Abstract
Δ6 fatty acyl desaturase (Fads2) is a rate-limiting enzyme in long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis. Comparative analysis of gene promoters of Fads2 between salmonids and carnivorous marine fish suggested that the lack of binding site for stimulatory protein 1 (Sp1) was responsible for the low expression of fads2 gene of carnivorous marine species. To confirm this speculation, the fads2 candidate promoter (2646 bp) was cloned from carnivorous marine teleost Epinephelus coioides, and 330 bp core regulatory region was identified. Several binding sites for transcriptional factors such as nuclear factor 1, nuclear factor Y, sterol regulatory element and hepatocyte nuclear factor 4γ were identified, while that for Sp1 was shown to be absent in the promoter by both bioinformatic analysis and site-directed mutation. Moreover, after the Sp1-binding site from the fads2 promoter of herbivorous Siganus canaliculatus, the first marine teleost demonstrated to have LC-PUFA biosynthetic ability, was inserted into the corresponding region of E. coioides fads2 promoter, activity was significantly increased. The results provided direct data for the importance of the Sp1-binding site in determining fads2 promoter activity, and indicated that its lack may be a reason for low expression of fads2 and poor LC-PUFA biosynthetic ability in E. coioides.
Highlights
The long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis pathway involves consecutive desaturation and elongation steps from LNA and LA catalyzed by fatty acyl desaturase (Fads) and elongation of very long-chain fatty acids (Elovl) enzymes[9]
The 790 bp 5′-UTR sequence consists of exon 1, intron 1 and partial sequence of exon 2, the schematic diagram of fads[2] promoter structure was shown in Fig. 2 and Supplementary Fig. S1
The results showed that mutation of binding sites for NF1, NF-Y, HNF4γ, RXR::VDR, TBP and YY1(two of three) caused significant reduction of promoter activity (Fig. 4), indicating these sites are key elements in fads[2] promoter of E. coioides
Summary
The LC-PUFA biosynthesis pathway involves consecutive desaturation and elongation steps from LNA and LA catalyzed by fatty acyl desaturase (Fads) and elongation of very long-chain fatty acids (Elovl) enzymes[9]. The comparative analysis of fads[2] gene promoter between Salmo salar (with LC-PUFA biosynthetic ability) and Gadus morhua (a carnivorous marine teleost with very limited LC-PUFA biosynthesis) suggested that low expression of fads[2] in the latter could be attributed, at least partly, to the lack of a binding site of stimulatory protein 1 (Sp1) in the fads[2] promoter[17]. It was speculated that the Sp1 site may play an important role in determining the fads[2] promoter activity and LC-PUFA biosynthetic ability in fish[11,17]. In order to investigate the underlying reasons, the present study was focused on clarifying the importance of the Sp1 binding site in determining the transcriptional activity of the fads[2] promoter, as speculated in marine carnivorous G. morhua and D. labrax. The results will be helpful for identifying the reasons underpinning the low LC-PUFA biosynthetic ability of E. coioides, and will provide novel insights into the regulatory mechanisms of LC-PUFA biosynthesis in vertebrates
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