Functional characterization of promoter regions in selenoprotein synthesis-relevant genes (sbp2, eefsec and sepsecs) and their selenium-dependent regulation in yellow catfish Pelteobagrus fulvidraco

Abstract

The study explored the transcriptional regulation of selenoprotein synthesis-relevant genes, such as selenocysteine insertion sequence element binding protein 2 (sbp2), eukaryotic elongation factor (eefsec) and o-phosphoserine selenocysteine tRNA synthase (sepsecs), and their selenium-mediated regulation in yellow catfish Pelteobagrus fulvidraco, an important fish with ecological and economic importance in several Asian countries. We cloned the sequences of sbp2, eefsec and sepsecs promoters, spanning from −2060 bp to +61 bp, −1910 bp to +53 bp and − 1456 bp to +51 bp relative to the TSS, respectively. Through sequential deletion and mutation analysis of their promoters, we identified several functional binding sites: the signal transducer and activator of transcription 1 (STAT1) binding site (−1308 bp to −1322 bp) and the forkhead box protein O1 (FOXO1) binding site (−1778 bp to −1788 bp) in the sbp2 promoter; the FOXO1 binding site (−1070 bp to −1080 bp) and the STAT3 binding site (−428 bp to −436 bp) in the eefsec promoter; and the FOXO1 binding site (−721 bp to −731 bp) in the sepsecs promoter. The activity of these binding sites was regulated by selenomethionine (Se-Met) incubation. Furthermore, electrophoretic mobility shift assay and chromatin immunoprecipitation experiments confirmed that these binding sites interact with their corresponding transcription factors above. For the first time, we demonstrated that STAT1 and FOXO1 regulate transcriptional activity of sbp2 promoter; STAT3 and FOXO1 regulate transcriptional activity of eefsec promoter; and FOXO1 regulates transcriptional activity of sepsecs promoter. These findings provide novel insights into regulatory mechanisms of selenoprotein synthesis in yellow catfish.