Hepatocyte nuclear factor 4α (Hnf4α) is involved in transcriptional regulation of Δ6/Δ5 fatty acyl desaturase (Fad) gene expression in marine teleost Siganus canaliculatus

As the first marine teleost demonstrated to biosynthesize long-chain polyunsaturated fatty acids (LC-PUFAs) from C-18 precursors such as linoleic acid (LOA, 18:2n-6) and alpha-linolenic acid (ALA, 18:3n-3), the rabbitfish (Siganus canaliculatus) contains the complete enzymatic system for LC-PUFA biosynthesis, including Delta 6/Delta 5 fatty acid desaturase (Fad), Delta 4 Fad, and elongase 5 (Elovl5). Previously, our group demonstrated that hepatocyte nuclear factor 4 alpha (Hnf4 alpha) is a transcription factor (TF) for rabbitfish Delta 4 fad and elovl5, and interacts with the core promoter of Delta 6/Delta 5 fad. To fully clarify the role of Hnf4 alpha in the regulation of LC-PUFA biosynthesis, the present study aimed to explore the regulatory role of Hnf4 alpha on Delta 6/Delta 5 fad gene expression. First, Hnf4 alpha overexpression and agonist assays identified the Hnf4 alpha response region in the Delta 6/Delta 5 fad core promoter as - 456 bp to + 51 bp. Bioinformatic analysis predicted four potential Hnf4 alpha binding elements in the core promoter, which were confirmed by site-directed mutation and functional assays in a dual luciferase assay system. Moreover, the mRNA expression levels of hnf4 alpha, Delta 6/Delta 5 fad, and Delta 4 fad were significantly increased in the S. canaliculatus hepatocyte line (SCHL) cells after treatment with Hnf4 alpha agonists (Alverine and Benfluorex) or its mRNA overexpression. By contrast, the expression levels of these three genes were markedly decreased after hnf4 alpha small interfering RNA (siRNA) transfection. The results indicated that Hnf4 alpha has a regulatory effect on rabbitfish Delta 6/Delta 5 fad gene transcription, identifying Hnf4 alpha as a TF of Delta 6/Delta 5 fad in vertebrates for the first time.