H H Arnold

Targeted inactivation of the muscle regulatory gene Myf-5 results in abnormal rib development and perinatal death

The Myf-5 gene, a member of the myogenic basic HLH factor family, has been inactivated in mice after homologous recombination in ES cells. Mice lacking Myf-5 were unable to breathe and died immediately after birth, owing to the absence of the major distal part of the ribs. Other skeletal abnormalities, except for complete ossification of the sternum, were not apparent. Histological examination of skeletal muscle from newborn mice revealed no morphological abnormalities. Northern blot analysis demonstrated normal levels of muscle-specific mRNAs including MyoD, myogenin, and Myf-6. However, the appearance of myotomal cells in early somites was delayed by several days. These results suggest that while Myf-5 plays a crucial role in the formation of lateral sclerotome derivatives, Myf-5 is dispensable for the development of skeletal muscle, perhaps because other members of the myogenic HLH family substitute for Myf-5 activity.

A novel human muscle factor related to but distinct from MyoD1 induces myogenic conversion in 10T1/2 fibroblasts.

We have isolated the cDNA encoding a novel human myogenic factor, Myf‐5, by weak cross‐hydridization to the mouse MyoD1 probe. Nucleotide sequence analysis and the identification of the corresponding gene indicate that human Myf‐5 is a member of a small gene family which also contains the human homologue to MyoD1. Although structurally related to the mouse factor, the human Myf‐5 constitutes a different protein which nevertheless is capable of inducing the myogenic phenotype in embryonic C3H mouse 10T1/2 ‘fibroblasts’. The existence of more than one MyoD1‐like protein in human skeletal muscle is further suggested by the detection of several similar but distinct cDNA clones. The phenotypic conversion of 10T1/2 cells by the human factor is recognized by the capacity of the cells to form multinucleated syncytia and synthesize sarcomeric myosin heavy chains. Moreover, transient expression of Myf‐5 in 10T1/2 cells leads to the activation of a co‐transfected muscle‐specific CAT reporter gene which by itself is transcriptionally silent in the non‐muscle cell background. The deduced amino acid sequence of clone Myf‐5 reveals a region which is highly similar to myc proteins and the developmental factors from Drosophila encoded by the achaete scute locus and the twist gene. The myc homology region and a preceding cluster of basic amino acids are located in a larger sequence domain with strong similarity to the mouse myogenic factor MyoD1. Two additional short segments with high serine and threonine content are conserved between the two proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Myf-6, a new member of the human gene family of myogenic determination factors: evidence for a gene cluster on chromosome 12.

The Myf‐6 gene, a novel member of the human gene family of muscle determination factors has been detected by its highly conserved sequence coding for a putative helix‐loop‐helix domain. This sequence motif is a common feature of all Myf factors and other regulatory proteins. The new Myf gene is located on human chromosome 12, approximately 6.5 Kb upstream of the Myf‐5 locus in a closely linked cluster of myogenic determination genes. Myf‐6 cDNAs were isolated from human and mouse skeletal muscle, the only tissue in which expression of the corresponding mRNA was observed. In contrast to human primary muscle cell cultures which express moderate levels of Myf‐6 mRNA, most established rodent muscle cell lines completely lack this mRNA. Myogenic 10T1/2 cells, however, induced by the expression of either pEMSV‐Myf‐4 or pEMSV‐Myf‐5 activate their endogenous mouse Myf‐6 gene. Constitutive expression of Myf‐6 cDNA in C3H 10T1/2 fibroblasts establishes the muscle phenotype at a similar frequency to the previously characterized myogenic factors. Moreover, muscle‐specific CAT reporter constructs containing either the human myosin light chain (MLC) enhancer or the promoter of the embryonic myosin light chain gene are activated in NIH 3T3 fibroblasts or in CV1 kidney cells by cotransfection of Myf‐6 expression vehicles. This transcriptional activation occurs in the absence of any apparent conversion of the cellular phenotype of the recipient cells. Glutathione‐S‐transferase fusion proteins with Myf‐3, Myf‐4 or Myf‐5 specifically bind to a MEF‐like consensus sequence present in the human MLC enhancer and the MLC1 emb promoter. In contrast, the Myf‐6 hybrid protein interacts weakly with the same sequences showing lower affinity and reduced specificity. Since co‐expressed pEMSV‐Myf‐6, nevertheless, is able to activate transcription of the MLC‐CAT reporter constructs in non‐muscle tissue culture cells, the different DNA binding properties in vitro might suggest that transactivation of gene expression by Myf‐6 involves distinct binding sites and/or additional protein factors.

Differential expression of myogenic determination genes in muscle cells: possible autoactivation by the Myf gene products.

The development of muscle cells involves the action of myogenic determination factors. In this report, we show that human skeletal muscle tissue contains, besides the previously described Myf‐5, two additional factors Myf‐3 and Myf‐4 which represent the human homologues of the rodent proteins MyoD1 and myogenin. The genes encoding Myf‐3, Myf‐4 and Myf‐5 are located on human chromosomes 11, 1, and 12 respectively. Constitutive expression of a single factor is sufficient to convert mouse C3H 10T1/2 fibroblasts to phenotypically normal muscle cells. The myogenic conversion of 10T1/2 fibroblasts results in the activation of the endogenous MyoD1 and Myf‐4 (myogenin) genes. This observation suggests that the expression of Myf proteins leads to positive autoregulation of the members of the Myf gene family. Individual myogenic colonies derived from MCA C115 cells (10T1/2 fibroblast transformed by methylcholanthrene) express various levels of endogenous MyoD1 mRNA ranging from nearly zero to high levels. The Myf‐5 gene was generally not activated in 10T1/2 derived myogenic cell lines but was expressed in some MCA myoblasts. In primary human muscle cells Myf‐3 and Myf‐4 mRNA but very little Myf‐5 mRNA is expressed. In mouse C2 and P2 muscle cell lines MyoD1 is abundantly synthesized together with myogenin. In contrast, the rat muscle lines L8 and L6 and the mouse BC3H1 cells express primarily myogenin and low levels of Myf‐5 but no MyoD1. Myf‐4 (myogenin) mRNA is present in all muscle cell lines at the onset of differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Inactivation of Myf-6 and Myf-5 genes in mice leads to alterations in skeletal muscle development.

Myf‐6, alternatively called MRF4 or herculin, is a member of a group of muscle‐specific transcription factors which also comprises Myf‐5, myogenin and MyoD. All family members show distinct expression patterns during skeletal muscle development and can convert a variety of cell lines to myocytes. We disrupted the Myf‐6 gene in mice to investigate its functional role in the network of regulatory factors controlling myogenesis. Homozygous mice carrying the disrupted Myf‐6 gene show pronounced down‐regulation of Myf‐5 transcription for reasons presently unknown. Consequently, these mice represent a double knock‐out model for Myf‐6 and Myf‐5. The mutants resemble most of the Myf‐5 phenotype with aberrant and delayed early myotome formation and lack of distal rib structures. In addition, we find a reduction in the size of axial muscles in the back. Apart from changes in the pattern of some contractile protein isoforms, the existing myofibers appear fairly normal. This suggests that Myf‐6 has no major role in the maturation of myotubes, as previously proposed. Our results provide evidence that skeletal myogenesis can proceed in the absence of two myogenic factors, Myf‐5 and Myf‐6, therefore they must exert largely non‐redundant functions in vivo.

Co-operativity of functional domains in the muscle-specific transcription factor Myf-5.

Myf‐5 is a member of a family of muscle‐specific transcription factors that activate myogenesis in 10T1/2 fibroblasts. Here we report the analysis of Myf‐5 structural domains that are responsible for its biological activity. Site‐directed mutagenesis revealed that two clusters of basic amino acids within a conserved basic region and two amphipathic helices within the adjacent HLH domain are essential for sequence‐specific DNA binding and hetero‐oligomerization, respectively. Transcriptional activation by Myf‐5 requires two additional domains located in the amino‐ and carboxyl‐termini. The two domains apparently co‐operate since deletion of either one results in inactivation. Chimeric proteins between DNA binding domain of the yeast transcription factor GAL4 and the separate Myf‐5 transactivator domains exhibit activity that is enhanced when both regions are combined. Dimerization of Myf‐5 with the ubiquitously expressed bHLH protein E12 not only increases the affinity for DNA but also stimulates transactivation independently of DNA binding. The Myf‐5 transactivator domains are dependent for activity on a specific amino acid sequence motif within the basic region when Myf‐5 activity is mediated through the E‐box DNA recognition sequence but not when DNA binding occurs through the GAL4 DNA binding domain. This demonstrates that muscle‐specific transactivation by Myf‐5 requires the collaboration of two activation domains and the DNA binding region in addition to sequence‐specific DNA binding. Transcriptional activation and interaction with DNA are executed by separable domains; however, transactivation is influenced by the basic region in a manner distinguishable from DNA binding.

Alkali myosin light chains in man are encoded by a multigene family that includes the adult skeletal muscle, the embryonic or atrial, and nonsarcomeric isoforms.

A set of cDNA clones coding for alkali myosin light chains (AMLC) was isolated from fetal human skeletal muscle. Nucleotide sequence analysis and RNA expression patterns of individual clones revealed related sequences corresponding to (i) fast fiber type MLC1 and MLC3; (ii) the embryonic MLC that is also expressed in fetal ventricle and adult atrium (MLCemb); and (iii) a nonsarcomeric MLC isoform that is found in all nonmuscle cell types and smooth muscle. The AMLC gene family in man comprises unique copies for MLC1, MLC3 and MLCemb, and multiple copies for the nonsarcomeric MLC genes. The gene coding for MLC1 and MLC3 is located on human chromosome 2.

The human MyoD1 (MYF3) gene maps on the short arm of chromosome 11 but is not associated with the WAGR locus or the region for the Beckwith-Wiedemann syndrome.

SummaryThe human gene encoding the myogenic determination factor myf3 (mouse MyoD1) has been mapped to the short arm of chromosome 11. Analysis of several somatic cell hybrids containing various derivatives with deletions or translocations revealed that the human MyoD (MYF3) gene is not associated with the WAGR locus at chromosomal band 11p13 nor with the loss of the heterozygosity region at 11p15.5 related to the Beckwith-Wiedemann syndrome. Subregional mapping by in situ hybridization with an myf3 specific probe shows that the gene resides at the chromosomal band 11p14, possibly at 11pl4.3.

Evidence for distinct phosphorylatable myosin light chains in avian heart and slow skeletal muscle

In mammalian organisms the regulatory or phosphorylatable myosin light chains in heart and slow skeletal muscle have been shown to be identical and presumable constitute the product of a single gene. We analyzed the expression of the avian cardiac myosin light chain (MLC) 2-A in heart and slow skeletal muscle by a combination of experimental approaches, e.g., two-dimensional gel electrophoresis of the protein and hybridization of mRNA to specific MLC 2-A sequences cloned from chicken. The investigations have indicated that, unlike in mammals, in avian organisms the phosphorylatable myosin light chains from heart and slow skeletal muscle are distinct proteins and therefore products of different genes. The expression of MLC 2-A is restricted to the myocardium and no evidence was found that it is shared with slow skeletal muscle.

Targeted Inactivation of the Muscle Regulatory Genes Myf-5 and MyoD: Effect on Muscle and Skeletal Development

MyoD is a member of a family of myogenic transcription factors with similar properties [1] including myogenin [2–4], Myf-5 [5], and Myf-6, also called MRF4 or herculin [6–8]. Transfection of the myogenic HLH genes into a wide range of cultured cells can induce the skeletal muscle differentiation program and activate muscle-specific genes. No substantial difference between the myogenic HLH genes has been revealed by these assays. The myogenic transcription factor genes are expressed solely in skeletal muscle, and thus, their role appears to be in determining the identity of the skeletal myocyte lineage [9–12].