Clustering of multiple specific genes and gene-rich R-bands around SC-35 domains: evidence for local euchromatic neighborhoods

Typically, eukaryotic nuclei contain 10-30 prominent domains (referred to here as SC-35 domains) that are concentrated in mRNA metabolic factors. Here, we show that multiple specific genes cluster around a common SC-35 domain, which contains multiple mRNAs. Nonsyntenic genes are capable of associating with a common domain, but domain "choice" appears random, even for two coordinately expressed genes. Active genes widely separated on different chromosome arms associate with the same domain frequently, assorting randomly into the 3-4 subregions of the chromosome periphery that contact a domain. Most importantly, visualization of six individual chromosome bands showed that large genomic segments ( approximately 5 Mb) have striking differences in organization relative to domains. Certain bands showed extensive contact, often aligning with or encircling an SC-35 domain, whereas others did not. All three gene-rich reverse bands showed this more than the gene-poor Giemsa dark bands, and morphometric analyses demonstrated statistically significant differences. Similarly, late-replicating DNA generally avoids SC-35 domains. These findings suggest a functional rationale for gene clustering in chromosomal bands, which relates to nuclear clustering of genes with SC-35 domains. Rather than random reservoirs of splicing factors, or factors accumulated on an individual highly active gene, we propose a model of SC-35 domains as functional centers for a multitude of clustered genes, forming local euchromatic "neighborhoods."     

Regulation of tropomyosin gene expression during myogenesis.

In skeletal muscle, tropomyosin has a critical role in transduction of calcium-induced contraction. Presently, little is known about the regulation of tropomyosin gene expression during myogenesis. In the present study, qualitative and quantitative changes in the nucleic acid populations of differentiating chicken embryo muscle cells in culture have been examined. Total nucleic acid content per nucleus increased about fivefold in fully developed myotubes as compared to mononucleated myoblasts. The contribution of deoxyribonucleic acid to the total nucleic acid population decreased from 24% in myoblasts to 5% of total nucleic acid in myotubes. Concomitant with the decrement in deoxyribonucleic acid contribution to total nucleic acid was an increase in polyadenylated ribonucleic acid (RNA) content per cell which reached levels in myotubes that were 17-fold higher than those of myoblasts. Specific changes in the RNA population during myogenesis were further investigated by quantitation of the synthetic capacity (messenger RNA levels) per cell for alpha- and beta-tropomyosin. Cell-free translation and immunoprecipitation demonstrated an approximately 40-fold increase in messenger RNA levels per nucleus for alpha- and beta-tropomyosin after fusion in the terminally differentiated myotubes. Indirect immunofluorescence with affinity-purified tropomyosin antibodies demonstrated the presence of tropomyosin-containing filaments in cells throughout myogenesis. Thus, the tropomyosin genes are constitutively expressed during muscle differentiation through the production of tropomyosin messenger RNA and translation into tropomyosin protein.     

Synthesis of rat myosin light chains in heterokaryons formed between undifferentiated rat myoblasts and chick skeletal myocytes

The control of gene expression during terminal myogenesis was explored in heterokaryons between differentiated and undifferentiated myogenic cells by analyzing the formation of species specific myosin light chains of chick and rat skeletal muscle. Dividing L6 rat myoblasts served as the biochemically undifferentiated parent. The differentiated parental cells were mononucleated muscle cells (myocytes) that were obtained from primary cultures of embryonic chick thigh muscle by blocking myotube formation with EGTA and later incubating the postimitotic cells in cytochalasin B. Heterokaryons were isolated by the selective rescue of fusion products between cells previously treated with lethal doses of different cell poisons. 95-99% pure populations of heterokaryons formed between undifferentiated rat myoblasts and differentiated chick myocytes were obtained. The cells were labeled with [35S]methionine, and whole cell extracts were analyzed on two-dimensional polyacrylamide gels. These heterokaryons synthesize the light chain of chick myosin and both embryonic and adult light chains of rat skeletal myosin. Control homokaryons formed by fusing undifferentiated cells to themselves did not synthesize skeletal myosin light chains. Control heterokaryons formed between undifferentiated rat myoblasts and chick fibroblasts also failed to synthesize myosin light chains. These results indicate that differentiated chick muscle cells provide some factor that induces L6 myoblasts to synthesize rat myosin light chains. This system provides a model for investigating the processes by which differentiated cell functions are induced.     

Association of lamin A/C with muscle gene-specific promoters in myoblasts

The A-type and B-type lamins form a filamentous meshwork underneath the inner nuclear membrane called the nuclear lamina, which is an important component of nuclear architecture in metazoan cells. The lamina interacts with large, mostly repressive chromatin domains at the nuclear periphery. In addition, genome–lamina interactions also involve dynamic association of lamin A/C with gene promoters in adipocytes. Mutations in the human lamin A gene cause a spectrum of hereditary diseases called the laminopathies which affect muscle, cardiac and adipose tissues. Since most mutations in lamin A/C affect skeletal muscle, we investigated lamin–chromatin interactions at promoters of muscle specific genes in both muscle and non-muscle cell lines by ChIP-qPCR. We observed that lamin A/C was specifically associated with promoter regions of muscle genes in myoblasts but not in fibroblasts. Lamin A/C dissociated from the promoter regions of the differentiation specific MyoD, myogenin and muscle creatine kinase genes when myoblasts were induced to differentiate. In the promoter regions of the myogenin and MyoD genes, the binding of lamin A/C in myoblasts inversely correlated with the active histone mark, H3K4me3. Lamin A/C binding on muscle genes was reduced and differentiation potential was enhanced on treatment of myoblasts with a histone deacetylase inhibitor. These findings suggest a role for lamina–chromatin interactions in muscle differentiation and have important implications for the pathological mechanisms of striated muscle associated laminopathies.     

Isolation and characterization of terminally differentiated chicken and rat skeletal muscle myoblasts

The ability of skeletal muscle myoblasts to differentiate in the absence of spontaneous fusion was studied in cultures derived from chicken embryo leg muscle, rat myoblast lines L6 and L8, and the mouse myoblast line G8. Following 48–96 hr of culture in a low-Ca²⁺ (25 μm), Mg²⁺-depleted medium, chicken myoblasts exhibited only 3–5% fusion whereas up to 64% of the cells fused in control cultures. Depletion of Mg²⁺ led to preferential elimination of fibroblasts, with the result that 97% of the mononucleated cells remaining at 120 hr exhibited a bipolar morphology and stained with antibodies directed against M-creatine kinase, skeletal muscle myosin, and desmin. Mononucleated myoblasts rarely showed visible cross-striations or M-line staining with anti-myomesin unless the medium was supplemented with 0.81 mM Mg²⁺, suggesting that Mg²⁺ plays a role in sarcomere assembly. Conditions of Ca²⁺ and Mg²⁺ depletion inhibited myoblast fusion in the rodent cell lines as well, but mononucleated myoblasts failed to differentiate under these conditions. Differentiated individual myoblasts from rat cell lines and from chicken cell cultures were obtained when fusion was inhibited by growth in cytochalasin B (CB). CB-treated rat myoblast cultures accumulated MM-CK to nearly twice the specific activity found in extensively fused control cultures of comparable age. Spherical cells which accumulated during CB treatment were isolated and shown to contain nearly eight times the CK specific activity present in nonspherical cells from the same cultures. Approximately 90% of these cells exhibited immunofluorescent staining with antibodies to skeletal muscle myosin, failed to incorporate [³H]thymidine or to form colonies in clonal subculture, and thus represent terminally differentiated rat myoblasts. Quantitative microfluorometric DNA measurements on individual nuclei demonstrated that the terminally differentiated myoblasts obtained in these experiments from both chicken and rat contain 2cDNA levels, suggesting arrest in the G₀ stage of the cell cycle.     

Tumor necrosis factor inhibits human myogenesis in vitro.

We examined the effects of human recombinant tumor necrosis factor-alpha (TNF) on human primary myoblasts. When added to proliferating myoblasts, TNF inhibited the expression of alpha-cardiac actin, a muscle-specific gene whose expression is observed at low levels in human myoblasts. TNF also inhibited muscle differentiation as measured by several parameters, including cell fusion and the expression of other muscle-specific genes, such as alpha-skeletal actin and myosin heavy chain. Muscle cells were sensitive to TNF in a narrow temporal window of differentiation. Northern (RNA) blot and immunofluorescence analyses revealed that human muscle gene expression became unresponsive to TNF coincident with myoblast differentiation. When TNF was added to differentiated myotubes, there was no effect on muscle gene expression. In contrast, TNF-inducible mRNAs such as interferon beta-2 still responded, suggesting that the signal mediated by TNF binding to its receptor had no effect on muscle-specific genes after differentiation.