Hepatocyte Nuclear Factor 4α (HNF4α) Is a Transcription Factor of Vertebrate Fatty Acyl Desaturase Gene as Identified in Marine Teleost Siganus canaliculatus

Abstract

Rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the capability of biosynthesizing long-chain polyunsaturated fatty acids (LC-PUFA) from C18 precursors, and to possess a Δ4 fatty acyl desaturase (Δ4 Fad) which was the first report in vertebrates, and is a good model for studying the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. In order to understand regulatory mechanisms of transcription of Δ4 Fad, the gene promoter was cloned and characterized in the present study. An upstream sequence of 1859 bp from the initiation codon ATG was cloned as the promoter candidate. On the basis of bioinformatic analysis, several binding sites of transcription factors (TF) including GATA binding protein 2 (GATA-2), CCAAT enhancer binding protein (C/EBP), nuclear factor 1 (NF-1), nuclear factor Y (NF-Y), hepatocyte nuclear factor 4α (HNF4α) and sterol regulatory element (SRE), were identified in the promoter by site-directed mutation and functional assays. HNF4α and NF-1 were confirmed to interact with the core promoter of Δ4 Fad by gel shift assay and mass spectrometry. Moreover, over-expression of HNF4α increased promoter activity in HEK 293T cells and mRNA level of Δ4 Fad in rabbitfish primary hepatocytes, respectively. The results indicated that HNF4α is a TF of rabbitfish Δ4 Fad. To our knowledge, this is the first report on promoter structure of a Δ4 Fad, and also the first demonstration of HNF4α as a TF of vertebrate Fad gene involved in transcription regulation of LC-PUFA biosynthesis.

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

“…Genomic DNA was extracted from muscle tissue of rabbitfish S. canaliculatus (Joseph Sambrook, 2001) and used for thermal asymmetric interlaced PCR (TAIL PCR) to clone the Δ6/Δ5 Fad gene promoter with forward primer AP4 in the Genome Walking Kit (TaKaRa, Dalian, China) and three reverse primers SP1, SP2, SP3 (Table 1), which was designed from the cloned Δ6/Δ5 Fad mRNA of S. canaliculatus (GenBank: EF424276.2). The PCR method was the same as we used before (Dong et al, 2016). Fragments of upstream sequence were purified and inserted into pMD18-T vector (TaKaRa, Dalian, China), and sequenced (Shanghai Sangon Biotech Co., Ltd, China).…”

“…canaliculatus (Dong et al, 2016), Δ6 Fad promoter sequences of Dicentrarchus labrax (Geay et al, 2012), Gadus morhua (Zheng et al, 2009), Salmo salar (Zheng et al, 2009), and Homo sapiens (Tang et al, 2003), and ∆6/∆5 desaturase promoter sequence from D. rerio genome Site-directed mutation of recombinant plasmids was carried out to detect the potential functions of predicted TF elements on core promoter activity. For the Δ6/Δ5 Fad promoter, site-directed mutants were designed and produced with Muta-direct TM site-directed mutagenesis kit (SBS Genetech, Shanghai, China) from the deletion mutant D2 construct (pGL4.10-D2) which was treated as wild-type.…”

“…These provide us a good model for investigating the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. Recently, studies at transcriptional levels have demonstrated the roles of HNF4α and Lxr-Srebp in Fad expression (Dong et al, 2016;Zhang et al, 2016a). At post-transcriptional level, miR17 targeted at the 3' untranslated region (3' UTR) of Δ4 Fad and downregulated its gene expression, and miR-33 was involved in the regulation of LC-PUFA biosynthesis probably through targeting insig1 in rabbitfish (Zhang et al, 2016b).…”

“…At post-transcriptional level, miR17 targeted at the 3' untranslated region (3' UTR) of Δ4 Fad and downregulated its gene expression, and miR-33 was involved in the regulation of LC-PUFA biosynthesis probably through targeting insig1 in rabbitfish (Zhang et al, 2016b). Moreover, the structure of rabbitfish Δ4 Fad promoter was characterized and HNF4α was identified as a TF of Fad genes, for the first time in vertebrates (Dong et al, 2016) .…”

Comparison of the growth performance and long-chain PUFA biosynthetic ability of the genetically improved farmed tilapia (Oreochromis niloticus) reared in different salinities

“…So investigation of upregulation of the expression of the desaturases and elongase to improve the endogenous LC-PUFA biosynthetic ability of fish is attracting more attention. At a molecular level, the expression of desaturases and elongases is reported to be modulated at the transcriptional level by key transcription factors such as PPAR, sterol regulatory element-binding protein-1 (SREBP-1), liver X receptor (LXR) and hepatocyte nuclear factor 4α (HNF4α) in vertebrates (10)(11)(12)(13)(14)(15) . For example, the expression of the Δ5 and Δ6 desaturases and Elovl5 is stimulated to promote the production of LC-PUFA through the LXRα-SREBP-1 pathway in mammals and fish (10,11) , while PPAR are reported to stimulate human SREBP-1, either by directly binding to the PPAR response elements on the promoter or via cross-regulation with the LXR in mice (12,13) .…”