C2C12 Myogenic Cells Research Articles

Cis-acting DNA elements regulating expression of the liver pyruvate kinase gene in hepatocytes and hepatoma cells. Evidence for tissue-specific activators and extinguisher.

To identify the DNA sequences that cis-regulate the expression of the rat liver pyruvate kinase (L-PK) genes, a series of constructs in which the chloramphenicol acetyltransferase reporter genes is driven by various deleted fragments of the 3200 base pairs (bp) upstream of the L-PK gene cap site have been assayed for transient expression after introduction into hepatoma HepG2 cells, rat hepatocytes in primary culture, fibroblast LTK- cells, myogenic C2C12 cells, and CHO cells. Four distinct regulatory domains have been characterized. A proximal promoter region containing a binding site for the hepatocyte nuclear factor 1 (HNF1) which is sufficient to confer liver specificity, even in the presence of a ubiquitous enhancer. A distal promoter region (-96 to -283 bp) containing binding sites for the liver-specific factor A1 (LFA1), the ubiquitous nuclear factor 1 (NF1), the major late transcriptional factor (MLTF), and so far unidentified proteins binding to the L5-PK region which is essential to maximally activate expression of the construct in HepG2 cells. An extinguisher region, located between positions -2082 and -1170 bp, which decreases efficiency of the L-PK promoter in HepG2 cells, but not in hepatocytes in primary culture. Finally, a far upstream region (-2900 to -2500 bp) which seems to correspond to a liver-specific DNase I hypersensitive site and which behaves in HepG2 cells as an activating sequence efficient in the absence of the extinguisher.

Multiple positive and negative elements regulate human brain creatine kinase gene expression

We characterized the developmental expression of the brain creatine kinase (BCK) gene in the C2C12 myogenic cell line with the use of isoenzyme, Western blot, and Northern blot analyses. The results show that both BCK subunit protein and mRNA are upregulated early in myogenesis, and then downregulated in fully differentiated myotubes. To characterize the transcriptional regulatory mechanisms, a chimeric construct containing 1.2 kilobase pairs of 5'-flanking DNA from the human BCK gene placed upstream of the chloramphenicol acetyltransferase gene in the promoterless plasmid pSVOCAT was transiently transfected into C2C12 cells. In myoblasts and differentiating myotubes, the time course of expression of the constructs paralleled that of endogenous BCK mRNA. Additional constructs prepared by deleting 5'-flanking DNA were also transfected into C2C12 cells. All constructs were preferentially expressed in myoblasts relative to myotubes with absolute levels of expression increasing with deletion of 5'-flanking DNA. In nonmyogenic cells expression of the plasmids also increased with deletion of 5'-flanking DNA. An element from -1150 to -388 was isolated and found to be capable of suppressing expression of the BCK promoter and of heterologous promoters independent of orientation and position and hence to function as a silencer. Thus, BCK expression is mediated by sequences contained in the 5'-flanking DNA, including negative elements active in both C2C12 cells and nonmyogenic cells and elements that mediate the developmental expression of the BCK gene in C2C12 myogenic cells.

OXBOX, a positive transcriptional element of the heart-skeletal muscle ADP/ATP translocator gene

Three positive transcriptional control regions have been identified in the promoter of the human heart-skeletal muscle adenine nucleotide translocator gene (ANT1). By transfecting promoter-chloramphenicol acetyltransferase fusion constructs into C2C12 myogenic cells, each positive region was found to increase transcription 2-3-fold. The first region spans from -123 to -674 base pairs (bp), the second from -2.6 to -3.1 kilobases, and the third from -3.1 to -8.8 kilobases. Linker-scanning mutants generated using the polymerase chain reaction and modified oligonucleotides have identified the OXBOX (5'-GGCTCTAAAGAGG) as the positive element within the -123 to -674-bp region. This element enhances transcription in muscle cells but not in HeLa cells, suggesting that it is muscle-specific. Gel retardation experiments have revealed a factor from C2C12 cells which specifically binds to a 40-bp piece of the ANT1 promoter containing the OXBOX. Since the OXBOX is also found in the promoter of the human ATP synthase beta subunit gene, it is the first tissue-specific element identified which could coordinately regulate mitochondrial oxidative phosphorylation genes.

The upstream muscle-specific enhancer of the rat muscle creatine kinase gene is composed of multiple elements.

A series of constructs that links the rat muscle creatine kinase promoter to the bacterial chloramphenicol acetyltransferase gene was generated. These constructs were introduced into differentiating mouse C2C12 myogenic cells to localize sequences that are important for up-regulation of the creatine kinase gene during myogenic differentiation. A muscle-specific enhancer element responsible for induction of chloramphenicol acetyltransferase expression during myogenesis was localized to a 159-base-pair region from 1,031 to 1,190 base pairs upstream of the transcription start site. Analysis of transient expression experiments using promoters mutated by deletion indicated the presence of multiple functional domains within this muscle-specific regulatory element. A DNA fragment spanning this region was used in DNase I protection experiments. Nuclear extracts derived from C2 myotubes protected three regions (designated E1, E2, and E3) on this fragment from digestion, which indicated there may be three or more trans-acting factors that interact with the creatine kinase muscle enhancer. Gel retardation assays revealed that factors able to bind specifically to E1, E2, and E3 are present in a wide variety of tissues and cell types. Transient expression assays demonstrated that elements in regions E1 and E3, but not necessarily E2, are required for full enhancer activity.

Differential expression of the Na,K-ATPase alpha 1 and alpha 2 subunit genes in a murine myogenic cell line. Induction of the alpha 2 isozyme during myocyte differentiation.

Expression of Na,K-ATPase catalytic alpha isoform (alpha 1, alpha 2, and alpha 3) and beta subunit genes in rodent muscle was investigated using the murine C2C12 myogenic cell line. RNA blot analyses of myoblasts revealed expression primarily of the alpha 1 mRNA and low levels of alpha 2 mRNA. Fusion of the proliferating myoblasts to form myotubes was accompanied by an approximate 12-fold induction of the alpha 2 mRNA. In contrast, expression of alpha 1 mRNA remained constant throughout myogenesis. The alpha 3 mRNA was not detected in either myoblasts or myotubes. The beta mRNA abundance also increased 2-3-fold during myotube formation. In rodent tissues, low and high affinity cardiac glycoside (e.g. ouabain) receptors have been shown to be associated with the Na,K-ATPase catalytic alpha 1 and alpha 2 isoform subunits, respectively. The existence of these two functional classes of Na,K-ATPase in myoblasts and myotubes correlated with the biphasic ouabain inhibition of Na,K-ATPase activity. Confluent myoblasts expressed primarily the alpha 1 isozyme (IC50 = 3.6 X 10(-5) M; 95% of total activity) and lesser amounts of the alpha 2 isozyme (IC50 = 1.1 X 10(-7) M; 5% of total activity). In contrast, the myotubes showed significant levels of the alpha 1 isozyme (IC50 = 4.0 X 10(-5) M; 68% of total activity) and, in addition, showed a 6-fold increase in the relative levels of the alpha 2 isozyme (IC50 = 1.1 X 10(-7) M; 32% of total activity). To quantitate further the expression of the high affinity, ouabain-sensitive alpha 2 isozyme, a whole cell [3H]ouabain-binding assay was used. Results revealed that myotubes have an approximately 6-fold greater concentration of [3H]ouabain-binding sites than myoblasts with an apparent dissociation constant (Kd) of 1.4 X 10(-7) M. The results indicate that muscle cells can express multiple isozymes of Na,K-ATPase and that expression of the alpha 2 isozyme is developmentally regulated during myogenesis.