Tissue expression profiles and transcriptional regulation of elongase of very long chain fatty acid 6 in bovine mammary epithelial cells

Abstract

In mammals, very long chain fatty acids (VLCFAs) perform pleiotropic roles in a wide range of biological processes, such as cell membrane formation, cell signal transduction, and endocrine regulation. Beef and milk are abundant of palmitic acid which can be further elongated into stearic acid for synthesizing VLCFAs. Elongase of very long chain fatty acid 6 (ELOVL6) is a rate-limiting enzyme for converting palmitic acid to stearic acid. Consequently, investigating the tissue expression patterns and transcriptional regulation of bovine ELOVL6 can provide new insights into improving the composition of beneficial fats in cattle and expanding the knowledge of transcriptional regulation mechanism among domestic animals. In the current study, we found that bovine ELOVL6 expressed ubiquitously. Dual-luciferase reporter assay identified that the core promoter region (-130/-41 bp) was located in the second CpG island. In addition, the deletion mutation of binding sites demonstrated that sterol regulatory element binding transcription factor 1 (SREBF1) and specific protein 1 (SP1) both were able to stimulate bovine ELOVL6 promoter activity independently, while resulting the similar effect. To confirm these findings, further RNA interference assays were executed in bovine mammary epithelial cells (BMECs). In summary, these data suggest that bovine ELOVL6 expressed ubiquitously and is activated by SREBF1 and SP1, via two binding sites present in the ELOVL6 promoter region between -130 bp to -41bp.

Fig 1. Tissue-specific expression patterns of bovine ELOVL6 mRNAs.

Each column represented the mean ± SD of three independent experiments which were performed in triplicate.

Fig 2. The phylogenetic analyses of bovine ELOVL6 promoter.

The phylogenetic relationship was analyzed by Neighbor Joining method (Mega program version 6.0) utilizing bovine ELOVL6 promoter and homologous sequences from other mammal species. Bootstrap values were obtained from 1000 repetitions and illustrated as percentages at the nodes. The evolutionary distance of 0.01 nucleic acid substitutions per position was represented at the scale bars.

Fig 3. The prediction and analyses of CpG islands in bovine ELOVL6 promoter.

(a) The predicted CpG islands in the bovine ELOVL6 promoter (+1 to -1000 bp). The red vertical illustrated the GC-rich regions. The blue shading regions indicated the predicated CpG islands; (b) Multiple sequence alignment of the second CpG island. Seven predicted transcription factors binding sites were underlined. Black shaded sequences indicated that the base pair was identical in all sequences of the alignment. Dark grey shadow indicated conserved substitutions and light grey shadow illustrated semi-conserved substitutions.

Fig 4. The structures and promoter activities of various truncation constructs of bovine ELOVL6 promoter region.

The promoter activities of 3T3-L1 cells (black bars) and 293A cells (white bars) were shown as the mean ± SD of the independent experiment performed in triplicate.

Fig 5. Luciferase activities of different deletion constructs.

(a) Schematic represented various deletion constructs in the core region of bovine ELOVL6 promoter. The transcription initiation site was designated as +1.; (b) Luciferase activities of different deletion constructs were indicated as mean ± SD of the independent experiment performed in triplicate.

Fig 6. siRNA decreased the mRNA expression of ELOVL6.

(a) The silencing of SREBF1 affected the mRNA expression of ELOVL6; (b) The silencing of SP1 affected the mRNA expression of ELOVL6. GAPDH was measured as the reference gene. Each column represented the mean ± SD of the independent experiment performed in triplicate. * p < 0.05; ** p < 0.01.