Barbara Winter

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.

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.

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.

Two Putative Protein Kinase CK2 Phosphorylation Sites Are Important for Myf-5 Activity

Myf-5, a member of a family of muscle-specific transcription factors, is important for myogenic cell determination and differentiation. Here, we report that Myf-5 protein constitutes a substrate for phosphorylation in vitro by protein kinase CK2. We identified two potential phosphorylation sites at serine49 and serine133, both of which seem to be necessary for Myf-5 activity. Mutants which can no longer be phosphorylated fail to transactivate E-box-dependent reporter genes and act as trans-dominant repressors of wild-type Myf-5. Normal activity can be restored by replacing the serine residues with glutamate suggesting that a negative charge at these sites is obligatory for Myf-5 activity. Although serine133 is part of helix 2 which mediates dimerization, we find no evidence for impaired DNA-binding or heterodimerization of the Ser-Ala133 mutant. Some serine49 mutations exhibit reduced nuclear localization and/or protein stability. Our data suggest that CK2-mediated phosphorylation of Myf-5 is required for Myf-5 activity.

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.

The cardiac myosin light chain (MLC-2A) gene in chicken is methylated in both expressing and nonexpressing tissues

Certain specific methylation sites of the chicken cardiac myosin light chain (MLC-2A) gene in DNA from embryonic heart tissue as well as from embryonic livers and brains were studied. MLC-2A specific mRAA was present in the heart only, indicating that the gene is transcriptionally active in this tissue and inactive in liver and brain. Using the methylation-sensitive restriction enzymes Hpa II and Hha I, the gene was found to be identically methylated in DNA from all three examined tissues, irrespective of its expression. Moreover, the gene was extensively methylated throughout, including the 5′ end and 3′ flanking regions, with the exception of 3 Hpa II sites, one located in the middle part of the gene, one in the last omtrpm and one in the 3′ flanking sequence. These results that specific genes can be actively expressed even when they are highly modified. and that changes in the methylation pattern from one tissue to another are not necessarily associated with the ene activation.