骨质疏松易感SNP rs4325274通过增强子远程调控SOX6基因的功能机制研究

doi:10.16288/j.yczz.20-098

遗传 ›› 2020, Vol. 42 ›› Issue (9): 889-897.doi: 10.16288/j.yczz.20-098

骨质疏松易感SNP rs4325274通过增强子远程调控SOX6基因的功能机制研究

妥晓梅¹, 朱东丽^1,², 陈晓峰¹, 荣誉¹, 郭燕¹, 杨铁林^1,²()

^1. 西安交通大学生命科学与技术学院,生物医学信息工程教育部重点实验室,生物医学信息与基因组学中心,西安 710049
^2. 浙江西安交通大学研究院,杭州 311215

收稿日期:2020-05-26 修回日期:2020-09-04 出版日期:2020-09-20 发布日期:2020-09-07
通讯作者: 杨铁林 E-mail:yangtielin@xjtu.edu.cn
作者简介:妥晓梅,在读硕士研究生,专业方向：疾病分子遗传机制的基础研究。E-mail: xmt18392079044@stu.xjtu.edu.cn
基金资助:
国家自然科学基金面上项目编号(31771399);国家自然科学基金面上项目编号(31970569);中国博士后基金项目编号(2019M650261);陕西省自然科学基础研究计划项目编号(2020JQ-026);浙江省自然科学基金资助编号(GF18C060003)

The osteoporosis susceptible SNP rs4325274 remotely regulates the SOX6 gene through enhancers

Xiaomei Tuo¹, Dongli Zhu^1,², Xiaofeng Chen¹, Yu Rong¹, Yan Guo¹, Tielin Yang^1,²()

^1. Biomedical Informatics & Genomics Center, Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an 710049;
^2. Research Institute of Zhejiang Xi'an Jiaotong University, Hangzhou 311215

Received:2020-05-26 Revised:2020-09-04 Online:2020-09-20 Published:2020-09-07
Contact: Yang Tielin E-mail:yangtielin@xjtu.edu.cn
Supported by:
Supported by the National Natural Science Foundation of China No(31771399);Supported by the National Natural Science Foundation of China No(31970569);China PostdoctoralScience Foundation No(2019M650261);Natural Science Basic Research Plan in Shaanxi Province of China No(2020JQ-026);the Zhejiang Provincial Natural Science Foundation of China No(GF18C060003)

摘要/Abstract

摘要：

骨质疏松症是一种典型的多基因复杂疾病,遗传力高达85%,其发病率已跃居常见疾病的第5位。尽管已经鉴定出大量骨质疏松易感SNP,但大多数SNP位点位于基因组非编码区,且功能机制未知。本研究旨在通过生物信息学分析和功能实验探究骨质疏松非编码功能性易感SNP rs4325274的分子调控机制。首先,通过表观注释发现该SNP所在区域处在增强子上,eQTL和Hi-C分析结果发现SNP调控的潜在靶基因是SOX6;然后,利用多种数据库进行Motif预测,并结合GEO数据库中的ChIP-seq数据分析进行了验证,结果发现转录因子HNF1A更倾向于结合SNP rs4325274-G碱基;进一步通过双荧光素酶报告基因实验验证了该SNP对SOX6基因表达的增强作用;最后,利用shRNA敲低转录因子HNF1A实验,检测靶基因SOX6的表达变化。以上研究结果初步解析了非编码区功能性SNPrs4325274作为增强子远程调控SOX6基因表达的分子机制,为复杂疾病非编码易感SNP的遗传调控研究提供新思路。

关键词: SNP rs4325274, SOX6基因, 转录因子, 骨质疏松症发生机制

Abstract:

Osteoporosis is a typical polygenic disease, and its heritability is as high as 85%. The incidence of osteoporosis has jumped to the fifth among the common diseases. Although a large number of osteoporosis-susceptible SNPs have been identified, most of them are in the non-coding regions of the genome and the functional mechanisms are unknown. The purpose of this study was to explore the function of non-coding osteoporosis-susceptible SNP rs4325274 and dissect the molecular regulatory mechanisms through integrating bioinformatics analysis and functional experiments. Firstly, we found the SNP rs4325274 resided in a putative enhancer element through functional annotation. eQTL and Hi-C analysis found that the SOX6 gene might be a potential distal target of rs4325274. We conducted the motif prediction using multiple databases and verified the result using ChIP-seq data from GEO database. The result showed that the transcription factor HNF1A could preferentially bind to SNP rs4325274-G allele. We further demonstrated that SNP rs4325274 acted as an enhancer regulating SOX6 gene expression by using dual-luciferase reporter assays. Knockdown of HNF1A decreased the SOX6 gene expression. Taken together, our results uncovered a new mechanism of a non-coding functional SNP rs4325274 as a distal enhancer to modulate SOX6 expression, which provides new insights into deciphering molecular regulatory mechanisms underlying non-coding susceptibility SNPs on complex diseases.

Key words: SNP rs4325274, SOX6 gene, transcription factor, mechanism of osteoporosis

妥晓梅, 朱东丽, 陈晓峰, 荣誉, 郭燕, 杨铁林. 骨质疏松易感SNP rs4325274通过增强子远程调控SOX6基因的功能机制研究[J]. 遗传, 2020, 42(9): 889-897.

Xiaomei Tuo, Dongli Zhu, Xiaofeng Chen, Yu Rong, Yan Guo, Tielin Yang. The osteoporosis susceptible SNP rs4325274 remotely regulates the SOX6 gene through enhancers[J]. Hereditas(Beijing), 2020, 42(9): 889-897.

图/表 8

表1

图1

图2

图3

图4

图5

图6

图7

参考文献 30

[1]	Lamichhane AP . Osteoporosis-an update. JNMA J Nepal Med Assoc, 2005,44(158):60-66. pmid: 16568580
[2]	Reginster JY, Burlet N . Osteoporosis: a still increasing prevalence. Bone, 2006,38(2 Suppl. 1):S4-9. doi: 10.1016/j.bone.2005.11.024 pmid: 16455317
[3]	Morales-Torres J , Gutiérrez-UreñaS.The burden of osteoporosis in latinamerica. OsteoporosInt, 2004,15(8):625-632.
[4]	Nguyen TV, Center JR, Eisman JA . Osteoporosis: underrated, underdiagnosed and undertreated. Med J Aust, 2004,180(S5):S18-22. pmid: 14984358
[5]	Smits P, Li P, Mandel J, Zhang ZP, Deng JM, Behringer RR, de Crombrugghe B, Lefebvre V,. The transcription factors L-Sox5 and Sox6 are essential for cartilage formation. Dev Cell, 2001,1(2):277-290. doi: 10.1016/s1534-5807(01)00003-x pmid: 11702786
[6]	Smits P, Dy P, Mitra S, Lefebvre V . Sox5 and Sox6 are needed to develop and maintain source, columnar, and hypertrophic chondrocytes in the cartilage growth plate. J Cell Biol, 2004,164(5):747-758. doi: 10.1083/jcb.200312045 pmid: 14993235
[7]	Renard E, Porée B, Chadjichristos C, Kypriotou M, Maneix L, Bigot N, Legendre F, Ollitrault D, De Crombrugghe B, Malléin-Gérin F, Moslemi S, Demoor M, Boumediene K, Galéra P . Sox9/Sox6 and Sp1 are involved in the insulin-like growth factor-I-mediated upregulation of human type II collagen gene expression in articular chondrocytes. J Mol Med (Berl), 2012,90(6):649-666. doi: 10.1007/s00109-011-0842-3
[8]	Liu J, Wang HW, Chen Y, Yu HL, Wang Q, Yang HF, Ma JX, Xiang LB . Regulatory effect of SOX6 and SOX9 on the growth and differentiation properties into chondrocytes of MPCs in primary OA articular cartilage. J Reg Anat Oper Surg, 2014, ( 5):477-481.
	刘军, 王洪伟, 陈语, 于海龙, 王琪, 杨会峰, 马骏雄, 项良碧 . SOX6和SOX9基因转染对人原发性骨关节炎关节软骨间充质祖细胞增殖和成软骨分化的调控作用. 局解手术学杂志, 2014, ( 5):477-481.
[9]	Zhang Y, Yang TL, Li X, Guo Y . Functional analyses reveal the essential role of SOX6 and RUNX2 in the communication of chondrocyte and osteoblast. OsteoporosInt, 2015,26(2):553-561.
[10]	Livshits G, Deng HW, Nguyen TV, Yakovenko K, Recker RR, Eisman JA, . Genetics of bone mineral density: evidence for a major pleiotropic effect from an intercontinental study. J Bone Miner Res, 2004,19(6):914-923. doi: 10.1359/JBMR.040132 pmid: 15125790
[11]	Peacock M, Turner CH, Econs MJ, Foroud T . Genetics of osteoporosis. Endocr Rev, 2002,23(3):303-326. doi: 10.1210/edrv.23.3.0464 pmid: 12050122
[12]	Rivadeneira F, Styrkársdottir U, Estrada K, Halldórsson BV, Hsu YH, Richards JB, Zillikens MC, Kavvoura FK, Amin N, Aulchenko YS, Cupples LA, Deloukas P, Demissie S, Grundberg E, Hofman A, Kong A, Karasik D, van Meurs JB, Oostra B, Pastinen T, Pols HA, Sigurdsson G, Soranzo N, Thorleifsson G, Thorsteinsdottir U, Williams FM, Wilson SG, Zhou YH, Ralston SH, van Duijn CM, Spector T, Kiel DP, Stefansson K, Ioannidis JP, Uitterlinden AG. Twenty bone-mineral-density loci identified by large-scale meta-analysis of genome-wide association studies. Nat Genet, 2009,41(11):1199-1206. doi: 10.1038/ng.446 pmid: 19801982
[13]	Hsu YH, Zillikens MC, Wilson SG, Farber CR, Demissie S, Soranzo N, Bianchi EN, Grundberg E, Liang LM, Richards JB, Estrada K, Zhou YH, van Nas A, Moffatt MF, Zhai GJ, Hofman A, van Meurs JB, Pols HA, Price RI, Nilsson O, Pastinen T, Cupples LA, Lusis AJ, Schadt EE, Ferrari S, Uitterlinden AG, Rivadeneira F, Spector TD, Karasik D, Kiel DP,. An integration of genome-wide association study and gene expression profiling to prioritize the discovery of novel susceptibility Loci for osteoporosis-related traits. PLoS Genet, 2010,6(6):e1000977. doi: 10.1371/journal.pgen.1000977 pmid: 20548944
[14]	Tan LJ, Liu R, Lei SF, Pan R, Yang TL, Yan H, Pei YF, Yang F, Zhang F, Pan F, Zhang YP, Hu HG, Levy S, Deng HW . A genome-wide association analysis implicates SOX6 as a candidate gene for wrist bone mass. Sci China Life Sci, 2010,53(9):1065-1072. doi: 10.1007/s11427-010-4056-7 pmid: 21104366
[15]	Villalobos-Comparán M, Jiménez-Ortega RF, Estrada K, Parra-Torres AY, González-Mercado A, Patiño N, Castillejos- López M, Quiterio M, Fernandez-López JC, Ibarra B, Romero-Hidalgo S, Salmerón J, Velázquez-Cruz R . A pilot genome-wide association study in postmenopausal mexican- mestizo women implicates the RMND1/CCDC170 locus is associated with bone mineral density. Int J Genomics, 2017,2017:5831020. doi: 10.1155/2017/5831020 pmid: 28840121
[16]	Morris JA, Kemp JP, Youlten SE, Laurent L, Logan JG, Chai RC, Vulpescu NA, Forgetta V, Kleinman A, Mohanty ST, Sergio CM, Quinn J, Nguyen-Yamamoto L, Luco AL, Vijay J, Simon MM, Pramatarova A, Medina-Gomez C, Trajanoska K, Ghirardello EJ, Butterfield NC, Curry KF, Leitch VD, Sparkes PC, Adoum AT, Mannan NS, Komla-Ebri DSK, Pollard AS, Dewhurst HF, Hassall TAD, Beltejar MG, Adams DJ, Vaillancourt SM, Kaptoge S, Baldock P, Cooper C, Reeve J, Ntzani EE, Evangelou E, Ohlsson C, Karasik D, Rivadeneira F, Kiel DP, Tobias JH, Gregson CL, Harvey NC, Grundberg E, Goltzman D, Adams DJ, Lelliott CJ, Hinds DA, Ackert-Bicknell CL, Hsu YH, Maurano MT, Croucher PI, Williams GR, Bassett JHD, Evans DM, Richards JB . An atlas of genetic influences on osteoporosis in humans and mice. Nat Genet, 2019,51(2):258-266. doi: 10.1038/s41588-018-0302-x pmid: 30598549
[17]	Medina-Gomez C, Kemp JP, Trajanoska K, Luan J, Chesi A, Ahluwalia TS, Mook-Kanamori DO, Ham A, Hartwig FP, Evans DS, Joro R, Nedeljkovic I, Zheng HF, Zhu K, Atalay M, Liu CT, Nethander M, Broer L, Porleifsson G, Mullin BH, Handelman SK, Nalls MA, Jessen LE, Heppe DHM, Richards JB, Wang C, Chawes B, Schraut KE, Amin N, Wareham N, Karasik D, Van der Velde N, Ikram MA, Zemel BS, Zhou YH, Carlsson CJ, Liu Y, McGuigan FE, Boer CG, Bønnelykke K, Ralston SH, Robbins JA, Walsh JP, Zillikens MC, Langenberg C, Li-Gao R, Williams FMK, Harris TB, Akesson K, Jackson RD, Sigurdsson G, den Heijer M, van der Eerden BCJ, van de Peppel J, Spector TD, Pennell C, Horta BL, Felix JF, Zhao JH, Wilson SG, de Mutsert R, Bisgaard H, Styrkársdóttir U, Jaddoe VW, Orwoll E, Lakka TA, Scott R, Grant SFA, Lorentzon M, van Duijn CM, Wilson JF, Stefansson K, Psaty BM, Kiel DP, Ohlsson C, Ntzani E, van Wijnen AJ, Forgetta V, Ghanbari M, Logan JG, Williams GR, Bassett JHD, Croucher PI, Evangelou E, Uitterlinden AG, Ackert-Bicknell CL, Tobias JH, Evans DM, Rivadeneira F. Life-Course genome-wide association study meta-analysis of total body BMD and assessment of age-specific effects. Am J Hum Genet, 2018,102(1):88-102. doi: 10.1016/j.ajhg.2017.12.005 pmid: 29304378
[18]	Chau D, Ng K, Chan TS, Cheng YY, Fong B, Tam S, Kwong YL, Tse E . Azacytidine sensitizes acute myeloid leukemia cells to arsenic trioxide by up-regulating the arsenic transporter aquaglyceroporin 9. J Hematol Oncol, 2015,8:46. doi: 10.1186/s13045-015-0143-3 pmid: 25953102
[19]	Pelletier L, Rebouissou S, Paris A, Rathahao-Paris E, Perdu E, Bioulac-Sage P, Imbeaud S, Zucman-Rossi J . Loss of hepatocyte nuclear factor 1alpha function in human hepatocellular adenomas leads to aberrant activationof signaling pathways involved in tumorigenesis. Hepatology, 2010,51(2):557-566. doi: 10.1002/hep.23362 pmid: 20041408
[20]	Krijger PHL, de Laat W . Regulation of disease-associated gene expression in the 3D genome. Nat Rev Mol Cell Biol, 2016,17(12):771-782. doi: 10.1038/nrm.2016.138 pmid: 27826147
[21]	Lane JM, Russell L, Khan SN . Osteoporosis. Clin Orthop Relat Res, 2000, ( 327):139-150.
[22]	Boudin E, Fijalkowski I, Hendrickx G, Van Hul W . Genetic control of bone mass. Mol Cell Endocrinol, 2016,432:3-13. doi: 10.1016/j.mce.2015.12.021 pmid: 26747728
[23]	Huang QY . Genetic study of complex diseases in the post-GWAS era. J Genet Genomics, 2015,42(3):87-98. doi: 10.1016/j.jgg.2015.02.001 pmid: 25819085
[24]	ENCODE Project Consortium . An integrated encyclopedia of DNA elements in the human genome. Nature, 2012,489(7414):57-74. doi: 10.1038/nature11247
[25]	Ding N, Qu HZ, Fang XD . The ENCODE project and functional genomics studies. Hereditas(Beijing), 2014,36(3):237-247.
	丁楠, 渠鸿竹, 方向东 . ENCODE计划和功能基因组研究. 遗传, 2014,36(3):237-247.
[26]	Bernstein BE, Stamatoyannopoulos JA, Costello JF, Ren B, Milosavljevic A, Meissner A, Kellis M, Marra MA, Beaudet AL, Ecker JR, Farnham PJ, Hirst M, Lander ES, Mikkelsen TS, Thomson JA . The NIH roadmap epigenomics mapping consortium. Nat Biotechnol, 2010,28(10):1045-1048. doi: 10.1038/nbt1010-1045 pmid: 20944595
[27]	Kilpinen H, Waszak SM, Gschwind AR, Raghav SK, Witwicki RM, Orioli A, Migliavacca E, Wiederkehr M, Gutierrez-Arcelus M, Panousis NI, Yurovsky A, Lappalainen T, Romano-Palumbo L, Planchon A, Bielser D, Bryois J, Padioleau I, Udin G, Thurnheer S, Hacker D, Core LJ, Lis JT, Hernandez N, Reymond A, Deplancke B, Dermitzakis ET . Coordinated effects of sequence variation on DNA binding, chromatin structure, and transcription. Science, 2013,342(6159):744-747. doi: 10.1126/science.1242463
[28]	Xiao SM, Gao Y, Cheung CL, Bow CH, Lau KS, Sham PC, Tan KC, Kung AW . Association of CDX1 binding site of periostin gene with bone mineral density and vertebral fracture risk. OsteoporosInt, 2012,23(7):1877-1887.
[29]	Xiao SM, Kung AW, Gao Y, Lau KS, Ma A, Zhang ZL, Liu JM, Xia W, He JW, Zhao L, Nie M, Fu WZ, Zhang MJ, Sun J, Kwan JS, Tso GH, Dai ZJ, Cheung CL, Bow CH, Leung AY, Tan KC, Sham PC . Post-genome wide association studies and functional analyses identify association of MPP7 gene variants with site-specific bone mineral density. Hum Mol Genet, 2012,21(7):1648-1657. doi: 10.1093/hmg/ddr586
[30]	Zhu DL, Chen XF, Hu WX, Dong SS, Lu BJ, Rong Y, Chen YX, Chen H, Thynn HN, Wang NN, Guo Y, Yang TL . Multiple functional variants at 13q14 risk locus for osteoporosis regulate RANKL expression through long- range super-enhancer. J Bone Miner Res, 2018,33(7):1335-1346. doi: 10.1002/jbmr.3419 pmid: 29528523

编辑推荐

Metrics

www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:

引物名称	引物序列(5?→3?)
SOX6启动子扩增引物	上游：CCCAAGCTTATGGTGCCGATAGACTTGCC(Hind III)
SOX6启动子扩增引物	下游：CCGCTCGAGCATCTTATGGGTTCCACGCCT(Xho I)
SNP扩增引物	上游：CGACGCGTAGAAGAGAAACGAGGTGTTGGT(Mlu I)
SNP扩增引物	下游：CCGCTCGAGACAAGTTTAGTGGGACAGGGTT(Xho I)
SNP突变引物	上游：GGGATCCTGCTTACTTACTATCTTTGGTCTTAAACTAGACC
SNP突变引物	下游：TAAGTAAGCAGGATCCCGATATCAAGTACCAGG
SNP分型引物	上游：AAGAGAAACGAGGTGTTGGTGAT
SNP分型引物	下游：GGTATCTTCCGTGCAGTTTTGG
SOX6qRT-PCR引物	上游：AGAACGCGCTTTGAGAATTT
SOX6qRT-PCR引物	下游：GCCCAGTTTTCCATCTTCAT
HNF1AqRT-PCR引物^[18]	上游：CCCCAGATTCAGGATCAGACA
HNF1AqRT-PCR引物^[18]	下游：CCATCATGTTCCATTTTTCGC

骨质疏松易感SNP rs4325274通过增强子远程调控SOX6基因的功能机制研究

The osteoporosis susceptible SNP rs4325274 remotely regulates the SOX6 gene through enhancers

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 8

参考文献 30

相关文章 15

编辑推荐

Metrics

[1]	孙凤宇, 许强华. 血液发生相关microRNAs研究进展[J]. 遗传, 2022, 44(9): 756-771.
[2]	慕蓉蓉, 牛晴晴, 孙玉强, 梅俊, 苗蒙. 陆地棉MYB类转录因子基因GhTT2克隆及功能初步分析[J]. 遗传, 2022, 44(8): 720-728.
[3]	吕孟冈, 刘艾嘉, 李庆伟, 苏鹏. RHR转录因子家族起源、功能以及进化机制的研究进展[J]. 遗传, 2021, 43(3): 215-225.
[4]	邱晓芬, 汤冬娥, 虞海燕, 廖秋燕, 胡芷洋, 周俊, 赵鑫, 何慧燕, 梁灼健, 许承明, 杨明, 戴勇. 基于单细胞ATAC测序技术对18-三体综合征染色质开放性区域转录因子的分析[J]. 遗传, 2021, 43(1): 74-83.
[5]	吴杰, 全建平, 叶勇, 吴珍芳, 杨杰, 杨明, 郑恩琴. 染色质转座酶可及性测序研究进展[J]. 遗传, 2020, 42(4): 333-346.
[6]	王涛涛, 杨勇, 魏唯, 林辰涛, 马留银. 互花米草NAC转录因子家族的鉴定与表达分析[J]. 遗传, 2020, 42(2): 194-211.
[7]	孙兆庆, 闫波. 转录因子GATA6在心血管疾病中的作用及其调控机制[J]. 遗传, 2019, 41(5): 375-383.
[8]	于好强,孙福艾,冯文奇,路风中,李晚忱,付凤玲. 转录因子BES1/BZR1调控植物生长发育及抗逆性[J]. 遗传, 2019, 41(3): 206-214.
[9]	鞠君毅,赵权. γ-珠蛋白基因表达调控机制与临床应用[J]. 遗传, 2018, 40(6): 429-444.
[10]	丁庆倩,王小婷,胡利琴,齐欣,葛林豪,徐伟亚,徐兆师,周永斌,贾冠清,刁现民,闵东红,马有志,陈明. 谷子MYB类转录因子SiMYB42提高转基因拟南芥低氮胁迫耐性[J]. 遗传, 2018, 40(4): 327-338.
[11]	任岚,肖茹丹,张倩,娄晓敏,张昭军,方向东. KLF1和KLF9对K562细胞红系分化的协同调控作用[J]. 遗传, 2018, 40(11): 998-1006.
[12]	张玲, 何建波. GATA6在肝脏发育中的作用及调控机制[J]. 遗传, 2018, 40(1): 22-32.
[13]	岳敏, 杨禹, 郭改丽, 秦曦明. 哺乳动物生物钟的遗传和表观遗传研究进展[J]. 遗传, 2017, 39(12): 1122-1137.
[14]	郭文雅,崔艳梅,王婷婷,喻德跃,黄方. 野生大豆花发育相关基因GsLFY的功能研究[J]. 遗传, 2017, 39(1): 56-65.
[15]	向小华, 吴新儒, 晁江涛, 杨明磊, 杨帆, 陈果, 刘贯山, 王元英. 普通烟草WRKY基因家族的鉴定及表达分析[J]. 遗传, 2016, 38(9): 840-856.