Transcription Factors Myogenin Research Articles

Functional interaction of the subunit 3 of RNA polymerase II (RPB3) with transcription factor-4 (ATF4)

RPB3 is a core subunit of RNA polymerase II (pol II) that, together with the RPB11 subunit, forms the heterodimer considered as a functional counterpart of the bacterial α subunit homodimer involved in promoter recognition. We previously employed the yeast two-hybrid system and identified an interaction between RPB3 and the myogenic transcription factor myogenin, demonstrating an involvement of this subunit in muscle differentiation. In this paper we report the interaction between RPB3 and another known transcription factor, ATF4. We found that the intensity of the interaction between RPB3 and ATF4 is similar to the one between RPB3 and myogenin. This interaction involves an RPB3 specific region not homologous to the prokaryotic α subunit. We demonstrated that RBP3 is able to enhance ATF4 transactivation, whereas the region of RPB3 (Sud) that contacts ATF4, when used as a dominant negative, markedly inhibits ATF4 transactivation activity. Interestingly, ATF4 protein level, as reported for its partner RPB3, increases during C2C7 cell line muscle differentiation.

The Interferon-inducible p202a Protein Modulates NF-κB Activity by Inhibiting the Binding to DNA of p50/p65 Heterodimers and p65 Homodimers While Enhancing the Binding of p50 Homodimers

p202a is a member of the interferon-inducible murine p200 family of proteins. These proteins share 1 or 2 partially conserved 200 amino acid segments of the a or the b type. The known biological activities of p202a include among others the regulation of muscle differentiation, cell proliferation, and apoptosis. These biological activities of p202a can be correlated with the inhibition of the activity of several transcription factors. Thus, the binding of p202a results in the inhibition of the sequence-specific binding to DNA of the c-Fos, c-Jun, E2F1, E2F4, MyoD, myogenin, and c-Myc transcription factors. This study concerns the mechanisms by which p202a inhibits the activity of NF-kappaB, a transcription factor involved among others in host defense, inflammation, immunity, and the apoptotic response. NF-kappaB consists of p50 and p65 subunits. We demonstrate that p202a can inhibit in vitro and in vivo the binding to DNA of p65 homodimers and p50/65 heterodimers, whereas it increases the binding of p50 homodimers. Thus p202a can impair NF-kappaB activity both by inhibiting the binding to DNA of the transcriptionally active p65 homodimers and p50/p65 heterodimers and by boosting the binding of the repressive p50 homodimers. p202a can bind p50 and p65 in vitro and in vivo, and p202a can be part of the p50 homodimer complex bound to DNA. p50 binds in p202a to the a type segment, whereas p65 binds to the b type segment. Transfected ectopic p202a increases the apoptotic effect of tumor necrosis factor (at least in part) by inhibiting NF-kappaB and its antiapoptotic activity.

EFFECTS OF ORAL CREATINE AND HEAVY RESISTANCE TRAINING ON MYOGENIC REGULATORY FACTOR AND CREATINE KINASE EXPRESSION

We have recently shown 12 wks of oral creatine (Cr) supplementation and heavy resistance training to increase myosin heavy chain mRNA and protein expression (MSSE, 33(10): 1674–1681, 2001). Using muscle samples from our previous study the purpose of this study was to further determine the effects of oral Cr and heavy resistance training on skeletal muscle creatine kinase (M-CK) mRNA expression and the mRNA and protein expression of the myogenic regulatory factors Myo-D, myogenin, MFR-4, and Myf5. Twenty-two untrained males were randomly assigned to either a control (CON), placebo (PLC), or Cr (CRT) group in a double blind fashion. Muscle biopsies were obtained before and after 12 wks of resistance training. PLC and CRT trained thrice weekly using 3 sets of 6–8 repetitions at 85%-90% 1-RM on the leg press, knee extension, and knee curl exercises. CRT ingested 6 g/d of Cr for 12 wks white PLC consumed the equal concentration of dextrose placebo. After training, M-CK mRNA expression, as well as myogenin and MRF-4 mRNA and protein expression, were found to be significantly greater for CRT compared to PLC and CON, while PLC was also significantly different from CON (P <0.05). For Myo-D mRNA and protein, both CRT and PLC were significantly different from CON (P <0.05) but CRT and PLC were not different from one another. No significant differences were located for Myf5 mRNA or protein (P >0.05). M-CK mRNA was correlated with myogenin and MRF-4 protein (P <0.05). Long-term Cr supplementation increases M-CK mRNA expression, likely due to concomitant increases in the protein expression of the transcription factors myogenin and MRF-4. The increase in myogenin and MRF-4 mRNA and protein may have also played a rote in the increases in myosin heavy chain expression that we have previously shown to occur with Cr supplementation. Supported in part by NutraSense, Inc., Shawnee Mission, KS

Repetitive stretch induces c- fos and myogenin mRNA within several hours in skeletal muscle removed from rats

Ikeda S, Yoshida A, Matayoshi S, Tanaka N. Repetitive stretch induces c- fos and myogenin mRNA within several hours in skeletal muscle removed from rats. Arch Phys Med Rehabil 2003;84:419-23. Objective: To investigate what effect repetitive stretch has on gene transcription in muscle tissues removed from rats. Design: Case-control study. Setting: University laboratory. Animals: Eight female 8-week-old Wistar rats. Interventions: Rat anterior tibial and soleus muscles were removed and repetitively stretched about 20% of the muscle length by using the Magnus method in Krebs-Ringer solution. Stretch-relax cycles 15 times per minute were continued for 2 hours in 1 group and 4 hours in another group. Contralateral muscles removed from the same rat were incubated without stretch as the control. Main Outcome Measures: After repetitive stretch, total ribonucleic acid (RNA) was extracted from the muscles and Northern blotting was carried out using c- fos and myogenin oligonucleotide as probes. Results: Repetitive stretch during 2 hours significantly increased the expression of c- fos messenger RNA (mRNA) in both the tibialis anterior and soleus muscles. Increased expression of myogenin mRNA in both muscles was observed with repetitive stretch for 4 hours. Because of the initial increase of immediate early gene c- fos mRNA and the subsequent increase of myogenic transcription factor myogenin mRNA, it appears that repetitive muscle stretch induces sequential progression of gene transcription toward muscle hypertrophy. Conclusions: Mechanical repetitive stretch for a few hours would be one of the stimulating factors for muscle growth and should be available for muscle training in clinical rehabilitation. [copy ] 2003 by the American Congress of Rehabilitation Medicine and the American Academy of Physical Medicine and Rehabilitation

Alteration of mesodermal cell differentiation by EWS/FLI-1, the oncogene implicated in Ewing's sarcoma.

The chimeric fusion gene EWS/FLI-1 is detected in most cases of Ewing's sarcoma (ES), the second most common malignant bone tumor of childhood. Although 80% of ES tumors develop in skeletal sites, the remainder can arise in almost any soft tissue location. The lineage of the cell developing the EWS/FLI-1 gene fusion has not been fully characterized but is generally considered to be of either mesenchymal or neural crest origin. To study this oncogene in a conceptually relevant target cell, EWS/FLI-1 was introduced into the murine cell line C2C12, a myoblast cell line capable of differentiation into muscle, bone, or fat. In this cellular context, EWS/FLI-1 profoundly inhibited the myogenic differentiation program. The block in C2C12 myogenic differentiation required the nuclear localization and DNA-binding functions of EWS/FLI-1 and was mediated by transcriptional and posttranscriptional suppression of the myogenic transcription factors MyoD and myogenin. Interestingly, C2C12-EWS/FLI-1 cells constitutively expressed alkaline phosphatase, a bone lineage marker, and were alkaline phosphatase positive by histochemistry but showed no other evidence of bone lineage commitment. Consistent with recent findings in human ES tumor cell lines, C2C12-EWS/FLI-1 cells constitutively expressed cyclin D1 and demonstrated decreased expression of the cell cycle regulator p21(cip1), even under differentiation conditions and at confluent density. This C2C12-EWS/FLI-1 cell model may assist in the identification of novel differentially expressed genes relevant to ES and provide further insight into the cell(s) of origin developing ES-associated genetic fusions.

The alpha-like RNA polymerase II core subunit 3 (RPB3) is involved in tissue-specific transcription and muscle differentiation via interaction with the myogenic factor myogenin.

RNA polymerase II core subunit 3 (RPB3) is an a-like core subunit of RNA polymerase II (pol II). It is selectively down-regulated upon treatment with doxorubicin (dox). Due to the failure of skeletal muscle cells to differentiate when exposed to dox, we hypothesized that RPB3 is involved in muscle differentiation. To this end, we have isolated human muscle RPB3-interacting proteins by using yeast two-hybrid screening. It is of interest that an interaction between RPB3 and the myogenic transcription factor myogenin was identified. This interaction involves a specific region of RPB3 protein that is not homologous to the prokaryotic a subunit. Although RPB3 contacts the basic helix-loop-helix (HLH) region of myogenin, it does not bind other HLH myogenic factors such as MyoD, Myf5, and MRF4. Coimmunoprecipitation experiments indicate that myogenin contacts the pol II complex and that the RPB3 subunit is responsible for this interaction. We show that RPB3 expression is regulated during muscle differentiation. Exogenous expression of RPB3 slightly promotes myogenin transactivation activity and muscle differentiation, whereas the region of RPB3 that contacts myogenin, when used as a dominant negative molecule (Sud), counteracts these effects. These results indicate for the first time that the RPB3 pol II subunit is involved in the regulation of tissue-specific transcription.

Selective repression of myoD transcription by v-Myc prevents terminal differentiation of quail embryo myoblasts transformed by the MC29 strain of avian myelocytomatosis virus.

We have investigated the mechanism by which expression of the v-myc oncogene interferes with the competence of primary quail myoblasts to undergo terminal differentiation. Previous studies have established that quail myoblasts transformed by myc oncogenes are severely impaired in the accumulation of mRNAs encoding the myogenic transcription factors Myf-5, MyoD and Myogenin. However, the mechanism responsible for such a repression remains largely unknown. Here we present evidence that v-Myc selectively interferes with quail myoD expression at the transcriptional level. Cis-regulatory elements involved in the auto-activation of qmyoD are specifically targeted in this unique example of transrepression by v-Myc, without the apparent participation of Myc-specific E-boxes or InR sequences. Transiently expressed v-Myc efficiently interfered with MyoD-dependent transactivation of the qmyoD regulatory elements, while the myogenin promoter was unaffected. Finally, we show that forced expression of MyoD in v-myc-transformed quail myoblasts restored myogenin expression and promoted extensive terminal differentiation. These data suggest that transcriptional repression of qmyoD is a major and rate-limiting step in the molecular pathway by which v-Myc severely inhibits terminal differentiation in myogenic cells.

Identification of new genes related to the myogenic differentiation arrest of human rhabdomyosarcoma cells

Rhabdomyosarcoma is a soft tissue tumor committed to the myogenic lineage but arrested prior to terminal differentiation. To identify new genes implicated in the block in myogenic differentiation of rhabdomyosarcoma cells and those responsible for their proceeding along the myogenic pathway we used cDNA microarrays to compare gene expression profiles of two clones of the human embryonal rhabdomyosarcoma cell line RD with different myogenic potentials: RD/12, which is unable to differentiate, and RD/18, which shows elements able to terminally differentiate, as defined by the expression of myosin heavy chain (up to 50% of the population) and the formation of multinucleated myotube-like structures. We identified 80 genes differentially expressed by the two clones. Differentiating RD/18 cells overexpressed the myogenic transcription factor myogenin along with known myogenic markers; myogenin transfection into undifferentiated RD/12 cells was able to revert the phenotype giving rise to 94% of clones displaying a differentiated morphology. RD/18 cells also expressed several genes not known to be expressed in rhabdomyosarcoma or muscle cells, such as pigment-epithelium derived factor and endothelin-3. Poorly differentiated RD/12 cells, along with genes related to mesenchymal lineage or early myogenic commitment, also expressed genes not previously known to be related to the differentiation block of human rhabdomyosarcoma, such as monocyte chemotactic protein-1, connective tissue growth factor and insulin-like growth factor binding protein-5. Differential expression of these genes in a time course of differentiation suggested their potential roles as either new myogenic markers or repressors of differentiation. These results identify a cluster of new genes related to the aberrant myogenic differentiation program of human rhabdomyosarcoma cells.

Critical Proliferation-independent Window for Basic Fibroblast Growth Factor Repression of Myogenesis via the p42/p44 MAPK Signaling Pathway

In many cell types including myoblasts, growth factors control proliferation and differentiation, in part, via the mitogen-activated protein kinase (MAPK) pathway (also known as the extracellular regulated kinase (Erk) pathway). In C2C12 myoblast cells, insulin-like growth factor-1 and basic fibroblast growth factor (bFGF) activate MAPK/Erk, and both growth factors promote myoblast proliferation. However, these factors have opposing roles with respect to differentiation; insulin-like growth factor-1 enhances muscle cell differentiation, whereas bFGF inhibits the expression of the muscle-specific transcription factors MyoD and myogenin. Cells treated with bFGF and PD98059, a specific inhibitor of the MAPK pathway, show enhanced expression of the muscle-specific transcription factors MyoD and myogenin as compared with cells not exposed to this inhibitor. Inhibiting MAPK activity also enhances myoblast fusion and the expression of the late differentiation marker myosin heavy chain. Basic FGF mediated repression of muscle-specific genes does not result from continued cell proliferation, since bFGF-treated cells progress through only one round of cell division. We have identified a critical boundary 16 to 20 h after plating during which bFGF induced MAPK activity is able to repress myogenic gene expression and differentiation. Thus, the targets of MAPK that regulate myogenesis are functional at this time and their identification is in progress.

The Basic Helix-Loop-Helix Transcription Factors Myogenin and Id2 Mediate Specific Induction of Caveolin-3 Gene Expression during Embryonic Development

Caveolin-3 protein is the only member of the caveolin family that shows a unique muscle-specific expression pattern, and loss of its functional activity causes muscular dystrophy. Caveolin-3 mRNA levels are dramatically increased during the formation of myotubes in the C2C12 cell line. In this study, we characterized the human caveolin-3 5'-flanking region. Promoter analyses demonstrate that the proximal E box element serves as a myogenin binding site and is both necessary and sufficient to control caveolin-3 gene transcription. Transient transfection assays indicated that overexpression of myogenin activates caveolin-3 reporter gene expression, whereas Id2 overexpression inhibited caveolin-3 promoter activation by myogenin. A mutant Id2 protein lacking the HLH domain was not capable of suppressing myogenin-mediated activation. Determination of caveolin-3 transcript distribution patterns in vivo revealed that mRNA was first detectable at day 10 of gestation in the developing somites and heart. Caveolin-3 protein in myoblasts and myotubes was expressed in both the plasma membrane and vesicular structures. During skeletal myogenesis the level of Id2, an inhibitor of differentiation, decreases, allowing the induced basic helix-loop-helix transcription factor myogenin to form transcriptionally active heterodimers that bind to the caveolin-3 promoter and thereby mediate its transcription.

Pulmonary hypoplasia in the myogenin null mouse embryo.

Although fetal breathing movements are required for normal lung development, there is uncertainty concerning the specific effect of absent fetal breathing movements on pulmonary cell maturation. We set out to evaluate pulmonary development in a genetically defined mouse model, the myogenin null mouse, in which there is a lack of normal skeletal muscle fibers and thus skeletal muscle movements are absent in utero. Significant decreases were observed in lung:body weight ratio and lung total DNA at embryonic days (E)14, E17, and E20. Reverse transcriptase/polymerase chain reaction, in situ immunofluorescence, and electron microscopy revealed early lung cell differentiation in both null and wild-type lungs as early as E14. However at E14, myogenin null lungs had decreased 5'-bromo-2-deoxyuridine incorporation compared with that of wild-type littermates, whereas at E17 and E20, increased Bax immunolabeling and terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick-end labeling staining were detected in the myogenin null mice but not in the wild-type littermates. These observations highlight the importance of skeletal muscle contractile activity in utero for normal lung organogenesis. Null mice lacking the muscle-specific transcription factor myogenin exhibit a secondary effect on lung development such that decreased lung cell proliferation and increased programmed cell death are associated with lung hypoplasia.

Helix-loop-helix transcription factors in electrically active and inactive skeletal muscles.

The muscle-specific helix-loop-helix (HLH) transcription factors myoD, myogenin, MRF4, and myf-5 are called the muscle regulatory factor family (MRF). Levels of MRFs are strongly regulated by muscle electrical activity and are thought to control downstream genes that are important for muscle phenotype such as the acetylcholine receptor (AChR) and possibly genes connected to muscle metabolic properties. The MRFs interact with ubiquitously expressed HLH factors such as E-proteins and Id-proteins to form heterodimers. In the present paper, we report the effects of paralysis obtained by nerve impulse block with tetrodotoxin (TTX) and denervation on messenger ribonucleic acid (mRNA) levels for Id-1, E47, myogenin, AChR alpha-subunit and beta-actin. Both Id-1 and E47 showed twofold increases in absence of nerve evoked electrical activity. These changes in the ubiquitously expressed HLH factors might have important functional implications for downstream gene expression, but in comparison, myogenin mRNA was increased 10-fold. We conclude that myogenin and the other muscle-specific MRFs remain the transcription factors with the strongest activity dependence that has so far been described in muscle.

Expression of a PKR Dominant-Negative Mutant in Myogenic Cells Interferes with the Myogenic Process

In this study, we explored the possibility that PKR, a dsRNA-activated regulatory protein, is an essential component in the differentiation program of myogenic cells in vitro. For this purpose, we used a retroviral expression vector pMV7, harboring the PKR dominant-negative mutant PKRΔ6 (pMV7-p68Δ6). Murine C2C12 myogenic cells were transfected either with pMV7 or with pMV7-p68Δ6. Neomycin-resistant clones from both types were isolated and expanded and the results obtained with the representative clones C2-NEO (transfected with pMV7) and clone 17 and clone 22 (both transfected with pMV7-p68Δ6) are presented. In clone 17 and 22 cells, regardless of IFN treatment, a similar level of the transfected human p68 PKR mutant was detected. This protein was absent in C2-NEO cells. In parallel, in all types of cells, a low basal level of the endogenous murine p65 PKR protein was observed, which was further induced by IFN. However, PKR enzymatic activity was significantly induced by IFN only in C2-NEO cells, while it was hardly detected in both clones 17 and 22, even after IFN treatment. Furthermore, in contrast to C2-NEO cells, only a slight to moderate increase in enzymatic activity was observed in clone 17 and 22 differentiating cells. Next, cells were grown either in growth medium (GM) or differentiation medium (DM), and the progression of the myogenic program was studied. An inhibition in myotube formation in clone 17 versus C2-NEO cells cultivated in DM was clearly observed. Furthermore, while the growth rate and thymidine incorporation were reduced in C2-NEO cells grown in DM, both clone 17 and 22 cells were less affected under the same conditions. Similarly, a delay in the accumulation of the transcription factors MyoD and myogenin, as well as in creatine kinase activity and accumulation of troponin T, was detected in DM-cultivated clone 17 and clone 22 cells. Moreover, a delay in the induction of p21 (WAF1), in down-regulation of cyclin D1 and c-myc, and in the accumulation of the underphosphorylated form of pRb was also observed in clone 17 cells. We conclude that inhibition of endogenous PKR activity by a PKR dominant-negative mutant interferes with the myogenic program of murine C2C12 myogenic cells.

Involvement of type 4 cAMP-phosphodiesterase in the myogenic differentiation of L6 cells.

Myogenic cell differentiation is induced by Arg(8)-vasopressin, whereas high cAMP levels and protein kinase A (PKA) activity inhibit myogenesis. We investigated the role of type 4 phosphodiesterase (PDE4) during L6-C5 myoblast differentiation. Selective PDE4 inhibition resulted in suppression of differentiation induced by vasopressin. PDE4 inhibition prevented vasopressin-induced nuclear translocation of the muscle-specific transcription factor myogenin without affecting its overall expression level. The effects of PDE4 inhibition could be attributed to an increase of cAMP levels and PKA activity. RNase protection, reverse transcriptase PCR, immunoprecipitation, Western blot, and enzyme activity assays demonstrated that the PDE4D3 isoform is the major PDE4 expressed in L6-C5 myoblasts and myotubes, accounting for 75% of total cAMP-hydrolyzing activity. Vasopressin cell stimulation caused a biphasic increase of PDE4 activity, which peaked at 2 and 15 min and remained elevated for 48 h. In the continuous presence of vasopressin, cAMP levels and PKA activity were lowered. PDE4D3 overexpression increased spontaneous and vasopressin-dependent differentiation of L6-C5 cells. These results show that PDE4D3 plays a key role in the control of cAMP levels and differentiation of L6-C5 cells. Through the modulation of PDE4 activity, vasopressin inhibits the cAMP signal transduction pathway, which regulates myogenesis possibly by controlling the subcellular localization of myogenin.

CDNA microarrays detect activation of a myogenic transcription program by the PAX3-FKHR fusion oncogene.

Alveolar rhabdomyosarcoma is an aggressive pediatric cancer of striated muscle characterized in 60% of cases by a t(2;13)(q35;q14). This results in the fusion of PAX3, a developmental transcription factor required for limb myogenesis, with FKHR, a member of the forkhead family of transcription factors. The resultant PAX3-FKHR gene possesses transforming properties; however, the effects of this chimeric oncogene on gene expression are largely unknown. To investigate the actions of these transcription factors, both Pax3 and PAX3-FKHR were introduced into NIH 3T3 cells, and the resultant gene expression changes were analyzed with a murine cDNA microarray containing 2,225 elements. We found that PAX3-FKHR but not PAX3 activated a myogenic transcription program including the induction of transcription factors MyoD, Myogenin, Six1, and Slug as well as a battery of genes involved in several aspects of muscle function. Notable among this group were the growth factor gene Igf2 and its binding protein Igfbp5. Relevance of this model was suggested by verification that three of these genes (IGFBP5, HSIX1, and Slug) were also expressed in alveolar rhabdomyosarcoma cell lines. This study utilizes cDNA microarrays to elucidate the pattern of gene expression induced by an oncogenic transcription factor and demonstrates the profound myogenic properties of PAX3-FKHR in NIH 3T3 cells.

Transcriptional Activation of the Myogenin Gene by MEF2-Mediated Recruitment of Myf5 Is Inhibited by Adenovirus E1A Protein

The basic helix-loop-helix (bHLH) transcription factor myogenin plays a crucial role in terminal differentiation of committed myoblasts into mature myocytes. Transcriptional activation of the myogenin gene requires coordinate action of myocyte enhancer factor 2 (MEF2) proteins and the myogenic bHLH regulators, MyoD or Myf5. Here we show that transcription of the myogenin gene in differentiated cells correlates with MEF2 and NF1 binding to their cognate sites in the proximal myogenin promoter but not with binding of Myf5 or MyoD to the E-box. The importance of MEF2 activity was further demonstrated by expression of antisense MEF2 RNA which repressed MEF2 and Myf5-mediated MEF2 site-dependent reporter gene activation and the synergistic transactivation of a myogenin CAT reporter by Myf5 and MEF2. Adenovirus E1A which has previously been shown to specifically interfere with myogenin gene transcription also inhibited the cooperative transactivation by Myf5/MEF2 and MEF2. Consistently, coimmunoprecipitation studies revealed impaired MEF2/Myf5 protein-protein interactions. These results support a model of transcriptional activation and stabilization of myogenin expression in which DNA-bound MEF2 recruits myogenic bHLH factors into an active but E1A-sensitive transcription factor complex.

The MADS domain containing transcription factor cMef2a is expressed in heart and skeletal muscle during embryonic chick development.

Muscle enhancer factor 2 (MEF2) proteins are important transcription factors for muscle-specific gene activation. Four family members are known in mammals, referred to as MEF2A, MEF2B, MEF2C, and MEF2D. Here we report the isolation and expression pattern of the chick Mef2a gene (cMef2a). cMef2a expression starts in precardiac mesoderm of HH stage 8 embryos. During further embryonic development expression continues in the heart tube and later in atrium and ventricle. A second cMef2a expression domain appears in somites of stage 13 embryos. Somitic cMef2a expression is limited to the myotome and is not found in newly formed somites until the muscle-specific transcription factors MyoD and myogenin are present. This suggests that activation of the cMef2a gene in skeletal muscle is dependent on these basic helix-loop-helix transcription factors. cMef2a expression in heart and skeletal muscle continues into adulthood when it is also seen in intestinal mesenchyme and in brain.

Cloning of Novel Injury-regulated Genes: IMPLICATIONS FOR AN IMPORTANT ROLE OF THE MUSCLE-SPECIFIC PROTEIN skNAC IN MUSCLE REPAIR

To gain insight into the molecular mechanisms underlying the wound repair process, we searched for genes that are regulated by skin injury. Using the differential display reverse transcription-polymerase chain reaction technique, we identified a gene that was strongly induced as early as 12 h after wounding. Sequence analysis revealed the identity of the corresponding protein with skeletal muscle nascent polypeptide-associated complex (skNAC), a recently identified muscle-specific transcription factor. By in situ hybridization and immunohistochemistry, we demonstrated the specific expression of skNAC in skeletal muscle cells of the panniculus carnosus at the wound edge. Furthermore, in vitro studies with cultured myoblasts revealed expression of skNAC in differentiating and differentiated, but not in proliferating, nondifferentiated cells. Differentiation of cultured myoblasts was accompanied by simultaneous expression of skNAC and the muscle-specific transcription factor myogenin. Our results provide the first evidence for a role of skNAC in muscle repair processes. Furthermore, they demonstrate the usefulness of our approach in identifying new players in wound repair.

A HypomorphicMyogeninAllele Reveals DistinctMyogeninExpression Levels Required for Viability, Skeletal Muscle Development, and Sternum Formation

The myogenic basic helix-loop-helix transcription factor myogenin plays an essential role in the differentiation of skeletal muscle and, secondarily, in rib and sternum formation during mouse development. However, virtually nothing is known about the quantitative requirements for myogenin in these processes. Here, we describe the generation of mice carrying a hypomorphic allele ofmyogenin,which expressesmyogenintranscripts at approximately one-fourth the level of the wild-typemyogeninallele. The hypomorphic allele in combination with wild-type andmyogenin-null alleles was used to create an allelic series. Embryos representing the complete range of genotypes from homozygous wild type to homozygous null were analyzed for their viability, ability to form normal ribs and sternum, and extent of skeletal muscle differentiation. Embryos carrying the hypomorphicmyogeninallele over a wild-type allele were normal. In embryos bearing homozygous hypomorphic alleles, the sternum developed normally and extensive skeletal muscle differentiation occurred. However, muscle hypoplasia and reduced muscle-specific gene expression were apparent in these embryos, and the mice were not viable as neonates. When the hypomorphic allele was placed over amyogenin-null allele, the resulting embryos had sternum defects resembling homozygousmyogenin-null embryos, and there was severe muscle hypoplasia. Our results demonstrate that skeletal muscle formation is highly sensitive to the absolute levels of myogenin and that correct sternum formation, skeletal muscle differentiation, and viability each require distinct threshold levels of myogenin.

P21(CIP1) and p57(KIP2) control muscle differentiation at the myogenin step.

Cell-cycle arrest is thought to be required for differentiation of muscle cells. However, the molecules controlling cell-cycle exit and the differentiation step(s) dependent on cell-cycle arrest are poorly understood. Here we show that two Cdk inhibitors, p21(CIP1) and p57(KIP2), redundantly control differentiation of skeletal muscle and alveoli in the lungs. Mice lacking both p21 and p57 fail to form myotubes, display increased proliferation and apoptotic rates of myoblasts, and display endoreplication in residual myotubes. This point of arrest during muscle development is identical to that of mice lacking the myogenic transcription factor myogenin, indicating a role for cell-cycle exit in myogenin function. Expression of myogenin, p21, and p57 is parallel but independent, and in response to differentiation signals, these proteins are coordinately regulated to trigger both cell-cycle exit and a dependent muscle-specific program of gene expression to initiate myoblast terminal differentiation and muscle formation.