Goat FADS2 controlling fatty acid metabolism is directly regulated by SREBP1 in mammary epithelial cells

Abstract

Goat milk provides benefits to human health due to its richness in bioactive components, such as polyunsaturated fatty acids (PUFAs). The fatty acid desaturase 2 (FADS2) is the first rate-limiting enzyme in PUFAs biosynthesis. However, its role and transcriptional regulation mechanisms in fatty acid metabolism in dairy goat remain unclear. Here, our study revealed that the FADS2 gene was highly expressed during the peak lactation compared with the dry period, early lactation, and late lactation. The content of triacylglycerol (TAG) was enhanced with the increasing mRNA expression of TAG synthesis genes (diacylglycerol acyltransferase 1/2, DGAT1/2) in FADS2-overexpressed goat mammary epithelial cells (GMECs). Overexpression of FADS2 was positively correlated with the elevated concentrations of dihomo-gamma-linolenic acid (DGLA) and docosahexaenoic acid (DHA) in GMECs. BODIPY staining showed that FADS2 promoted lipid droplet accumulation in GMECs. To clarify the transcriptional regulatory mechanisms of FADS2, 2,226 bp length of FADS2 promoter was obtained. Deletion mutation assays revealed that the core region of FADS2 promoter was located between the -375 and -26 region, which contained SRE1 (-361/-351) and SRE2 (-191/-181) cis-acting elements of transcription factor sterol regulatory element-binding protein 1 (SREBP1). Overexpression of SREBP1 enhanced relative luciferase activity of the single mutant of SRE1 or SRE2, vice versa, and failed to alter the relative luciferase activity of the joint mutant of SRE1 and SRE2. Chromatin immunoprecipitation (ChIP) and site-directed mutation assays further demonstrated that SREBP1 regulated the transcription of the FADS2 gene by binding to SRE sites in vivo and in vitro. In addition, the mRNA levels of FADS2 were significantly decreased by targeting SRE1 and SRE2 sites in the genome via the CRISPR interference (CRISPRi) system. These findings establish a direct role for FADS2 regulating TAG and fatty acid synthesis by SREBP1 transcriptional regulation in dairy goat, providing new insights into fatty acid metabolism in mammary gland of ruminants.

Keywords: FADS2; SREBP1; GMECs; fatty acid synthesis; transcriptional regulation.

Figure 1.

Overexpression of FADS2 increases TAG content and promotes PUFAs synthesis. (A) The DHA biosynthetic pathway from C18:3 n-3 through desaturases and elongases in mammals. (B) RT-qPCR analysis of FADS2 mRNA expression in different lactation stages (N = 3 biological replicates). FADS2 expression values were normalized to the expression of the dry period. (C) FADS2 mRNA expression was quantified in Ad-FADS2 or Ad-GFP virus-infected GMECs for 48 h. (D and E) Western blotting assays (D) and protein quantitative(E) analysis of FADS2 protein levels in Ad-FADS2 or Ad-GFP virus-infected GMECs for 48 h. (F and G) The content of (F) TAG and (G) expression of genes related to TAG synthesis were analyzed. (H, K, and L) The mRNA levels of genes related to (H) lipid accumulation (ADRP) and fatty acid transport (FABP3), (K) PUFAs synthesis (FADS1, SCD1, ELOVL2, ELOVL5, ELOVL6), and (L) fatty-acid synthesis (SREBP1, FASN, ACACA) were measured in GMECs following treatment with Ad-GFP or Ad-FADS2 virus infection for 48 h. (I) Confocal images BODIPY 493/503 staining of lipid droplets after transiently transfection of FADS2 overexpression plasmid (pEF1a-FLAG-NEO-FADS2 plasmid) in GMECs for 24 h. Scale bar, 10 μm. (J) Quantitative analysis of lipid droplet staining was based on the confocal images; mean ± SEM were shown. Different lowercase letters represented significant differences among groups (P < 0.05); the same lowercase letters denoted no significant difference between the groups (P > 0.05); **P<0.01; *P < 0.05; ns, P >0.05.

Figure 2.

Sequence cloning and identification of FADS2 promoter core region. (A) Relative luciferase activity analysis of FADS2 promoter. The FADS2 promoter was cloned into the pGL3-basic vector and co-transfected with a TK-Renilla luciferase vector into GMECs for 48 h. (B) The schematic map of the FADS2 promoter sequence and important transcription factor predicted binding sites. The position of transcription factor predicted binding sites were underlined, and the transcription start site was indicated by a black arrow. (C) Relative luciferase activity analysis of deletion fragments of FADS2 promoter. The −1,649/+102, −1,310/+102, −701/+102, −375/+102, −180/+102, and −26/+102 deletion fragments of FADS2 promoter were cloned into the pGL3-basic vector and cotransfected with a TK-Renilla luciferase vector into GMECs for 48 h. Three independent replicates were shown as mean ± SEM. Different lowercase letters represented significant differences among groups (P < 0.05); the same lowercase letters denoted no significant difference between the groups (P > 0.05); **P < 0.01.

Figure 3.

FADS2 expression is promoted by SREBP1. (A and E) The mRNA levels analysis of SREBP1 and FADS2 in (A) SREBP1-overexpression or (E and F) knockdown GMECs. (B and C) Western blotting (B) and relative protein abundance (C) analysis of FADS2 protein level in SREBP1-overexpression GMECs. (D and F) Relative luciferase activity analysis of FADS2 promoter in (D) SREBP1-overexpression or (E) knockdown GMECs. (G) Relative luciferase activity analysis of deletion fragments of the FADS2 promoter in SREBP1-overexpression GMECs. **P < 0.01; *P < 0.05; ns, P > 0.05.

Figure 4.

The SRE sites play important roles in maintaining FADS2 promoter activity. (A) The canonical binding motif of sterol regulatory element (SRE) for transcriptional factor SREBP1. (B) Analysis of SRE1 and SRE2 conserved sequence in the FADS2 promoter region of dairy goat, cow, human, and mouse. The conserved regions were highlighted in blue, while the same nucleotides were marked with asterisks. (C) Relative luciferase activity analysis of SRE1, SRE2, or SRE1/2 mutants in −375/+102 region of FADS2 promoter. The SRE1, SRE2, or SRE1/2 mutants in the −375/+102 region of the FADS2 promoter were cloned into the pGL3-basic vector and cotransfected with a TK-Renilla luciferase vector in GMECs for 48 h (N = 3 biological replicates). Mutation sites were marked in red. Different lowercase letters represented significant differences among groups (P < 0.05); the same lowercase letters denoted no significant difference between the groups (P > 0.05).

Figure 5.

SREBP1 regulates FADS2 transcription via directly binding SRE sites in GMECs. (A) Relative luciferase activity analysis of SREBP1-overexpression effect on SRE1, SRE2, or SRE1/2 mutants in −375/+102 region of FADS2 promoter. The SRE1, SRE2, or SRE1/2 mutants cloned into the pGL3-basic vector were cotransfected with the TK-Renilla luciferase vector and pEGFPc1-SREBP1 overexpression plasmid (or pEGFPc1 empty vector) in GMECs for 48 h (N = 3 biological replicates). Mutation sites were marked in red. (B) Relative luciferase activity analysis of SREBP1-interference effect on SRE1, SRE2, or SRE1/2 mutants in −375/+102 region of FADS2 promoter. The SRE1, SRE2, or SRE1/2 mutants cloned into pGL3-basic vector were cotransfected with TK-Renilla luciferase vector into pretreated GMECs with si-SREBP1 or scramble. Mutation sites were marked in red. (C and D) Relative enrichment assessment of SREBP1 at (C) SRE1 site and (D) SRE2 site in FADS2 promoter by ChIP-PCR and ChIP-qPCR assays. **P<0.01; *P < 0.05; ns, P >0.05.

Figure 6.

FADS2 is silenced by dCas9/KRAB system with SRE sites in genome. (A and B) Schematic showing (A) inhibition of constitutive promoter of FADS2 using a catalytically-dead dCas9 fused to a transcriptional repressor domain (KRAB) and (B) packaging and infection of lentiviral. sgRNA was designed targeting SRE1 or targeting SRE2. (C) RT-qPCR analysis mRNA levels of FADS2 after infection either dCas9/KRAB empty vector lentivirus (No sgRNA), dCas9/KRAB infusion with SRE1-sgRNA lentivirus or SRE2-sgRNA lentivirus in GMECs. (D) RT-qPCR analysis of FADS2 mRNA levels of sgRNAs lentivirus infection with overexpression of SREBP1 or not in GMECs. **P < 0.01; *P < 0.05; ns, P > 0.05.

Figure 7.

Model of the function of FADS2 gene in fatty acid metabolism and its expression is directly regulated by SREBP1 in dairy goat. TAG content and lipid droplet accumulation were promoted in FADS2-overexpressed GMECs, and PUFAs (DHA and DGLA) contents were also increased. FADS2 was transcriptional regulated by SREBP1 via binding to SRE1 (−361/−351) and SRE2 (−191/−181) sites in site-directed mutation and ChIP assays. In addition, the mRNA level of FADS2 was significantly decreased by targeting SRE1 and SRE2 sites in the genome via the CRISPR interference (CRISPRi) system.