In vivo analysis of two striated muscle actin promoters reveals combinations of multiple regulatory modules required for skeletal and cardiac muscle-specific gene expression

We isolated two striated muscle actin genes from medaka Oryzias latipes. OIMA1 is a skeletal muscle actin gene expressed in somitic muscle and head muscle and OIMA2 is probably a cardiac muscle actin gene expressed in both somitic and cardiac muscle. The differential transcription mechanisms for these two genes were examined in embryos by introducing fusion genes in which the OIMA1 or OIMA2 upstream region was connected to the green fluorescent protein gene. Embryos were injected with these fusion genes at the 2-cell stage. A fusion gene containing the region up to -949 of OIMA1 exhibited strong expression in somitic muscle. The coexistence of two regions, -949/-662 and -421/-201, is necessary for skeletal muscle-specific expression of OIMA1. Two E boxes and other unidentified sequences cooperatively function to achieve the full activity of the enhancer -949/-662. As for OIMA2, the region up to -520 is sufficient for strong muscle-specific expression. The region between -520 and -174 of OIMA2 is necessary for specific expression in both skeletal and cardiac muscles. In addition to the CArG box located at -140, an E-box at -430 is important for the expression in cardiac muscle as well as skeletal muscle. When the enhancers for the two muscle actin genes were switched and combined with each other's promoter, they were able to upregulate tissue-specific expression according to their origin. These results suggest that distinct expression patterns of OIMA land OIMA2 are regulated by combination of regulatory modules, each of which contains multiple regulatory elements.