Identification and characterization of a cardiac-specific transcriptional regulatory element in the slow/cardiac troponin C gene.

Abstract

The slow/cardiac troponin C (cTnC) gene has been used as a model system for defining the molecular mechanisms that regulate cardiac and skeletal muscle-specific gene expression during mammalian development. cTnC is expressed continuously in both embryonic and adult cardiac myocytes but is expressed only transiently in embryonic fast skeletal myotubes. We have reported previously that cTnC gene expression in skeletal myotubes is controlled by a developmentally regulated, skeletal muscle-specific transcriptional enhancer located within the first intron of the gene (bp 997 to 1141). In this report, we show that cTnC gene expression in cardiac myocytes both in vitro and in vivo is regulated by a distinct and independent transcriptional promoter and enhancer located within the immediate 5' flanking region of the gene (bp -124 to +32). DNase I footprint and electrophoretic mobility shift assay analyses demonstrated that this cardiac-specific promoter/enhancer contains five nuclear protein binding sites (designated CEF1, CEF-2, and CPF1-3), four of which bind novel cardiac-specific nuclear protein complexes. Functional analysis of the cardiac-specific cTnC enhancer revealed that mutation of either the CEF-1 or CEF-2 nuclear protein binding site abolished the activity of the cTnC enhancer in cardiac myocytes. Taken together, these results define a novel mechanism for developmentally regulating a single gene in multiple muscle cell lineages. In addition, they identify previously undefined cardiac-specific transcriptional regulatory motifs and trans-acting factors.

The GATA-4 transcription factor transactivates the cardiac muscle-specific troponin C promoter-enhancer in nonmuscle cells.

“…The cTnC enhancer contains two functionally important nuclear protein-binding sites designated CEF-1 and CEF-2. Both sites have been shown to bind cardiac muscle-restricted nuclear protein complexes (54). In this report, we demonstrate that GATA-4 binds specifically to the CEF-1 site of the cTnC enhancer and that GATA-4 is the major CEF-1-binding activity present in cardiac nuclear extracts.…”

“…The murine slow/cardiac troponin C (cTnC) gene has been used as a model system with which to examine the molecular mechanisms that regulate cardiac muscle-specific transcription during mammalian development (53,54,66). cTnC gene expression is restricted to adult slow skeletal and cardiac muscle and is controlled at the level of transcription (53,66).…”

“…We have previously identified a cardiac muscle-specific transcriptional enhancer located within the immediate 5' flanking region of the cTnC gene (bp -79 to -124). This enhancer functions in concert with the cTnC promoter to program high-level transcription both in cultured cardiac myocytes and in cardiac muscle in vivo (54). The cTnC enhancer contains two functionally important nuclear protein-binding sites designated CEF-1 and CEF-2.…”

“…NIH 3T3 and COS-7 cells were grown in Dulbecco's modified Eagle's medium-10% fetal bovine serum-1% penicillin and streptomycin (GIBCO, Gaithersburg, Md.). Primary cultures of neonatal rat cardiac myocytes and fibroblasts were isolated and grown as described previously (54).…”

“…The promoterless control plasmid pCAT-Basic (Promega, Madison, Wis.) and plasmid p-124CAT, containing the 156-bp (bp -124 to +32) cTnC cardiac muscle-specific promoter and enhancer subcloned 5' of the chloramphenicol acetyltransferase (CAT) reporter gene, have been described previously (54). Plasmid p-124CATmCEF-1, containing five nucleotide substitutions within the CEF-1 GATA-4-binding site within the context of the 156-bp cTnC promoter-enhancer, was constructed by using PCR-mediated site-directed mutagenesis (28) and subcloned into HindIII-XbaI-digested pCAT-Basic as described previously (54). The sequence of the mutated 156-bp fragment was confirmed by dideoxy sequence analysis.…”