特殊順式調(diào)控元件與染色質(zhì)三維結構
發(fā)布時間：2018-08-23 10:50
【摘要】：對于真核生物,染色質(zhì)是最大的單一生物大分子,是基因組的存在形式,是遺傳和表觀遺傳的共同載體,而細胞核是染色質(zhì)的儲存空間。在有絲分裂間期,不同的染色質(zhì)在細胞核內(nèi)分別占據(jù)一個相對獨立但又相對固定的空間位置即染色體領域CT(Chromatin Territory),每條染色質(zhì)進一步分層級折疊成復雜而有序的三維高級結構,這些穩(wěn)定的高級結構及其核內(nèi)空間定位與DNA重組與復制、基因的轉(zhuǎn)錄甚至翻譯等密切相關。大量研究發(fā)現(xiàn),很多調(diào)控因子都是依賴著染色質(zhì)三維空間結構來發(fā)揮功能。染色體構象捕獲技術3C(Chromosome Conformation Capture)是2002年出現(xiàn)的研究染色質(zhì)折疊的核心生化技術,近年來隨著3C的衍生技術如4C、5C、Hi-C、Capture-C以及考察特定蛋白介導染色質(zhì)互作的ChIA-PET等分子圖譜(Mapping)以及DNA-FISH等不同類型的成像(Imaging)技術的迅速發(fā)展,人們對染色質(zhì)折疊的認識不斷加深。目前普遍認為:有絲分裂間期染色質(zhì)的折疊包含染色質(zhì)領域(chromosome territory)、拓撲結構域(topological associated domain,TAD)、染色質(zhì)環(huán)(chromatin loop)等從大到小不同層級的結構單元,作為基因組序列及其DNA和組蛋白修飾、染色質(zhì)調(diào)控蛋白的結合等的最終載體和響應器(carrierresponser)或“集大成者”(moderator),基因組的復雜三維高級結構與組蛋白修飾、DNA甲基化、DNase敏感性等基因組其它組學特征以及基因的復制、轉(zhuǎn)錄等基因組功能特征密切相關。順式調(diào)控元件CREs(cis-regulatory elements)往往指的是與結構基因串聯(lián)的特定非編碼DNA序列,它們通過與轉(zhuǎn)錄因子等的結合而調(diào)控基因轉(zhuǎn)錄的精確起始和轉(zhuǎn)錄效率,從而參與基因表達的調(diào)控。根據(jù)序列組成等特性,可以將其分為常見的啟動子、終止子、增強子、絕緣子、邊界元件、基因組座位調(diào)控元件以及重復元件、核基質(zhì)附著區(qū)等不同類型。其中重復元件(repeat elements)在基因組中多次重復出現(xiàn),包含了大量的有著不同的結構和來源的DNA元件,在真核生物基因組中的比例可達到一半以上。根據(jù)片段長度及生物學特性,重復元件可進一步分成串聯(lián)重復序列及轉(zhuǎn)座元件等。由于受到測序讀長、比對等技術手段的限制,人們對于重復元件在基因組中發(fā)揮的功能和機制目前還知之甚少,需要進一步發(fā)掘。核基質(zhì)附著區(qū)(Matrix Attachment Regions,MARs)是一類在不同物種與組織細胞的基因組中廣泛存在的、富含AT序列且與核基質(zhì)緊密結合的DNA元件。目前認為MARs元件一方面可能作為染色質(zhì)三維折疊的結構單元,對于建立和維持染色質(zhì)領域發(fā)揮著作用;另一方面可能作為基因表達調(diào)控的功能性單元,通過與核基質(zhì)等結合,調(diào)節(jié)基因表達。本研究以染色質(zhì)三維結構實驗數(shù)據(jù)為基礎,通過多組學數(shù)據(jù)整合分析,從基因組序列特征出發(fā),考察了多種不同順式調(diào)控元件在染色質(zhì)三維折疊中的可能作用,以及染色質(zhì)三維結構對這些元件演化等潛在影響,為進一步深入了解不同元件在染色質(zhì)三維高階結構的建立、維持以及傳遞等過程中可能的作用奠定了基礎。本論文的主要研究內(nèi)容如下:開發(fā)了Hi-C數(shù)據(jù)處理的一站式軟件HBP。建立了針對特殊順式調(diào)控元件的研究方法,開發(fā)了能夠系統(tǒng)考察特殊順式調(diào)控元件參與的染色質(zhì)相互作用的處理軟件HBP(Hi-C BED file analysis Pipeline),通過簡單的操作即可實現(xiàn)針對不同元件的處理和分析。HBP作為一款開源、靈活、高度優(yōu)化的一站式處理平臺,能夠有效地分析全基因組三維高級結構特點及特殊序列元件參與的染色質(zhì)相互作用,大大方便了針對特定相關區(qū)域染色質(zhì)折疊的系統(tǒng)研究。作為一個簡便、高效、可靠的工具,HBP能夠通過整合組蛋白修飾一級Ch IP-seq、RNA-seq等其它組學數(shù)據(jù),針對特定區(qū)域的染色質(zhì)相互作用進行大規(guī)模系統(tǒng)挖掘與分析,對潛在分子機制以及生物學意義等進行多方面的研究。基于染色質(zhì)三維結構對部分重復元件進行了研究。重復元件在各類物種基因組中廣泛分布且種類繁多,不同種類之間序列功能各異。本研究探索了與染色質(zhì)三維結構高度相關的一些重復元件子類,發(fā)現(xiàn)了包括Alu等在內(nèi)的重復元件廣泛地參與了染色質(zhì)的三維折疊。首次從染色質(zhì)三維結構的空間距離層面,系統(tǒng)考察了相鄰重復元件子類間的演化過程,發(fā)現(xiàn)在一維序列演化關系上相對靠近的子類,在三維空間結構上的相互作用強度相對較高,提示重復元件的進化歷程可能與基因組三維高級結構的形成密切相關�；谌旧|(zhì)三維結構對核基質(zhì)附著區(qū)進行了研究。核基質(zhì)附著區(qū)MARs(Matrix Associated Regions)在不同的物種間廣泛存在,雖然存在AT序列相對富集等共同序列特征,卻沒有發(fā)現(xiàn)顯著保守性等其它結構特征,此外其在細胞核內(nèi)的功能和機制也尚無定論。本研究針對MARs元件,從相互作用頻率分布、網(wǎng)絡拓撲結構及潛在生物學功能等三方面進行了探索。發(fā)現(xiàn)MARs元件與染色質(zhì)三維結構高度相關,且在高強度相互作用中占據(jù)著較大的比例,提示MARs元件對染色質(zhì)折疊具有重要影響。同時,拓撲結構聚類分析證實MARs元件可以分為不同類型,包括了維持染色質(zhì)領域及高級空間構象等方面的結構單元部分以及調(diào)控基因表達等方面的功能單元部分,提示不同類型MARs元件在基因組細胞核高級結構及功能上可能發(fā)揮不同的作用。綜上所述,本課題以染色質(zhì)三維結構為基礎,以研究不同順式調(diào)控元件在染色質(zhì)三維結構中發(fā)揮的作用為目的,開發(fā)了特定的Hi-C數(shù)據(jù)分析處理軟件HBP,建立了多組學數(shù)據(jù)的整合分析方法,并且對部分重復元件及核基質(zhì)附著區(qū)與基因組三維高級結構的關系等進行了分析,發(fā)現(xiàn)了一些與染色質(zhì)三維結構高度相關的現(xiàn)象,為進一步的染色質(zhì)高階構象及構成元件結構與功能等方面的研究奠定了基礎。
[Abstract]:In eukaryotes, chromatin is the largest single biological macromolecule, a form of genome existence, a common carrier of heredity and epigenetics, and the nucleus is the storage space of chromatin. Chromatin Territory (CT), in which each chromatin is folded into complex and ordered three-dimensional higher-order structures, is closely related to DNA recombination and replication, gene transcription and even translation. Chromosome Conformation Capture (3C) is the core biochemical technique for studying chromatin folding that emerged in 2002. In recent years, with the development of 3C derivatives such as 4C, 5C, Hi-C, Capture-C, and molecular maps such as CHIA-PET for investigating specific protein-mediated chromatin interactions (Mapping) and DNA-FISH With the rapid development of various imaging techniques, people have a deeper understanding of chromatin folding. It is generally believed that chromatin folding in mitotic interphase includes chromatin domain, topological associated domain (TAD), chromatin loop and so on. Homologous structural units, as the ultimate carriers and responders of genome sequences, DNA and histone modifications, chromatin-regulated protein binding, etc., complex three-dimensional high-level structure of the genome and histone modification, DNA methylation, DNase sensitivity and other genomic characteristics Cis-regulatory elements (CREs) often refer to specific non-coding DNA sequences linked to structural genes, which regulate the precise initiation and transcriptional efficiency of gene transcription by binding to transcription factors, and thus participate in the regulation of gene expression. According to the characteristics of sequence composition, it can be divided into common promoters, terminators, enhancers, insulators, boundary elements, genomic locus regulatory elements and repeat elements, nuclear matrix attachment regions and other different types. Repeat elements are repeated in genomes, including a large number of different junctions. Repetitive elements can be further divided into tandem repeats and transposable elements according to their length and biological characteristics. Due to the limitation of sequencing, length reading, alignment and other technical means, the functions of repetitive elements in genomes are limited. Matrix Attachment Regions (MARs) are a class of DNA elements that exist widely in the genomes of different species and tissues and cells and are rich in AT sequences and closely bound to the nuclear matrix. Construction units play an important role in the establishment and maintenance of chromatin; on the other hand, they may act as functional units of gene expression regulation, regulating gene expression by binding to nuclear matrix and so on. The possible roles of various cis-regulatory elements in the three-dimensional folding of chromatin and the potential effects of the three-dimensional structure of chromatin on the evolution of these elements lay the foundation for further understanding the possible roles of different elements in the establishment, maintenance and transmission of three-dimensional high-order chromatin structures. The main contents are as follows: Hi-C data processing one-stop software HBP is developed. The research methods for special cis-regulatory elements are established. The processing software HBP (Hi-C BED file analysis Pipeline) is developed, which can systematically investigate the interaction of chromatin with special cis-regulatory elements. Different cis-regulatory elements can be realized by simple operation. As an open source, flexible and highly optimized one-stop processing platform, HBP can effectively analyze the three-dimensional high-level structural characteristics of the whole genome and chromatin interactions involving special sequence elements, greatly facilitating the systematic study of chromatin folding for specific related regions. By integrating histone-modified first-order Ch IP-seq, RNA-seq and other histological data, HBP can conduct large-scale systematic mining and analysis of chromatin interactions in specific regions, and study potential molecular mechanisms and biological significance in many aspects. Based on the three-dimensional structure of chromatin, some repetitive elements were studied. Repetitive elements are widely distributed in the genomes of various species, and their sequence functions vary from species to species. This study explored some subclasses of repetitive elements highly related to the three-dimensional structure of chromatin. Repetitive elements, including Alu, were found to be involved extensively in the three-dimensional folding of chromatin. On the level of spatial distance, we systematically investigated the evolutionary process between subclasses of adjacent duplicate elements, and found that the subclasses which are relatively close to each other in the evolutionary relationship of one-dimensional sequences have relatively high interaction intensity in the three-dimensional structure, suggesting that the evolutionary process of duplicate elements may be closely related to the formation of three-dimensional high-level structures of genomes. Matrix Associated Regions (MARs) are ubiquitous among different species. Although there are common sequence features such as relative AT sequence enrichment, no other structural features such as significant conservatism have been found. In addition, the function and mechanism of MARs in the nucleus are still absent. The results show that the MARs are highly correlated with the three-dimensional structure of chromatin and occupy a large proportion in the high-intensity interaction, suggesting that MARs play an important role in chromatin folding. Topological cluster analysis confirmed that MARs elements could be classified into different types, including structural units for maintaining chromatin domain and advanced spatial conformation, and functional units for regulating gene expression, suggesting that different types of MARs might play different roles in the higher structure and function of genomic nuclei. In summary, based on the three-dimensional structure of chromatin, and for the purpose of studying the role of different cis-regulatory elements in the three-dimensional structure of chromatin, a specific Hi-C data analysis and processing software, HBP, was developed. The integrated analysis method of multi-group data was established, and some duplicate elements and nuclear matrix attachment regions and genes were analyzed. Some phenomena highly correlated with the three-dimensional structure of chromatin were found, which laid a foundation for further study on the high-order conformation of chromatin and the structure and function of chromatin components.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q78
，
本文編號：2198855
[Abstract]:In eukaryotes, chromatin is the largest single biological macromolecule, a form of genome existence, a common carrier of heredity and epigenetics, and the nucleus is the storage space of chromatin. Chromatin Territory (CT), in which each chromatin is folded into complex and ordered three-dimensional higher-order structures, is closely related to DNA recombination and replication, gene transcription and even translation. Chromosome Conformation Capture (3C) is the core biochemical technique for studying chromatin folding that emerged in 2002. In recent years, with the development of 3C derivatives such as 4C, 5C, Hi-C, Capture-C, and molecular maps such as CHIA-PET for investigating specific protein-mediated chromatin interactions (Mapping) and DNA-FISH With the rapid development of various imaging techniques, people have a deeper understanding of chromatin folding. It is generally believed that chromatin folding in mitotic interphase includes chromatin domain, topological associated domain (TAD), chromatin loop and so on. Homologous structural units, as the ultimate carriers and responders of genome sequences, DNA and histone modifications, chromatin-regulated protein binding, etc., complex three-dimensional high-level structure of the genome and histone modification, DNA methylation, DNase sensitivity and other genomic characteristics Cis-regulatory elements (CREs) often refer to specific non-coding DNA sequences linked to structural genes, which regulate the precise initiation and transcriptional efficiency of gene transcription by binding to transcription factors, and thus participate in the regulation of gene expression. According to the characteristics of sequence composition, it can be divided into common promoters, terminators, enhancers, insulators, boundary elements, genomic locus regulatory elements and repeat elements, nuclear matrix attachment regions and other different types. Repeat elements are repeated in genomes, including a large number of different junctions. Repetitive elements can be further divided into tandem repeats and transposable elements according to their length and biological characteristics. Due to the limitation of sequencing, length reading, alignment and other technical means, the functions of repetitive elements in genomes are limited. Matrix Attachment Regions (MARs) are a class of DNA elements that exist widely in the genomes of different species and tissues and cells and are rich in AT sequences and closely bound to the nuclear matrix. Construction units play an important role in the establishment and maintenance of chromatin; on the other hand, they may act as functional units of gene expression regulation, regulating gene expression by binding to nuclear matrix and so on. The possible roles of various cis-regulatory elements in the three-dimensional folding of chromatin and the potential effects of the three-dimensional structure of chromatin on the evolution of these elements lay the foundation for further understanding the possible roles of different elements in the establishment, maintenance and transmission of three-dimensional high-order chromatin structures. The main contents are as follows: Hi-C data processing one-stop software HBP is developed. The research methods for special cis-regulatory elements are established. The processing software HBP (Hi-C BED file analysis Pipeline) is developed, which can systematically investigate the interaction of chromatin with special cis-regulatory elements. Different cis-regulatory elements can be realized by simple operation. As an open source, flexible and highly optimized one-stop processing platform, HBP can effectively analyze the three-dimensional high-level structural characteristics of the whole genome and chromatin interactions involving special sequence elements, greatly facilitating the systematic study of chromatin folding for specific related regions. By integrating histone-modified first-order Ch IP-seq, RNA-seq and other histological data, HBP can conduct large-scale systematic mining and analysis of chromatin interactions in specific regions, and study potential molecular mechanisms and biological significance in many aspects. Based on the three-dimensional structure of chromatin, some repetitive elements were studied. Repetitive elements are widely distributed in the genomes of various species, and their sequence functions vary from species to species. This study explored some subclasses of repetitive elements highly related to the three-dimensional structure of chromatin. Repetitive elements, including Alu, were found to be involved extensively in the three-dimensional folding of chromatin. On the level of spatial distance, we systematically investigated the evolutionary process between subclasses of adjacent duplicate elements, and found that the subclasses which are relatively close to each other in the evolutionary relationship of one-dimensional sequences have relatively high interaction intensity in the three-dimensional structure, suggesting that the evolutionary process of duplicate elements may be closely related to the formation of three-dimensional high-level structures of genomes. Matrix Associated Regions (MARs) are ubiquitous among different species. Although there are common sequence features such as relative AT sequence enrichment, no other structural features such as significant conservatism have been found. In addition, the function and mechanism of MARs in the nucleus are still absent. The results show that the MARs are highly correlated with the three-dimensional structure of chromatin and occupy a large proportion in the high-intensity interaction, suggesting that MARs play an important role in chromatin folding. Topological cluster analysis confirmed that MARs elements could be classified into different types, including structural units for maintaining chromatin domain and advanced spatial conformation, and functional units for regulating gene expression, suggesting that different types of MARs might play different roles in the higher structure and function of genomic nuclei. In summary, based on the three-dimensional structure of chromatin, and for the purpose of studying the role of different cis-regulatory elements in the three-dimensional structure of chromatin, a specific Hi-C data analysis and processing software, HBP, was developed. The integrated analysis method of multi-group data was established, and some duplicate elements and nuclear matrix attachment regions and genes were analyzed. Some phenomena highly correlated with the three-dimensional structure of chromatin were found, which laid a foundation for further study on the high-order conformation of chromatin and the structure and function of chromatin components.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q78
，
本文編號：2198855
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