绵羊CCL19基因启动子活性分析及其转录调控元件筛选

doi:10.16431/j.cnki.1671-7236.2022.04.002

摘要/Abstract

摘要： 【目的】鉴定绵羊趋化因子C-C基序配体19(C-C motif chemokine ligand 19,CCL19)基因启动子的核心启动子区域和关键转录因子,探究该基因在转录调控方面的作用机制。【方法】选取绵羊CCL19基因5'-侧翼序列1 000 bp,PCR扩增启动子的7个不同长度的截短片段,并连接至pGL3-Basic质粒;将重组质粒与pRL-TK质粒共转染到293T细胞中,结合双荧光素酶报告基因检测系统分析不同截短片段的相对荧光活性。利用在线预测软件分析和筛选CCL19基因核心启动子区域内的转录因子结合位点。采用定点突变技术构建转录因子结合位点缺失的荧光素酶报告载体,与pRL-TK质粒共转染到293T细胞,分析转录因子结合位点缺失质粒的相对荧光活性。【结果】成功构建了7个不同长度(pGL3-P、pGL3-P1、pGL3-P2、pGL3-P3、pGL3-P4、pGL3-P5及pGL3-P6)的CCL19基因启动子片段的荧光素酶报告载体;采用双荧光素酶报告基因检测系统鉴定出转录起始位点上游－256/－186 bp为CCL19基因启动子核心启动子区域,表明该区域对CCL19基因转录调控有重要作用。生物信息学分析预测到该区域存在POU5F1(-201/-189 bp)、ZBTB26(-228/-217 bp)、FOXI1(-239/-228 bp)、GLI2(-255/-243 bp)和SP2(-219/-211 bp) 5个转录因子的结合位点,并成功构建了转录因子结合位点缺失的荧光素酶报告载体。双荧光素酶报告基因检测系统分析显示,POU5F1转录因子的结合位点缺失后绵羊CCL19基因转录活性极显著降低(P<0.01),FOXI1、ZBTB26、SP2转录因子结合位点缺失后绵羊CCL19基因转录活性均极显著升高(P<0.01)。【结论】试验成功构建CCL19基因启动子荧光素酶报告载体,确定CCL19基因启动子的核心启动子区域为转录起始位点上游－256/－186 bp,并鉴定出转录因子POU5F1结合位点可能是CCL19基因转录的重要调控位点,为下一步研究绵羊CCL19基因在先天性免疫、适应性免疫和淋巴细胞迁移等方面的功能提供理论基础。

关键词: 绵羊; CCL19基因; 启动子; 转录调控

Abstract: 【Objective】 The objective of this study was to identify the core promoter region and key transcription factors of sheep C-C motif chemokine ligand 19(CCL19) gene,and explore the transcription regulation mechanism of CCL19 gene.【Method】 Using sheep genomic DNA as template,the 5'-flanking sequence 1 000 bp of CCL19 gene was amplified by PCR.Moreover,seven active region sequences of CCL19 gene promoter were amplified and connected to the pGL3-Basic promoter vector.The recombined plasmid and pRL-TK plasmid were co-transfected into 293T cells,and the activity of relative luciferin enzymes was measured through dual-luciferase detection system.Transcription binding factor sites(TFBSs)prediction was made in the CCL19 gene promoter region by bioinformational method.The luciferase reporter vectors with TFBSs deletion were constructed using site mutation technology and co-transfected into 293T cells,the relative fluorescence activity of these deletion plasmids were analyzed.【Result】 The results showed that seven different length of CCL19 gene promoter fragments were successfully amplified and their dual-luciferase reporting vector (pGL3-P,pGL3-P1,pGL3-P2,pGL3-P3,pGL3-P4,pGL3-P5 and pGL3-P6) were constructed.The active detection of different length promoter fragments found that －256/－186 bp was the core promoter region of CCL19 gene,indicating that this region played an important role in the transcription regulation of CCL19 gene.Bio-information analysis of the CCL19 gene promoter region sequence found that there were 5 TFBSs in this region,including POU5F1(-201/-189 bp),ZBTB26(-228/-217 bp),FOXI1(-239/-228 bp),GLI2(-255/-243 bp) and SP2(-219/-211 bp),and the luciferase reporter vectors with TFBSs deletion were constructed.Dual-luciferase report results showed that the deletion of POU5F1 binding site extremely significantly reduced the transcriptional activity of CCL19 gene in sheep (P<0.01),and FOXI1,ZBTB26 and SP2 extremely significantly enhanced the transcriptional activity of CCL19 gene in sheep (P<0.01).【Conclusion】 This study indicated that the luciferase reporter vector of CCL19 gene promoter was successfully constructed and the core region of CCL19 gene promoter was determined.TFBSs of POU5F1 might be the transcriptional regulation site of CCL19 gene.This result could provide the theoretical basis for further exploring the function of CCL19 gene in lymphocyte migration,innate and adaptive immunity of sheep.

Key words: sheep; CCL19 gene; promoter; transcriptional regulation

中图分类号:

翟哲, 陈思, 吴艳茹, 王雪梅, 李崇瑞, 刘志勇, 陈巧玲, 王凤阳, 杜丽, 李昌, 金宁一. 绵羊CCL19基因启动子活性分析及其转录调控元件筛选[J]. 中国畜牧兽医, 2022, 49(4): 1213-1222.

ZHAI Zhe, CHEN Si, WU Yanru, WANG Xuemei, LI Chongrui, LIU Zhiyong, CHEN Qiaoling, WANG Fengyang, DU Li, LI Chang, JIN Ningyi. Analysis of Promoter Activity and Screening of Transcription Regulatory Elements of CCL19 Gene in Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(4): 1213-1222.

参考文献

[1] YAN Y,CHEN R F,WANG X,et al.CCL19 and CCR7 expression,signaling pathways,and adjuvant functions in viral infection and prevention[J].Frontiers in Cell and Developmental Biology,2019,7:212.
[2] 李博.趋化因子CCL19对小鼠认知及情绪样行为的影响[D].南宁:广西医科大学,2018. LI B.Effect of chemokine CCL19 on cognition and emotion-like behavior in mice[D].Nanning:Guangxi Medical University,2018.(in Chinese)
[3] 吴诗佳.共表达IL-7和CCL19的PSMA-CAR-T细胞对前列腺癌的治疗效果的研究[D].上海:华东师范大学,2020. WU S J.Study on the therapeutic effect of PSMA-CAR-T cells co-expressing IL-7 and CCL19 on prostate cancer[D].Shanghai:East China Normal University,2020.(in Chinese)
[4] PURVANOV V,MATTI C,SAMSON G P B,et al.Fluorescently tagged CCL19 and CCL21 to monitor CCR7 and ACKR4 functions[J].International Journal of Molecular Sciences,2018,19(12):3876.
[5] MORTEN H,ZCAN M,NIELS B L,et al.Autocrine CCL19 blocks dendritic cell migration toward weak gradients of CCL21[J].Cytotherapy,2016,18(9):1187-1196.
[6] XU Z,ZHU C,CHEN C,et al.CCL19 suppresses angiogenesis through promoting miR-206 and inhibiting Met/ERK/Elk-1/HIF-1α/VEGF-A pathway in colorectal cancer[J].Cell Death and Disease,2018,9(10):974.
[7] SHEN Y B,SUN Z F,MAO S,et al.IRF-1 contributes to the pathological phenotype of VSMCs during atherogenesis by increasing CCL19 transcription[J].Aging,2020,13(1):933-943.
[8] ZHANG X S,WANG Y,CAO Y N,et al.Increased CCL19 expression is associated with progression in cervical cancer[J].Oncotarget,2017,8(43):73817-73825.
[9] SHIHAB K,FATEMA A R,MOHAMED A F,et al.Adipose tissue expression of CCL19 chemokine is positively associated with insulin resistance[J].Diabetes/Metabolism Research and Reviews,2019,35(2):e3087.
[10] 罗荣松.奶绵羊与蒙古羊全基因组选择信号和DNA甲基化差异研究[D].呼和浩特:内蒙古大学,2020. LUO R S.Study on the difference of whole genome selection signal and DNA methylation between milk sheep and Mongolian sheep[D].Hohhot:Inner Mongolia University,2020.(in Chinese)
[11] 张灿.湿热应激对藏绵羊和山羊生产性能、瘤胃发酵及血液生化指标影响的比较研究[D].雅安:四川农业大学,2016. ZHANG C.Comparative study of the effects of damp-heat stress on performance,rumen fermentation and blood biochemical indicators of Tibetan sheep and goat[D].Ya’an:Sichuan Agricultural University,2016.(in Chinese)
[12] 王开胜.杜泊羊、湖羊MHC-DRB1基因外显子2多态性与绵羊支原体肺炎抗性关联分析[D].石河子:石河子大学,2017. WANG K S.Association analysis of MHC-DRB1 gene second exon polymorphism of Dorper and Hu sheep with hydatidosis resistance[D].Shihezi:Shihezi University,2017.(in Chinese)
[13] 李清.藏系绵羊抗病免疫基因筛选、分析及功能研究[D].西宁:青海大学,2020. LI Q.Screening,analysis and functional study of disease-resistant immune genes in Tibetan sheep[D].Xining:Qinghai University,2020.(in Chinese)
[14] HAUSER M A AND LEGLER D F.Common and biased signaling pathways of the chemokine receptor CCR7 elicited by its ligands CCL19 and CCL21 in leukocytes[J].Journal of Leukocyte Biology,2016,99(6):869-882.
[15] 孟昭源.miR-493-5p在CCL19促进肺癌细胞侵袭转移中的作用及机制研究[D].昆明:昆明医科大学,2019. MENG Z Y.The role and mechanism of miR-493-5p in CCL19 promoting invasion and metastasis of lung cancer cells[D].Kunming:Kunming Medical University,2019.(in Chinese)
[16] LIU Z W,LI F X,PAN A X,et al.Elevated CCL19/CCR7 expression during the disease process of primary Sjgren’s syndrome[J].Frontiers in Immunology,2019,10:795.
[17] ZHANG R Y,ZHU W Y,MAO S Y,et al.High-concentrate feeding upregulates the expression of inflammation-related genes in the ruminal epithelium of dairy cattle[J].Journal of Animal Science and Biotechnology,2016,7(4):599-611.
[18] MATHES A L,CHRISTMANN R B,STIFANO G,et al.Global chemokine expression in systemic sclerosis (SSc):CCL19 expression correlates with vascular inflammation in SSc skin[J].Annals of the Rheumatic Diseases,2014,73(10):1864-1872.
[19] PIETIL T E,VILLE V,ANNE L,et al.Multiple NF-kappaB and IFN regulatory factor family transcription factors regulate CCL19 gene expression in human monocyte-derived dendritic cells[J].Journal of Immunology,2007,178(1):253-261.
[20] AIME M D,ADRIAN B.CpG islands and the regulation of transcription[J].Genes & Development,2011,25(10):1010-1022.
[21] HARDIVILL S,PARTHA S B,EBRU S S A,et al.TATA-box binding protein O-GlcNAcylation at T114 regulates formation of the B-TFIID complex and is critical for metabolic gene regulation[J].Molecular Cell,2019,77(5):1143-1152.e7.
[22] ZHANG Y J,ZHANG H.RNAa induced by TATA box——Targeting microRNAs[J].Advances in Experimental Medicine and Biology,2017,983:91-111.
[23] LI Y Q.Networks of transcription factors for Oct4 expression in mice[J].DNA and Cell Biology,2017,36(9):725-736.
[24] 蔡东晖.兔HPRT基因和POU5F1基因的克隆及其打靶载体的构建[D].苏州:苏州大学,2007. CAI D H.Cloning of rabbit HPRT gene and POU5F1 gene and construction of its targeting vector[D].Suzhou:Soochow University,2007.(in Chinese)
[25] HEINZ G B,BJRN W,KAREN L,et al.Pou5f1 contributes to dorsoventral patterning by positive regulation of vox and modulation of FGF8A expression[J].Developmental Biology,2011,356(2):323-336.
[26] KUANG W B,DENG Q C,LI Y G,et al.miRNA regulates OCT4 expression in breast cancer cells[J].European Review for Medical and Pharmacological Sciences,2018,22(5):1351-1357.
[27] LU C S,SHIAU A L,SU B H,et al.Oct4 promotes M2 macrophage polarization through upregulation of macrophage colony-stimulating factor in lung cancer[J].Journal of Hematology & Oncology,2020,13(1):62.
[28] LI F,WANG T,HUANG Y,et al.POU2F1 induces the immune escape in lung cancer by up-regulating PD-L1[J].American Journal of Translational Research,2021,13(2):672-683.
[29] LATCHMAN D S.Inhibitory transcription factors[J].The International Journal of Biochemistry & Cell Biology,1996,9(28):965-974.