Gene name - Myocyte enhancer factor 2
Synonyms - D-Mef2
Cytological map position - 46C
Function - transcription factor
Keyword(s) - myogenesis
Symbol - Mef2
Genetic map position - 2-
Classification - MADS box
Cellular location - nuclear
|Recent literature||Lovato, T. L., Sensibaugh, C. A., Swingle, K. L., Martinez, M. M. and Cripps, R. M. (2015). The Drosophila transcription factors Tinman and Pannier activate and collaborate with Myocyte enhancer factor-2 to promote heart cell fate. PLoS One 10: e0132965. PubMed ID: 26225919
Expression of the MADS domain transcription factor Myocyte Enhancer Factor 2 (MEF2) is regulated by numerous and overlapping enhancers which tightly control its transcription in the mesoderm. To understand how Mef2 expression is controlled in the heart, this study has identified a late stage Mef2 cardiac enhancer that is active in all heart cells beginning at stage 14 of embryonic development. This enhancer is regulated by the NK-homeodomain transcription factor Tinman, and the GATA transcription factor Pannier through both direct and indirect interactions with the enhancer. Since Tinman, Pannier and MEF2 are evolutionarily conserved from Drosophila to vertebrates, and since their vertebrate homologs can convert mouse fibroblast cells to cardiomyocytes in different activator cocktails, tests were performed to see whether over-expression of these three factors in vivo could ectopically activate known cardiac marker genes. Mesodermal over-expression of Tinman and Pannier resulted in approximately 20% of embryos with ectopic Hand and Sulphonylurea receptor (Sur) expression. By adding MEF2 alongside Tinman and Pannier, a dramatic expansion in the expression of Hand and Sur was observed in almost all embryos analyzed. Two additional cardiac markers were also expanded in their expression. These results demonstrate the ability to initiate ectopic cardiac fate in vivo by the combination of only three members of the conserved Drosophila cardiac transcription network, and provide an opportunity for this genetic model system to be used to dissect the mechanisms of cardiac specification.
|Chechenova, M. B., Maes, S. and Cripps, R. M. (2015). Expression of the Troponin C at 41C gene in adult Drosophila tubular muscles depends upon both positive and negative regulatory inputs.PLoS One 10: e0144615. PubMed ID: 26641463
Most animals express multiple isoforms of structural muscle proteins to produce tissues with different physiological properties. In Drosophila, the adult muscles include tubular-type muscles and the fibrillar indirect flight muscles. This study analyzed the transcriptional regulation of TpnC41C, a Troponin C gene expressed in the tubular jump muscles, but not in the fibrillar flight muscles. A 300-bp promoter fragment of TpnC41C is sufficient for the fiber-specific reporter expression. Two sites necessary for the activation of the enhancer were identified. Mutations in each resulted in 70% reduction of enhancer activity. One was characterized as a binding site for Myocyte Enhancer Factor-2. In addition, a repressive element was identified that prevents activation of the enhancer in other muscle fiber types. Mutation of this site increased jump muscle-specific expression of the reporter, but more importantly reporter expression expanded into the indirect flight muscles. These findings demonstrate that expression of the TpnC41C gene in jump muscles requires integration of multiple positive and negative transcriptional inputs. Identification of the transcriptional regulators binding the cis-elements that were identified will reveal the regulatory pathways controlling muscle fiber differentiation.
|Arredondo, J. J., Vivar, J., Laine-Menendez, S., Martinez-Morentin, L. and Cervera, M. (2017). CF2 transcription factor is involved in the regulation of Mef2 RNA levels, nuclei number and muscle fiber size. PLoS One 12(6): e0179194. PubMed ID: 28617826
CF2 and Mef2 influence a variety of developmental muscle processes at distinct stages of development. Nevertheless, the exact nature of the CF2-Mef2 relationship and its effects on muscle building remain yet to be resolved. This study explored the regulatory role of CF2 in the Drosophila embryo muscle formation. To address this question and not having proper null CF2 mutants, loss or gain of function strategies were employed to study the contribution of CF2 to Mef2 transcription regulation and to muscle formation. The data point to CF2 as a factor involved in the regulation of muscle final size and/or the number of nuclei present in each muscle. This function is independent of its role as a Mef2 collaborative factor in the transcriptional regulation of muscle-structural genes. Although Mef2 expression patterns do not change, reductions or increases in parallel in CF2 and Mef2 transcript abundance were observed in interfered and overexpressed CF2 embryos. Since CF2 expression variations yield altered Mef2 expression levels but with correct spatio-temporal Mef2 expression patterns, it can be concluded that only the mechanism controlling expression levels is de-regulated. It is proposed that CF2 regulates Mef2 expression through a Feedforward Loop circuit.
|Crittenden, J. R., Skoulakis, E. M. C., Goldstein, E. S. and Davis, R. L. (2018). Drosophila mef2 is essential for normal mushroom body and wing development. Biol Open. PubMed ID: 30115617
MEF2 (myocyte enhancer factor 2) transcription factors are found in the brain and muscle of insects and vertebrates and are essential for the differentiation of multiple cell types. In Drosophila, MEF2 is essential for the formation of mushroom bodies in the embryonic brain and for the normal development of wings in the adult. In embryos mutant for mef2, there is a striking reduction in the number of mushroom body neurons and their axon bundles are not detectable. The onset of MEF2 expression in neurons of the mushroom bodies coincides with their formation in the embryo and, in larvae, expression is restricted to post-mitotic neurons. In flies with a mef2 point mutation that disrupts nuclear localization, MEF2 was found to be restricted to a subset of Kenyon cells that project to the alpha/beta, and gamma axonal lobes of the mushroom bodies, but not to those forming the alpha'/beta' lobes.
|Lin, W. H. and Baines, R. A. (2019). Myocyte enhancer factor-2 and p300 interact to regulate expression of the homeostatic regulator Pumilio in Drosophila. Eur J Neurosci. PubMed ID: 30687963
Pumilio (Pum), an RNA-binding protein, is a key component of neuron firing-rate homeostasis that likely maintains stability of neural circuit activity in all animals, from flies to mammals. Whilst Pum is ubiquitously expressed, little is understood about how synaptic excitation regulates its expression in the CNS. This study characterized the Drosophila dpum promoter and identified multiple Myocyte enhancer factor-2 (Mef2)-binding elements. Twelve dmef2 splice variants were identifiedf and a luciferase-based assay was used to monitor dpum promoter activity. Whilst all 12 dMef2 splice variants enhance dpum promoter activity, exon 10-containing variants induce greater transactivation. Previous work shows dPum expression increases with synaptic excitation. However, no change was observed in dMef2 transcript in larval CNS, of both sexes, exposed to the proconvulsant picrotoxin. The lack of activity-dependence is indicative of additional regulation. p300 was identified as a potential candidate. By binding to dMef2, p300 represses dpum transactivation. Significantly, p300 transcript is down-regulated by enhanced synaptic excitation (picrotoxin) which, in turn, increases transcription of dpum through de-repression of dMef2. These results advance understanding of dpum by showing activity-dependent expression is regulated by an interaction between p300 and dMef2.
|Schmitt, R. E., Shell, B. C., Lee, K. M., Shelton, K. L., Mathies, L. D., Edwards, A. C. and Grotewiel, M. (2019). Convergent evidence from humans and Drosophila melanogaster implicates the transcription factor MEF2B/Mef2 in alcohol sensitivity. Alcohol Clin Exp Res. PubMed ID: 31241765
Self-rating of the effects of alcohol (SRE) measures level of response to ethanol in humans. Interestingly, there is a positive relationship between the SRE and risk for abusing alcohol, suggesting mechanistic connections between SRE and alcohol abuse. To identify candidate genes with a role in SRE and alcohol-related behavior more generally, this study coupled human genetic analyses with studies in Drosophila melanogaster. First, a gene-based analysis was performed of GWAS summary statistics for SRE in the Avon Longitudinal Study of Parents and Children (ALSPAC) sample. Based on prior findings in humans, orthology to fly genes and the availability of genetic reagents, a subset of genes was selected for studies on ethanol behavior in Drosophila. This study found 37 genes with nominal associations in the SRE GWAS. The role was explored of 6 orthologous genes in Drosophila ethanol sedation and rapid tolerance. The transcription factor Mef2 was found to be required for normal ethanol sedation in flies. Pan-neuronal expression of two independent Mef2 RNAi transgenes significantly reduced Mef2 expression and made flies resistant to ethanol sedation. Additionally, flies with multiple independent mutant alleles of Mef2 were also resistant to ethanol sedation, confirming a role for Mef2 in this behavior. Altered expression of Mef2 did not change ethanol rapid tolerance or cause a net change in internal ethanol concentrations. It is concluded that these studies indicate that MEF2B influences SRE in humans and that Mef2 impacts ethanol sedation in Drosophila.
|Zhao, X., Li, X., Shi, X. and Karpac, J. (2020). Diet-MEF2 interactions shape lipid droplet diversification in muscle to influence Drosophila lifespan. Aging Cell 19(7). PubMed ID: 32537848
Using Drosophila, a role for was uncovered for myocyte enhancer factor 2 (MEF2) in modulating diet-dependent lipid droplet diversification within adult striated muscle, impacting mortality rates. Muscle-specific attenuation of MEF2, whose chronic activation maintains glucose and mitochondrial homeostasis, leads to the accumulation of large, cholesterol ester-enriched intramuscular lipid droplets in response to high calorie, carbohydrate-sufficient diets. The diet-dependent accumulation of these lipid droplets also correlates with both enhanced stress protection in muscle and increases in organismal lifespan. Furthermore, MEF2 attenuation releases an antagonistic regulation of cell cycle gene expression programs, and up-regulation of Cyclin E is required for diet- and MEF2-dependent diversification of intramuscular lipid droplets. The integration of MEF2-regulated gene expression networks with dietary responses thus plays a critical role in shaping muscle metabolism and function, further influencing organismal lifespan. Together, these results highlight a potential protective role for intramuscular lipid droplets during dietary adaptation (Zhao, 2020).
|Harsh, S. and Eleftherianos, I. (2020). Tumor induction in Drosophila imaginal epithelia triggers modulation of fat body lipid droplets. Biochimie 179: 65-68. PubMed ID: 32946989
Understanding of cancer-specific metabolic changes is currently unclear. In recent years, the fruit fly Drosophila melanogaster with its powerful genetic tools has become an attractive model for studying both tumor autonomous and the systemic processes resulting from the tumor growth. This study investigated the effect of tumorigenesis on the modulation of lipid droplets (LDs) in the larval fat bodies (mammalian equivalent of adipose tissue). Notch signaling was overexpressed alone or in combination with the developmental regulator Myocyte enhancer factor 2 (Mef2) using wing-specific and eye-specific drivers, the size of LDs in the fat body of the different tumor bearing larvae was quantified, and the expression of genes associated with lipolysis and lipogenesis was estimated. Hyperplastic and neoplastic tumor induced by overexpression of Notch and co-expression of Notch and Mef2 respectively were found to trigger impaired lipid metabolism marked by increased size of fat body LDs. The impaired lipid metabolism in tumor carrying larvae is linked to the altered expression of genes that participate in lipolysis and lipogenesis. These findings reveal modulation of LDs as one of the host's specific response upon tumor initiation. This information could potentially uncover mechanisms for designing innovative approaches to modulate cancer growth.
The quest for the gene responsible for regulation of muscle differentiation in Drosophila is a hot topic, as is the search for its vertebrate counterpart. Nautilus isn't the answer, since it is found only in somatic cell precursors, and not in heart muscle precursors. Tinman fails the test for the opposite reasons; it is found in heart precursors but not in skeletal muscle precursors. Does Mef2 provide an answer?
Mef2 is expressed earlier than nautilus, and it is expressed in presumptive heart and skeletal muscle precursors. It is dependent on twist , the gene that determines mesodermal fate. What prevents an otherwise neat answer is that nautilus and Mef2 expression are mutually independent of one another. Additionally, Mef2 mutants express mesodermal markers like even-skipped, that appear in precursors of one dorsal somatic muscle and a number of pericardial cells. There are fewer EVE-positive cells in Mef2 mutants but they don't disappear. nautilus expressing cells are present as well, but not in multinucleate syncytia, meaning that in Mef2 mutants nautilus expressing cells cannot mature to become muscle fibers.
Therefore, Mef2 is not necessary for the initial specification of muscle cells, but for their continued differentiation (Ranganayakulu, 1995). Muscle precursors undergo programmed cell death in Mef2 mutants, a sure indicator of defective differentiation (Bour, 1995).
mef2 and myoblast city mutations have been used to study requirements for neuromuscular junction (NMJ) formation during Drosophila development. myoblast city is required for myoblast fusion to establish syncytial muscles; its loss results in the formation of mononucleate muscles within a field of unfused myoblasts (Rushton, 1995). In myoblast city mutants, mononucleate muscles make functional neuromuscular synapses with correctly localized presynaptic active zones (presynaptic densities with clear synaptic vesicles). In mbc mutant embryos, as in wild type, only a fraction of the muscle surface is devoted to the NMJ. This fraction in mbc mutants represents a far smaller surface for the localization of active zones than the surface encountered by equivalent motorneurons in wild-type muscle, suggesting that muscles are capable of supporting a limited number of boutons, relative to their size. Motorneurons innervating the smaller mononucleate muscles in mbc mutant embryos fail to establish many active zones, as half the active zones are in axonal swellings not attached to muscle. It is clear that active zones are able to form in the absense of a NMJ.
In mef2 mutants, the pattern of muscle founder cells is largely normal in the absence of mef2 function. Myoblasts also fail to fuse but still attract appropriate innervation. Motorneurons that establish contact are always attracted by the correct founder cells, as judged by the branch pattern of innervation. There is no evidence of motor axons contacting any myoblasts other than those that become founders. These myoblasts, however, fail to differentiate into muscles. Concomitantly, active zones are present at frequencies comparable with wild type, however, nearly all of them are neurohemal, neuroneural, or on glial cells. Even in the rare instances where active zones are located at a neuromuscular contact, the pre- and postsynaptic membranes fail to form the tight apposition that is typical of synapse. In most cases the presynaptic active zones fail to localize at neuromuscular contacts. It is concluded that the localization of presynaptic active zones necessary to form a NMJ requires mef2-dependent muscle differentiation (Prokop, 1996).
In addition to essential myogenic functions, mutant Mef2 adult females are weakly fertile and produce defective eggs. Mef2 is expressed in nurse and follicle cells of the wild-type egg chamber. The Mef2 oogenic phenotype has been analyzed and it has been shown that the gene is required for the normal patterning and differentiation of the centripetally migrating follicle cells (CMFCs) that are crucial for development of the anterior chorionic structures. Mef2 alleles exhibit a genetic interaction with a dominant-negative allele of thick veins (tkv), which encodes a type I receptor of the Decapentaplegic-signaling pathway. TKV mRNA is overexpressed in Mef2 mutant egg chambers, and, conversely, forced expression of Mef2 represses tkv expression. These results indicate roles for Mef2 in the regulation of tkv gene expression and Decapentaplegic signal transduction that are essential for proper determination and/or differentiation of the anterior follicle cells. Mef2 is also expressed in both nurse and follicle cells. No defects have been observed in the germ line, either the number of germ cells or the location of the oocyte within the egg chamber. Therefore, a possible requirement for Mef2 in germ-line cells remains to be elucidated (Mantrova, 1999).
Mef2 appears to function in the somatic follicle cells, particularly in subpopulations of the oocyte-associated follicle cells (O-FCs), by negatively regulating TKV mRNA levels. It is not known whether Mef2 directly represses tkv gene transcription. Curiously, the expression patterns of Mef2 and TKV RNA are not mutually exclusive. Whereas Mef2 is expressed in all follicle cells, TKV is absent only in different populations of follicle cells at different times. Perhaps subtle changes in Mef2 levels can have different effects on tkv expression. For example, at stage 10A, Mef2 is more abundant in the leading CMFC than in the other O-FCs, whereas tkv is not expressed in CMFCs and expressed at a low level in the rest of the follicle cells. Alternatively, Mef2 may be a constitutive repressor of tkv, whereas other tissue-specific factors can counteract Mef2 and induce tkv expression (Mantrova, 1999).
In wild type, tkv expression is dynamic during oogenesis and appears to highlight a specific group of follicle cells, the leading front of the CMFCs. At stage 10A just before the commencement of centripetal migration, these cells form a ring marking the boundary between the oocyte and the nurse cell complex. After stage 10B, this ring of cells migrates inward until it reaches the border cells located at the center of the oocyte anterior. At stage 10A, tkv is expressed in O-FC but not in the leading CMFCs. This pattern is opposite that of the dpp expression pattern, which is highly expressed in the leading CMFCs but not in the rest of the O-FCs. It will be of interest to examine whether or not tissue-specific expression of dpp and tkv in the egg chamber is autoregulated by DPP signaling (Mantrova, 1999).
At stage 10B, tkv is expressed in the ventral half of the CMFC in addition to two short stripes in the dorsal region of the oocyte-associated follicular epithelium. This expression pattern appears to be complementary to that of the Egfr blocker argos, which forms a T-shaped pattern along the dorsal CMFCs and dorsal midline. argos expression is induced by the highest level of Egfr signaling; Egfr in turn, reduces the signaling strength by blocking the interaction between the receptor and its ligands. Thus, the initial graded distribution of Egfr signaling, extending laterally from the anterodorsal midline of the O-FCs, is transformed into two ridges of the Egfr-signaling level just lateral to the dorsal midline. These two ridges define the two lines of O-FCs that ultimately produce the two dorsal appendages. Interestingly, argos expression is diminished in the Mef2 mutant, consistent with the observed mutant egg chambers possessing broad and fused appendages. Although the notion is favored that argos expression is modulated by Mef2 through the action of Tkv, it cannot be ruled out that Mef2 may directly control the transcription of argos (Mantrova, 1999).
In addition to regulating the expression pattern of argos, Mef2 may play a more general role in modulating the Egfr-signaling level. This is suggested by the presence of Mef2 mutant egg chambers with reduced and fused dorsal appendages, a phenotype typical of hypomorphic Egfr-signaling pathway mutants. Indeed, reduced expression of Egfr-signaling components such as rhomboid has been observed in Mef2 mutants. More detailed and expansive studies are needed to elucidate the possible interaction between the Dpp- and Egfr-signaling pathways with Mef2 as a potential mediator (Mantrova, 1999).
Nevertheless, this report does demonstrate that the dpp-expressing CMFCs are poorly defined in D-mef2 mutant egg chambers. CMFCs are responsible for forming the operculum and, together with the border cells, specifying the construction of the micropile. Formation of these structures is also essential to closing the anterior end of the egg chamber. Because Dpp is critical for specifying anterior chorion production, the disrupted patterning of CMFCs in the Mef2 mutant may explain, at least in part, the chorion phenotypes observed (Mantrova, 1999).
Earlier studies have shown that Mef2 is a late-acting component of the genetic network controlling embryonic myogenesis. In the cardiac lineage, Mef2 is a direct target of Tinman, a homeodomain transcription factor essential for heart formation. tin function is required for the specification of cardiac precursor cells within the dorsal mesoderm, and its expression within this domain is induced by Dpp, produced by cells of the dorsal ectoderm. Additionally, Mef2 expression in a broader dorsal mesodermal domain is controlled by Dpp and the transcription factor Medea. Thus, Mef2 can be considered a downstream regulator within the Dpp-signaling pathway needed for cardiogenesis in the fly (Mantrova, 1999).
In the current study, it has been shown that Mef2 modulates the dissemination of a Dpp signal in the egg chamber through its control of tkv expression levels. Because multifunctional proteins often are members of conserved gene cassettes that function in different developmental processes, it is possible that Mef2 provides an additional critical function for Dpp signaling in the mesoderm. tkv is required for, and expressed during, inductive events occurring in the dorsal mesoderm. Likewise, Mef2 is present throughout the mesoderm at this stage and may have a comparable function in regulating tkv expression in mesodermal cells as has been elucidated in follicle cells. This could occur through a possible feedback regulatory loop from Mef2 to tkv. It will be important to investigate potential interactions of the two genes in the mesoderm, perhaps providing new insights into the specificity of Dpp signaling during Drosophila development (Mantrova, 1999).
Dissecting components of key transcriptional networks is essential for understanding complex developmental processes and phenotypes. Genetic studies have highlighted the role of members of the Mef2 family of transcription factors as essential regulators in myogenesis from flies to man. To understand how these transcription factors control diverse processes in muscle development, chromatin immunoprecipitation analysis was combined with gene expression profiling to obtain a temporal map of Mef2 activity during Drosophila embryonic development. This global approach revealed three temporal patterns of Mef2 enhancer binding, providing a glimpse of dynamic enhancer use within the context of a developing embryo. These results provide mechanistic insight into the regulation of Mef2's activity at the level of DNA binding and suggest cooperativity with the bHLH protein Twist. The number and diversity of new direct target genes indicates a much broader role for Mef2, at all stages of myogenesis, than previously anticipated (Sandmann, 2006).
To identify enhancer regions bound by Mef2 in vivo, chromatin immunoprecipitation followed by microarray analysis (ChIP-on-chip) was performed at five consecutive time points of embryogenesis spanning key stages of muscle development. To systematically identify Mef2 bound genomic regions in an unbiased manner, a Drosophila genomic tiling array was constructed, taking advantage of genomic clones generated by the Berkeley Drosophila Genome Project (BDGP) to sequence the Drosophila genome. The array consists of overlapping 3 kb fragments tiling across ~50% of the Drosophila genome (Sandmann, 2006).
These experiments identified 1015 significantly enriched Mef2 bound fragments, with less than 1% estimated false positives. Due to the overlapping nature of the array, this represents 670 nonoverlapping genomic regions bound by Mef2 at one or more developmental time points. To assess the quality of this data set, it was determined if regions previously reported to be bound by Mef2 were recovered. Eight of the previously characterized Mef2 direct target genes are covered by the arrays. Mef2 binding was identified in the proximity of all eight genes assayed: Actin 57B, Muscle LIM protein at 84B and Muscle LIM protein at 60A, β-tubulin60D, Tropomyosin I, inflated, mir-1, and Mef2 itself. In many cases, the study not only identified the previously reported Mef2 bound enhancer, but also identified additional ones (Sandmann, 2006).
Five genes are known to be genetically downstream of Mef2, though the mechanism of regulation remains unclear: Myosin heavy chain, meso18E, muscleblind, nautilus, and Chorion factor 2. The results show Mef2 binding to genomic regions close to four of these genes (Mhc, mbl, nau, and meso18E) and to a genomic region further 5′ of CF2. This indicates that these genes are directly regulated by Mef2 and identifies the location of at least one of their enhancer regions. In summary, the successful identification of Mef2 binding in the vicinity of all known and suspected target genes suggests a high accuracy of the approach taken. In addition to the 8 known enhancers, over 650 new Mef2 bound regions were identified (Sandmann, 2006).
As a complementary approach to assess the molecular function of Mef2, which genes depend on Mef2 activity for their correct expression during embryonic development was determined. The gene expression profile of wild-type embryos was compared to that of stage-matched Mef2 mutant embryos throughout a developmental time series. Pure populations of Mef2 homozygous mutant embryos were isolated. For each developmental time point assayed, four independent replicates were analyzed on microarrays containing at least one probe for every predicted gene in the genome (Sandmann, 2006).
The expression profiles of Mef2 mutant embryos was examined at 11 consecutive 1 hr time points of embryogenesis, spanning from 5 to 16 hr of development (stages 9–16). An additional time point was added at 18–19 hr, stage 17, to identify genes that are expressed in differentiated muscle. This allowed the generation of a high-resolution map of Mef2-dependent temporal changes in gene expression, spanning the stages of mesoderm subdivision, myoblast specification, myoblast fusion, and the initiation of terminal muscle differentiation (Sandmann, 2006).
This study identified 700 genes with significant changes in gene expression in Mef2 mutant embryos at 2 or more consecutive time points. The eight known Mef2 protein-coding target genes are among them, with Act57B, Mlp84B, Mlp60A, and TmI showing a greater than 4-fold decrease in expression at multiple stages of development. The differentially expressed genes are significantly enriched in genes expressed in muscle, indicating that many of the misregulated genes are expressed in the same cells as Mef2 (Sandmann, 2006).
The function of a substantial number of the differentially expressed genes is unknown. This study indicates that they are genetically downstream of Mef2 as either direct or indirect targets, and provides a useful resource to identify genes likely to be involved in muscle development. The combination of ChIP-on-chip results with this expression profiling data allowed determination of which genes are directly regulated by Mef2 (Sandmann, 2006).
Genomic tiling arrays provide an unbiased method to identify new regulatory regions independent of their distance to the gene. While this offers a great advantage over promoter arrays, it raises a new challenge for ChIP-on-chip studies: how to accurately match transcription factor bound regions to their correct target genes. Metazoan enhancers have been identified at large distances from their target genes, including within introns of neighboring loci. Assuming that the enhancer is regulating the closest proximal gene will, especially in gene-dense regions, often cause the wrong target gene to be selected (Sandmann, 2006).
Different sources of metadata were used to systematically link ChIP-enriched regions to their target genes. The genes in the vicinity of each Mef2 bound region received a cumulative score based on: (1) the distance between a gene and a Mef2 bound region, (2) a change in expression in Mef2 mutant embryos, and (3) supporting information, for example about the gene's expression patterns (BDGP in situ database, Flybase, literature). Genes were not assigned based on proximity alone. Using this approach, 211 Mef2 direct target genes were identified with high confidence, including all known targets covered by the tiling array (Sandmann, 2006).
To estimate the accuracy of the automated gene assignment, a collection was used of characterized enhancers from single gene studies. A total of 33 of the Mef2 bound regions assigned to target genes with a high confidence score overlap with a previously identified gene's enhancer. In 28 cases (84.8%), the regulated locus was correctly identified, illustrating the accuracy of the gene assignment strategy. The remaining five sequences map to the Enhancer of split region and were assigned to a different member of this gene cluster. A total of 29 additional Mef2 bound regions overlapped with known enhancer regions. The 12 regulated target genes were not assigned by using the automated approach, since no additional supporting evidence was available. Combining the automatic assignments with information about known regulatory relationships yielded 234 unique genes directly regulated by Mef2 (Sandmann, 2006).
Rather than potentially making an incorrect gene assignment, the remaining 574 Mef2 bound fragments were not assigned to genes, although they are equally significantly enriched with a stringent statistical cutoff. The vast majority (87.3%) of unassigned Mef2 bound fragments were in an intron or within 5 kb of one or more genes; therefore, they likely contain regulatory modules. To enable other researchers to make their own gene assignments, a searchable web site was created, displaying all Mef2 bound regions in their genomic context together with the results from the expression profiling experiments (Sandmann, 2006).
Of the 1015 Mef2 bound regions, 62 were previously identified as active enhancers. The vast majority of these regulatory regions were not known to bind to Mef2, but they have been shown to bind to a number of other transcription factors in vitro, revealing interesting insights into combinatorial gene regulation with Mef2. For example, Mef2 binding to an Antp bound enhancer region of the apterous and teashirt genes was identified. Many of these regions were shown to function as muscle enhancers in vivo, providing additional evidence that the identified Mef2 bound regions are active enhancers. To test if additional Mef2 bound regions can function as enhancers in vivo, five regions were selected with representative levels of enrichment in the ChIP-on-chip experiments that were assigned to target genes with known expression. This allowed evaluation of their ability to drive reporter gene expression in a pattern similar to that of the target gene. Using conservation in different Drosophila species as a guide, regions between 0.4 and 2.5 kb within the bound fragments were assayed (Sandmann, 2006).
All five Mef2 bound regions tested were able to drive GFP expression in Mef2-expressing cells. The enhancer region of pnt initiates expression of GFP early in the mesoderm, at stages 9–10. This mirrors the expression of the pnt transcripts in wild-type embryos. Since the GFP protein has a long half-life in Drosophila embryos, it can subsequently be detected in differentiating myoblasts. The enhancer regions of CG14687 and CG5080 initiate GFP expression slightly later. The enriched region upstream of CG14687 is sufficient to direct expression of GFP initially in the visceral muscle (stage 11) and later in the somatic muscle (stage 12), closely resembling the spatial and temporal expression pattern of the gene itself (Sandmann, 2006).
Myosin light chain 2 (Mlc2) is a target of Mef2 proteins in vertebrates. The data show that this regulation is conserved in flies. A Mef2 bound region 5′ of the Mlc2 locus reproduces the gene's expression, driving GFP expression in differentiating somatic muscle cells from stage 13 onward. Finally, the endogenous expression of CG9416 initiates in the longitudinal visceral muscle precursors at stage 10 and in the somatic muscle at stage 13. A 372 bp enhancer region is sufficient to direct GFP expression in both tissues, reproducing the full expression pattern of CG9416 (Sandmann, 2006).
In summary, all five Mef2 bound genomic regions are sufficient to direct reporter gene expression resembling the temporal and spatial patterns of the respective predicted target gene. These results indicate that the ChIP-on-chip approach was very successful in identifying new muscle enhancers in vivo, and that the assignment of target genes to Mef2 bound regions is accurate (Sandmann, 2006).
Many of the identified direct target genes are misexpressed in Mef2 mutant embryos, showing a requirement for Mef2 for their normal expression. To determine if Mef2 is sufficient to regulate its target genes in nonmuscle cells, Mef2 was ectopically expressed in the ectoderm by using the UAS/Gal4 system. As putative cofactors are likely to be absent, this assay is a very stringent test of a regulatory relationship. Remarkably, Mef2 could induce ectopic expression of 5 of the 13 genes tested in nonmuscle cells. Overexpression of Mef2 in engrailed stripes in the ectoderm is sufficient to cause ectopic expression of Him, CG9416, and CG30080. Overexpression of Mef2 with lmd, a transcription factor known to regulate Mef2, is sufficient to drive ectopic expression of CG5080 and delilah in the ectoderm. Ectopic expression of either transcription factor alone could not induce expression of these genes, suggesting that Mef2 and Lmd cooperatively regulate their expression and are sufficient to do so in nonmuscle cells. These results, in combination with the expression profiling data, confirm that Mef2 is essential and sufficient for the expression of a large percentage of its target genes (Sandmann, 2006).
While Mef2 is found in all muscle cells from gastrulation to the end of embryogenesis, its known target genes show temporally and spatially different expression patterns. For example, Act57B and β3-tub60D RNA are not transcribed until about stage 11, while Mhc and Mlp84B RNAs are not detectable until stages 13–14. Moreover, while Mef2 is expressed in the entire myogenic lineage, some of its known targets are expressed in smaller subsets of cells (Sandmann, 2006).
Clearly, there must be additional ways to control Mef2's regulatory activity. To determine if regulation occurs at the level of DNA binding, the temporal information from the ChIP time course was used to investigate if there are distinct patterns of Mef2 enhancer occupancy. K-means clustering analysis was used to subdivide the 1015 enriched genomic regions according to their temporal profile of Mef2 binding. Three major groups of temporally bound enhancers were identified (Sandmann, 2006).
Binding to the first group of enhancers was initially detected at 4–6 hr of development, after which Mef2 remained bound through the three subsequent developmental time points assayed. This group, representing almost half of the enriched fragments, suggests that these enhancers remain occupied by Mef2 throughout development. This temporal binding pattern of Mef2 matches its broad expression during all stages of muscle development (Sandmann, 2006).
The second group, representing 21% of the enhancers, was bound by Mef2 at 4–6 hr of development, but it was not bound at later developmental time points. Since Mef2 continues to be expressed, and is capable of binding to other enhancers, the transient occupancy of the early enhancers demonstrates that Mef2's ability to bind to DNA is tightly regulated (Sandmann, 2006).
The third group, containing 32% of the enhancers, is only bound by Mef2 late in development, with maximal binding at the last time point assayed. This group contains enhancers for many genes involved in late aspects of muscle differentiation, e.g., Mhc, Mlc1, Mlc2, TmI, TmII, act57B, β3-tub60D, Mlp60A, Mlp84b, Mp20, mbl, and wupA. Although Mef2 protein is present at high levels early in development, it does not occupy these enhancers until much later, demonstrating additional specificity in the regulation of Mef2 binding (Sandmann, 2006).
An investigation examined whether the temporal binding of Mef2 to a target gene's enhancer coincides with the onset of that gene's expression. Remarkably, the first time point when Mef2 binds to an enhancer is significantly correlated with the onset of that gene's expression during embryogenesis. While this trend holds for all time points assayed, the correlation is particularly strong for late bound enhancers, mirroring the coordinated expression of late muscle differentiation genes. Although additional levels of “post binding” regulation cannot be excluded, these results demonstrate that Mef2's DNA binding is tightly regulated and is a trigger for target gene expression (Sandmann, 2006).
These results provide the first evidence that, while Mef2 is broadly expressed during muscle development, its ability to bind to DNA is temporally regulated depending on the context of the enhancer. This finding is intriguingly similar to what has been shown for MyoD in fibroblasts and Pha-4 in C. elegans. While both transcription factors have broad temporal expression, they regulate temporally restricted enhancers. This highlights a potentially general mechanism for encoding spatiotemporal specificity within the context of a regulatory region (Sandmann, 2006).
To explain the three temporal patterns of Mef2 binding, regulatory motifs were sought within each group of enhancers. It was first determined whether the number of Mef2 sites was equally distributed between the three temporal groups of Mef2 bound enhancers. Interestingly, enhancers in the continuously bound and the late bound groups were significantly enriched in single and multiple Mef2 sites per fragment. This significant enrichment of Mef2 sites is conserved in the orthologous sequences of the related species, Drosophila pseudoobscura. In contrast, the early cluster of transiently bound enhancers contains a similar number of Mef2 sites as the rest of the genome (Sandmann, 2006).
The bHLH transcription factor Twist is essential for all aspects of early mesoderm development in Drosophila. Twist has a transient expression pattern, with peak expression at stage 11 (~6–7 hr) mirroring the peak binding of Mef2 to the early enhancers. In vitro studies have shown cooperative regulation between vertebrate Mef2 proteins and bHLH transcription factors via direct protein-protein intereactions (e.g., MyoD and Hand). Given the temporal expression of Twist protein and the ability of vertebrate Mef2 proteins to bind to bHLH proteins, cooperative binding of Mef2 and Twist is an attractive model to explain the transient Mef2 binding to the early group of enhancers (Sandmann, 2006).
Two lines of evidence indicate that this hypothesis is correct. First, Twist sites are significantly enriched in the early bound enhancers, and not in the continuous and late bound enhancers. This significant enrichment of Twist sites is conserved in the ortologous sequences of D. pseudoobscura. Second, Twist and Mef2 cobind to the early enhancers at the same stage of development. ChIP-on-chip studies were performed of Twist at 4–6 hr of development. Comparing the results of both studies showed in vivo binding of Twist to a large percentage of the early enhancers, demonstrating that Mef2 and Twist can cooccupy the early enhancers. Five examples were given of Mef2 bound early enhancers cobound by Twist at 4–6 hr of development (Sandmann, 2006).
While bHLH proteins have a central role in muscle development in all species examined to date, the predominant roles of individual family members have diverged. In vertebrates, MyoD family members play a central role in activating muscle gene expression, while Twist represses myogenesis. In Drosophila, the only MyoD family member, nau, is not essential for general muscle development. It has been speculated that Twist is the central bHLH regulator of Drosophila muscle development, as it is sufficient to activate the myogenic program upon ectopic expression in the ectoderm. The results provide further evidence for the evolutionary similar roles of Twist in flies and vertebrate MRFs; Drosophila Twist and Mef2 proteins may cooperatively regulate muscle gene expression in a similar manner to MyoD and Mef2 proteins in vertebrates (Sandmann, 2006).
Embryos with loss-of-function mutations in Drosophila Mef2 show a block of myoblast fusion and lack expression of a number of contractile muscle proteins. This study identified a number of Mef2 target genes involved in both processes; e.g., blow and lmd, two genes essential for myoblast fusion, as well as numerous cytoskeletal proteins (Sandmann, 2006).
In addition to these severe phenotypes, defects in neuromuscular junction (NMJ) formation and muscle attachments have been observed in Mef2 mutant embryos, the molecular basis of which is not understood. A significant enrichment was found of Mef2 target genes involved in both processes, providing a molecular understanding of the observed phenotypes (Sandmann, 2006).
The results also indicate that Mef2 is required for muscle function in differentiated myofibers. A number of Mef2 target genes involved in muscle energy production or storage were identified: Pfrx, GluRIIA, GlyP, Gpdh, Glycogenin, Pgi, and Pgk. This role may be further strengthened by the direct regulation of Ptx1, a transcription factor thought to regulate muscle physiology (Sandmann, 2006).
Drosophila body wall muscles are formed from progenitor cells that are selected through the action of Ras signaling and Notch-Delta lateral inhibition. The data show in vivo binding of Mef2 to enhancer regions of a striking number of genes that are essential for this process, in addition to expression changes for some genes in Mef2 mutant embryos. This includes components of the Notch-Delta pathway (Delta, mam, bib, E(spl) complex, and Neur), which is essential for specification of the somatic muscle and heart. Since these experiments were analyzed with tiling arrays covering 50% of the genome, Mef2 likely regulates even more genes involved in these signaling pathways, which could not be identifyied. This is in agreement with Junion (2005), who identified sfl, spen, and argos as Mef2 targets (Sandmann, 2006).
Once specified, founder cells express a characteristic set of transcription factors called identity genes. In vivo binding of Mef2 to enhancer regions of eight of the ten known muscle identity genes was identified. Importantly, the temporal binding of Mef2 correlates with the initiation of identity gene expression in founder cells (stages 9–11; time points 4–6 and 6–8 hr). Mef2 also binds to enhancer regions of a number of genes previously reported to be enriched in founder cells, suggesting that Mef2 acts in concert with the identity genes to regulate the transcriptional program within these myoblasts (Ubx, htl, CG14207, CG9520, CG17492, CG8417, and krT95D in the VO5 muscle (Sandmann, 2006).
Genetic studies looking at the interplay between motor neuron and muscle development observed a consistent reduction in the number of somatic muscles in Mef2 mutants. It has not been clear if the loss of muscles is a secondary defect due to a general failure of the muscle to differentiate, and the molecular mechanism was not been explored. The current results indicate that Mef2 directly regulates founder cell identity gene expression. It is suggested that Mef2 provides an extra layer of regulation to buffer this key step in muscle development from stochastic fluctuations in the levels of key regulators. When this regulation is absent in Mef2 mutant embryos, founder cells are not specified or maintained in a robust manner, leading to the observed low penetrance loss-of-muscles phenotype (Sandmann, 2006).
Traditionally, Mef2 is placed toward the bottom of the myogenic transcriptional hierarchy, due to its well-characterized role as a regulator of muscle effector proteins. Because of this, it was surprising to find that Mef2 regulates a large number of transcription factors, many of which are involved in early aspects of myogenesis. For example, Mef2 regulates transcription factors essential for visceral muscle development (bap, slp1, HLH54F) and cardioblast specification (nmr2, Zfh1, ush). These results implicate a role for Mef2 in the subdivision of the dorsal mesoderm and place Mef2 at the center of the transcriptional program required for Drosophila muscle development (Sandmann, 2006).
Vertebrate Mef2 proteins regulate the expression of the transcription factors MyoD and cJun. Direct regulation was found of the orthologs of these genes (nau, jra) by Drosophila Mef2, indicating that Mef2 is part of an evolutionary conserved genetic program. This suggests that many of the additional transcriptional connections identified in Drosophila may also be conserved (Sandmann, 2006).
One of the most surprising findings of this study was the large number of enhancer regions that are bound by Mef2. Previous studies searching for Mef2 targets focused on a limited part of the genome. The present study provides a unique opportunity to get a more accurate view of the total number of Mef2-regulated enhancers and genes. Using a tiling array covering ~50% of the genome, at least 670 unique Mef2 bound genomic regions were found. A total of 600 of these enhancer regions are within 5 kb of a gene locus and are therefore likely to represent active enhancers of a gene. Extrapolating to the whole genome, this indicates that Mef2 regulates as many as 1000 genes during the course of muscle development (Sandmann, 2006).
The current view of Mef2 in the literature is of a transcription factor required late in development for muscle differentiation. However, given the diversity of Mef2 target genes, its ability to regulate genes during all stages of muscle development, and the huge number of enhancer regions bound by Mef2, the view of the function of the transcription factor needs to be adjusted. Mef2 is likely to bind to enhancer regions of most, if not all, muscle genes, not just structural muscle proteins, and may thereby act as a “general” muscle transcription factor. The presence of feed-forward loops regulated by Mef2 as well as of low-penetrance phenotypes in Mef2 mutants suggests that one of the functions of this transcription factor is to provide robustness within the myogenic program (Sandmann, 2006).
The N-terminal MADS box, a mediator for DNA binding, is adjacent to a Mef2 domain present in mouse and human homologs (Lilly, 1994).
The serum response factor (see Drosophila SRF or Blistered) and myocyte enhancer factor 2A (MEF2A) represent two human members of the MADS-box transcription factor family. Each protein has a distinct biological function that is reflected by the distinct specificities of the proteins for their coregulatory protein partners and DNA-binding sites. The mechanism of DNA binding utilized by these two related transcription factors was examined. Although SRF and MEF2A belong to the same family and contain related DNA-binding domains, their DNA-binding mechanisms differ in several key aspects. In contrast to the dramatic DNA bending induced by SRF, MEF2A induces minimal DNA distortion. A combination of loss- and gain-of-function mutagenesis identifies a single amino acid residue located at the N terminus of the recognition helices as the critical mediator of this differential DNA bending. This residue is also involved in determining DNA-binding specificity, thus indicating a link between DNA bending and DNA-binding specificity determination. Different basic residues within the putative recognition alpha-helices are critical for DNA binding, and the role of the C-terminal extensions to the MADS box in dimerization between SRF and MEF2A also differs. These important differences in the molecular interactions of SRF and MEF2A are likely to contribute to their differing roles in the regulation of specific gene transcription (West, 1997).
date revised: 26 FEB 97
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