Analysis of star promoter in common carp and catfish testis: Role of c-jun and its association with testicular function as a transcription factor

Steroidogenic acute regulatory protein (Star) is a key factor that predominantly mediates the rate-limiting step of steroidogenesis which involved in the translocation of cholesterol from the outer to inner mitochondrial membrane in most of the vertebrates. To understand the transcriptional regulation of star (star1) in fish, the 5’ upstream region of the star was cloned from both common carp and catfish genomic libraries and in silico promoter motif analysis predicted putative binding sites of C-jun, AP-1, Gata2, and Oct1 in common carp while C-jun, Gata1, Gata2, and Oct1 binding sites were predicted in star promoter of catfish. Luciferase reporter assay through sequential deletion constructs in TM3 cells revealed significantly high promoter activity (P < 0.001) having C-jun site in both common carp and catfish but not with other motifs largely. Increased activity of star promoter was observed upon hCG induction, in vitro, indicating gonadotropin dependency. Site-directed mutagenesis and chromatin immunoprecipitation assays further confirmed the binding of C-jun to the upstream promoter region of star in both species. Expression of c-jun was significantly (P < 0.05) higher during the spawning phase of the testicular cycle in both species. Both c-jun and star were found to be gonadotropin inducible and androgen-dependent in both species. Further, transient gene silencing through c-jun-siRNA in both the species decreased expression of c-jun, star, and other AP-1 factors such as c-fos, junB, sox family genes, steroidogenic enzyme genes, sperm-related factor, testis-related genes, along with reduced testosterone and 11-ketotestosterone levels. The protein level of C-jun was decreased upon transient gene silencing in both species. This was the first report to show that C-jun binds to the upstream promoter motif of stars in both common carp and catfish and also adds valuable new insights that define the molecular mechanisms of c-jun regulating star transcription during steroidogenesis in teleost and probably testicular function.