Possible involvement of Jun, Foxo1, Nobox in the regulation of smad2/3 transcription in the protogynous hermaphroditic ricefield eel (Monopterus albus)

Small mothers against decapentaplegic2/3 (Smad2/3) are two signalling intermediates in the transforming growth factor beta (TGF-β) signalling pathway, which plays a critical role in gonad development. However, a systemic analysis of Smad2/3 in Monopterus albus (M. albus) has not been performed. The smad2 sequence in M. albus was characterized in our previous publication. In this study, we characterized the nucleotide sequence of smad3 and reported that smad3 is widely expressed in different tissues of M. albus. Importantly, the expression of smad3 significantly increased at the early vitellogenic stage in the ovary and decreased with oocyte maturation. Next, we aimed to identify the upstream factors that regulate Smad2/3 transcription. The 5′ flanking sequences of smad2/3 were cloned, and their core promoter regions were determined to be between −447 bp and −100 bp and between −1520 bp and −927 bp, respectively. The Jun and Foxo1 binding sites were conserved in the smad2 promoter region, and the Sp1, Bcl-6, and Nobox binding sites were conserved in the smad3 promoter region. Through dual-luciferase reporter gene experiments, we showed that Jun and Foxo1 binding sites may be involved in the regulation of smad2 transcription, whereas the Nobox binding site may be involved in the activation of the smad3 promoter.