Functional analysis of two MyoDs revealed their role in the activation of myomixer expression in yellowfin seabream (Acanthopagrus latus) (Hottuyn, 1782).

ABSTRACT

Myoblast determination protein (MyoD), a muscle-specific basic helix-loop-helix (bHLH) transcription factor, plays a pivotal role in regulating skeletal muscle growth and development. However, the regulation mechanism of MyoD has not been determined in marine fishes. In the present study, we isolated the MyoD1 (AlMyoD1) and MyoD2 (AlMyoD2) genomic sequences and analyzed the expression patterns in different tissues of yellowfin seabream (Acanthopagrus latus). The open reading frame (ORF) sequences of AlMyoD1 and AlMyoD2 encoded 297 and 271 amino acids possessing three common characteristic domains, respectively, containing a myogenic basic domain, a bHLH domain, and a ser-rich region (helix III). Phylogenetic and genome structure analyses exhibited classic phylogeny and highly conserved exon/intron architecture. Furthermore, the AlMyoD1 and AlMyoD2 transcription levels were higher in white muscle than in the other tissues. In order to further study AlMyoD function in muscle, promoter sequence analysis found that several E-box binding sites were present. Additionally, binding sites of Almyomixer involved in mammal myoblast fusion, which expression was also the highest in white muscle, were found in the promoter of AlMyoD. Pomoter activity assays further confirmed that both AlMyoD1 and AlMyoD2 can dramatically activate Almyomixer expression, and the AlMyoD1 M2 and AlMyoD2 M5 E-box binding sites were functionally important for Almyomixer transcription based on mutation analysis and electrophoretic mobile shift assays (EMSA). In summary, two MyoDs play a core role in Almyomixer regulation and may promote myofibre formation during muscle development and growth by regulating Almyomixer expression.