Cloning and Activity Analysis of Transcription Factor FoxO1 Promoter in Dairy Goat (Capra hircus)
HE Qiu-Ya, LUO Jun*, GAO Liang-Jia-Hui, WU Jiao, YAO Wei-Wei
College of Animal Science and Technology/Shaanxi Provincial Key Laboratory of Molecular Biology for Agriculture, Northwest A& F University, Yangling 712100, China
Abstract Forkhead box protein O1 (FoxO1) is a key transcription factor regulating fatty acid metabolism. Although FoxO1 promoter has been shown to be regulated by upstream transcription factors in non-ruminants, the mechanism of FoxO1 regulation during lactation in dairy goats (Capra hircus) remains unclear. This study designed primers based on the genome sequence of Yunnan black goat and cloned the 5' flanking sequence of FoxO1 gene. Bioinformatic analysis of FoxO1 promoter sequence was performed using online software to predict the binding sites of transcription factors. Double luciferase reporter vectors containing different lengths of FoxO1 promoter fragments were constructed by deletion mutation method, and double luciferase reporter vectors containing mutations of cis-acting elements were constructed by overlapping extension PCR method. The recombinant constructs and reference vectors were co-transfected into goat mammary epithelial cells (GMEC), and the promoter activity was detected by dual luciferase system. The results showed that 1 500 bp FoxO1 promoter sequence was cloned from the blood genome of dairy goats. Bioinformatics analysis revealed 2 binding sites of FoxO1 on the FoxO1 promoter. Deletion mutation results showed that region from -95 to -24 bp was necessary for basal transcription activity of FoxO1 promoter. Overexpression of FoxO1 significantly increased the activity of FoxO1 promoter. Mutation of the forkhead transcription factor 1 (FKH1) (-359 bp) site showed a significant decrease in FoxO1 promoter activity compared to wild type, but unchanged after mutation of FKH1 and FKH2 sites. Above results suggest that FoxO1 might promote FoxO1 transcription by binding to the FKH elements located at the promoter. This study provides theoretical reference for the functional study of the FoxO1 gene promoter and basic data for constructing the fatty acid metabolism network in mammary gland of dairy goats.
HE Qiu-Ya,LUO Jun,GAO Liang-Jia-Hui, et al. Cloning and Activity Analysis of Transcription Factor FoxO1 Promoter in Dairy Goat (Capra hircus)[J]. 农业生物技术学报, 2024, 32(3): 578-586.
[1] 赫秋亚, 罗军, 姚大为, 等. 2016. 奶山羊ELOVL6基因启动子的克隆及活性区域分析[J]. 农业生物技术学报, 24(6): 790-798. (He Q Y, Luo J, Yao D W, et al.2016. Cloning and analysis of active region of the promoter of elovl6 gene in dairy goats[J]. Journal of Agricultural Biotechnology, 24(6): 790-798.) [2] 张晓, 罗军, 李建华, 等. 2010. 西农萨能奶山羊脂肪酸合酶基因启动子的克隆及活性测定[J]. 中国农业科学, 43(3): 640-647. (Zhang X, Luo J, Li J H, et al.2010. Cloning and activity determination of fatty acid synthase gene promoter in Xinong Sanen dairy goats[J]. Agricultural Science in China, 43(3): 640-647.) [3] Bhaskar P, Guangyu D, Dana T G.2012. Role of forkhead transcription factors in diabetes-induced oxidative stress[J]. Experimental Diabetes Research, 2012(Pt.2): 939751. [4] Chakrabarti P, Kandror K V.2009. Foxo1 controls insulin-dependent adipose triglyceride lipase (ATGL) expression and lipolysis in adipocytes[J]. Journal of Biological Chemistry, 284(20): 13296-13300. [5] Daisuke H, Keisuke Y, Katsuyuki N, et al.2020. Hepatic levels of DHA-containing phospholipids instruct SREBP1-mediated synthesis and systemic delivery of polyunsaturated fatty acids[J]. Iscience, 23(9): 101495. [6] Deng X, Zhang W, O-Sullivan I, et al.2012. Foxo1 inhibits sterol regulatory element-binding protein-1c (SREBP-1c) gene expression via transcription factors SP1 and SREBP-1c[J]. The Journal of Biological Chemistry, 287(24): 20132-20143. [7] Dong X C.2017. Foxo transcription factors in non-alcoholic fatty liver disease[J]. Liver Research, 1(3): 168-173. [8] Dong X C, Copps K D, Guo S, et al.2008. Inactivation of hepatic foxo1 by insulin signaling is required for adaptive nutrient homeostasis and endocrine growth regulation[J]. Cell Metabolism, 8(1): 65-76. [9] Haenlein G F W.2004. Goat milk in human nutrition[J]. Small Ruminant Research, 51(2): 155-163. [10] Haeusler R A, Domenico A.2008. The double life of irs[J]. Cell Metabolism, 8(1): 7-9. [11] Haeusler R A, Han S, Accili D.2010. Hepatic foxo1 ablation exacerbates lipid abnormalities during hyperglycemia[J]. Journal of Biological Chemistry, 285(35): 26861. [12] He Q, Luo J, Wu J, et al.2021. Elovl6 promoter binding sites directly targeted by sterol regulatory element binding protein 1 in fatty acid synthesis of goat mammary epithelial cells[J]. Journal of Dairy Science, 104(5): 6253-6266. [13] He Q, Luo J, Wu J, et al.2020. Foxo1 knockdown promotes fatty acid synthesis via modulating SREBP1 activities in the dairy goat mammary epithelial cells[J]. Journal of Agricultural and Food Chemistry, 68(43): 12067-12078. [14] Huishou Z, Fuyang Z, Dan S, et al.2020. Branched-chain amino acids exacerbate obesity-related hepatic glucose and lipid metabolic disorders via attenuating Akt2 signaling[J]. Diabetes, 69(6): 1164-1177. [15] Jang H, Lee G Y, Selby C P, et al.2016. Srebp1c-cry1 signalling represses hepatic glucose production by promoting foxo1 degradation during refeeding[J]. Nature Communications, 7(1): 12180. [16] Liu W, Zhou Y, Sun H, et al.2021. Front cover: Goat milk improves glucose homeostasis via enhancement of hepatic and skeletal muscle AMP‐activated protein kinase activation and modulation of gut microbiota in streptozocin‐induced diabetic rats[J]. Molecular Nutrition & Food Research, 65(6): e2000888. [17] Liu X, Qiao A, Ke Y, et al.2010. Foxo1 represses lxralpha-mediated transcriptional activity of srebp-1c promoter in hepg2 cells[J]. FEBS Letters, 584(20): 4330-4334. [18] Li Y, Pang Y, Zhao Z, et al.2020. Molecular characterization, nutritional and insulin regulation of elovl6 in rainbow trout (Oncorhynchus mykiss)[J]. Biomolecules, 10(2): 264. [19] Ono H, Shimano H, Katagiri H, et al.2003. Hepatic akt activation induces marked hypoglycemia, hepatomegaly, and hypertriglyceridemia with sterol regulatory element binding protein involvement[J]. Diabetes, 52(12): 2905-2913. [20] Ponugoti B, Dong G, Graves D T.2012. Role of forkhead transcription factors in diabetes-induced oxidative stress[J]. Experimental Diabetes Research, 2012: 939751. [21] Qin W, Pan J, Qin Y, et al.2014. Identification of functional glucocorticoid response elements in the mouse foxO1 promoter[J]. Biochemical and Biophysical Research Communications, 450(2): 979-983. [22] Shi H, Shi H, Luo J, et al.2014. Establishment and characterization of a dairy goat mammary epithelial cell line with human telomerase (ht-mecs)[J]. Animal Science Journal, 85(7): 735-743. [23] Tian Y, Chen H, Tian C, et al.2020. Polymerase independent repression of foxo1 transcription by sequence-specific PARP1 binding to foxo1 promoter[J]. Cell Death & Disease, 11(1): 71. [24] Vaziri-Gohar A, Zheng Y, Houston K D.2017. Igf-1 receptor modulates foxo1-mediated tamoxifen response in breast cancer cells[J]. Molecular Cancer Research, 15(4): 489-497. [25] Wang Z, Luo J, Wang W, et al.2010. Characterization and culture of isolated primary dairy goat mammary gland epithelial cells[J]. Chinese Journal of Biotechnology, 26(8): 1123-1127. [26] Xu H F, Luo J, Wang H P, et al.2016. Sterol regulatory element binding protein-1 (SREBP-1) c promoter: Characterization and transcriptional regulation by mature SREBP-1 and liver X receptor a in goat mammary epithelial cells[J]. Journal of Dairy Science, 99: 1595-1604. [27] Yao D, Luo J, He Q, et al.2017. SCD1 alters long-chain fatty acid (LCFA) composition and its expression is directly regulated by SREBP-1 and PPAR gamma 1 in dairy goat mammary cells[J]. Journal of Cellular Physiology, 232(3): 635-649. [28] Zhang W, Bu S Y, Mara T M, et al.2016. Integrated regulation of hepatic lipid and glucose metabolism by adipose triacylglycerol lipase and foxo proteins[J]. Cell Reports, 15(2): 349-359. [29] Zhang Y, Yuan T, Su Z, et al.2020. Reduced methylation of PP2AC promotes ethanol-induced lipid accumulation through foxo1 phosphorylation in vitro and in vivo[J]. Toxicology Letters, 331(1): 65-74. [30] Zhao H, Zhang F, Sun D, et al.2020. Branched-chain amino acids exacerbate obesity-related hepatic glucose and lipid metabolic disorders via attenuating AKT2 signaling[J]. Diabetes, 69(6): 1164-1177. [31] Zhao N, Tan H, Wang L, et al.2021. Palmitate induces fat accumulation via repressing foxo1-mediated atgl-dependent lipolysis in HEPG2 hepatocytes[J]. PLOS ONE, 16(1): e0243938. [32] Zhu J, Sun Y, Luo J, et al.2015. Specificity protein 1 regulates gene expression related to fatty acid metabolism in goat mammary epithelial cells[J]. International Journal of Molecular Sciences, 16(1): 1806-1820.
