Structural And Functional Analysis Of Fatty Acid Synthase Gene Promoter Of Xinong Saanen Dairy Goat

Fatty acid synthase (FASN) plays a key role in de novo synthesis of fatty acids in mammals. FASN helps to catalyze all the reaction steps in the conversion of acety1-CoA, malonyl-CoA and NADPH to saturated fatty acids. Biosynthesis of short- and medium-chain fatty acids in goat mammary gland is functioned with a transferase reaction by the region of Acetyl-CoA and malonyl-CoA transacylases (AT/MT) in FASN. Change in FASN expression would result in changes of fatty acid composition in goat mammary gland and milk flavor. Therefore, study of the structure and functions of FASN promoter can further reveal regulatory mechanisms of FASN on transcription level.The present study cloned FASN gene promoter of Xinong Saanen Dairy Goat by PCR, and conducted sequence analysis upon the promoter. Fragments of the promoter of different lengths were obtained by deletion and sub-cloned into expression vectors with a luciferase reporter gene. The vectors were transfected into goat mammary epithelial cells and MCF-7 cells, and their activities of expression were measured using dual-luciferase reporter assay system. The core region of the promoter with basal transcriptional activity was determined with dual-luciferase reporter assays. The effect of transcriptional factor binding sites on promoter activity was detected via overlap extension PCR. The results are as follows:(1) The cloned sequence of FASN gene promoter is 2589 bp in length, including a 591 bp region upstream of transcription initiation site (+1), exonⅠ, intronⅠand partial exonⅠ. Translation initiation site ATG was located in exonⅠ. Homology of nucleotide sequences between Xinong Saanen Goat FASN gene promoter and bovine, human and rat FASN promoters was over 90%. TATA box and CAAT box, which are typical eukaryotic promoter elements, were located at -41 bp and -74 bp, respectively. 76% of the upstream region is G/C, with four GC boxes (GGGCGG and CCGCCC) found upstream of the TATA box. Potential transcription factor binding sites of PPAR, AP-2, LXR, ER, Sp1, SREBP-1c, NF-Y and USF were found in goat FASN promoter via bioinformatic analysis.(2) The promoting activities of deletion fragments were determined with dual-luciferase reporter assay system. The core region of FASN promoter was from–293 bp to–14 bp conferring basal transcriptional activity. The potential negative regulatory element was located in forepart of FASN gene promoter. Transcriptional factor binding sites of Sp1, NF-Y, USF and SREBP-1c were identified in this core region.(3) The results of site-directed mutagenesis showed that mutation in LXR(-400 bp), SREBP-1c(-150 bp) and E-box(-65 bp) binding sites significantly decreased the promoter activity (P<0.05, P<0.01 and P<0.01); mutation in ER(-390 bp) binding site increased activity of FASN promoter significantly (P<0.05); However, NF-Y binding site (-90 bp) had no effect on promoter activity (P>0.05). The results showed that transcription factor LXR, SREBP-1c and ER regulate FASN gene on transcriptional level through binding onto the FASN promoter.In conclusion, the present study cloned the sequence of goat FASN promoter from Xinong Saanen goat; the sequence of which is highly homological with bovine, human and rat FASN promoters. Deletion analysis showed that the core region of FASN promoter is from–293 bp to–14 bp. The results of site-directed mutagenesis showed LXR, SREBP-1c and ER participate in regulating of FASN gene transcription. The research results could be the basis for to further study of transcriptional regulatory mechanism of FASN gene.