Myocyte enhancer factor 2 regulates expression of medaka Oryzias latipes fast skeletal myosin heavy chain genes in a temperature-dependent manner.

We characterized the promoter activity of fast skeletal myosin heavy chain genes (MYHs) from medaka Oryzias latipes. The 5'-flanking region of approximately 6 kb in medaka MYHs, mMYH10 and mMYH30, predominantly expressed in medaka acclimated to 10 degrees C and 30 degrees C, respectively, contained various cis-elements that are supposed to bind to transcriptional regulatory factors such as MyoD and myocyte enhancer factor 2 (MEF2) family members and nuclear factor of activated T cells. To localize functional regions responsible for the mMYH expression in a temperature-dependent manner, a series of deletion and site mutation constructs prepared from the 5'-flanking regions were fused to the luciferase gene in a commercially available plasmid and directly injected into the dorsal fast muscle of medaka acclimated to 10 degrees C and 30 degrees C. The truncation of MEF2 binding site located at -966 to -957 in the 5'-flanking region of mMYH10 resulted in distinct gene expression at 10 degrees C. The activation effect by the removal of this binding site was further confirmed by the mutation construct. One of the E box sites, to which MyoD family members are supposed to bind, was located at -613 to -607 of mMYH10, and found to be responsible for the transcriptional activity. In contrast, the MEF2 binding site located at -960 to -951 of mMYH30 was involved in the activation at 30 degrees C. Thus, these transient transfection assays demonstrated that the MEF2 binding site is crucial for a temperature-dependent expression of mMYHs.