Tissue Expression of ACACA Gene and Methylation Pattern Analysis of Adipose Tissue in Tianzhu White Yak (Bos grunniens)
SHI Xue-Hong1, SHI Bin-Gang1, ZUO Zhi1, ZHAO Shi-Jie1, HE Zhao-Hua1, ZHANG Xue-Ping1, ZHAO Zhi-Dong1, WANG Ji-Qing1, ZHOU Zhi-De2, HU Jiang1,*
1 Faculty of Animal Science and Technology, Gansu Agricultural University/Gansu Key Laboratory of Herbivorous Animal Biotechnology/Gansu Engineering Lab of Genetic Improvement in Ruminants, Lanzhou 730070, China; 2 Tianzhu County Songshan Town Animal Husbandry Veterinary Station, Tianzhu 733200, China
Abstract：Acetyl-CoA carboxylases alpha (ACACA) is a key rate-limiting enzyme in the de novo synthesis of fatty acids, which plays an important role in the regulation of fatty acid biosynthesis. In this study, 0.5-year-old and 4.5-year-old Tianzhu white yaks (Bos grunniens) were studied using quantitative real-time PCR (qRT-PCR) and heavy sulfite sequencing (BSP) methods. The expression of ACACA gene in 13 tissues such as heart, liver, and subcutaneous fat, as well as the methylation level of CpG island in PⅠ promoter region in subcutaneous adipose tissue were detected, and the gene expression regular and epigenetic mechanism in subcutaneous adipose tissue were studied. The results showed that the expression of ACACA gene in yaks were different in tissues and ages.The liver, brain, dorsal longest muscle and kidney tissues of 0.5-year-old yaks were highly expressed and the liver expression was significantly higher than other tissues (P<0.05);The expression of subcutaneous adipose and liver in 4.5-year-old yaks was extremely significantly higher than other tissues (P<0.01); The expression in the kidney of 0.5-year-old yaks were extremely significantly higher than that of 4.5-year-old yaks (P<0.01), but the expression of subcutaneous adipose, liver and testicular was significantly or extremely significantly less than that 4.5-year-old yaks (P<0.05 or P<0.01).In the subcutaneous adipose tissue, the ACACA gene PⅠ promoter 1st CpG island area (g.-795 bp~-193 bp) was highly demethylated and there were age differences. The average methylation level of BSP-1 primer amplification zone in 0.5-year-old yaks was extremely significantly higher than that in 4.5-year-old yaks (P<0.01). 0.5-year-old yaks BSP-1 primer amplification zone CpG11, CpG31, CpG35, CpG37 site and BSP-2 primer amplification zone CpG5', CpG13', CpG15', CpG27', CpG28' site methylation level significantly or extremely significantly higher than 4.5-year-old yaks (P<0.05 or P< 0.01); The level of methylation in the 1st CpG island region of the PⅠ promoter was negatively correlated with the expression of the gene in subcutaneous adipose tissue. This study revealed the differences of ACACA gene expression and PⅠ promoter methylation in yaks of different ages, which provide theoretical basis for molecular genetic research of yaks.
[1] 房希碧. 2017. 基于转录组和全基因组甲基化联合分析的牛肉质性状候选基因的鉴定与功能验证[D]. 博士学位论文,吉林大学, 导师: 赵志辉, pp. 2-3. (Fang X B.2017. Screening and identification of candidate genes related to bovine meat quality by conjoint analysis with RNA-seq and whole genome methylation[D]. Thesis for Ph.D., Jilin University, Supervisor: Zhao Z H, pp. 2-3.) [2] 韩玉娇. 2016. 秦川牛LCORL基因的多态性、mRNA表达及启动子区甲基化研究[D]. 硕士学位论文, 西北农林科技大学, 导师: 刘小林. pp. 1-2. (Han Y J.2016. The single nucleotide polymorphisms, mRNA expression and promoter region methylation patterns of LCORL in Qin Chuan cattle[D]. Thesis for M.S., Northwest A&F University, Supervisor: Liu X L. pp. 1-2.) [3] 沈秀平, 林月霞, 徐琪. 2012. DNA甲基化及其生物学功能[J]. 中国畜牧兽医, 39(6): 83-86. (Shen X P, Lin Y X, Xu Q.2012. DNA methylation and its biological functions[J]. Animal Husbandry and Veterinary Medicine, 39(6): 83-86.) [4] 田卫华, 杨丽玉, 李红, 等. 2019. 蛋鸡脂肪代谢相关基因ACACA和FASN表达及调控特性[J]. 农业生物技术学报, 27(02): 280-288. (Tian W H, Yang L Y, Li H, et al.2019. Expression and regulation characteristics of lipid metabolism-related ACACA and FASN Genes in laying hens[J]. Journal of Agricultural Biotechnology, 27(02):280-288.) [5] 杨勤, 柴志欣, 王会, 等. 2018. DNA甲基化在牛遗传育种中的应用[J]. 青海畜牧兽医杂志, 48(06): 55-59. (Yang Q, Chai Z X, Wang H, et al.2018. Application of DNA methylation in cow genetic breeding[J]. Qinghai Animal Husbandry Veterinary Magazine, 48(06): 55-59.) [6] 赵海谕. 2014. 奶山羊PITX1、PITX2基因的遗传变异、mRNA表达及DNA甲基化修饰对泌乳性能的影响[D].硕士学位论文,西北农林科技大学, 导师: 蓝贤勇, pp. 1-2. (Zhao Y H.2014. Genetic variation, mRNA expression and DNA methylation modification of PITX1 and PITX2 genes and their influence on milk production traits in dairy goats[D]. Thesis for M.S.,Northwest Agriculture Forestry University, Supervisor: Lan X Y, pp. 1-2.) [7] 周顺武. 2004. 动物生物化学[M]. 中国农业出版社, pp. 126-127. (Zhou S W.2004. Animal Biochemistry[M]. China Agricultural Press, pp. 126-127.) [8] Arnandine H, PhilippeA.2010. Genome-wide identification of new imprinted genes[J]. Briefings in Functional Genomics, 9(4): 304-314. [9] Baik M, Vu T T T, Piao M Y, et al.2014. Association of DNA methylation levels with tissue-specific expression of adipogenic and lipogenic genes in longissimus dorsi muscle of Korean cattle[J]. Asian-Australasian Journal of Animal Sciences, 27(10): 1493-1498. [10] Barber M C, Price N T, Travers M T.2005. Structure and regulation of acetyl-CoA carboxylase genes of metazoan[J]. Biochimicaet Biophysica Acta, 1733(1): 1-28. [11] Barber M C, Travers M T.1998. Elucidation of a promoter activity that directs the expression of acetyl-CoA carboxylase α with an alternative N-terminus in a tissue-restricted fashion[J]. Biochemical Journal, 333(1): 17-25. [12] Di C, Xu X.2018. DNA methyltransferases, DNA methylation, and age-associated cognitive function[J]. International Journal of Molecular Sciences, 19(5): 1315-1331. [13] Feng J, Fan G.2009. The role of DNA methylation in the central nervous system and neuropsychiatric disorders[J]. International Review of Neurobiology, 89: 67-84. [14] FujikiK, Kano F, Shiota K, et al.2009. Expression of the peroxisome proliferator activated receptor gamma gene is repressed by DNA methylation in visceral adipose tissue of mouse models of diabetes[J]. BMC Biology, 7(1): 38: 38-52. [15] Gore A, Li Z, Fung H L, et al.2011. Somatic coding mutations in human induced pluripotent stem cells[J]. Nature, 471(7336): 63-67. [16] Ichimura T, Watanabe S, Sakamoto Y, et al.2005. Transcriptional repression and heterochromatin formation by MBD1 and MCAF/AM family proteins[J]. Journal of Biological Chemistry, 280(14): 13928-13935. [17] Laird, Peter W.2010. Principles and challenges of genome-wide DNA methylation analysis[J]. Nature Reviews Genetics, 11(3): 191-203. [18] Mao J.2002. STAT5 binding contributes to lactational stimulation of promoter Ⅲ expressing the bovine acetyl-CoA carboxylase alpha-encoding gene in the mammary gland[J]. Journal of Molecular Endocrinology, 29(1): 73-88. [19] Mao J, Marcos S, Davis S K, et al.2001. Genomic distribution of three promoters of the bovine gene encoding acetyl-CoA carboxylase α and evidence that the nutritionally regulated promoter I contains a repressive element different from that in rat[J]. Biochemical Journal, 358(1): 127. [20] Mao J, Seyfert H M.2002. Promoter Ⅱ of the bovine acetyl-coenzyme A carboxylase-α-encoding gene is widely expressed and strongly active in different cells[J]. BiochimicaetBiophysicaActa, 1576(3): 324-329. [21] Masashi K, Ayako T, Kasumi N, et al.2016. DNA Methylation Suppresses Leptin Gene in 3T3-L1 Adipocytes[J]. PLOS ONE, 11(8): e0160532. [22] Monika,Stachowiak, Krzysztof, et al.2019. Analysis of allele-specific expression of seven candidate genes involved in lipid metabolism in pig skeletal muscle and fat tissues reveals allelic imbalance of ACACA, LEP, SCD, and TNF[J]. Journal of Applied Genetics, 60(1): 97-101. [23] Moore L D, Le T, Fan G.2013. DNA methylation and its basic function[J]. Neuropsychopharmacology, 38(1): 23-38. [24] Neri F, Rapelli S, Krepelova A, et al.2017. Intragenic DNA methylation prevents spurious transcription initiation[J]. Nature, 543(7643): 72-77. [25] Shi X, Liu S, Metges C C, et al.2010. C/EBP-beta drives expression of the nutritionally regulated promoter IA of the acetyl-CoA carboxylase-alpha gene in cattle[J]. Biochimicaet Biophysica Acta-Gene Regulatory Mechanisms, 1799(8): 561-567. [26] Shi X, Metges C C, Seyfert H M.2012. Interaction of C/EBP-beta and NF-Y factors constrains activity levels of the nutritionally controlled promoter IA expressing the acetyl-CoA carboxylase-alpha gene in cattle[J]. BMC Molecular Biology, 13(1): 21. [27] Shi X, Metges C C, Seyfert H M.2013. Characterization of a far upstream located promoter expressing the acetyl-CoA carboxylase-alpha in the brain of cattle[J]. Gene, 515(2): 266-271. [28] Tong L.2005. Acetyl-coenzyme A carboxylase:crucial metabolic enzyme and attractive target for drug discovery[J]. Cellular and Molecular Life Sciences, 62(16): 1784-1803. [29] Travers M T, Barber M C.2001. Acetyl-CoA carboxylase-α: Gene structure-function relationships[J]. Journal of Animal Science, 79(E-Suppl): E136-E143. [30] Unterberg M, Kreuzer M J, Simon Thomas Schäfer, et al.2016. NFKB1 promoter DNA from nt+402 to nt+99 is hypomethylated in different human immune cells[J]. PLOS ONE, 11(6): 1-14. [31] Wakil S J, Abu-Elheiga L A.2008. Fatty acid metabolism: Target for metabolic syndrome[J]. The Journal of Lipid Research, 50(Supplement): S138-S143. [32] Zhang S, Knight T J, Reecy J M, et al.2010. Associations of polymorphisms in the promoterⅠof bovine acetyl-CoA carboxylase-α gene with beef fatty acid composition[J]. Animal Genetics, 41(4): 417-420.
