Regulation Mechanism Of Fatty Acid Synthase Gene Promoter In Dairy Goat

The high content of short- and medium-chain fatty acids confers a high nutrition value to goat milk. Their biochemical nature greatly influences the flavor and quality of the milk. Fatty acid synthase(FASN) is the central enzyme of the de novo fatty acid biosynthesis of milk. It catalyzes all of the reaction steps for synthesizing saturated fatty acids from acetyl-Co A and malonyl-Co A in an NADPH-dependent manner. In the goat mammary gland, the acetyl-Co A and malonyl-Co A transacylase(AT/MT) domain of the FASN gene show medium-chain thioesterase activity, which reportedly influences the specific fatty acid composition of goat milk. Therefore, studying the regulation of FASN in goat mammary epithelial cells(GMECs) will provide a theoretical and practical basis for genetically regulating beneficial fatty acids in milk. However, the mechanisms regulating goat FASN transcription remain elusive. The aim of this study was to characterize the goat FASN promoter and investigate the transcriptional regulatory mechanisms of goat FASN in GMECs. In this study, we cloned the sequence of the goat FASN promoter and analyzed its structure and function. We also investigated the transcription regulation mechanisms of goat FASN promoter by transcriptional factors SREBP-1, LXR? and USF1. The current work will contribute valuable information to understanding the molecular regulatory mechanisms of FASN during lactation. Main results obtained in this study are as follows: 1. Cloning and bioinformatics analysis of goat FASN promoterWe cloned and sequenced a 1.8-kb fragment of the FASN 5? flanking region from goat genomic DNA. The bioinformatics analysis showed that there was a TATA-box at-41 bp of transcription start site(+1). Some transcriptional factor binding sites including PPAR, AP-2, LXR, ER, Sp1, SREBP-1, NF-Y and USF1 were identified in FASN promoter. Deletion analysis revealed a putative core promoter region located in-297/-14 bp upstream of the transcription site. Multiple alignment analysis demonstrated that the entire 1.8-kb fragment has little homology but that the sub-section nearest the transcriptional start site(-203 to +1) is more conserved across species, in particular the binding motifs for transcriptional regulation. The binding sites of Sp1, SREBP-1, NF-Y and USF1 were identified in FASN core promoter. 2. Regulation of goat FASN promoter by SREBP1Mutations of either sterol response element(SRE1 and SRE2) or NF-Y binding site appeared to significantly down-regulate the FASN basal promoter activity in goat mammary epithelial cells(P<0.05). However, the overexpression of SREBP-1 markedly enhanced FASN promoter activity when each of these binding sites was individually mutated. The mutation of both SREs caused a marked reduction of its basal promoter activity and completely abolished the stimulatory effect by SREBP-1. These observations indicated that both SRE sites responded to SREBP-1. Further analysis showed that SREBP-1 overexpression and knockdown by si RNA influenced the FASN promoter activity and endogenous FASN m RNA abundance. These data suggested that the transcriptionalregulation of FASN by SREBP-1 may required the presence of the two SREs elements in goat mammary epithelial cells. 3. Direct and indirect mechanism for regulation of FASN expression by LXR?T0901317(T09), an agonist for LXR?, significantly enhanced the m RNA expression and promoter activity of FASN. Sequence analysis revealed the presence of one LXR response element(LXRE) and two SREs in FASN promoter. Deletion or mutation of the LXRE site could reduce, but did not eliminate the transcriptional response of FASN to T09. While the LXRE and the SREs were both disrupted, the basal transcriptional activity was severely reduced and showed no response to T09 treatment. This suggested that a complete response required one LXRE and two SREs. Knockdown of LXR? by si RNA did not alter the basal or T09-induced transcriptional activity of FASN. However, when SREBP1 was silenced, T09 significantly increased FASN transcription by wild-type promoter, but had no effect on cells transfected with LXRE-mutant promoters. The results demonstrated that LXR? regulates FASN promoter activity through direct interaction with the LXRE as well as through increasing SREBP1 abundance. Taken together, these results suggested that both transcription factors are significant regulators of FASN transcription, with SREBP1 being necessary for maintaining basal levels and LXR? being capable of inducing higher expression of FASN upon activation. 4. Regulation of goat FASN promoter by USF1The entire coding sequence of goat upstream stimulation factor 1(USF1) was cloned, sequenced and subcloned into p EF-Neo-Flag vector. Overexpression of USF1 increased the promoter activity and m RNA abundance of FASN. Deletion analysis found that the E-box(-70 bp) of FASN promoter played an important role in response to USF1, while the E-box(-340 bp) had little effect on USF1 activation. Knockdown of USF1 by si RNA significantly decreased the m RNA and promoter activity of FASN. Deletion analysis also indicated that the E-box(-340 bp) was not required for the activation of FASN by USF1, and-70 bp E-box was capable of inducing expression of FASN upon USF1 activation. 5. USF1 and SREBP1 synergistically regulated FASN promoter activityCo-transfection of USF1 and SREBP1 overexpression vectors indicated that the two transcription factors synergistically activated the promoter activity of goat FASN. The p Bi FC-VN155-USF1 and p Bi FC-VC155-SREBP1 eukaryotic expression vectors were constructed and transfected into goat mammary epithelial cells to detect the interaction of USF1 and SREBP1 by bimolecular fluorescence complementation(Bi FC) assay. The strong Bi FC signals could be detected in cells co-transfected with p Bi FC-VN155-USF1 and p Bi FC-VC155-SREBP1, the fluorescence signal was located in the cell nucleus and cytoplasm, suggesting that USF1 and SREBP1 interact with each other in goat mammary epithelial cells. These results indicated that USF1 and SREBP1 synergistically regulated goat FASN transcription through interaction of the two factors.In summary, this study illustrated the regulation mechanisms of FASN transcription in goat mammary epithelial cells. The obtained results demonstrated that SREBP1 and LXR? play an important role in regulating the transcription of goat FASN gene. SREBP1 regulated FASN transcription not only by binding SRE elements presented in FASN promoter, but also interacted with USF1 synergistically to exert regulation effect. LXR? regulates FASN transcription through direct interaction with the FASN promoter, and indirect mechanism was through inducing the expression of SREBP1. The current work will contribute valuable information to understanding the molecular regulatory mechanisms of FASN during lactation, and it will bring new insights into the effects of fatty acid composition on nutrition value and the flavor of goat milk.