Promoter Function And Transcriptional Regulation Mechanism Of Stearoyl-Coa Desaturase 1 Gene Of 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). Lipid synthesis metabolism during lactation is of great importance to the fatty acid content in goat milk. Stearoyl-coenzyme A desaturase 1(SCD1) is the rate-limiting enzyme catalyzing the ?9-cis desaturation of palmitoyl- and stearoyl-CoA to MUFA. These products are the most abundant MUFA and serve as substrates for the synthesis of triacylglycerol in milk. Therefore, exploring the function of SCD1 and the mechanisms underlying the regulation of SCD1 in goat mammary epithelial cells(GMEC) will help to improve the nutritional value of goat milk. However, the function of SCD1 and the molecular mechanism underlying SCD1 transcriptional regulation have not yet been well characterized in the lactating ruminant mammary gland.The main goal of this study was to better understand the function of SCD1, clone and analyze goat SCD1 promoter, and finally elucidate the regulatory mechanism of the goat SCD1 promoter. The function of SCD1 was verified by overexpression mediated by adenovirus and RNAi mediated interference. We cloned the sequence of the goat SCD1 promoter and analyzed its structure and function. We also investigated the transcription regulation mechanisms of goat SCD1 promoter by transcriptional factors including SREBP1, LXRα, PPARG and linoleic acid. Main results obtained in this study are as follows:1. Ad-SCD1 adenovirus was constructed and packed for overexpressing SCD1. Overexpression of SCD1 increased the intracellular MUFA content and lipid accumulation along with the up-regulation of FASN, DGAT2, GPAM, FABP3 and PPARΑ(P< 0.05). In contrast, overexpression of SCD1 led to the down-regulation of PNPLA2, LIPE, CD36 and CPT1B(P< 0.05). The desaturation indices of C16:1 and C18:1 was markedly increased upon SCD1 overexpression(P< 0.05).2. The efficiency of siRNA-SCD1 was tested before the following experiments. The SCD1 knockdown decreased the expression of FASN, ACACA, LPIN1, DGAT1, DGAT2, LIPE, FABP3, PPARΑ and ACOX1(P< 0.05). The expression of CD36 and CPT1B(P< 0.05) was up-regulated by silencing SCD1. The knockdown of SCD1 led to a significant decrease in the desaturation indices of C18:1.3. We used goat genomic DNA to clone and sequence a 1778 bp fragment of the SCD1 5’ flanking region. A TATA box-like sequence was located at 49 bp upstream of the TSS. Bioinformatics analysis revealed a series of consensus binding sites for transcription factors such as NF-Y, Sp1, AP-2, PPAR, SREBP, C/EBP and ER. Progressive deletion analysis revealed the core region of the SCD1 promoter spans from –415 to –109 bp which contains the binding sites for Sp1, SREBP1 and NF-Y.4. Site-deleted mutation analysis revealed that deletion of SRE or NF-Y significantly reduced the basal promoter activity of SCD1. Overexpression of sterol regulatory element binding factor 1(SREBF1) enhanced SCD1 expression and its promoter activity, but that effect was abolished in GMEC in which SREBF1 was silenced. Furthermore, deletion of SRE andNF-Y binding sites within a –1713~+65-base pair region of the SCD1 promoter completely abolished SREBP1-induced SCD1 transcription. These data demonstrated that the transcriptional regulation of SCD1 by SREBP1 is directly through SRE and NF-Y in GMEC.5. The LXRα agonist T0901317(T09) markedly enhanced the mRNA expression of SCD1 and SREBF1. The concentration of C16:1 and C18:1 and their desaturation indices also were increased by LXRα activation. However, knockdown of LXRα did not alter the mRNA expression of SCD1. Although SCD1 was repressed by SREBF1 knockdown, T09 significantly increased SCD1 expression. Further analysis revealed that the SCD1 promoter activity was activated by LXRα overexpression. The goat SCD1 promoter contains two LXREs, one SRE and one NF-Y binding site. Site-directed mutagenesis of LXRE1 or LXRE2 did not eliminate the upregulation of SCD1 when LXRα was overexpressed. In contrast, when NF-Y alone or in combination with SRE was mutated simultaneously, the activity of the SCD1 promoter did not respond to LXRα overexpression. Furthermore, when SREBF1 was knocked down, overexpression of LXRα did not affect the promoter activity of SCD1. Together, these data suggested that SREBP1 is necessary for maintaining basal transcriptional activity of SCD1, whereas LXRα regulated the expression of SCD1 in an indirect manner.6. The mRNA expression of SCD1 and SREBF1 was significantly reduced by linoleic acid along with decreased promoter activity of SCD1 in GMEC. The PUFA response region is highly-conserved among goat, human and mice. Progressive deletion and site-directed mutation analysis revealed that the repressive effect induced by linoleic acid was abrogated once NF-Y was deleted. When GMEC were cultured with linoleic acid, addition of T09 markedly increased SCD1 transcription compared with the control, but had no effect on cells with a deleted SRE promoter. These results demonstrated that linoleic acid can regulate SCD1 expression at the transcriptional level through impeding SREBP1 binding on the promoter of SCD1 in an LXR-dependent fashion in the goat mammary gland.7. PPARG1 overexpression also stimulated the expression of SCD1 and its transcriptional activity. Progressive deletion and bioinformatics analysis revealed that PPRE located in-29 bp upstream of the TSS in the SCD1 promoter which is responsible for the inductive effect by PPARG. In addition, this PPRE site is conserved between species. Mutation analysis demonstrated that PPARG directly regulated the expression of goat SCD1 via this PPRE site.In summary, SCD1 promotes the lipid accumulation in GMEC. SREBP1 directly regulates the transcriptional activity of SCD1, whereas LXRα regulates the expression of SCD1 through increasing SREBP1 abundance to promote interaction with the SRE and NF-Y binding sites. Linoleic acid affects the promoter activity of SCD1 through repressing the active form of SREBP1 in various mechanisms. PPARG regulates SCD1 transcription through direct interaction with the SCD1 promoter. The current work will provide valuable information to understand the molecular regulatory mechanisms of SCD1 during lactation, and it will bring new insights in modifying beneficial fatty acids and improving nutritional value of goat milk.