Myogenic bHLH Factors Research Articles

MyoR: a muscle-restricted basic helix-loop-helix transcription factor that antagonizes the actions of MyoD.

Skeletal muscle development is controlled by a family of muscle-specific basic helix-loop-helix (bHLH) transcription factors that activate muscle genes by binding E-boxes (CANNTG) as heterodimers with ubiquitous bHLH proteins, called E proteins. Myogenic bHLH factors are expressed in proliferating undifferentiated myoblasts, but they do not initiate myogenesis until myoblasts exit the cell cycle. We describe a bHLH protein, MyoR (for myogenic repressor), that is expressed in undifferentiated myoblasts in culture and is down-regulated during differentiation. MyoR is also expressed specifically in the skeletal muscle lineage between days 10.5 and 16.5 of mouse embryogenesis and down-regulated thereafter during the period of secondary myogenesis. MyoR forms heterodimers with E proteins that bind the same DNA sequence as myogenic bHLH/E protein heterodimers, but MyoR acts as a potent transcriptional repressor that blocks myogenesis and activation of E-box-dependent muscle genes. These results suggest a role for MyoR as a lineage-restricted transcriptional repressor of the muscle differentiation program.

Muscle differentiation: more complexity to the network of myogenic regulators

Recent genetic and biochemical approaches have advanced our understanding of control mechanisms underlying myogenesis in vertebrate organisms. In particular, systematic combinations of targeted gene disruptions in mice have revealed unique and overlapping functions of members of the MyoD family of transcription factors within the regulatory network that establishes skeletal muscle cell lineages. Moreover, Pax3 has been identified as a key regulator of myogenesis which seems to act genetically upstream of MyoD. In addition, novel genes have been discovered that modulate myogenesis and the activity of myogenic basic helix-loop-helix (bHLH) proteins in positive or negative ways. The molecular mechanisms of these interactions and cooperativity are being elucidated, most notably between the myogenic bHLH factors and MEF2 transcription factors.

Overlapping functions of the myogenic bHLH genes MRF4 and MyoD revealed in double mutant mice.

The myogenic basic helix-loop-helix (bHLH) genes - MyoD, Myf5, myogenin and MRF4 - exhibit distinct, but overlapping expression patterns during development of the skeletal muscle lineage and loss-of-function mutations in these genes result in different effects on muscle development. MyoD and Myf5 have been shown to act early in the myogenic lineage to establish myoblast identity, whereas myogenin acts later to control myoblast differentiation. In mice lacking myogenin, there is a severe deficiency of skeletal muscle, but some residual muscle fibers are present in mutant mice at birth. Mice lacking MRF4 are viable and have skeletal muscle, but they upregulate myogenin expression, which could potentially compensate for the absence of MRF4. Previous studies in which Myf5 and MRF4 null mutations were combined suggested that these genes do not share overlapping myogenic functions in vivo. To determine whether the functions of MRF4 might overlap with those of myogenin or MyoD, we generated double mutant mice lacking MRF4 and either myogenin or MyoD. MRF4/myogenin double mutant mice contained a comparable number of residual muscle fibers to mice lacking myogenin alone and myoblasts from those double mutant mice formed differentiated multinucleated myotubes in vitro as efficiently as wild-type myoblasts, indicating that neither myogenin nor MRF4 is absolutely essential for myoblast differentiation. Whereas mice lacking either MRF4 or MyoD were viable and did not show defects in muscle development, MRF4/MyoD double mutants displayed a severe muscle deficiency similar to that in myogenin mutants. Myogenin was expressed in MRF4/MyoD double mutants, indicating that myogenin is insufficient to support normal myogenesis in vivo. These results reveal unanticipated compensatory roles for MRF4 and MyoD in the muscle differentiation pathway and suggest that a threshold level of myogenic bHLH factors is required to activate muscle structural genes, with this level normally being achieved by combinations of multiple myogenic bHLH factors.

Identification of a developmentally regulated Trichinella spiralis protein that inhibits MyoD-specific protein: DNA complexes in vitro

Development of the infective L1 larva of Trichinella spiralis occurs as an intracellular parasite of skeletal muscle and leads to the dedifferentiation of the host cell. A novel Trichinella gene, tsJ5, has been identified from a cDNA library screen for sequences encoding Trichinella proteins related to the myogenic bHLH factors. The tsJ5 gene is developmentally regulated, showing preferential expression in the infective muscle stage larva. The product of the tsJ5 gene is not a bHLH protein but represents a novel protein with properties in common with some myogenic repressors. A recombinant TsJ5 protein affects the formation of MyoD:DNA complexes in vitro.

HSP90α Gene Expression May Be a Conserved Feature of Vertebrate Somitogenesis

We have previously demonstrated that thehsp90α andhsp90β genes in zebrafish are expressed in dramatically different spatial and temporal patterns in early embryos. In the case ofhsp90α, expression is spatially restricted within the somites to putative myogenic cells which also express mRNA encoding the myogenic bHLH transcription factor myoD and is downregulated along withmyoDfollowing myogenesis. In the present study, we have examinedhsp90α gene expression in developing chicken embryos using a gene-specific probe. We show thathsp90α gene expression is also localized to a subset of cells within the somites of chicken embryos and that the expression pattern correlates closely to that observed formyoD.Furthermore, expression of thehsp90α gene is strongly upregulated throughout the embryo following heat shock in a manner similar to that observed in heat-shocked zebrafish embryos. The data suggest that thehsp90α gene may play an evolutionarily conserved role during somitogenesis in vertebrates in addition to providing protection to all cells of the embryo following stress.

Repression of Muscle-Specific Gene Activation by the Murine Twist Protein

Inhibition of myogenic differentiation can be achieved by various mechanisms. The murine bHLH protein Twist has been shown to inhibit muscle differentiation in mammalian cells. Here we demonstrate that this inhibition is cell autonomous and does not alter cell proliferation. By overexpression of E12, we can distinguish the inhibitory mechanisms of Twist and the dominant negative HLH factor Id. A difference is seen both for the native muscle-specific enhancers of myogenin and myosin light chain 1/3 and for an enhancer consisting only of four E-boxes. Mutagenesis experiments revealed that both the basic region and an evolutionarily conserved carboxy-terminal domain are required for the Twist-specific type of inhibition. Loss of either of these regions renders Twist less efficient and more similar to Id. Gel mobility shift assays demonstrate that Twist can bind to the muscle creatine kinase E-box and inhibit DNA binding of heterodimers of E12 with myogenic bHLH transcription factors like MyoD. However, a fourfold excess of Twist compared to MyoD is required for both effects. Our results suggest that Twist inhibits muscle-specific gene activation by formation of actively inhibitory complexes rather than by sequestering E-proteins.

Defining the regulatory networks for muscle development

The formation of skeletal muscle during vertebrate embryogenesis requires commitment of mesodermal precursor cells to the skeletal muscle lineage, withdrawal of myoblasts from the cell cycle, and transcriptional activation of dozens of muscle structural genes. The myogenic basic helix-loop helix (bHLH) factors — MyoD, myogenin, Myf5, and MRF4 — act at multiple points in the myogenic lineage to establish myoblast identity and to control terminal differentiation. Recent studies have begun to define the inductive mechanisms that regulate myogenic bHLH gene expression and muscle cell determination in the embryo. Myogenic bHLH factors interact with components of the cell cycle machinery to control withdrawal from the cell cycle and act combinatorially with other transcription factors to induce skeletal muscle transcription. Elucidation of these aspects of the myogenic program is leading to a detailed understanding of the regulatory circuits controlling muscle development.

Inhibition of myogenic bHLH and MEF2 transcription factors by the bHLH protein Twist.

The myogenic basic helix-loop-helix (bHLH) and MEF2 transcription factors are expressed in the myotome of developing somites and cooperatively activate skeletal muscle gene expression. The bHLH protein Twist is expressed throughout the epithelial somite and is subsequently excluded from the myotome. Ectopically expressed mouse Twist (Mtwist) was shown to inhibit myogenesis by blocking DNA binding by MyoD, by titrating E proteins, and by inhibiting trans-activation by MEF2. For inhibition of MEF2, Mtwist required heterodimerization with E proteins and an intact basic domain and carboxyl-terminus. Thus, Mtwist inhibits both families of myogenic regulators and may regulate myotome formation temporally or spatially.

Cooperative activation of muscle gene expression by MEF2 and myogenic bHLH proteins

Members of the myocyte enhancer factor-2 (MEF2) family of MADS domain transcription factors cannot induce myogenesis in transfected fibroblasts, but when coexpressed with the myogenic basic-helix-loop-helix (bHLH) proteins MyoD or myogenin they dramatically increase the extent of myogenic conversion above that seen with either myogenic bHLH factor alone. This cooperativity required direct interactions between the DNA-binding domains of MEF2 and the myogenic bHLH factors, but only one of the factors needed a transactivation domain, and only one of the factors needed to be bound to DNA. These interactions allow either factor to activate transcription through the other's binding site and reveal a novel mechanism for indirect activation of gene expression via protein-protein interactions between the DNA-binding domains of heterologous classes of transcription factors.

Interactions of myogenic bHLH transcription factors with calcium-binding calmodulin and S100a (alpha alpha) proteins.

MyoD belongs to a family of myogenic basic helix-loop-helix (bHLH) transcription factors that activate muscle-specific genes. The basic helix I sequence of the bHLH motif contains a consensus sequence for protein kinase C (PKC) substrates. We show here that MyoD is indeed phosphorylated by PKC in vitro on Thr 115 within the basic part of the bHLH motif. By analogy with calmodulin-target peptide models, we also identified within the consensus basic helix I motif of myogenic proteins a conserved putative calmodulin/S100-binding domain. Calcium-dependent interaction between MyoD with calmodulin and the abundant muscle S100a(alpha alpha) proteins was demonstrated by affinity chromatography and cross-linking experiments. The binding of calmodulin and S100a inhibited MyoD phosphorylation by PKC as well as MyoD DNA binding activity. S100a was found to be more efficient than calmodulin in antagonizing DNA binding to MyoD. We next developed a rapid purification method for bacterial recombinant MyoD-bHLH domain by affinity chromatography using a calmodulin-Sepharose column and investigated the phosphorylation of that peptide by PKC and its interactions with calmodulin and S100a. We confirmed the phosphorylation of the threonine residue 115 in the MyoD-bHLH by PKC with a Km of 0.8 microM. Calmodulin and S100a binding inhibited MyoD-bHLH phosphorylation by PKC. A strict calcium-dependent interaction between calcium binding proteins and the MyoD-bHLH was identified by native gel electrophoresis and fluorescence spectroscopy with 5-(dimethylamino)naphthalene-1-sulfonylcalmodulin. The MyoD-bHLH bound to fluorescently labeled 5-(dimethylamino)naphthalene-1-sulfonylcalmodulin with a dissociation constant around 20 nM. S100a inhibited stoichiometrically the binding of the bHLH peptide for labeled calmodulin, suggesting an affinity of S100a for the bHLH peptide at least 1 order of magnitude higher than calmodulin. In favor of an in vivo interaction between S100a and MyoD, we report that S100a- and MyoD-like immunoreactivities colocalize in H9c2 cells, and that a significant amount of MyoD-like immunoreactivity is recovered in the S100a immunoprecipitate from crude H9c2 cell extract in the presence of calcium. We propose that myogenic proteins represent a new family of calmodulin/S100-binding PKC substrates and that calmodulin/S100a could participate in the regulation of the bHLH myogenic protein activities.

Inactivation of the myogenic bHLH gene MRF4 results in up-regulation of myogenin and rib anomalies.

The myogenic basic helix-loop-helix (bHLH) proteins MyoD, myf5, myogenin, and MRF4 can initiate myogenesis when expressed in nonmuscle cells. During embryogenesis, each of the myogenic bHLH genes is expressed in a unique temporospatial pattern within the skeletal muscle lineage, suggesting that they play distinct roles in muscle development. Gene targeting has shown that MyoD and myf5 play partially redundant roles in the genesis of myoblasts, whereas myogenin is required for terminal differentiation. MRF4 is expressed transiently in the somite myotome during embryogenesis and then becomes up-regulated during late fetal development to eventually become the predominant myogenic bHLH factor expressed in adult skeletal muscle. On the basis of its expression pattern, it has been proposed that MRF4 may regulate skeletal muscle maturation and aspects of adult myogenesis. To determine the function of MRF4, we generated mice carrying a homozygous germ-line mutation in the MRF4 gene. These mice showed only a subtle reduction in expression of a subset of muscle-specific genes but showed a dramatic increase in expression of myogenin, suggesting that it may compensate for the absence of MRF4 and demonstrating that MRF4 is required for the down-regulation of myogenin expression that normally occurs in postnatal skeletal muscle. Paradoxically, MRF4-null mice exhibited multiple rib anomalies, including extensive bifurcations, fusions, and supernumerary processes. These results demonstrate an unanticipated regulatory relationship between myogenin and MRF4 and suggest that MRF4 influences rib outgrowth through an indirect mechanism.

The Mouse MRF4 Promoter Is trans-Activated Directly and Indirectly by Muscle-specific Transcription Factors

MRF4 is a member of the basic helix-loop-helix (bHLH) family of muscle-specific transcription factors, which also includes MyoD, myogenin, and myf5. The myocyte enhancer binding factor 2 (MEF2) proteins also serve as important muscle-specific transcription factors. In addition to activating the expression of many muscle-specific structural genes, various members of these two classes of proteins activate their own expression and the expression of each other in a complex transcriptional network that results in the establishment and maintenance of the muscle phenotype. To begin to determine how the expression of MRF4 is regulated by other muscle-specific transcription factors, we have isolated a region of the MRF4 gene that confers muscle-specific expression and have analyzed this promoter region for cis-acting elements involved in trans-activation by the myogenic bHLH and MEF2 transcription factors. Here, we show that in 10T1/2 fibroblasts the MRF4 promoter is trans-activated by myogenin, MyoD, myf5, and by the MEF2 factors, but that MRF4 does not activate expression of its own promoter. Myogenin activated the MRF4 promoter directly by an E box-dependent mechanism, while MEF2 factors activated the promoter through an indirect pathway. The E box-dependent regulation of the MRF4 promoter is in contrast to the regulation of the myogenin and MyoD promoters and may represent a mechanism for the differential expression of these factors during myogenesis.

Inhibition of cellular differentiation by the SCL/tal oncoprotein: transcriptional repression by an Id-like mechanism

In cases of T-cell acute lymphoblastic leukemia (T-ALL), the basic helix-loop-helix (bHLH) oncogene SCL/tal undergoes frequent rearrangements activating ectopic expression. Despite the compelling epidemiological association of SCL/tal expression with T-ALL, no specific transforming function has been attributable to the protein product. However, investigators have recently demonstrated that forced overexpression of SCL/tal can block monocytic differentiation of M1 murine myeloid leukemia cells. Thus, inappropriate expression of wild- type SCL/tal protein may in part account for the maturation arrest phenotype observed in T-ALL cells. In this study, ectopic expression of the SCL/tal gene blocked the differentiation of C2C12 muscle precursor cells. Characterization of the mechanism of differentiation blockade showed that the SCL/tal protein repressed transcriptional activation by the myogenic bHLH factor MyoD. Protein interaction analysis showed that SCL/tal and MyoD compete for common partners (E bHLH proteins) but do not directly bind one other. A model is thus proposed in which ectopic SCL/tal protein, by its ability to titrate out E proteins, prevents the formation of bHLH complexes that drive cellular differentiation: the “Id-like” mechanism.

Inhibition of Cellular Differentiation by the SCL/tal Oncoprotein: Transcriptional Repression by an Id-Like Mechanism

In cases of T-cell acute lymphoblastic leukemia (T-ALL), the basic helix-loop-helix (bHLH) oncogene SCL/tal undergoes frequent rearrangements activating ectopic expression. Despite the compelling epidemiological association of SCL/tal expression with T-ALL, no specific transforming function has been attributable to the protein product. However, investigators have recently demonstrated that forced overexpression of SCL/tal can block monocytic differentiation of M1 murine myeloid leukemia cells. Thus, inappropriate expression of wild-type SCL/tal protein may in part account for the maturation arrest phenotype observed in T-ALL cells. In this study, ectopic expression of the SCL/tal gene blocked the differentiation of C2C12 muscle precursor cells. Characterization of the mechanism of differentiation blockade showed that the SCL/tal protein repressed transcriptional activation by the myogenic bHLH factor MyoD. Protein interaction analysis showed that SCL/tal and MyoD compete for common partners (E bHLH proteins) but do not directly bind one other. A model is thus proposed in which ectopic SCL/tal protein, by its ability to titrate out E proteins, prevents the formation of bHLH complexes that drive cellular differentiation: the "Id-like" mechanism.

Cloning and characterization of a basic helix-loop-helix protein expressed in early mesoderm and the developing somites.

Basic helix-loop-helix (bHLH) heterodimer protein complexes regulate transcription of genes during the processes of differentiation and development. To study the molecular basis of early mesodermal differentiation, we sought to identify bHLH proteins from cells of mesodermal origin. By using an interaction cloning strategy with a radiolabled recombinant bHLH protein, E12, a clone encoding a potential heterodimer partner was isolated from an endothelial cell library. This gene (bHLH-EC2) is most homologous to Twist but shares similarity within the bHLH domain with TAL1 and other members of this family. bHLH-EC2 is expressed in cultured endothelial cells and in embryonic stem cell, erythroleukemia, and muscle cell lines in a differentiation-dependent manner. In situ hybridization studies of mouse embryos reveal that bHLH-EC2 is expressed throughout the primitive mesoderm as early as 7.5 days postcoitum. Expression then becomes restricted to the paraxial mesoderm and to the dermamyotome of the developing somite. Expression of bHLH-EC2 in cells destined to become myoblasts thus predates expression of myogenic bHLH factors. bHLH-EC2 is expressed in early endothelial and hematopoietic cells in vivo, as shown by RNA studies of embryonic yolk sac and cultured cells derived from yolk sac explants. These findings suggest that bHLH-EC2 plays a role in the development of multiple cell types derived from the primitive mesoderm.

Expression of AMD 1, a gene for a MyoD 1-related factor in the ascidian Halocynthia roretzi.

We have isolated and cloned a gene, designated AMD1 (ascidian MyoD-related factor 1), and its cDNAs that encode a member of the family of myogenic basic helix-loop-helix (bHLH) factors from the ascidian Halocynthia roretzi. The AMD1 gene consists of four exons and is transcribed into at least two distinct mRNAs, which differ in their 3' untranslated region. The gene encodes a protein of 435 amino acids, which exhibits homology to the bHLH domain of other myogenic bHLH factors including vertebrate MyoDt. A reverse transcription-polymerase chain reaction (RT-PCR) assay revealed that transcripts of the gene were not detectable in fertilized eggs or in very early embryos. They were first detected at the 64-cell stage, a few hours prior to the accumulation of mRNAs for embryonic muscle-specific proteins. In addition, expression of the AMD1 gene was evident in adult body-wall muscle but not in heart and other nonmuscle tissues. These results suggest the possibility that, in ascidians as in vertebrates, the myogenic bHLH factor is involved in the specification of embryonic cells as myogenic cells.

The regulation of myogenin gene expression during the embryonic development of the mouse.

The myogenic program can be activated in cultured cells by each of four basic-helix-loop-helix (bHLH) transcription factors, the expression of which is strictly controlled, both temporally and spatially, during embryonic development. To begin to understand the mechanisms by which these regulators are regulated themselves, we have used transgenic animals to define the minimal sequences required for the complete recapitulation of the temporal and spatial expression pattern of the myogenin gene during embryogenesis. We show that this can be achieved with only 133 bp of 5'-flanking DNA and identify two essential motifs, which are consensus binding sites for the bHLH proteins and for the proteins of the RSRF family. We show further that these sequences, when juxtaposed to a heterologous promoter, are capable of imposing the myogenin expression pattern. We conclude that the proper regulation of myogenin requires a bHLH protein, most probably Myf-5, the only myogenic bHLH factor known to be present in the embryo at the time that myogenin is activated, and an RSRF-like binding activity. Furthermore, the expression pattern of a mutant myogenin promoter lacking the RSRF site reveals the existence of at least two populations of cells within the myotomes and of novel rostrocaudal gradients of expression.