Nonmyogenic Cells Research Articles

Laminin α4 and Integrin α6 Are Upregulated in Regenerating dy/dy Skeletal Muscle: Comparative Expression of Laminin and Integrin Isoforms in Muscles Regenerating after Crush Injury

The expression of laminin isoforms and laminin-binding integrin receptors known to occur in muscle was investigated during myogenic regeneration after crush injury. Comparisons were made between dystrophic 129ReJ dy/dy mice, which have reduced laminin α2 expression, and their normal littermates. The overall histological pattern of regeneration after crush injury was similar in dy/dy and control muscle, but proceeded faster in dy/dy mice. In vitro studies revealed a greater yield of mononuclear cells extracted from dy/dy muscle and a reduced proportion of desmin-positive cells upon in vitro cultivation, reflecting the presence of inflammatory cells and “preactivated” myoblasts due to ongoing regenerative processes within the endogenous dystrophic lesions. Laminin α1 was not detectable in skeletal muscle. Laminin α2 was present in basement membranes of mature myofibers and newly formed myotubes in control and dy/dy muscles, albeit weaker in dy/dy. Laminin α2-negative myogenic cells were detected in dy/dy and control muscle, suggesting the involvement of other laminin α chains in early myogenic differentiation, such as laminin α4 and α5 which were both transiently expressed in basement membranes of newly formed myotubes of dy/dy and control mice. Integrin β1 was expressed on endothelial cells, muscle fibers, and peripheral nerves in uninjured muscle and broadened after crush injury to the interstitium where it occurred on myogenic and nonmyogenic cells. Integrin α3 was not expressed in uninjured or regenerating muscle, while integrin α6 was expressed mainly on endothelial cells and peripheral nerves in uninjured muscle. Upon crush injury integrin α6 increased in the interstitium mainly on nonmyogenic cells, including infiltrating leukocytes, endothelial cells, and fibroblasts. In dy/dy muscle, integrin α6 occurred on some newly formed myotubes. Integrin α7 was expressed on muscle fibers at the myotendinous junction and showed weak and irregular expression on muscle fibers. After crush injury, integrin α7 expression extended to the newly formed myotubes and some myoblasts. However, many myoblasts and newly formed myotubes were integrin α7 negative. No marked difference was observed in integrin α7 expression between dy/dy and control muscle, either uninjured or after crush injury. Only laminin α4 and integrin α6 expression patterns were notably different between dy/dy and control muscle. Expression of both molecules was more extensive in dy/dy muscle, especially in the interstitium of regenerating areas and on newly formed myotubes. In view of the faster myogenic regeneration observed in dy/dy mice, the data suggest that laminin α4 and integrin α6 support myogenic regeneration. However, whether these accelerated myogenic effects are a direct consequence of the reduced laminin α2 expression in dy/dy mice, or an accentuation of the ongoing regenerative events in focal lesions in the muscle, requires further investigation.

Expression of Myostatin Gene in Regenerating Skeletal Muscle of the Rat and Its Localization

The expression of myostatin mRNA was examined in regenerating skeletal muscle of the rat. Skeletal muscle regeneration was induced by injecting bupivacaine or hypertonic saline solution into the femoral muscle, and the tissues were collected 48 h after the treatment. In situ hybridization analysis revealed that the cells positive for myostatin message were localized in the regenerating area of the bupivacaine-treated tissues, where a numerous number of mononucleated cells were present. The myostatin-positive mononucleated cells contained both myogenic and nonmyogenic cells, as revealed by immunohistochemical staining for desmin and vimentin. Bupivacaine treatment to the testes resulted in no myostatin message expression in the testicular vimentin-positive cells, suggesting that the expression of myostatin message in vimentin-positive cells is a skeletal muscle-specific phenomenon. Furthermore, crushed muscle extract prepared from regenerating skeletal muscle had induced myostatin mRNA expression in skeletal muscle-derived fibroblasts in a dose-dependent manner. These results indicated that myostatin is expressed during skeletal muscle regeneration both in myogenic and nonmyogenic cells, and suggested that some factor(s) capable of inducing myostatin expression in fibroblasts are present in regenerating skeletal muscle.

Rapid purification of transfected porcine muscle cells.

A detailed methodology is described for fluorescence-activated cell sorting (FACS) of porcine muscle cells that have been transfected to express green fluorescent protein (GFP). Cells are liberated from porcine skeletal muscle and primary cultures are transfected with DNA encoding GFP. Primary cultures are subjected to immunocytochemistry using a primary muscle-specific monoclonal antibody followed by incubation with a phycoerythrin-conjugated second antibody. Transfected myoblasts are sorted from fibroblasts using forward angle light scatter and ninety degree light scatter, phycoerythrin fluorescence, and GFP fluorescence. These procedures allow for isolation of genetically- engineered porcine muscle cells more rapidly than traditional clonal selection procedures. Consequently, FACS provides porcine myoblast populations that retain the majority of their replicative capacity and are not contaminated with non-myogenic cells.

Identification of skeletal muscle satellite cells by transfecting EGFP driven by skeletal alpha-actin promoter.

In isolating skeletal muscle satellite cells, sometimes a problem is encountered in removing contaminating nonmyogenic cells. In the present study, we constructed a novel vector, pSKA-EGFP, which achieves the expression of enhanced green fluorescent protein (EGFP) exclusively in myogenic cells under the control of skeletal alpha-actin promoter when transfected to primary cultured cells from skeletal muscle. Cells from rat skeletal muscle positive for EGFP after transfecting with pSKA-EGFP were all positive for desmin and none of the nonmyogenic cells expressed EGFP, indicating that the expression of EGFP is specific to myogenic cells. Among the cells positive for EGFP were proliferating cells, presumably satellite cells. In addition, EGFP positive cells derived from horse skeletal muscle after transfecting pSKA-EGFP in vitro formed multinuclear myotubes, indicating that myogenic expression of EGFP driven by skeletal alpha-actin was achieved also in the equine cells. These results indicated that pSKA-EGFP vector will be useful in identifying and following up the satellite cells in real time, and also permit us to isolate satellite cells in combination with fluorescence-activated cell sorting (FACS).

Expression of the sea urchin MyoD homologue, SUM1, is not restricted to the myogenic lineage during embryogenesis

SUM1 (sea urchin myogenic factor 1) is a sea urchin homologue of the myogenic basic helix-loop-helix transcription factors of the MyoD family. SUM1 was initially cloned from Lytechinus variegatus where immunocytochemistry demonstrated restricted expression in precursors of the circumesophageal muscles, the only identified muscle cells in the early embryo. Subsequent in situ hybridization analysis indicates that SUM1 embryonic expression is not restricted to the myogenic lineage; a distinct population of nonmyogenic cells also expresses SUM1. For comparative purposes, we cloned the SUM1 orthologue in the distantly related sea urchin, Strongylocentrotus purpuratus, where we found SpSUM1 transcripts in the same population of nonmyogenic cells.

Dystrophy and myogenesis in mdx diaphragm muscle.

In order to determine why the diaphragm is more severely affected by progressive dystrophy than limb muscles in the mdx mouse, we examined how regional variations in diaphragm dystrophy, the measures of disease and repair, proliferation by committed myogenic cells, and the expression of mitogenic basic fibroblast growth factor (bFGF) could contribute to muscle-specific disease phenotypes. There were regional variations in new myotube formation in the diaphragm, with disease more severe in crural than costal leaflets. New repair increased in hyperthyroidism without changes in accumulated repair, probably due to fiber loss. General proliferation was nearly twofold higher in limb than diaphragm mononuclear cells. Since only 2.5-8.4% of committed muscle precursors were proliferating, the higher proliferation by myf5+ myogenic cells in diaphragm did not account for muscle-specific differences. Proliferation by bFGF+ mononuclear cells and an immunogold labeling index for bFGF protein in diaphragm myoblasts were lower in diaphragm than limb muscle. In culture, mixed limb myoblast and fibroblasts contained more S phase cells than diaphragm cells, although myoblasts cycled similarly between muscles. Therefore while muscle architecture and the formation and number of new myotubes certainly affect disease phenotype, the differential outcome of regeneration in mdx diaphragm and limb muscle appears to be contributed by both nonmyogenic and myogenic cells.

Skeletal and Cardiac α-Actin Isoforms Exhibit Unanticipated Temporal and Tissue-Specific Gene Expression Patterns in Developing Avian Limbs and Embryos

The initial expression of skeletal muscle structural genes typically occurs after myogenic determination factor gene expression. We investigated this temporal relationship via a reverse transcription–polymerase chain reaction (RT–PCR) analysis of skeletal and cardiac α-actin (s- and c-actin) mRNA during chick limb development. c-actin transcripts were first detected at the beginning of muscle cytodifferentiation in stage 24/25 limbs, shortly after the initial appearance of MyoD and myogenin mRNAs, and were not detected in nonmyogenic tissues. In contrast, s-actin mRNA was detected in limbs at stage 15–16, periods when myf5 and MRF4 but not MyoD and myogenin transcripts are present (Lin-Jones, J., and Hauschka, S. D.,Dev. Biol.174, 407–422, 1996). While s-actin mRNA was not detected in E7 neural retina and was at variable levels in stage 9–15 neural tube, significant levels were consistently detected in mesodermal tissues which contribute nonmyogenic cells to the limb: stage 9–12 lateral plate and distal portions of stage 25/26 limbs. s-actin transcripts detected in the earliest limbs could thus be in myogenic and/or nonmyogenic cells. These data indicate that while c-actin expression is activated at the onset of limb muscle cytodifferentiation, s-actin expression occurs much earlier, as well as in some nonmyogenic tissues. Whether the precocious expression of s-actin plays a functional role in muscle development remains to be determined.

Evidence for multiple satellite cell populations and a non-myogenic cell type that is regulated differently in regenerating and growing skeletal muscle

We have performed studies to determine if different populations of satellite cells provide nuclei to growing and regenerating skeletal muscle fibers. Satellite cells were isolated from regenerating or growing anterior tibialis muscles, and their phenotypic properties were compared in vitro. Isolates from regenerating muscle contained 31% satellite cells, and those from control muscle contained 66% satellite cells, as determined by their expression of desmin. Among the desmin-positive satellite cells present from each preparation, two distinct populations of satellite cells were evident. Approximately 28% of satellite cell colonies were composed of only large cells, contained less than 50 cells/colony, and were designated as type 1 colonies. The remainder of satellite cell colonies isolated from either regenerating or control muscles were primarily composed of small cells, contained from 60 to 150 cells/colony, and were designated as type 2 colonies. Despite dramatic differences in the ratio of myogenic to non-myogenic cell types, satellite cells from regenerating and control muscles formed myotubes and expressed myosin heavy chain at similar levels. Treatment of regenerating cultures with dexamethasone resulted in a 16% increase in the number of desminpositive colonies and dramatically decreased the proliferation of non-myogenic cells. These results suggest that at least two distinct populations of satellite cells can be isolated from regenerating and control skeletal muscles, and that non-myogenic cells are differentially regulated in regenerating versus non-regenerating environments.

Early expression of the different isoforms of the myocyte enhancer factor-2 (MEF2) protein in myogenic as well as non-myogenic cell lineages during mouse embryogenesis

MEF2 proteins are a family of transcription factors that have muscle-specific DNA binding activity and bind to conserved A/T rich elements in the regulatory regions of numerous muscle-specific genes. They are thought have an important role in the development and differentiation of skeletal muscle. Recent in situ hybridization studies using mouse MEF2 probes have shown that MEF2 gene transcripts are detected very early in development at high levels in the myogenic cells of the myotome and embryonic heart. However, MEF2A and MEF2D transcripts have been detected in many adult tissues where they are not translated or the corresponding proteins are rapidly degraded. Therefore, it is important to establish the temporal and spatial correlation between MEF2 RNA and protein expression. In the present study we have performed in situ immunohistochemistry of whole mount mouse embryos at different stages of development using polyclonal antibodies specific for the MEF2A, MEF2C and MEF2D isoforms. At day 8.5 of development, all three MEF2 isoforms are expressed in the heart. MEF2A and C are detected in a few cells in the rostral-most somite by day 9 of development. Their expression then proceeds in a rostro-caudal direction concurrent with somite maturation. The pattern of expression of the MEF2D isoform is similar to that of MEF2C but the amount detected is much lower. Interestingly, MEF2A is also detected as early as day 8.5 p.c. in cells of the embryonic vasculature and non-myogenic cells. These results demonstrate that MEF2 proteins are detected early in development in the somites and heart, thus supporting their importance in the early stages of the hierarchical cascade of myogenesis. Their presence in non-muscle cells further suggests they could also play a role in the determination of other mesodermal derivatives, including cells of the vasculature.

Correlation of Terminal Cell Cycle Arrest of Skeletal Muscle with Induction of p21 by MyoD

Skeletal muscle differentiation entails the coordination of muscle-specific gene expression and terminal withdrawal from the cell cycle. This cell cycle arrest in the G0 phase requires the retinoblastoma tumor suppressor protein (Rb). The function of Rb is negatively regulated by cyclin-dependent kinases (Cdks), which are controlled by Cdk inhibitors. Expression of MyoD, a skeletal muscle-specific transcriptional regulator, activated the expression of the Cdk inhibitor p21 during differentiation of murine myocytes and in nonmyogenic cells. MyoD-mediated induction of p21 did not require the tumor suppressor protein p53 and correlated with cell cycle withdrawal. Thus, MyoD may induce terminal cell cycle arrest during skeletal muscle differentiation by increasing the expression of p21.

Dermal fibroblasts convert to a myogenic lineage in mdx mouse muscle.

Duchenne muscular dystrophy is a primary muscle disease that manifests itself in young boys as a result of a defect in a gene located on the X-chromosome. This gene codes for dystrophin, a normal muscle protein that is located beneath the sarcolemma of muscle fibres. Therapies to alleviate this disease have centred on implanting normal muscle precursor cells into dystrophic fibres to compensate for the lack of this gene and its product. To date, donor cells for implantation in such therapy have been of myogenic origin, derived from paternal biopsies. Success in human muscle, however, has been limited and may reflect immune rejection problems. To overcome this problem the patient's own myogenic cells, with the dystrophin gene inserted, could be used, but this could lead to other problems, since these cells are those that are functionally compromised by the disease. Here, we report the presence of high numbers of dystrophin-positive fibres after implanting dermal fibroblasts from normal mice into the muscle of the mdx mouse-the genetic homologue of Duchenne muscular dystrophy. Dystrophin-positive fibres were also abundant in mdx muscle following the implantation of cloned dermal fibroblasts from the normal mouse. Our results suggest the in vivo conversion of these non-myogenic cells to the myogenic pathway resulting in the formation of dystrophin-positive muscle fibres in the deficient host. The use of dermal fibroblasts may provide an alternative approach to the previously attempted myoblast transfer therapy, which in human trials has yielded disappointing results.

Desmin sequence elements regulating skeletal muscle-specific expression in transgenic mice.

During the development of the mouse embryo, desmin is one of the first muscle proteins detected in both the heart and the somites. The expression of the desmin gene differs from most other muscle genes, since it is initiated in replicating myoblasts and accumulates as the muscle differentiates. We have characterized a muscle-specific enhancer which directs the expression of desmin in vitro in the myoblasts and myotubes of C2 cells but not in non-myogenic cells. We report here on the generation and characterization of transgenic mice bearing a transgene in which the 1 kb DNA 5' regulatory sequence of the desmin gene is linked to a reporter gene coding for Escherichia coli beta-galactosidase (Des1-nlacZ). The enhancer activity of the desmin promoter is very strong and the reporter gene expression is easily detected in tissue sections. We have demonstrated that the regulatory elements present in the transgene Des1-nlacZ are sufficient to direct muscle-specific and developmentally regulated expression of nlacZ in skeletal muscles. Endogenous desmin expression and transgene activity were found to be correlated during the development of skeletal muscles. The transgene was expressed in the committed mononucleate myoblasts as well as in the myotubes. In addition, we have shown that the desmin-derived sequences direct a highly selective expression of nlacZ in cells that leave the somites and invade the limb bud, indicating that the cells that migrate from the somites are already predetermined for myogenesis. In contrast, smooth and cardiac muscle cells were beta-galactosidase negative both during embryonic and foetal development. Interestingly, the transgene was found to be expressed in the conduction system of the heart, which exhibits many features characteristic of skeletal muscles.

A possible regulatory role for conserved promoter motifs in an adult-specific muscle myosin gene from mouse.

The mouse gene encoding a myosin heavy chain (MHC) protein expressed in type IIB fibers of adult skeletal muscle has been cloned and its promoter isolated. A number of DNA sequence motifs are found within the first 2.5 kilobases of the promoter which are similar to motifs present in the promoters of other muscle-specific genes. One sequence located at approximately -940 base pairs corresponds to the motif called MEF1 which has been shown in other muscle genes to bind the myogenic regulatory factors of which MyoD is one example. The MEF1 site of this adult IIB MHC promoter does indeed bind MyoD although this factor is normally thought to be involved in early muscle cell differentiation. The IIB MHC promoter also has several motifs located in the first 200 base pairs which are strikingly conserved between this mouse gene and several chicken skeletal MHC genes. Of these evolutionarily conserved sequences, two motifs rich in A and T residues appear to be major contributors to the muscle-specific transcriptional activity of the mouse IIB MHC promoter when transfected into quail myogenic and non-myogenic cells. These observations suggest an important functional role for these AT-rich sequence motifs in the regulation of genes of the MHC family.

An evaluation of cell separation techniques in a model mixed cell population.

Muscle precursor cells may act not only as a means of inserting normal genes into diseased muscle fibres, in order to correct or alleviate a genetically inherited myopathy, but recent demonstrations have shown they may prove an invaluable tool for the expression of, and systemic dissemination of, non-muscle gene products. If muscle precursor cells are proved to act as such widespread vectors in terms of gene therapy, then it is imperative that methods are properly elucidated to produce large populations of pure viable myogenic cells for such purposes. In the past, many methods of cell separation have been investigated but carry with them the problems of either a lack of myogenic purity of the population or poor percentage recovery of the original cell population. In the present work we have investigated two methods for segregating myogenic from non-myogenic cells and have critically reviewed the efficiency of separation of the two techniques used. To obtain a quantitative measure of separation efficiency, segregation was carried out on a 1:1 mixture of murine C2 myogenic and murine 3T3 fibroblastic cells. To distinguish between C2 and 3T3 cells, the latter were prelabelled with the fluorescent strain carboxyfluorescein diacetate succinimyl ester (CFSE). Once incorporated into the cell, CFSE remains there, thus preventing transfer of the label to C2 cells. Both methods of separation used depend on the affinity of myogenic cells for the monoclonal antibody Mab H28, which specifically binds to the mouse neuronal cell adhesion molecule N-CAM, but differ in that one method, "panning", completes segregation by adherence of N-CAM positive cells to a dish precoated with secondary IgG antibody whereas in the other separation proceeds by the use of commercially available IgG-coated magnetic beads. Results indicate magnetic bead separation to be more efficient than panning if the beads are precoated with 0.1% gelatin.

Differentiation of smooth muscle cells in the fetal rat testis and ovary: localization of alkaline phosphatase, smooth muscle myosin, F-actin, and desmin.

The histochemical demonstration of alkaline phosphatase (AP) activity and localization of smooth muscle myosin (SMM), F-actin, and desmin were carried out on frozen sections of testes and ovaries from 15-day-old fetal to newborn rats. The presence of immunocytochemically localized SMM and desmin was confirmed by Western blot analysis of proteins from isolated gonads. The development of smooth muscle cells was predominant in the testis. The first SMM-positive cells with an increasing intensity for F-actin and desmin appeared in the testicular tunica albuginea and around the testicular cords by the age of 16 days. A continuous layer of SMM- and F-actin-positive (but not uniformly desmin-positive) myoid cells was detected in the newborn testis. In the early gonads and in the newborn ovary, a majority of the interstitial cells expressed desmin, indicating that, in undifferentiated tissues, non-myogenic cells may also express desmin. During fetal development, male and female gonocytes showed a decrease in F-actin content but retained their high AP activity. In the cortex of the newborn rat ovary, the observed high AP activity and the presence of desmin may be associated with the postnatal histogenesis of the follicles. The presence of SMM-containing cells in the hilus of the ovary may be required for the demarcation of the ovary from the mesonephros by the constriction of the mesovarium. The occurrence of SMM-positive cells predominantly in male fetuses suggests that the development of the contractile cells in the fetal testis may be induced by testicular androgens.

Fos and Jun repress transcriptional activation by myogenin and MyoD: the amino terminus of Jun can mediate repression.

Myogenin and MyoD belong to a family of muscle-specific helix-loop-helix (HLH) proteins that have the potential to activate muscle-specific genes in nonmyogenic cells. Peptide growth factors can block the ability of myogenin and MyoD to activate their target genes. Here, we show that the growth factor-inducible proto-oncogenes c-fos, c-jun, and junB mimic the effects of exogenous growth factors and suppress trans-activation of the muscle creatine kinase (MCK) enhancer by myogenin and MyoD. In contrast, JunD, which shares DNA-binding specificity with JunB and c-Jun but is expressed constitutively in muscle cells, is an inefficient inhibitor of the trans-activating capacity of myogenin and MyoD. Transcriptional repression by Fos and Jun is specific to myogenic HLH proteins and is not observed with the widely expressed HLH protein E47, which recognizes the same DNA sequence. Repression of the MCK enhancer by Fos and Jun is targeted at the myogenin and MyoD DNA recognition sequence and can be mediated by the amino terminus of c-Jun. Comparison of several myogenin mutants for their responsiveness to Fos and Jun shows that repression is directed at the basic-HLH region. These results indicate that members of the Jun family can be distinguished on the basis of their effects on muscle-specific transcription and suggest there is cross talk between transcription factors that control myogenesis and those involved in cell proliferation.

Regulation of sarcoplasmic reticulum gene expression during cardiac and skeletal muscle development.

The expression of major sarcoplasmic reticulum proteins during cardiac and fast-twitch skeletal muscle development was examined using gene-specific probes. Through the use of S1 nuclease mapping, Northern blot, and RNA slot-blot analysis, sarcoplasmic reticulum proteins were shown to exhibit both narrow tissue specificity and plasticity in their expression during muscle development. In fast-twitch skeletal muscle, the cardiac/slow-twitch isoforms of Ca(2+)-ATPase and calsequestrin were detected at high levels in fetal stages but were gradually replaced by fast-twitch isoforms in adult muscle. In contrast, cardiac muscle expressed exclusively cardiac/slow-twitch isoforms of Ca(2+)-ATPase and calsequestrin at all stages. Both fast-twitch and slow-twitch skeletal muscle expressed the same skeletal muscle ryanodine receptor isoform, whereas cardiac muscle expressed a cardiac isoform. Phospholamban expression was restricted to cardiac and slow-twitch skeletal muscle and did not appear in developing fast-twitch skeletal muscle. During in vitro myogenesis of C2C12 cells, the mRNA transcripts encoding sarcoplasmic reticulum proteins were found to be coordinately induced in synchrony with that of contractile protein mRNA. The myogenic factor "myogenin" induced sarcoplasmic reticulum gene transcripts along with contractile protein mRNAs in nonmyogenic cells. These data suggest that the induction of both sarcoplasmic reticulum and contractile protein gene families is under the control of a common myogenic differentiation program.

Cell Culture as a Tool for the Study of Poultry Skeletal Muscle Development ,

Postnatal development of skeletal muscle is the responsibility of the myogenic satellite cells. Satellite cells, isolated from the pectoralis major muscle of young growing tom turkeys, have been cultured in vitro to provide a system for studying cellular and hormonal aspects of poultry skeletal muscle development. Satellite cell clones derived from primary cultures have been developed so that in vitro observations would not be confounded by the presence of nonmyogenic cells. Likewise, a serum-free medium that promotes proliferation of the turkey satellite cell has been developed to provide a hormonally controlled environment for in vitro developmental studies. These two techniques have enabled us to examine the following: 1) factors that influence satellite cell proliferation and differentiation, 2) the interaction of hormones with cellular receptors, 3) secretion of biologically important proteins from cells and 4) the expression of genes important to muscle development.

Domains outside of the DNA-binding domain impart target gene specificity to myogenin and MRF4.

Myogenin and MRF4 belong to the MyoD family of muscle-specific transcription factors, which can activate myogenesis when introduced into nonmyogenic cells. These proteins share homology within a basic-helix-loop-helix motif that mediates DNA binding and dimerization, but they are divergent in their amino and carboxyl termini. Although myogenin and MRF4 bind the same sequence within the muscle creatine kinase enhancer, only myogenin efficiently transactivates this enhancer. By creating chimeras of myogenin and MRF4, we show that the specificities of these factors for transactivation of the muscle creatine kinase enhancer can be interchanged by swapping their amino and carboxyl termini. Within these chimeras, strong cooperation between the amino and carboxyl termini was observed. These findings suggest that myogenin and MRF4 discriminate between muscle-specific enhancers and that target gene specificity is determined by domains surrounding the basic-helix-loop-helix region.

Domains outside of the DNA-binding domain impart target gene specificity to myogenin and MRF4.

Myogenin and MRF4 belong to the MyoD family of muscle-specific transcription factors, which can activate myogenesis when introduced into nonmyogenic cells. These proteins share homology within a basic-helix-loop-helix motif that mediates DNA binding and dimerization, but they are divergent in their amino and carboxyl termini. Although myogenin and MRF4 bind the same sequence within the muscle creatine kinase enhancer, only myogenin efficiently transactivates this enhancer. By creating chimeras of myogenin and MRF4, we show that the specificities of these factors for transactivation of the muscle creatine kinase enhancer can be interchanged by swapping their amino and carboxyl termini. Within these chimeras, strong cooperation between the amino and carboxyl termini was observed. These findings suggest that myogenin and MRF4 discriminate between muscle-specific enhancers and that target gene specificity is determined by domains surrounding the basic-helix-loop-helix region.