Multiple transcription factors mediating the expressional regulation of myosin heavy chain gene involved in the indeterminate muscle growth of fish

Torafugu myosin heavy chain gene, MYHM2528-1, is specifically expressed in neonatal slow and fast muscle fibers, suggesting its functional role in indeterminate muscle growth in fish. However, the transcriptional regulatory mechanisms of MYHM2528-1 involved in indeterminate muscle growth in fish remained unknown. We previously isolated a 2100 bp 5'-flanking sequence of torafugu MYHM2528-1 that showed sufficient promoter activity to allow specific gene expression in neonatal muscle fibers of zebrafish. Here, we examined the cis-regulatory mechanism of 2100 bp 5'-flanking region of torafugu MYHM2528-1 using deletion-mutation analysis in zebrafish embryo. We discovered that myoblast determining factor (MyoD) binding elements play a key role and participate in the transcriptional regulation of MYHM2528-1 expression in zebrafish embryos. We further discovered that paired box protein (Pax3) are required for promoting MYHM2528-1 expression and myocyte enhancer factor 2 (MEF2) binding sites participate in the transcriptional regulation of MYHM2528-1 expression in slow/fast skeletal muscles. Our study also confirmed that the nuclear factor of activated T-cell (NFAT) binding sites take part in the transcriptional regulation of MYHM2528-1 expression in slow and fast muscles fiber in relation to indeterminate muscle growth. These results obviously confirmed that multiple cis-elements in the 5'-flanking region of MYHM2528-1 function in the transcriptional regulation of its expression.