Cloning and characterization of Delta 6/Delta 5 fatty acyl desaturase (Fad) gene promoter in the marine teleost Siganus canaliculatus

The rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the ability of biosynthesizing long-chain polyunsaturated fatty acids (LC-PUFA) from C-18 PUFA precursors, and all genes encoding the key enzymes for LC-PUFA biosynthesis have been cloned and functionally characterized, which provides us a potential model to study the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. As the primary step to clarify such mechanisms, present research focused on promoter analysis of gene encoding Delta 6/Delta 5 fatty acyl desaturase (Fad), a rate-limiting enzyme catalyzing the first step in the conversion of C18 PUFA to LC-PUFA. First, 2044 bp promoter sequence was cloned by genome walking, and the sequence from -456 bp to +51 bp was determined as core promoter by progressive deletion mutation. Moreover, binding sites of transcription factors (TF) such as CCAAT enhancer binding protein (C/EBP), nuclear factor 1 (NF-1), stimulatory protein 1 (Sp1), nuclear factor Y (NF-Y), activated protein 1 (AP1), sterol regulatory element (SRE), hepatocyte nuclear factor 4 alpha (HNF4 alpha) and peroxisome proliferator activated receptor gamma (PPAR gamma) were identified in the core promoter by site-directed mutation and functional assays. Moreover, NF-1 and HNF4 alpha were confirmed to interact with the core promoter region by gel shift assay and mass spectrometry. This is the first report of the promoter structure of a Delta 6/Delta 5 Fad in a marine teleost, and a novel discovery of NF-1 and HNF4 alpha binding to the Delta 6/Delta 5 Fad promoter.