Myocyte enhancer factor 2

EVOLUTIONARY HOMOLOGS (part 1/2)

Invertebrate MEF2 homologs

Podocoryne carnea is a typical representative of the class Hydrozoa (jellyfish). With few exceptions, hydrozoans are marine and exhibit a life cycle that consists of the free swimming planula larva, the sessile polyp and the sexual stage, the medusam which is formed from polyps through budding. In some hydrozoans, the medusa generation has become secondarily reduced. It is generally assumed that all cnidarians are formed of an outer and an inner layer of multifunctional myoepithelial cells. The other cell types, interstitial cells, nerve cells or nematocytes are interspersed in either of the two layers. Therefore, Cnidaria are classified as diploblasts or simple bilayered animals. While this classification accurately describes the basic tissue organization of the planula larva and the polyp, the anatomy of the medusa is more complex. Most of the differences are found in the medusa bell, which not only can carry complicated sense organs such as lens eyes, statocysts, and nerve rings but also consists of two nonmyoepithelial cell layers and additionally a third layer of epithelial mononucleated striated muscle cells. In the entire phylum, the planula larva and the polyp lack these medusa-specific cell types and sense organs (Spring, 2002 and references therein).

In bilaterians, the striated and smooth muscle tissues are in general a derivative of the third or middle germ layer, the mesoderm. In the hydrozoan jellyfish, the striated muscle is a derivative of the entocodon, a tissue layer that separates from the ectodermal layer early in medusa development. The entocodon is located between the distal ectodermal and the endodermal tissue, and is separated from both layers by an extracellular matrix. Entocodon cells in early bud stages are embryonic in appearance and highly proliferative. Later the entocodon forms a cavity, which finally connects to the outside by the developing velar opening. In older bud stages mitotic activity in the bell gradually stops and the outer wall of the entocodon differentiates into the striated muscle while the inner wall forms the smooth muscle of the feeding and sex organ of the animal (Spring, 2002 and references therein).

The histology and developmental pattern of muscle formation in medusa development has led to the idea that the entocodon could be a mesoderm-like layer. Striated myofilaments are usually found in cells derived from the mesoderm, however, exceptions are known. Tentacles of entoprocts contain flagellated ectodermal epithelia that contain striated myofilaments. This indicates that the appropriate structural genes can be activated independent of the germ layer and that the analysis of the striated muscle-specific structural genes alone would not be sufficient. The molecular analysis of muscle development in Podocoryne has demonstrated that the structural genes for a tropomyosin and a myosin heavy chain are structurally and functionally conserved and specific for the striated muscle tissue. Furthermore, the presence of the homeobox gene Otx, a head and gastrulation regulator in bilaterians, in jellyfish striated muscle and the basic helix-loop-helix (bHLH) factor Twist during the formation of the entocodon in medusa development, indicates that genes with specific roles in mesoderm patterning of bilaterians are already present in the common ancestor with bilaterians. Next to homeodomain and bHLH transcription factors, the best-studied regulatory genes are members of the T-box, MADS-box and zinc finger families, such as Brachyury, Mef2, and Snail, respectively. These three gene families are involved at different levels in the specification of the mesodermal and myogenic lineage of bilaterian animals from Drosophila to vertebrates (Spring, 2002 and references therein).

To investigate the hypothesis that the entocodon of jellyfish is homologous to the mesoderm of bilaterians, a Podocoryne homolog of each of the three gene families was isolated and structure and expression patterns were studied throughout the life cycle and specifically during muscle development. The results demonstrate that all three genes are expressed during myogenic differentiation. Additionally, as is true for their bilataterian cognates, they appear to have other functions as well. The sequence and expression data demonstrate that the genes are structurally and functionally conserved and even more similar to humans or other deuterostomes than to protostome model organisms such as Drosophila or Caenorhabditis elegans. The data further strengthen the hypothesis that the common ancestor of cnidarians and bilaterians already used the same regulatory and structural genes and comparable developmental patterns to build muscle systems (Spring, 2002).

MEF2 is an evolutionarily conserved MADS (MCM1, Agamous, Deficiens, and serum response factor) box-type transcription factor that plays a critical role in vertebrate and Drosophila melanogaster myogenesis. This study addresses the developmental role of the single MEF2-like factor, CeMEF2, in C. elegans. Using expression assays and two mef-2 deletion alleles, it has been shown that CeMEF2 is not required for proper myogenesis or development. Moreover, a putative null mef-2 allele fails to enhance or suppress the phenotypes of mutants in CeMyoD or CeTwist. These results suggest that despite its evolutionary conservation of sequence and DNA binding properties, CeMEF2 has adopted a divergent role in development in the nematode compared with Drosophila and vertebrates (Dichoso, 2000).

C. elegans appears to have evolved a developmental program of myogenesis that is independent of MEF2 functions. In Drosophila, all myogenesis is dependent on MEF2, and mutant studies in the mouse suggest an essential role for MEF2 in at least some aspects of muscle development. Apparently C. elegans myogenesis has diverged during evolution. Several other unique aspects of C. elegans myogenesis have been described previously. In vertebrates, heterodimers of members of the bHLH transcription factor families MyoD and E are required for muscle cell fate determination and differentiation. The activity of these heterodimeric complexes is negatively regulated by HLH Id factors that are capable of competing for binding to E factors but fail to bind DNA as heterodimeric complexes. In contrast, CeMyoD is not required for muscle cell fate identity in C. elegans and is not required for expression of most muscle-specific genes. Moreover, the C. elegans E/Daughterless-related factor CeE/DA is not coexpressed with CeMyoD during embryogenesis in differentiating striated muscles and fails to heterodimerize efficiently with CeMyoD in vitro. No Id-like gene has been identified among the 24 known HLH genes present in the C. elegans genomic sequence. Although CeMEF2, CeMyoD, and CeE/DA all display a high level of evolutionary conservation across the core functional domains, the precise roles in nematode muscle development are apparently divergent from those in other systems. These divergent roles presumably reflect functions and protein-protein interactions that are mediated by the substantial regions of these proteins that are not conserved (Dichoso, 2000).

In triploblastic animals, mesoderm gives rise to many tissues and organs, including muscle. By contrast, the representatives of the diploblastic phylum Cnidaria (corals, sea anemones, jellyfish and hydroids) lack mesoderm but possess muscle. In vertebrates and insects, the transcription factor Mef2 plays a pivotal role in muscle differentiation; however, it is also an important regulator of neuron differentiation and survival. In the sea anemone Nematostella vectensis, an organism that lacks mesoderm but has muscles and neurons, Mef2 (Nvmef2) has been reported in single ectodermal cells of likely neural origin. Surprisingly Nvmef2 was found to be alternatively spliced, forming differentially expressed variants. Using morpholino-mediated knockdown and mRNA injection, it was demonstrated that specific splice variants of Nvmef2 are required for the proliferation and differentiation of endodermal cells and for the development of ectodermal nematocytes, a neuronal cell type. Moreover, a small conserved motif was identified in the transactivation domain that is crucially involved in the endodermal function of Nvmef2. The identification of a crucial and conserved motif in the transactivation domain predicts a similarly important role in vertebrate Mef2 function. This is the first functional study of a determinant of several mesodermal derivatives in a diploblastic animal. These data suggest that the involvement of alternative splice variants of Mef2 in endomesoderm and neuron differentiation predates the cnidarian-bilaterian split (Genikhovich, 2011).

Cloning and characterization of vertebrate Mef2 family members

The four members of the MEF2 family of MADS-box transcription factors, MEF2-A, MEF2-B, MEF2-C and MEF2-D, are expressed in overlapping patterns in developing muscle and neural cell lineages during embryogenesis. However, during late fetal development and postnatally, MEF2 transcripts are also expressed in a wide range of cell types. Because MEF2 expression is controlled by translational and post-translational mechanisms, it has been unclear whether the presence of MEF2 transcripts in the embryo reflects transcriptionally active MEF2 proteins. To define the temporospatial expression pattern of transcriptionally active MEF2 proteins during mouse embryogenesis, transgenic mice were generated harboring a lacZ reporter gene controlled by three tandem copies of the MEF2 dependent site and flanking sequences from the desmin MEF2-binding enhancer, which is active in cardiac, skeletal and smooth muscle cells. Expression of this MEF2-dependent transgene parallels expression of MEF2 mRNAs in developing myogenic lineages and regions of the adult brain. However, it is not expressed in other cell types that express MEF2 transcripts. Tandem copies of the MEF2 site from the c-jun promoter direct expression in a similar pattern to the desmin MEF2 site, suggesting that transgene expression reflects the presence of transcriptionally active MEF2 proteins, rather than other factors specific for DNA sequences flanking the MEF2 site. These results demonstrate the presence of transcriptionally active MEF2 proteins in the early muscle and neural cell lineages during embryogenesis and argue against the existence of lineage-restricted MEF2 cofactors that discriminate between MEF2 sites with different immediate flanking sequences. The discordance between MEF2 mRNA expression and MEF2 transcriptional activity in nonmuscle cell types of embryos and adults also supports the notion that post-transcriptional mechanisms regulate the expression of MEF2 proteins (Naya, 1999).

The myocyte enhancer factor (MEF) 2 family of transcription factors has been implicated in the regulation of muscle transcription in vertebrates. Vertebrate Mef2 recognizes a conserved A+T rich element associated with numerous muscle specific genes. D-MEF2 shares extensive amino acid homology with the MADS (MCM1, Agamous, Deficiens, and serum-response factor) domains of the vertebrate MEF2 proteins (Lilly, 1994). Recent evidence has shown that vertebrate Mef2 induces the expression of MyoD and myogen (Kaushal, 1992). The fly protein can function in mice: it can bind to and activate mouse muscle creative kinase promoter (Lilly, 1994).

Members of the MEF2 family interact with a set of AT-rich sequences commonly found in the promoters and enhancers of muscle-specific genes. A MEF2 binding site precisely overlaps the TFIID binding site (TATA box) in the Xenopus MyoDa (XMyoDa) promoter and appears to play an important role in the muscle-specific activity of this promoter. Proteins that bind specifically to this site are present at low levels during early development and increase in abundance during gastrulation and neurulation. Two related cDNAs have been isolated that encode proteins that recognize the XMyoDa TATA motif. Both proteins are highly homologous to each other, belong to the MADS (MCM1 agamous deficiens SRF) protein family, and are most highly related to the mammalian MEF2A gene products. Xenopus MEF2A (XMEF2A) transcripts accumulate preferentially in forming somites after the appearance of XMyoD transcripts. Transcriptional activation of the XMyoDa promoter requires only the conserved DNA binding domain of XMEF2A. An independent domain of XMEF2A is necessary for activity when the protein is bound to multiple upstream sites. Apparently transcriptional activation occurs by different mechanisms depending on the location of the MEF2 binding site (Wong, 1994).

Paradoxically, M-twist, the vertebrate homolog of Twist inhibits myogenesis by blocking DNA binding by MyoD, by titrating E proteins (the vertebrate homologs of Daughterless), and by inhibiting trans-activation by murine MEF2. For inhibition of MEF2, M-twist requires heterodimerization with E proteins and an intact basic domain and carboxyl-terminus. The inhibitory role of M-twist is consistent with exclusion of M-twist from the myotome (Spicer, 1996).

Four members of the myocyte enhancer binding factor 2 (MEF2) family of transcription factors (MEF2A, -B, -C, and -D) have homology within an amino-terminal MADS box and an adjacent MEF2 domain that together mediate dimerization and DNA binding. MEF2A, -C, and -D bind an A/T-rich DNA sequence in the control regions of numerous muscle-specific genes, whereas MEF2B is reported to be unable to bind this sequence unless the carboxyl terminus is deleted. In fact MEF2B binds the same DNA sequence as other members of the MEF2 family and acts as a strong transactivator through that sequence. Transcriptional activation by MEF2B is dependent on the carboxyl terminus, which contains two conserved sequence motifs found in all vertebrate MEF2 factors. During mouse embryogenesis, MEF2B transcripts are expressed in the developing cardiac and skeletal muscle lineages in a temporospatial pattern distinct from but overlapping with those of the other Mef2 genes. The mouse Mef2b gene maps to chromosome 8 and is unlinked to other Mef2 genes; its intron-exon organization is similar to that of the other vertebrate Mef2 genes and the single Drosophila Mef2 gene, consistent with the notion that these different Mef2 genes evolved from a common ancestral gene (Molkentin, (1996a).

Myogenic bHLH factors and MEF2 in vertebrates act as cofactors, suggesting that Nautilus and MEF2 act on the same basis in Drosophila. Evidence suggests that MEF2s of vertebrates (here called vMEF2s) recognize the basic regions of bHLH factors. Immediately C-terminal to the MADS-box is a 29-amino acid domain known as the MEF2 domain. Mutational analysis of the vMEF2s demonstrate that the MADS and MEF2 domains are necessary and sufficient for dimerization and DNA binding, while the C-terminal regions of the vMEF2s cause transactivation. vMEF2s and myogenic bHLH factors show overlapping expression patterns in the skeletal muscle lineage, and evidence suggests that both factors regulate each other, and act cooperatively to regulate muscle specific gene expression. There are muscle genes that lack E-boxes (the target of bHLH factors), but can be induced by bHLH factors. Transactivation of the myogenin promoter by myogenin and MyoD, for example, requires an MEF2 site, but not an E-box. The skeletal muscle-specific enhancer from the troponin C gene also contains a single MEF2 site but no E-boxes, yet this enhancer can be strongly activated by either MyoD or myogenin. Although vMEF2s are unable to induce myogenesis alone, they might function as cofactors for myogenic bHLH proteins. Both MADS and the MEF2 domains of vMEFs are required for interaction with the myogenic bHLH/E12 heterodimer (E12 is the vertebrate homolog of Daughterless). The synergy between myogenic bHLH and vMEF2s in activation of the endogenous myogenic program depends on the myogenic residues (alanine and threonine) in the DNA binding domains of myogenic bHLH proteins and is not observed with a MyoD mutant containg the E12 basic region (Molkentin, 1996b).

Developmental gene regulation in vertebrate somatic muscles involves the cooperative interaction of MEF2 (myocyte-specific enhancer-binding factor 2) and members of the b-HLH (basic helix-loop-helix) family of myogenic factors. Until recently, however, nothing was know about the factors that control the developmental regulation of muscle genes during embryogenesis in Drosophila. The Drosophila Tropomyosin I (TmI) gene contains a proximal and distal muscle enhancer within the first intron that regulates its expression in embryonic/larval and adult muscles. The 355-bp proximal enhancer contains a binding site for the Drosophila homolog of vertebrate MEF2, which acts cooperatively with a basal level muscle activator region to direct high level muscle expression in transgenic flies. The 92-bp muscle activator region, however, contains no consensus E-box (CANNTG) binding site sequences for b-HLH myogenic factors, suggesting that MEF2 may interact with other factors to regulate muscle genes in Drosophila. Mutation analysis and germ-line transformation were used to analyze cis-acting elements within the muscle activator region that regulate TmI's expression in transgenic flies. A 71-bp region has been identified that is sufficient for low basal level temporal- and muscle-specific expression in the embryo, larva, and adult. Substitution mutations within the muscle activator region have identified several cis-element regions spanning 60-bp that are required for either full or partial muscle activator function. An analysis of proteins that bind to this region by gel mobility shift assay and copper nuclease footprinting has identified the sites in this region at which multiple proteins complex and interact. It is propose that these cis-elements and the proteins that they bind regulate muscle activator function and together with MEF2 are capable of regulating high level muscle expression (Lin, 1997b).

Myogenic regulatory factors (MRF) of the MyoD family regulate the skeletal muscle differentiation program. Non-muscle cells transfected with exogenous MRF either are converted to the myogenic lineage or fail to express the muscle phenotype, depending on the cell type analysed. MRF-induced myogenic conversion of NIH3T3 cells results in an incomplete reprogramming of these cells. Transfected cells withdraw from the cell cycle and undergo biochemical differentiation but, surprisingly, terminally differentiated myocytes absolutely fail to fuse into multinucleated myotubes. Analysis of muscle regulatory and structural gene expression fails to provide an explanation for the fusion defectiveness. However, myogenic derivatives of NIH3T3 cells are unable to accumulate the transcripts encoding muscle-specific isoforms of the integrin subunit ss1D and the transcription factor MEF2D1b2, that depend on muscle-specific alternative splicing. These results suggest that the fusion into myotubes is under a distinct genetic control that might depend, at least partially, on differential splicing (Russo, 1998).

Structure of the MEF2A-DNA complex

The solution structure of the 33 kDa complex between the dimeric DNA-binding core domain of the transcription factor MEF2A (residues 1-85) and a 20mer DNA oligonucleotide comprising the consensus sequence CTA(A/T)4TAG has been solved by NMR. The protein comprises two domains: a MADS-box (residues 1-58) and a MEF2S domain (residues 59-73). Recognition and specificity are achieved by interactions between the MADS-box and both the major and minor grooves of the DNA. A number of critical differences in protein-DNA contacts observed in the MEF2A-DNA complex and the DNA complexes of the related MADS-box transcription factors SRF and MCM1 provide a molecular explanation for modulation of sequence specificity and extent of DNA bending (~15° versus ~70°). The structure of the MEF2S domain is entirely different from that of the equivalent SAM domain in SRF and MCM1, accounting for the absence of cross-reactivity with other proteins that interact with these transcription factors (Huang, 2000).

Biochemical work has also suggested an important role for residues 11–14 as determinants of specificity in MEF2A and a critical role in DNA bending played by the N-terminal residue of helix alpha1, namely Glu13 and Lys13 in MEF2A and SRF, respectively. Indeed, the introduction of the mutation Lys13 to Glu into SRF severely disrupts its ability to mediate DNA bending. The overall bending of the DNA in SRF is achieved by three individual bends that co-add: one bend of ~15° at the dyad axis of the DNA, and two bends of ~30° on either side of the central 8 bp. The latter two bends direct the path of the DNA along the side of SRF such that Lys13, Thr18 and Ser21, and Lys24 of helix alpha1 contact the phosphate of C10', the base of T8' and the phosphate of T8', respectively, while Thr50 and His52 of the ß-loop contact the phosphate of A9'. In contrast, in the MEF2A complex the bends outside the central 8 bp do not co-add, and the overall bend is contributed by only a single bend of ~15° at the dyad axis of the DNA. Associated with this is the absence of any contacts between the ß-loop of MEF2A and the DNA. Indeed, the only contacts outside the central 10 bp in the MEF2A complex involve the side chain of Arg14 and the bases of C8' and G7' (including two hydrogen bonds from the guanidino group of Arg 14 to G7') and the sugar–phosphate of C8' (including a hydrogen bond from the Nepsilon atom of Arg14 to the phosphate of C8'). Hydrogen bonding interactions involving residue 14 are precluded in SRF since this position is occupied by a Leu. The electrostatic surfaces of MEF2A and SRF highlight the role of the N-terminal residues of helix alpha1: in MEF2A the presence of a negatively charged Glu at position 13 precludes an upward path of the DNA along the side of the protein owing to unfavorable electrostatic interactions with the phosphate backbone of the DNA, while the presence of the positively charged Arg14 directs the DNA along an essentially linear path. In contrast, for SRF the positively charged Lys13 can readily interact with the phosphate backbone, thereby bringing the DNA into close proximity with residues in the ß-loop. These interactions are entirely consistent with the observation that the introduction of negatively charged residues into the end of helix alpha1 and the ß-loop of SRF and other MADS-box proteins severely disrupts protein-induced DNA bending (Huang, 2000 and references therein).

Protein interactions of Mef2 family members

Big MAP kinase 1 (BMK1), also known as ERK5, is a mitogen-activated protein (MAP) kinase member whose biological role is largely undefined. The activity of BMK1 in rat smooth muscle cells is up-regulated by oxidants. A constitutively active form of the MAP kinase kinase, MEK5(D), is described which selectively activates BMK1 but not other MAP kinases in vivo. Through utilization of MEK5(D), it has been determined that a member of the MEF2 transcription factor family, MEF2C, is a protein substrate of BMK1. BMK1 dramatically enhances the transactivation activity of MEF2C by phosphorylating a serine residue at amino acid position 387 in this transcription factor. Serum is also a potent stimulator of BMK1-induced MEF2C phosphorylation, since a dominant-negative form of BMK1 specifically inhibits serum-induced activation of MEF2C. One consequence of MEF2C activation is increased transcription of the c-jun gene. Taken together, these results strongly suggest that in some cell types the MEK5/BMK1 MAP kinase signaling pathway regulates serum-induced early gene expression through the transcription factor MEF2C (Kato, 1997).

Mitogen-activated protein (MAP) kinase-mediated signaling to the nucleus is an important event in the conversion of extracellular signals into a cellular response. However, the existence of multiple MAP kinases that phosphorylate similar phosphoacceptor motifs poses a problem in maintaining substrate specificity and hence the correct biological response. Both the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) subfamilies of MAP kinases use a second specificity determinant and require docking to their transcription factor substrates to achieve maximal substrate activation. Among the different MAP kinases, the MADS-box transcription factors MEF2A and MEF2C are preferentially phosphorylated and activated by the p38 subfamily members p38alpha and p38beta2. The efficiency of phosphorylation in vitro and transcriptional activation in vivo of MEF2A and MEF2C by these p38 subtypes requires the presence of a kinase docking domain (D-domain). Furthermore, the D-domain from MEF2A is sufficient to confer p38 responsiveness on different transcription factors, and reciprocal effects are observed upon the introduction of alternative D-domains into MEF2A. These results therefore contribute to understanding of signaling to MEF2 transcription factors and demonstrate that the requirement for substrate binding by MAP kinases is an important facet of three different subclasses of MAP kinases (ERK, JNK, and p38) (Yang, 1999).

Differentiation of muscle cells is regulated by extracellular growth factors that transmit largely unknown signals into the cells. Some of these growth factors induce mitogen-activated protein kinase (MAPK) cascades within muscle cells. The kinase activity of p38 MAPK is induced early during terminal differentiation of L8 cells. Addition of a specific p38 inhibitor (SB 203580) to myoblasts blocks their fusion to multinucleated myotubes and prevents the expression of MyoD and MEF2 family members and myosin light chain 2. The expression of MKK6, a direct activator of p38, or of p38 itself enhances the activity of MyoD in converting 10T1/2 fibroblasts to muscle, whereas treatment with SB 203580 inhibits MyoD. Several lines of evidence suggesting that the involvement of p38 in MyoD activity is mediated via its co-activator MEF2C, a known substrate of p38, are presented. In these experiments MEF2C protein and MEF2-binding sites are shown to be necessary for the p38 MAPK pathway to regulate the transcription of muscle creatine kinase reporter gene. These results indicate that the p38 MAPK pathway promotes skeletal muscle differentiation at least in part via activation of MEF2C (Zetser, 1999).

Myocyte enhancer factor 2 (MEF2) is in the MADS family of transcription factors. Although MEF2 is known as a myogenic factor, the expression pattern of the MEF2 family of genes (MEF2A-D) in developing brain also suggests a role in neurogenesis. Transfection with MEF2C, the predominant form in mammalian cerebral cortex, induces a mixed neuronal/myogenic phenotype in undifferentiated P19 precursor cells. During retinoic acid-induced neurogenesis of these cells, a dominant negative form of MEF2 enhances apoptosis but does not affect cell division. The mitogen-activated protein kinase p38alpha activates MEF2C. Dominant negative p38alpha also enhances apoptotic death of differentiating neurons, but these cells can be rescued from apoptosis by coexpression of constitutively active MEF2C. These findings suggest that the p38alpha/MEF2 pathway prevents cell death during neuronal differentiation (Okamoto, 2000).

In mammals, the earliest site of MEF2 expression is the heart where the MEF2C isoform is detectable as early as embryonic day 7.5. Inactivation of the MEF2C gene causes cardiac developmental arrest and severe downregulation of a number of cardiac markers, including atrial natriuretic factor (ANF). However, most of these promoters contain no or low affinity MEF2 binding sites and they are not significantly activated by any MEF2 proteins in heterologous cells, suggesting a dependence on a cardiac-enriched cofactor for MEF2 action. Evidence is provided that MEF2 proteins are recruited to target promoters by the cell-specific GATA transcription factors, and that MEF2 potentiates the transcriptional activity of this family of tissue-restricted zinc finger proteins. Functional MEF2/GATA-4 synergy involves physical interaction between the MEF2 DNA-binding domain and the carboxy zinc finger of GATA-4, and requires the activation domains of both proteins. However, neither MEF2 binding sites nor MEF2 DNA binding capacity are required for transcriptional synergy. The results unravel a novel pathway for transcriptional regulation by MEF2 and provide a molecular paradigm for elucidating the mechanisms of action of MEF2 in muscle and non-muscle cells (Morin, 2000).

Nuclear receptor-mediated activation of transcription involves coactivation by cofactors collectively denoted as the steroid receptor coactivators (SRCs). The process also involves the subsequent recruitment of p300/CBP and PCAF to a complex that synergistically regulates transcription and remodels the chromatin. PCAF and p300 have also been demonstrated to function as critical coactivators for the muscle-specific basic helix-loop-helix (bHLH) protein MyoD during myogenic commitment. Skeletal muscle differentiation and the activation of muscle-specific gene expression is dependent on the concerted action of another bHLH factor, myogenin, and the MADS protein, MEF-2, which function in a cooperative manner. An examination was carried out of the functional role of one SRC, GRIP-1, in muscle differentiation, an ideal paradigm for the analysis of the determinative events that govern the cell's decision to divide or differentiate. The mRNA encoding GRIP-1 is expressed in proliferating myoblasts and post-mitotic differentiated myotubes, and protein levels increase during differentiation. Exogenous/ectopic expression studies with GRIP-1 sense and antisense vectors in myogenic C2C12 cells demonstrate that this SRC is necessary for (1) induction/activation of myogenin, MEF-2, and the crucial cell cycle regulator, p21, and (2) contractile protein expression and myotube formation. Furthermore, the SRC GRIP-1 coactivates MEF-2C-mediated transcription. GRIP-1 also coactivates the synergistic transactivation of E box-dependent transcription by myogenin and MEF-2C. GST-pulldowns, mammalian two-hybrid analysis, and immunoprecipitation demonstrate that the mechanism involves direct interactions between MEF-2C and GRIP-1 and is associated with the ability of the SRC to interact with the MADS domain of MEF-2C. The HLH region of myogenin mediates the direct interaction of myogenin and GRIP-1. Interestingly, interaction with myogenic factors is mediated by two regions of GRIP-1, an amino-terminal bHLH-PAS region and the carboxy-terminal region between amino acids 1158 and 1423 (that encodes an activation domain, has HAT activity, and interacts with the coactivator-associated arginine methyltransferase). This work demonstrates that GRIP-1 potentiates skeletal muscle differentiation by acting as a critical coactivator for MEF-2C-mediated transactivation and is the first study to ascribe a function to the amino-terminal bHLH-PAS region of SRCs (Chen, 2000).

The myocyte enhancer factor 2 (MEF2) consists of a family of transcription factors that play important roles in a number of physiological processes from muscle cell differentiation to neuronal survival and T cell apoptosis. MEF2 has been reported to be associated with several distinct repressors including Cabin1(cain), MEF2-interacting transcriptional repressor (MITR), and HDAC4. Cabin1 is associated with MEF2 in a calcium-sensitive manner; activated calmodulin binds to Cabin1 and releases it from MEF2. However, it is not known whether the binding of HDAC4 and MITR to MEF2 is also regulated by calcium. HDAC4 and MITR contain calmodulin-binding domains that overlap with their MEF2-binding domains. Binding of calmodulin to HDAC4 leads to its dissociation from MEF2, relieving MEF2 from the transcriptional repression by HDAC4. Together, HDAC4, MITR, and Cabin1 constitute a family of calcium-sensitive transcriptional repressors of MEF2 (Youn, 2000a).

TCR signaling leading to thymocyte apoptosis is mediated through the expression of the Nur77 family of orphan nuclear receptors. MEF2 has been shown to be the major transcription factor responsible for calcium-dependent Nur77 transcription. Cabin1 was recently identified as a transcriptional repressor of MEF2, which can be released from MEF2 in a calcium-dependent fashion. The molecular basis of repression of MEF2 by Cabin1, however, has remained unknown. Cabin1 is shown to represse MEF2 by two distinct mechanisms. Cabin1 recruits mSin3 and its associated histone deacetylases 1 and 2; Cabin1 also competes with p300 for binding to MEF2. Thus, activation of MEF2 and the consequent transcription of Nur77 are controlled by the association of MEF2 with the histone deacetylases via the calcium-dependent repressor Cabin1 (Youn, 2000b).

T-cell antigen receptor (TCR)-induced thymocyte apoptosis is mediated by calcium-dependent signal transduction pathways leading to the transcriptional activation of members of the Nur77 family (Drosophila homolog HR38). The major calcium- and calcineurin-responsive elements in the Nur77 promoter are binding sites for myocyte enhancer factor-2 (MEF2). It has been shown that nuclear factor of activated T cells (NFAT: Drosophila homolog: CG11172) interacts with MEF2D and enhances its transcriptional activity, offering a plausible mechanism of activation of MEF2D by calcineurin. NFATp synergizes with MEF2D to recruit the coactivator p300 for the transcription of Nur77. Surprisingly, the enhancement of transcriptional activity of MEF2D by NFATp does not require NFATp's DNA-binding activity, suggesting that NFATp acts as a coactivator for MEF2D. Transient co-expression of p300, MEF2D, NFATp and constitutively active calcineurin is sufficient to recapitulate TCR signaling for the selective induction of the endogenous Nur77 gene. These results implicate NFAT as an important mediator of T-cell apoptosis and suggest that NFAT is capable of integrating the calcineurin signaling pathway and other pathways through direct protein-protein interaction with other transcription factors (Youn, 2000c).

Calcineurin-dependent pathways have been implicated in the hypertrophic response of skeletal muscle to functional overload (OV). Skeletal muscles overexpressing an activated form of calcineurin (CnA*) exhibit a phenotype indistinguishable from wild-type counterparts under normal weightbearing conditions and respond to OV with a similar doubling in cell size and slow fiber number. These adaptations occur despite the fact that CnA* muscles display threefold higher calcineurin activity and enhance dephosphorylation of the calcineurin targets NFATc1, MEF2A, and MEF2D. Moreover, when calcineurin signaling is compromised with cyclosporin A, muscles from OV wild-type mice display a lower molecular weight form of CnA, originally detected in failing hearts, whereas CnA* muscles are spared this manifestation. OV-induced growth and type transformations are prevented in muscle fibers of transgenic mice overexpressing a peptide that inhibits calmodulin from signaling to target enzymes. Taken together, these findings provide evidence that both calcineurin and its activity-linked upstream signaling elements are crucial for muscle adaptations to OV and that, unless significantly compromised, endogenous levels of this enzyme can accommodate large fluctuations in upstream calcium-dependent signaling events (Dunn, 2000).

Regarding the potential identity of contractile activity-dependent signal transduction events, there is mounting evidence that calcineurin must interact with parallel calcium-sensitive signaling pathways in order to fully activate downstream target genes. For instance, calcineurin synergizes with phorbol ester-dependent pathways to stimulate the IL-2 promoter in T lymphocytes and the expression of atrial natriuretic factor in cardiomyocytes. Similarly, calcineurin acts in conjunction with CaM-dependent kinase IV to fully activate the myoglobin promoter in cultured skeletal myocytes and the Nur77 promoter in T lymphocytes. Moreover, retroviral-mediated gene transfer of CnA* induces skeletal myogenesis in vitro only in the presence of extracellular Ca2+. Additionally, there is evidence that MAP kinase pathways are activated in response to increased contractile activity and play a role in regulation of the slow fiber phenotype. In this context, MEF2 is an enticing candidate as an integrator of calcineurin and other activation-linked signal transduction pathways, since this transcription factor is both dephosphorylated by calcineurin and phosphorylated by various CaM kinases, ERK5, p38, and PKC (Dunn, 2000 and references therein).

An alternative possibility is that calcineurin signaling may converge with other activity-linked pathways via the association of GATA with NFAT. Indeed, activation of calcineurin promotes the association of these two transcription factors via the dephosphorylation of NFATc1 and increased expression of GATA-2 under conditions of skeletal myocyte growth. Consistent with findings from hypertrophic myocytes, this protein is upregulated in the plantaris in response to muscle overload, but not lowered by CsA treatment, suggesting that this transcription factor may be important for growth but not necessarily a gene target of calcineurin. The fact that GATA is also known to associate with MEF2, and that fiber hypertrophy is observed only when NFATc1 and MEF2 are dephosphorylated and GATA-2 increases, leads to the idea that NFAT, MEF2, and GATA proteins act in synergy to transactivate target genes that lead to fiber growth in response to OV. Future studies should help identify the particular permutations of these transcription factors involved in the activation of slow fiber-specific genes versus those modulating adult fiber size (Dunn, 2000 and references therein).

Gene expression in skeletal muscles of adult vertebrates is altered profoundly by changing patterns of contractile work. The functional activity of MEF2 transcription factors is stimulated by sustained periods of endurance exercise or motor nerve pacing, as assessed by expression in transgenic mice of a MEF2-dependent reporter gene (desMEF2-lacZ). This response is accompanied by transformation of specialized myofiber subtypes, and is blocked either by cyclosporin A, a specific chemical inhibitor of calcineurin, or by forced expression of the endogenous calcineurin inhibitory protein: myocyte-enriched calcineurin interacting protein 1. Calcineurin removes phosphate groups from MEF2, and augments the potency of the transcriptional activation domain of MEF2 fused to a heterologous DNA binding domain. Across a broad range, the enzymatic activity of calcineurin correlates directly with expression of endogenous genes that are transcriptionally activated by muscle contractions. These results delineate a molecular pathway in which calcineurin and MEF2 participate in the adaptive mechanisms by which skeletal myofibers acquire specialized contractile and metabolic properties as a function of changing patterns of muscle contraction (Wu, 2001).

D-type cyclin-cdk4 complexes, which are only active in proliferating cells, can suppress the skeletal muscle differentiation program in proliferating myoblasts. Cyclin D-cdk activity can block the activity of the MEF2 family of transcriptional regulators, which are crucial regulators of skeletal muscle gene expression. Cyclin D-cdk activity blocks the association of MEF2C with the coactivator protein GRIP-1 and thereby inhibits the activity of MEF2. During skeletal muscle differentiation, GRIP-1 is localized to punctate nuclear structures and can apparently tether MEF2 to such structures. Cotransfection of GRIP-1 can both potentiate the transcriptional activity of a Gal4-MEF2C construct and induce MEF2C localization to punctate nuclear structures. Consistent with the absence of punctate nuclear GRIP-1 in proliferating myoblasts, it was found that ectopic cyclin D-cdk4 expression disrupts the localization of both GRIP-1 and MEF2C to these punctate subnuclear structures. These findings indicate that cyclin D-cdk4 activity represses skeletal muscle differentiation in proliferating cells by blocking the association of MEF2 with the coactivator GRIP-1 and concomitantly disrupts the association of these factors with punctate nuclear subdomains within the cell (Lazaro, 2002).

Expression of many skeletal muscle-specific genes depends on TEF-1 (transcription enhancer factor-1) and MEF2 transcription factors. In Drosophila, the TEF-1 homolog Scalloped interacts with the cofactor Vestigial to drive differentiation of the wing and indirect flight muscles. Three mammalian vestigial-like genes, Vgl-1, Vgl-2, and Vgl-3, have been identified that share homology in a TEF-1 interaction domain. Vgl-1 and Vgl-3 transcripts are enriched in the placenta, whereas Vgl-2 is expressed in the differentiating somites and branchial arches during embryogenesis and is skeletal muscle-specific in the adult. During muscle differentiation, Vgl-2 mRNA levels increase and Vgl-2 protein translocates from the cytoplasm to the nucleus. In situ hybridization revealed co-expression of Vgl-2 with myogenin in the differentiating muscle of embryonic myotomes but not in newly formed somites prior to muscle differentiation. Like Vgl-1, Vgl-2 interacts with TEF-1. In addition, Vgl-2 interacts with MEF2 in a mammalian two-hybrid assay and Vgl-2 selectively binds to MEF2 in vitro. Co-expression of Vgl-2 with MEF2 markedly co-activates an MEF2-dependent promoter through its MEF2 element. Overexpression of Vgl-2 in MyoD-transfected 10T(1/2) cells markedly increases myosin heavy chain expression, a marker of terminal muscle differentiation. These results identify Vgl-2 as an important new component of the myogenic program (Maeda, 2002).

Neurotoxic insults deregulate Cdk5 activity, which leads to neuronal apoptosis and may contribute to neurodegeneration. The biological activity of Cdk5 has been ascribed to its phosphorylation of cytoplasmic substrates. However, its roles in the nucleus remain unknown. The mechanism by which Cdk5 promotes neuronal apoptosis has been investigated.The prosurvival transcription factor MEF2 has been identified as a direct nuclear target of Cdk5. Cdk5 phosphorylates MEF2 at a distinct serine in its transactivation domain to inhibit MEF2 activity. Neurotoxicity enhances nuclear Cdk5 activity, leading to Cdk5-dependent phosphorylation and inhibition of MEF2 function in neurons. MEF2 mutants resistant to Cdk5 phosphorylation restore MEF2 activity and protect primary neurons from Cdk5 and neurotoxin-induced apoptosis. These studies reveal a nuclear pathway by which neurotoxin/Cdk5 induces neuronal apoptosis through inhibiting prosurvival nuclear machinery (Gong, 2003).

The myocyte enhancer factor-2 (MEF2) family of transcription factors has important roles in the development and function of T cells, neuronal cells and muscle cells. MEF2 is capable of repressing or activating transcription by association with a variety of co-repressors or co-activators in a calcium-dependent manner. Transcriptional repression by MEF2 has attracted particular attention because of its potential role in hypertrophic responses of cardiomyocytes. Several MEF2 co-repressors, such as Cabin1/Cain and class II histone deacetylases (HDACs), have been identified. However, the molecular mechanism of their recruitment to specific promoters by MEF2 remains largely unknown. This study reports a crystal structure of the MADS-box/ MEF2S domain of human MEF2B bound to a motif of the transcriptional co-repressor Cabin1 and DNA at 2.2 Å resolution. The crystal structure reveals a stably folded MEF2S domain on the surface of the MADS box. Cabin1 adopts an amphipathic alpha-helix to bind a hydrophobic groove on the MEF2S domain, forming a triple-helical interaction. These studies of the ternary Cabin1/MEF2/DNA complex show a general mechanism by which MEF2 recruits transcriptional co-repressor Cabin1 and class II HDACs to specific DNA sites (Han, 2003).

Structure of p300 bound to MEF2 on DNA reveals a mechanism of enhanceosome assembly

Transcription co-activators CBP and p300 are recruited by sequence-specific transcription factors to specific genomic loci to control gene expression. A highly conserved domain in CBP/p300, the TAZ2 domain, mediates direct interaction with a variety of transcription factors including the myocyte enhancer factor 2 (MEF2). This study reports the crystal structure of a ternary complex of the p300 TAZ2 domain bound to MEF2 on DNA at 2.2A resolution. The structure reveals three MEF2:DNA complexes binding to different sites of the TAZ2 domain. Using structure-guided mutations and a mammalian two-hybrid assay, this study shows that all three interfaces contribute to the binding of MEF2 to p300, suggesting that p300 may use one of the three interfaces to interact with MEF2 in different cellular contexts and that one p300 can bind three MEF2:DNA complexes simultaneously. These studies, together with previously characterized TAZ2 complexes bound to different transcription factors, demonstrate the potency and versatility of TAZ2 in protein-protein interactions. These results also support a model wherein p300 promotes the assembly of a higher-order enhanceosome by simultaneous interactions with multiple DNA-bound transcription factors (J. He, 2011).

Mef2 interaction with histone deacetylases

Skeletal muscle differentiation is controlled by associations between myogenic basic-helix-loop-helix and MEF2 transcription factors. Chromatin associated with muscle genes regulated by these transcription factors becomes acetylated during myogenesis and class II histone deacetylases (HDACs), which interact with MEF2, specifically suppress myoblast differentiation. These HDACs do not interact directly with MyoD, yet they suppress its myogenic activity through association with MEF2. Elevating the level of MyoD can override the repression imposed by HDACs on muscle genes. HDAC-mediated repression of myogenesis also can be overcome by CaM kinase and insulin-like growth factor (IGF) signaling. These findings reveal central roles for HDACs in chromatin remodeling during myogenesis and as intranuclear targets for signaling pathways controlled by IGF and CaM kinase (Lu, 2000).

The results of this study demonstrate that HDACs 4 and 5 inhibit myogenesis by repressing MyoD activity through association with MEF2 and support a model in which the decision of a myoblast to differentiate is dictated by a balance of positive and negative influences on the transcriptional activity of MEF2. Consistent with the conclusion that HDACs 4 and 5 repress muscle transcription by deacetylating core histones associated with muscle gene regulatory regions, the level of acetylated histone H4 associated with the MCK enhancer and myogenin promoter, both of which are regulated directly by MEF2, increases during myogenesis (Lu, 2000).

HDACs 4 and 5, classified as Class II HDAC enzymes, have been shown to deacetylate all four core histones in vitro. These HDACs interact with amino acids 39-72 of MEF2 factors, spanning the junction of the MADS and MEF2 domains. This region of MEF2 encompasses the residues that mediate MEF2 homodimerization, but interaction with HDACs does not affect dimerization or DNA binding of MEF2. In contrast to the relatively confined region of MEF2 recognized by HDACs, MyoD interacts with an extended surface of MEF2 factors that includes residues throughout the MADS and MEF2 domains. Based on the strength of interactions in two-hybrid and coimmunoprecipitation assays, HDACs appear to exhibit a much higher affinity than MyoD for MEF2. Whether MyoD overcomes HDAC-mediated repression by competing with HDAC for interaction with MEF2 has been investigated, but no evidence has been found for such competition. Thus, a model is favored in which high concentrations of MyoD result in greater occupancy of E boxes, resulting in opposition to the inhibitory activity of HDACs by the strong transcription activation domain of MyoD or by recruitment of additional coactivators by MyoD (Lu, 2000).

MyoD and MEF2 have been shown to interact with the p300/CBP coactivators that possess HAT activity and would therefore be expected to antagonize the actions of HDACs. It is possible that HDACs 4 and 5 diminish MyoD activity by direct deacetylation. However, this would have to require binding of MEF2 to sites adjacent to MyoD binding sites in muscle gene control regions, since these HDACs do not affect MyoD activity on genes lacking MEF2 sites. Deacetylation of MyoD alone by HDACs 4 and 5 also cannot account for their ability to completely block MyoD activity because MyoD mutants that cannot be acetylated retain substantial activity (Lu, 2000).

Overexpression of HDACs 4 and 5 can block myoblast differentiation and repress the myogenic activity of MyoD. Conversely, increasing the ratio of MyoD to HDACs, as occurs during normal myogenesis, counterbalances the inhibitory effects of HDACs and promotes differentiation. HDACs can only inhibit MyoD activity on target genes that contain both MyoD and MEF2 sites. This suggests that the key MyoD target genes required for activation of the skeletal muscle differentiation program contain MEF2 sites. Myogenin is a likely downstream gene in this pathway since it is regulated by an essential E box and MEF2 site in its promoter and is required for myogenesis. MEF2D is expressed in myoblasts, and that MyoD activates the myogenin promoter in combination with preexisting MEF2 at the onset of myogenesis. Thus, recruitment of HDACs 4 and 5 to the myogenin promoter by MEF2 in myoblasts would be expected to prevent differentiation (Lu, 2000).

Experiments with native and artificial promoters have identified three potential types of target genes for myogenic bHLH and MEF2 factors that differ in their responsiveness to class II HDACs. Muscle genes that contain E boxes but not MEF2 sites would be activated by myogenic bHLH factors and would be insulated from the inhibitory effects of HDACs. This type of MyoD target gene might be expressed in proliferating myoblasts. Other genes contain MEF2 sites but no MyoD sites. This class of gene would be activated by MEF2 and repressed by HDACs and would be unaffected by the presence of MyoD. Finally, many muscle genes such as MCK are controlled by E boxes and MEF2 sites. Expression of these genes would be dependent on the balance between MyoD and HDAC activity (Lu, 2000).

These results show that MEF2 is a signal-dependent activator of skeletal myogenesis that responds to CaMK and MAP kinase pathways. CaMK signaling overcomes the inhibitory activity of HDAC by preventing association of HDAC with MEF2, whereas MKK6, which activates p38, stimulates MEF2 activity by phosphorylation of the carboxy-terminal transcription activation domain. MKK6 can only activate MEF2 in cardiac myocytes if HDAC is dissociated from the DNA binding domain. Thus, the CaMK and MAP kinase pathways synergize to activate MEF2-dependent transcription by targeting different domains of MEF2 (Lu, 2000).

Remarkably, a CaMK dominant-negative mutant completely blocks the ability of MyoD to activate myogenesis, revealing an essential role for CaMK signaling in the transcriptional pathway for muscle gene activation. What might be the target for CaMK? The possibility is favored that HDACs are targets, either directly or indirectly, for CaMK signaling and that in the absence of a CaMK signal, MEF2 activity is repressed by association with HDACs. Myoblast differentiation has been shown to be accompanied by an increase in CaMK activity that would be predicted to stimulate MEF2 activity (Lu, 2000).

A simple model is proposed for the role of HDAC and MEF2 in myogenesis. According to this model, HDACs 4 and 5 associate with MEF2 in myoblasts and repress muscle-specific genes. When myoblasts are triggered to differentiate, MyoD upregulates expression of MEF2, and together MEF2 and MyoD activate myogenin transcription and establish a positive feedback loop that amplifies expression of both factors as well as other myogenic bHLH factors. Thus, although HDAC expression remains constant in myoblasts and myotubes, increasing levels of myogenic bHLH and MEF2 factors in differentiating muscle cells would exceed the capacity of HDAC to repress MEF2-dependent genes, resulting in muscle gene activation. CaMK signaling stimulates myogenesis by dissociating HDACs from MEF2, and MAPKs further enhance MEF2 activity by phosphorylation of the activation domain. Dissociation of HDACs from MEF2 in response to CaMK signaling may result in activation of MEF2 not only through relief from HDAC-mediated repression but may also facilitate association with CBP and p300 coactivators that also interact with the DNA binding domain of MEF2C (Lu, 2000).

In addition to regulating skeletal muscle differentiation, MEF2 factors have been implicated in cardiac morphogenesis, vascular development, and neuronal differentiation, as well as in the control of growth factor-inducible genes. Calcium-dependent signals have also been shown to connect MEF2 to cell survival and apoptotic pathways. How MEF2 discriminates between the different sets of target genes involved in these processes and whether the type of signal-dependent derepression of HDACs described here for IGF-1 and CaMK participate in these gene regulatory programs remains to be determined. Given the selective expression of Class II HDACs and MEF2 in skeletal muscle, heart, and brain, and the importance of calcium signaling in these tissues, it seems likely that the type of regulatory circuitry through which these transcriptional regulators connect extracellular signaling with chromatin remodeling in skeletal muscle cells will have relevance to multiple aspects of gene expression in these tissues (Lu, 2000).

Members of the myocyte enhancer factor-2 (MEF2) family of transcription factors associate with myogenic basic helix-loop-helix transcription factors such as MyoD to activate skeletal myogenesis. MEF2 proteins also interact with the class II histone deacetylases HDAC4 and HDAC5, resulting in repression of MEF2-dependent genes. Execution of the muscle differentiation program requires release of MEF2 from repression by HDACs, which are expressed constitutively in myoblasts and myotubes. HDAC5 shuttles from the nucleus to the cytoplasm when myoblasts are triggered to differentiate. Calcium/calmodulin-dependent protein kinase (CaMK) signalling, which stimulates myogenesis and prevents formation of MEF2-HDAC complexes, also induces nuclear export of HDAC4 and HDAC5 by phosphorylation of these transcriptional repressors. An HDAC5 mutant lacking two CaMK phosphorylation sites is resistant to CaMK-mediated nuclear export and acts as a dominant inhibitor of skeletal myogenesis, whereas a cytoplasmic HDAC5 mutant is unable to block efficiently the muscle differentiation program. These results highlight a mechanism for transcriptional regulation through signal- and differentiation-dependent nuclear export of a chromatin-remodelling enzyme, and suggest that nucleo-cytoplasmic trafficking of HDACs is involved in the control of cellular differentiation (McKinsey, 2000).

The heart responds to stress signals by hypertrophic growth, which is accompanied by activation of the MEF2 transcription factor and reprogramming of cardiac gene expression. Class II histone deacetylases (HDACs), which repress MEF2 activity, are substrates for a stress-responsive kinase specific for conserved serines that regulate MEF2-HDAC interactions. Signal-resistant HDAC mutants lacking these phosphorylation sites are refractory to hypertrophic signaling and inhibit cardiomyocyte hypertrophy. Conversely, mutant mice lacking the class II HDAC, HDAC9, are sensitized to hypertrophic signals and exhibit stress-dependent cardiomegaly. Thus, class II HDACs act as signal-responsive suppressors of the transcriptional program governing cardiac hypertrophy and heart failure (Zhang, 2002).

The class II deacetylase histone deacetylase 4 (HDAC4) negatively regulates the transcription factor MEF2. HDAC4 is believed to repress MEF2 transcriptional activity by binding to MEF2 and catalyzing local histone deacetylation. HDAC4 also controls MEF2 by a novel SUMO E3 ligase activity. HDAC4 interacts with the SUMO E2 conjugating enzyme Ubc9 and is itself sumoylated (see Drosophila SUMO). The overexpression of HDAC4 leads to prominent MEF2 sumoylation in vivo, whereas recombinant HDAC4 stimulates MEF2 sumoylation in a reconstituted system in vitro. Importantly, HDAC4 promotes sumoylation on a lysine residue that is also subject to acetylation by a MEF2 coactivator, the acetyltransferase CBP, suggesting a possible interplay between acetylation and sumoylation in regulating MEF2 activity. Indeed, MEF2 acetylation is correlated with MEF2 activation and dynamically induced upon muscle cell differentiation, while sumoylation inhibits MEF2 transcriptional activity. Unexpectedly, it was found that HDAC4 does not function as a MEF2 deacetylase. Instead, the NAD+-dependent deacetylase SIRT1 can potently induce MEF2 deacetylation. These studies reveal a novel regulation of MEF2 transcriptional activity by two distinct classes of deacetylases that affect MEF2 sumoylation and acetylation (Zhao, 2005).

Klf5 regulates muscle differentiation by directly targeting muscle-specific genes in cooperation with MyoD in mice

Kruppel-like factor 5 (Klf5) (see Drosophila dar1) is a zinc-finger transcription factor that controls various biological processes, including cell proliferation and differentiation. This study shows that Klf5 is also an essential mediator of skeletal muscle regeneration and myogenic differentiation (see myogenesis in Drosophila). During muscle regeneration after injury (cardiotoxin injection), Klf5 is induced in the nuclei of differentiating myoblasts and newly formed myofibers expressing myogenin in vivo. Satellite cell-specific Klf5 deletion severely impairs muscle regeneration, and myotube formation is suppressed in Klf5-deleted cultured C2C12 myoblasts and satellite cells. Klf5 knockdown suppresses induction of muscle differentiation-related genes, including myogenin. Klf5 ChIP-seq revealed that Klf5 binding overlaps that of MyoD (see Drosophila nau) and Mef2, and Klf5 physically associates with both MyoD and Mef2. In addition, MyoD recruitment is greatly reduced in the absence of Klf5. These results indicate that Klf5 is an essential regulator of skeletal muscle differentiation, acting in concert with myogenic transcription factors such as MyoD and Mef2 (Hayashi, 2016).

Cooperative activation of cardiac transcription through myocardin bridging of paired MEF2 sites.

Enhancers frequently contain multiple binding sites for the same transcription factor. These homotypic binding sites often exhibit synergy, whereby the transcriptional output from two or more binding sites is greater than the sum of the contributions of the individual binding sites alone. Although this phenomenon is frequently observed, the mechanistic basis for homotypic binding site synergy is poorly understood. This study identified a bona fide cardiac-specific Prkaa2 enhancer that is synergistically activated by homotypic MEF2 (see Drosophila Mef2) binding sites. Two MEF2 sites in the enhancer function cooperatively due to bridging of the MEF2C-bound sites by the SAP domain-containing co-activator protein myocardin, and paired sites were shown to buffer the enhancer from integration site-dependent effects on transcription in vivo Paired MEF2 sites are prevalent in cardiac enhancers, suggesting that this might be a common mechanism underlying synergy in the control of cardiac gene expression in vivo (Anderson, 2017).

Mef2 homologs and heart and vasculature development

Continued: Evolutionary Homologs part 2/2

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