牛CART基因核心启动子鉴定及转录调控分析

doi:10.11843/j.issn.0366-6964.2023.09.010

摘要/Abstract

摘要： 旨在筛选牛CART基因核心启动子区并鉴定调控CART表达的转录因子,探究其转录调控机制。本研究采集3头健康母牛下丘脑组织,提取基因组DNA,通过PCR扩增、测序获得牛CART启动子序列,EMBOSS、MethPrimer、New PLACE数据库分析启动子结构特征;构建4个包含不同截短长度的CART启动子报告基因载体,双荧光素酶报告基因活性检测鉴定核心启动子区;应用DNA pull down结合质谱分析(n=3),功能聚类及结合位点预测分析筛选核心启动子区候选转录因子;构建转录因子过表达载体,转染至293T细胞,分析转录因子对牛CART核心启动子区的转录调控功能(n=3)。结果表明,牛CART基因-1 200 bp~+22 bp区域存在CpG岛和TATA box、CAAT box等顺式作用元件;构建的4个截短启动子报告基因载体均具有转录起始活性,-292 bp~+22 bp片段转录起始活性最强,为牛CART基因核心启动子区;转录因子RFX5、CREB、RFX1、JUND、TEAD4、TFAP2D、RELA可与牛CART基因核心启动子区特异性结合;进一步研究证实RFX5、RFX1、TEAD4抑制CART转录(P<0.01),CREB与RELA激活CART转录(P<0.001)。本研究成功扩增获得牛CART启动子区域,筛选鉴定-292 bp~+22 bp为牛CART基因的核心启动子区,证实RFX5、RFX1、TEAD4为CART转录抑制因子,CREB、RELA为CART转录激活因子。研究结论为丰富牛下丘脑CART表达调控机制,深入研究CART调控卵泡发育机理提供依据。

关键词: 牛, CART, 核心启动子, 转录调控, DNA pull down

Abstract: The present study aimed to screen the core promoter of bovine CART gene and identify the transcription factors regulating CART expression, and explore its transcriptional regulation mechanism.The hypothalamus of 3 healthy cow were collected for genomic DNA extraction. The promoter of bovine CART gene was obtained through PCR amplification and clone sequencing, and its sequence characteristics were analyzed by EMBOSS, MethPrimer, New PLACE database. The 4 CART gene promoter reporter vectors containing different truncated lengths were constructed to identify the core promoter region by dual luciferase reporter gene activity assay. DNA pull down and mass spectrometry, functional cluster and binding site prediction analysis were used to screen the candidate transcription factors in the core promoter region (n=3). The transcription factors overexpression vectors were constructed and transfected to 293T cells to analyze the regulatory function of transcription factors on bovine CART gene transcription (n=3). The results showed that there were CpG island and cis-acting elements such as TATA box and CAAT box in the -1 200 bp-+22 bp region of bovine CART gene. All the 4 reporter vectors with truncated promoters had transcription initiation activity and the -292 bp-+22 bp was the highest, this region was the core promoter of bovine CART gene. RFX5, CREB, RFX1, JUND, TEAD4, TFAP2D, RELA could specifically bind to the core promoter region of the bovine CART gene. Furthermore, it was proved that RFX5, RFX1, TEAD4 could inhibit CART gene transcription (P<0.01); CREB and RELA could activate CART gene transcription (P<0.001). In this study, bovine CART promoter region was successfully amplified, and identify the -292 bp-+22 bp was core region of bovine CART gene promoter. And it was proved that the RFX5, RFX1, TEAD4 were inhibiting transcription factors of CART gene; the CREB and RELA were activating transcription factors of CART gene. These findings will enrich the regulatory mechanism of CART gene expression in bovine hypothalamus, and strengthen the understanding of the mechanism of CART regulating follicular development.

Key words: bovine, CART, core promoter, transcriptional regulation, DNA pull down

中图分类号:

S823.2

王栋梁, 任静, 郝琴琴, 李鹏飞. 牛CART基因核心启动子鉴定及转录调控分析[J]. 畜牧兽医学报, 2023, 54(9): 3689-3699.

WANG Dongliang, REN Jing, HAO Qinqin, LI Pengfei. Identification and Transcriptional Regulation Analysis of Core Promoter of Bovine CART Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3689-3699.

参考文献

[1] RAJAKOSKI E.The ovarian follicular system in sexually mature heifers with special reference to seasonal,cyclical,and left-right variations[J].Acta Endocrinol,1960,34(S3):S7-S68.
[2] SAMSON W K,SALVEMINI D,YOSTEN G L C.Overcoming stress,hunger,and pain:cocaine-and amphetamine-regulated transcript peptide's promise[J].Endocrinology,2021,162(8):bqab108.
[3] ONG Z Y,MCNALLY G P.CART in energy balance and drug addiction:Current insights and mechanisms[J].Brain Res,2020, 1740:146852.
[4] BARANOWSKA B,WOLIŃSKA-WITORT E,MARTYŃSKA L,et al.Effects of cocaine-amphetamine regulated transcript (CART) on hormone release[J].Regul Pept,2004,122(2):55-59.
[5] SMITH G W,SEN A,FOLGER J K,et al.Putative role of cocaine-and amphetamine-regulated transcript (CARTPT) in dominant follicle selection in cattle[J].Soc Reprod Fertil Suppl,2010,67:105-117.
[6] LV L H,JIMENEZ-KRASSEL F,SEN A,et al.Evidence supporting a role for cocaine-and amphetamine-regulated transcript (CARTPT) in control of granulosa cell estradiol production associated with dominant follicle selection in cattle[J].Biol Reprod,2009,81(3):580-586.
[7] SHOBATAKE R,TAKASAWA K,OTA H,et al.Up-regulation of POMC and CART mRNAs by intermittent hypoxia via GATA transcription factors in human neuronal cells[J].Int J Biochem Cell Biol,2018,95:100-107.
[8] ZHANG J,WANG S H,YUAN L,et al.Neuron-restrictive silencer factor (NRSF) represses cocaine-and amphetamine-regulated transcript (CART) transcription and antagonizes cAMP-response element-binding protein signaling through a dual NRSE mechanism[J].J Biol Chem,2012,287(51):42574-42587.
[9] ROGGE G A,JONES D C,GREEN T,et al.Regulation of CART peptide expression by CREB in the rat nucleus accumbens in vivo[J].Brain Res,2009,1251:42-52.
[10] ROGGE G A,SHEN L L,KUHAR M J.Chromatin immunoprecipitation assays revealed CREB and serine 133 phospho-CREB binding to the CART gene proximal promoter[J].Brain Res,2010,1344:1-12.
[11] 朱芷葳,侯淑宁,郝庆玲,等.牛卵泡AGTR2序列结构及表达特性分析[J].中国农业科学,2020,53(7):1482-1490.
ZHU Z W,HOU S N,HAO Q L,et al.Sequence structure and expression characteristics analysis of AGTR2 in bovine follicle[J].Scientia Agricultura Sinica,2020,53(7):1482-1490.(in Chinese)
[12] 成俊丽,郝庆玲,侯淑宁,等.牛卵泡颗粒细胞CART相互作用蛋白鉴定及受体筛选[J].畜牧兽医学报,2020,51(12):3046-3056.
CHENG J L,HAO Q L,HOU S N,et al.Identification and receptor screening of CART interacting proteins in bovine follicular granulosa cells[J].Acta Veterinaria et Zootechnica Sinica,2020,51(12):3046-3056.(in Chinese)
[13] LI P F,YU X J,XIE J S,et al.Expression of cocaine-and amphetamine-regulated transcript (CART) in hen ovary[J].Biol Res,2017,50(1):18.
[14] REGAN S L P,KNIGHT P G,YOVICH J L,et al.Granulosa cell apoptosis in the ovarian follicle-a changing view[J].Front Endocrinol,2018,9:61.
[15] SEN A,BETTEGOWDA A,JIMENEZ-KRASSEL F,et al.Cocaine-and amphetamine-regulated transcript regulation of follicle-stimulating hormone signal transduction in bovine granulosa cells[J].Endocrinology,2007,148(9):4400-4410.
[16] TANIMOTO R,SEKII K,MOROHAKU K,et al.Blocking estrogen-induced AMH expression is crucial for normal follicle formation[J].Development,2021,148(6):dev197459.
[17] CASARINI L,RICCETTI L,DE PASCALI F,et al.Estrogen modulates specific life and death signals Induced by LH and hCG in human primary granulosa cells in vitro[J].Int J Mol Sci,2017,18(5):926.
[18] 李鹏飞,孟金柱,郝庆玲,等.PDF2和ODF1转录组测序筛选牛卵泡发育相关基因[J].畜牧兽医学报,2018,49(2):300-309.
LI P F,MENG J Z,HAO Q L,et al.Screening and analysing of genes associated with follicular development in bovine ODF1 and PDF2 transcriptome[J].Acta Veterinaria et Zootechnica Sinica,2018,49(2):300-309.(in Chinese)
[19] 任静,郝琴琴,成俊丽,等.bta-miR-377靶向调控牛下丘脑CART基因表达的研究[J].中国畜牧兽医,2022,49(9):3301-3309.
REN J,HAO Q Q,CHENG J L,et al.Expression analysis of bta-miR-377 regulate CART gene in bovine hypothalamus[J].China Animal Husbandry & Veterinary Medicine,2022,49(9):3301-3309.(in Chinese)
[20] YAMADA K,YUAN X,OTABE S,et al.Sequencing of the putative promoter region of the cocaine-and amphetamine-regulated-transcript gene and identification of polymorphic sites associated with obesity[J].Int J Obes,2002,26(1):132-136.
[21] DOMINGUEZ G,LAKATOS A,KUHAR M J.Characterization of the cocaine-and amphetamine-regulated transcript (CART) peptide gene promoter and its activation by a cyclic AMP-dependent signaling pathway in GH3 cells[J].J Neurochem,2002,80(5):885-893.
[22] LING F,WEI L Q,WANG T,et al.Cloning and characterization of the 5'-flanking region of the pig cocaine-and amphetamine-regulated transcript gene[J].DNA Cell Biol,2011,30(2):91-97.
[23] DEATON A M,BIRD A.CpG islands and the regulation of transcription[J].Genes Dev,2011,25(10):1010-1022.
[24] ANGELONI A,BOGDANOVIC O.Sequence determinants,function,and evolution of CpG islands[J].Biochem Soc Trans,2021, 49(3):1109-1119.
[25] SHARMA V K,SINGH T G.CREB:a multifaceted target for Alzheimer's disease[J].Curr Alzheimer Res,2020,17(14):1280-1293.
[26] WANG L,HU X H,HUANG Z X,et al.Regulation of CREB functions by phosphorylation and sumoylation in nervous and visual systems[J].Curr Mol Med,2017,16(10):885-892.
[27] DIMBERG J,HUGANDER A,HÄLL-KARLSSON B M,et al.RFX-B,a MHC class II transcription factor,suppressed in human colorectal adenocarcinomas[J].Int J Mol Med,2002,9(3):213-216.
[28] SUGIAMAN-TRAPMAN D,VITEZIC M,JOUHILAHTI E M,et al.Characterization of the human RFX transcription factor family by regulatory and target gene analysis[J].BMC Genomics,2018,19(1):181.
[29] CHEN D B,XIE X W,ZHAO Y J,et al.RFX5 promotes the progression of hepatocellular carcinoma through transcriptional activation of KDM4A[J].Sci Rep,2020,10(1):14538.
[30] SU J C,TSENG P H,HSU C Y,et al.RFX1-dependent activation of SHP-1 induces autophagy by a novel obatoclax derivative in hepatocellular carcinoma cells[J].Oncotarget,2014,5(13):4909-4919.
[31] ISSAC J,RAVEENDRAN P S,DAS A V.RFX1:a promising therapeutic arsenal against cancer[J].Cancer Cell Int,2021,21(1):253.
[32] 王娜,甲芝莲,吴强.RFX5调控原钙粘蛋白α基因簇的表达[J].遗传,2020,42(8):760-774.
WANG N,JIA Z L,WU Q.RFX5 regulates gene expression of the Pcdhα cluster[J].Hereditas,2020,42(8):760-774.(in Chinese)
[33] LIN K C,MOROISHI T,MENG Z P,et al.Regulation of Hippo pathway transcription factor TEAD by p38 MAPK-induced cytoplasmic translocation[J].Nat Cell Biol,2017,19(8):996-1002.
[34] SHI Z,HE F,CHEN M,et al.DNA-binding mechanism of the Hippo pathway transcription factor TEAD4[J].Oncogene, 2017, 36(30):4362-4369.
[35] CHI M,LIU J,MEI C X,et al.TEAD4 functions as a prognostic biomarker and triggers EMT via PI3K/AKT pathway in bladder cancer[J].J Exp Clin Cancer Res,2022,41(1):175.
[36] WANG C Y,NIE Z,ZHOU Z M,et al.The interplay between TEAD4 and KLF5 promotes breast cancer partially through inhibiting the transcription of p27^Kip1[J].Oncotarget,2015,6(19):17685-17697.
[37] HERNANDEZ J M,FLOYD D H,WEILBAECHER K N,et al.Multiple facets of junD gene expression are atypical among AP-1 family members[J].Oncogene,2008,27(35):4757-4767.
[38] FRAUNE C,HARMS L,BÜSCHECK F,et al.Upregulation of the transcription factor TFAP2D is associated with aggressive tumor phenotype in prostate cancer lacking the TMPRSS2:ERG fusion[J].Mol Med,2020,26(1):24.
[39] NAKAZATO A,MOCHIZUKI M,SHIBUYA-TAKAHASHI R,et al.RELA is required for CD271 expression and stem-like characteristics in hypopharyngeal cancer[J].Sci Rep,2022,12(1):17751.
[40] BASU P,ALTUVIA S.RelA binding of mRNAs modulates translation or sRNA-mRNA basepairing depending on the position of the GGAG site[J].Mol Microbiol,2022,117(1):143-159.