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Exploring how the genome folds through proximity ligation and sequencing

通过邻近连接和测序探索基因组如何折叠

基本信息

批准号：
8879882
负责人：
Erez Lieberman-Aiden
金额：
$ 51.51万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-30 至 2017-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8879882
关键词：
Architecture Cataloging Catalogs Cell Nucleus Cell physiology ChIP-seq Chimera organism Communities Complex Computer Analysis Coupled Couples Coupling DNA DNA Binding DNA-Binding Proteins Data Data Set Development Drug Targeting Enhancers Fractals Generations Genes Genetic Genetic Transcription Genome Genomic DNA High-Throughput Nucleotide Sequencing Human Genome Informatics Ligation Maps Methods Molecular Molecular Biology Movement Mus Nucleic Acids Oligonucleotides Pathway interactions Play Primary Neoplasm Process Proteins RNA RNA immunoprecipitation sequencing RNA-Binding Proteins Regulation Research Resolution Resources Role Sampling Science Series Structure Techniques Technology Work base biological systems density embryonic stem cell genome-wide human embryonic stem cell human subject improved in vivo mammalian genome new technology new therapeutic target physical model promoter protein complex relating to nervous system simulation tool tumorigenesis

项目摘要

Biological systems contain a large number of components whose physical interactions bring about cellular processes. A fundamental problem in molecular biology is to catalog these interactions and to decipher their functional consequences. High throughput sequencing has made it possible to characterize some of these interactions rapidly, at high-resolution, and in vivo (e.g., protein-DNA binding via ChIP-Seq and protein-RNA binding via RIP-Seq). But many interactions are not susceptible to these methods (e.g., RNARNA complexes, ncRNA-DNA binding, and - aside from recent work described below - DNA-DNA contacts and genome folding.) This gap may be bridged by coupling high-throughput sequencing with proximity-ligation-based methods. In proximity ligation, spatially proximate nucleic acids ligate to one another, forming a chimeric oligo. Observation of a chimera composed of X and Y suggests that X and Y must have been near one another in the original sample. As a result, questions about spatial arrangement become questions about sequence composition, making it possible to take advantage of high-throughput sequencing. Nevertheless, the development of these approaches is challenging: they involve subtle molecular biology and produce massive high-dimensional datasets requiring wholly new analytical paradigms including extensive physical modeling. We recently developed Hi-C, the first technology that couples proximity ligation and high- throughput sequencing in an unbiased, genome-wide fashion (Lieberman-Aiden et al., Science, 2009). Hi-C uses a DNA-DNA proximity ligation step to identify long-range physical contacts between genomic DNA loci in vivo. We used Hi-C to create a low-resolution three-dimensional map of the human genome, and made two significant discoveries: (1) genetic regulation is accompanied by the three-dimensional movement of genes from an 'on' compartment to an 'off' compartment, and vice-versa; (2) a never-before-seen macromolecular state, the fractal globule, which couples extraordinary spatial density and a total absence of knots. Here, we propose to dramatically extend the above work, by building a new generation of tools for systematically exploring the spatial organization of genomes, RNAs, and proteins, and by applying these tools to explore how RNAs and proteins establish and regulate the three- dimensional architecture of the genome. We will accomplish this through three specific research aims: (1) We will create an ensemble of new technologies combining proximity ligation and sequencing to enable comprehensive mapping of (a) DNA-RNA contacts [via DNA-RNA proximity ligation]; (b) RNA-RNA complexes [via RNA-RNA proximity ligation]; (c) selected protein-protein complexes [via probe-coupled proximity ligation]. We will use these methods to generate maps of biomolecular contacts in vivo. (2) We will create high-resolution Hi-C maps of mammalian genomes, comprehensively mapping promoter-enhancer contacts and exploring large-scale organizational features such as transcription factories. (3) We will develop new analytical approaches that combine the data produced by (1) and (2) with new (a) informatic tools, (b) computational analyses, (c) physical simulations, and (d) rigorous theoretical methods. We will characterize how physical interactions change during differentiation and tumorigenesis; identify the RNAs, proteins and pathways that that are most crucial in regulating genome folding, and produce detailed physical models of these pathways and how they modulate the physical structure of the genome. We plan to initially apply these techniques to characterize murine ES cells differentiating down a neural lineage, and later to differentiating human ES cells and to primary tumors. This effort will produce powerful new molecular methods which will dramatically improve our ability to assess the spatial arrangement of cellular components. It will transform our understanding of how mammalian genomes fold inside the nucleus. It will reveal how specific physical interactions between DNA, RNA, and protein play a role in differentiation, tumorigenesis, and genome folding, and suggest new drug targets in the process. Finally, this work will generate a series of datasets that will serve as valuable resources for the scientific community as a whole.

生物系统包含大量的组成部分，它们的物理相互作用关于细胞过程。分子生物学的一个基本问题是将这些相互作用并破译其功能后果。高通量测序具有使我们能够快速、高分辨率地描述其中一些相互作用，体内（例如，通过ChIP-Seq的蛋白质-DNA结合和通过RIP-Seq的蛋白质-RNA结合）。但许多相互作用对这些方法不敏感（例如，RNA RNA复合物，ncRNA-DNA 结合，以及-除了下面描述的最近的工作- DNA-DNA接触和基因组 folding.）这个缺口可以通过将高通量测序与基于邻位连接的测序相结合来桥接。方法.在邻近连接中，空间上邻近的核酸彼此连接，形成邻近的连接。嵌合寡核苷酸对由X和Y组成的嵌合体的观察表明，X和Y必须在最初的样本中彼此接近。因此，关于空间的问题排列变成了关于序列组成的问题，使得有可能采取高通量测序的优势。然而，这些方法的发展是具有挑战性的：它们涉及微妙的分子生物学，并产生大量的高维数据集需要全新的分析范式，包括广泛的物理建模。我们最近开发了Hi-C，这是第一种将邻近连接和高- 以无偏的全基因组方式进行通量测序（Lieberman-Aiden等，科学、 2009年）。Hi-C使用DNA-DNA邻近连接步骤来识别长距离物理接触基因组DNA位点之间的联系我们用Hi-C制作了一个低分辨率的三维图像人类基因组图谱，并取得了两个重大发现：（1）遗传调控是伴随着基因从“开”到"关“的三维运动，（2）一种从未见过的大分子状态，分形球，它结合了非凡的空间密度和完全没有结。在这里，我们建议通过构建新一代的工具，系统地探索基因组、RNA和蛋白质的空间组织，应用这些工具来探索RNA和蛋白质是如何建立和调节这三个- 基因组的三维结构我们将通过三项具体研究来实现这一目标。目的： (1)我们将创造一个新技术的集合，测序以实现（a）DNA-RNA接触[通过DNA-RNA 邻近连接];（B）RNA-RNA复合物[通过RNA-RNA邻近连接];（c）选择的蛋白质-蛋白质复合物[通过探针偶联的邻近连接]。我们将使用这些方法，在体内生成生物分子接触的地图。 (2)我们将创建高分辨率的哺乳动物基因组Hi-C图谱，绘制启动子-增强子接触图，并探索大规模的组织特征，转录工厂 (3)我们将开发新的分析方法，联合收割机（1）和（2）产生的数据。利用新的（a）信息工具，（B）计算分析，（c）物理模拟，以及（d）严格的理论方法。我们将描述物理相互作用如何在分化和肿瘤发生;确定RNA，蛋白质和途径，在调节基因组折叠中至关重要，并产生这些途径的详细物理模型以及它们如何调节基因组的物理结构。我们计划首先应用这些技术来表征小鼠ES细胞分化成神经谱系，分化人ES细胞和原发性肿瘤。这一努力将产生强大的新分子方法，这将大大改善我们的研究。评估细胞成分的空间排列的能力。它将改变我们的了解哺乳动物基因组在细胞核内如何折叠。它将揭示出 DNA、RNA和蛋白质之间的物理相互作用在分化中起作用，肿瘤发生和基因组折叠，并在此过程中提出新的药物靶点。最后这项工作将产生一系列数据集，这些数据集将作为科学研究的宝贵资源。整个社区。