2008).

The basic regulatory design described in this study appears to differ in muscle genes with a more restricted expression profiles. This is the case for mPM, which is almost exclusively expressed in adults, as well as for actin 88F or flightin that are flight muscle-specific proteins. Recent functional studies to identify regulatory elements that control mPM expression indicated that the structure and organization of such regulatory elements is completely different to that identified in this study. The three elements identified in mPM are smaller and none of them contain conserved putative MEF2 or CF2 binding sites. The search for similar regulatory elements in actin 88F and flightin genes has been unsuccessful, possibly because these two genes are expressed in distinct muscle subsets than mPM (García-Zaragoza, 2008).

DNA microarray studies in Drosophila have offered an overview of genomic activity during development, grouping together thousands of genes with similar expression profiles. More recently this technology is being directed towards the understanding of the mechanisms driving simultaneous activation of each of these different groups of genes. The studies performed here on the structure of the different URE and IRE enhancer-like elements reveal that they share common organizational features. Thus, the systematic comparison of six Drosophila structural muscle genes and the large-genome scans identified clusters of MEF2-CF2 binding sites within 700-bp fragments. Moreover, these clusters were always located both upstream and downstream of the transcription initiation site of each of the genes analysed. Since these co-regulated genes share expression profiles, attention of this study was focused on other muscle genes that also share similar patterns of gene expression, such as actin 57B or actin 87E. Interestingly, similar clusters of MEF2 and CF2 binding sites were identified close to the transcription initiation sites of these two genes. In fact, it has been recently demonstrated that although both MEF2 and CF2 can individually activate actin gene expression, these two factors collaborate in regulating the actin 57B target gene in vitro and in vivo (Tanaka, 2008). However, no clusters were identified in transcriptional units with distinct developmental expression patterns, such as the mPM, actin 88F, flightin or TnC 41C genes, which are expressed almost exclusively in different subsets of the adult musculature. Furthermore, no MEF2-CF2 clusters were found in 'non-relevant' genes that are expressed in other organs or tissues, such as yellow-e2 that is expressed in the fly cuticle or proneural genes. These results demonstrate that the enhancer organization identified and the conserved clusters in whole-genome scans will be useful for guiding functional analyses. Indeed, this approach may serve to identify new functional components or members of co-expressed groups of genes, as well as grouping together genes with similar patterns of activity (García-Zaragoza, 2008).

A study was recently performed combining chromatin immunoprecipitation with microarray analysis to identify the in vivo 'occupied' MEF2 binding sites present in the whole Drosophila genome (Sandmann, 2006). 1015 overlapping genomic fragments were identified that were grouped as 670 independent DNA fragments, containing 1975 predicted MEF2 binding sites. In order to test the hypothesis that MEF2-CF2 clusters play an important role in the transcriptional regulation of a subset of muscle genes, MEF2/CF2 clusters were sought within these fragments. Significantly, it was found that 15.30% of the MEF2 binding sites have at least one CF2 binding site less than 200 bp away, a frequency that is 9 times higher than it would be expected for a random distribution. Hence, the location of CF2 sites in the vicinity of MEF2 binding sites appears to be conserved in this group of muscle enhancer-like elements. This conservation strongly suggests that these two transcription factors act together to fulfil a central role in regulating muscle development (García-Zaragoza, 2008).

It has been demonstrated that MEF2 is essential for muscle differentiation in both Drosophila and vertebrates, directly activating structural muscle genes. Recent studies demonstrated that MEF2 maintains its own expression in all differentiated muscle cell types, suggesting that it is required for muscle maintenance and growth during myogenesis. In vertebrate skeletal muscle, MEF2 cooperates with the myogenic basic helix-loop-helix (bHLH) transcription factors to activate and maintain the muscle phenotype. Indeed, in Drosophila Twist activates MEF2 expression and its expression declines during myogenesis. To date, no other factors are expressed in all muscles that might help maintain muscle-specific gene transcription and the muscle phenotype (García-Zaragoza, 2008).

Functional and computational analysis suggests that CF2 is a good candidate to collaborate with complexes containing MEF2 in maintaining muscle-specific gene transcription. Since no null CF2 mutants are available, a detailed study was conducted of two hypomorph CF2 mutants. Q-RT-PCR assays, flight tests and EM microscopy confirmed that depletion of the CF2 protein disturbs muscle gene expression, particularly the relative expression of thick and thin filament components. At distinct stages of development, a reduction in the expression of thin filament genes was mostly paralleled by an increase of thick filament gene transcription in both CF2 mutants. This imbalance was more pronounced as the flies aged. In summary, a strong reduction of CF2 alters the correct expression of thin and thick filament components through an unknown mechanism, affecting the stoichiometry of contractile proteins and consequently, myofibril assembly (García-Zaragoza, 2008).

It is concluded that CF2, together with MEF2 and other unknown factors, is an essential component of the macromolecular multi-protein complexes that bind to the URE and IRE enhancer-like elements. Supporting this hypothesis, a very weak interaction between MEF2 and CF2 proteins has been observed in GST-Pull Down assays. Within this complex MEF2 may maintain the muscle phenotype while CF2 guarantees the correct levels of expression of the genes encoding the protein components of thin and thick filaments (García-Zaragoza, 2008).

This study has demonstrated the presence of functionally similar enhancer-like duplicates in Drosophila muscle genes. Direct or indirect interactions between these elements guarantee rapid and strong responses of structural muscle gene expression in muscle formation within each muscle type. It is proposed that this mechanism, or a similar one, might be a general mechanism to control the correct levels of gene expression during development of functionally related co-expressed genes in all cell types (García-Zaragoza, 2008).

Combinatorial binding leads to diverse regulatory responses: Lmd is a tissue-specific modulator of Mef2 activity

Understanding how complex patterns of temporal and spatial expression are regulated is central to deciphering genetic programs that drive development. Gene expression is initiated through the action of transcription factors and their cofactors converging on enhancer elements leading to a defined activity. Specific constellations of combinatorial occupancy are therefore often conceptualized as rigid binding codes that give rise to a common output of spatio-temporal expression. This study assessed this assumption using the regulatory input of two essential transcription factors within the Drosophila myogenic network. Mutations in either Myocyte enhancing factor 2 (Mef2) or the zinc-finger transcription factor Lame duck (Lmd) lead to very similar defects in myoblast fusion, yet the underlying molecular mechanism for this shared phenotype is not understood. Using a combination of ChIP-on-chip analysis and expression profiling of loss-of-function mutants, a global view was obtained of the regulatory input of both factors during development. The majority of Lmd-bound enhancers are co-bound by Mef2, representing a subset of Mef2's transcriptional input during these stages of development. Systematic analyses of the regulatory contribution of both factors demonstrate diverse regulatory roles, despite their co-occupancy of shared enhancer elements. These results indicate that Lmd is a tissue-specific modulator of Mef2 activity, acting as both a transcriptional activator and repressor, which has important implications for myogenesis. More generally, this study demonstrates considerable flexibility in the regulatory output of two factors, leading to additive, cooperative, and repressive modes of co-regulation (Cunha, 2010).

Genes that are co-regulated by the same two (or more) transcription factors are generally expected to have very similar spatio-temporal expression profiles. In fact, this assumption has been used by many studies to computationally predict the location of enhancer elements by searching for common TF binding motifs in the vicinity of clusters of co-expressed genes (or synexpression groups). It was therefore surprising when a comparison of experimentally-identified enhancer regions bound by the same two transcription factors uncovered a diverse range of regulatory responses. The 59 genes with enhancer elements co-bound by Lmd and Mef2 at the same stages of development are regulated either in a cooperative, additive or repressive manner depending on the individual enhancers. These data suggest that enhancer regions integrate regulatory inputs more flexibly than previously anticipated. By focusing on individual enhancer elements, how Lmd and Mef2 influence regulatory activity in different contexts was evaluated both in vivo and in vitro. Combining a number of complementary approaches allowed identification of three different modes of TF integration at developmental enhancers leading to additive, cooperative or repressive regulation (Cunha, 2010).

Mef2 and Lmd provide an additive positive input to the regulation of the Act57B locus. Ectopic Mef2 expression in the ectoderm is sufficient to induce Act57B expression, while providing Lmd alone is not. Conversely, enhancer-reporter gene expression is completely lost in lmd mutant embryos and only slightly reduced in Mef2 loss-of-function mutant embryos. Together, these data reveal a role for both transcription factors at this enhancer. Previous studies demonstrated that the initiation of Act57B expression at stage 11 requires Mef2 for its activation. Yet, artificially increasing Mef2 levels at this stage does not lead to premature activation of this locus. The current findings offer an explanation for this result: at this stage of development, combined input from Lmd and Mef2 is required to drive gene expression, while the presence of Mef2 alone is not sufficient to activate transcription. At later stages, when lmd expression is lost, Mef2 concentration has increased sufficiently to maintain Act57B expression. Conversely, the CG14687 locus can be activated by ectopic Lmd in the ectoderm, but not by Mef2 alone and requires lmd, but not Mef2, for its expression in the somatic muscle. Combined ectopic expression of the two TFs, in contrast, leads to a marked increase of reporter signal, again indicating combinatorial positive regulation by both TFs. These findings are supported by the ability of both Lmd and Mef2 to separately activate reporter gene expression in vitro and to yield additive reporter activity in combination (Cunha, 2010).

The blow enhancer shows a different mode of regulation and is synergistically activated by both factors. While neither Mef2 nor Lmd alone are sufficient to activate ectopic gene expression in vivo, supplying both factors simultaneously leads to robust target gene expression. Assaying for reporter gene activation in the two mutant backgrounds yields a complementary result; Mef2 and Lmd activity is required to activate transcription in the somatic mesoderm via the blow enhancer. Moreover, the in vitro reporter assay reveals a positive interaction between the two proteins, indicating that the blow enhancer functions as a cooperative switch (Cunha, 2010).

Analysis of the CG9416 enhancer revealed an antagonistic interaction between Lmd and Mef2. While ectopic expression of Mef2 leads to enhancer activation, simultaneous expression of Lmd markedly attenuates the transcriptional output from this locus. This effect can be reproduced in vitro: while providing Mef2 alone leads to robust activation of the CG9416 enhancer, Lmd is not able to activate gene expression. Instead, Lmd antagonizes the activating input of Mef2 in a concentration-dependent manner. This is the first example of direct negative regulation by Lmd. To identify additional examples of a repressive role for Lmd, the expression profiles of lmd and Mef2 mutant embryos was re-examined. CG9416 is markedly upregulated in lmd mutants, but shows reduced expression in embryos lacking Mef2. Another direct target gene with similar expression changes was selected in these genetic backgrounds, CG30035, and after determining the limits of the ChIP-enriched region its ability to drive reporter gene expression in vitro was assayed. Similar to the CG9416 enhancer, the CG30035 enhancer is robustly activated by Mef2, and this activation is inhibited by Lmd in a dose-dependent manner. This provides a second, independent example for Lmd-mediated repression of gene expression (Cunha, 2010).

In summary, starting from a genomic perspective, a large cohort of genes co-regulated by a pair of tissue-specific transcription factors was identified. Lmd modulates the activity of Mef2 at different enhancers in a context-dependent fashion, allowing for additive, cooperative or antagonistic interactions in the same cells. In this way, the timing and expression levels of Mef2 target genes can be further refined, as exemplified by the Act57B locus, which may owe its early activation during embryonic development to the combined activity of both proteins. Lmd shows homology with the Gli superfamily of transcription factors, which can act both as transcriptional activators and repressors, depending on proteolytic cleavage regulated by the hedgehog signaling pathway. To date, there is no evidence for proteolytic cleavage of Lmd and an irreversible conversion of Lmd from a transcriptional activator to an inhibitor is difficult to reconcile with the observation that Lmd can perform both roles at different loci at the same time, in the same tissue. For the same reason, it is also considered unlikely that Lmd interferes with transcriptional activation simply by binding to Mef2 and sequestering the protein in the cytoplasm. Instead, it is proposed that Lmd exerts a dominant inhibitory influence over a transcriptional activator, either by locally quenching Mef2's activity or through direct repression of the locus, similar to transcriptional repressors described in other developmental networks. These results provide a molecular understanding for the genetic observation that restoring Mef2 activity in lmd mutant embryos is not sufficient to rescue muscle differentiation. Both transcription factors are required to provide different regulatory inputs to a large number of co-regulated target genes during myogenesis. Their associated enhancers have revealed considerable flexibility in integrating regulatory inputs from these two TFs at individual cis-regulatory regions (Cunha, 2010).

Notch and Mef2 synergize to promote proliferation and metastasis through JNK signal activation in Drosophila

Genetic analyses in Drosophila revealed a synergy between Notch and the pleiotropic transcription factor Mef2 (myocyte enhancer factor 2), which profoundly influences proliferation and metastasis. This study shows that these hyperproliferative and invasive Drosophila phenotypes are attributed to upregulation of eiger, a member of the tumour necrosis factor superfamily of ligands, and the consequent activation of Jun N-terminal kinase signalling, which in turn triggers the expression of the invasive marker MMP1. Expression studies in human breast tumour samples demonstrate correlation between Notch and Mef2 paralogues and support the notion that Notch-MEF2 synergy may be significant for modulating human mammary oncogenesis (Pallavi, 2012).

A genetic modifier screen was undertaken in Drosophila and a number of genetic modifiers of Notch signals were identified that affect proliferation. Further examination of one of these modifiers, Mef2, established that its synergy with Notch signals directly triggers expression of the Drosophila JNK pathway ligand eiger, consequently activating JNK signalling that profoundly influences proliferation and metastatic behaviour. It might perhaps be worth noting that metastatic behaviour in Drosophila may not be completely equivalent to mammalian metastasis, notwithstanding the fact that they share molecular signatures, for example, MMP activation (Pallavi, 2012).

Cancer is characterized by the deregulation of the balance between differentiation, proliferation and apoptosis; thus, it is not surprising that the Notch signalling pathway, which plays a central role in all these developmental events, is increasingly implicated in oncogenic events. The rationale of this study is based on the fact that synergy between Notch and other genes is key in understanding how Notch signals contribute to oncogenesis. It remains a remarkable fact that while activating mutations in the Notch receptor have been associated with >50% of T-cell lymphoblastic leukemias (T-ALLs), a search for mutations in other cancers, despite a few suggestive reports, remains essentially unfruitful. Yet, correlative studies have linked Notch activity with a broad spectrum of human cancers and work in mice suggests that while Notch activation promotes proliferation, it is the synergy between Notch and other factors that eventually leads to cancer. Similar synergies have been identified before but the extraordinary complexity of the gene circuitry that modulates the Notch pathway suggests that more such relationships will be uncovered as exemplified by the discovery of Mef2 as a Notch synergistic partner affecting proliferation (Pallavi, 2012).

The transcription factor Mef2 plays an essential role in myogenic differentiation, but several studies have also shown a broad pleiotropic role of Mef2. Mef2 can integrate signals from several signalling cascades through chromatin remodelling factors and other transcriptional regulators to control differentiation events. This study extends the functionality of Mef2 by uncovering the profound effect it can have on proliferation and metastatic cell migration in synergy with Notch signals (Pallavi, 2012).

This is not the first study to link Mef2 with Notch. They have been linked before in the context of myogenesis both in Drosophila and in vertebrates. A ChIP-on-chip analysis of Mef2 target regions identified several Notch pathway components as potential Mef2 targets during Drosophila myogenesis. In human myoblasts, Mef2C was suggested to bind directly to the intracellular domain of Notch via the ankyrin repeat region, suppressing Mef2C-induced myogenic differentiation. Mef2 has also been reported to interact with the Notch coactivator MAML1 and suppress differentiation (Pallavi, 2012).

While upregulation of Mef2 alone does not show overt proliferation effects, these analyses demonstrate that in vivo it can activate MMP1. Even though Mef2 was ectopically expressed in the whole wing pouch, MMP1 expression was confined around the D/V boundary, where endogenous Notch signals are active. This effect of Mef2 overexpression depends on Notch signals, a notion corroborated by the fact that inhibiting Notch activity by RNAi reverses the effects of Mef2 on MMP1 (Pallavi, 2012).

The polarity gene scribble cooperates with Ras signalling to upregulate the JNK pathway, promoting invasiveness and hyperplasticity. However, the synergy seen in this study appears to be scribble independent. The fact that both the scribbled/Ras and the Notch/Mef2 metastatic pathways converge at the level of JNK signal activation suggests that JNK is a crucial regulator of oncogenic behaviour, which is controlled by inputs from multiple signals. Even though there is little evidence that twist activates JNK signalling, it is a crucial regulator of epithelial-to-mesenchymal transition and metastasis and has also been independently linked to both Mef2 and Notch in myogenesis. However, it is noted that the Notch-Mef2 synergy seems to be independent of twist, as Twist cannot replace Mef2 in the synergistic relationship (Pallavi, 2012).

Numerous reports link JNK signalling to normal developmental events requiring cell movement and to metastatic phenomena both in Drosophila and in vertebrates. JNK signals seem to be crucial for controlling gene activities involved in epithelial integrity and the observations from Drosophila suggest that the Nact and Mef2 synergy may be important in JNK-linked carcinogenesis. A role for Notch in controlling JNK signals has been reported previously. While studies carried out using breast cancer samples can only be correlative now, the observations suggest that metastatic breast tumours harbour higher levels of Notch and Mef2 paralogue pairs, consistent with observations in Drosophila (Pallavi, 2012).

Although the majority of studies on Mef2 are focused on muscle development/differentiation, some intriguing links between Mef2C and leukaemias are noteworthy. MEF2C and Sox4 synergize to cause myeloid leukaemia in mice. Analysis of T-ALL patient samples revealed increased levels of Mef2C; however, Mef2C alone could not cause cellular transformation of NIH3T3 cells, but it could do so in the presence of RAS or myc. Given the role of Notch in T-ALL it will be important to examine how activated Notch mutations, often the causative oncogenic mutation, correlate with Mef2 family members. The functional differences between the different Mef2 homologues in humans are not well understood and the specific role each may play in the Notch synergy remains to be elucidated (Pallavi, 2012).

This analysis clearly indicates that, in Drosophila, the underlying molecular mechanism of the Notch/Mef2 synergy relies on the direct upregulation of expression of the prototypical TNF ligand egr through the binding of Mef2 and Su(H), the effector of Notch signals, to regulatory sequences on the egr promoter. In Drosophila, egr is the only JNK ligand while in humans, the superfamily is large and includes the cytokines TNFα (TNF), TRAIL and RANKL which have been associated with tumour progression in numerous human cancers including breast. RANKL plays a key role in bone metastasis of breast cancer, and is the target of a therapeutically effective monoclonal antibody. In breast cancer cells, TNFα, which can signal through several pathways, including JNK and NF-κB, affects proliferation and promotes invasion and metastasis (Pallavi, 2012).

In human breast cancer, clinical relapse after initial treatment is almost always accompanied by metastatic spread and it is almost invariably lethal. ER- tumours tend to respond well to first-line chemotherapy, but a significant subset of these tumours recur. Recurrent ER- tumours are typically resistant to chemotherapy and radiation, and are highly lethal. The current data suggest that ER- tumours that recur but not ER- tumours that do not recur show significant positive correlation between NOTCH1 and all four MEF2 paralogues. Further, the data show that even within the recurrent subset, NOTCH1 expression predicts poor survival but MEF2 expression does not. While these observations do not establish causality, they are consistent with the hypothesis that NOTCH1/MEF2 coexpression identifies a set of breast cancers that are more likely to relapse, and that MEF2 genes act as NOTCH cofactors rather than independently of NOTCH (Pallavi, 2012).

In conclusion, this study in Drosophila uncovers a new functional role for Mef2, which in synergy with Notch affects proliferation and metastasis. Mechanistically, this synergy relies on the direct upregulation of the JNK pathway ligand eiger. The correlation analysis and tumour staining of human cancer samples suggests that the observations in Drosophila may well be valid in humans, defining Notch-Mef2 synergy as a critical oncogenic parameter, one that may be associated with metastatic behaviour, emphasizing the value of model systems in gaining insight into human pathobiology (Pallavi, 2012).

Protein Interactions

In vertebrates, transcriptional control of skeletal muscle genes during differentiation is regulated by enhancers that direct the combinatorial binding and/or interaction of Mef2 and the bHLH MyoD family of myogenic factors. Drosophila Mef2 plays a role similar to its vertebrate counterpart in the regulation of the Tropomyosin I gene in the development of Drosophila somatic muscles, however, unlike vertebrates, Drosophila Mef2 interacts with a muscle activator region that does not have binding sites for myogenic bHLH-like factors or any other known Drosophila transcription factors. This study describes the isolation and characterization of a component of the muscle activator region named PDP1 (PAR-domain protein 1). PDP1 is a novel transcription factor, highly homologous to the PAR subfamily of mammalian bZIP transcription factors HLF, DBP and VBP/TEF. This is the first member of the PAR subfamily of bZIP transcription factors to be identified in Drosophila. PDP1 is involved in regulating expression of the Tropomyosin I gene in somatic body-wall and pharyngeal muscles by binding to DNA sequences within the muscle activator that are required for activator function. Mutations that eliminate PDP1 binding eliminate muscle activator function and severely reduce expression of a muscle activator plus Mef2 mini-enhancer. These and previous results suggest that PDP1 may function as part of a larger protein/DNA complex that interacts with Mef2 to regulate transcription of Drosophila muscle genes. In addition to being expressed in the mesoderm that gives rise to the somatic muscles, PDP1 is also expressed in the mesodermal fat body, the developing midgut endoderm, the hindgut and Malpighian tubules, and the epidermis and central nervous system, suggesting that PDP1 is also involved in the terminal differentiation of these tissues (S. C. Lin, 1997a).


Myocyte enhancer factor 2: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

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