奶山羊<i>ATGL</i>基因启动子鉴定与转录活性分析

doi:10.7668/hbnxb.20192556

摘要/Abstract

摘要：

脂肪甘油三酯水解酶(ATGL)是脂肪水解过程中的主要限速酶,是调控细胞中甘油三酯和脂肪酸之间平衡的关键因子。为了分析奶山羊ATGL基因启动子的结构及转录调控机制,以奶山羊全血DNA为模板,通过PCR技术扩增出ATGL基因5'侧翼序列并进行生物信息学分析;构建了5个不同长度的启动子缺失片段报告基因载体,通过转染奶山羊乳腺上皮细胞并检测其活性,确定其核心区域;分别用PPARG激动剂罗格列酮和SREBP1激动剂T0901317处理细胞,检测其对ATGL启动子活性的影响。结果表明,克隆得到奶山羊ATGL基因5'侧翼序列2 721 bp,包含转录起始位点上游2 024 bp。经在线软件分析发现,ATGL启动子含有真核生物启动子元件TATA-box和CpG岛;对转录因子结合位点预测发现,其上含有FOXO、SREBF1、PPARG、C/EBPα和E2F1等结合位点。启动子活性分析结果表明,ATGL启动子核心区域位于转录起始位点上游-256—+1位,且在-527—-256 位存在负调控元件。用罗格列酮和T0901317处理细胞后发现,二者均能显著上调ATGL启动子活性,且罗格列酮作用的区域为-527—-256 位,T0901317在-882—-527 位发挥作用,表明PPARG和SREBP1调控ATGL启动子活性。

关键词: 奶山羊, ATGL, 启动子, 核心区域, 转录活性

Abstract:

Adipose triglyceride lipase(ATGL)is the main rate limiting enzyme in the process of fat hydrolysis.In order to analyze the structure and transcriptional regulation mechanism of ATGL promoter in dairy goat,the 5' flanking sequence of ATGL was amplified by PCR and analyzed by bioinformatics.Luciferase reporter gene vectors of five deletion fragments with different lengths of promoter were constructed and transfected into mammary epithelial cells of dairy goat.The cells were treated with rosiglitazone and T0901317 respectively to detect the effects of PPARG and SREBP1 on ATGL promoter activity.The results showed that the 5' flanking sequence of ATGL gene of dairy goat was 2 721 bp,including 2 024 bp upstream from the transcription start site.The ATGL promoter contained eukaryotic promoter elements TATA-box and CpG island.It was also found that there were transcription factors-binding sites of FOXO,SREBF1,PPARG,C/EBPα and E2F1 by the online software prediction.The core region of ATGL promoter was located at -256—+1,and there were negative regulatory elements at -527—-256.When cells were treated with rosiglitazone and T0901317,it was found that both of them could significantly up-regulate ATGL promoter activity.The response regions of rosiglitazone and T0901317 were -527—-256 and -882—-527,respectively,indicating that PPARG and SREBP1 could regulate ATGL promoter activity.

Key words: Dairy goat, Adipose triglyceride lipase, Promoter, Core region, Transcriptional activity

李君, 闫志浩, 贾万里, 许蒙蒙, 张景锋, 徐秋良, 韩浩园, 权凯. 奶山羊ATGL基因启动子鉴定与转录活性分析[J]. 华北农学报, 2022, 37(2): 223-230. doi: 10.7668/hbnxb.20192556.

LI Jun, YAN Zhihao, JIA Wanli, XU Mengmeng, ZHANG Jingfeng, XU Qiuliang, HAN Haoyuan, QUAN Kai. Identification and Transcriptional Activity Analysis of ATGL Gene Promoter in Dairy Goat[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(2): 223-230. doi: 10.7668/hbnxb.20192556.

http://www.hbnxb.net/CN/Y2022/V37/I2/223

图/表 7

表1 奶山羊ATGL基因启动子克隆引物序列

Tab.1 The sequences of primers used for cloning ATGL gene promoter of dairy goat

引物名称 Primer name	引物序列 Primer sequence	结合位点 Binding site	产物大小/bp Product length
APF	GGCAGAACTCGCAATCCTA	-2 024	2 721
APR	CTAGCACCCGGTCTTACA	+697
APS1	ATTACGCGTGGCAGAACTCGCAATCCTA	-2 024	2 240
APS2	ATTACGCGTCTCAGTCCTTACTTGAACCT	-1 399	1 615
APS3	ATTACGCGTTCAGAACAGAGACAGGACT	-882	1 098
APS4	ATTACGCGTGATTCTGTGATGAGTCTCG	-527	743
APS5	ATTACGCGTCCTGGCTACTGTCTTTGG	-256	472
APA	GGAAGATCTCACGCCGATATGGTAGAC	+216

图1 ATGL基因启动子的PCR扩增产物

Fig.1 The PCR amplification products of ATGL gene promoter

图2 奶山羊ATGL部分启动子序列及潜在的转录因子结合位点
A.ATGL基因5'侧翼序列图示;B.ATGL启动子潜在的转录因子结合位点分析(+1为转录起始位点)。

Fig.2 The partial nucleotide sequence and potential transcription factor binding sites of ATGL promoter
A.Schematic representation of ATGL 5'flanking region;B.Analysis of the putative transcription factor binding sites of the ATGL promoter(+1 is the first base at transcription start site).

图3 ATGL基因启动子不同缺失片段的扩增与载体构建
A.不同缺失片段PCR扩增产物:1.-2 024—+216位;2.-1 399—+216位;3.-882—+216位;4.-527—+216位;5.-256—+216位;B.不同缺失片段重组质粒酶切鉴定:1.pGL-APS1(-2 024—+216位);2.pGL-APS2(-1 399—+216 位);3.pGL-APS3(-882—+216位);4.pGL-APS4(-527—+216位);5.pGL-APS5(-256—+216位)。

Fig.3 Amplification products and vector construction of various deletion fragments of ATGL promoter
A.PCR amplification products of various deletion fragments:1.-2 024—+216;2.-1 399—+216;3.-882—+216;4.-527—+216;5.-256—+216;B.Restriction enzyme digestion of recombinant plasmids of various deletion fragments:1.pGL-APS1(-2 024—+216);2.pGL-APS2(-1 399—+216);3.pGL-APS3(-882—+216);4.pGL-APS4(-527—+216);5.pGL-APS5(-256—+216).

图4 ATGL启动子不同缺失片段活性分析
pGL3-Basic.荧光素酶报告基因空载体;LUC.不同缺失片段的荧光素酶报告基因载体;不同字母表示差异显著(P<0.05)。图5—6同。

Fig.4 Relative activity of the various deletion fragments of ATGL promoter
pGL3-Basic.Luciferase reporter gene empty vector;LUC.Luciferase reporter gene vector with various deletion fragments;Different letters indicate significant differences(P<0.05).The same as Fig.5—6.

图5 罗格列酮对ATGL启动子活性的影响
A.罗格列酮对ATGL启动子全长活性的影响;B.罗格列酮对ATGL启动子不同片段的影响。

Fig.5 Effect of ROSI on the activity of ATGL promoter
A.Effect of ROSI on the activity of ATGL promoter in full length;B.Effect of ROSI on the activity of various deletion fragments of ATGL promoter.

图6 T0901317对ATGL启动子活性的影响
A.T0901317对ATGL启动子全长活性的影响;B.T0901317对ATGL启动子不同片段的影响。

Fig.6 Effect of T0901317 on the activity of ATGL promoter
A.Effect of T0901317 on the activity of ATGL promoter in full length;B.Effect of T0901317 on the activity of various deletion fragments of ATGL promoter.

参考文献 28

[1]	Cerk I K,Wechselberger L,Oberer M.Adipose triglyceride lipase regulation:An overview[J].Current Protein & Peptide Science,2018,19(2):221-233.doi:10.2174/1389203718666170918160110.
[2]	Lake A C,Sun Y,Li J L,Kim J E,Johnson J W,Li D M,Revett T,Shih H H,Liu W,Paulsen J E,Gimeno R E.Expression,regulation,and triglyceride hydrolase activity of adiponutrin family members[J].Journal of Lipid Research,2005,46(11):2477-2487.doi:10.1194/jlr.M500290-JLR200.
[3]	Zimmermann R,Strauss J G,Haemmerle G,Schoiswohl G,Birner-Gruenberger R,Riederer M,Lass A,Neuberger G,Eisenhaber F,Hermetter A,Zechner R.Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase[J].Science,2004,306(5700):1383-1386.doi:10.1126/science.1100747.
[4]	Li J,Luo J,Wang H,Shi H B,Zhu J J,Sun Y T,Yu K,Yao D W.Adipose triglyceride lipase regulates lipid metabolism in dairy goat mammary epithelial cells[J].Gene,2015,554(1):125-130.doi:10.1016/j.gene.2014.10.020.
[5]	Kershaw E E,Hamm J K,Verhagen L A W,Peroni O,Katic M,Flier J S.Adipose triglyceride lipase:Function,regulation by insulin,and comparison with adiponutrin[J].Diabetes,2006,55(1):148-157.doi:10.2337/diabetes.55.01.06.db05-0982.
[6]	Roy D,Farabaugh K T,Wu J,Charrier A,Smas C,Hatzoglou M,Thirumurugan K,Buchner D A.Coordinated transcriptional control of adipocyte triglyceride lipase(Atgl)by transcription factors Sp1 and peroxisome proliferator-activated receptor γ(PPARγ)during adipocyte differentiation[J].Journal of Biological Chemistry,2017,292(36):14827-14835.doi:10.1074/jbc.M117.783043.
[7]	Kaltenecker D,Mueller K M,Benedikt P,Feiler U,Themanns M,Schlederer M,Kenner L,Schweiger M,Haemmerle G,Moriggl R.Adipocyte STAT5 deficiency promotes adiposity and impairs lipid mobilisation in mice[J].Diabetologia,2017,60(2):296-305.doi:10.1007/s00125-016-4152-8.
[8]	Wang X Y,Xu M X,Peng Y,Naren Q,Xu Y T,Wang X,Yang G S,Shi X E,Li X.Triptolide enhances lipolysis of adipocytes by enhancing ATGL transcription via upregulation of p53[J].Phytotherapy Research,2020,34(12):3298-3310.doi:10.1002/ptr.6779.
[9]	Chakrabarti P,Kandror K V.FoxO1 controls insulin dependent ATGL expression and lipolysis in adipocytes[J].Journal of Biological Chemistry,2009,284(20):13296-13300.doi:10.1074/jbc.C800241200.
[10]	Eguchi J,Wang X,Yu S T,Kershaw E E,Chiu P C,Dushay J,Estall J L,Klein U,Maratos-Flier E,Rosen E D.Transcriptional control of adipose lipid handling by IRF4[J]. Cell Metabolism,2011,13(3):249-259.doi:10.1016/j.cmet.2011.02.005.
[11]	汪亮,李忠秋,唐晓东,张海峰,刘娣,张冬杰.猪ATP6V0A4基因启动子核心区的筛选与分析[J].华北农学报,2021,36(3):216-221.doi: 10.7668/hbnxb.20191591.
	Wang L,Li Z Q,Tang X D,Zhang H F,Liu D,Zhang D J.Screening and analysis of the core region of porcine ATP6V0A4 gene promoter[J].Acta Agriculturae Boreali-Sinica,2021,36(3):216-221.
[12]	Schweiger M,Schreiber R,Haemmerle G,Lass A,Fledelius C,Jacobsen P,Tornqvist H,Zechner R,Zimmermann R.Adipose triglyceride lipase and hormone-sensitive lipase are the major enzymes in adipose tissue triacylglycerol catabolism[J].Journal of Biological Chemistry,2006,281(52):40236-40241.doi:10.1074/jbc.M608048200.
[13]	李圣彦,郎志宏,黄大昉.真核生物启动子研究概述[J].生物技术进展,2014,4(3):158-164.doi:10.3969/j.issn.2095-2341.2014.03.02.
	Li S Y,Lang Z H,Huang D F.Research progress on eukaryotic promoter[J].Current Biotechnology,2014,4(3):158-164.
[14]	Kim J Y,Tillison K,Lee J H,Rearick D A,Smas C M.The adipose tissue triglyceride lipase ATGL/PNPLA2 is downregulated by insulin and TNF-α in 3T3-L1 adipocytes and is a target for transactivation by PPARγ[J].American Journal of Physiology Endocrinology and Metabolism,2006,291(1):E115-E127.doi:10.1152/ajpendo.00317.2005.
[15]	Kim S J,Tang T Y,Abbott M,Viscarra J A,Wang Y H,Sul H S.AMPK phosphorylates desnutrin/ATGL and hormone-sensitive lipase to regulate lipolysis and fatty acid oxidation within adipose tissue[J].Molecular and Cellular Biology,2016,36(14):1961-1976.doi:10.1128/MCB.00244-16.
[16]	孙健. 营养限制条件下ATGL维持草鱼脂肪细胞脂质代谢稳态的转录调控机制研究[D].杨凌:西北农林科技大学,2019:49-51.
	Sun J.Transcriptional of ATGL and its role in maintaining adipocyte lipid homeostasis in grass carp(tenopharyngodon idellus)adipocytes under nutritional restriction[D].Yangling:Northwest A&F University, 2019:49-51.
[17]	王小宇. 转录因子p53调控猪脂肪酸甘油三酯水解酶ATGL转录的机制研究[D].杨凌:西北农林科技大学,2020:11-14.doi:10.27409/d.cnki.gxbnu.2020.001552.
	Wang X Y.Mechanism research of transcription factor p53 regulation pig ATGL transcription[D].Yangling:Northwest A&F University,2020:11-14.
[18]	Wang M Q,Zhou C H,Cong S,Han D X,Wang C J,Tian Y,Zhang J B,Jiang H,Yuan B.Lipopolysaccharide inhibits triglyceride synthesis in dairy cow mammary epithelial cells by upregulating miR-27a-3p,which targets the PPARG gene[J].Journal of Dairy Science,2021,104(1):989-1001.doi:10.3168/jds.2020-18270.
[19]	Shi H B,Luo J,Yao D W,Zhu J J,Xu H F,Shi H P,Loor J J.Peroxisome proliferator-activated receptor-γ stimulates the synthesis of monounsaturated fatty acids in dairy goat mammary epithelial cells via the control of stearoyl-coenzyme A desaturase[J].Journal of Dairy Science,2013,96(12):7844-7853.doi:10.3168/jds.2013-7105.
[20]	Yu K,Shi H B,Luo J,Li J,Zhao W S,Tian H B,Shi H P.PPARG modulated lipid accumulation in dairy GMEC via regulation of ADRP gene[J].Journal of Cellular Biochemistry,2015,116(1):192-201.doi:10.1002/jcb.24958.
[21]	辛慧杰,刘建新,石恒波.关键转录因子调节反刍动物乳腺脂肪酸代谢的研究进展[J].农业生物技术学报,2020,28(5):918-924.doi:10.3969/j.issn.1674-7968.2020.05.016.
	Xin H J,Liu J X,Shi H B.Research progress on the key transcription factors regulating mammary fatty acid metabolism in ruminants[J].Journal of Agricultural Biotechnology,2020,28(5):918-924.
[22]	Shi H B,Luo J,Zhu J J,Li J,Sun Y T,Lin X Z,Zhang L P,Yao D W,Shi H P.PPARγ regulates genes involved in triacylglycerol synthesis and secretion in mammary gland epithelial cells of dairy goats[J].PPAR Research,2013,2013:310948.doi:10.1155/2013/310948.
[23]	Nielsen R,Pedersen T A,Hagenbeek D,Moulos P,Siersbaek R,Megens E,Denissov S,Børgesen M,Francoijs K J,Mandrup S,Stunnenberg H G.Genome-wide profiling of PPARγ:RXR and RNA polymerase Ⅱ occupancy reveals temporal activation of distinct metabolic pathways and changes in RXR dimer composition during adipogenesis[J].Genes & Development,2008,22(21):2953-2967.doi:10.1101/gad.501108.
[24]	Xu H F,Luo J,Tian H B,Li J,Zhang X Y,Chen Z,Li M,Loor J J.Rapid communication:Lipid metabolic gene expression and triacylglycerol accumulation in goat mammary epithelial cells are decreased by inhibition of SREBP-1[J].Journal of Animal Science,2018,96(6):2399-2407.doi:10.1093/jas/sky069.
[25]	Xu H F,Luo J,Ma G Z,Zhang X Y,Yao D W,Li M,Loor J J.Acyl-CoA synthetase short-chain family member 2(ACSS2)is regulated by SREBP-1 and plays a role in fatty acid synthesis in caprine mammary epithelial cells[J].Journal of Cellular Physiology,2018,233(2):1005-1016.doi:10.1002/jcp.25954.
[26]	Oppi-Williams C,Suagee J K,Corl B A.Regulation of lipid synthesis by liver X receptor α and sterol regulatory element-binding protein 1 in mammary epithelial cells[J].Journal of Dairy Science,2013,96(1):112-121.doi:10.3168/jds.2011-5276.
[27]	Xu H F,Luo J,Zhang X Y,Li J,Bionaz M.Activation of liver X receptor promotes fatty acid synthesis in goat mammary epithelial cells via modulation of SREBP1 expression[J].Journal of Dairy Science,2019,102(4):3544-3555.doi:10.3168/jds.2018-15538.
[28]	Xu H F,Luo J,Zhao W S,Yang Y C,Tian H B,Shi H B,Bionaz M.Overexpression of SREBP1(sterol regulatory element binding protein 1)promotes de novo fatty acid synthesis and triacylglycerol accumulation in goat mammary epithelial cells[J].Journal of Dairy Science,2016,99(1):783-795.doi:10.3168/jds.2015-9736.

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奶山羊ATGL基因启动子鉴定与转录活性分析

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奶山羊ATGL基因启动子鉴定与转录活性分析

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