Myocyte Enhancer Factor 2 Function Research Articles

Phosphorylation of the Myogenic Factor Myocyte Enhancer Factor-2 Impacts Myogenesis InVivo.

Activity of the myogenic regulatory protein myocyte enhancer factor-2 (MEF2) is modulated by post-translational modification. We investigated the invivo phosphorylation of Drosophila MEF2, and identified serine 98 (S98) as a phosphorylated residue. Phospho-mimetic (S98E) and phospho-null (S98A) isoforms of MEF2 did not differ from wild-type in their activity invitro, so we used CRISPR/Cas9 to generate an S98A allele of the endogenous gene. In mutant larvae we observed phenotypes characteristic of reduced MEF2 function, including reduced body wall muscle size and reduced expression of myofibrillar protein genes; conversely,S98A homozygotes showed enhanced MEF2 function through muscle differentiation within the adult myoblasts associated with the wing imaginal disc. In adults, S98A homozygotes were viable with normal mobility, yet showed patterning defects in muscles that were enhanced when the S98A allele was combined with a Mef2 null allele. Overall our data indicate that blocking MEF2 S98 phosphorylation in myoblasts enhances its myogenic capability, whereas blocking S98 phosphorylation in differentiating muscles attenuates MEF2 function. Our studies are among the first to assess the functional significance of MEF2 phosphorylation sites in the intact animal, and suggest that the same modification can have profoundly different effects upon MEF2 function depending upon the developmental context.

A conserved MADS-box phosphorylation motif regulates differentiation and mitochondrial function in skeletal, cardiac, and smooth muscle cells.

Exposure to metabolic disease during fetal development alters cellular differentiation and perturbs metabolic homeostasis, but the underlying molecular regulators of this phenomenon in muscle cells are not completely understood. To address this, we undertook a computational approach to identify cooperating partners of the myocyte enhancer factor-2 (MEF2) family of transcription factors, known regulators of muscle differentiation and metabolic function. We demonstrate that MEF2 and the serum response factor (SRF) collaboratively regulate the expression of numerous muscle-specific genes, including microRNA-133a (miR-133a). Using tandem mass spectrometry techniques, we identify a conserved phosphorylation motif within the MEF2 and SRF Mcm1 Agamous Deficiens SRF (MADS)-box that regulates miR-133a expression and mitochondrial function in response to a lipotoxic signal. Furthermore, reconstitution of MEF2 function by expression of a neutralizing mutation in this identified phosphorylation motif restores miR-133a expression and mitochondrial membrane potential during lipotoxicity. Mechanistically, we demonstrate that miR-133a regulates mitochondrial function through translational inhibition of a mitophagy and cell death modulating protein, called Nix. Finally, we show that rodents exposed to gestational diabetes during fetal development display muscle diacylglycerol accumulation, concurrent with insulin resistance, reduced miR-133a, and elevated Nix expression, as young adult rats. Given the diverse roles of miR-133a and Nix in regulating mitochondrial function, and proliferation in certain cancers, dysregulation of this genetic pathway may have broad implications involving insulin resistance, cardiovascular disease, and cancer biology.

Pro-survival function of MEF2 in cardiomyocytes is enhanced by β-blockers.

β1-Adrenergic receptor (β1-AR) stimulation increases apoptosis in cardiomyocytes through activation of cAMP/protein kinase A (PKA) signaling. The myocyte enhancer factor 2 (MEF2) proteins function as important regulators of myocardial gene expression. Previously, we reported that PKA signaling directly represses MEF2 activity. We determined whether (a) MEF2 has a pro-survival function in cardiomyocytes, and (b) whether β-adrenergic/PKA signaling modulates MEF2 function in cardiomyocytes. Initially, we observed that siRNA-mediated gene silencing of MEF2 induces cardiomyocyte apoptosis as indicated by flow cytometry. β1-AR activation by isoproterenol represses MEF2 activity and promotes apoptosis in cultured neonatal cardiomyocytes. Importantly, β1-AR mediated apoptosis was abrogated in cardiomyocytes expressing a PKA-resistant form of MEF2D (S121/190A). We also observed that a β1-blocker, Atenolol, antagonizes isoproterenol-induced apoptosis while concomitantly enhancing MEF2 transcriptional activity. β-AR stimulation modulated MEF2 cellular localization in cardiomyocytes and this effect was reversed by β-blocker treatment. Furthermore, Kruppel-like factor 6, a MEF2 target gene in the heart, functions as a downstream pro-survival factor in cardiomyocytes. Collectively, these data indicate that (a) MEF2 has an important pro-survival role in cardiomyocytes, and (b) β-adrenergic signaling antagonizes the pro-survival function of MEF2 in cardiomyocytes and β-blockers promote it. These observations have important clinical implications that may contribute to novel strategies for preventing cardiomyocyte apoptosis associated with heart pathology.

Apelin-APJ Signaling Is a Critical Regulator of Endothelial MEF2 Activation in Cardiovascular Development

The peptide ligand apelin and its receptor APJ constitute a signaling pathway with numerous effects on the cardiovascular system, including cardiovascular development in model organisms such as xenopus and zebrafish. This study aimed to characterize the embryonic lethal phenotype of the Apj-/- mice and to define the involved downstream signaling targets. We report the first characterization of the embryonic lethality of the Apj-/- mice. More than half of the expected Apj-/- embryos died in utero because of cardiovascular developmental defects. Those succumbing to early embryonic death had markedly deformed vasculature of the yolk sac and the embryo, as well as poorly looped hearts with aberrantly formed right ventricles and defective atrioventricular cushion formation. Apj-/- embryos surviving to later stages demonstrated incomplete vascular maturation because of a deficiency of vascular smooth muscle cells and impaired myocardial trabeculation and ventricular wall development. The molecular mechanism implicates a novel, noncanonical signaling pathway downstream of apelin-APJ involving Gα13, which induces histone deacetylase (HDAC) 4 and HDAC5 phosphorylation and cytoplasmic translocation, resulting in activation of myocyte enhancer factor 2. Apj-/- mice have greater endocardial Hdac4 and Hdac5 nuclear localization and reduced expression of the myocyte enhancer factor 2 (MEF2) transcriptional target Krüppel-like factor 2. We identify a number of commonly shared transcriptional targets among apelin-APJ, Gα13, and MEF2 in endothelial cells, which are significantly decreased in the Apj-/- embryos and endothelial cells. Our results demonstrate a novel role for apelin-APJ signaling as a potent regulator of endothelial MEF2 function in the developing cardiovascular system.

Postsynaptic FMRP bidirectionally regulates excitatory synapses as a function of developmental age and MEF2 activity

Rates of synapse formation and elimination change over the course of postnatal development, but little is known of molecular mechanisms that mediate this developmental switch. Here, we report that the dendritic RNA-binding protein fragile X mental retardation protein (FMRP) bidirectionally and cell autonomously regulates excitatory synaptic function, which depends on developmental age as well as function of the activity-dependent transcription factor myocyte enhancer factor 2 (MEF2). The acute postsynaptic expression of FMRP in CA1 neurons of hippocampal slice cultures (during the first postnatal week, P6-P7) promotes synapse function and maturation. In contrast, the acute expression of FMRP or endogenous FMRP in more mature neurons (during the second postnatal week; P13-P16) suppresses synapse number. The ability of neuronal depolarization to stimulate MEF2 transcriptional activity increases over this same developmental period. Knockout of endogenous MEF2 isoforms causes acute postsynaptic FMRP expression to promote, instead of eliminate, synapses onto 2-week-old neurons. Conversely, the expression of active MEF2 in neonatal neurons results in a precocious FMRP-dependent synapse elimination. Our findings suggest that FMRP and MEF2 function together to fine tune synapse formation and elimination rates in response to neuronal activity levels over the course of postnatal development.

MEF2 negatively regulates learning-induced structural plasticity and memory formation

Memory formation is thought to be mediated by dendritic-spine growth and restructuring. Myocyte enhancer factor 2 (MEF2) restricts spine growth in vitro, suggesting that this transcription factor negatively regulates the spine remodeling necessary for memory formation. Here we show that memory formation in adult mice was associated with changes in endogenous MEF2 levels and function. Locally and acutely increasing MEF2 function in the dentate gyrus blocked both learning-induced increases in spine density and spatial-memory formation. Increasing MEF2 function in amygdala disrupted fear-memory formation. We rescued MEF2-induced memory disruption by interfering with AMPA receptor endocytosis, suggesting that AMPA receptor trafficking is a key mechanism underlying the effects of MEF2. In contrast, decreasing MEF2 function in dentate gyrus and amygdala facilitated the formation of spatial and fear memory, respectively. These bidirectional effects indicate that MEF2 is a key regulator of plasticity and that relieving the suppressive effects of MEF2-mediated transcription permits memory formation.

Modulation of Neuronal Survival Factor MEF2 by Kinases in Parkinson’s Disease

Parkinson’s disease (PD) is the second most common neurodegenerative disorder due to selective death of neurons in the substantia nigra pars compacta. The cause of cell death remains largely unknown. Myocyte enhancer factor 2 (MEF2) is a group of transcriptional factors required to regulate neuronal development, synaptic plasticity, as well as survival. Recent studies show that MEF2 functions are regulated in multiple subcellular organelles and suggest that dysregulation of MEF2 plays essential roles in the pathogenesis of PD. Many kinases associated with transcription, translation, protein misfolding, autophagy, and cellular energy homeostasis are involved in the neurodegenerative process. Following the first demonstration that mitogen-activated protein kinase p38 (p38 MAPK) directly phosphorylates and activates MEF2 to promote neuronal survival, several other kinase regulators of MEF2s have been identified. These include protein kinase A and extracellular signal regulated kinase 5 as positive MEF2 regulators, and cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3β as negative regulators in response to diverse toxic signals relevant to PD. It is clear that MEF2 has emerged as a key point where survival and death signals converge to exert their regulatory effects, and dysregulation of MEF2 function in multiple subcellular organelles may underlie PD pathogenesis. Moreover, several other kinases such as leucine-rich repeat kinase 2 and PTEN-induced putative kinase 1 (PINK1) are of particular interest due to their potential interaction with MEF2.

Regulation of myocyte enhancer factor-2 transcription factors by neurotoxins

Various isoforms of myocyte enhancer factor-2 (MEF2) constitute a group of nuclear proteins found to play important roles in increasing types of cells. In neurons, MEF2s are required to regulate neuronal development, synaptic plasticity, as well as survival. MEF2s promote the survival of several types of neurons under different conditions. In cellular models, negative regulation of MEF2s by stress and toxic signals contributes to neuronal death. In contrast, enhancing MEF2 activity not only protects cultured primary neurons from death in vitro but also attenuates the loss of dopaminergic neurons in substantia nigra pars compacta in a 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease. In this work, the mechanisms of regulation of MEF2 function by several well-known neurotoxins and their implications in various neurodegenerative diseases are reviewed.

Fragile X Mental Retardation Protein Is Required for Synapse Elimination by the Activity-Dependent Transcription Factor MEF2

Fragile X syndrome (FXS), the most common genetic form of mental retardation and autism, is caused by loss-of-function mutations in an RNA-binding protein, Fragile X Mental Retardation Protein (FMRP). Neurons from patients and the mouse Fmr1 knockout (KO) model are characterized by an excess of dendritic spines, suggesting a deficit in excitatory synapse elimination. In response to neuronal activity, myocyte enhancer factor 2 (MEF2) transcription factors induce robust synapse elimination. Here, we demonstrate that MEF2 activation fails to eliminate functional or structural excitatory synapses in hippocampal neurons from Fmr1 KO mice. Similarly, inhibition of endogenous MEF2 increases synapse number in wild-type but not Fmr1 KO neurons. MEF2-dependent synapse elimination is rescued in Fmr1 KO neurons by acute postsynaptic expression of wild-type but not RNA-binding mutants of FMRP. Our results reveal that active MEF2 and FMRP function together in an acute, cell-autonomous mechanism to eliminate excitatory synapses.

Signalling GLUTtony keeps the sugar coming through glucose transporter expression

Skeletal muscle is one of the major sites of insulin-stimulated glucose disposal via the glucose transporter isoform 4 (GLUT4).

Direct Interaction between Myocyte Enhancer Factor 2 (MEF2) and Protein Phosphatase 1α Represses MEF2-Dependent Gene Expression

The myocyte enhancer factor 2 (MEF2) transcription factors play important roles in neuronal, cardiac, and skeletal muscle tissues. MEF2 serves as a nuclear sensor, integrating signals from several signaling cascades through protein-protein interactions with kinases, chromatin remodeling factors, and other transcriptional regulators. Here, we report a novel interaction between the catalytic subunit of protein phosphatase 1alpha (PP1alpha) and MEF2. Interaction occurs within the nucleus, and binding of PP1alpha to MEF2 potently represses MEF2-dependent transcription. The interaction utilizes uncharacterized domains in both PP1alpha and MEF2, and PP1alpha phosphatase activity is not obligatory for MEF2 repression. Moreover, a MEF2-PP1alpha regulatory complex leads to nuclear retention and recruitment of histone deacetylase 4 to MEF2 transcription complexes. PP1alpha-mediated repression of MEF2 overrides the positive influence of calcineurin signaling, suggesting PP1alpha exerts a dominant level of control over MEF2 function. Indeed, PP1alpha-mediated repression of MEF2 function interferes with the prosurvival effect of MEF2 in primary hippocampal neurons. The PP1alpha-MEF2 interaction constitutes a potent locus of control for MEF2-dependent gene expression, having potentially important implications for neuronal cell survival, cardiac remodeling in disease, and terminal differentiation of vascular, cardiac, and skeletal muscle.

Myocyte Enhancer Factor 2 and Microphthalmia-associated Transcription Factor Cooperate with NFATc1 to Transactivate the V-ATPase d2 Promoter during RANKL-induced Osteoclastogenesis

The V-ATPase d2 protein constitutes an important subunit of the V-ATPase proton pump, which regulates bone homeostasis; however, currently little is known about its transcriptional regulation. Here, in an attempt to understand regulation of the V-ATPase d2 promoter, we identified the presence of NFATc1, microphthalmia-associated transcription factor (MITF)- and myocyte enhancer factor 2 (MEF2)-binding sites within the V-ATPase d2 promoter using complementary bioinformatic analyses, chromatin immunoprecipitation, and electromobility shift assay. Intriguingly, activation of the V-ATPase d2 promoter by NFATc1 was enhanced by either MEF2 or MITF overexpression. By comparison, coexpression of MITF and MEF2 did not further enhance V-ATPase d2 promoter activity above that of expression of MITF alone. Consistent with a role in transcriptional regulation, both NFATc1 and MITF proteins translocated from the cytosol to the nucleus during RANKL-induced osteoclastogenesis, whereas MEF2 persisted in the nucleus of both osteoclasts and their mononuclear precursors. Targeted mutation of the putative NFATc1-, MITF-, or MEF2-binding sites in the V-ATPase d2 promoter impaired its transcriptional activation. Additionally retroviral overexpression of MITF or MEF2 in RAW264.7 cells potentiated RANKL-induced osteoclastogenesis and V-ATPase d2 gene expression. Based on these data, we propose that MEF2 and MITF function cooperatively with NFATc1 to transactivate the V-ATPase d2 promoter during RANKL-induced osteoclastogenesis.

The deacetylase HDAC4 controls myocyte enhancing factor‐2‐dependent structural gene expression in response to neural activity

Histone deacetylase 4 (HDAC4) binds and inhibits activation of the critical muscle transcription factor myocyte enhancer factor-2 (MEF2). However, the physiological significance of the HDAC4-MEF2 complex in skeletal muscle has not been established. Here we show that in skeletal muscle, HDAC4 is a critical modulator of MEF2-dependent structural and contractile gene expression in response to neural activity. We present evidence that loss of neural input leads to concomitant nuclear accumulation of HDAC4 and transcriptional reduction of MEF2-regulated gene expression. Cell-based assays show that HDAC4 represses structural gene expression via direct binding to AT-rich MEF2 response elements. Notably, using both surgical denervation and the neuromuscular disease amyotrophic lateral sclerosis (ALS) model, we found that elevated levels of HDAC4 are required for efficient repression of MEF2-dependent structural gene expression, indicating a link between the pathological induction of HDAC4 and subsequent MEF2 target gene suppression. Supporting this supposition, we show that ectopic expression of HDAC4 in muscle fibers is sufficient to induce muscle damage in mice. Our study identifies HDAC4 as an activity-dependent regulator of MEF2 function and suggests that activation of HDAC4 in response to chronically reduced neural activity suppresses MEF2-dependent gene expression and contributes to progressive muscle dysfunction observed in neuromuscular diseases.

Cardiac expression of the drosophila Transglutaminase (CG7356) gene is directly controlled by myocyte enhancer factor‐2

The myocyte enhancer factor-2 (MEF2) family of transcription factors plays key roles in the activation of muscle structural genes. In Drosophila, MEF2 accumulates at high levels in the embryonic muscles, where it activates target genes throughout the mesoderm. Here, we identify the Transglutaminase gene (Tg; CG7356) as a direct transcriptional target of MEF2 in the cardiac musculature. Tg is expressed in cells forming the inflow tracts of the dorsal vessel, and we identify the enhancer responsible for this expression. The enhancer contains three binding sites for MEF2, and can be activated by MEF2 in tissue culture and in vivo. Moreover, loss of MEF2 function, or removal of the MEF2 binding sites from the enhancer, results in loss of Tg expression. These studies identify a new MEF2 target in the cardiac musculature. These studies provide a possible mechanism for the activation of transglutaminase genes.

Histone Deacetylase 3 Interacts with and Deacetylates Myocyte Enhancer Factor 2

The myocyte enhancer factor 2 (MEF2) family of transcription factors is not only important for controlling gene expression in normal cellular programs, like muscle differentiation, T-cell apoptosis, neuronal survival, and synaptic differentiation, but has also been linked to cardiac hypertrophy and other pathological conditions. Lysine acetylation has been shown to modulate MEF2 function, but it is not so clear which deacetylase(s) is involved. We report here that treatment of HEK293 cells with trichostatin A or nicotinamide upregulated MEF2D acetylation, suggesting that different deacetylases catalyze the deacetylation. Related to the trichostatin A sensitivity, histone deacetylase 4 (HDAC4) and HDAC5, two known partners of MEF2, exhibited little deacetylase activity towards MEF2D. In contrast, HDAC3 efficiently deacetylated MEF2D in vitro and in vivo. This was specific, since HDAC1, HDAC2, and HDAC8 failed to do so. While HDAC4, HDAC5, HDAC7, and HDAC9 are known to recognize primarily the MEF2-specific domain, we found that HDAC3 interacts directly with the MADS box. In addition, HDAC3 associated with the acetyltransferases p300 and p300/CBP-associated factor (PCAF) to reverse autoacetylation. Furthermore, the nuclear receptor corepressor SMRT (silencing mediator of retinoid acid and thyroid hormone receptor) stimulated the deacetylase activity of HDAC3 towards MEF2 and PCAF. Supporting the physical interaction and deacetylase activity, HDAC3 repressed MEF2-dependent transcription and inhibited myogenesis. These results reveal an unexpected role for HDAC3 and suggest a novel pathway through which MEF2 activity is controlled in vivo.

PI3K/Akt‐dependent regulation of the transcription factor myocyte enhancer factor‐2 in insulin‐like growth factor‐1‐ and membrane depolarization‐mediated survival of cerebellar granule neurons

Survival signals such as insulin-like growth factor-1 (IGF-1) or membrane depolarization convey their neuronal protective effects through the activation of signaling networks and nuclear factors. In cerebellar granule neurons, IGF-1 mediates survival primarily through the PI3K/Akt pathway. The function of the transcription factor myocyte enhancer factor-2 (MEF2) is required for mediating membrane depolarization-dependent neuronal survival. However, whether PI3K/Akt regulates MEF2 and the role of MEF2 in IGF-1-mediated survival of neurons are unknown. In addition, the contribution of the PI3K/Akt pathway in membrane depolarization-induced neuronal survival remains undefined. We show here that the PI3K/Akt pathway promotes the survival of cerebellar granule neurons derived from Long-Evans rats following IGF-1 stimulation or membrane depolarization through regulation of MEF2 activity. IGF-1 stimulated the gene transactivation activity of MEF2 and its DNA binding potential. Moreover, regulation of MEF2 function by IGF-1 was dependent on the activity of the PI3K/Akt signaling pathway. Blocking MEF2 function reduced IGF-1-induced survival of cerebellar granule neurons. Membrane depolarization stimulated phosphorylation of Akt in cerebellar granule neurons. Blocking of the PI3K/Akt pathway with either a pharmacological inhibitor of PI3K, LY294002, or dominant negative mutants of PI3K and Akt inhibited the membrane depolarization-induced increase in MEF2 transactivation as well as its DNA binding activity and reduced neuronal survival. Together, these findings provide clear evidence to support an important role of the PI3K/Akt pathway in the regulation of nuclear survival factor MEF2 upon either IGF-1 stimulation or membrane depolarization, thus placing MEF2 as a novel downstream effector of the PI3K/Akt pathway in neurons.

Regulation of Neuroprotective Activity of Myocyte-enhancer Factor 2 by cAMP-Protein Kinase A Signaling Pathway in Neuronal Survival

The transcription factor myocyte-enhancer factor 2 (MEF2) has been shown to be required for the survival of different types of neurons. However, the death- or survival-inducing second messenger pathways that regulate MEF2 activity remain to be fully elucidated. Membrane depolarization by KCl induces neuronal survival that is dependent upon MEF2-mediated gene transactivation. Here we report that membrane depolarizationinduced activation of MEF2 requires the cAMP-protein kinase A (PKA) pathway. Inhibition of the activity of cAMP-PKA pathway attenuates membrane depolarization-induced activation of MEF2 activity and neuronal survival, whereas enhancing the activity of this pathway prevents KCl withdrawal-induced inhibition of MEF2 and neuronal apoptosis. Moreover, PKA directly phosphorylates MEF2 at Thr-20 in vitro to increase MEF2 DNA binding activity. A mutation of Thr-20 to Ala renders MEF2 resistant to PKA phosphorylation in vitro and reduces its DNA binding activity. Transfection of this T20A mutant blocks survival and induces apoptosis in cultured cortical and cerebellar granule neurons. This study identifies the transcription factor MEF2 as a target of cAMP-PKA pathway and demonstrates that PKA phosphorylation of MEF2 is a key step in modulating its DNA binding activity and ability to promote neuronal survival.

TGF-beta-activated Smad3 represses MEF2-dependent transcription in myogenic differentiation.

Transforming growth factor beta (TGF-beta) inhibits myogenesis and associated gene expression. We previously reported that the TGF-beta signaling effector Smad3 mediates this inhibition, by interfering with the assembly of myogenic bHLH transcription factor heterodimers on E-box sequences in the regulatory regions of muscle-specific genes. We now show that TGF-beta-activated Smad3 suppresses the function of MEF2, a second class of essential myogenic factors. TGF-beta signaling through Smad3 represses myogenin expression independently of E-boxes, and prevents a tethered MyoD-E47 dimer to activate transcription indirectly through MEF2-binding sites. In addition, Smad3 interacts with MEF2C, which requires its MADS domain, and disrupts its association with the SRC-family coactivator GRIP-1, thus diminishing the transcription activity of MEF2C. Consistent with this physical displacement, TGF-beta signaling blocks the GRIP-1-induced redistribution of MEF2C to discrete nuclear subdomains in 10T1/2 cells, and the recruitment of GRIP-1 to the myogenin promoter in differentiating myoblasts. These findings indicate that the TGF-beta/Smad3 pathway targets two critical components of the myogenic transcription machinery to inhibit terminal differentiation.

A Dominant-Negative Form of Transcription Factor MEF2 Inhibits Myogenesis

A biological role for MEF2 (myocyte enhancer factor 2) activity during mammalian myogenesis has been inferred but not directly proven because of its role in the transcriptional activation of many muscle-specific genes. Therefore, our purpose was to determine whether MEF2 activity is absolutely required for mammalian myogenesis. Using a dominant-negative approach to address this question, we constructed a mutated MEF2A protein comprised of the amino-terminal DNA binding/dimerization domain of MEF2A without its trans-activation domain as a bacterial fusion protein (GST-131) or in a eukaryotic expression vector (pcDNA-131). GST-131 and the protein encoded by pcDNA-131 bind specifically to the MEF2 cis element and abrogate trans-activation of a MEF2-responsive luciferase reporter gene by wild type MEF2A, thus serving a role as trans-dominant inhibitors of MEF2 function. In congruence with their ability to interfere with wild type MEF2 function, microinjection of GST-131 or pcDNA-131 into L6E9 or C2C12 myoblasts inhibited myotube formation. Immunofluorescence analysis showed that the expression of myogenin, myosin heavy chain, and MEF2A were inhibited in the GST-131 or pcDNA-131-injected cells compared with GST or pcDNA-injected controls. We also document that this trans-dominant MEF2 inhibitor impairs the myogenic conversion of C3H10T1/2 fibroblasts by MyoD. Thus, these data provide evidence that the trans-activation function of the MEF2 proteins during mammalian myogenesis is required for muscle-specific gene expression and differentiation.