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 m yotubes, 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 g ene (bp -124 to +1). In this report, we demonstrate that the cTnC gene contains a second distinct and independent transcriptional enhancer w hich is located in the first intron. This second enhancer is skeletal myotube specific and is developmentally up-regulated during the differ entiation of myoblasts to myotubes. This enhancer contains three funct ionally important nuclear protein binding sites: a CACCC box, a MEF-2 binding site, and a previously undescribed nuclear protein binding sit e, designated MEF-3, which is also present in a large number of skelet al muscle-specific transcriptional enhancers. Unlike most skeletal mus cle-specific transcriptional regulatory elements, the cTnC enhancer do es not contain a consensus binding site (CANNTG) for the basic helix-l oop-helix (bHLH) family of transcription factors and does not directly bind MyoD-E12 protein complexes. Despite these findings, the cTnC enh ancer can be transactivated by overexpression of the myogenic bHLH pro teins, 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 10 T1/2 cells into myoblasts and myotubes by stable transfection with a M yoD expression vector. In particular, MEF-2 binding activity was up-re gulated in 10T1/2 cells stably transfected with a MyoD expression vect or only after these cells fused and differentiated into skeletal myotu bes. 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 muscl e. In addition, they show that bHLH transcription factors can indirect ly transactivate the expression of some muscle-specific genes.