Myocyte enhancer factor 2


Mef2 homologs and heart and vasculature development

The muscle-specific regulatory region of the alpha-cardiac myosin heavy-chain (MHC) gene contains the thyroid hormone response element (TRE) and two A/T-rich DNA sequences (designated A/T1 and A/T2), binding sites for the putative myocyte-specific enhancer factor 2 (MEF2). An investigation was carried out of the roles of the TRE and MEF2 binding sites and the potential interaction between thyroid hormone receptor (TR) and MEF2 proteins regulating the alpha-MHC promoter. Deletion mutation analysis indicates that both the A/T2 motif and TRE are required for muscle-specific expression of the alpha-MHC gene. The alpha-MHC enhancer containing both the A/T2 motif and TRE is synergistically activated by coexpression of MEF2 and TR in nonmuscle cells, whereas neither factor by itself activates the alpha-MHC reporters. The reporter construct containing the A/T2 sequence and the TRE linked to a heterologous promoter also shows synergistic activation by coexpression of MEF2 and TR in nonmuscle cells. Protein binding assays demonstrate that MEF2 and TR specifically bind to one another in vitro and in vivo. The MADS domain of MEF2 and the DNA-binding domain of TR are necessary and sufficient to mediate their physical interaction. These results suggest that the members of the MADS family (MEF2) and steroid receptor superfamily (TR) interact with one another to synergistically activate the alpha-cardiac MHC gene expression (Lee, 1997).

The Nkx2-5 homeodomain protein (Drosophila homolog: Tinman) plays a key role in cardiomyogenesis. Ectopic expression in frog and zebrafish embryos results in an enlarged myocardium; however, expression of Nkx2-5 in fibroblasts is not able to trigger the development of beating cardiac muscle. In order to examine the ability of Nkx2-5 to modulate endogenous cardiac specific gene expression in cells undergoing early stages of differentiation, P19 cell lines overexpressing Nkx2-5 were differentiated in the absence of Me2SO. Nkx2-5 expression induces cardiomyogenesis in these cultures aggregated without Me2SO. During differentiation into cardiac muscle, Nkx2-5 expression results in the activation of myocyte enhancer factor 2C (MEF2C), but not MEF2A, -B, or -D. In order to compare the abilities of Nkx2-5 and MEF2C to induce cellular differentiation, P19 cells overexpressing MEF2C were aggregated in the absence of Me2SO. Similar to Nkx2-5, MEF2C expression initiates cardiomyogenesis, resulting in the up-regulation of Brachyury T, bone morphogenetic protein-4, Nkx2-5, GATA-4, cardiac alpha-actin, and myosin heavy chain expression. These findings indicate the presence of a positive regulatory network between Nkx2-5 and MEF2C and show that both factors can direct early stages of cell differentiation into a cardiomyogenic pathway (Skerjanc, 1998).

The embryonic vasculature develops from endothelial cells that form a primitive vascular plexus that recruits smooth muscle cells to form the arterial and venous systems. The MADS-box transcription factor MEF2C is expressed in developing endothelial cells and smooth muscle cells (SMCs), as well as in surrounding mesenchyme, during embryogenesis. Targeted deletion of the mouse MEF2C gene results in severe vascular abnormalities and lethality in homozygous mutants by embryonic day 9.5. Endothelial cells are present and are able to differentiate, but fail to organize normally into a vascular plexus, and smooth muscle cells do not differentiate in MEF2C mutant embryos. These vascular defects resemble those in mice lacking the vascular-specific endothelial cell growth factor VEGF or its receptor Flt-1, both of which are expressed in MEF2C mutant embryos. These results reveal multiple roles for MEF2C in vascular development and suggest that MEF2-dependent target genes mediate endothelial cell organization and SMC differentiation (Lin, 1998).

The MEF2 family of transcription factors has been implicated in transcriptional regulation in a number of different cell types. Targeted deletion of the MEF2C gene, in particular, reveals its importance for early cardiogenesis. This deletion also results in vascular anomalies characterized by extreme variability in lumen size and defects in remodeling. While primary vascular networks form in the yolk sac of the mutants, they fail to remodel into more complex vascular structures. Likewise, although the primordia of the dorsal aortae form normally, anomalies are observed in these vessels later in development. Dorsal and anterior to the heart, the aortae exhibit abnormally small lumens, as do the anterior cardinal veins and intersegmental arteries. In contrast, the dorsal aortae and intersegmental arteries caudal to the heart are grossly enlarged. Cranial vessels are also enlarged and less branched than normal. Endocardiogenesis in the mutant is abnormal with the endothelial cells exhibiting a number of aberrant phenotypes. These endocardial defects are accompanied by a notable reduction in angiopoietin 1 and VEGF mRNA production by the myocardium, indicating that MEF2C is required for myocardial expression of these important endothelial-directed cytokines and thus for correct endocardial morphogenesis (Bi, 1999).

The vertebrate heart forms initially as a linear tube derived from a primary heart field in the lateral mesoderm. Recent studies in mouse and chick have demonstrated that the outflow tract and right ventricle originate from a separate source of mesoderm that is anterior to the primary heart field. The discovery of this anterior, or secondary, heart field has led to a greater understanding of the morphogenetic events involved in heart formation; however, many of the underlying molecular events controlling these processes remain to be determined. The MADS domain transcription factor MEF2C is required for proper formation of the cardiac outflow tract and right ventricle, suggesting a key role in anterior heart field development. Therefore, as a first step toward identifying the transcriptional pathways upstream of MEF2C, a lacZ reporter gene was introduced into a bacterial artificial chromosome (BAC) encompassing the murine Mef2c locus and this recombinant was used to generate transgenic mice. This BAC transgene was sufficient to recapitulate endogenous Mef2c expression, and comparative sequence analyses revealed multiple regions of significant conservation in the noncoding regions of the BAC. One of these conserved noncoding regions represents a transcriptional enhancer that is sufficient to direct expression of lacZ exclusively to the anterior heart field throughout embryonic development. This conserved enhancer contains two consensus GATA binding sites that are efficiently bound by the zinc finger transcription factor GATA4 and are completely required for enhancer function in vivo. This enhancer also contains two perfect consensus sites for the LIM-homeodomain protein ISL1. These elements are specifically bound by ISL1 and are essential for enhancer function in transgenic embryos. Thus, these findings establish Mef2c as the first direct transcriptional target of ISL1 in the anterior heart field and support a model in which GATA factors and ISL1 serve as the earliest transcriptional regulators controlling outflow tract and right ventricle development (Dodou, 2004).

Mef2 family members - Transcriptional targets

Serum induction of c-jun (Drosophila homolog: Jun-related antigen) expression in HeLa cells requires a MEF2 site at -59 in the c-jun promoter. MEF2 sites, found in many muscle-specific enhancers, are bound by a family of transcription factors, MEF2A through -D, which are related to serum response factor in their DNA binding domains. MEF2D is the predominant protein in HeLa cells that binds to the c-jun MEF2 site. Serum induction of a MEF2 reporter gene is not observed in a line of NIH 3T3 cells that contains low MEF2 site binding activity. Transfection of MEF2D into NIH 3T3 cells reconstitutes serum induction, demonstrating that MEF2D is required for the serum response. Deletion analysis of MEF2D shows that its DNA binding domain, when fused to a heterologous transcriptional activation domain, is sufficient for serum induction of a MEF2 reporter gene. This is the domain homologous to that in the serum response factor that is required for serum induction of the c-fos serum response element, suggesting that serum regulation of c-fos and c-jun may share a common mechanism (Han, 1995).

Important regulatory elements responsible for regulated expression of the human GLUT4 promoter are located between -1154 and -412 relative to transcription initiation. A perfectly conserved myocyte enhancer factor 2 (MEF2)-binding domain (-CTAAAAATAG-) has been identified that is necessary, but not sufficient, to support tissue-specific expression of a reporter gene in transgenic mice. Biochemical analysis of this DNA element demonstrates the formation of a specific DNA-protein complex using nuclear extracts isolated from heart, hindquarter skeletal muscle, and adipose tissue but not from liver. DNA binding studies indicate that this element functionally interacts with the MEF2A and/or MEF2C MADS family of DNA binding transcription factors. MEF2 DNA binding activity is substantially reduced in nuclear extracts isolated from both heart and skeletal muscle of diabetic mice; this decrease in binding activity correlates with a decreased transcription rate of the GLUT4 gene. MEF2 binding activity completely recovers to control levels following insulin treatment. Together these data demonstrated that MEF2 binding activity is necessary for regulation of the GLUT4 gene promoter in muscle and adipose tissue (Thai, 1998).

The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor plays important roles in neuronal development, plasticity, and cell death. NMDA receptor subunit 1 (NR1) is an essential subunit of the NMDA receptor and is developmentally expressed in postnatal neurons of the central nervous system. A binding site has been identified on the NR1 promoter for myocyte enhancer factor 2C (MEF2C), a developmentally expressed neuron/muscle transcription factor found in cerebrocortical neurons. Co-expression of MEF2C and Sp1 cDNAs in primary neurons or cell lines synergistically activates the NR1 promoter. Disruption of the MEF2 site or the MEF2C DNA binding domain moderately reduces this synergism. Mutation of the Sp1 sites or the activation domains of Sp1 protein strongly reduces the synergism. Results of yeast two-hybrid and co-immunoprecipitation experiments reveal a physical interaction between MEF2C and Sp1 proteins. The MEF2C DNA binding domain is sufficient for this interaction. Dominant-negative MEF2C interferes with expression of NR1 mRNA in neuronally differentiated P19 cells. Growth factors, including epidermal growth factor and basic fibroblast growth factor, can up-regulate NR1 promoter activity in stably transfected PC12 cells, even in the absence of the MEF2 site, but the Sp1 sites are necessary for this growth factor regulation, suggesting that Sp1 sites may mediate these effects (Krainc, 1998).

MEF2C is a MADS-box transcription factor required for cardiac myogenesis and morphogenesis. In MEF2C mutant mouse embryos, heart development arrests at the looping stage (embryonic day 9.0), the future right ventricular chamber fails to form, and cardiomyocyte differentiation is disrupted. To identify genes regulated by MEF2C in the developing heart, differential array analysis coupled with subtractive cloning was performed using RNA from heart tubes of wild-type and MEF2C-null embryos. A novel MEF2C-dependent gene is described that encodes a cardiac-restricted protein, called CHAMP (cardiac helicase activated by MEF2 protein), that contains seven conserved motifs characteristic of helicases involved in RNA processing, DNA replication, and transcription. During mouse embryogenesis, CHAMP expression commences in the linear heart tube at embryonic day 8.0, shortly after initiation of MEF2C expression in the cardiogenic region. Thereafter, CHAMP is expressed specifically in embryonic and postnatal cardiomyocytes. At the trabeculation stage of heart development, CHAMP expression is highest in the trabecular region in which cardiomyocytes have exited the cell cycle and is lowest in the proliferative compact zone. These findings suggest that CHAMP acts downstream of MEF2C in a cardiac-specific regulatory pathway for RNA processing and/or transcriptional control (Liu, 2001).

Muscle tissue is the major site for insulin-stimulated glucose uptake in vivo, due primarily to the recruitment of the insulin-sensitive glucose transporter (GLUT4) to the plasma membrane. Surprisingly, virtually all cultured muscle cells express little or no GLUT4. Adenovirus-mediated expression of the transcriptional coactivator PGC-1, which is expressed in muscle in vivo but is also deficient in cultured muscle cells, causes the total restoration of GLUT4 mRNA levels to those observed in vivo. This increased GLUT4 expression correlates with a 3-fold increase in glucose transport, although much of this protein is transported to the plasma membrane even in the absence of insulin. PGC-1 mediates this increased GLUT4 expression, in large part, by binding to and coactivating the muscle-selective transcription factor MEF2C. These data indicate that PGC-1 is a coactivator of MEF2C and can control the level of endogenous GLUT4 gene expression in muscle (Michael, 2001).

Genome-wide transcription factor binding and expression profiling has been used to assemble a regulatory network controlling the myogenic differentiation program in mammalian cells. A cadre of overlapping and distinct targets of the key myogenic regulatory factors (MRFs) -- MyoD and myogenin -- and Myocyte Enhancer Factor 2 (MEF2) have been identified. MRFs and MEF2 regulate a remarkably extensive array of transcription factor genes that propagate and amplify the signals initiated by MRFs. MRFs play an unexpectedly wide-ranging role in directing the assembly and usage of the neuromuscular junction. Interestingly, these factors also prepare myoblasts to respond to diverse types of stress. Computational analyses identified novel combinations of factors that, depending on the differentiation state, might collaborate with MRFs. These studies suggest unanticipated biological insights into muscle development and highlight new directions for further studies of genes involved in muscle repair and responses to stress and damage (Blais, 2005).

One of the most striking observations was that transcription factors represent the largest cluster of MRF targets. Although consistent with a cascade model of gene activation, the markedly high number of transcription factors regulated by MRFs and MEF2 suggests that the cascade may be more extensive than expected. These analyses suggest the existence of new nodal points from which the transcriptional output of MyoD is relayed, greatly expanding the repertoire of indirect targets of MyoD. The role of MRFs in differentiation is contrasted with E2F4, a repressor that plays a role in cell cycle exit: only a handful of transcription factor genes are bound by E2F4, suggesting that gene regulatory programs involved in cell cycle control (and cell cycle exit) may be wired in fundamentally different ways from terminal differentiation (Blais, 2005).

It is proposed that transcriptional regulators (Eya1 and TEAD4/TEF-3) relay the differentiation signal initiated by MyoD. Several biochemical, computational, and genetic observations suggest that the Eya1/Six1 pathway is associated with MRF function. (1) ChIP-on-chip results indicate that Eya1 is a direct target of MyoD in growing myoblasts. Eya1 has the ability to switch the activity of Six1, a homeobox transcriptional regulator, from repressor to activator. (2) The MEF3 PWM, a binding site for Six1, is specifically enriched among myogenin target genes that are induced during differentiation. (3) Mice lacking Six1 display defects in embryonic myogenesis that are exacerbated when Eya1 function is also ablated (Blais, 2005).

TEAD4 is closely related to TEAD1 (TEF-1), the founding member of a family of transcriptional regulators that bind M-CAT DNA elements (GGAATG). By binding M-CAT sites, TEAD4 participates in muscle-fiber-type switching and mediates in part the transcriptional effects of hypoxia and alpha-adrenergic-stimulated muscular hypertrophy. Together with the observation that the M-CAT sequence is enriched among MRF targets, this suggests that, besides regulating additional genes during the muscle hypertrophic response, TEAD4 propagates the myogenic signal originating from MyoD and cooperates with MRFs to induce the expression of their targets (Blais, 2005).

These studies identified other transcription factors likely to be involved in propagating gene expression cascades during myogenesis. These MRF targets include Naca (skNAC), a muscle-specific transcription factor involved in muscle repair, and Ankrd1 and Ankrd2, muscle-specific transcriptional modulators involved in myofibril-based hypertrophic response signaling. Identifying their as yet unknown targets will be essential to elucidate their role in response to activation signals originating from MyoD (Blais, 2005).

The vertebrate heart is assembled during embryogenesis in a modular manner from different populations of precursor cells. The right ventricular chamber and outflow tract are derived primarily from a population of progenitors known as the anterior heart field. These regions of the heart are severely hypoplastic in mutant mice lacking the myocyte enhancer factor 2C (MEF2C) and BOP transcription factors, suggesting that these cardiogenic regulatory factors may act in a common pathway for development of the anterior heart field and its derivatives. bop encodes a muscle-restricted transcriptional repressor and putative histone methyltransferase. Bop expression in the developing heart depends on the direct binding of MEF2C to a MEF2-response element in the Bop promoter that is necessary and sufficient to recapitulate endogenous Bop expression in the anterior heart field and its cardiac derivatives during mouse development. The element contains a single MEF2 consensus-binding site [CTA(A/T)4TAA/G]. The Bop promoter also directs transcription in the skeletal muscle lineage, but only cardiac expression is dependent on MEF2. Expression of the reporter required the three E-boxes, targetted by MYOD1. These findings identify Bop as an essential downstream effector gene of MEF2C in the developing heart, and reveal a transcriptional cascade involved in development of the anterior heart field and its derivatives (Phan, 2005).

Myocyte enhancer factor 2 (MEF2) plays essential roles in transcriptional control of muscle development. However, signaling pathways acting downstream of MEF2 are largely unknown. A microarray analysis has been performed using Mef2c-null mouse embryos and a novel MEF2-regulated gene has been identified encoding a muscle-specific protein kinase, Srpk3, belonging to the serine arginine protein kinase (SRPK) family, which phosphorylates serine/arginine repeat-containing proteins. The Srpk3 gene is specifically expressed in the heart and skeletal muscle from embryogenesis to adulthood and is controlled by a muscle-specific enhancer directly regulated by MEF2. Srpk3-null mice display a new entity of type 2 fiber-specific myopathy with a marked increase in centrally placed nuclei; while transgenic mice overexpressing Srpk3 in skeletal muscle show severe myofiber degeneration and early lethality. It is concluded that normal muscle growth and homeostasis require MEF2-dependent signaling by Srpk3 (Nakagawa, 2005).

Co-occupancy by multiple cardiac transcription factors identifies transcriptional enhancers active in heart

Identification of genomic regions that control tissue-specific gene expression is currently problematic. ChIP and high-throughput sequencing (ChIP-seq) of enhancer-associated proteins such as p300 identifies some but not all enhancers active in a tissue. This study shows that co-occupancy of a chromatin region by multiple transcription factors (TFs) identifies a distinct set of enhancers. GATA-binding protein 4 (GATA4), NK2 transcription factor-related, locus 5 (NKX2-5), T-box 5 (TBX5), serum response factor (SRF), and myocyte-enhancer factor 2A (MEF2A), referred to as 'cardiac TFs,' have been hypothesized to collaborate to direct cardiac gene expression. Using a modified ChIP-seq procedure, chromatin occupancy by these TFs and p300 were defined genome wide and unbiased support for this hypothesis is provided. This principle was used to show that co-occupancy of a chromatin region by multiple TFs can be used to identify cardiac enhancers. Of 13 such regions tested in transient transgenic embryos, seven (54%) drove cardiac gene expression. Among these regions were three cardiac-specific enhancers of Gata4, Srf, and swItch/sucrose nonfermentable-related, matrix-associated, actin-dependent regulator of chromatin, subfamily d, member 3 (Smarcd3), an epigenetic regulator of cardiac gene expression. Multiple cardiac TFs and p300-bound regions were associated with cardiac-enriched genes and with functional annotations related to heart development. Importantly, the large majority (1,375/1,715) of loci bound by multiple cardiac TFs did not overlap loci bound by p300. These data identify thousands of prospective cardiac regulatory sequences and indicate that multiple TF co-occupancy of a genomic region identifies developmentally relevant enhancers that are largely distinct from p300-associated enhancers (A. He, 2011).

The cardiac transcription network modulated by Gata4, Mef2a, Nkx2.5, Srf, histone modifications, and microRNAs

The transcriptome, as the pool of all transcribed elements in a given cell, is regulated by the interaction between different molecular levels, involving epigenetic, transcriptional, and post-transcriptional mechanisms. However, many previous studies investigated each of these levels individually, and little is known about their interdependency. A systems biology study is presented integrating mRNA profiles with DNA-binding events of key cardiac transcription factors (Gata4, Mef2a, Nkx2.5, and Srf), activating histone modifications (H3ac, H4ac, H3K4me2, and H3K4me3), and microRNA profiles obtained in wild-type and RNAi-mediated knockdown. Finally, conclusions primarily obtained in cardiomyocyte cell culture were confirmed in a time-course of cardiac maturation in mouse around birth. Insights are provided into the combinatorial regulation by cardiac transcription factors and show that they can partially compensate each other's function. Genes regulated by multiple transcription factors are less likely differentially expressed in RNAi knockdown of one respective factor. In addition to the analysis of the individual transcription factors, it was found that histone 3 acetylation correlates with Srf- and Gata4-dependent gene expression and is complementarily reduced in cardiac Srf knockdown. Further, it was found that altered microRNA expression in Srf knockdown potentially explains up to 45% of indirect mRNA targets. Considering all three levels of regulation, an Srf-centered transcription network is presented providing on a single-gene level insights into the regulatory circuits establishing respective mRNA profiles. In summary, this study shows the combinatorial contribution of four DNA-binding transcription factors in regulating the cardiac transcriptome and provide evidence that histone modifications and microRNAs modulate their functional consequence. This opens a new perspective to understand heart development and the complexity cardiovascular disorders (Schlesinger, 2011; full text of article).

MEF2 transcription factors are key regulators of sprouting angiogenesis

Angiogenesis, the fundamental process by which new blood vessels form from existing ones, depends on precise spatial and temporal gene expression within specific compartments of the endothelium. However, the molecular links between proangiogenic signals and downstream gene expression remain unclear. During sprouting angiogenesis (see Drosophila dorsal vessel), the specification of endothelial cells into the tip cells that lead new blood vessel sprouts is coordinated by vascular endothelial growth factor A (VEGFA) (see Drosophila Pvf1) and Delta-like ligand 4 (Dll4)/Notch signaling (see Drosophila Notch) and requires high levels of Notch ligand DLL4 (see Drosophila Dl). This study identifies MEF2 (see Drosophila Mef2) transcription factors as crucial regulators of sprouting angiogenesis directly downstream from VEGFA. Through the characterization of a Dll4 enhancer directing expression to endothelial cells at the angiogenic front, it was found that MEF2 factors directly transcriptionally activate the expression of Dll4 and many other key genes up-regulated during sprouting angiogenesis in both physiological and tumor vascularization. Unlike ETS-mediated regulation, MEF2-binding motifs are not ubiquitous to all endothelial gene enhancers and promoters but are instead overrepresented around genes associated with sprouting angiogenesis. MEF2 target gene activation is directly linked to VEGFA-induced release of repressive histone deacetylases and concurrent recruitment of the histone acetyltransferase EP300 (see Drosophila nej) to MEF2 target gene regulatory elements, thus establishing MEF2 factors as the transcriptional effectors of VEGFA signaling during angiogenesis (Sacilotto, 2016).

Mef2 family members: transcriptional regulation

A vertebrate homolog of Drosophila Zfh-1, called ZEB, is a negative regulator of muscle differentiation. ZEB binds to a subset of E boxes in muscle genes and functions by actively repressing transcription. One target of this repression is the members of the MEF-2 family, which synergize with proteins of the myogenic basic helix-loop-helix family (bHLH) (myoD, myf-5, myogenin and MRF-4) to induce myogenic differentiation. As muscle differentiation proceeds, myogenic bHLH proteins accumulate to levels sufficient to displace ZEB from the E boxes, releasing the repression and allowing bHLH proteins to further activate transcription. This mechanism of active transcriptional repression distinguishes ZEB from other negative regulators of myogenesis (Id, Twist and I-mfa) that inhibit muscle differentiation by simply binding and inactivating myogenic factors. The relative affinity of ZEB versus myogenic bHLH proteins varies for E boxes in different genes such that ZEB would be displaced from different genes at distinct times as myogenic bHLH proteins accumulate during myogenesis, thus providing a mechanism to regulate temporal order of gene expression (Postigo, 1997).

Different patterns of motor nerve activity drive distinctive programs of gene transcription in skeletal muscles, thereby establishing a high degree of metabolic and physiological specialization among myofiber subtypes. It has been proposed that the influence of motor nerve activity on skeletal muscle fiber type is transduced to the relevant genes by calcineurin, which controls the functional activity of NFAT (nuclear family of activated T cell) proteins. Calcineurin-dependent gene regulation in skeletal myocytes is mediated also by MEF2 transcription factors, and is integrated with additional calcium-regulated signaling inputs, specifically calmodulin-dependent protein kinase activity. In skeletal muscles of transgenic mice, both NFAT and MEF2 binding sites are necessary for properly regulated function of a slow fiber-specific enhancer, and either forced expression of activated calcineurin or motor nerve stimulation up-regulates a MEF2-dependent reporter gene. Functional activation of MEF2 correlates with its dephosphorylation by a calcium-regulated, calcineurin-dependent mechanism. These results provide new insights into the molecular mechanisms by which specialized characteristics of skeletal myofiber subtypes are established and maintained (Wu, 2000).

Promoter/reporter constructions were prepared using transcriptional control elements that direct fiber type-specific expression of slow and fast isoforms of troponin I. In transgenic mice, these SURE (slow upstream regulatory element) and FIRE (fast intronic regulatory element) enhancers direct expression of reporter genes selectively to slow and fast myofibers, respectively. Forced expression of MEF2 proteins in C2C12 myoblasts stimulate the transcriptional activity of the SURE enhancer to a greater degree than the FIRE enhancer, and this difference is accentuated in the presence of constitutively active calcineurin. All of the major isoforms of MEF2 (A, B, C and D) act preferentially on the slow fiber-specific transcriptional control region. This selective effect of calcineurin to up-regulate the SURE enhancer is not attributable to a generalized effect on muscle differentiation in this cultured cell system. On the contrary, the expression of the FIRE enhancer is activated 145-fold in the transition of C2C12 myoblasts to myotubes while the corresponding effect on the SURE enhancer plasmid is 25-fold. If calcineurin were simply accelerating muscle differentiation, then the FIRE enhancer would be expected to exhibit greater calcineurin-dependent activation than SURE, whereas the converse response was observed (Wu, 2000).

The premise that changes in regulatory pools of intracellular calcium constitute a proximate stimulus by which the firing pattern of motor neurons controls specific programs of gene expression in skeletal myofibers suggests that other calcium-regulated signaling pathways, in addition to calcineurin, may be pertinent to fiber type-specific gene transcription. As an initial test of this concept, functional interactions between calcineurin and calmodulin-dependent protein kinase IV (CaMKIV) in the control of myoglobin and desMEF2 enhancer function were assessed. CaMKIV was shown previously to modify calcineurin-regulated transcriptional responses mediated by the CREB transcription factor in hippocampal neurons, but studies of combinatorial interactions between calcineurin- and calmodulin-dependent protein kinases in skeletal myocytes have not been reported previously. A pronounced enhancement of calcineurin-induced stimulation of MEF2 transactivator function in the presence of a constitutively active form of CaMKIV was observed. This synergistic effect is evident using either the native myoglobin promoter/enhancer or the synthetic desMEF2 enhancer to detect the response. CaMKIV had only small effects on transcription of either of these MEF2-dependent enhancers in the absence of concomitant calcineurin activity. In combination, however, CaMKIV and calcineurin evoke a large (35- to 60-fold) response in this assay. These results indicate that MEF2 serves to integrate signaling inputs derived both from calcineurin and from other calcium-regulated pathways. Although these initial studies focused on CaMKIV, it is likely that other isoforms of CaMK that are abundant in skeletal muscle may also prove pertinent to calcium-regulated gene expression in this cell background (Wu, 2000).

Members of the MEF2 family of transcription factors are upregulated during skeletal muscle differentiation and cooperate with the MyoD family of myogenic basic helix-loop-helix (bHLH) transcription factors to control the expression of muscle-specific genes. To determine the mechanisms that regulate MEF2 gene expression during skeletal muscle development, the mouse Mef2c gene was analyzed for cis-regulatory elements that direct expression in the skeletal muscle lineage in vivo. A skeletal muscle-specific control region is described for Mef2c that is sufficient to direct lacZ reporter gene expression in a pattern that recapitulates that of the endogenous Mef2c gene in skeletal muscle during pre- and post-natal development. This control region is a direct target for the binding of myogenic bHLH and MEF2 proteins. Mutagenesis of the Mef2c control region shows that a binding site for myogenic bHLH proteins is essential for expression at all stages of skeletal muscle development, whereas an adjacent MEF2 binding site is required for maintenance but not for initiation of Mef2c transcription. These findings reveal the existence of a regulatory circuit between these two classes of transcription factors that induces, amplifies and maintains their expression during skeletal muscle development (Wang, 2001).

The development of skeletal muscle in the vertebrate embryo is controlled by a transcriptional cascade that includes the four myogenic regulatory factors Myf-5, MyoD, Myogenin, and MRF4. The dynamic expression pattern of myf-5 during myogenesis is thought to be consistent with its role during early determination of the myogenic lineage. To study the factors and mechanisms that regulate myf-5 transcription in Xenopus, a genomic DNA clone containing 4858 bp of Xmyf-5 5' flanking region was isolated. Using a transgenic reporter assay, this genomic contig was shown to be sufficient to recapitulate the dynamic stage- and tissue-specific expression pattern of Xmyf-5 from the gastrula to tail bud stages. For the primary induction of myf-5 transcription, three main regulatory elements were identified; these are responsible for (1) activation in dorsal mesoderm, (2) activation in ventral mesoderm, and (3) repression in midline mesoderm, respectively. Their combined activities define the two-winged expression domain of myf-5 in the preinvoluted mesoderm. Repression in midline mesoderm is mediated by a single TCF binding site located in the 5' end of the -4.8 kbp sequence, which binds XTcf-3 protein in vitro. Endogenous Wnt signaling in the lateral mesoderm is required to overcome the long-range repression through this distal TCF site, and to stimulate myf-5 transcription independently from it. The element for ventral mesoderm activation responds to Activin. Together, these results describe a regulatory mosaic of repression and activation, which defines the myf-5 expression profile in the frog gastrula (Yang, 2002).

Myf5 is the first myogenic regulatory factor to be expressed in the mouse embryo and it determines the entry of cells into the skeletal muscle program. A region situated between -58 kb and -48 kb from the gene directs Myf5 transcription at sites where muscles will form. This region consists of a number of distinct regulatory elements that specifically target sites of myogenesis in the somite, limbs and hypoglossal cord, and also sites of Myf5 transcription in the central nervous system. Deletion of these sequences in the context of the locus shows that elements within the region are essential, and also reveals the combinatorial complexity of the transcriptional regulation of Myf5. Both within the -58 kb to -48 kb region and elsewhere in the locus, multiple sequences are present that direct transcription in subdomains of a single site during development, thus revealing distinct phases of myogenesis when subpopulations of progenitor cells enter the program of skeletal muscle differentiation (Hadchoue, 2003).

The anterior heart field (AHF) mediates formation of the outflow tract (OFT) and right ventricle (RV) during looping morphogenesis of the heart. Foxh1 is a forkhead DNA binding transcription factor in the TGFß-Smad pathway. Foxh1−/− mutant mouse embryos form a primitive heart tube, but fail to form OFT and RV and display loss of outer curvature markers of the future working myocardium, similar to the phenotype of Mef2c−/− mutant hearts. Further, Mef2c is shown to be a direct target of Foxh1, which physically and functionally interacts with Nkx2-5 to mediate strong Smad-dependent activation of a TGFß response element in the Mef2c gene. This element directs transgene expression to the presumptive AHF, as well as the RV and OFT, a pattern that closely parallels endogenous Mef2c expression in the heart. Thus, Foxh1 and Nkx2-5 functionally interact and are essential for development of the AHF and its derivatives, the RV and OFT, in response to TGFß-like signals (von Both, 2004).

Members of the Myocyte Enhancer Factor 2 (MEF2) family of transcription factors play key roles in the development and differentiation of numerous cell types during mammalian development, including the vascular endothelium. mef2c is expressed very early in the development of the endothelium, and genetic studies in mice have demonstrated that mef2c is required for vascular development. However, the transcriptional pathways involving MEF2C during endothelial cell development have not been defined. As a first step towards identifying the transcriptional factors upstream of MEF2C in the vascular endothelium, a screened was carried out for transcriptional enhancers from the mouse mef2c gene that regulate vascular expression in vivo. In this study, a transcriptional enhancer was identified from the mouse mef2c gene sufficient to direct expression to the vascular endothelium in transgenic embryos. This enhancer is active in endothelial cells within the developing vascular system from very early stages in vasculogenesis, and the enhancer remains robustly active in the vascular endothelium during embryogenesis and in adulthood. This mef2c endothelial cell enhancer contains four perfect consensus Ets transcription factor binding sites that are efficiently bound by Ets-1 protein in vitro and are required for enhancer function in transgenic embryos. Members of the Ets family are defined by the presence of a conserved DNA binding domain, which folds into a winged helix-turn-helix motif and binds to the consensus core DNA sequence GGAW with variable flanking sequences. Of the nearly 30 mammalian family members so far identified, at least five, Ets-1, Erg, Fli-1, TEL, and NERF-2, are expressed in the vasculature during embryonic development and each has been shown to play an important role in endothelial-restricted gene expression. Thus, these studies identify mef2c as a direct transcriptional target of Ets factors via an evolutionarily conserved transcriptional enhancer and establish a direct link between these two early regulators of vascular gene expression during endothelial cell development in vivo (De Val, 2004).

To elucidate the function of the T-box transcription factor Tbx20 in mammalian development, a graded loss-of-function series was generated by transgenic RNA interference in entirely embryonic stem cell-derived mouse embryos. Complete Tbx20 knockdown results in defects in heart formation, including hypoplasia of the outflow tract and right ventricle, which derive from the anterior heart field (AHF), and decrease in the expression of Nkx2-5 and Mef2c, transcription factors required for AHF formation. A mild knockdown led to persistent truncus arteriosus (unseptated outflow tract) and hypoplastic right ventricle, entities similar to human congenital heart defects; this demonstrates a critical requirement for Tbx20 in valve formation. Finally, an intermediate knockdown revealed a role for Tbx20 in motoneuron development, specifically in the regulation of the transcription factors Isl2 and Hb9, which are important for terminal differentiation of motoneurons. Tbx20 can activate promoters/enhancers of several genes in cultured cells, including the Mef2c AHF enhancer and the Nkx2-5 cardiac enhancer. The Mef2c AHF enhancer relies on Isl1- and Gata-binding sites. A similar Isl1 binding site has been identified in the Nkx2-5 AHF enhancer, which in transgenic mouse embryos is essential for activity in a large part of the heart, including the outflow tract. Tbx20 synergizes with Isl1 and Gata4 to activate both the Mef2c and Nkx2-5 enhancers, thus providing a unifying mechanism for gene activation by Tbx20 in the AHF. It is thus concluded that Tbx20 is positioned at a critical node in transcription factor networks required for heart and motoneuron development where it dose-dependently regulates gene expression (Takeuchi, 2005).

Nuclear respiratory factors NRF1 and NRF2 regulate the expression of nuclear genes encoding heme biosynthetic enzymes, proteins required for mitochondrial genome transcription and protein import, and numerous respiratory chain subunits. NRFs thereby coordinate the expression of nuclear and mitochondrial genes relevant to mitochondrial biogenesis and respiration. Only two of the nuclear-encoded respiratory chain subunits have evolutionarily conserved tissue-specific forms: the cytochrome c oxidase (COX) subunits VIa and VIIa heart/muscle (H) and ubiquitous (L) isoforms. Genome comparisons were used to conclude that the promoter regions of COX6A(H) and COX7A(H) lack NRF sites but have conserved myocyte enhancer factor 2 (MEF2) elements. MEF2A mRNA is induced with forced expression of NRF1, and the MEF2A 5'-regulatory region contains an evolutionarily conserved canonical element that binds endogenous NRF1 in chromatin immunoprecipitation (ChIP) assays. NRF1 regulates MEF2A promoter-reporters according to overexpression, RNA interference, underexpression, and promoter element mutation studies. As there are four mammalian MEF2 isotypes, an isoform-specific antibody was used in ChIP to confirm MEF2A binding to the COX6A(H) promoter. These findings support a role for MEF2A as an intermediary in coordinating respiratory chain subunit expression in heart and muscle through a NRF1 --> MEF2A --> COX(H) transcriptional cascade. MEF2A also bound the MEF2A and PPARGC1A promoters in ChIP, placing it within a feedback loop with PGC1alpha in controlling NRF1 activity. Interruption of this cascade and loop may account for striated muscle mitochondrial defects in mef2a null mice. These findings also account for the previously described indirect regulation by NRF1 of other MEF2 targets in muscle such as GLUT4 (Ramachandran, 2008).

Waardenburg syndromes are characterized by pigmentation and autosensory hearing defects, and mutations in genes encoding transcription factors that control neural crest specification and differentiation are often associated with Waardenburg and related disorders. For example, mutations in SOX10 result in a severe form of Waardenburg syndrome, Type IV, also known as Waardenburg-Hirschsprung disease, characterized by pigmentation and other neural crest defects, including defective innervation of the gut. SOX10 controls neural crest development through interactions with other transcription factors. The MADS box transcription factor MEF2C is an important regulator of brain, skeleton, lymphocyte and cardiovascular development and is required in the neural crest for craniofacial development. This study established a novel role for MEF2C in melanocyte development. Inactivation of Mef2c in the neural crest of mice results in reduced expression of melanocyte genes during development and a significant loss of pigmentation at birth due to defective differentiation and reduced abundance of melanocytes. WA transcriptional enhancer of Mef2c was identified that directs expression to the neural crest and its derivatives, including melanocytes, in transgenic mouse embryos. This novel Mef2c neural crest enhancer contains three functional SOX binding sites and a single essential MEF2 site. Mef2c is a direct transcriptional target of SOX10 and MEF2 via this evolutionarily conserved enhancer. Furthermore, it was shown that SOX10 and MEF2C physically interact and function cooperatively to activate the Mef2c gene in a feed-forward transcriptional circuit, suggesting that MEF2C might serve as a potentiator of the transcriptional pathways affected in Waardenburg syndromes (Agarwal, 2011).

Global transcriptional regulation of the locus encoding the skeletal muscle determination genes Mrf4 and Myf5

The linked Mrf4 and Myf5 genes encode two transcription factors essential for the determination and differentiation of skeletal muscle in the embryo. The locus is controlled by a multitude of interdigitated enhancers that activate gene expression at different times and in precisely defined progenitor cell populations. Manipulation of the enhancer-promoter composition of the locus reveals a novel mechanism for the regulation of such a gene cluster. Enhancers, promoters, and a new class of elements called transcription balancing sequences, which can act as cryptic promoters, exist in a series of equilibria to ensure that enhancers and promoters together produce the highly dynamic and exquisitely specific expression patterns of the two genes. The proposed model depends upon nonproductive interactions between enhancers and both minimal and cryptic promoters, and is distinct from those developed for the β-globin and Hox clusters. Moreover, it provides an explanation for the unexpected phenotypes of the three Mrf4 knockout alleles (Carvajal, 2008).

There are now a number of examples of genes with regulatory elements located far from the promoter, and it is noteworthy that they tend to encode either signaling molecules (e.g., Shh) or transcription factors (e.g., Sox9) involved in the control of cell fate decisions. The extremely complicated enhancer organization in the Mrf4/Myf5 locus is also not unique. Sox2 is expressed throughout the early nervous system, but this overall expression pattern is the summation of the activities of a number of dispersed enhancers, each of which operates in a particular population of progenitor cells at a particular time. Structuring the regulatory elements in this way, together with the novel regulatory mechanism described in this study, would seem to be particularly appropriate for genes encoding transcription factors, like Myf5 and Sox2, which determine cell fate and have to be induced widely in the embryo in response to a variety of inductive signals (Carvajal, 2008).

The data lead to a model that is quite distinct from those developed from analyses of the β-globin and Hox loci. They imply that the enhancers operate in what might be thought of as a neural network model. They are continuously interacting with not only the two conventional promoters in the locus but also with a number of sequences that can act as cryptic promoters, which have been called TRABS. These interactions must be modulated by the inductive signals that determine skeletal muscle identity and lead to the activation of the two genes in an exquisite temporal and spatial pattern involving many different progenitor cell populations at many stages of development (Carvajal, 2008).

Mef2 family members and chromatin

p300 and CBP, two related molecules that act as transcriptional adapters, coactivate the myogenic basic-helix-helix-loop (bHLH) proteins. Coactivation by p300 induces novel physical interactions between p300 and the amino-terminal activation domain of MyoD. In particular, disruption of the FYD domain, a group of three amino acids coserved in the activation domains of other myogenic bHLH proteins, drastically diminishes the transactivation potential of MyoD and abolishes both p300-mediated coactivation and the physical interaction between MyoD and p300. Two domains of p300, at its amino and carboxy terminals, independently function to both mediate coactivation and physical interaction with MyoD. A truncated segment of p300, unable to bind MyoD, acts as a dominant negative mutation and abrogates both myogenic conversion and transactivation by MyoD, suggesting that endogenous p300 is a required coactivator for MyoD functions. The p300 dominant negative peptide forms multimers with intact p300. p300 and CBP serve as coactivators of another class of transcriptional activators critical for myogenesis, known as myocyte enhancer factor 2 (MEF2). In fact, transactivation mediated by the MEF2C protein is potentiated by the two coactivators; this phenomenon is associated with the ability of p300 to interact with the MADS domain of MEF2. These results suggest that p300 and CBP may positively influence myogenesis by reinforcing the transcriptional autoregulatory loop established between the myogenic bHLH and the MEF2 factors. The fact that p300 and CBP are histone acetyltransferases suggests that MyoD and MEF2 act to alter the acetylation state of chromatin adjacent to transcription factor binding sites thus affecting the access of the the transcription apparatus to promoter regions (Sartorelli, 1997).

The acetylation state of histones can influence transcription. Acetylation, carried out by acetyltransferases such as CBP/p300 and P/CAF, is commonly associated with transcriptional stimulation, whereas deacetylation, mediated by the three known human deacetylases HDAC1, 2 and 3, causes transcriptional repression. The known human deacetylases represent a single family and are homologs of the yeast RPD3 deacetylase. HDAC4, a representative of a new human histone deacetylase family, which is homologous to the yeast HDA1 deacetylase, has been identified and characterized. HDAC4, unlike other deacetylases, shuttles between the nucleus and the cytoplasm in a process involving active nuclear export. In the nucleus, HDAC4 associates with the myocyte enhancer factor MEF2A. Binding of HDAC4 to MEF2A results in the repression of MEF2A transcriptional activation, a function that requires the deacetylase domain of HDAC4. These results identify MEF2A as a nuclear target for HDAC4-mediated repression and suggests that compartmentalization may be a novel mechanism for controlling the nuclear activity of this new family of deacetylases. HDAC4 may contribute to this silencing of MEF2-regulated genes during the process of terminal differentiation. Control of the nuclear distribution of HDAC4 during the differentiation process could provide the signal for the selective silencing of such genes (Miska, 1999).

A transcriptionally inactive mutant of Xenopus MEF2D has been used in a yeast two-hybrid screen. This approach has identified a novel protein expressed in the early embryo that binds to XMEF2D and XMEF2A. The MEF-2 interacting transcription repressor (MITR) protein binds to the N-terminal MADS/MEF-2 region of the MEF-2 proteins but does not bind to the related Xenopus MADS protein serum response factor. In the early embryo, MITR expression commences at the neurula stage within the mature somites and is subsequently restricted to the myotomal muscle. In functional assays, MITR negatively regulates MEF-2-dependent transcription and this repression is mediated by direct binding of MITR to the histone deacetylase HDAC1. Thus, it is proposed that MITR acts as a co-repressor, recruiting a specific deacetylase to downregulate MEF-2 activity (Sparrow, 1999).

Database comparisons reveal that MITR is related to a recently reported family of proteins. (1) The human cDNA KIAA0744 contains sequences with high homology to the entire MITR coding region, and thus probably represents hMITR. (2) Two other related proteins contain an N-terminal domain similar to the entire MITR coding region. These have been named KIAA0600/NY-Co-9/mHDA1/HDACB/HDAC5 , and KIAA0288/HDACA/HDAC4. Interestingly, both of these proteins also possess a C-terminal domain, unrelated to MITR, that has been demonstrated to be a functional histone deacetylase. This is an intriguing result, since histone deacetylase domains have been demonstrated to be involved in negative regulation of transcription. In summary, MITR belongs to a family of three proteins represented in Xenopus, mouse and human. Two family members consist of an N-terminal MITR domain and a C-terminal histone deacetylase domain, whereas MITR itself lacks the C-terminal deacetylase domain. A fourth protein, mHDA2/HDAC6, has been placed in this family by virtue of homology within the histone deacetylase domain. Uniquely, it consists of two adjacent histone deacetylase domains. However, it has no region with homology to MITR, and thus may instead represent the founder member of a novel class of histone deacetylase. Comparison of the Xenopus MITR amino acid sequence with that of the MITR domain in other family members reveals a number of blocks of very highly conserved residues. Searches of the database with either the full amino acid sequence or with any of the conserved domains do not reveal any significant homologies to any other protein. A minimal fragment comprising amino acids 172-222 of MITR is sufficient for MITR-MEF-2 interaction. In reciprocal experiments, the first 100 amino acids of XMEF2D or XMEF2A are both necessary and sufficient for the interaction with the original 222 residue MITR fragment. This corresponds to the MADS/MEF-2 domain which contains distinct regions for DNA binding, MEF-2 homo- and hetero-dimerization and interaction with myogenic bHLH factors (Sparrow, 1999).

In Xenopus, the onset of myogenesis occurs during gastrulation in the paraxial mesoderm prior to somite formation, and it is in this pattern that XMEF2D transcripts are first detected. MITR transcripts are not detected until some hours later in the more mature somites of early neurula embryos, coincident with transcripts of XMEF2A and terminal differentiation markers. Similarly, the MITR-related mouse protein mHDA1 has been shown to accumulate in cells only after they have been induced to differentiate. Together, these observations suggest a relatively late function for the MITR-MEF-2 complex during myogenesis. Since MITR represses MEF-2 activity, it is possible that MITR acts as part of a molecular switch, repressing the early functions of XMEF2D and XMEF2A in the mature myotome, allowing the onset of the next stage in skeletal muscle differentiation to occur. A puzzling result is the apparent failure to block the onset of myogenesis in early embryos by ectopic expression of MITR. One explanation may be the absence of HDAC1 or other MITR cofactors in early embryos at gastrulation when myogenesis begins. Alternatively, there may be translational control of the injected MITR RNA, preventing accumulation of MITR protein until after myogenesis has been initiated (Sparrow, 1999).

The class II histone deacetylases (HDACs) 4, 5, and 7 share a common structural organization, with a carboxyl-terminal catalytic domain and an amino-terminal extension that mediates interactions with members of the myocyte enhancer factor-2 (MEF2) family of transcription factors. Association of these HDACs with MEF2 factors represses transcription of MEF2 target genes. MEF2-interacting transcription repressor (MITR) shares homology with the amino-terminal extensions of class II HDACs and also acts as a transcriptional repressor, but lacks a histone deacetylase catalytic domain. This suggests that MITR represses transcription by recruiting other corepressors. The amino-terminal regions of MITR and class II HDACs interact with the transcriptional corepressor, COOH-terminal-binding protein (CtBP), through a CtBP-binding motif (P-X-D-L-R) conserved in MITR and HDACs 4, 5, and 7. Mutation of this sequence in MITR abolishes interaction with CtBP and impairs, but does not eliminate, the ability of MITR to inhibit MEF2-dependent transcription. The residual repressive activity of MITR mutants that fail to bind CtBP can be attributed to association with other HDAC family members. These findings reveal CtBP-dependent and -independent mechanisms for transcriptional repression by MITR and show that MITR represses MEF2 activity through recruitment of multicomponent corepressor complexes that include CtBP and HDACs (Zhang, 2001).

The development and differentiation of distinct cell types is achieved through the sequential expression of subsets of genes; yet, the molecular mechanisms that temporally pattern gene expression remain largely unknown. In skeletal myogenesis, gene expression is initiated by MyoD and includes the expression of specific Mef2 isoforms and activation of the p38 mitogen-activated protein kinase (MAPK) pathway. p38 activity facilitates MyoD and Mef2 binding at a subset of late-activated promoters, and the binding of Mef2D recruits Pol II. Most importantly, expression of late-activated genes can be shifted to the early stages of differentiation by precocious activation of p38 and expression of Mef2D, demonstrating that a MyoD-mediated feed-forward circuit temporally patterns gene expression (Penn, 2004).

Temporally patterned gene expression in a complex program of cell differentiation is achieved through a feed-forward mechanism. MyoD initiates the expression of specific Mef2 isoforms and activates the p38 MAPK pathway. p38 activity facilitates MyoD and Mef2 binding at genes expressed late in the myogenic program, and the binding of Mef2D recruits Pol II and correlates with the transcription of these genes. Most importantly, expression of some late-stage genes can be shifted to the early stages of differentiation by precocious activation of p38 and expression of Mef2D, demonstrating that the timing of expression is programmed by an intrinsic delay while Mef2 isoforms and p38 activity accumulate, and substantiating the role of a transcriptional feed-forward circuit in temporally patterning gene expression. Because p38 and Mef2D cooperate with MyoD to regulate only a subset of late-stage genes, it is likely that additional sets of genes might require other MyoD-regulated intermediate factors (Penn, 2004).

This study suggests two distinct roles of p38 kinase: (1) as a rate limiting factor in the binding of Mef2 and MyoD, and (2) in facilitating phosphorylation and progression of Pol II. The role of p38 in facilitating the binding of MyoD and Mef2 is likely to be through an effect on chromatin, since it does not alter the binding of these factors in gel-shift assays, and the recent demonstration that the p38 pathway targets the SWI/SNF complex to muscle loci through an interaction with MyoD might account for its effect on factor binding, although other mechanisms, such as histone phosphorylation, might also effect factor binding. The role of p38 in facilitating Pol II phosphorylation and progression is likely to be through the phosphorylation of Mef2D, because prior studies have shown that p38 phosphorylation of the Mef2 activation domain greatly potentiates the transcriptional activity of Mef2. This study shows that the Mef2D isoform is rate limiting for transcription at a subset of late promoters. This suggests that the Mef2D isoform has promoter-specific activities and that the relative abundance of Mef2 isoforms determines which subsets of promoters are actively transcribed (Penn, 2004).

Mef2s are required for thick filament formation in nascent muscle fibres

During skeletal muscle differentiation, the actomyosin motor is assembled into myofibrils, multiprotein machines that generate and transmit force to cell ends. How expression of muscle proteins is coordinated to build the myofibril is unknown. Zebrafish Mef2d and Mef2c proteins are required redundantly for assembly of myosin-containing thick filaments in nascent muscle fibres, but not for the earlier steps of skeletal muscle fibre differentiation, elongation, fusion or thin filament gene expression. mef2d mRNA and protein is present in myoblasts, whereas mef2c expression commences in muscle fibres. Knockdown of both Mef2s with antisense morpholino oligonucleotides or in mutant fish blocks muscle function and prevents sarcomere assembly. Cell transplantation and heat-shock-driven rescue reveal a cell-autonomous requirement for Mef2 within fibres. In nascent fibres, Mef2 drives expression of genes encoding thick, but not thin, filament proteins. Among genes analysed, myosin heavy and light chains and myosin-binding protein C require Mef2 for normal expression, whereas actin, tropomyosin and troponin do not. These findings show that Mef2 controls skeletal muscle formation after terminal differentiation and define a new maturation step in vertebrate skeletal muscle development at which thick filament gene expression is controlled (Hinits, 2007).

The microRNA miR-1 regulates a MEF-2-dependent retrograde signal at neuromuscular junctions

miR-1 regulates aspects of both pre- and postsynaptic function at C. elegans neuromuscular junctions. miR-1 regulates the expression level of two nicotinic acetylcholine receptor (nAChR) subunits (UNC-29 and UNC-63), thereby altering muscle sensitivity to acetylcholine (ACh). miR-1 also regulates the muscle transcription factor MEF-2, which results in altered presynaptic ACh secretion, suggesting that MEF-2 activity in muscles controls a retrograde signal. The effect of the MEF-2-dependent retrograde signal on secretion is mediated by the synaptic vesicle protein RAB-3. Finally, acute activation of levamisole-sensitive nAChRs stimulates MEF-2-dependent transcriptional responses and induces the MEF-2-dependent retrograde signal. It is proposed that miR-1 refines synaptic function by coupling changes in muscle activity to changes in presynaptic function (Simon, 2008).

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). 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. Reducing MEF2 activity in the NAc in vivo is required for the cocaine-induced increases in dendritic spine density. Surprisingly, increasing MEF2 activity in the NAc, which blocks the cocaine-induced increase in dendritic spine density, enhances sensitized behavioral responses to cocaine. Together, these 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 (Pulipparacharuvil, 2008).

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Myocyte enhancer factor 2: Biological Overview | Regulation | Targets of Activity and Protein Interactions | Developmental Biology | Effects of Mutation | References

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