果蝇基因组沉默子鉴定和特征研究

生物生长发育过程中，基因表达受到时空特异的精准调控。基因组顺式调控元件是在转录水平参与基因调控的DNA序列，包括启动子、增强子、沉默子和隔离子等，它们分别通过起始、促进、抑制目标基因以及阻隔调控元件对目标基因的调控而影响基因的转录。调控元件的突变会造成基因表达的异常，进而导致生物体发生表型的改变，甚至导致疾病和死亡。因此，研究调控元件及对目标基因的调控，对于了解细胞增殖和分化、生物生长发育过程至关重要。沉默子作为一类重要的调控元件，通常不依赖位置和方向发挥抑制基因转录的功能。但是，目前人们对于它的功能和特征缺少认识，一方面是因为对沉默子的研究不足，另一方面则是现有研究往往是针对单个沉默子或者染色质开放区的基因组序列，限制了对沉默子更全面和系统的认识。

为全面分析沉默子的特征和功能机制，本研究首先建立了全基因组沉默子的鉴定方法，通过将随机打断的基因组序列插入包含强启动子的报告载体中，鉴定插入序列的转录抑制活性。本论文利用此方法，在果蝇基因组鉴定沉默子，分析了沉默子在基因组上的分布。本研究发现果蝇的沉默子与已报道的增强子均不在外显子区富集，增强子主要富集在转录起始区，而沉默子则在转录起始区和转录终止区都富集。此外，沉默子的报告基因转录抑制活性与它们所处的基因组位置有一定的相关性，外显子区的沉默子活性最低，而启动子附近的沉默子活性较高。进一步的分析发现沉默子相邻基因表达水平较低。有趣的是，不同类型的基因对沉默子的响应不同，组织特异性基因比看家基因更易受到沉默子的抑制，且其表达水平与沉默子的数量和抑制活性呈正相关。此外，组织特异性基因拥有的沉默子比例更高，而看家基因中增强子的比例更高。

其次，对沉默子表观遗传特征研究显示，沉默子缺乏特异的组蛋白修饰，但富集部分隔离子结合蛋白。分析结果还显示沉默子的活性与基因组上的染色质状态无关，而与其结合的转录因子相关。进一步的分析表明，调控元件、染色质状态、所结合的转录因子种类都可能与沉默子靶基因的转录调控有关。另外，本研究还发现当基因与临近沉默子分别位于不同的拓扑结构域时，基因的表达水平会明显高于与沉默子位于同一拓扑结构域的基因，这表明三维染色质结构限制了调控元件的功能范围，阻碍调控元件跨拓扑结构域发挥功能。

再次，本研究同时从沉默子鉴定文库中鉴定出促进转录的增强子，部分新鉴定的增强子与已报道的增强子重叠，且新鉴定增强子的序列特征以及其表观遗传特征也与已报道的增强子相似。但是，新鉴定增强子的组蛋白修饰和转录因子的富集较弱，其在TAD边缘的富集程度也偏低。本研究还发现，新鉴定增强子的靶基因的表达水平低于已知增强子的靶基因，提示这些新鉴定的增强子在基因组上可能处于被抑制的状态。

最后，本研究基于自转录水平检测实验的数据特征，结合测序读段数差异和曲线相似性开发了高效的沉默子识别软件Fast-NR。相比其他潜在或已报道的沉默子识别软件，Fast-NR在模拟数据以及实验数据中都拥有着更好的表现力，且其拥有的曲线相似性分析可以提高识别沉默子的能力。Fast-NR在果蝇沉默子鉴定文库中的应用提示这一软件可以提供强健的识别沉默子的计算方法。

综上所述，本论文首先在果蝇全基因组范围内完成了功能性沉默子的鉴定，避免因为仅分析部分序列而可能带来的特征分析偏差。其次，本论文系统地分析了沉默子在基因组的分布特点、以及表观遗传特征。沉默子的特征分析是发展沉默子预测方法的重要前提，可以促进对沉默子作用机制的更深理解。第三，本论文沉默子鉴定基于报告质粒体系，独立于基因组环境，因此所鉴定的沉默子并不完全反映其在真实的基因组环境中的活性和功能。实际上在真实的基因组环境中，所鉴定的沉默子附近的基因表达水平明显更低，侧面验证了本研究所鉴定的沉默子的可信度。最后，本论文还分析了调控元件受多种表观遗传状态影响，协同调控目标基因的可能性，结果表明基因表达水平应该是多种调控的综合结果。总而言之，本论文表明所鉴定的沉默子可以抑制基因的表达，并为将来沉默子功能机制的研究提供了数据支撑。

[1] 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.
[2] NORD A S, WEST A E. Neurobiological functions of transcriptional enhancers[J]. Nat Neurosci, 2020, 23(1):5-14.
[3] ZENG W, MIN X, JIANG R. EnDisease: a manually curated database for enhancer-disease associations[J]. Database (Oxford), 2019, 2019:baz020.
[4] RICE G, REBEIZ M. Evolution: How Many Phenotypes Do Regulatory Mutations Affect?[J]. Curr Biol, 2019, 29(1):R21-R23.
[5] CHENG C K, WONG T H Y, YUNG Y L, et al. Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells[J]. J Vis Exp, 2019, (151):e60130.
[6] KARIMI M M, GOYAL P, MAKSAKOVA I A, et al. DNA methylation and SETDB1/H3K9me3 regulate predominantly distinct sets of genes, retroelements, and chimeric transcripts in mESCs[J]. Cell Stem Cell, 2011, 8(6):676-687.
[7] MATHARU N, AHITUV N. Modulating gene regulation to treat genetic disorders[J]. Nat Rev Drug Discov, 2020, 19(11):757-775.
[8] OGBOURNE S, ANTALIS T M. Transcriptional control and the role of silencers in transcriptional regulation in eukaryotes[J]. Biochem J, 1998, 331 ( Pt 1):1-14.
[9] SANKARAN V G, XU J, BYRON R, et al. A functional element necessary for fetal hemoglobin silencing[J]. N Engl J Med, 2011, 365(9):807-814.
[10] OGIYAMA Y, SCHUETTENGRUBER B, PAPADOPOULOS G L, et al. Polycomb-Dependent Chromatin Looping Contributes to Gene Silencing during Drosophila Development[J]. Mol Cell, 2018, 71(1):73-88 e75.
[11] 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.
[12] GOLDMAN J A, POSS K D. Gene regulatory programmes of tissue regeneration[J]. Nat Rev Genet, 2020, 21(9):511-525.
[13] HABERLE V, STARK A. Eukaryotic core promoters and the functional basis of transcription initiation[J]. Nat Rev Mol Cell Biol, 2018, 19(10):621-637.
[14] HABERLE V, ARNOLD C D, PAGANI M, et al. Transcriptional cofactors display specificity for distinct types of core promoters[J]. Nature, 2019, 570(7759):122-126.
[15] CALO E, WYSOCKA J. Modification of enhancer chromatin: what, how, and why?[J]. Mol Cell, 2013, 49(5):825-837.
[16] SHLYUEVA D, STAMPFEL G, STARK A. Transcriptional enhancers: from properties to genome-wide predictions[J]. Nat Rev Genet, 2014, 15(4):272-286.
[17] CUBENAS-POTTS C, ROWLEY M J, LYU X, et al. Different enhancer classes in Drosophila bind distinct architectural proteins and mediate unique chromatin interactions and 3D architecture[J]. Nucleic Acids Res, 2017, 45(4):1714-1730.
[18] WEI G, LIU D, LIANG C. Chromatin domain boundaries insulators and beyond[J]. Cell Research, 2005, 15:292-300.
[19] BRASSET E, VAURY C. Insulators are fundamental components of the eukaryotic genomes[J]. Heredity (Edinb), 2005, 94(6):571-576.
[20] DEBRUYNE D N, DRIES R, SENGUPTA S, et al. BORIS promotes chromatin regulatory interactions in treatment-resistant cancer cells[J]. Nature, 2019, 572(7771):676-680.
[21] OZDEMIR I, GAMBETTA M C. The Role of Insulation in Patterning Gene Expression[J]. Genes (Basel), 2019, 10(10):767.
[22] TARJAN D R, FLAVAHAN W A, BERNSTEIN B E. Epigenome editing strategies for the functional annotation of CTCF insulators[J]. Nat Commun, 2019, 10(1):4258.
[23] ARZATE-MEJIA R G, JOSUE CERECEDO-CASTILLO A, GUERRERO G, et al. In situ dissection of domain boundaries affect genome topology and gene transcription in Drosophila[J]. Nat Commun, 2020, 11(1):894.
[24] 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.
[25] LAIMINS L, HOLMGREN-KöNIG M, KHOURY G. Transcriptional “silencer” element in rat repetitive sequences associated with the rat insulin 1 gene locus[J]. Proc Natl Acad Sci U S A, 1986, 83(10):3151-3155.
[26] ZHAO L, XIE L, ZHANG Q, et al. Integrative analysis of reference epigenomes in 20 rice varieties[J]. Nat Commun, 2020, 11(1):2658.
[27] ANDERSSON R, SANDELIN A. Determinants of enhancer and promoter activities of regulatory elements[J]. Nat Rev Genet, 2020, 21(2):71-87.
[28] ANDERSSON R, SANDELIN A, DANKO C G. A unified architecture of transcriptional regulatory elements[J]. Trends Genet, 2015, 31(8):426-433.
[29] 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.
[30] HALFON M S. Silencers, Enhancers, and the Multifunctional Regulatory Genome[J]. Trends Genet, 2020, 36(3):149-151.
[31] 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, 77:917-929.
[32] KOJO S, YASMIN N, MUROI S, et al. Runx-dependent and silencer-independent repression of a maturation enhancer in the Cd4 gene[J]. Nat Commun, 2018, 9(1):3593.
[33] MORI N, SCHOENHERR C, VANDENBERGH D J, et al. A common silencer element in the SCG10 and type II Na+ channel genes binds a factor present in nonneuronal cells but not in neuronal cells[J]. Neuron, 1992, 9:45-54.
[34] BESSIS A, CHAMPTIAUX N, CHATELIN L, et al. The neuron-restrictive silencer element: a dual enhancer/silencer crucial for patterned expression of a nicotinic receptor gene in the brain[J]. Proc Natl Acad Sci U S A, 1997, 94(11):5906-5911.
[35] YE J, GHOSH P, CIPPITELLI M, et al. Characterization of a silencer regulatory element in the human interferon-gamma promoter[J]. J Biol Chem, 1994, 269(41):25728-25734.
[36] RAY B K, DHAR S, SHAKYA A, et al. Z-DNA-forming silencer in the first exon regulates human ADAM-12 gene expression[J]. Proc Natl Acad Sci U S A, 2011, 108(1):103-108.
[37] SUN N, ZHAO H. Transcription activator-like effector nucleases (TALENs): a highly efficient and versatile tool for genome editing[J]. Biotechnol Bioeng, 2013, 110(7):1811-1821.
[38] HSU P D, LANDER E S, ZHANG F. Development and applications of CRISPR-Cas9 for genome engineering[J]. Cell, 2014, 157(6):1262-1278.
[39] CROCKER J, STERN D L. TALE-mediated modulation of transcriptional enhancers in vivo[J]. Nat Methods, 2013, 10(8):762-767.
[40] GILBERT L A, LARSON M H, MORSUT L, et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes[J]. Cell, 2013, 154(2):442-451.
[41] AKHTAR W, DE JONG J, PINDYURIN A V, et al. Chromatin position effects assayed by thousands of reporters integrated in parallel[J]. Cell, 2013, 154(4):914-927.
[42] 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.
[43] PANG B, SNYDER M P. Systematic identification of silencers in human cells[J]. Nat Genet, 2020, 52(3):254-263.
[44] DELLA ROSA M, SPIVAKOV M. Silencers in the spotlight[J]. Nat Genet, 2020, 52(3):244-245.
[45] PENGELLY A R, COPUR O, JACKLE H, et al. A histone mutant reproduces the phenotype caused by loss of histone-modifying factor Polycomb[J]. Science, 2013, 339(6120):698-699.
[46] CATARINO R R, STARK A. Assessing sufficiency and necessity of enhancer activities for gene expression and the mechanisms of transcription activation[J]. Genes Dev, 2018, 32(3-4):202-223.
[47] BOSSELUT R. CD4/CD8-lineage differentiation in the thymus: from nuclear effectors to membrane signals[J]. Nat Rev Immunol, 2004, 4(7):529-540.
[48] CORCES M R, GRANJA J M, SHAMS S, et al. The chromatin accessibility landscape of primary human cancers[J]. Science, 2018, 362(6413).
[49] GUAN X, DENG H, CHOI U L, et al. EZH2 overexpression dampens tumor-suppressive signals via an EGR1 silencer to drive breast tumorigenesis[J]. Oncogene, 2020, 39(48):7127-7141.
[50] SOTTNIK J L, VANDERLINDEN L, JOSHI M, et al. Androgen Receptor Regulates CD44 Expression in Bladder Cancer[J]. Cancer Res, 2021, 81(11):2833-2846.
[51] MATHARU N, RATTANASOPHA S, TAMURA S, et al. CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency[J]. Science, 2019, 363(6424).
[52] WOODRUFF K A, ROSENBLATT J D, MOORE T B, et al. Cell type-specific activity of the N-myc promoter in human neuroblastoma cells is mediated by a downstream silencer[J]. Oncogene, 1995, 10(7):1335-1341.
[53] YE J, YOUNG H A, ZHANG X, et al. Regulation of a cell type-specific silencer in the human interleukin-3 gene promoter by the transcription factor YY1 and an AP2 sequence-recognizing factor[J]. J Biol Chem, 1999, 274(38):26661-26667.
[54] NATESAN S, GILMAN M Z. DNA bending and orientation-dependent function of YY1 in the c-fos promoter[J]. Genes Dev, 1993, 7(12B):2497-2509.
[55] ZOU Y, YU Q, CHIU Y H, et al. Position effect on the directionality of silencer function in Saccharomyces cerevisiae[J]. Genetics, 2006, 174(1):203-213.
[56] NAKABAYASHI H, HASHIMOTO T, MIYAO Y, et al. A position-dependent silencer plays a major role in repressing alpha-fetoprotein expression in human hepatoma[J]. Mol Cell Biol, 1991, 11(12):5885-5893.
[57] CHEN D, MCKEARIN D M. A discrete transcriptional silencer in the bam gene determines asymmetric division of the Drosophila germline stem cell[J]. Development, 2003, 130(6):1159-1170.
[58] TRUJILLO M A, SAKAGASHIRA M, EBERHARDT N L. The human growth hormone gene contains a silencer embedded within an Alu repeat in the 3'-flanking region[J]. Mol Endocrinol, 2006, 20(10):2559-2575.
[59] BIRE S, CASTERET S, PIEGU B, et al. Mariner Transposons Contain a Silencer: Possible Role of the Polycomb Repressive Complex 2[J]. PLoS Genet, 2016, 12(3):e1005902.
[60] ORTIZ E M, DUSETTI N J, DAGORN J C, et al. Characterization of a silencer regulatory element in the rat PAP I gene which confers tissue-specific expression and is promoter-dependent[J]. Archives of Biochemistry and Biophysics, 1997, 340(1):111-116.
[61] KIM M K, LESOONWOOD L A, WEINTRAUB B D, et al. A soluble transcription factor, Oct-1, is also found in the insoluble nuclear matrix and possesses silencing activity in its alanine-rich domain[J]. Molecular and Cellular Biology, 1996, 16(8):4366-4377.
[62] STARK K, KIRK D L, SCHMITT R. Two enhancers and one silencer located in the introns of regA control somatic cell differentiation in Volvox carteri[J]. Genes Dev, 2001, 15(11):1449-1460.
[63] DONG J M, LIM L. The human neuronal alpha 1-chimaerin gene contains a position-dependent negative regulatory element in the first exon[J]. Neurochem Res, 1996, 21(9):1023-1030.
[64] LI Y P, CHEN W, STASHENKO P. Characterization of a silencer element in the first exon of the human osteocalcin gene[J]. Nucleic Acids Res, 1995, 23(24):5064-5072.
[65] YANNOUTSOS N, BARRETO V, MISULOVIN Z, et al. A cis element in the recombination activating gene locus regulates gene expression by counteracting a distant silencer[J]. Nature Immunology, 2004, 5(4):443-450.
[66] BANDARA T, OTSUKA K, MATSUBARA S, et al. A dual enhancer-silencer element, DES-K16, in mouse spermatocyte-derived GC-2spd(ts) cells[J]. Biochem Biophys Res Commun, 2020, 534:1007-1012.
[67] LI Z, WANG M, LIN K, et al. The bread wheat epigenomic map reveals distinct chromatin architectural and evolutionary features of functional genetic elements[J]. Genome Biol, 2019, 20(1):139.
[68] HEINTZMAN N D, STUART R K, HON G, et al. Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome[J]. Nature Genetics, 2007, 39(3):311-318.
[69] RADA-IGLESIAS A, BAJPAI R, SWIGUT T, et al. A unique chromatin signature uncovers early developmental enhancers in humans[J]. Nature, 2011, 470(7333):279-283.
[70] 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.
[71] PETERS A H F M, MERMOUD J E, O'CARROLL D, et al. Histone H3 lysine 9 methylation is an epigenetic imprint of facultative heterochromatin[J]. Nature Genetics, 2002, 30(1):77-80.
[72] YOUNG M D, WILLSON T A, WAKEFIELD M J, et al. ChIP-seq analysis reveals distinct H3K27me3 profiles that correlate with transcriptional activity[J]. Nucleic Acids Res, 2011, 39(17):7415-7427.
[73] STENDER J D, PASCUAL G, LIU W, et al. Control of proinflammatory gene programs by regulated trimethylation and demethylation of histone H4K20[J]. Mol Cell, 2012, 48(1):28-38.
[74] 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.
[75] MULLER J. Transcriptional silencing by the Polycomb protein in Drosophila embryos[J]. EMBO J, 1995, 14(6):1209-1220.
[76] GUO Y, ZHAO S, WANG G G. Polycomb Gene Silencing Mechanisms: PRC2 Chromatin Targeting, H3K27me3 'Readout', and Phase Separation-Based Compaction[J]. Trends Genet, 2021, 37(6):547-565.
[77] TANG Y, JIA Z, XU H, et al. Mechanism of REST/NRSF regulation of clustered protocadherin alpha genes[J]. Nucleic Acids Res, 2021, 49(8):4506-4521.
[78] TANIUCHI I, LITTMAN D R. Epigenetic gene silencing by Runx proteins[J]. Oncogene, 2004, 23(24):4341-4345.
[79] RIGGS K J, SALEQUE S, WONG K K, et al. Yin-yang 1 activates the c-myc promoter[J]. Mol Cell Biol, 1993, 13(12):7487-7495.
[80] WU G, LAI E, HUANG N, et al. Oct-1 and CCAATenhancer-binding protein (CEBP) bind to overlapping elements within the interleukin-8 promoter[J]. J Biol Chem, 1997, 272(4):2396-2403.
[81] 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.
[82] FRIEDMAN R Z, GRANAS D M, MYERS C A, et al. Information content differentiates enhancers from silencers in mouse photoreceptors[J]. Elife, 2021, 10:e67403.
[83] LEVINE L, MANLEY J L. Transcriptional repression of eukaryotic promoters[J]. cell, 1989, 59:405-408.
[84] TIWARI V K, MCGARVEY K M, LICCHESI J D, et al. PcG proteins, DNA methylation, and gene repression by chromatin looping[J]. PLoS Biol, 2008, 6(12):2911-2927.
[85] LIN X, LEICHER R, LIU S, et al. Cooperative DNA looping by PRC2 complexes[J]. Nucleic Acids Res, 2021, 49(11):6238-6248.
[86] HEENAN P R, WANG X, GOODING A R, et al. Bending and looping of long DNA by Polycomb repressive complex 2 revealed by AFM imaging in liquid[J]. Nucleic Acids Res, 2020, 48(6):2969-2981.
[87] ARNOLD R, BURCIN M, KAISER B, et al. DNA bending by the silencer protein NeP1 is modULATED BY TR and RXR[J]. Nucleic Acids Research, 1996, 24(14):2640-2647.
[88] DREW L R, TANG D C, BERG P E, et al. The role of trans-acting factors and DNA-bending in the silencing of human beta-globin gene expression[J]. Nucleic Acids Res, 2000, 28(14):2823-2830.
[89] YANG H, LUAN Y, LIU T, et al. A map of cis-regulatory elements and 3D genome structures in zebrafish[J]. Nature, 2020, 588(7837):337-343.
[90] GALUPA R, NORA E P, WORSLEY-HUNT R, et al. A Conserved Noncoding Locus Regulates Random Monoallelic Xist Expression across a Topological Boundary[J]. Mol Cell, 2020, 77(2):352-367.e358.
[91] CAVALLI G. A RING to rule them all: RING1 as silencer and activator[J]. Dev Cell, 2014, 28(1):1-2.
[92] WHITE M A, KWASNIESKI J C, MYERS C A, et al. A Simple Grammar Defines Activating and Repressing cis-Regulatory Elements in Photoreceptors[J]. Cell Rep, 2016, 17(5):1247-1254.
[93] SHI Y, SETO E, CHANG L S, et al. Transcriptional repression by YY1, a human GLI-Krüppel-related protein, and relief of repression by adenovirus E1A protein[J]. Cell, 1991, 67(2):377-388.
[94] PANKRATZ M, SEIFERT E, GERWIN N, et al. Gradients of Krüppel and knirps gene products direct pair-rule gene stripe patterning in the posterior region of the Drosophila embryo[J]. Cell, 1990, 61(2):309-317.
[95] LICHT J D, GROSSEL M J, FIGGE J, et al. Drosophila Krüppel protein is a transcriptional represser[J]. Nature, 1990, 346(6279):76-79.
[96] SAUER F, JäCKLE H. Concentration-dependent transcriptional activation or repression by Krüppel from a single binding site[J]. Nature, 1991, 353(6344):563-566.
[97] YAN W, CHEN D, SCHUMACHER J, et al. Dynamic control of enhancer activity drives stage-specific gene expression during flower morphogenesis[J]. Nat Commun, 2019, 10(1):1705.
[98] SIMA J, CHAKRABORTY A, DILEEP V, et al. Identifying cis Elements for Spatiotemporal Control of Mammalian DNA Replication[J]. Cell, 2019, 176(4):816-830.e818.
[99] TANIUCHI I, SUNSHINE M J, FESTENSTEIN R, et al. Evidence for distinct CD4 silencer functions at different stages of thymocyte differentiation[J]. Molecular Cell, 2002, 10(5):1083-1096.
[100] ZOU Y-R, SUNSHINE M-J, TANIUCHI I, et al. Epigenetic silencing of CD4 in T cells committed to the cytotoxic lineage[J]. Nature Genetics, 2001, 29(3):332-336.
[101] AVISAR N, SHIFTAN L, BEN-DROR I, et al. A silencer element in the regulatory region of glutamine synthetase controls cell type-specific repression of gene induction by glucocorticoids[J]. J Biol Chem, 1999, 274(16):11399-11407.
[102] KONDO T, ISONO K, KONDO K, et al. Polycomb potentiates meis2 activation in midbrain by mediating interaction of the promoter with a tissue-specific enhancer[J]. Dev Cell, 2014, 28(1):94-101.
[103] MCEACHERN L A, LLOYD V K. The maize b1 paramutation control region causes epigenetic silencing in Drosophila melanogaster[J]. Mol Genet Genomics, 2012, 287(7):591-606.
[104] DREWELL R A, GODDARD C J, THOMAS J O, et al. Methylation-dependent silencing at the H19 imprinting control region by MeCP2[J]. Nucleic Acids Research, 2002, 30(5):1139-1144.
[105] LYKO F, BRENTON J D, SURANI M A, et al. An imprinting element from the mouse H19 locus functions as a silencer in Drosophila[J]. Nature Genetics, 1997, 16(2):171-173.
[106] ARNEY K L, BAE E, OLSEN C, et al. The human and mouse H19 imprinting control regions harbor an evolutionarily conserved silencer element that functions on transgenes in Drosophila[J]. Dev Genes Evol, 2006, 216(12):811-819.
[107] 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.
[108] CHEN Q, DENG X, HU X, et al. Breast Cancer Risk-Associated SNPs in the mTOR Promoter Form De Novo KLF5- and ZEB1-Binding Sites that Influence the Cellular Response to Paclitaxel[J]. Mol Cancer Res, 2019, 17(11):2244-2256.
[109] RAICH N, PAPAYANNOPOULOU T, STAMATOYANNOPOULOS G, et al. Demonstration of a human epsilon-globin gene silencer with studies in transgenic mice[J]. Blood, 1992, 79(4):861-864.
[110] CONSTANCIA M, DEAN W, LOPES S, et al. Deletion of a silencer element in Igf2 results in loss of imprinting independent of H19[J]. Nat Genet, 2000, 26(2):203-206.
[111] KRASNOPOLSKY S, KUZMINA A, TAUBE R. Genome-wide CRISPR knockout screen identifies ZNF304 as a silencer of HIV transcription that promotes viral latency[J]. PLoS Pathog, 2020, 16(9):e1008834.
[112] ROSCITO J G, SAMEITH K, PARRA G, et al. Phenotype loss is associated with widespread divergence of the gene regulatory landscape in evolution[J]. Nat Commun, 2018, 9(1):4737.
[113] JOHNSON W C, ORDWAY A J, WATADA M, et al. Genetic Changes to a Transcriptional Silencer Element Confers Phenotypic Diversity within and between Drosophila Species[J]. PLoS Genet, 2015, 11(6):e1005279.
[114] HARDISON R C, TAYLOR J. Genomic approaches towards finding cis-regulatory modules in animals[J]. Nat Rev Genet, 2012, 13(7):469-483.
[115] ROBERTSON G, HIRST M, BAINBRIDGE M, et al. Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing[J]. Nat Methods, 2007, 4:651-657.
[116] GREIL F, MOORMAN C, VAN STEENSEL B. DamID: mapping of in vivo protein-genome interactions using tethered DNA adenine methyltransferase[J]. Methods Enzymol, 2006, 410:342-359.
[117] KLEMM S L, SHIPONY Z, GREENLEAF W J. Chromatin accessibility and the regulatory epigenome[J]. Nat Rev Genet, 2019, 20(4):207-220.
[118] BOYLE A P, DAVIS S, SHULHA H P, et al. High-resolution mapping and characterization of open chromatin across the genome[J]. Cell, 2008, 132(2):311-322.
[119] BUENROSTRO J D, GIRESI P G, ZABA L C, et al. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position[J]. Nature Methods, 2013, 10(12):1213-1218.
[120] SHASHIKANT T, ETTENSOHN C A. Genome-wide analysis of chromatin accessibility using ATAC-seq[J]. Methods Cell Biol, 2019, 151:219-235.
[121] LIEBERMAN-AIDEN E, VAN BERKUM N L, WILLIAMS L, et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome[J]. Science (New York, NY), 2009, 326(5950):289-293.
[122] FULLWOOD M J, LIU M H, PAN Y F, et al. An oestrogen-receptor-α-bound human chromatin interactome[J]. Nature, 2009, 462(7269):58-64.
[123] ZHENG M, TIAN S Z, CAPURSO D, et al. Multiplex chromatin interactions with single-molecule precision[J]. Nature, 2019, 566(7745):558-562.
[124] MELNIKOV A, MURUGAN A, ZHANG X, et al. Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay[J]. Nature Biotechnology, 2012, 30(3):271-277.
[125] INOUE F, AHITUV N. Decoding enhancers using massively parallel reporter assays[J]. Genomics, 2015, 106(3):159-164.
[126] INOUE F, KREIMER A, ASHUACH T, et al. Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction[J]. Cell Stem Cell, 2019, 25(5):713-727.e710.
[127] WANG X, HE L, GOGGIN S M, et al. High-resolution genome-wide functional dissection of transcriptional regulatory regions and nucleotides in human[J]. Nat Commun, 2018, 9(1):5380.
[128] ARNOLD C D, NEMCKO F, WOODFIN A R, et al. A high-throughput method to identify trans-activation domains within transcription factor sequences[J]. EMBO J, 2018, 37(16).
[129] ARNOLD C D, GERLACH D, STELZER C, et al. Genome-wide quantitative enhancer activity maps identified by STARR-seq[J]. Science, 2013, 339(6123):1074-1077.
[130] NEUMAYR C, PAGANI M, STARK A, et al. STARR-seq and UMI-STARR-seq: Assessing Enhancer Activities for Genome-Wide-, High-, and Low-Complexity Candidate Libraries[J]. Curr Protoc Mol Biol, 2019, 128(1):e105.
[131] KWASNIESKI J C, MOGNO I, MYERS C A, et al. Complex effects of nucleotide variants in a mammalian cis-regulatory element[J]. Proc Natl Acad Sci U S A, 2012, 109(47):19498-19503.
[132] MASTON G A, EVANS S K, GREEN M R. Transcriptional regulatory elements in the human genome[J]. Annu Rev Genomics Hum Genet, 2006, 7:29-59.
[133] GASPERINI M, HILL A J, MCFALINE-FIGUEROA J L, et al. A Genome-wide Framework for Mapping Gene Regulation via Cellular Genetic Screens[J]. Cell, 2019, 176(1-2):377-390.e319.
[134] RAJAGOPAL N, SRINIVASAN S, KOOSHESH K, et al. High-throughput mapping of regulatory DNA[J]. Nat Biotechnol, 2016, 34(2):167-174.
[135] ZENG W, CHEN S, CUI X, et al. SilencerDB: a comprehensive database of silencers[J]. Nucleic Acids Res, 2020, 49(D1):D221-D228.
[136] GHANDI M, MOHAMMAD-NOORI M, GHAREGHANI N, et al. gkmSVM: an R package for gapped-kmer SVM[J]. Bioinformatics, 2016, 32(14):2205-2207.
[137] BARAKAT T S, HALBRITTER F, ZHANG M, et al. Functional Dissection of the Enhancer Repertoire in Human Embryonic Stem Cells[J]. Cell Stem Cell, 2018, 23(2):276-288.e278.
[138] ZHANG Y, LIU T, MEYER C A, et al. Model-based analysis of ChIP-Seq (MACS)[J]. Genome Biol, 2008, 9(9):R137.
[139] HEINZ S, BENNER C, SPANN N, et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities[J]. Mol Cell, 2010, 38(4):576-589.
[140] BUERGER A. BasicSTARRseq: Basic peak calling on STARR-seq data[EB/OL]. 2019: (2021-10-26)
[2022-02-23]. http://www.bioconductor.org/packages/release/bioc/html/BasicSTARRseq.html.
[141] LEE D, SHI M, MORAN J, et al. STARRPeaker: uniform processing and accurate identification of STARR-seq active regions[J]. Genome Biol, 2020, 21(1):298.
[142] ANDERS S, PYL P T, HUBER W. HTSeq--a Python framework to work with high-throughput sequencing data[J]. Bioinformatics, 2015, 31(2):166-169.
[143] LI W, XU H, XIAO T, et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens[J]. Genome Biol, 2014, 15(12):554.
[144] ROBINSON M D, MCCARTHY D J, SMYTH G K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data[J]. Bioinformatics, 2010, 26(1):139-140.
[145] ANDERS S, HUBER W. Differential expression analysis for sequence count data[J]. Genome Biol, 2010, 11(10):R106.
[146] GASPAR J M, HART R P. DMRfinder: efficiently identifying differentially methylated regions from MethylC-seq data[J]. BMC Bioinformatics, 2017, 18(1):528.
[147] PARK Y, WU H. Differential methylation analysis for BS-seq data under general experimental design[J]. Bioinformatics, 2016, 32(10):1446-1453.
[148] FAROOQ A, GRONMYR S, ALI O, et al. HMST-Seq-Analyzer: A new python tool for differential methylation and hydroxymethylation analysis in various DNA methylation sequencing data[J]. Comput Struct Biotechnol J, 2020, 18:2877-2889.
[149] ASHOOR H, LOUIS-BRENNETOT C, JANOUEIX-LEROSEY I, et al. HMCan-diff: a method to detect changes in histone modifications in cells with different genetic characteristics[J]. Nucleic Acids Res, 2017, 45(8):e58.
[150] KIM Y S, JOHNSON G D, SEO J, et al. Correcting signal biases and detecting regulatory elements in STARR-seq data[J]. Genome Res, 2021, 31(5):877-889.
[151] LUN A T, SMYTH G K. csaw: a Bioconductor package for differential binding analysis of ChIP-seq data using sliding windows[J]. Nucleic Acids Res, 2016, 44(5):e45.
[152] LIENHARD M, GRIMM C, MORKEL M, et al. MEDIPS: genome-wide differential coverage analysis of sequencing data derived from DNA enrichment experiments[J]. Bioinformatics, 2014, 30(2):284-286.
[153] ZHANG Y, LIN Y H, JOHNSON T D, et al. PePr: a peak-calling prioritization pipeline to identify consistent or differential peaks from replicated ChIP-Seq data[J]. Bioinformatics, 2014, 30(18):2568-2575.
[154] SHEN L, SHAO N Y, LIU X, et al. diffReps: detecting differential chromatin modification sites from ChIP-seq data with biological replicates[J]. PLoS One, 2013, 8(6):e65598.
[155] CREMONA M A, XU H, MAKOVA K D, et al. Functional data analysis for computational biology[J]. Bioinformatics, 2019, 35(17):3211-3213.
[156] THOMAS R, THOMAS S, HOLLOWAY A K, et al. Features that define the best ChIP-seq peak calling algorithms[J]. Brief Bioinform, 2017, 18(3):441-450.
[157] YAN F, POWELL D R, CURTIS D J, et al. From reads to insight: a hitchhiker's guide to ATAC-seq data analysis[J]. Genome Biol, 2020, 21(1):22.
[158] WU H, JI H. PolyaPeak: detecting transcription factor binding sites from ChIP-seq using peak shape information[J]. PLoS One, 2014, 9(3):e89694.
[159] ZHANG X, ROBERTSON G, KRZYWINSKI M, et al. PICS: probabilistic inference for ChIP-seq[J]. Biometrics, 2011, 67(1):151-163.
[160] STRINO F, LAPPE M. Identifying peaks in *-seq data using shape information[J]. BMC Bioinformatics, 2016, 17 Suppl 5:206.
[161] QUINLAN A R, HALL I M. BEDTools: a flexible suite of utilities for comparing genomic features[J]. Bioinformatics, 2010, 26(6):841-842.
[162] LANGMEAD B, TRAPNELL C, POP M, et al. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome[J]. Genome Biol, 2009, 10(3):R25.
[163] LI H, HANDSAKER B, WYSOKER A, et al. The Sequence Alignment/Map format and SAMtools[J]. Bioinformatics, 2009, 25(16):2078-2079.
[164] TEAM R C. R: A language and environment for statistical computing.[EB/OL]. R Foundation for Statistical Computing, 2018 (2022-02-28)
[2022-02-23]. https://www.R-project.org/.
[165] MACHANICK P, BAILEY T L. MEME-ChIP: motif analysis of large DNA datasets[J]. Bioinformatics, 2011, 27(12):1696-1697.
[166] KENT W J, SUGNET C W, FUREY T S, et al. The human genome browser at UCSC[J]. Genome Res, 2002, 12(6):996-1006.
[167] WICKHAM H. ggplot2: Elegant Graphics for Data Analysis[EB/OL]. Springer-Verlag New York, 2016: (2021-06-25)
[2022-02-23]. https://ggplot2.tidyverse.org.
[168] YU G, WANG L G, HAN Y, et al. clusterProfiler: an R package for comparing biological themes among gene clusters[J]. OMICS, 2012, 16(5):284-287.
[169] YU G, WANG L G, HE Q Y. ChIPseeker: an R/Bioconductor package for ChIP peak annotation, comparison and visualization[J]. Bioinformatics, 2015, 31(14):2382-2383.
[170] ZHU L J, GAZIN C, LAWSON N D, et al. ChIPpeakAnno: a Bioconductor package to annotate ChIP-seq and ChIP-chip data[J]. BMC Bioinformatics, 2010, 11:237.
[171] NAVARRO GONZALEZ J, ZWEIG A S, SPEIR M L, et al. The UCSC Genome Browser database: 2021 update[J]. Nucleic Acids Res, 2021, 49(D1):D1046-D1057.
[172] CHARRAD M, GHAZZALI N, BOITEAU V, et al. Nbclust: An R Package for Determining the Relevant Number of Clusters in a Data Set[J]. J Stat Softw, 2014, 61(6):1-36.
[173] RAMIREZ F, RYAN D P, GRUNING B, et al. deepTools2: a next generation web server for deep-sequencing data analysis[J]. Nucleic Acids Res, 2016, 44(W1):W160-165.
[174] DE HOON M J, IMOTO S, NOLAN J, et al. Open source clustering software[J]. Bioinformatics, 2004, 20(9):1453-1454.
[175] SALDANHA A J. Java Treeview--extensible visualization of microarray data[J]. Bioinformatics, 2004, 20(17):3246-3248.
[176] THORVALDSDOTTIR H, ROBINSON J T, MESIROV J P. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration[J]. Brief Bioinform, 2013, 14(2):178-192.
[177] BROAD I. Picard Toolkit[EB/OL]. Broad Institute, 2019: (2022-01-04)
[2022-02-23]. https://broadinstitute.github.io/picard/.
[178] ILLUMINA. bcl2fastq [EB/OL]. Illumina, 2019: (2019-02-01)
[2022-02-23]. https://support.illumina.com/sequencing/sequencing_software/bcl2fastq-conversion-software.html.
[179] THURMOND J, GOODMAN J L, STRELETS V B, et al. FlyBase 2.0: the next generation[J]. Nucleic Acids Res, 2019, 47(D1):D759-D765.
[180] CELNIKER S E, DILLON L A L, GERSTEIN M B, et al. Unlocking the secrets of the genome[J]. Nature, 2009, 459(7249):927-930.
[181] CHERBAS L, WILLINGHAM A, ZHANG D, et al. The transcriptional diversity of 25 Drosophila cell lines[J]. Genome Res, 2011, 21(2):301-314.
[182] CHINTAPALLI V R, WANG J, DOW J A T. Using FlyAtlas to identify better Drosophila melanogaster models of human disease[J]. Nature Genetics, 2007, 39(6):715-720.
[183] NIU L, SHEN W, HUANG Y, et al. Amplification-free library preparation with SAFE Hi-C uses ligation products for deep sequencing to improve traditional Hi-C analysis[J]. Commun Biol, 2019, 2:267.
[184] SEXTON T, YAFFE E, KENIGSBERG E, et al. Three-dimensional folding and functional organization principles of the Drosophila genome[J]. Cell, 2012, 148(3):458-472.
[185] KHARCHENKO P V, ALEKSEYENKO A A, SCHWARTZ Y B, et al. Comprehensive analysis of the chromatin landscape in Drosophila melanogaster[J]. Nature, 2011, 471(7339):480-485.
[186] SCHOENFELDER S, FRASER P. Long-range enhancer-promoter contacts in gene expression control[J]. Nat Rev Genet, 2019, 20(8):437-455.
[187] RICCI W A, LU Z, JI L, et al. Widespread long-range cis-regulatory elements in the maize genome[J]. Nat Plants, 2019, 5(12):1237-1249.
[188] DELANEAU O, ZAZHYTSKA M, BOREL C, et al. Chromatin three-dimensional interactions mediate genetic effects on gene expression[J]. Science, 2019, 364(6439):eaat8266.
[189] GREENWALD W W, LI H, BENAGLIO P, et al. Subtle changes in chromatin loop contact propensity are associated with differential gene regulation and expression[J]. Nat Commun, 2019, 10(1):1054.
[190] NICETTO D, ZARET K S. Role of H3K9me3 heterochromatin in cell identity establishment and maintenance[J]. Curr Opin Genet Dev, 2019, 55:1-10.
[191] JIANG J G, DEFRANCES M C, MACHEN J, et al. The repressive function of AP2 transcription factor on the hepatocyte growth factor gene promoter[J]. Biochem Biophys Res Commun, 2000, 272(3):882-886.
[192] SCHNEIDERMAN J I, GOLDSTEIN S, AHMAD K. Perturbation analysis of heterochromatin-mediated gene silencing and somatic inheritance[J]. PLoS Genet, 2010, 6(9):e1001095.
[193] GRAY S, LEVINE M. Short-range transcriptional repressors mediate both quenching and direct repression within complex loci in Drosophila[J]. Gene Dev, 1996, 10(6):700-710.
[194] ANDERSON J, SALZER C L, KUMAR J P. Regulation of the retinal determination gene dachshund in the embryonic head and developing eye of Drosophila[J]. Dev Biol, 2006, 297(2):536-549.
[195] CHONG J H A, TAPIARAMIREZ J, KIM S, et al. Rest - a Mammalian Silencer Protein That Restricts Sodium-Channel Gene-Expression to Neurons[J]. Cell, 1995, 80(6):949-957.
[196] STROSCHEIN-STEVENSON S L, FOLEY E, O'FARRELL P H, et al. Identification of Drosophila Gene Products Required for Phagocytosis of Candida albicans[J]. PLOS Biology, 2005, 4(1):e4.
[197] DIXON J R, SELVARAJ S, YUE F, et al. Topological domains in mammalian genomes identified by analysis of chromatin interactions[J]. Nature, 2012, 485(7398):376-380.
[198] AN L, YANG T, YANG J, et al. OnTAD: hierarchical domain structure reveals the divergence of activity among TADs and boundaries[J]. Genome Biol, 2019, 20(1):282.
[199] WANG G, MENG Q, XIA B, et al. TADsplimer reveals splits and mergers of topologically associating domains for epigenetic regulation of transcription[J]. Genome Biol, 2020, 21(1):84.
[200] SANTINI S, BOORE J L, MEYER A. Evolutionary conservation of regulatory elements in vertebrate Hox gene clusters[J]. Genome Res, 2003, 13(6A):1111-1122.
[201] ARNOLD C D, GERLACH D, SPIES D, et al. Quantitative genome-wide enhancer activity maps for five Drosophila species show functional enhancer conservation and turnover during cis-regulatory evolution[J]. Nat Genet, 2014, 46(7):685-692.
[202] 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.
[203] HOWER V, EVANS S N, PACHTER L. Shape-based peak identification for ChIP-Seq[J]. BMC Bioinformatics, 2011, 12:15.
[204] JOHNSON G D, BARRERA A, MCDOWELL I C, et al. Human genome-wide measurement of drug-responsive regulatory activity[J]. Nat Commun, 2018, 9(1):5317.
[205] CONSORTIUM E P. An integrated encyclopedia of DNA elements in the human genome[J]. Nature, 2012, 489(7414):57-74.
[206] (NCBI) N C F B I. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information[EB/OL]. National Center for Biotechnology Information, 1988:
[2022-2-23]. https://www.ncbi.nlm.nih.gov/.
[207] LANGMEAD B, SALZBERG S L. Fast gapped-read alignment with Bowtie 2[J]. Nat Methods, 2012, 9(4):357-359.
[208] DANECEK P, BONFIELD J K, LIDDLE J, et al. Twelve years of SAMtools and BCFtools[J]. Gigascience, 2021, 10(2):giab008.
[209] DAVIS C A, HABERLAND M, ARNOLD M A, et al. PRISM/PRDM6, a transcriptional repressor that promotes the proliferative gene program in smooth muscle cells[J]. Mol Cell Biol, 2006, 26(7):2626-2636.
[210] WU Y, FERGUSON J E, 3RD, WANG H, et al. PRDM6 is enriched in vascular precursors during development and inhibits endothelial cell proliferation, survival, and differentiation[J]. J Mol Cell Cardiol, 2008, 44(1):47-58.
[211] ZHAO C, MENG A. Sp1-like transcription factors are regulators of embryonic development in vertebrates[J]. Dev Growth Differ, 2005, 47(4):201-211.