A novel myogenic regulatory circuit controls slow/cardiac troponin C gene transcription in skeletal muscle.

Abstract

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) The expression of many muscle-specific proteins is developmentally regulated at the level of transcription (7,34,39,66,85). Thus, one approach to understanding the molecular basis of mammalian myogenesis is to elucidate the transcriptional mechanisms that regulate the expression of muscle-specific genes. The identification and characterization of the basic helix-loop-helix (bHLH) family of muscle-determining transcription factors, including MyoD, myogenin, myf-5, and MRF4/herculin/myf-6, has added significantly to our understanding of skeletal myogenesis (1,3,10,17,49,61,80). Expression of each of these transcription factors appears to be sufficient to activate the skeletal muscle phenotype in many types of cultured cells (77), and hexanucleotide binding sites for these factors (CANNTG), termed E boxes, have been identified in most but not all skeletal muscle-specific transcriptional regulatory elements studied to date (reviewed in reference 71). bHLH proteins each contain a basic domain which is required for DNA binding and an HLH region which is involved in the formation of homo-and heterodimers (6,9,74). The basic domain of each myogenic bHLH family member contains conserved alanine and threonine residues that distinguish them from the nonmyogenic bHLH transcription factors (5, 9).

Differentiation-dependent mechanisms of transcriptional regulation of the catalytic subunit of phosphorylase kinase

“…These have been shown to be important for skeletal-and cardiac-muscle development [26,27]. The MEF-2 motif has been shown to regulate the cardiac troponin C gene [43] as well as the myogenin gene itself [44]. In the Phk γ subunit they appear to be providing yet a further differentiationdependent signal for expression (Figure 7).…”

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

“…While relatively little is understood about the molecular mechanisms that regulate cardiac muscle-specific gene expression, current developmental paradigms suggest that the regulation of cardiac musclespecific genes is likely to be controlled by the activity of cardiac muscle-specific transcription factors. We have used the cTnC gene as a model system with which to examine the molecular mechanisms that regulate cardiac and skeletal muscle transcription (52,54). The expression of the cTnC gene in cardiac myocytes both in vitro and in vivo is controlled by a transcriptional enhancer located in the immediate 5' flanking region of the gene (bp -124 to -79).…”

Identification of Weight-bearing-responsive Elements in the Skeletal Muscle Sarco(endo)plasmic Reticulum Ca2+ ATPase (SERCA1) Gene

“…Functional E-boxes are required for expression of many genes in adult tissue, including muscle creatine kinase (38), myosin light chain 1/3 fast (39), myosin heavy chain IIB (40), cardiac ␣-actin (27), and slow skeletal troponin I (31,32). Studies with cardiac troponin C have shown that overexpression of MyoD can stimulate transcription even in the absence of E-boxes (25). This can be explained by the fact that MyoD/E12 (or myogenein/ FIG.…”

“…These include 21 CACC sites (CACCC), 17 myogenic E-boxes (CAGNTG), 2 M-CAT sites (GGAATG), 4 NFAT sites (WGGAAANH), 1 Sp1 site (GGGCGG), 10 Sp1-like sites (GGGAGG), and 12 TRE half-sites (RGGTSA). Interestingly there are no MEF2 sites (YTAWWWWTAR), MEF3 sites (SSTCAGGTTWC), or CArG boxes (CCWWWWWWGG) previously shown to be important for transcription of some muscle genes (22)(23)(24)(25)(26)(27). Given the relatively high number of CACC sites and E-boxes, it is not surprising that these two elements were found to be necessary for transcriptional regulation of the SERCA1 gene with unloading.…”

“…Given the relatively high number of CACC sites and E-boxes, it is not surprising that these two elements were found to be necessary for transcriptional regulation of the SERCA1 gene with unloading. There is precedence in the literature for the regulatory pairing of CACC and MEF2 sites necessary for transcription of many muscle genes, including rat myosin light chain 2 slow (28), human myoglobin (24), rat muscle creatine kinase (29), mouse troponin C slow (25), and human ␤-enolase (30), but the regulatory pairing of CACC sites and E-boxes is less well defined (31,32).…”