The structure and regulation of expression of the murine fast skeletal troponin C gene. Identification of a developmentally regulated, muscle-specific transcriptional enhancer.
Fast skeletal muscle troponin C (sTnC) is the calcium-binding subunit of the myofibrillar thin filament that regulates excitation-contraction coupling. Utilizing a polymerase chain reaction cloning strategy, we have isolated cDNA clones encoding murine sTnC. The 160-amino acid sTnC protein shares 70% amino acid sequence identity with the slow/cardiac isoform of troponin C (cTnC). However, three areas of significant sequence divergence were identified. Southern blot analyses demonstrated that murine sTnC is encoded by a single copy gene that is distinct from that which encodes cTnC. Northern blot analyses showed that the sTnC gene is expressed exclusively in skeletal muscle (extensor digitorum and anterior tibialis) and not in neonatal or adult heart, brain, kidney, liver, lung, or testes. Studies of the murine C2C12 myoblast cell line demonstrated that sTnC gene expression is developmentally regulated during the differentiation of these myoblasts into myotubes. A full-length murine sTnC genomic clone was isolated and characterized by DNA sequence, primer extension, and S1 nuclease protection analyses. The sTnC gene is composed of six exons spanning 2.6 kilobase pairs of genomic DNA. Although the introns do not divide the gene into functional domains, the intron-exon borders are nearly identical to those of the other members of the troponin C multigene family. Transient transfection assays using chloramphenicol acetyltransferase reporter plasmids demonstrated that the sTnC promoter alone is relatively inactive in muscle cells and that high level sTnC gene expression in these cells is controlled by a potent transcriptional enhancer element located within the first intron of the gene. In additional transfection experiments, the sTnC enhancer was shown to display three important biological activities. (i) It was required for high level transcription from the sTnC promoter in muscle cells; (ii) its activity was muscle cell specific; and (iii) its activity was developmentally regulated during the differentiation of C2C12 myoblasts to myotubes. Taken together, these data define the sTnC gene as an excellent model system for studies of developmentally regulated gene expression in skeletal muscle.
Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor.
M S
MS
Parmacek
A R
AR
Bengur
A J
AJ
Vora
J M
JM
Leiden
D000375:Aging; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D005796:Genes; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D024510:Muscle Development; D015345:Oligonucleotide Probes; D009928:Organ Specificity; D015183:Restriction Mapping; D013045:Species Specificity; D014158:Transcription, Genetic; D014162:Transfection; D014336:Troponin / Q000235:genetics*; D019209:Troponin C
D015345:Oligonucleotide Probes; D014336:Troponin; D019209:Troponin C; D004247:DNA