The rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the ability for the biosynthesis of long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors, and all the catalytic enzymes including two fatty acyl desaturase 2 (Δ4 Fads2 and Δ6/Δ5 Fads2) and two elongases (Elovl4 and Elovl5) have been identified, providing a good model for studying the regulatory mechanisms of LC-PUFA biosynthesis in fish. Stimulatory protein 1 (Sp1) has been speculated to be a vital transcription factor in determining the promoter activity of Fads-like genes in fish, however its regulatory effects on gene expression and LC-PUFA biosynthesis have not been demonstrated. Bioinformatic analysis predicted potential Sp1 binding sites in the promoters of the rabbitfish Δ6/Δ5 fads2 and elovl5, but not in Δ4 fads2 promoter. Here we cloned full-length cDNA of the rabbitfish sp1 gene, which encoded a putative protein of 701 amino acids, and was expressed in all tissues studied with highest levels in gill and eyes. The dual luciferase reporter assay in HepG2 line cells demonstrated the importance of the Sp1 binding site for the promoter activities of both Δ6/Δ5 fads2 and elovl5. Moreover, the electrophoretic mobility shift assay confirmed the direct interaction of Sp1 with the two promoters. Insertion of the Sp1 binding site of Δ6/Δ5 fads2 promoter into the corresponding region of the Δ4 fads2 promoter significantly increased activity of the latter. In the Siganus canaliculatus hepatocyte line (SCHL) cells, mRNA levels of Δ6/Δ5 fads2 and elovl5 were positively correlated with the expression of sp1 when sp1 was overexpressed or knocked-down by RNAi or antagonist (mithramycin) treatment. Moreover, overexpression of sp1 also led to a higher conversion of 18:2n−6 to 18:3n−6, 18:2n−6 to 20:2n−6, and 18:3n−3 to 20:3n−3, which related to the functions of Δ6/Δ5 Fads2 and Elovl5, respectively. These results indicated that Sp1 is involved in the transcriptional regulation of LC-PUFA biosynthesis by directly targeting Δ6/Δ5 fads2 and elovl5 in rabbitfish, which is the first report of Sp1 involvement in the regulation of LC-PUFA biosynthesis in vertebrates.

Elongases of very-long-chain fatty acids (Elovls) are critical rate-limiting enzymes that are involved in LC-PUFA biosynthesis through catalyzing the two-carbon elongation of a pre-existing fatty acyl chain. Thus far, several Elovls have been extensively studied in teleost. However, the functional and physiological roles of Elovls in chondrichthyans have rarely been reported. In this study, we identified and characterized elovl2 from the endangered Chinese sturgeon (Acipenser sinensis) by whole genome scanning. The results show that the coding sequence of elovl2 was 894 bp in length, for a putative protein of 297 amnio acids. Comparative genomic analyses indicated that Chinese sturgeon elovl2 was evolutionarily conserved. Functional characterization in yeast demonstrated that the Chinese sturgeon Elovl2 could efficiently elongate C₂₀ (ARA and EPA) and C₂₂ (22:4n-6 and 22:5n-3) substrates, confirming its critical roles in LC-PUFA biosynthesis. Spatial and temporal expression analyses showed high elovl2 mRNA levels were detected in the liver and brain and showed an increase trend both in embryonic and post-hatching stages. Interestingly, diets with vegetable oils as lipid sources could significantly induce the high expression of elovl2 in Chinese sturgeon, implying that the endogenous LC-PUFA biosynthesis pathway was stimulated by lack of LC-PUFA in their diets. Our findings will enhance our understanding about the evolutionary and functional roles of elovl2 and provide novel insights into the LC-PUFA biosynthesis mechanism in vertebrates.