Multiple cis-elements in the 5'-flanking region of embryonic/larval fast-type of the myosin heavy chain gene of torafugu, MYH(M743-2), function in the transcriptional regulation of its expression

Abstract

The myosin heavy chain gene, MYH(M743-2), is highly expressed in fast muscle fibers of torafugu embryos and larvae, suggesting its functional roles for embryonic and larval muscle development. However, the transcriptional regulatory mechanism involved in its expression remained unknown. Here, we analyzed the 2075bp 5'-flanking region of torafugu MYH(M743-2) to examine the spatial and temporal regulation by using transgenic and transient expression techniques in zebrafish embryos. Combining both transient and transgenic analyses, we demonstrated that the 2075bp 5'-flanking sequences was sufficient for its expression in skeletal, craniofacial and pectoral fin muscles. The immunohistochemical observation revealed that the zebrafish larvae from the stable transgenic line consistently expressed enhanced green fluorescent protein (EGFP) in fast muscle fibers. Promoter deletion analyses demonstrated that the minimum 468bp promoter region could direct MYH(M743-2) expression in zebrafish larvae. We discovered that the serum response factor (SRF)-like binding sites are required for promoting MYH(M743-2) expression and myoblast determining factor (MyoD) and myocyte enhancer factor-2 (MEF2) binding sites participate in the transcriptional control of MYH(M743-2) expression in fast skeletal muscles. We further discovered that MyoD binding sites, but not MEF2, participate in the transcriptional regulation of MYH(M743-2) expression in pectoral fin and craniofacial muscles. These results clearly demonstrated that multiple cis-elements in the 5'-flanking region of MYH(M743-2) function in the transcriptional control of its expression.