Transcriptional Regulation Mechanism Of ELOVL6 Gene Promoter Of Xinong Saanen Dairy Goat

The nutritional and treatment value of goat milk in the human diet has been recently underscored, particularly for its high content of short, medium-chain fatty acids and monounsaturated fatty acids(MUFA). Their biochemical nature greatly influences the flavor and quality of the milk. Elongase of very long chain fatty acids 6(ELOVL6) is the rate-limiting enzyme that catalyzes the elongation of long-chain saturated and monounsaturated fatty acids with 12, 14, and 16 carbons. Especially, ELOVL6 play an important role in the synthesis of stearate(18:0) and oleate(18:1, n-9). Exploring the mechanisms underlying the regulation of ELOVL6 will help to improve the nutritional value of goat milk.Therefore, study of the structure and functions of FASN promoter can further reveal regulatory mechanisms of ELOVL6 on transcription level.The objective of this study was to clone the promoter region of ELOVL6 of Xinong Saanen dairy goat and analyzed its structure and function. Ten promoter fragments with different length were obtained by progressive deletion. Then, the promoter fragments were cloned into pGL-3 luciferase reporter gene vectors. The constructs and the pRL-TK were transiently co-transfected into goat mammary epithelial cells. The core region of the promoter with basal transcriptional activity was determined with dual-luciferase reporter assays. We also investigated the transcription regulation mechanisms of goat ELOVL6 promoter by transcriptional factors SREBP-1.The results are as follows:(1) We obtained a 2 360 bp of the 5’ flanking sequence, containing 2 168 bp upstream of transcription start site(+1), the first exon(+1 to +87), and part of the first itron. An intiation codon ATG was located in exon 2 and the region is high GC-rich, but TATA-box was not found in the promter of ELOVL6. Multiple alignment analysis indicated that the homology of ELOVL6 promoter compared with bovine, homo and mouse was 95%, 81% and 80%, respectively. Morever, some consensus bingding sites for transcription factors such as PPARG, PPARA, LXR, SREBP1, Sp1, and E-box were observed upon bioinformatics analysis.(2) Using the dual-lueiferase reporter assay system to detect the promoting activities of deletion fragments.The regulatory elements for goat ELOVL6 positive transcription are located in forepart of ELOVL6 promoter. The region from-109 to-40 is necessary and required for basal trsnscription activity of goat ELOVL6 gene. Transcriptional factor binding sites of Sp1, E-box, and SREBP-1c were identified in this core region.(3) The results of site-directed mutagenesis showed that mutation in SRE1(-70 bp) and SRE3(-1 105 bp) binding sites significantly decreased the promoter activity. Knockdown of sterol regulatory element binding factor 1(SREBF1) by siRNA decreased ELOVL6 expression and its promoter activity. However,mutation in SRE binding sites of the ELOVL6 promoter completely abolished SREBP1-induced ELOVL6 transcription. These data suggested that the transcriptional regulation of ELOVL6 by SREBP-1 may required SREs elements in goat mammary epithelial cells.(4) The relative mRNA expression of SREBF1 and ELOVL6 decreased dramatically after GMEC were treated with linoleic acid. Morever, Consistent with this effect, linoleic acid repressed the ELOVL6 gene promoter activity. However, site-directed mutation of SREs completely abolished the repressive effect of linoleic acid. These results suggested that linoleic acid can regulate ELOVL6 expression by disrupting SREBP1 binding on the promoter of ELOVL6.In conclusion, the present study cloned the 5 ’flanking sequences of goat ELOVL6 promoter from Xinong Saanen goat; the sequence of which is highly homological with bovine, human and mouse ELOVL6 promoters. The core region of ELOVL6 promoter is from-109 to-40 bp by deletion analysis. The results of site-directed mutagenesis showed that SREBP play a important role in transcriptional regulation of ELOVL6. Linoleic acid affect the promoter activity of ELOVL6 by repressing the activity of SREBP1. These results would lay a foundation for the study function mechanism of ELOVL6 promoter,and it will provide information in improving the quality of milk in dairy goat.