Isolation of a spontaneously fusing BC3H1 muscle cell line: fusion alters the response to serum stimulation

Summary Differentiation of skeletal muscle cells involves two distinct events: exit from the cell cycle and expression of musclespecific contractile genes and formation of multinucleated myocytes. Although many studies have shown that growth factors regulate the initial step of differentiation, little is known about regulation of fusion. BC3H1 cells are a skeletal muscle cell line characterized by a nonfusing phenotype and an ability to dedifferentiate. When subjected to serum or growth factors, differentiated BC3H1 cells lose muscle-specific gene expression and re-enter the cell cycle. In this study, we describe a spontaneously fusing clone of BC3H1 cells. We demonstrate that this fusion capability is not due to altered muscle regulatory factor or adhesion molecule expression. Furthermore, we show that fusion inhibits dedifferentiation. Multinucleated BC3H1 cells do not lose myosin expression, nor do they re-enter the cell cycle. Fused BC3H1 cells react to serum stimulation with a hypertrophic response. Our results suggest that the state of differentiation, mono- or multinucleated, is essential to how myocytes react to growth stimulation and may provide a mechanism for how differentiation, fusion, and hypertrophy are regulated in vivo.     

Identification and characterization of genes controlled by the sporulation-regulatory gene spo0H in Bacillus subtilis.

We describe a general strategy for the identification of genes that are controlled by a specific regulatory factor in vivo and the use of this strategy to identify genes in Bacillus subtilis that are controlled by spo0H, a regulatory gene required for the initiation of sporulation. The general strategy makes use of a cloned regulatory gene fused to an inducible promoter to control expression of the regulatory gene and random gene fusions to a reporter gene to monitor expression in the presence and absence of the regulatory gene product. spo0H encodes a sigma factor of RNA polymerase, sigma H, and is required for the extensive reprograming of gene expression during the transition from growth to stationary phase and during the initiation of sporulation. We identified 18 genes that are controlled by sigma H (csh genes) in vivo by monitoring expression of random gene fusions to lacZ, made by insertion mutagenesis with the transposon Tn917lac, in the presence and absence of sigma H. These genes had lower levels of expression in the absence of sigma H than in the presence of sigma H. Patterns of expression of the csh genes during growth and sporulation in wild-type and spo0H mutant cells indicated that other regulatory factors are probably involved in controlling expression of some of these genes. Three of the csh::Tn917lac insertion mutations caused noticeable phenotypes. One caused a defect in vegetative growth, but only in combination with a spo0H mutation. Two others caused a partial defect in sporulation. One of these also caused a defect in the development of genetic competence. Detailed characterization of some of the csh genes and their regulatory regions should help define the role of spo0H in the regulation of gene expression during the transition from growth to stationary phase and during the initiation of sporulation.