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

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 them 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.