Table of contents

Interaction of MyoD with MEK1

To elucidate the mechanism through which MAPK signaling regulates the MyoD family of transcription factors, the role of the signaling intermediate MEK1 in myogenesis was investigated. Transfection of activated MEK1 strongly represses gene activation and myogenic conversion by the MyoD family. This repression is not mediated by direct phosphorylation of MyoD or by changes in MyoD stability or subcellular distribution. Deletion mapping has revealed that MEK1-mediated repression requires the MyoD amino-terminal transactivation domain. Moreover, activated MEK1 is nuclearly localized and binds a complex containing MyoD in a manner that is dependent on the presence of the MyoD amino terminus. Together, these data demonstrate that MEK1 signaling has a strong negative effect on MyoD activity via a novel mechanism involving binding of MEK1 to the nuclear MyoD transcriptional complex (Perry, 2001).

Interaction of Nautilus homologs with transcription factors

The tumor suppressor retinoblastoma protein (pRB) plays an important role in the production and maintenance of the terminally differentiated phenotype of muscle cells. pRB inactivation, through either phosphorylation, binding to T antigen, or genetic alteration, inhibits myogenesis. Moreover, inactivation of pRB in terminally differentiated cells allows them to reenter the cell cycle. In addition to its involvement in the myogenic activities of MyoD, pRB is also required for the cell growth-inhibitory activity of this myogenic factor. pRB and MyoD directly bind to each other, both in vivo and in vitro, through a region that involves the pocket and the basic-helix-loop-helix domains, respectively. All the results obtained are consistent with the proposal that the effects of MyoD on the cell cycle and of pRB on the myogenic pathway result from the direct binding of the two molecules (Gu, 1993).

The formation of striated muscle in both vertebrates and invertebrates involves the activity of the MyoD family of basic-helix-loop-helix (bHLH) transcription factors. The high degree of evolutionary conservation of MyoD-related proteins, both in the sequence of their bHLH domains and in their general developmental expression patterns, suggests that these factors are also conserved at the level of function. This question was addressed directly using MyoD and E protein factors from vertebrates, Drosophila, and Caenorhabditis elegans. Various MyoD and E factor combinations were tested for their ability to interact in vitro and to function in vivo in the myogenic conversion of 10T1/2 mouse fibroblasts. The ability of different homo- and heterodimers to bind DNA in vitro is an accurate measure of biological activity in vivo. The inability of C. elegans CeE/DA or any other E protein to augment the activity of CeMyoD is consistent with the notion that CeMyoD functions as a homodomer rather than a heterdimer. A second assessment of conserved function comes from the ability of these factors to rescue a C. elegans hlh-1 (CeMyoD) null mutation. Both Drosophila and chicken MyoD-related factors are able to rescue a C. elegans CeMyoD loss-of-function mutation. These results demonstrate a remarkable degree of functional conservation of these myogenic factors despite differences in E-protein interactions (Zhang, 1999a).

Dimerization of three Id proteins (Id1, Id2, and Id3) with the four class A E proteins (E12, E47, E2-2, and HEB) and two groups of class B proteins, the myogenic regulatory factors (MRFs: MyoD, myogenin, Myf-5 and MRF4/Myf-6), and the hematopoietic factors (Scl/Tal-1, Tal-2, and Lyl-1) were tested in a quantitative yeast 2-hybrid assay. All three Ids (homologs of Drosophila Extra machrochaetae) bind with high affinity to E proteins, but a much broader range of interactions is observed between Ids and the class B factors. Id1 and Id2 interact strongly with MyoD and Myf-5 and weakly with myogenin and MRF4/Myf-6, whereas Id3 interacts weakly with all four MRFs. Similar specificities are observed in co-immunoprecipitation and mammalian 2-hybrid analyses. No interactions are found between the Ids and any of the hematopoietic factors. Each Id is able to disrupt the ability of E protein-MyoD complexes to transactivate from a muscle creatine kinase reporter construct in vivo. Mutagenesis experiments show that the differences between Id1 and Id3 binding all map to three amino acids in the first helix and to a small cluster of upstream residues. The Id proteins thus display a signature range of interactions with all of their potential dimerization partners and may play a role in myogenesis which is distinct from that in hematopoiesis (Langlands, 1997).

In a search for novel factors that interact with Id1, a component of the 26 S proteasome, S5a, was identified that has previously been implicated only in the recognition of ubiquitinated polypeptides destined for proteolysis. S5a interacts strongly with Id1, less strongly with the basic helix-loop-helix proteins MyoD and E12, and not at all with other Id proteins. S5a restores DNA binding by MyoD-Id1 and E12-Id1 heterodimers, enhances DNA binding by MyoD and E12 homodimers, and reverses Id1-mediated repression of the muscle creatine kinase promoter during myogenic differentiation. Amino acids flanking the helix-loop-helix domain plus three residues in the first helix of Id1 impart S5a recognition. This requires only the NH2-terminal half of S5a. S5a thus appears to promote the positive regulation of myogenic genes through ubiquitin-independent mechanisms involving inhibition of Id1 and the enhancement of DNA binding by MyoD and E12. This latter property may permit the selection of novel promoter binding sites during myogenesis (Anand, 1997).

The muscle LIM protein (MLP) is a muscle-specific LIM-only factor that exhibits a dual subcellular localization, being present in both the nucleus and in the cytoplasm. Overexpression of MLP in C2C12 myoblasts enhances skeletal myogenesis, whereas inhibition of MLP activity blocks terminal differentiation. Thus, MLP functions as a positive developmental regulator, although the mechanism through which MLP promotes terminal differentiation events remains unknown. While examining the distinct roles associated with the nuclear and cytoplasmic forms of MLP, it was found that nuclear MLP functions through a physical interaction with the muscle basic helix-loop-helix (bHLH) transcription factors MyoD, MRF4, and myogenin. This interaction is highly specific since MLP does not associate with nonmuscle bHLH proteins E12 or E47 or with the myocyte enhancer factor-2 (MEF2) protein, which acts cooperatively with the myogenic bHLH proteins to promote myogenesis. The first LIM motif in MLP and the highly conserved bHLH region of MyoD are responsible for mediating the association between these muscle-specific factors. MLP also interacts with MyoD-E47 heterodimers, leading to an increase in the DNA-binding activity associated with this active bHLH complex. Although MLP lacks a functional transcription activation domain, it is proposed that it serves as a cofactor for the myogenic bHLH proteins by increasing their interaction with specific DNA regulatory elements. Thus, the functional complex of MLP-MyoD-E protein reveals a novel mechanism for both initiating and maintaining the myogenic program and suggests a global strategy for how LIM-only proteins may control a variety of developmental pathways (Kong, 1997).

In vertebrates, the basic helix-loop-helix (bHLH) protein Twist may be involved in the negative regulation of cellular determination and in the differentiation of several lineages, including myogenesis, osteogenesis, and neurogenesis. Although it has been shown that mouse twist (M-Twist) (1) sequesters E proteins, thus preventing formation of myogenic E protein-MyoD complexes and (2) inhibits the MEF2 transcription factor, a cofactor of myogenic bHLH proteins, overexpression of E proteins and MEF2 fails to rescue the inhibitory effects of M-Twist on MyoD. M-Twist physically interacts with the myogenic bHLH proteins in vitro and in vivo; this interaction is required for the inhibition of MyoD by M-Twist. In contrast to the conventional HLH-HLH domain interaction formed in the MyoD/E12 heterodimer, this novel type of interaction uses the basic domains of the two proteins. While the MyoD HLH domain without the basic domain fails to interact with M-Twist, a MyoD peptide containing only the basic and helix 1 regions is sufficient to interact with M-Twist, suggesting that the basic domain contacts M-Twist. The replacement of three arginine residues by alanines in the M-Twist basic domain is sufficient to abolish both the binding and inhibition of MyoD by M-Twist, while the domain retains other M-Twist functions, such as heterodimerization with an E protein and inhibition of MEF2 transactivation. These findings demonstrate that M-Twist interacts with MyoD through the basic domains, thereby inhibiting MyoD (Hamamori, 1997).

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 containing the E12 basic region (Molkentin, 1996).

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).

Skeletal muscle development is controlled by a family of muscle-specific basic helix-loop-helix (bHLH) transcription factors that activate muscle genes by binding E-boxes (CANNTG) as heterodimers with ubiquitous bHLH proteins, called E proteins. Myogenic bHLH factors are expressed in proliferating undifferentiated myoblasts, but they do not initiate myogenesis until myoblasts exit the cell cycle. A bHLH protein, MyoR (for myogenic repressor), is described that is expressed in undifferentiated myoblasts in culture and is down-regulated during differentiation. MyoR is also expressed specifically in the skeletal muscle lineage between days 10.5 and 16.5 of mouse embryogenesis and down-regulated thereafter during the period of secondary myogenesis. MyoR forms heterodimers with E proteins that bind the same DNA sequence as myogenic bHLH/E protein heterodimers, but MyoR acts as a potent transcriptional repressor that blocks myogenesis and activation of E-box-dependent muscle genes. These results suggest a role for MyoR as a lineage-restricted transcriptional repressor of the muscle differentiation program. At least three types of mechanisms are suggested whereby MyoR can block muscle gene expression: (1) MyoR can compete with myogenic bHLH/E-protein heterodimers for E-box binding sites in muscle gene control regions; (2) MyoR bound to E-boxes in muscle control regions can actively repress transcription through its transcriptional repression domain. (3) MyoR can compete with myogenic bHLH proteins for limiting quantities of E protein dimerization partners. However, because excess E12 does not rescue the ability of MyoD or myogenin to activate muscle genes in the presence of MyoR, and MyoR shows strong inhibition of a MyoD~E47 tethered heterodimer, the latter mechanism for repression appears to be of lesser importance. While one could imagine other mechanisms whereby MyoR might inhibit myogenesis for example, by inducing cell death or stimulating cell proliferation, experimental results argue against these types of mechanisms (Lu, 1999).

Homodimeric complexes of members of the E protein family of basic helix-loop-helix (bHLH) transcription factors are important for tissue-specific activation of genes in B lymphocytes. A novel cis-acting transcriptional repression domain is present in the E protein family of bHLH transcription factors. This domain, the Rep domain, is present in each of the known vertebrate E proteins. Extensive mapping analysis demonstrates that this domain is an acidic region of 30 amino acids with a predicted loop structure. Fusion studies indicate that the Rep domain can repress both of the E protein transactivation domains (AD1 and AD2). Physiologically, the Rep domain plays a key role in maintaining E protein homodimers in an inactive state on myogenic enhancers. In addition, Rep domain mediated repression of AD1 is a necessary for the function of MyoD-E protein heterodimeric complexes. These studies demonstrate that the Rep domain is important for modulating the transcriptional activity of E proteins and provide key insights into both the selectivity and mechanism of action of E protein containing bHLH protein complexes (Markus, 2002).

Selective recognition of the E-box sequences on muscle gene promoters by heterodimers of myogenic basic helix-loop-helix (bHLH) transcription factors, such as MyoD, with the ubiquitous bHLH proteins E12 and E47 is a key event in skeletal myogenesis. However, homodimers of MyoD or E47 are incapable of binding to and activating muscle chromatin targets, suggesting that formation of functional MyoD/E47 heterodimers is pivotal in controlling muscle transcription. p38 MAPK, whose activity is essential for myogenesis, regulates MyoD/E47 heterodimerization. Phosphorylation of E47 at Ser140 by p38 induces MyoD/E47 association and activation of muscle-specific transcription, while the nonphosphorylatable E47 mutant Ser140Ala fails to heterodimerize with MyoD and displays impaired myogenic potential. Moreover, inhibition of p38 activity in myocytes precludes E47 phosphorylation at Ser140, which results in reduced MyoD/E47 heterodimerization and inefficient muscle differentiation, as a consequence of the impaired binding of the transcription factors to the E regulatory regions of muscle genes. These findings identify a novel pro-myogenic role of p38 in regulating the formation of functional MyoD/E47 heterodimers that are essential for myogenesis (Lluis, 2005).

p38 has been shown to induce the activity of the muscle coactivator MEF2 and to target the SWI/SNF complex to muscle promoters through the functional MyoD transcription factor. It is proposed that p38 may complex two timely and closely linked muscle transcription mechanisms. By binding to and phosphorylating E47, p38 promotes formation of the functional MyoD/E47 heterodimer, which will allow subsequent recruitment of the SWI/SNF complex on myogenic promoters (Lluis, 2005).

The basic helix-loop-helix (bHLH) transcription factor Myod directly regulates gene expression throughout the program of skeletal muscle differentiation. It is not known how a Myod-driven myogenic program is modulated to achieve muscle fiber-type-specific gene expression. Pbx homeodomain proteins mark promoters of a subset of Myod target genes, including myogenin (Myog); thus, Pbx proteins might modulate the program of myogenesis driven by Myod. By inhibiting Pbx function in zebrafish embryos, this study has shown that Pbx proteins are required in order for Myod to induce the expression of a subset of muscle genes in the somites. In the absence of Pbx function, expression of myog and of fast-muscle genes is inhibited, whereas slow-muscle gene expression appears normal. By knocking down Pbx or Myod function in combination with another bHLH myogenic factor, Myf5, it was shown that Pbx is required for Myod to regulate fast-muscle, but not slow-muscle, development. Furthermore, this study shows that Sonic hedgehog requires Myod in order to induce both fast- and slow-muscle markers but requires Pbx only to induce fast-muscle markers. These results reveal that Pbx proteins modulate Myod activity to drive fast-muscle gene expression, thus showing that homeodomain proteins can direct bHLH proteins to establish a specific cell-type identity (Maves, 2007).

Glucocorticoid-induced gene-1 (Gig1) was identified in a yeast one-hybrid screen for factors that interact with the MyoD core enhancer. The Gig1 gene encodes a novel C2H2 zinc finger protein that shares a high degree of sequence similarity with two known DNA binding proteins in humans, Glut4 enhancer factor and papillomavirus binding factor (PBF). The mouse ortholog of PBF was also isolated in the screen. The DNA binding domain of Gig1, which contains TCF-E-tail CR1 and CR2 motifs shown to mediate promoter specificity of TCF-E-tail isoforms, was mapped to a C-terminal domain that is highly conserved in Glut4 enhancer factor and PBF. In mouse embryos, in situ hybridization revealed a restricted pattern of expression of Gig1 that overlaps with MyoD expression. A nuclear-localized lacZ knockin null allele of Gig1 was produced to studyGig1 expression with greater resolution and to assess Gig1 functions. X-gal staining of Gig1nlacZ heterozygous embryos revealed Gig1 expression in myotomal myocytes, skeletal muscle precursors in the limb, and in nascent muscle fibers of the body wall, head and neck, and limbs through E14.5 (latest stage examined). Gig1 was also expressed in a subset of Scleraxis-positive tendon precursors/rudiments of the limbs, but not in the earliest tendon precursors of the somite (syndetome) defined by Scleraxis expression. Additional regions of Gig1 expression included the apical ectodermal ridge, neural tube roof plate and floor plate, apparent motor neurons in the ventral neural tube, otic vesicles, notochord, and several other tissues representing all three germ layers. Gig1 expression was particularly well represented in epithelial tissues and in a number of cells/tissues of neural crest origin. Expression of both the endogenous MyoD gene and a reporter gene driven by MyoD regulatory elements was similar in wild-type and homozygous null Gig1nlacZ embryos, and mutant mice were viable and fertile, indicating that the functions of Gig1 are redundant with other factors (Yamamoto, 2007).

Regulation of expression of Nautilus homologs

Gene targeting has indicated that the bHLH transcription factors Myf-5 and MyoD are required for myogenic determination because skeletal myoblasts and myofibers are entirely ablated in mouse embryos lacking both Myf-5 and MyoD. Entrance into the skeletal myogenic program during development occurs following the independent transcriptional induction of either Myf-5 or MyoD. To identify sequences required for the de novo induction of MyoD transcription during development, the expression patterns of MyoD-lacZ transgenes were investigated in embryos deficient in both Myf-5 and MyoD. A 258-bp fragment containing the core of the -20-kb MyoD enhancer activates expression in newly formed somites and limb buds in compound mutant embryos lacking both Myf-5 and MyoD. Importantly, Myf-5- and MyoD-deficient presumptive muscle precursor cells expressing beta-galactosidase are observed to assume nonmuscle fates primarily as precartilage primordia in the trunk and the limbs, suggesting that these cells are multipotential. Therefore, cells are recruited into the MyoD-dependent myogenic lineage through activation of the -20-kb MyoD enhancer. This occurs independently in somites and limb buds (Kablar, 1999).

Skeletal muscle lineage determination is regulated by the myogenic regulatory genes, MyoD and Myf-5. A 258 bp core enhancer element 20 kb 5' of the MyoD gene has been identified that regulates MyoD gene activation in mouse embryos. To elucidate the cis control mechanisms that regulate MyoD transcription, the entire core enhancer was mutagenized using linker-scanner mutagenesis, and the transcriptional activity of enhancer mutants using lacZ reporter gene expression was tested in transgenic mouse embryos. In total, 83 stable transgenic lines representing 17 linker-scanner mutations were analyzed in midgestational mouse embryos. Eight linker-scanner mutations resulted in a partial or complete loss of enhancer activity, demonstrating that MyoD is primarily under positive transcriptional control. Six of these mutations reduce or abolish transgene expression in all skeletal muscle lineages, indicating that activation of MyoD expression in trunk, limb and head musculature is regulated, in part, by shared transcriptional mechanisms. Interestingly, however, two adjacent linker-scanner mutations (LS-14 and LS-15) result in a dramatic reduction in transgene expression specifically in myotomes at 11.5 days. At later stages, transgene expression is absent or greatly reduced in myotomally derived muscles, including epaxial muscles (deep back muscles) and hypaxial muscles of the body wall (intercostal muscles, abdominal wall musculature). In contrast, head muscles, as well as muscles of the body derived from migrating muscle progenitor cells (e.g. limb, diaphragm), are unaffected by these mutations. In Pax-3-mutant mice, LS-14 and LS-15 transgene expression is eliminated in the body, but is unaffected in the head, yielding an identical expression pattern to the endogenous MyoD gene in mice mutant for both Myf-5 and Pax-3. These data support the hypothesis that LS-14 and LS-15 define the core enhancer targets for Myf-5-dependent activation of MyoD in myotomal muscles (Kucharczuk, 1999).

In Myf-5-mutant mice, MyoD expression in myotomal muscles is greatly delayed, but is eventually initiated by a Pax-3- dependent mechanism. In LS-14 and LS-15 transgenic lines, however, reporter gene expression in myotomally derived muscles is impaired from 11.5 to 14.5 days of development, and Pax-3-dependent compensation is not observed. The mechanism by which Pax-3 rescues myogenesis and MyoD expression in myotomal muscles in the absence of Myf-5 is currently unknown. Analogous to its required function for migration of limb muscle progenitor cells, Pax-3 might promote the local migration of myogenic precursor cells such that they are positioned properly in relation to embryonic inducing signals. Alternatively, Pax-3 might have a more direct transcriptional effect on MyoD expression, as indicated by the ability of Pax-3 to induce expression of MyoD and other muscle markers in several types of non-muscle embryonic cells in culture (Kucharczuk, 1999 and references).

Sonic hedgehog (Shh), produced by the notochord and floor plate, is proposed to function as an inductive and trophic signal that controls somite and neural tube patterning and differentiation. To investigate Shh functions during somite myogenesis in the mouse embryo, an analysis was carried out of the expression of the myogenic determination genes, Myf5 and MyoD, and other regulatory genes in somites of Shh null embryos and in explants of presomitic mesoderm from wild-type and Myf5 null embryos. Shh has an essential inductive function in the early activation of the myogenic determination genes, Myf5 and MyoD, in the epaxial somite cells that give rise to the progenitors of the deep back muscles. Shh is not required for the activation of Myf5 and MyoD at any of the other sites of myogenesis in the mouse embryo, including the hypaxial dermomyotomal cells that give rise to the abdominal and body wall muscles, or the myogenic progenitor cells that form the limb and head muscles. Shh also functions in somites to establish and maintain the medio-lateral boundaries of epaxial and hypaxial gene expression. Myf5, and not MyoD, is the target of Shh signaling in the epaxial dermomyotome, since MyoD activation by recombinant Shh protein in presomitic mesoderm explants is defective in Myf5 null embryos. In further support of the inductive function of Shh in epaxial myogenesis, Shh has been shown to be not essential for the survival or the proliferation of epaxial myogenic progenitors. However, Shh is required specifically for the survival of sclerotomal cells in the ventral somite as well as for the survival of ventral and dorsal neural tube cells. It is concluded, therefore, that Shh has multiple functions in the somite, including inductive functions in the activation of Myf5, leading to the determination of epaxial dermomyotomal cells to myogenesis, as well as trophic functions in the maintenance of cell survival in the sclerotome and adjacent neural tube. These findings, therefore, indicate that other Shh-independent signals function to induce myogenesis at other sites in the embryo. In vitro explant studies have shown that different Wnts produced by surface ectoderm and dorsal neural tube can differentially induce Myf5 and MyoD, and therefore are candidates for Shh-independent signals that regulate epaxial and hypaxial myogenesis (Borycki, 1999).

Cyclin-dependent kinase 5, coupled with its activator p35, is required for normal neuronal differentiation and patterning. Xp35.1, a novel member of the p35 family that can activate cdk5, has been isolated from Xenopus embryos. Xp35.1 is expressed in both proliferating and differentiated neural and mesodermal cells and is particularly high in developing somites where cdk5 is also expressed. Using dominant-negative cdk5 (cdk5 DN), cdk5 kinase activity has been shown to be required for normal somitic muscle development: expression of cdk5 DN results in disruption of somitic muscle patterning, accompanied by stunting of the embryos. Using explants of animal pole tissue from blastula embryos that will differentiate into mesoderm in response to activin, it has been shown that blocking cdk5 kinase activity down-regulates the expression of the muscle marker muscle actin in response to activin, whereas the pan-mesodermal marker Xbra is unaffected. Expression of MyoD and MRF4 (master regulators of myogenesis) is suppressed in the presence of cdk5 DN, indicating that these myogenic genes may be targets for cdk5 regulation, whereas the related factor Myf5 is largely unaffected. In addition, overexpression of Xp35.1 disrupts muscle organization. Thus, a novel role for cdk5 has been demonstrated in regulating myogenesis in the early embryo (Philpott, 1997).

Myogenin, one of the MyoD family of proteins, is expressed early during somitogenesis and is required for myoblast fusion in vivo. Previous studies in transgenic mice have shown that a 184-bp myogenin promoter fragment is sufficient to correctly drive expression of a beta-galactosidase transgene during embryogenesis. Mutation of one of the DNA motifs present in this region, the MEF3 motif, abolishes correct expression of this beta-galactosidase transgene. MEF3 motifs are found in many other skeletal muscle-specific regulatory regions and have been shown to be involved in the transcriptional regulation of the cardiac troponin C gene (11) and the aldolase A muscle-specific promoter. The proteins that bind to the MEF3 site are homeoproteins of the Six/sine oculis family. Antibodies directed specifically against Six1 or Six4 proteins reveal that each of these proteins is present in the embryo when myogenin is activated and constitutes a muscle-specific MEF3-binding activity in adult muscle nuclear extracts. Both of these proteins accumulate in the nucleus of C2C12 myogenic cells, and transient transfection experiments confirm that Six1 and Six4 are able to transactivate a reporter gene containing MEF3 sites. Altogether these results establish Six homeoproteins as a family of transcription factors controlling muscle formation through activation of one of its key regulators, myogenin (Spitz, 1998).

Six proteins are the mammalian homologs of Drosophila sine oculis (so), which is required for the different steps of eye formation. Together with eyeless (a protein homolog of vertebrate Pax-6 protein) and eye absent (eya), so has been shown to act within a network of regulators, which synergistically drive Drosophila eye morphogenesis. In addition to the finding reported here, that Six proteins play an important role in the early steps of myogenesis, it has been demonstrated that Pax3 is required to activate somitic myogenesis. It is thus possible that Pax, Six, and Eya proteins, all of which are coexpressed during vertebrate somitogenesis, cooperate during vertebrate muscle development, in a manner reminiscent of eyeless, so, and eya in Drosophila. In this developmental context, SO has been found to interact physically with Eya through protein motifs conserved between Drosophila and mammals. Interestingly, Eya proteins are expressed in mouse somites: these proteins, which possess a powerful transcription activation domain, but are devoid of any known DNA binding domain, could similarly contribute to the transcriptional regulation mediated by Six proteins through MEF3 sites. Such a requirement for a synergistic interaction with Eya may account for the relatively limited reporter gene transactivation by Six4 and Six1 alone in transfection assays (Spitz, 1998 and references).

In vertebrates, somite differentiation is mediated in part by Sonic Hedgehog (Shh), secreted by the notochord and the floor plate. However, Shh-null mice display close to normal expression of molecular markers for dermomytome, myotome, and sclerotome, indicating that Shh might not be required for their initial induction. This paper addresses the capacity of Shh to regulate in vivo the expression of the somite differentiation markers Pax-1, MyoD, and Pax-3 after separation of paraxial mesoderm from axial structures. Pax-1, which is lost under these experimental conditions, is rescued by Shh. In contrast, Shh maintains, but cannot induce MyoD expression, while Pax-3 expression is independent of the presence of axial structures or Shh. Shh is a potent mitogen for somitic cells, supporting the idea that it may serve to expand subpopulations of cells within the somite (Marcelle, 1999).

Differentiation is a coordinated process of irreversible cell cycle exit and tissue-specific gene expression. To probe the functions of the retinoblastoma protein (RB) family in cell differentiation, HBP1 was isolated as a specific target of RB and p130. HBP1 is a transcriptional repressor and a cell cycle inhibitor. The induction of HBP1, RB, and p130 upon differentiation in the muscle C2C12 cells suggests a coordinated role. The expression of HBP1 unexpectedly blocks muscle cell differentiation without interfering with cell cycle exit. Moreover, the expression of MyoD and myogenin, but not Myf5, is inhibited in HBP1-expressing cells. HBP1 inhibits transcriptional activation by the MyoD family members. The inhibition of MyoD family function by HBP1 requires binding to RB and/or p130. Since Myf5 might function upstream of MyoD, these data suggest that HBP1 probably blocks differentiation by disrupting Myf5 function, thus preventing expression of MyoD and myogenin. Consistent with this, the expression of each MyoD family member can reverse the inhibition of differentiation by HBP1. Further investigation implicates the relative ratio of RB to HBP1 as a determinant of whether cell cycle exit or full differentiation occurs. At a low RB/HBP1 ratio cell cycle exit occurs but there is no tissue-specific gene expression. At elevated RB/HBP1 ratios, full differentiation occurs. Similar changes in the RB/HBP1 ratio have been observed in normal C2 differentiation. Thus, it is postulated that the relative ratio of RB to HBP1 may be one signal for activation of the MyoD family. A model is proposed in which a checkpoint of positive and negative regulation may coordinate cell cycle exit with MyoD family activation to give fidelity and progression in differentiation (Shih, 1998).

During Drosophila myogenesis, Notch signaling acts at multiple steps of the muscle differentiation process. In vertebrates, Notch activation has been shown to block MyoD activation and muscle differentiation in vitro, suggesting that this pathway may act to maintain the cells in an undifferentiated proliferative state. In this paper, the role of Notch signaling has been addressed in vivo during chick myogenesis. The Notch1 receptor is expressed in postmitotic cells of the myotome and the Notch ligands Delta1 and Serrate2 are detected in subsets of differentiating myogenic cells and are thus in position to signal to Notch1 during myogenic differentiation. The expression of MyoD and Myf5 during avian myogenesis was investigated, and Myf5 was shown to be expressed earlier than MyoD. Forced expression of the Notch ligand, Delta1, during early myogenesis, using a retroviral system, has no effect on the expression of the early myogenic markers Pax3 and Myf5, but causes strong down-regulation of MyoD in infected somites. Although Delta1 overexpression results in the complete lack of differentiated muscles, detailed examination of the infected embryos shows that initial formation of a myotome is not prevented, indicating that exit from the cell cycle has not been blocked. These results suggest that Notch signaling acts in postmitotic myogenic cells to control a critical step of muscle differentiation (Hirsinger, 2001).

The effect of Notch activation on the expression of the myogenic factors MyoD and Myf5 was assessed 48 hours after infection. In infected somites, MyoD expression is strongly down-regulated in the myotome, whereas Myf5 is still normally expressed. The infected dermomyotome maintains its epithelial structure after it should have undergone an epithelio-mesenchymal transition, allowing the release of dermal precursors. Myf5 is expressed in proliferative cells of the dermomyotome and the dorsal lip in addition to the myotome, whereas MyoD is essentially found in the postmitotic cells of myotome. The absence of MyoD in the infected embryos could be due to an accumulation of proliferative Myf5-expressing cells that are unable to proceed further in their differentiation. This situation would be reminiscent of that in the nervous system where widespread Delta1 overexpression blocks exit of neural progenitor cells from the cell cycle. To examine whether myogenic progenitors are also prevented from exiting the cell cycle, BrdU incorporation together with the expression of MyoD and Myf5 were examined in infected embryos. Postmitotic myogenic cells were found in both infected and uninfected myotomes, indicating that ectopic Notch signaling does not block exit from the cell cycle in this context. This is consistent with the retention of normal, and not dramatically widespread, Pax3 and Myf5 expression in the dermomyotome and myotome. The loss of MyoD expression but maintenance of Myf5 expression in postmitotic cells in the myotomal layer implies that constitutive Notch activation does not affect the production of postmitotic Myf5-expressing cells, but specifically blocks subsequent MyoD expression by these cells (Hirsinger, 2001).

Hedgehog proteins have been implicated in the control of myogenesis in the medial vertebrate somite. In the mouse, normal epaxial expression of the myogenic transcription factor gene myf5 is dependent on Sonic hedgehog. The interaction between Hedgehog signals, the expression of myoD family genes, including the newly cloned zebrafish myf5, and slow myogenesis have been examined in zebrafish. Sonic hedgehog is necessary for normal expression of both myf5 and myoD in adaxial slow muscle precursors, but not in lateral paraxial mesoderm. Expression of both genes is initiated normally in rostral presomitic mesoderm in sonic you mutants, which lack all Sonic hedgehog. Similar initiation continues during tailbud outgrowth when the cells forming caudal somites are generated. However, adaxial cells in sonic you embryos are delayed in terminal differentiation and caudal adaxial cells fail to maintain myogenic regulatory factor expression. Despite these defects, other signals are able to maintain, or reinitiate, some slow muscle development in sonic you mutants. In the cyclops mutant, the absence of floorplate-derived Tiggywinkle hedgehog and Sonic hedgehog has no discernible effect on slow adaxial myogenesis. Similarly, the absence of notochord-derived Sonic hedgehog and Echidna hedgehog in mutants lacking notochord delays, but does not prevent, adaxial slow muscle development. In contrast, removal of both Sonic hedgehog and a floorplate signal, probably Tiggywinkle hedgehog, from the embryonic midline in cyclops;sonic you double mutants essentially abolishes slow myogenesis. It is concluded that several midline signals, likely to be various Hedgehogs, collaborate to maintain adaxial slow myogenesis in the zebrafish embryo. Moreover, the data demonstrate that, in the absence of this required Hedgehog signaling, expression of myf5 and myoD is insufficient to commit cells to adaxial myogenesis (Coutelle, 2001).

Hedgehog proteins have been implicated in the control of myogenesis in the medial vertebrate somite. In the mouse, normal epaxial expression of the myogenic transcription factor gene myf5 is dependent on Sonic hedgehog. The interaction between Hedgehog signals, the expression of myoD family genes, including the newly cloned zebrafish myf5, and slow myogenesis have been examined in zebrafish. Sonic hedgehog is necessary for normal expression of both myf5 and myoD in adaxial slow muscle precursors, but not in lateral paraxial mesoderm. Expression of both genes is initiated normally in rostral presomitic mesoderm in sonic you mutants, which lack all Sonic hedgehog. Similar initiation continues during tailbud outgrowth when the cells forming caudal somites are generated. However, adaxial cells in sonic you embryos are delayed in terminal differentiation and caudal adaxial cells fail to maintain myogenic regulatory factor expression. Despite these defects, other signals are able to maintain, or reinitiate, some slow muscle development in sonic you mutants. In the cyclops mutant, the absence of floorplate-derived Tiggywinkle hedgehog and Sonic hedgehog has no discernible effect on slow adaxial myogenesis. Similarly, the absence of notochord-derived Sonic hedgehog and Echidna hedgehog in mutants lacking notochord delays, but does not prevent, adaxial slow muscle development. In contrast, removal of both Sonic hedgehog and a floorplate signal, probably Tiggywinkle hedgehog, from the embryonic midline in cyclops;sonic you double mutants essentially abolishes slow myogenesis. It is concluded that several midline signals, likely to be various Hedgehogs, collaborate to maintain adaxial slow myogenesis in the zebrafish embryo. Moreover, the data demonstrate that, in the absence of this required Hedgehog signaling, expression of myf5 and myoD is insufficient to commit cells to adaxial myogenesis (Coutelle, 2001).

Hedgehog proteins have been implicated in the control of myogenesis in the medial vertebrate somite. In the mouse, normal epaxial expression of the myogenic transcription factor gene myf5 is dependent on Sonic hedgehog. The interaction between Hedgehog signals, the expression of myoD family genes, including the newly cloned zebrafish myf5, and slow myogenesis have been examined in zebrafish. Sonic hedgehog is necessary for normal expression of both myf5 and myoD in adaxial slow muscle precursors, but not in lateral paraxial mesoderm. Expression of both genes is initiated normally in rostral presomitic mesoderm in sonic you mutants, which lack all Sonic hedgehog. Similar initiation continues during tailbud outgrowth when the cells forming caudal somites are generated. However, adaxial cells in sonic you embryos are delayed in terminal differentiation and caudal adaxial cells fail to maintain myogenic regulatory factor expression. Despite these defects, other signals are able to maintain, or reinitiate, some slow muscle development in sonic you mutants. In the cyclops mutant, the absence of floorplate-derived Tiggywinkle hedgehog and Sonic hedgehog has no discernible effect on slow adaxial myogenesis. Similarly, the absence of notochord-derived Sonic hedgehog and Echidna hedgehog in mutants lacking notochord delays, but does not prevent, adaxial slow muscle development. In contrast, removal of both Sonic hedgehog and a floorplate signal, probably Tiggywinkle hedgehog, from the embryonic midline in cyclops;sonic you double mutants essentially abolishes slow myogenesis. It is concluded that several midline signals, likely to be various Hedgehogs, collaborate to maintain adaxial slow myogenesis in the zebrafish embryo. Moreover, the data demonstrate that, in the absence of this required Hedgehog signaling, expression of myf5 and myoD is insufficient to commit cells to adaxial myogenesis (Coutelle, 2001).

Transcription factors Myf5 and MyoD are critical for myoblast determination. Myogenin is a direct transcriptional target of these factors and its expression is associated with commitment to terminal differentiation. Here, myogenic derivatives of human U20S cells have been used that express Myf5 or MyoD under control of a tetracycline-sensitive promoter in order to study expression of endogenous myogenin (myf4). Myf5-mediated induction of myogenin shows striking dependence on cell density. At high cell density, Myf5 is a potent inducer of myogenin expression. At low cell density, Myf5 (unlike MyoD) is a poor inducer of myogenin expression, while retaining the capacity to direct expression of other muscle-specific genes. The permissive influence of high cell density on myogenin induction by Myf5 is not a consequence of serum depletion or cell cycle arrest, but is mimicked by a disruption adjacent to the basic region of Myf5 (Myf5/mt) which reduces its DNA binding affinity for E-boxes without compromising its ability to transactivate a reporter gene driven by the myogenin promoter. Coculture of cells expressing wild-type Myf5 and Myf5/mt leads to reduced myogenin induction in Myf5/mt cells. It has been proposed that at low cell density, Myf5 inhibits induction of myogenin (Lindon, 2001).

Vertebrate myogenesis is controlled by four transcription factors known as the myogenic regulatory factors (MRFs): Myf5, Mrf4, myogenin and MyoD. During mouse development Myf5 is the first MRF to be expressed and it acts by integrating multiple developmental signals to initiate myogenesis. Numerous discrete regulatory elements are involved in the activation and maintenance of Myf5 gene expression in the various muscle precursor populations, reflecting the diversity of the signals that control myogenesis. Focus was placed on the enhancer that recapitulates the first phase of Myf5 expression in the epaxial domain of the somite, in order to identify the subset of cells that first transcribes the gene and therefore gain insight into molecular, cellular and anatomical facets of early myogenesis. Deletion of this enhancer from a YAC reporter construct that recapitulates the Myf5 expression pattern demonstrates that this regulatory element is necessary for expression in the early epaxial somite but in no other site of myogenesis. Importantly, Myf5 is subsequently expressed in the epaxial myotome under the control of other elements located far upstream of the gene. These data suggest that the inductive signals that control Myf5 expression switch rapidly from those that impinge on the early epaxial enhancer to those that impinge on the other enhancers that act later in the epaxial somite, indicating that there are significant changes in either the signaling environment or the responsiveness of the cells along the rostrocaudal axis. It is proposed that the first phase of Myf5 epaxial expression, driven by the early epaxial enhancer in the dermomyotome, is necessary for early myotome formation, while the subsequent phases are associated with cytodifferentiation within the myotome (Teboul, 2002).

Transgenic analyses have defined two MyoD enhancers in mammals -- the core enhancer and distal regulatory region (DRR); these enhancers exhibit complementary activities and together are sufficient to recapitulate MyoD expression in developing and mature skeletal muscle. DRR activity is restricted to differentiated muscle and persists postnatally, suggesting an important role in maintaining MyoD expression in myocytes and muscle fibers. The DRR is five kilobases upstream of the MyoD structural gene, and is unrelated in sequence to the core enhancer and exhibits largely complementary activity in transgenic mice. DRR activity is entirely MyoD and Myf-5-dependent and is restricted to differentiated skeletal muscle in vivo, which is reflected as a significant delay in DRR-driven transgene expression in several sites of myogenesis relative to the endogenous MyoD gene. Unlike the core enhancer, the DRR remains active in adult muscle, showing a similar expression profile as the endogenous MyoD gene. Collectively, these data indicate that the core enhancer and DRR have distinct activation and maintenance functions, respectively, that collaborate to establish the dynamic pattern of MyoD expression in embryonic and adult skeletal muscle (Chen, 2002).

Targeted mutagenesis was used in the mouse to define essential functions of the DRR in its normal chromosomal context. Surprisingly, deletion of the DRR results in reduced MyoD expression in all myogenic lineages at E10.5, at least 1 day prior to detection of DRR activity in limb buds and branchial arches of transgenic mice. At later embryonic and fetal stages, however, no defect in MyoD expression is observed, indicating that the DRR is dispensable for regulating MyoD during muscle differentiation. Expression analyses in wild-type and Myf-5 mutant embryos also indicate that the DRR is not an obligate target for Myf-5- and Pax-3-dependent regulation. In contrast to embryonic and fetal stages, deletion of the DRR results in a pronounced reduction in MyoD mRNA levels in adults, showing a functional requirement for DRR activity in mature muscle. These data reveal essential and redundant functions of the DRR and underscore the importance of loss-of-function enhancer analyses for understanding cis transcriptional circuitry (Chen, 2002).

Gene targeting has indicated that Myf5 and MyoD are required for myogenic determination because skeletal myoblasts and myofibers are missing in mouse embryos lacking both Myf5 and MyoD. To investigate the fate of Myf5:MyoD-deficient myogenic precursor cells during embryogenesis, the sites of epaxial, hypaxial, and cephalic myogenesis were examined at different developmental stages. In newborn mice, excessive amounts of adipose tissue were found in the place of muscles whose progenitor cells have undergone long-range migrations as mesenchymal cells. Analysis of the expression pattern of Myogenin-lacZ transgene and muscle proteins reveals that myogenic precursor cells are not able to acquire a myogenic fate in the trunk (myotome) nor at sites of MyoD induction in the limb buds. Importantly, the Myf5-dependent precursors, as defined by Myf5nlacZ-expression, deficient for both Myf5 and MyoD, were observed early in development to assume nonmuscle fates (e.g., cartilage) and, later in development, to extensively proliferate without cell death. Their fate appears to significantly differ from the fate of MyoD-dependent precursors, as defined by 258/-2.5lacZ-expression (-20 kb enhancer of MyoD), of which a significant proportion failed to proliferate and underwent apoptosis. Taken together, these data strongly suggest that Myf5 and MyoD regulatory elements respond differentially in different compartments (Kablar, 2003).

Cytokines, such as tumor necrosis factor-alpha (TNFalpha), potently inhibit the differentiation of mesenchymal cells and down-regulate the expression of Sox9 and MyoD, transcription factors required for chondrocyte and myocyte development. NF-kappaB controls TNFalpha-mediated suppression of myogenesis through a mechanism involving MyoD mRNA down-regulation. NF-kappaB also suppresses chondrogenesis and destabilizes Sox9 mRNA levels. Multiple copies of an mRNA cis-regulatory motif (5'-ACUACAG-3') are necessary and sufficient for NF-kappaB-mediated Sox9 and MyoD down-regulation. These results suggest that the ACTACAG motifs represent, presumably indirect, NF-kappaB-responsive elements in the MyoD and Sox9 mRNAs. Thus, in response to cytokine signaling, NF-kappaB modulates the differentiation of mesenchymal-derived cell lineages via RNA sequence-dependent, posttranscriptional down-regulation of key developmental regulators (Sitcheran, 2003).

The distal regulatory region (DRR) of the mouse and human MyoD gene contains a conserved SRF binding CArG-like element. In electrophoretic mobility shift assays with myoblast nuclear extracts, this CArG sequence, although slightly divergent, binds two complexes containing, respectively, the transcription factor YY1 and SRF associated with the acetyltransferase CBP and members of C/EBP family. A single nucleotide mutation in the MyoD-CArG element suppresses binding of both SRF and YY1 complexes and abolishes DRR enhancer activity in stably transfected myoblasts. This MyoD-CArG sequence is active in modulating endogeneous MyoD gene expression because microinjection of oligonucleotides corresponding to the MyoD-CArG sequence specifically and rapidly suppress MyoD expression in myoblasts. In vivo, the expression of a transgenic construct comprising a minimal MyoD promoter fused to the DRR and beta-galactosidase is induced with the same kinetics as MyoD during mouse muscle regeneration. In contrast induction of this reporter is no longer seen in regenerating muscle from transgenic mice carrying a mutated DRR-CArG. These results show that an SRF binding CArG element present in MyoD gene DRR is involved in the control of MyoD gene expression in skeletal myoblasts and in mature muscle satellite cell activation during muscle regeneration (L'honore, 2003).

The Ezh2 protein endows the Polycomb PRC2 and PRC3 complexes with histone lysine methyltransferase (HKMT) activity that is associated with transcriptional repression. Ezh2 expression is developmentally regulated in the myotome compartment of mouse somites and its down-regulation coincides with activation of muscle gene expression and differentiation of satellite-cell-derived myoblasts. Increased Ezh2 expression inhibits muscle differentiation, and this property is conferred by its SET domain, required for the HKMT activity. In undifferentiated myoblasts, endogenous Ezh2 is associated with the transcriptional regulator YY1. Both Ezh2 and YY1 are detected, with the deacetylase HDAC1, at genomic regions of silent muscle-specific genes: their presence correlates with methylation of K27 of histone H3. YY1 is required for Ezh2 binding because RNA interference of YY1 abrogates chromatin recruitment of Ezh2 and prevents H3-K27 methylation. Upon gene activation, Ezh2, HDAC1, and YY1 dissociate from muscle loci, H3-K27 becomes hypomethylated and MyoD and SRF are recruited to the chromatin. These findings suggest the existence of a two-step activation mechanism whereby removal of H3-K27 methylation, conferred by an active Ezh2-containing protein complex, followed by recruitment of positive transcriptional regulators at discrete genomic loci are required to promote muscle gene expression and cell differentiation (Cartetti, 2004).

These results indicate that Ezh2 is recruited at the chromatin of selected muscle regulatory regions by the transcriptional regulator YY1. Both can be coimmunoprecipitated from myoblast and not myotube cell extracts, and the proteins colocalize at the same muscle chromatin regions in a developmentally regulated manner. The interaction of endogenous YY1 and Ezh2 is likely to be mediated by the PcG EED protein because recombinant YY1 and Ezh2 do not directly associate. Previous reports have demonstrated a negative role for YY1 in regulating muscle gene expression through interaction with distinct nucleotides within the CarG-box [CC(A+T-rich)6GG], one of the DNA elements required for muscle-specific gene transcription. Transcriptional activation coincides with replacement of YY1 by the serum response factor (SRF), whose interaction with the CarG-box is required for muscle-specific transcription to proceed. These data suggest a two-step activation model of muscle gene expression. In the repressed state, YY1 recruits a complex containing both Ezh2 and HDAC1 that silences transcription through histone methylation (H3-K27) and deacetylation. Transcriptional activation entails the initial removal of the YY1-Ezh2-HDAC1 repressive complex and subsequent recruitment of the activators SRF (which replaces YY1) and the MyoD family of transcription factors and associated acetyltransferases. Since YY1 binding tolerates a substantial nucleotide heterogeneity in its DNA recognition sites, muscle and non-muscle-specific CarG-less regulatory regions may be also occupied and regulated in a similar manner. In contrast, Ezh2 does not appear to promiscuously regulate expression of all muscle-specific genes as indicated by the transient coexpression of Ezh2 and myogenin in the myotome of developing embryos and lack of Ezh2 recruitment and H3-K27 methylation at the myogenin promoter. Distinct histone methyltransferases and deacetylases have been shown to modify histones at the myogenin promoter (Cartetti, 2004 and references therein).

The myogenic regulatory factor Myf5 is integral to the initiation and control of skeletal muscle formation. In adult muscle, Myf5 is expressed in satellite cells, stem cells of mature muscle, but not in the myonuclei that sustain the myofibre. Using the Myf5nlacZ/+ mouse, Myf5 was shown to be constitutively expressed in muscle spindles-stretch-sensitive mechanoreceptors, while muscle denervation induces extensive reactivation of the Myf5 gene in myonuclei. To identify the elements involved in the regulation of Myf5 in adult muscle, reporter gene expression was analyzed in a transgenic bacterial artificial chromosome (BAC) deletion series of the Mrf4/Myf5 locus. A BAC carrying 140 kb upstream of the Myf5 transcription start site was sufficient to drive all aspects of Myf5 expression in adult muscle. In contrast, BACs carrying 88 and 59 kb upstream were unable to drive consistent expression in satellite cells, although expression in muscle spindles and reactivation of the locus in myonuclei were retained. Therefore, as during development, multiple enhancers are required to generate the full expression pattern of Myf5 in the adult. Together, these observations show that elements controlling adult Myf5 expression are genetically separable and possibly distinct from those that control Myf5 during development. These studies are a first step towards identifying cognate transcription factors involved in muscle stem cell regulation (Zammit, 2004).

Recruitment of multipotent mesodermal cells to the myogenic lineage is mediated by the transcription factor Myf5, the first of the myogenic regulatory factors to be expressed in most sites of myogenesis in the mouse embryo. Among numerous elements controlling the spatiotemporal pattern of Myf5 expression, the -58/-56 kb distal Myf5 enhancer directs expression in myogenic progenitor cells in limbs and in somites. This study shows by site-directed mutagenesis within this enhancer that a predicted homeobox adjacent to a putative paired domain-binding site is required for the activity in muscle precursor cells in limbs and strongly contributes to expression in somites. By contrast, predicted binding sites for Tcf/Lef, Mef3 and Smad transcription factors play no apparent role for the expression in limbs but might participate in the control in somites. A 30mer oligonucleotide sequence containing and surrounding the homeo and paired domain-binding motifs directs faithful expression in myogenic cells in limbs and also enhances myotomal expression in somites. Pax3 and Meox2 transcription factors can bind to these consensus sites in vitro and therefore constitute potential regulators. However, genetic evidence in the Meox2-deficient mouse mutant argues against a role for Meox2 in the regulation of Myf5 expression. The data presented in this study demonstrate that a composite homeo and paired domain-binding motif within the -58/-56 enhancer is required and sufficient for activation of the Myf5 gene in muscle progenitor cells in the limb. Although Pax3 constitutes a potential cognate transcription factor for the enhancer, it fails to transactivate the site in transfection experiments (Buchberger, 2007).

Postnatal growth and regeneration of skeletal muscle requires a population of resident myogenic precursors named satellite cells. The transcription factor Pax7 is critical for satellite cell biogenesis and survival and has been also implicated in satellite cell self-renewal; however, the underlying molecular mechanisms remain unclear. Pax7 overexpression in adult primary myoblasts has been shown to down-regulate MyoD and prevent myogenin induction, inhibiting myogenesis. This study shows that Pax7 prevents muscle differentiation independently of its transcriptional activity, affecting MyoD function. Conversely, myogenin directly affects Pax7 expression and may be critical for Pax7 down-regulation in differentiating cells. These results provide evidence for a cross-inhibitory interaction between Pax7 and members of the muscle regulatory factor family. This could represent an additional mechanism for the control of satellite cell fate decisions resulting in proliferation, differentiation, and self-renewal, necessary for skeletal muscle maintenance and repair (Olguin, 2007).

Although FoxO and Pax proteins represent two important families of transcription factors in determining cell fate, they had not been functionally or physically linked together in mediating regulation of a common target gene during normal cellular transcription programs. This study identified MyoD, a key regulator of myogenesis, as a direct target of FoxO3 and Pax3/7 in myoblasts. Cell-based assays and in vitro studies reveal a tight codependent partnership between FoxO3 and Pax3/7 to coordinately recruit RNA polymerase II and form a preinitiation complex (PIC) to activate MyoD transcription in myoblasts. The role of FoxO3 in regulating muscle differentiation is confirmed in vivo by observed defects in muscle regeneration caused by MyoD downregulation in FoxO3 null mice. These data establish a mutual interdependence and functional link between two families of transcription activators serving as potential signaling sensors and regulators of cell fate commitment in directing tissue specific MyoD transcription (Hu, 2008).

Sequence analysis revealed four potential FRE sites in a 6 kb region upstream of the myod transcription initiation site, which are between the DRR and PRR elements important for MyoD regulation during embryonic muscle development. ChIP experiments were performed to determine promoter occupancy of FoxOs at the myod gene using antibodies specifically directed against each of the three FoxO proteins. FoxO3 was efficiently detected at the myod promoter regions overlapping putative FRE -940 and -1598, but not at -1928 and -2351. In marked contrast to FoxO3, neither FoxO1 nor FoxO4 was detected significantly above background at the four potential FREs in C2C12 cells. As a control, the same FoxO1 and FoxO4 antibodies were used to successfully detect their occupancy at the p21 promoter, which is known to be regulated by these FoxOs. Indeed, it appears that FoxO3 selectively binds two of the putative FREs at the myod promoter in myoblasts, suggesting that FoxO3 binding may correspond to one of the important steps regulating MyoD expression. RNA polymerase II was also found at the myod promoter by ChIP, further confirming the correlation between FoxO3 promoter occupancy and transcription activation. Although FoxO4 appeared to have low transcriptional activity, little, if any, FoxO4 was detected at the myod promoter by ChIP in myoblasts. Taken together, these results suggest that at least FoxO3 is likely part of the active transcription machinery directly recruited to the myod promoter in myoblasts (Hu, 2008).

Since FoxO3 binds to putative FRE -940 and FRE -1598 of the myod promoter in C2C12 cells, whether FoxO3 is also able to recognize and bind these putative FREs in vitro was tested by electrophoretic mobility shift assays (EMSA). Purified recombinant FoxO3 efficiently bound to both the FRE -940 and FRE -1598 probes, but not to mutant probes, confirming that FoxO3 can bind to both FRE sequences specifically in vitro. Consistent with ChIP results, in luciferase reporter assays, mutations in the FREs at -940 or -1598 significantly debilitated myod transcription, while mutations in FRE -1928 and FRE -2351 had little or no effect on transcription activity. These results suggest that FRE -940 and -1598 bound by FoxO3 are critical for myod transcription activation. Taken together, these assays indicate that FoxO3 can specifically bind two FREs in the myod promoter and activate transcription (Hu, 2008).

Although FoxOs have previously been implicated functioning in muscle differentiation, skeletal muscle-specific genes directly targeted by FoxOs had not been identified. The current findings indicate that FoxO3 (but not FoxO1 or FoxO4) binds a subset of FREs in the myod promoter to work in concert with Pax3/7 in regulating cell type-specific transcription activation in myoblasts. The contribution of FoxO3 in directing myod transcription activation in vivo was further confirmed by the observed muscle regeneration defects in FoxO3 null mice (Hu, 2008).

Identification of FoxO3 as an important myod transcription activator may provide a handle to explore the potential signaling pathways governing muscle regeneration. Interestingly, FoxO1 was found to negatively regulate MyoD expression indirectly through the Delta-Notch pathway. The potential repression of MyoD by FoxO1 together with the results of direct activation of MyoD by FoxO3 suggests an intriguing mechanism to fine tune MyoD expression. Muscle differentiation may therefore utilize selected FoxOs in partnership with Pax3/7 to integrate inputs from multiple signaling pathways (Hu, 2008).

The apparent Kd of FoxO3 and Pax3/7 binding individually to the DNA elements in the myod promoter is on the order of 10−7M in vitro, which is rather modest compared to a typical DNA binding protein, such as GAL4 (apparent Kd, ~10−11M). This is consistent with the finding that neither FoxO3 nor Pax3/7 alone binds promoter DNA efficiently to form a stable DNA-activator complex capable of recruiting a functional PIC. It appears that to efficiently assemble an active PIC via FoxO3 and Pax3/7 at the myod promoter both protein-DNA and protein-protein interactions mediated by this hitherto unknown activator partnership must take place to trigger transcription activation synergistically. This may therefore represent a useful and efficient combinatorial mechanism to direct cell type-specific transcription while utilizing two activators shared by many cell types. This codependence is reminiscent of the mechanism utilized by Sox2 and Pax6 to drive lens-specific transcription of δ-crystallin gene (Hu, 2008).

Although under normal conditions FoxO3 is predominantly detected occupying the FRE at -1598 of the myod promoter, curiously, it was found that some FoxO4 can be detected at this site in myoblasts when Pax3/7 is depleted. It is known that Pax3/7 is not expressed in myotubes, but MyoD expression persists in myotubes. It will be interesting to survey the identity of FoxOs binding to the myod promoter in myotubes versus myoblasts and explore additional mechanisms involved in cell type-specific transcription activation during later stages of myogenesis. Intriguingly, there is a switching of the core transcription machinery from the canonical holo-TFIID to a TRF3/TAF3 complex during myoblasts differentiation to myotubes (Deato, 2007). While FoxO3 and Pax3/7 appear to work in concert with TFIID at the MyoD promoter in myoblasts, it will be important to identify the key activator(s) that function together with the TRF3/TAF3 complex in myotubes (Hu, 2008).

Recent findings implicate alternate core promoter recognition complexes in regulating cellular differentiation. This study reports a spatial segregation of the alternative core factor TAF3, but not canonical TFIID subunits, away from the nuclear periphery, where the key myogenic gene MyoD is preferentially localized in myoblasts. This segregation is correlated with the differential occupancy of TAF3 versus TFIID at the MyoD promoter. Loss of this segregation by modulating either the intranuclear location of the MyoD gene or TAF3 protein leads to altered TAF3 occupancy at the MyoD promoter. Intriguingly, in differentiated myotubes, the MyoD gene is repositioned to the nuclear interior, where TAF3 resides. The specific high-affinity recognition of H3K4Me3 by the TAF3 PHD finger appears to be required for the sequestration of TAF3 to the nuclear interior. It is suggested that intranuclear sequestration of core transcription components and their target genes provides an additional mechanism for promoter selectivity during differentiation (Yao, 2011).

Zic genes encode a conserved family of zinc finger proteins with essential functions in neural development and axial skeletal patterning in the vertebrate embryo. Zic proteins also function as Gli co-factors in Hedgehog signaling. This study reports that Zic genes have a role in Myf5 regulation for epaxial somite myogenesis in the mouse embryo. In situ hybridization studies show that Zic1, 2, and 3 transcripts are expressed in Myf5-expressing epaxial myogenic progenitors in the dorsal medial dermomyotome of newly forming somites, and immunohistological studies show that Zic2 protein is co-localized with Myf5 and Pax3 in the dorsal medial lip of the dermomyotome, but is not expressed in the forming myotome. In functional reporter assays, Zic1 and Zic2, but not Zic3, potentiate the transactivation of Gli-dependent Myf5 epaxial somite-specific (ES) enhancer activity in 3T3 cells, and Zic1 activates endogenous Myf5 expression in 10T1/2 cells and in presomitic mesoderm explants. Zic2 also co-immunoprecipitates with Gli2, indicating that Zic2 forms complexes with Gli2 to promote Myf5 expression. Genetic studies show that, although Zic2 and Zic1 are activated normally in sonic hedgehog−/− mutant embryos, Myf5 expression in newly forming somites is deficient in both sonic hedgehog−/− and in Zic2kd/kd mutant mouse embryos, providing further evidence that these Zic genes are upstream regulators of Hedgehog-mediated Myf5 activation. Myf5 activation in newly forming somites is delayed in Zic2 mutant embryos until the time of Zic1 activation, and both Zic2 and Myf5 require noggin for their activation (Pan, 2011).

Post-transcriptonal regulation of Nautilus homologs

Helicase UPF1 functions in both Staufen 1 (STAU1)-mediated mRNA decay (SMD) and nonsense-mediated mRNA decay (NMD), which are competitive pathways. STAU1- and UPF2-binding sites within UPF1 overlap so that STAU1 and UPF2 binding to UPF1 appear to be mutually exclusive. Furthermore, down-regulating the cellular abundance of STAU1, which inhibits SMD, increases the efficiency of NMD, whereas down-regulating the cellular abundance of UPF2, which inhibits NMD, increases the efficiency of SMD. Competition under physiological conditions is exemplified during the differentiation of C2C12 myoblasts to myotubes: The efficiency of SMD increases and the efficiency of NMD decreases, consistent with the finding that more STAU1 but less UPF2 bind UPF1 in myotubes compared with myoblasts. Moreover, an increase in the cellular level of UPF3X during myogenesis results in an increase in the efficiency of an alternative NMD pathway that, unlike classical NMD, is largely insensitive to UPF2 down-regulation. The remarkable balance NCC SMD and the two types of NMD are discussed in view of data indicating that PAX3 mRNA is an SMD target whose decay promotes myogenesis whereas myogenin mRNA is a classical NMD target encoding a protein required for myogenesis (Gong, 2009).

Nautilus homologs and cell cycle regulation

The muscle regulators MyoD and Myf-5 control cell cycle withdrawal and induction of differentiation in skeletal muscle cells. By immunofluorescence analysis, it has been shown that MyoD and Myf-5 expression patterns become mutually exclusive when C2 cells are induced to differentiate with Myf-5 staining present in cells that fail to differentiate. Isolation of these undifferentiated cells reveals that upon serum stimulation they reenter the cell cycle, express MyoD and downregulate Myf-5. Similar regulations of MyoD and Myf-5 are observed using cultured primary myoblasts derived from satellite cells. To further analyze these regulations of MyoD and Myf-5 expression, proliferating myoblasts were synchronized. Analysis of MyoD and Myf-5 expression during cell cycle progression reveals distinct and contrasting profiles of expression. MyoD is absent in G0, peaks in mid-G1, falls to its minimum level at G1/S and reaugments from S to M. In contrast, Myf-5 protein is high in G0, decreases during G1 and reappears at the end of G1 to remain stable until mitosis. These data demonstrate that the two myogenic factors MyoD and Myf-5 undergo specific and distinct cell cycle-dependent regulation, thus establishing a correlation between the cell cycle-specific ratios of MyoD and Myf-5 and the capacity of cells to differentiate: (a) in G1, when cells express high levels of MyoD and enter differentiation; (b) in G0, when cells express high levels of Myf-5 and fail to differentiate (Kitzmann, 1998).

MyoD has been proposed to facilitate terminal myoblast differentiation by binding to and inhibiting phosphorylation of the retinoblastoma protein (pRb). MyoD can interact with cyclin-dependent kinase 4 (cdk4) through a conserved 15 amino acid (aa) domain in the C-terminus of MyoD. MyoD, its C-terminus lacking the basic helix-loop-helix (bHLH) domain, or the 15 aa cdk4-binding domain all inhibit the cdk4-dependent phosphorylation of pRb in vitro. Cellular expression of full-length MyoD or fusion proteins containing either the C-terminus or just the 15 aa cdk4-binding domain of MyoD inhibits cell growth and pRb phosphorylation in vivo. The minimal cdk4-binding domain of MyoD fused to GFP can also induce differentiation of C2C12 muscle cells in growth medium. The defective myogenic phenotype in MyoD-negative BC3H1 cells can be rescued completely only when MyoD contains the cdk4-binding domain. It is proposed that a regulatory checkpoint in the terminal cell cycle arrest of the myoblast during differentiation involves the modulation of the cyclin D cdk-dependent phosphorylation of pRb through the opposing effects of cyclin D1 and MyoD. Attempts to detect interaction between MyoD or myogenin and pRb in the two-hybrid assay were unsuccessful. In contrast to the suggestion from in vitro immunoprecipitation studies, neither MyoD nor myogenin were found to interact with pRb. Thus, if there is any direct interaction between pRb and the myogenic factors in C2C12 cells it appears to be weak. In support of these observations, early targets of MyoD transcription are fully induced in cells lacking pRb, making the significance of the in vitro MyoD-pRb interaction questionable. Preliminary experiments indicate that the MyoD homologs from Drosophila (Nautilus) and Caenorhabditis elegans (hlh-1) can specifically bind vertebrate cdk4 and can inhibit cell growth in the BrdU incorporation assay. Virtually all cyclin D1-dependent kinase activity in proliferating mouse fibroblasts can be attributed to cdk4; cyclin D1 is the only ectopically expressed cyclin that will inhibit myogenesis, consistent with a unique role for the MyoD-cdk4 interaction. These results, however, do not rule out a similar interaction with cdk6, since binding between the 15 aa MyoD cdk4-binding site and cdk6 was also observed (Zhang, 1999b).

The dynamics of the MyoD-cdk4 interaction in the myoblast can be represented by the following model. The forced overexpression of MyoD inhibits the cdk4-dependent phosphorylation of pRb to trigger growth arrest and the exit from the cell cycle (likely to include cdk6 as well). Dephosphorylated pRb is thought to be required to maintain cell cycle arrest by inhibiting the growth promoting actions of E2F/DP family members, the induction of apoptosis, and DNA replication in myotubes. Excess cyclin D1 activates more cdk4 and the complex is translocated to the nucleus where it inhibits MyoD and the activation of the myogenic program. Excess expression of nuclear cdk4 triggers phosphorylation of pRb, allowing dissociation of E2F/DP and cell growth. In contrast, excess ectopic expression of MyoD depletes active cdk4, prevents the phosphorylation of pRb and induces growth arrest while activating target genes to drive myogenesis. Thus, excess cyclin D1 and cdk4 induce growth and block differentiation, whereas excess MyoD induces exit from the cell cycle and differentiation. D-type cyclins act as growth factor sensors and levels of cyclin D1 appear to be rate limiting in the formation of active cdk4, based upon the half-life of each protein. The ectopic expression of cyclin D1 can increase nuclear levels of cdk4 to shorten the G1 phase of the cell cycle. Thus, the relative nuclear ratios of MyoD and cyclin D cdks in the cell would appear to be key determinants in the cell cycle decisions of the myoblast during terminal differentiation, and this ratio is determined by growth factor modulation of cyclin D1 expression levels and the cyclin D1-dependent translocation of active cdk4 to the nucleus (Zhang, 1999b and references therein).

Table of contents

nautilus: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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