Structural Characterization and Functional Analysis of the Follistatin Promoter of Larimichthys crocea

Follistatin is a secreted glycoprotein, which involved in numerous physiological activities as an antagonist of the transforming growth factor-beta superfamily. However, little is known about the regulation mechanism of follistatin in fish. In this study we cloned and analyzed part of the 5' flanking region of the follistatin gene in Larimichthys crocea. Sequence analysis revealed several putative binding sites for transcription factors including activator protein 1 (AP1), myogenic differentiation factor (MyoD), stimulating protein 1 (SP1), and sex determining gene on the mammalian Y chromosome (SRY) in the cloned fragment. Transcriptional activities of two fragments (485 and 261 bp) truncated from follistatin upstream region were examined in vitro, using transient transfection in Ctenopharyngodon idella kidney (CIK) and Rattus norvegicus skeletal muscle myoblast (L6) cells. The result showed that the promoter activity correlated positively with the length of truncated fragments in both CIK and L6 cells. To study the regulation of follistatin expression in L. crocea, we cloned MyoD and SRY-box 8 (Sox8) genes and examined their action on the follistatin promoter by co-transfection in CIK and L6 cells. The results showed MyoD and Sox8 could suppress the activities of follistatin promoter at different levels in CIK and L6 cells.