In vivo regulation of the chicken cardiac troponin T gene promoter in zebrafish embryos

Abstract

The chicken cardiac troponin T (cTnT) gene is representative of numerous cardiac and skeletal muscle-specific genes that contain muscle-CAT (MCAT) elements within their promoters. We examined the regulation of the chicken cTnT gene in vivo in zebrafish embryos, and in vitro in cardiomyocyte, myoblast, and fibroblast cultures. Defined regions of the cTnT promoter were linked to the green fluorescent protein (GFP) gene for in vivo analysis, and the luciferase gene for in vitro analysis. Injection of the cTnT promoter constructs into fertilized zebrafish eggs resulted in GFP expression in both heart and skeletal muscle cells reproducing the pattern of expression of the endogenous cTnT gene in the chicken embryo. Promoter deletion analysis revealed that the cis-regulatory regions responsible for cardiac and skeletal muscle-specific expression functioned in an equivalent manner in both in vitro and in vivo environments. In addition, we show that mutation of the poly-ADP ribose polymerase-I (PARP-I) binding site adjacent to the distal MCAT element in the chicken cTnT promoter produced a non-cell-specific promoter in vitro and in the zebrafish. Thus, the PARP-I transcriptional regulatory mechanism that governs muscle specificity of the chicken cTnT promoter is conserved across several chordate classes spanning at least 350 million years of evolution.

Transcription Enhancer Factor-1-dependent Expression of the α-Tropomyosin Gene in the Three Muscle Cell Types

“…For instance, the coactivators TONDU and poly(ADP-ribose) polymerase have been described to interact with TEF-1 (69,73,74). In the latter case, the coactivator poly(ADP-ribose) polymerase has been shown to be essential for correct in vivo expression of the cardiac muscle TnT gene in zebrafish embryos (75). The potential involvement of such coactivators in activation of the ␣-Tm gene awaits additional experiments.…”

Sp3 inhibits Sp1-mediated activation of the cardiac troponin T promoter and is downregulated during pathological cardiac hypertrophy in vivo

“…Sp proteins bind GC boxes and the related GT/CACC-box motifs and are implicated in the recruitment and assembly of the basal transcriptional complex. GC boxes have been implicated in the regulation of cardiac promoters, but the direct effects of Sp family members on cardiac promoter activity have not been defined.We use the cardiac troponin T (cTnT) gene promoter as a model of cardiac-specific gene expression during myocardial differentiation (3,5,22,27,29). The cTnT promoter is developmentally regulated by numerous promoter motifs and transacting factors, including MCAT or transcription enhancer factor-1 (TEF-1) binding sites, AT-rich/monocyte enhancer factor-2 (MEF-2) binding sites, GATA motifs, and Nkx2.5.…”

“…We use the cardiac troponin T (cTnT) gene promoter as a model of cardiac-specific gene expression during myocardial differentiation (3,5,22,27,29). The cTnT promoter is developmentally regulated by numerous promoter motifs and transacting factors, including MCAT or transcription enhancer factor-1 (TEF-1) binding sites, AT-rich/monocyte enhancer factor-2 (MEF-2) binding sites, GATA motifs, and Nkx2.5.…”

Divergent transcriptional enhancer factor-1 regulates the cardiac troponin T promoter

“…The MCAT motif (CATTCCT) is present in two copies in the cTnT gene promoter, both of which are important for cardiac and skeletal muscle gene activation in vitro (21)(22)(23)(24) and in vivo (36). MCAT motifs are necessary for the activity of other cardiac promoters, including skeletal ␣-actin, ␤-MHC, ␣-MHC, troponin C, and myosin light chain (11-13, 17, 28 -30).…”