The Regulatory Mechanism Of FOXO1 Gene On Fatty Acid Metabolism In Dairy Goat

Forkhead box protein O1(FOXO1)is a key cellular regulatory factor,exerting its biological function mainly by transcription and transmitting various cytokine signals.In the liver of mammals,FOXO1 inhibits the expression of SREBP1 to suppress the de novo fatty acid synthesis and promotes the degradation of fatty acids,thereby changing the composition of fatty acids in cells and maintaining the balance of lipid metabolism.However,it remains unclear whether FOXO1 regulates fatty acid metabolism during lactation in ruminants.Goat milk is rich in a variety of fatty acids,especially short,medium-chain and unsaturated fatty acids that are beneficial to human health.More than half of the fatty acids come from de novo synthesis.Therefore,studying the basic functions of FOXO1 in the mammary gland of dairy goats and the transcriptional regulation mechanism of FOXO1 on fatty acid metabolism genes has important theoretical and practical significance for dairy goat breeding and improving the nutritional value of goat milk.In the present study,we using CHIP-seq technology to obtain the enrichment data of histone H3K27 ac in goat mammary gland at different lactation period.Function of FOXO1 in goat mammary epithelial cells(GMECs)was validated through adenovirus-mediated overexpression and interference technology.The transcriptional activity of FOXO1 was explored by exogenous addition of insulin.We explored the effect of ATGL,a key enzyme fatty acid decomposition,on lipid droplet synthesis and triglyceride degradation by using overexpression technology.The transcriptional regulation mechanism of FOXO1 on SREBP1 gene and ATGL gene was studied by mutagenesis.The main results were as follows:1.FOXO1 screening of dairy goat by CHIP-Seq.The mammary gland tissues were pretreated by liquid nitrogen grinding.The 100-500 bp DNA fragments were obtained by using Bioruptor Pico sonicator machine in which the parameters were set at 30 s ON,30 s OFF,and 24 cycles;It showed that 5 ?L antibody was optimal for improving enrichment efficiency according to the content of enriched DNA.Histone CHIP-seq data from the mammary tissue at different lactation period indicated that the enrichment of H3K27 ac in the FOXO1 enhancer is higher in the lactation period than that in the dry period.These speculated that FOXO1 plays an important role in lactation of mammary gland.2.Overexpression of goat FOXO1 gene.Goat FOXO1 gene(2004 bp)was cloned and construct the Ad-FOXO1 adenovirus overexpression vector.High-titer Ad-FOXO1 adenovirus was packaged and expanded by transfecting into 293 A cells.After infection with Ad-FOXO1,the m RNA level of FOXO1 gene was 500-fold higher than control group,and the protein level was also significantly increased.The expression of fatty acid synthesis-related genes FASN,ACACA,SCD1,PLIN3,XDH,ACOX1 and CD36 were significantly down-regulated after overexpression of FOXO1.Meanwhile,overexpression of FOXO1 also significantly down-regulated the content of triglycerides,the m RNA levels of triglyceride synthesis related genes DGAT1 and LPIN1 and the contents of C16:0 and C18:1in cells,while the content of C18:0 increased significantly.3.RNA interference of goat FOXO1 gene.Three pairs of specific sh RNA targeting FOXO1 were designed and synthesized.Sh RNA-FOXO1 adenovirus vector was constructed and high-titer Ad-sh FOXO1 adenovirus was obtained.The expression of FOXO1 m RNA was reduced by more than 70% after infection Ad-sh RNA2,and the protein level was also significantly reduced.Interference with FOXO1 significantly increased the expression of FASN,ELOVL6,SCD1,CD36,DGAT2 and GPAM genes,and down-regulated the m RNA levels of PPARA and ATGL.At the same time,the interference of FOXO1 gene increased the accumulation of intracellular lipid droplets and the content of triglycerides.In addition,interference with FOXO1 could significantly promote cell proliferation and had no effect on the protein expression of Caspase 3.4.Effects of insulin on transcriptional activity of FOXO1 gene.Insulin treatment of GMECs could significantly up-regulate the expression of fatty acid de novo synthesis related genes SREBP1,FASN and ACACA,and the intracellular triglyceride content.Meanwhile,insulin could activate the PI3K/AKT signaling pathway and significantly increase the phosphorylated PI3K(p-PI3K)and phosphorylated AKT(p AKT)protein,and then the phosphorylation of FOXO1 protein was transferred from the nucleus to the cytoplasm resulting in a significant decrease of FOXO1 protein in the nucleus and increase of phosphorylation FOXO1(p FOXO1)protein in the cytoplasm.However,the total protein of FOXO1 did not change significantly.After adding PI3K/AKT signaling pathway inhibitor(LY294002),the expression of p-PI3 K and p-AKT protein were significantly reduced and the protein level of p FOXO1 was also influenced.Treatment with insulin after overexpression of FOXO1 could significantly down regulate the expression of SREBP1,FASN and ACACA and the content of intracellular lipid droplets compared with insulin treatment alone.However,the expression was still higher than the overexpression of FOXO1 group,indicating that insulin could attenuate the inhibitory effect of FOXO1 on lipid synthesis.5.Transcriptional regulation of goat SREBP1 gene by FOXO1.Overexpression of FOXO1 significantly down-regulated SREBP1 gene expression and promoter activity.Interference with FOXO1 could significantly up-regulate the m RNA and protein expression of SREBP1 gene and the activity of SREBP1 promoter.The results of deleted mutation showed that overexpression of FOXO1 had inhibitory effect on the SREBP1 promoter activity of different deleted fragments,and the inhibitory effect was the strongest in the core region of the promoter.T0901317(LXR? agonist)treatment significantly increased the promoter activity of SREBP1,while overexpression of FOXO1 weakened the induction of SREBP1 promoter by T0901317.Bioinformatics analysis revealed that there were two LXRE sites and two SRE sites on the SREBP1 promoter.After site-directed mutation of the LXRE site,overexpression of FOXO1 could still down-regulate the activity of the SREBP1 promoter,but the mutation of the LXRE site could not eliminate the induction of SREBP1 promoter by T0901317.The simultaneous mutation of two LXRE sites and SRE sites resulted in the disappearance of the inhibitory effect of FOXO1 on SREBP1 promoter.These results revealed that FOXO1 regulated the transcriptional activity of SREBP1 promoter through LXRE and SRE elements.The activity of SREBP1 promoter was significantly up-regulated after insulin treatment with cells,however,overexpression of FOXO1 attenuated the induction of SREBP1 promoter by insulin.6.Effects of ATGL overexpression on TAG and lipid droplet synthesis.The m RNA level of ATGL gene was up-regulated by more than 1000-fold and the protein level was up-regulated by about 20-fold after infection high-titer Ad-ATGL adenovirus.Overexpression of ATGL could significantly down-regulate the expression of lipid metabolism-related genes PPARA,ADRP,BTN1A1 and CD36 and significantly up-regulate the expression of lipolytic gene HSL.Meanwhile,overexpression of ATGL reduced the accumulation of intracellular lipid droplets and the content of triglycerides and increased the content of free fatty acids in the cells and had no effect on the free fatty acids in the medium.7.Study on transcriptional regulation of goat ATGL gene by FOXO1.We cloned a 2 240 bp fragment of goat ATGL promoter.Bioinformatics analysis revealed that ATGL promoter had binding sites for transcription factors such as FOXO1,STAT5 A,LXR,NF-Y and SP1.The double luciferase activity experiment showed that interference with FOXO1 could significantly down regulate the activity of ATGL promoter.The results of the deletion mutation indicated that the core region of the ATGL promoter was located between-882 bp and-524 bp and there were two FOXO1 binding sites(FKH1 and FKH2)in the core region.Single mutation or simultaneous mutation of FKH element could significantly down-regulate the activity of ATGL promoter and weaken the activation of FOXO1 on ATGL promoter.Chromatin immunoprecipitation experiments showed that FOXO1 could directly bind to the FKH element of the ATGL promoter in vivo,and had a higher affinity for FKH2 than FKH1.In summary,we determined the optimal method for chromatin immunoprecipitation of goat mammary gland and analyzed the enrichment of H3K27 Ac on FOXO1.The exogenous insulin inhibited FOXO1 transcriptional activity by changing the localization of FOXO1.Meanwhile,FOXO1 could directly regulate the transcription of SREBP1 and ATGL,and regulating the expression of genes related to lipid metabolism,leading to inhibiting the de novo synthesis of fatty acids and promoting the lipolysis process of fatty acids.Our study will provide theoretical and experimental support for further elucidating the regulation mechanism of fatty acid content in goat milk.