Myogenin is an essential regulator of adult myofibre growth and muscle stem cell homeostasis.

Growth and maintenance of skeletal muscle fibres depend on coordinated activation and return to quiescence of resident muscle stem cells (MuSCs). The transcription factor Myogenin (Myog) regulates myocyte fusion during development, but its role in adult myogenesis remains unclear. In contrast to mice, myog-/-zebrafish are viable, but have hypotrophic muscles. By isolating adult myofibres with associated MuSCs, we found that myog-/- myofibres have severely reduced nuclear number, but increased myonuclear domain size. Expression of fusogenic genes is decreased, Pax7 upregulated, MuSCs are fivefold more numerous and mis-positioned throughout the length of myog-/-myofibres instead of localising at myofibre ends as in wild-type. Loss of Myog dysregulates mTORC1 signalling, resulting in an 'alerted' state of MuSCs, which display precocious activation and faster cell cycle entry ex vivo, concomitant with myod upregulation. Thus, beyond controlling myocyte fusion, Myog influences the MuSC:niche relationship, demonstrating a multi-level contribution to muscle homeostasis throughout life.

Inositol 1,4,5 trisphosphate receptor 1 is a key player of human myoblast differentiation.

Cytosolic Ca(2+) signals are fundamental for the early and late steps of myoblast differentiation and are, as in many cells, generated by Ca(2+) release from internal stores as well as by plasma membrane Ca(2+) entry. Our recent studies identified the store-operated Ca(2+) channels, Orai1 and TRPC1&C4, as crucial for the early steps of human myogenesis and for the late fusion events. In the present work, we assessed the role of the inositol-1,4,5 tris-phosphate receptor (IP3R) type 1 during human myoblast differentiation. We demonstrated, using siRNA strategy that IP3R1 is required for the expression of muscle-specific transcription factors such as myogenin and MEF2 (myocyte enhancer factor 2), and for the formation of myotubes. The knockdown of IP3R1 strongly reduced endogenous spontaneous Ca(2+) transients, and attenuated store-operated Ca(2+) entry. As well, two Ca(2+)-dependent key enzymes of muscle differentiation, NFAT and CamKII are down-regulated upon siIP3R1 treatment. On the contrary, the overexpression of IP3R1 accelerated myoblasts differentiation. These findings identify Ca(2+) release mediated by IP3R1 as an essential mechanism during the early steps of myoblast differentiation.

Myogenin is a positive regulator of MEGF10 expression in skeletal muscle.

MEGF10 is known to function as a myogenic regulator of satellite cells in skeletal muscle. Mutations in MEGF10 gene cause a congenital myopathy called early onset myopathy, areflexia, respiratory distress and dysphagia (EMARDD). Despite its biological importance in muscle physiology, transcriptional regulation of the MEGF10 gene is unknown. Here, we characterized the 5' flanking region of the human MEGF10 gene and showed that the role of myogenic basic helix-loop-helix factor (bHLH) myogenin in MEGF10 transcription in muscle cells. Myogenin was found to share a similar expression pattern with MEGF10 during muscle regeneration and to increase the promoter activity of the MEGF10 gene in C2C12 cells. Overexpression of myogenin led to upregulation of MEGF10 mRNA in C2C12 cells. Site-directed mutagenesis assays revealed that the conserved E-box element at the region -114/-108 serves as a myogenin-binding motif. Promoter enzyme immunoassays and chromatin immunoprecipitation analysis showed direct interaction between myogenin and the myogenin-binding motif in the MEGF10 promoter. Taken together, these results indicate that myogenin is a positive regulator in transcriptional regulation of MEGF10 in skeletal muscle.

Identification of the STAC3 gene as a skeletal muscle-specifically expressed gene and a novel regulator of satellite cell differentiation in cattle.

Recent studies in mice and zebrafish suggest that the unannotated Src homology 3 and cysteine rich domain 3 (STAC3) gene plays an important role in skeletal muscle development and contraction. The objective of this study was to determine the tissue specificity of the bovine STAC3 gene and its potential role in the proliferation and differentiation of bovine satellite cells. The STAC3 mRNA was detected only in skeletal muscle among 18 bovine tissues examined by reverse transcription PCR. Western blotting revealed the expression of STAC3 protein in bovine skeletal muscle and the absence of it in 6 bovine tissues analyzed. Transfection of the bovine satellite cells with a pool of 2 STAC3 small interfering RNA (siRNA) caused a 90% reduction in STAC3 mRNA. Cell proliferation assays revealed that STAC3 knockdown had no effect on the proliferation rate of bovine satellite cells. Approximately 60% of bovine satellite cells transfected with STAC3 siRNA formed myotubes by 72 h of differentiation, whereas that percentage was 40% for those transfected with negative control siRNA (P < 0.05). At 24, 48, and 72 h of differentiation, bovine satellite cells transfected with STAC3 siRNA had greater mRNA expression of myogenin, myosin heavy chain 3, and myosin heavy chain 7, markers of myotubes, than those transfected with negative control siRNA (P < 0.05). These results suggest that the STAC3 gene is a negative regulator of the differentiation and fusion of bovine satellite cells into myotubes. However, STAC3 expression was increased during the differentiation of bovine satellite cells into myotubes. This suggests that STAC3 might have different functions in bovine myotubes than in bovine satellite cells.

Chronic binge alcohol consumption alters myogenic gene expression and reduces in vitro myogenic differentiation potential of myoblasts from rhesus macaques.

Chronic alcohol abuse is associated with skeletal muscle myopathy. Previously, we demonstrated that chronic binge alcohol (CBA) consumption by rhesus macaques accentuates skeletal muscle wasting at end-stage of simian immunodeficiency virus (SIV) infection. A proinflammatory, prooxidative milieu and enhanced ubiquitin proteasome activity were identified as possible mechanisms leading to loss of skeletal muscle. The possibility that impaired regenerative capacity, as reflected by the ability of myoblasts derived from satellite cell (SCs) to differentiate into myotubes has not been examined. We hypothesized that the inflammation and oxidative stress in skeletal muscle from CBA animals impair the differentiation capacity of myoblasts to form new myofibers in in vitro assays. We isolated primary myoblasts from the quadriceps femoris of rhesus macaques that were administered CBA or isocaloric sucrose (SUC) for 19 mo. Proliferation and differentiation potential of cultured myoblasts were examined in vitro. Myoblasts from the CBA group had significantly reduced PAX7, MYOD1, MYOG, MYF5, and MEF2C expression. This was associated with decreased myotube formation as evidenced by Jenner-Giemsa staining and myonuclei fusion index. No significant difference in the proliferative ability, cell cycle distribution, or autophagy was detected between myoblasts isolated from CBA and SUC groups. Together, these results reflect marked dysregulation of myoblast myogenic gene expression and myotube formation, which we interpret as evidence of impaired skeletal muscle regenerative capacity in CBA-administered macaques. The contribution of this mechanism to alcoholic myopathy warrants further investigation.

Molecular characterization and expression patterns of myogenin in compensatory growth of Megalobrama amblycephala.

Myogenin (myog) is a muscle-specific basic helix-loop-helix (bHLH) transcription factor that plays an essential role in regulating skeletal muscle development and growth. To investigate molecular characterization of myog and the effect of starvation/refeeding on the gene expression, we isolated the myog cDNA sequence and analyzed the expression patterns using quantitative real-time polymerase chain reaction in Megalobrama amblycephala. Sequence analysis indicated that M. amblycephala myog shared an analogous structure with the highly conserved His/Cys-rich, bHLH and C-terminal helix III domains with other vertebrates. Sequence alignment and phylogenetic tree showed that M. amblycephala myog had the highest identity with the homologues of Ctenopharyngodon idella and Cyprinus carpio. Spatio-temporal expression patterns revealed that myog mRNA levels at the segmentation period and 12 h post-hatching (hph) were significantly higher than at other development stages (P<0.05). Furthermore, the highest myog expression level was predominantly observed in white muscle compared with the other types of muscle. Fish body weight continuously decreased during 21-day starvation and then significantly increased after 7days of refeeding and reached the similar level to the control at 21days of refeeding, indicating that the pattern of complete compensatory growth possibly occurred in M. amblycephala; meanwhile, the relative somatic growth rate after refeeding was also dramatically higher than the control group. In addition, the myog expression decreased during 21days of starvation and then exhibited a strong rebound effect after 7days of refeeding and subsequently declined gradually to the control level by 21days of refeeding.

Caprine sex affects skeletal muscle profile and MRFs expression during postnatal development.

The important roles of myogenic regulatory factors (MRFs) have been well addressed in the process of mammalian skeletal myogenesis, while limited research has been performed in small ruminants. Furthermore, the effects of gender on the development of skeletal muscle and MRFs expression remain unknown. In this study, we identified the caprine Myf5, Myf6, MyoD and myogenin genes and quantified their expressions at six different postnatal time points by real-time RT-PCR. The sex has a marked effect on the expression differences of Myf5, MyoD and myogenin in the five investigated skeletal muscles, while minor influence on the expression difference of Myf6 except for Semitendinosus and Quadriceps femoris tissues (P < 0.001). The histological sections of muscles revealed a constant increase of muscle fiber diameter with aging but non-significant differences between genders. We provide novel evidence for MRFs expression in age- and gender-dependent manners, which will contribute to prioritizing these genes as potential candidate genes for trait-associated study and provide a foundation for understanding the molecular control of skeletal muscle growth in goat species.

Myogenin recruits the histone chaperone facilitates chromatin transcription (FACT) to promote nucleosome disassembly at muscle-specific genes.

Facilitates chromatin transcription (FACT) functions to reorganize nucleosomes by acting as a histone chaperone that destabilizes and restores nucleosomal structure. The FACT complex is composed of two subunits: SSRP1 and SPT16. We have discovered that myogenin interacts with the FACT complex. Transfection of FACT subunits with myogenin is highly stimulatory for endogenous muscle gene expression in 10T1/2 cells. We have also found that FACT subunits do not associate with differentiation-specific genes while C2C12 cells are proliferating but are recruited to muscle-specific genes as differentiation initiates and then dissociate as differentiation proceeds. The recruitment is dependent on myogenin, as knockdowns of myogenin show no recruitment of the FACT complex. These data suggest that FACT is involved in the early steps of gene activation through its histone chaperone activities that serve to open the chromatin structure and facilitate transcription. Consistent with this hypothesis, we find that nucleosomes are depleted at muscle-specific promoters upon differentiation and that this activity is dependent on the presence of FACT. Our results show that the FACT complex promotes myogenin-dependent transcription and suggest that FACT plays an important role in the establishment of the appropriate transcription profile in a differentiated muscle cell.

HMGB1-RAGE regulates muscle satellite cell homeostasis through p38-MAPK- and myogenin-dependent repression of Pax7 transcription.

Expression of the paired-box 7 (PAX7) transcription factor is regulated during both myoblast proliferation and differentiation: high levels of PAX7 compromise myogenic differentiation because of excess and prolonged proliferation, whereas low levels of PAX7 result in precocious differentiation. We showed that myogenin repressed Pax7 transcription in differentiating myoblasts by binding to specific recognition sites in the Pax7 promoter, and that high-mobility group box 1 (HMGB1)-receptor for advanced glycation end-products (RAGE) signaling was required for myogenin induction and myogenin-dependent repression of Pax7 transcription. In addition, PAX7 negatively and myogenin positively regulated RAGE expression. RAGE, a multiligand receptor of the immunoglobulin superfamily, was not expressed in adult skeletal muscles, and was transiently expressed in activated, proliferating and differentiating satellite cells (SCs) in injured muscles. Compared with wild-type muscles, Rage(-/-) muscles exhibited increased numbers of basal SCs that were further increased in injured Rage(-/-) muscles following elevated myoblast asymmetric division; complete regeneration of injured Rage(-/-) muscles was found to be delayed by ~1 week. Thus, RAGE signaling physiologically repressed Pax7 transcription in SCs by upregulating myogenin, thereby accelerating muscle regeneration and limiting SC self-renewal.

Myogenin regulates exercise capacity but is dispensable for skeletal muscle regeneration in adult mdx mice.

Duchenne muscular dystrophy (DMD) is the most prevalent inherited childhood muscle disorder in humans. mdx mice exhibit a similar pathophysiology to the human disorder allowing for an in-depth investigation of DMD. Myogenin, a myogenic regulatory factor, is best known for its role in embryonic myogenesis, but its role in adult muscle maintenance and regeneration is still poorly understood. Here, we generated an mdx:Myog(flox/flox) mouse harboring a tamoxifen-inducible Cre recombinase transgene, which was used to conditionally delete Myog during adult life. After tamoxifen treatment, three groups of mice were created to study the effects of Myog deletion: mdx:Myog(flox/flox) mice (mdx), Myog(flox/flox) mice (wild-type), and mdx:Myog(floxΔ/floxΔ):Cre-ER mice (mdx:Myog-deleted). mdx:Myog-deleted mice exhibited no adverse phenotype and behaved normally. When run to exhaustion, mdx:Myog-deleted mice demonstrated an enhanced capacity for exercise compared to mdx mice, running nearly as far as wild-type mice. Moreover, these mice showed the same signature characteristics of muscle regeneration as mdx mice. Unexpectedly, we found that myogenin was dispensable for muscle regeneration. Factors associated with muscle fatigue, metabolism, and proteolysis were significantly altered in mdx:Myog-deleted mice, and this might contribute to their increased exercise capacity. Our results reveal novel functions for myogenin in adult muscle and suggest that reducing Myog expression in other muscle disease models may partially restore muscle function.

Expression of Gα(z) in C2C12 cells restrains myogenic differentiation.

The recent identification of Gα(z) expression in C2C12 myoblasts and its demonstrated interaction with the transcription factor Eya2 inferred an unanticipated role of Gα(z) in muscle development. In the present study, endogenous Gα(z) mRNA and protein expressions in C2C12 cells increased upon commencement of myogenesis and peaked at around 4-6days after induction but were undetectable in adult skeletal muscle. Surprisingly, stable expression of recombinant Gα(z) in C2C12 myoblasts strongly suppressed myotube formation upon serum deprivation, and the constitutively active mutant Gα(z)QL exerted more pronounced effects. Transcriptional activities of reporter genes responsive to early (MyoD, MEF2 and myogenin) and late (muscle creatine kinase and myosin heavy chain) myogenic markers were reduced by transiently expressed Gα(z)QL. Membrane attachment of Gα(z) was apparently required for the suppressive effects because a fatty acylation-deficient Gα(z) mutant could not inhibit myogenin expression. Introduction of siRNA against Gα(z) enhanced myogenin-driven luciferase activity and increased myosin heavy chain expression. Immunostaining of C2C12 cells over-expressing Gα(z) showed delayed nuclear expression of myogenin and severe myotube deformation. Gα(z) expression was accompanied by reduced levels of Rock2, RhoA and RhoGAP, enhanced expression of Rnd3, and a reduction of serum-responsive factor-driven reporter activity. These results support a novel role of Gα(z) in restraining myogenic differentiation through the disruption of Rho signaling.

Myogenin regulates exercise capacity and skeletal muscle metabolism in the adult mouse.

Although skeletal muscle metabolism is a well-studied physiological process, little is known about how it is regulated at the transcriptional level. The myogenic transcription factor myogenin is required for skeletal muscle development during embryonic and fetal life, but myogenin's role in adult skeletal muscle is unclear. We sought to determine myogenin's function in adult muscle metabolism. A Myog conditional allele and Cre-ER transgene were used to delete Myog in adult mice. Mice were analyzed for exercise capacity by involuntary treadmill running. To assess oxidative and glycolytic metabolism, we performed indirect calorimetry, monitored blood glucose and lactate levels, and performed histochemical analyses on muscle fibers. Surprisingly, we found that Myog-deleted mice performed significantly better than controls in high- and low-intensity treadmill running. This enhanced exercise capacity was due to more efficient oxidative metabolism during low- and high-intensity exercise and more efficient glycolytic metabolism during high-intensity exercise. Furthermore, Myog-deleted mice had an enhanced response to long-term voluntary exercise training on running wheels. We identified several candidate genes whose expression was altered in exercise-stressed muscle of mice lacking myogenin. The results suggest that myogenin plays a critical role as a high-level transcriptional regulator to control the energy balance between aerobic and anaerobic metabolism in adult skeletal muscle.

Myogenic regulatory factors transactivate the Tceal7 gene and modulate muscle differentiation.

Recurrent injuries eventually exhaust the capacity of skeletal muscle to fully restore or regenerate its cellular architecture. Therefore a comprehensive understanding of the muscle regeneration programme is needed to provide a platform for new therapies for devastating diseases such as Duchenne muscular dystrophy. To begin to decipher the molecular programme that directs muscle regeneration, we undertook an unbiased strategy using microarray analysis of cardiotoxin-injured skeletal muscle at defined time periods in the adult mouse. Using this strategy, we identified Tceal7 [transcription elongation factor A (SII)-like 7], which was dynamically regulated during muscle regeneration. Our studies revealed that Tceal7 was restricted to the skeletal muscle lineage during embryogenesis. Using transgenic technologies and transcriptional assays, we defined an upstream 0.7 kb fragment of the Tceal7 gene that directed the LacZ reporter to the developing skeletal muscle lineage. Analysis of the Tceal7 promoter revealed evolutionarily conserved E-box motifs within the 0.7 kb upstream fragment that were essential for promoter activity, as mutation of the E-box motifs resulted in the loss of reporter expression in the somites of transgenic embryos. Furthermore, we demonstrated that MRFs (myogenic regulatory factors) were Tceal7 upstream transactivators using transcriptional assays, EMSAs (electrophoretic mobility-shift assays), and ChIP (chromatin immunoprecipitation) assays. Overexpression of Tceal7 in C2C12 myoblasts decreased cellular proliferation and enhanced differentiation. Further studies revealed that p27 expression was up-regulated following Tceal7 overexpression. These studies support the hypothesis that MRFs transactivate Tceal7 gene expression and promote muscle differentiation during muscle development and regeneration.

An unconventional myosin required for cell polarization and chemotaxis.

MyTH/FERM (myosin tail homology 4/band 4.1, ezrin, radixin, and moesin) myosins have roles in cellular adhesion, extension of actin-filled projections such as filopodia and stereocilia, and directional migration. The amoeba Dictyostelium discoideum expresses a simple complement of MyTH/FERM myosins, a class VII (M7) myosin required for cell-substrate adhesion and a unique myosin named MyoG. Mutants lacking MyoG exhibit a wide range of normal actin-based behaviors, including chemotaxis to folic acid, but have a striking defect in polarization and chemotaxis to cAMP. Although the myoG mutants respond to cAMP stimulation by increasing persistence and weakly increasing levels of cortical F-actin, they do not polarize; instead, they maintain a round shape and move slowly and randomly when exposed to a chemotactic gradient. The mutants also fail to activate and localize PI3K to the membrane closest to the source of chemoattractant. These data reveal a role for a MyTH/FERM myosin in mediating early chemotactic signaling and suggest that MyTH/FERM proteins have conserved roles in signaling and the generation of cell polarity.

Myogenic regulatory factors regulate M-cadherin expression by targeting its proximal promoter elements.

M- and N-cadherin are members of the Ca(2+)-dependent cell-cell adhesion molecule family. M-cadherin is expressed predominantly in developing skeletal muscles and has been implicated in terminal myogenic differentiation, particularly in myoblast fusion. N-cadherin-mediated cell-cell adhesion also plays an important role in skeletal myogenesis. In the present study, we found that both genes were differentially expressed in C2C12 and Sol8 myoblasts during myogenic differentiation and that the expression of M-cadherin was preferentially enhanced in slow-twitch muscle. Interestingly, most MRFs (myogenic regulatory factors) significantly activated the promoter of M-cadherin, but not that of N-cadherin. In line with this, overexpression of MyoD in C3H10T1/2 fibroblasts strongly induced endogenous M-cadherin expression. Promoter analysis in silico and in vitro identified an E-box (from -2 to +4) abutting the transcription initiation site within the M-cadherin promoter that is bound and differentially activated by different MRFs. The activation of the M-cadherin promoter by MRFs was also modulated by Bhlhe40 (basic helix-loop-helix family member e40). Finally, chromatin immunoprecipitation proved that MyoD as well as myogenin binds to the M-cadherin promoter in vivo. Taken together, these observations identify a molecular mechanism by which MRFs regulate M-cadherin expression directly to ensure the terminal differentiation of myoblasts.

S. macrurus myogenic regulatory factors (MRFs) induce mammalian skeletal muscle differentiation; evidence for functional conservation of MRFs.

The current-producing cells of the electric organ, i.e., electrocytes, in Sternopygus macrurus derive from skeletal muscle fibers. Mature electrocytes are not contractile, but they do retain some muscle proteins, are multinucleated, and receive cholinergic innervation. Electrocytes express the myogenic regulatory factors (MRFs) MyoD, myogenin, Myf5 and MRF4 despite their incomplete muscle phenotype. Although S. macrurus MRFs share functional domains which are highly conserved and their expression is confined to the myogenic lineage, their capability to induce the muscle phenotype has not been determined. To test the functional conservation of S. macrurus MRFs to transcriptionally activate skeletal muscle gene expression and induce the myogenic program, we transiently over-expressed S. macrurus MyoD (SmMyoD) and myogenin (SmMyoG) in mouse C3H/10T1/2 and NIH3T3 embryonic cells. RT-PCR and immunolabeling studies showed that SmMyoD and SmMyoG can efficiently convert these two cell lines into multinucleated myotubes which expressed differentiated muscle markers. The levels of myogenic induction by SmMyoD and SmMyoG were comparable to those obtained with mouse MRF homologs. Furthermore, SmMyoD and SmMyoG proteins were able to induce mouse MyoD and myogenin in C3H/10T1/2 cells. We conclude that S. macrurus MRFs are functionally conserved as they can transcriptionally activate skeletal muscle gene expression and induce the myogenic program in mammalian non-muscle cells. Hence, these data suggest that the partial muscle phenotype of electrocytes is not likely due to differences in the MRF-dependent transcriptional program between skeletal muscle and electric organ.

The transcription factor Six1a plays an essential role in the craniofacial myogenesis of zebrafish.

Transcription factor Six1a plays important roles in morphogenesis, organogenesis, and cell differentiation. However, the role of Six1a during zebrafish cranial muscle development is still unclear. Here, we demonstrated that Six1a was required for sternohyoideus, medial rectus, inferior rectus, and all pharyngeal arch muscle development. Although Six1a was also necessary for myod and myogenin expression in head muscles, it did not affect myf5 expression in cranial muscles that originate from head mesoderm. Overexpression of myod enabled embryos to rescue all the defects in cranial muscles induced by injection of six1a-morpholino (MO), suggesting that myod is directly downstream of six1a in controlling craniofacial myogenesis. However, overexpression of six1a was unable to rescue arch muscle defects in the tbx1- and myf5-morphants, suggesting that six1a is only involved in myogenic maintenance, not its initiation, during arch muscle myogenesis. Although the craniofacial muscle defects caused by pax3-MO phenocopied those induced by six1a-MO, injection of six1a, myod or myf5 mRNA did not rescue the cranial muscle defects in pax3 morphants, suggesting that six1a and pax3 do not function in the same regulatory network. Therefore, we proposed four putative regulatory pathways to understand how six1a distinctly interacts with either myf5 or myod during zebrafish craniofacial muscle development.

Myogenin regulates a distinct genetic program in adult muscle stem cells.

In contrast to the detailed understanding we have for the regulation of skeletal muscle gene expression in embryos, similar insights into postnatal muscle growth and regeneration are largely inferential or do not directly address gene regulatory mechanisms. Muscle stem cells (satellite cells) are chiefly responsible for providing new muscle during postnatal and adult life. The purpose of this study was to determine the role that the myogenic basic helix-loop-helix regulatory factor myogenin has in postnatal muscle growth and adult muscle stem cell gene expression. We found that myogenin is absolutely required for skeletal muscle development and survival until birth, but it is dispensable for postnatal life. However, Myog deletion after birth led to reduced body size implying a role for myogenin in regulating body homeostasis. Despite a lack of skeletal muscle defects in Myog-deleted mice during postnatal life and the efficient differentiation of cultured Myog-deleted adult muscle stem cells, the loss of myogenin profoundly altered the pattern of gene expression in cultured muscle stem cells and adult skeletal muscle. Remarkably, these changes in gene expression were distinct from those found in Myog-null embryonic skeletal muscle, indicating that myogenin has separate functions during postnatal life.

A highly conserved molecular switch binds MSY-3 to regulate myogenin repression in postnatal muscle.

Myogenin is the dominant transcriptional regulator of embryonic and fetal muscle differentiation and during maturation is profoundly down-regulated. We show that a highly conserved 17-bp DNA cis-acting sequence element located upstream of the myogenin promoter (myogHCE) is essential for postnatal repression of myogenin in transgenic animals. We present multiple lines of evidence supporting the idea that repression is mediated by the Y-box protein MSY-3. Electroporation in vivo shows that myogHCE and MSY-3 are required for postnatal repression. We further show that, in the C2C12 cell culture system, ectopic MSY-3 can repress differentiation, while reduced MSY-3 promotes premature differentiation. MSY-3 binds myogHCE simultaneously with the homeodomain protein Pbx in postnatal innervated muscle. We therefore propose a model in which the myogHCE motif operates as a switch by specifying opposing functions; one that was shown previously is regulated by MyoD and Pbx and it specifies a chromatin opening, gene-activating function at the time myoblasts begin to differentiate; the other includes MYS-3 and Pbx, and it specifies a repression function that operates during and after postnatal muscle maturation in vivo and in myoblasts before they begin to differentiate.

Reciprocal inhibition between Pax7 and muscle regulatory factors modulates myogenic cell fate determination.

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. Previously, we showed that Pax7 overexpression in adult primary myoblasts down-regulates MyoD and prevents myogenin induction, inhibiting myogenesis. We show 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. Our 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.