中文版 | English
题名

人类基因组沉默子元件的功能验证及机制研究

其他题名
VALIDATION AND MECHANISTIC STUDIES OF SILENCERS IN THE HUMAN GENOME
姓名
姓名拼音
ZHANG Zhe
学号
12032147
学位类型
硕士
学位专业
0710 生物学
学科门类/专业学位类别
07 理学
导师
陈炜
导师单位
生命科学学院
论文答辩日期
2023-05-15
论文提交日期
2023-06-30
学位授予单位
南方科技大学
学位授予地点
深圳
摘要

真核生物细胞发育和分化离不开精确的基因表达调控。基因组中存在多种顺式调控元件调控基因表达,如启动子(promoter)、增强子(enhancer)、绝缘子(insulator)、沉默子(silencer)等。沉默子是一类转录负调控元件,能够降低基因的表达水平。目前对沉默子特征和功能机制的研究比较薄弱,仍需深入探索。在完成人类沉默子全基因组鉴定的基础上,本研究选取了部分沉默子进行体外克隆和功能验证,并探究其发挥作用的机制。首先,本研究选取通过不同方法鉴定的沉默子,克隆构建含有沉默子序列的瞬时表达质粒,通过检测双荧光素酶和GFP报告基因信号变化验证沉默子在细胞核内环境中的功能。结果显示,不同方法鉴定的沉默子具有不同程度的报告基因沉默活性,甚至部分沉默子表现出增强子的功能。为进一步探究沉默子在基因组中的功能,本研究使用CRISPR/Cas9技术在K562细胞系中成功敲除了多个沉默子片段,通过实时荧光定量PCR、RNA-seq技术检测细胞内基因的表达变化情况,结果显示沉默子序列敲除后,周围基因表达水平发生了显著改变,部分基因上调,而同时部分基因下调,证明了沉默子在基因组环境下对基因表达具有更为复杂的影响。为了进一步探究沉默子对差异表达基因的作用机制,我们通过染色质构象捕获实验和染色质免疫沉淀法检测敲除沉默子后基因组三维结构和染色质表观修饰的变化,初步揭示了沉默子发挥作用的可能的复杂机制。本研究为进一步探究沉默子的作用机制打下了基础,也加深了对沉默子与增强子等顺式调控元件关系的理解。

关键词
语种
中文
培养类别
独立培养
入学年份
2020
学位授予年份
2023-06
参考文献列表

[1] RICCI M A, MANZO C, GARCIA-PARAJO M F, et al. Chromatin fibers are formed by heterogeneous groups of nucleosomes in vivo [J]. Cell, 2015, 160(6): 1145 -1158.
[2] AKALIN A. Computational Genomics with r [M]. Chapman and Hall/CRC, 2020.
[3] STERN D L, ORGOGOZO V. The loci of evolution: how predictable is genetic evolution? [J]. Evolution: International Journal of Organic Evolution, 2008, 62(9): 2155-2177.
[4] BRAND A H, BREEDEN L, ABRAHAM J, et al. Characterization of a "silencer" in yeast: A DNA sequence with properties opposite to those of a transcriptional enhancer [J]. Cell, 1985, 41(1): 41-48.
[5] HUANG D, OVCHARENKO I. Enhancer-silencer transitions in the human genome [J]. Genome Res, 2022, 32(3):437-448.
[6] JUVEN-GERSHON T, KADONAGA J T. Regulation of gene expression via the core promoter and the basal transcriptional machinery [J]. Developmental Biology, 2010, 339(2): 225-229.
[7] BANERJI J, RUSCONI S, SCHAFFNER W. Expression of a beta-globin gene is enhanced by remote SV40 DNA sequences [J]. Cell, 1981, 27(2): 299 -308.
[8] GASPERINI M, TOME J M, SHENDURE J. Towards a comprehensive catalogue of validated and target-linked human enhancers [J]. Nature Reviews Genetics, 2020, 21(5): 292-310.
[9] 谢骏辉, 孙艳, 王昇, et al. 增强子功能鉴定及其在农业动物中的研究进展 [J]. 中国细胞生物学学报, 2019, 41(07): 1395-1400.
[10] FURLONG E E M, LEVINE M. Developmental enhancers and chromosome topology [J]. Science, 2018, 361(6409): 1341-1345.
[11] CHEN F X, XIE P, COLLINGS C K, et al. PAF1 regulation of promoter-proximal pause release via enhancer activation [J]. Science, 2017, 357(6357): 1 294-1298.
[12] ENGREITZ J M, HAINES J E, PEREZ E M, et al. Local regulation of gene expression by lncRNA promoters, transcription and splicing [J]. Nature, 2016, 539(7629): 452 -455.
[13] GROSVELD F, ANTONIOU M, BERRY M, et al. The regulation of human globin gene switching [J]. Philos Trans R Soc Lond B Biol Sci, 1993, 339(1288): 183 -191.
[14] GASZNER M, FELSENFELD G. Insulators: exploiting transcriptional and epigenetic mechanisms [J]. Nat Rev Genet, 2006, 7(9): 703-713.
[15] RAAB J R, KAMAKAKA R T. Insulators and promoters: closer than we think [J]. Nat Rev Genet, 2010, 11(6): 439-446.
[16] ROSEMAN R R, PIRROTTA V, GEYER P K. The su(Hw) protein insulates expression of the Drosophila melanogaster white gene from chromosomal position -effects [J]. The EMBO Journal, 1993, 12(2): 435-442.
[17] MAJUMDER P, ROY S, BELOZEROV V E, et al. Diverse transcription influences can be insulated by the Drosophila SF1 chromatin boundary [J]. Nucleic Acids Research, 2009, 37(4227 - 4233.
[18] GOHL D, AOKI T, BLANTON J, et al. Mechanism of chromosomal boundary action: roadblock, sink, or loop? [J]. Genetics, 2011, 187(3): 731-748.
[19] 王海, 阮修艳, 方向东. 位点控制区(LCR)调控基因表达的研究进展 [J]. 生命科学, 2010, 22(01): 74-78.
[20] THURTLE D M, RINE J. The molecular topography of silenced chromatin in Saccharomyces cerevisiae [J]. Genes Dev, 2014, 28(3): 245-258.
[21] LAIMINS L, HOLMGREN-KONIG M, KHOURY G. Transcriptional "silencer" element in rat repetitive sequences associated with the rat insulin 1 gene locus [J]. Proceedings of the National Academy of Sciences, 1986, 83(10): 3151-3155.
[22] JIANG J, CAI H, ZHOU Q, et al. Conversion of a dorsal-dependent silencer into an enhancer: evidence for dorsal corepressors [J]. The EMBO Journal, 1993, 12(8): 3201 -3209.
[23] SAWADA S, SCARBOROUGH J D, KILLEEN N, et al. A lineage-specific transcriptional silencer regulates CD4 gene expression during T lymphocyte development [J]. Cell, 1994, 77(6): 917-929.
[24] TANIUCHI I, SUNSHINE M J, FESTENSTEIN R, et al. Evidence for distinct CD4 silencer functions at different stages of thymocyte differentiation [J]. Mol Cell, 2002, 10(5): 1083-1096.
[25] DONDA A, SCHULZ M, BüRKI K, et al. Identification and characterization of a human CD4 silencer [J]. Eur J Immunol, 1996, 26(2): 493-500.
[26] MORI N, STEIN R, SIGMUND O, et al. A cell type-preferred silencer element that controls the neural-specific expression of the SCG10 gene [J]. Neuron, 1990, 4(4): 583-594.
[27] BRUCE A W, DONALDSON I J, WOOD I C, et al. Genome-wide analysis of repressor element 1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) target genes [J]. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(28): 10458-10463.
[28] MORTAZAVI A, LEEPER THOMPSON E C, GARCIA S T, et al. Comparative genomics modeling of the NRSF/REST repressor network: from single conserved sites to genome-wide repertoire [J]. Genome Res, 2006, 16(10): 1208-1221.
[29] JOHNSON R, GAMBLIN R J, OOI L, et al. Identification of the REST regulon reveals extensive transposable element-mediated binding site duplication [J]. Nucleic Acid Res, 2006, 34(14): 3862-3877.
[30] SCHOENHERR C J, ANDERSON D J. The neuron-restrictive silencer factor (NRSF): a coordinate repressor of multiple neuron-specific genes [J]. Science, 1995, 267(5202): 1360-1363.
[31] 单春燕, 李圆君, 张东惠, et al. SCG10 在神经系统发育中的功能 [J]. 2017.
[32] GISSELBRECHT S S, PALAGI A, KURLAND J V, et al. Transcriptional Silencers in Drosophila Serve a Dual Role as Transcriptional Enhancers in Alternate Cellular Contexts [J]. Mol Cell, 2020, 77(2): 324-337 e328.
[33] PANG B, SNYDER M P. Systematic identification of silencers in human cells [J]. Nat Genet, 2020, 52(3): 254-263.
[34] DONI JAYAVELU N, JAJODIA A, MISHRA A, et al. Candidate silencer elements for the human and mouse genomes [J]. Nat Commun, 2020, 11(1): 1061.
[35] HUANG D, PETRYKOWSKA H M, MILLER B F, et al. Identification of human silencers by correlating cross-tissue epigenetic profiles and gene expression [J]. Genome Res, 2019, 29(4): 657-667.
[36] NGAN C Y, WONG C H, TJONG H, et al. Chromatin interaction analyses elucidate the roles of PRC2-bound silencers in mouse development [J]. Nat Genet, 2020, 52(3): 264-272.
[37] CAI Y, ZHANG Y, LOH Y P, et al. H3K27me3-rich genomic regions can function as silencers to repress gene expression via chromatin interactions [J]. Nat Commun, 2021, 12(1): 719.
[38] GISSELBRECHT S S, BARRERA L A, PORSCH M, et al. Highly parallel assays of tissue-specific enhancers in whole Drosophila embryos [J]. Nat Methods, 2013, 10(8): 774-780.
[39] MOZZETTA C, BOYARCHUK E, PONTIS J, et al. Sound of silence: the properties and functions of repressive Lys methyltransferases [J]. Nat Rev Mol Cell Biol, 2015, 16(8): 499-513.
[40] HEINTZMAN N D, STUART R K, HON G, et al. Distinct and predictive chromati n signatures of transcriptional promoters and enhancers in the human genome [J]. Nature Genetics, 2007, 39(3): 311-318.
[41] ARANDA S, MAS G, DI CROCE L. Regulation of gene transcription by Polycomb proteins [J]. Science advances, 2015, 1(11): e1500737.
[42] LEE S W, OH Y M, LU Y L, et al. MicroRNAs Overcome Cell Fate Barrier by Reducing EZH2-Controlled REST Stability during Neuronal Conversion of Human Adult Fibroblasts [J]. Dev Cell, 2018, 46(1): 73-84 e77.
[43] SEGERT J A, GISSELBRECHT S S, BULYK M L. Transcriptional Silencers: Driving Gene Expression with the Brakes On [J]. Trends Genet, 2021, 37(6): 514 -527.
[44] SUNDARAM V, WYSOCKA J. Transposable elements as a potent source of diverse cis-regulatory sequences in mammalian genomes [J]. Philos Trans R Soc Lond B Biol Sci, 2020, 375(1795): 20190347.
[45] BOURQUE G, LEONG B, VEGA V B, et al. Evolution of the mammalian transcription factor binding repertoire via transposable elements [J]. Genome Res, 2008, 18(11): 1752-1762.
[46] LYNCH V J, NNAMANI M C, KAPUSTA A, et al. Ancient transposable elements transformed the uterine regulatory landscape and transcriptome during the evolution of mammalian pregnancy [J]. Cell Rep, 2015, 10(4): 551-561.
[47] TRIZZINO M, PARK Y, HOLSBACH-BELTRAME M, et al. Transposable elements are the primary source of novelty in primate gene regulation [J]. Genome Res, 2017, 27(10): 1623-1633.
[48] CANO-GAMEZ E, TRYNKA G. From GWAS to Function: Using Functional Genomics to Identify the Mechanisms Underlying Complex Diseases [J]. Fron t Genet, 2020, 11(424.
[49] CAMPBELL T M, CASTRO M A A, DE SANTIAGO I, et al. FGFR2 risk SNPs confer breast cancer risk by augmenting oestrogen responsiveness [J]. Carcinogenesis, 2016, 37(8): 741-750.
[50] DUNNING A M, MICHAILIDOU K, KUCHENBAECKER K B, et al. Breast cancer risk variants at 6q25 display different phenotype associations and regulate ESR1, RMND1 and CCDC170 [J]. Nat Genet, 2016, 48(4): 374-386.
[51] ZHANG Z H, BARAJAS-MARTíNEZ H, XIA H, et al. Distinct Features of Probands With Early Repolarization and Brugada Syndromes Carrying SCN5A Pathogenic Variants [J]. J Am Coll Cardiol, 2021, 78(16): 1603-1617.
[52] KAPOOR A, LEE D, ZHU L, et al. Multiple SCN5A variant enhancers modulate its cardiac gene expression and the QT interval [J]. Proc Natl Ac ad Sci U S A, 2019, 116(22): 10636-10645.
[53] WANG Y, JIANG T, TANG P, et al. Family-based whole-genome sequencing identifies compound heterozygous protein-coding and noncoding mutations in tetralogy of Fallot [J]. Gene, 2020, 741(144555.
[54] DEMIRCI S, LEONARD A, ESSAWI K, et al. CRISPR-Cas9 to induce fetal hemoglobin for the treatment of sickle cell disease [J]. Mol Ther Methods Clin Dev, 2021, 23(276-285.
[55] PANG B, VAN WEERD J H, HAMOEN F L, et al. Identification of non-coding silencer elements and their regulation of gene expression [J]. Nat Rev Mol Cell Biol, 2023, 24(6): 383-95.
[56] BAI X, HUANG Y, HU Y, et al. Duplication of an upstream silencer of FZP increases grain yield in rice [J]. Nat Plants, 2017, 3(11): 885-893.
[57] RAN F A, HSU P D, WRIGHT J, et al. Genome engineering using the CRISPR-Cas9 system [J]. Nat Protoc, 2013, 8(11): 2281-2308.
[58] BONEV B, CAVALLI G. Organization and function of the 3D genome [J]. Nat Rev Genet, 2016, 17(11): 661-678.
[59] RAO S S, HUNTLEY M H, DURAND N C, et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping [J]. Cell, 2014, 159(7): 1665-1680.
[60] YE R, CAO C, XUE Y. Enhancer RNA: biogenesis, function, and regulation [J]. Essays Biochem, 2020, 64(6): 883-894.
[61] CUI P, LIU W, ZHAO Y, et al. The association between H3K4me3 and antisense transcription [J]. Genomics Proteomics Bioinformatics, 2012, 10(2): 74 -81.
[62] ZHENG H, XIE W. The role of 3D genome organization in development and cell differentiation [J]. Nat Rev Mol Cell Biol, 2019, 20(9): 535-550.
[63] ZHANG T, ZHANG Z, DONG Q, et al. Histone H3K27 acetylation is dispensable for enhancer activity in mouse embryonic stem cells [J]. Genome Biol, 2020, 21(1): 45.
[64] WANG H, FAN Z, SHLIAHA P V, et al. H3K4me3 regulates RNA polymerase II promoter-proximal pause-release [J]. Nature, 2023, 615(7951): 339-348.
[65] MARTIN B J E, BRIND'AMOUR J, KUZMIN A, et al. Transcription shapes genome -wide histone acetylation patterns [J]. Nat Commun, 2021, 12(1): 210.
[66] HOWE F S, FISCHL H, MURRAY S C, et al. Is H3K4me3 instructive for transcription activation? [J]. Bioessays, 2017, 39(1): 1-12.

所在学位评定分委会
生物学
国内图书分类号
Q341
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条目标识符http://sustech.caswiz.com/handle/2SGJ60CL/544748
专题生命科学学院_生物系
推荐引用方式
GB/T 7714
张哲. 人类基因组沉默子元件的功能验证及机制研究[D]. 深圳. 南方科技大学,2023.
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