A novel myogenic regulatory circuit controls slow/cardiac troponin C gene transcription in skeletal muscle.
The slow/cardiac troponin C (cTnC) gene is expressed in three distinct striated muscle lineages: cardiac myocytes, embryonic fast skeletal myotubes, and adult slow skeletal myocytes. We have reported previously that cTnC gene expression in cardiac muscle is regulated by a cardiac-specific promoter/enhancer located in the 5' flanking region of the gene (bp -124 to +1). In this report, we demonstrate that the cTnC gene contains a second distinct and independent transcriptional enhancer which is located in the first intron. This second enhancer is skeletal myotube specific and is developmentally up-regulated during the differentiation of myoblasts to myotubes. This enhancer contains three functionally important nuclear protein binding sites: a CACCC box, a MEF-2 binding site, and a previously undescribed nuclear protein binding site, designated MEF-3, which is also present in a large number of skeletal muscle-specific transcriptional enhancers. Unlike most skeletal muscle-specific transcriptional regulatory elements, the cTnC enhancer does not contain a consensus binding site (CANNTG) for the basic helix-loop-helix (bHLH) family of transcription factors and does not directly bind MyoD-E12 protein complexes. Despite these findings, the cTnC enhancer can be transactivated by overexpression of the myogenic bHLH proteins, MyoD and myogenin, in C3H10T1/2 (10T1/2) cells. Electrophoretic mobility shift assays demonstrated changes in the patterns of MEF-2, CACCC, and MEF-3 DNA binding activities following the conversion of 10T1/2 cells into myoblasts and myotubes by stable transfection with a MyoD expression vector. In particular, MEF-2 binding activity was up-regulated in 10T1/2 cells stably transfected with a MyoD expression vector only after these cells fused and differentiated into skeletal myotubes. Taken together, these results demonstrated that distinct lineage-specific transcriptional regulatory elements control the expression of a single myofibrillar protein gene in fast skeletal and cardiac muscle. In addition, they show that bHLH transcription factors can indirectly transactivate the expression of some muscle-specific genes.
Parmacek
M S
MS
Department of Medicine, University of Chicago, Illinois 60637.
Ip
H S
HS
Jung
F
F
Shen
T
T
Martin
J F
JF
Vora
A J
AJ
Olson
E N
EN
Leiden
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D018257:Helix-Loop-Helix Motifs; D007438:Introns; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D009132:Muscles / Q000502:physiology*; D017570:MyoD Protein / Q000378:metabolism; D018008:Myogenin / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014336:Troponin / Q000235:genetics*; D019209:Troponin C
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D017570:MyoD Protein; C503801:Myog protein, mouse; D018008:Myogenin; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D014336:Troponin; D019209:Troponin C