Myocyte Enhancer Factor 2 Proteins Research Articles

Structural insights into the HDAC4-MEF2A-DNA complex and its implication in long-range transcriptional regulation.

Class IIa Histone deacetylases (HDACs), including HDAC4, 5, 7and 9, play key roles in multiple important developmental and differentiation processes. Recent studies have shown that class IIa HDACs exert their transcriptional repressive function by interacting with tissue-specific transcription factors, such as members of the myocyte enhancer factor 2 (MEF2) family of transcription factors. However, the molecular mechanism is not well understood. In this study, we determined the crystal structure of an HDAC4-MEF2A-DNA complex. This complex adopts a dumbbell-shaped overall architecture, with a 2:4:2 stoichiometry of HDAC4, MEF2A and DNA molecules. In the complex, two HDAC4 molecules form a dimer through the interaction of their glutamine-rich domain (GRD) to form the stem of the 'dumbbell'; while two MEF2A dimers and their cognate DNA molecules are bridged by the HDAC4 dimer. Our structural observations were then validated using biochemical and mutagenesis assays. Further cell-based luciferase reporter gene assays revealed that the dimerization of HDAC4 is crucial in its ability to repress the transcriptional activities of MEF2 proteins. Taken together, our findings not only provide the structural basis for the assembly of the HDAC4-MEF2A-DNA complex but also shed light on the molecular mechanism of HDAC4-mediated long-range gene regulation.

Decreased Myocyte Enhancer Factor 2 Levels in the Hippocampus of Huntington's Disease Mice Are Related to Cognitive Dysfunction.

People suffering from Huntington's disease (HD) present cognitive deficits. Hippocampal dysfunction has been involved in the HD learning and memory impairment, but proteins leading this dysregulation are not fully characterized. Here, we studied the contribution of the family of transcription factors myocyte enhancer factor 2 (MEF2) to the HD cognitive deficits. To this aim, we first analyzed MEF2 protein levels and found that they are reduced in the hippocampus of exon-1 (R6/1) and full-length (HdhQ7/Q111) mutant huntingtin (mHTT) mice at the onset of cognitive dysfunction. By the analysis of MEF2 mRNA levels and mHTT-MEF2 interaction, we discarded that reduced MEF2 levels are due to changes in the transcription or sequestration in mHTT aggregates. Interestingly, we showed in R6/1 primary hippocampal cultures that reduction of MEF2 is strongly related to a basal and non-apoptotic caspase activity. To decipher the involvement of hippocampal decreased MEF2 in memory impairment, we used the BML-210 molecule that activates MEF2 transcriptional activity by the disruption MEF2-histone deacetylase class IIa interaction. BML-210 treatment increased the number and length of neurites in R6/1 primary hippocampal cultures. Importantly, this effect was prevented by transduction of lentiviral particles containing shRNA against MEF2. Then, we demonstrated that intraperitoneal administration of BML-210 (150mg/Kg/day) for 4days in R6/1 mice improved cognitive performance. Finally, we observed that BML-210 treatment also promoted the activation of MEF2-dependent memory-related genes and the increase of synaptic markers in the hippocampus of R6/1 mice. Our findings point out that reduced hippocampal MEF2 is an important mediator of cognitive dysfunction in HD and suggest that MEF2 slight basal activation could be a good therapeutic option.

Neuronal Myocyte-Specific Enhancer Factor 2D (MEF2D) Is Required for Normal Circadian and Sleep Behavior in Mice.

The transcription factor, myocyte enhancer factor-2 (MEF2), is required for normal circadian behavior in Drosophila; however, its role in the mammalian circadian system has not been established. Of the four mammalian Mef2 genes, Mef2d is highly expressed in the suprachiasmatic nucleus (SCN) of the hypothalamus, a region critical for coordinating peripheral circadian clocks. Using both conventional and brain-specific Mef2d KO (Mef2d-/-) mouse lines, we demonstrate that MEF2D is essential for maintaining the length of the circadian free-running period of locomotor activity and normal sleep patterns in male mice. Crossing Mef2d-/- with Per2::luc reporter mice, we show that these behavioral changes are achieved without altering the endogenous period of the master circadian oscillator in the SCN. Together, our data suggest that alterations in behavior in Mef2d-/- mice may be the result of an effect on SCN output, rather than an effect on timekeeping within the SCN itself. These findings add to the growing body of evidence that MEF2 proteins play important roles in the brain.SIGNIFICANCE STATEMENT These studies are the first to show a role for MEF2 proteins in the brain outside of the hippocampus, and our findings suggest that these proteins may play diverse roles in the CNS. It is important to continue to build on our understanding of the roles of proteins acting in the SCN because SCN dysfunction underlies jet lag in humans and influences the response to shift work schedules, which are now known as risk factors for the development of cancer. Our work on MEF2D could be the basis for opening new lines of research in the development and regulation of circadian rhythms.

Crystal Structure of Apo MEF2B Reveals New Insights in DNA Binding and Cofactor Interaction.

The myocyte enhancer factor 2 (MEF2) family of transcription factors plays important roles in developmental processes and adaptive responses. Although MEF2 proteins are known to bind DNA in the nucleus to regulate specific gene expression, there are reports that show that MEF2 also functions in the cytoplasm. Previous structural studies of MEF2 focused exclusively on DNA-bound MEF2 with and without various corepressors or coactivators. While these studies have established a comprehensive structural model of DNA recognition and cofactor recruitment by MEF2, the structure of MEF2 not bound to DNA, which include cytoplasmic MEF2 and free MEF2 in the nucleus, is unknown. Here we determined the structure of the MADS-box/MEF2 domain of MEF2B without DNA nor cofactor. The Apo structure of MEF2B reveals a largely preformed DNA binding interface that may be important for recognizing the shape of DNA from the minor groove side. In addition, our structure also reveals that the C-terminal helix of the MEF2-specific domain could flip up to bind to the hydrophobic groove that serves as the binding sites of MEF2 transcription cofactors. These observations shed new insights into DNA binding and cofactor interaction by MEF2 proteins.

Ca2+/Calmodulin-Dependent Protein Kinase II and Androgen Signaling Pathways Modulate MEF2 Activity in Testosterone-Induced Cardiac Myocyte Hypertrophy.

Testosterone is known to induce cardiac hypertrophy through androgen receptor (AR)-dependent and -independent pathways, but the molecular underpinnings of the androgen action remain poorly understood. Previous work has shown that Ca2+/calmodulin-dependent protein kinase II (CaMKII) and myocyte-enhancer factor 2 (MEF2) play key roles in promoting cardiac myocyte growth. In order to gain mechanistic insights into the action of androgens on the heart, we investigated how testosterone affects CaMKII and MEF2 in cardiac myocyte hypertrophy by performing studies on cultured rat cardiac myocytes and hearts obtained from adult male orchiectomized (ORX) rats. In cardiac myocytes, MEF2 activity was monitored using a luciferase reporter plasmid, and the effects of CaMKII and AR signaling pathways on MEF2C were examined by using siRNAs and pharmacological inhibitors targeting these two pathways. In the in vivo studies, ORX rats were randomly assigned to groups that were administered vehicle or testosterone (125 mg⋅kg-1⋅week-1) for 5 weeks, and plasma testosterone concentrations were determined using ELISA. Cardiac hypertrophy was evaluated by measuring well-characterized hypertrophy markers. Moreover, western blotting was used to assess CaMKII and phospholamban (PLN) phosphorylation, and MEF2C and AR protein levels in extracts of left-ventricle tissue from control and testosterone-treated ORX rats. Whereas testosterone treatment increased the phosphorylation levels of CaMKII (Thr286) and phospholambam (PLN) (Thr17) in cardiac myocytes in a time- and concentration-dependent manner, testosterone-induced MEF2 activity and cardiac myocyte hypertrophy were prevented upon inhibition of CaMKII, MEF2C, and AR signaling pathways. Notably, in the hypertrophied hearts obtained from testosterone-administered ORX rats, both CaMKII and PLN phosphorylation levels and AR and MEF2 protein levels were increased. Thus, this study presents the first evidence indicating that testosterone activates MEF2 through CaMKII and AR signaling. Our findings suggest that an orchestrated mechanism of action involving signal transduction and transcription pathways underlies testosterone-induced cardiac myocyte hypertrophy.

Freeze-responsive regulation of MEF2 proteins and downstream gene networks in muscles of the wood frog, Rana sylvatica

The wood frog survives frigid North American winters by retreating into a state of suspended animation characterized by the freezing of up to 65% of total body water as extracellular ice and displaying no heartbeat, breathing, brain activity, or movement. Physiological and biochemical adaptations are in place to facilitate global metabolic depression and protect against the consequences of whole body freezing. This study examined the myocyte enhancer factor 2 (MEF2) transcription factor family, proteins responsible for coordinating selective gene expression of a myriad of cellular functions from muscle development and remodelling to various stress responses. Immunoblotting, subcellular localization, and RT-PCR were used to analyze the regulation of MEF2A and MEF2C transcription factors and selected downstream targets under their control at transcriptional, translational, and post-translational levels in skeletal and cardiac muscles from control, frozen and thawed frogs. Both MEF2A/C proteins were freeze-responsive in skeletal muscle, displaying increases of 1.7–2 fold for phosphorylated MEF2AThr312 and MEF2CThr300 during freezing with an enrichment of nuclear phosphorylated MEF2 proteins (by 1.7–2.1 fold) observed as early as 4h post-freezing. Despite the reduced response of total and phosphorylated MEF2A/C protein levels observed in cardiac muscle, the MEF2 downstream gene targets (glucose transporter-4, calreticulin, and creatine kinase brain and muscle isozymes) displayed similar increases in transcript levels (1.7–4.8 fold) after 24h freezing in both muscle types. This study describes a novel freeze-responsive function for MEF2 transcription factors and further elaborates our understanding of the molecular mechanisms underlying natural freeze tolerance. This novel freeze-responsive regulation suggests a role for MEF2s and downstream genes in cryoprotectant glucose distribution, calcium homeostasis, and maintenance of energy reserves vital for successful freeze tolerance.

MEF2 Is Restricted to the Male Gonad and Regulates Expression of the Orphan Nuclear Receptor NR4A1

Leydig cell steroidogenesis is controlled by the pituitary gonadotropin LH that activates several signaling pathways, including the Ca(2+)/calmodulin kinase I (CAMKI) pathway. In other tissues, CAMKI regulates the activity of the myocyte enhancer factor 2 (MEF2) transcription factors. MEF2 factors are essential regulators of cell differentiation and organogenesis in numerous tissues but their expression and role in the mammalian gonad had not been explored. Here we show that MEF2 factors are expressed in a sexually dimorphic pattern in the mouse gonad. MEF2 factors are present in the testis throughout development and into adulthood but absent from the ovary. In the testis, MEF2 was localized mainly in the nucleus of both somatic lineages, the supporting Sertoli cells and the steroidogenic Leydig cells. In Leydig cells, MEF2 was found to activate the expression of Nr4a1, a nuclear receptor important for hormone-induced steroidogenesis. In these cells MEF2 also cooperates with forskolin and CAMKI to enhance Nr4a1 promoter activity via two MEF2 elements (-318 and -284 bp). EMSA confirmed direct binding of MEF2 to these elements whereas chromatin immunoprecipitation revealed that MEF2 recruitment to the proximal Nr4a1 promoter was increased following hormonal stimulation. Modulation of endogenous MEF2 protein level (small interfering RNA-mediated knockdown) or MEF2 activity (MEF2-Engrailed active dominant negative) led to a significant decrease in Nr4a1 mRNA levels in Leydig cells. All together, our results identify MEF2 as a novel testis-specific transcription factor, supporting a role for this factor in male sex differentiation and function. MEF2 was also positioned upstream of NR4A1 in a regulatory cascade controlling Leydig cell gene expression.

50. Muscle disuse atrophy: The expression of Myocyte Enhancer Factor 2 in the skeletal muscle of Spermophilus tridecemlineatus during hibernation

Thirteen-lined ground squirrels, Spermophlus tridecemlineatus , are true hibernators that survive the winter by hibernating in underground burrows from August through to March. During hibernation, squirrels experience little or no muscle atrophy despite a reduced work load. Atrophy is minimized by changing the composition of muscle proteins to provide an optimal mix of isoforms. This study analyzed protein levels of Myocyte Enhancer Factor-2 (MEF-2) in skeletal muscle. MEF-2 proteins are transcription factors that regulate the expression of genes responsible for muscle remodeling in skeletal muscles. Western immunoblotting showed enhanced levels of MEF-2 and an increase in its nuclear localization in muscle of hibernating animals. Finally, Western blots of MEF-2 downstream genes were analyzed. An increase in protein levels of downstream genes supports the activation of MEF-2 transcription factors and the subsequent transcription of genes necessary for muscle remodeling. Funded by NSERC Canada. For more information go to: www.carleton.ca/∼kbstorey .

Mef2A, a homologue of animal Mef2 transcription factors, regulates cell differentiation in Dictyostelium discoideum

BackgroundTranscription factors from the MADS-box family play a relevant role in cell differentiation and development and include the animal SRF (serum response factor) and MEF2 (myocyte enhancer factor 2) proteins. The social amoeba Dictyostelium discoideum contains four genes coding for MADS-box transcription factors, two of these genes code for proteins that are more similar to SRF, and the other two code for proteins that are more similar to MEF2 animal factors.ResultsThe biological function of one of the two genes that codes for MEF2-related proteins, a gene known as mef2A, is described in this article. This gene is expressed under the transcriptional control of two alternative promoters in growing cells, and its expression is induced during development in prespore cells. Mutant strains where the mef2A gene has been partially deleted were generated to study its biological function. The mutant strains showed reduced growth when feeding on bacteria and were able to develop and form fruiting bodies, but spore production was significantly reduced. A study of developmental markers showed that prespore cells differentiation was impaired in the mutant strains. When mutant and wild-type cells were set to develop in chimeras, mutant spores were underrepresented in the fruiting bodies. The mutant cells were also unable to form spores in vitro. In addition, mutant cells also showed a poor contribution to the formation of the tip-organizer and the upper region of slugs and culminant structures. In agreement with these observations, a comparison of the genes transcribed by mutant and wild-type strains during development indicated that prestalk gene expression was enhanced, while prespore gene expression decreased in the mef2A- strain.ConclusionsOur data shows that mef2A plays a role in cell differentiation in D. discoideum and modulates the expression of prespore and prestalk genes.

Class IIa Histone Deacetylases and Myocyte Enhancer Factor 2 Proteins Regulate the Mesenchymal-to-Epithelial Transition of Somatic Cell Reprogramming

Class IIa histone deacetylases (HDACs) and myocyte enhancer factor 2 (MEF2) proteins compose a signaling module that orchestrates lineage specification during embryogenesis. We show here that this module also regulates the generation of mouse induced pluripotent stem cells by defined transcription factors. Class IIa HDACs and MEF2 proteins rise steadily during fibroblast reprogramming to induced pluripotent stem cells. MEF2 proteins tend to block the process by inducing the expression of Tgfβ cytokines, which impairs the necessary phase of mesenchymal-to-epithelial transition (MET). Conversely, class IIa HDACs endeavor to suppress the activity of MEF2 proteins, thus enhancing the MET and colony formation efficiency. Our work highlights an unexpected role for a developmental axis in somatic cell reprogramming and provides new insight into how the MET is regulated in this context.

FMRP: a triple threat to PSD-95

FMRP: a triple threat to PSD-95

Translation of Myocyte Enhancer Factor-2 is induced by hypertrophic stimuli in cardiomyocytes through a Calcineurin-dependent pathway

The Myocyte Enhancer Factor-2 (MEF2) family of transcription factors regulates gene expression during cardiomyocyte differentiation and adaptation of the myocardium to stress. MEF2 activity is enhanced by increasing its transcription and by MAPK-dependent phosphorylation, and is reduced by binding to class-II Histone Deacetylases and by miR-1-mediated degradation of its transcript. Here we show that MEF2 protein abundance is regulated at the translational level, determining myocyte size, during hypertrophy. In order to reduce MEF2 protein expression, its silencing through RNA interference required serum deprivation and, even in this condition, MEF2 protein abundance recovered to basal levels in presence of phenylephrine. Hypertrophic agonist stimulation of neonatal ventricular cardiomyocytes increased Mef2 expression by enhancing its translation, without changing its transcription or blocking degradation of the protein. MEF2 abundance was increased by Calcineurin overexpression in vivo and was reduced by Calcineurin inhibition in vitro, without affecting Mef2 mRNA levels. Calcineurin activity influenced expression of Polypyrimidine Tract Protein (PTB), contributing to MEF2 translation. Thus, our results show a previously unrecognized but relevant level of MEF2 activity regulation through the control of its translation that involves Calcineurin and PTB.

Identification of HZF1 as a novel target gene of the MEF2 transcription factor

The MADS box-containing transcription factors MEF2A-D regulate the expression of most skeletal and cardiac muscle structural genes. Recently, a zinc finger protein called HZF1 was identified as a transcription factor, and found to be highly expressed in cardiac muscle. By screening the transcription regulatory region of HZF1, we found that myocyte enhancer factor 2 (MEF2) potentially recognizes and binds the regulatory region of the HZF1 gene. We also found that the knockdown of MEF2A endogenous expression in human aortic smooth muscle cells decreases the expression of HZF1, while the enforced expression of MEF2A in turn promotes the expression of HZF1. Furthermore, we employed the luciferase reporter system and chromatin immunoprecipitation (ChIP) to demonstrate that MEF2A does in fact bind the regulatory region of HZF1, which suggests that HZF1 is the direct target of MEF2A. Based on the fact that MEF2 proteins function as essential regulators of cardiac development and as important pathological factors for certain cardiac diseases, our finding that MEF2 positively regulates the expression of HZF1 may contribute to further research investigating the role of the zinc finger protein HZF1 in cardiac development and disease.

Vertebrate Paralogous MEF2 Genes: Origin, Conservation, and Evolution

BackgroundThe myocyte enhancer factor 2 (MEF2) gene family is broadly expressed during the development and maintenance of muscle cells. Although a great deal has been elucidated concerning MEF2 transcription factors' regulation of specific gene expression in diverse programs and adaptive responses, little is known about the origin and evolution of the four members of the MEF2 gene family in vertebrates.Methodology/Principal FindingsBy phylogenetic analyses, we investigated the origin, conservation, and evolution of the four MEF2 genes. First, among the four MEF2 paralogous branches, MEF2B is clearly distant from the other three branches in vertebrates, mainly because it lacks the HJURP_C (Holliday junction recognition protein C-terminal) region. Second, three duplication events might have occurred to produce the four MEF2 paralogous genes and the latest duplication event occurred near the origin of vertebrates producing MEF2A and MEF2C. Third, the ratio (Ka/Ks) of non-synonymous to synonymous nucleotide substitution rates showed that MEF2B evolves faster than the other three MEF2 proteins despite purifying selection on all of the four MEF2 branches. Moreover, a pair model of M0 versus M3 showed that variable selection exists among MEF2 proteins, and branch-site analysis presented that sites 53 and 64 along the MEF2B branch are under positive selection. Finally, and interestingly, substitution rates showed that type II MADS genes (i.e., MEF2-like genes) evolve as slowly as type I MADS genes (i.e., SRF-like genes) in animals, which is inconsistent with the fact that type II MADS genes evolve much slower than type I MADS genes in plants.ConclusionOur findings shed light on the relationship of MEF2A, B, C, and D with functional conservation and evolution in vertebrates. This study provides a rationale for future experimental design to investigate distinct but overlapping regulatory roles of the four MEF2 genes in various tissues.

Proline Isomerase Pin1 Represses Terminal Differentiation and Myocyte Enhancer Factor 2C Function in Skeletal Muscle Cells

Reversible proline-directed phosphorylation at Ser/Thr-Pro motifs has an essential role in myogenesis, a multistep process strictly regulated by several signaling pathways that impinge on two families of myogenic effectors, the basic helix-loop-helix myogenic transcription factors and the MEF2 (myocyte enhancer factor 2) proteins. The question of how these signals are deciphered by the myogenic effectors remains largely unaddressed. In this study, we show that the peptidyl-prolyl isomerase Pin1, which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds to induce conformational changes of its target proteins, acts as an inhibitor of muscle differentiation because its knockdown in myoblasts promotes myotube formation. With the aim of clarifying the mechanism of Pin1 function in skeletal myogenesis, we investigated whether MEF2C, a critical regulator of the myogenic program that is the end point of several signaling pathways, might serve as a/the target for the inhibitory effects of Pin1 on muscle differentiation. We show that Pin1 interacts selectively with phosphorylated MEF2C in skeletal muscle cells, both in vitro and in vivo. The interaction with Pin1 requires two novel critical phospho-Ser/Thr-Pro motifs in MEF2C, Ser98 and Ser110, which are phosphorylated in vivo. Overexpression of Pin1 decreases MEF2C stability and activity and its ability to cooperate with MyoD to activate myogenic conversion. Collectively, these findings reveal a novel role for Pin1 as a regulator of muscle terminal differentiation and suggest that Pin1-mediated repression of MEF2C function could contribute to this function.

CREB‐dependent Nur77 induction following depolarization in PC12 cells and neurons is modulated by MEF2 transcription factors

Expression of the nuclear orphan receptor gene Nur77 in neuronal cells is induced by activity-dependent increases in intracellular Ca2+ ions. Ca2+ responsiveness of the Nur77 gene has been attributed to two distinct DNA regulatory regions that recruit the transcription factors cAMP response element binding protein (CREB) and myocyte enhancer factor-2 (MEF2). Here we used dominant interfering and constitutively active mutants of CREB and MEF2 proteins to assess their relative contribution to depolarization-induced Nur77 expression in undifferentiated PC12 cells and hippocampal neurons. We show that while CREB is necessary for Ca2+-activated Nur77 expression MEF2 functions to modulate CREB-dependent Nur77 expression by acting as a repressor in quiescent cells.

Chronic ethanol feeding impairs AMPK and MEF2 expression and is associated with GLUT4 decrease in rat myocardium

Chronic and heavy alcohol consumption is one of the causes of heart diseases. However, the effects of ethanol on insulin sensitivity in myocardium has been unclear. To investigate the effects of ethanol on the expression of AMP-activated protein kinase (AMPK), myocyte enhancer factor 2 (MEF2) and glucose transporter 4 (GLUT4), all of which are involved in the regulation of insulin sensitivity, in the myocardium, we performed three parts of experiments in vivo and in vitro. I: Rats were injected with 5-amino-4-imidazolecarboxamide ribonucleotide (AICAR, 0.8 mg.kg(-l)) for 2 h. II: Rats received different dose (0.5, 2.5 or 5 g.kg(-l).d(-l)) of ethanol for 22-week. III: Primary neonatal rat cardiomyocytes were isolated and treated with or without 100 mM ethanol or 1 mM AICAR for 4 h. The cardiac protein and mRNA expression of AMPKalpha subunits, MEF2 and GLUT4 were observed by western-blotting and RT-PCR, respectively. Serum TNFalpha levels were assessed by ELISA. The results showed chronic ethanol exposure induced insulin resistance. Ethanol decreased the mRNA levels of AMPKalpha1 and alpha2, the protein levels of total- and phospho-AMPKalpha in cardiomyocytes. Similarly, ethanol showed inhibitory effects on both the mRNA and protein levels of MEF2A and 2D, and GLUT4 in a dose-response-like fashion. Correlation analysis implied an association between phospho-AMPKalpha and MEF2A or MEF2D, and between the levels of MEF2 protein and GLUT4 transcription. In addition, ethanol elevated serum TNFalpha level. Taken together, chronic ethanol exposure decreases the expression of AMPKalpha and MEF2, and is associated with GLUT4 decline in rat myocardium.

The cardiac pacemaker-specific channel Hcn4 is a direct transcriptional target of MEF2

Hcn4, which encodes the hyperpolarization-activated, cyclic nucleotide-sensitive channel (I(h)), is a well-established marker of the cardiac sino-atrial node. We aimed to identify cis-elements in the genomic locus of the Hcn4 gene that regulate the transcription of Hcn4. We screened evolutionarily conserved non-coding sequences (CNSs) that are often involved in the regulation of gene expression. The VISTA Enhancer Browser identified 16 regions, termed CNS 1-16, within the Hcn4 locus. Using the luciferase reporter assay in primary neonatal rat cardiomyocytes, we found that CNS13 conferred a prominent enhancer activity (more than 30-fold) on the Hcn4 promoter. Subsequent mutation analysis revealed that the Hcn4 enhancer function was dependent on myocyte enhancer factor-2 (MEF2) and activator protein-1 (AP1) binding sequences located in CNS13. Electrophoretic mobility shift assay and chromatin immunoprecipitation confirmed that MEF2 and AP1 proteins bound CNS13. Furthermore, overexpression of a dominant negative MEF2 mutant inhibited the enhancer activity of CNS13, decreased Hcn4 mRNA expression and also decreased the amplitude of I(h) current in myocytes isolated from the inflow tract of embryonic heart. These results suggest that the novel enhancer CNS13 and MEF2 may play a critical role in the transcription of Hcn4 in the heart.

Regulation of GLUT4 expression in denervated skeletal muscle

Denervation by sciatic nerve resection causes decreased muscle glucose transporter 4 (GLUT4) expression, but little is known about the signaling events that cause this decrease. Experiments were designed to test the hypothesis that decreased GLUT4 expression in denervated muscle occurs because of decreased calcium/CaMK activity, which would then lead to decreased activation of the transcription factors myocyte enhancer factor 2 (MEF2) and GLUT4 enhancer factor (GEF), which are required for normal GLUT4 expression. GLUT4 mRNA was elevated in mice expressing constitutively active CaMK isoform IV (CaMKIV) and decreased by denervation. Denervation decreased GEF binding to the promoter and the content of GEF in the nucleus, but there was no change in either MEF2 binding or MEF2 protein content. Expression of a MEF2-dependent reporter gene did not change in denervated skeletal muscle. To determine the domains of the GLUT4 promoter that respond to denervation, transgenic mice expressing the chloramphenicol acetyl transferase (CAT) reporter gene driven by different lengths of the human GLUT4 promoter were denervated. Using several different promoter/reporter gene constructs, we found that all areas of the GLUT4 promoter were truncated or missing, except for the MEF2 binding domain and the basal promoter. All of the GLUT4 promoter/CAT reporter constructs evaluated responded normally to denervation. Our data lead us to conclude that decreased CaMK activity is not the reason for decreased GLUT4 content in denervated muscle and that negative control of GLUT4 expression is not mediated through the MEF2 or GEF-binding domains. These findings indicate that withdrawal of a GEF- or MEF2-dependent signal is not likely a major determinant of the denervation effect on GLUT4 expression. Thus, the response to denervation may be mediated by other elements present in the basal promoter of the GLUT4 gene.

Cocaine Regulates MEF2 to Control Synaptic and Behavioral Plasticity

Repeated exposure to cocaine causes sensitized behavioral responses and increased dendritic spines on medium spiny neurons of the nucleus accumbens (NAc). We find that cocaine regulates myocyte enhancer factor 2 (MEF2) transcription factors to control these two processes in vivo. Cocaine suppresses striatal MEF2 activity in part through a mechanism involving cAMP, the regulator of calmodulin signaling (RCS), and calcineurin. We show that reducing MEF2 activity in the NAc in vivo is required for the cocaine-induced increases in dendritic spine density. Surprisingly, we find that increasing MEF2 activity in the NAc, which blocks the cocaine-induced increase in dendritic spine density, enhances sensitized behavioral responses to cocaine. Together, our findings implicate MEF2 as a key regulator of structural synapse plasticity and sensitized responses to cocaine and suggest that reducing MEF2 activity (and increasing spine density) in NAc may be a compensatory mechanism to limit long-lasting maladaptive behavioral responses to cocaine.