Expression of Muscle Genes in Heterokaryons Depends on Gene Dosage

We report that gene dosage, or the ratio of nuclei from two cell types fused to form a heterokaryon, affects the t ime course of differentiation-specific gene expression. The rate of appearance of the human muscle antigen, 5.1H 11, is significantly faster in heterokaryons with equal or near-equal numbers of mouse muscle and human fibroblast nuclei than in heterokaryons with increased numbers of nuclei from either cell type. By 4 d after fusion, a high frequency of gene expression is evident at all ratios and >75% of heterokaryons express the antigen even when the nonmuscle nuclei greatly ou tnumber the muscle nuclei. The kinetic differences observed with different nuclear ratios suggest that the concentrat ion of putative trans-acting factors significantly influences the rate of muscle gene expression: a threshold concentrat ion is necessary, but an excess may be inhibitory. elucidation of the mechanisms that regulate changes in gene expression is of central importance to an understanding of the development of complex multicellular eukaryotes. Cellular differentiation does not appear to be due to loss of genomic material (9, 11, 21). Instead, to generate and maintain the specialized characteristics of cells of differentiated tissues, genes must be activated and repressed in an orderly fashion. There are some indications that the differential regulation of genes is mediated by the interaction of specific trans-acting factors with cis-acting genomic sequences, such as promoters and enhancers (4). Although the mechanism of action of certain general mediators of transcription has been demonstrated to be concentration dependent (16, 17, 19, 22, 29, 30), relatively little is known regarding the amounts required for tissue-specific gene expression in mammalian cells. Experiments with somatic cell hybrids suggested that tissuespecific trans-acting molecules might play a role in the expression of differentiated functions in mammalian cells. This is now known to be true from studies of the expression of certain cloned genes following transfection. Cell fusion results in the combination of the entire genomes and cytoplasms of two cells of different functional states. This leads either to the repression of the differentiated functions of the expressing cell type or to the activation of silent genes in the non-expressing cell type. The different outcomes obtained with hybrids are correlated with gene dosage, or the relative ratio of the chromosomes contributed by each of the parental cell types. Repression is typically observed with equivalent gene dosage or with increased genetic input from the non-expressing cell type; activation is usually detected only when the genetic input from the expressing cell type is increased (for review see references 10, 20, 28). However, a limitation of such studies is that the initial fusion products can rarely be analyzed for more than short periods of time after their production; cell division and chromosome loss and rearrangement rapidly ensue. Consequently, a kinetic analysis of the influence of gene dosage on gene expression is not possible. To examine concentration requirements in the activation and expression of differentiation-specific eukaryotic genes over time, we have used a stable heterokaryon system in which cell division does not occur. Gene dosage can be manipulated in these heterokaryons by altering the ratio of intact nuclei so that the normal location of genes within the genome and the stoichiometry between regulatory and structural genes per nucleus are maintained. The detailed kinetic analysis of the relationship between nuclear ratio and gene expression presented here is possible because the initial fusion product, a heterokaryon, survives for up to 2 wk in culture. We have previously shown that when mouse muscle cells are fused with human fibroblasts to form heterokaryons, human muscle genes are activated. The expression of genes coding for seven diverse functions is detected: a cell surface component (5.1HI 1), an enzyme essential for energy production (creatine kinase), and five structural proteins of the contractile apparatus (actins and myosin light chains) (1, 2, 6, 7, 12). Furthermore, the frequency and efficiency ofgene activation is high; 95 ___ 1% of all heterokaryons express human muscle genes and the relative amounts of the muscle gene products are typical of pure muscle cultures. Examples of gene repression and activation using similar muscle heterokaryons have been reported by others (14, 31, 32). We show here that regardless of nuclear ratio, muscle gene activation is observed; nuclear ratio, however, significantly ~c. The Rockefeller University Press, 0021-9525/86/01/0124/07 $ 1.00 124 The Journal of Cell Biology Volume 102, Janua~, 1986 124-130 on Jne 6, 2017 D ow nladed fom Published January 1, 1986