We characterized the promoter activity of fast skeletal myosin heavy chain genes (MYHs) from medaka Oryzias latipes. The 5′-flanking region of ∼ 6 kb in medaka MYHs, mMYH10 and mMYH30, predominantly expressed in medaka acclimated to 10 °C and 30 °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 °C and 30 °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 °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 °C. Thus, these transient transfection assays demonstrated that the MEF2 binding site is crucial for a temperature-dependent expression of mMYHs.
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