Bacteriophage Mu as Tool to Study Genome Organization in Bacteria and Eukaryotes

噬菌体 Mu 作为研究细菌和真核生物基因组组织的工具

基本信息

批准号：
10265837
负责人：
Lydia Freddolino
金额：
$ 44.71万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-22 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10265837
关键词：
3-Dimensional Architecture Bacillus subtilis Bacteria Bacteriophage mu Bacteriophages Binding Sites Biological Cell Line Cell physiology Cells Chemicals Chromatin Chromosomes Complex DNA DNA Transposable Elements Data Dependence Development Developmental Biology Diagnostic Disease Distant Electroporation Elephants Escherichia coli Eukaryota Evolution Family Fluorescence Fluorescence Microscopy Fluorescent in Situ Hybridization Frequencies Gene Expression Gene Expression Regulation Gene Family Gene Rearrangement Genes Genetic Recombination Genome Genomics Gram-Positive Bacteria HU Protein Hi-C Histones Human In Situ In Vitro Length Ligation Location Malignant Neoplasms Mammalian Cell Maps Measurement Measures Mediating Methodology Methods Microscopy Modeling Molecular Conformation Monitor Mutation Nuclear Operon Pathway interactions Physiology Play Property Proteins Resolution Ribosomal RNA Role Site Specificity Structure Techniques Work Yeasts base chemical fixation chromosome conformation capture computerized data processing condensin crosslink design experimental study genomic locus insight mammalian genome mu transposase three dimensional structure tool vector

项目摘要

Bacteriophage Mu as Tool to Study Genome Organization in Bacteria and Eukaryotes The 3D configuration of the genome is complex, dynamic and crucial for gene regulation. The majority of recent insights into genome conformations have been made using proximity-ligation based chromosome conformation capture methods (e.g., 3C and HiC) and fluorescent in situ hybridization (FISH) techniques. Drawbacks of both approaches are that they require chemical fixation, and in many cases require specification of a small number of target sites on the genome to be tracked. Proximity ligation approaches selectively probe only DNA-protein mediated interactions, have different efficiencies of detecting contacts with varying spatial distances either within the same chromosome or between different chromosomes, and require several additional in vitro steps after chemical crosslinking for obtaining and processing the data. We have developed a new methodology that requires no chemical fixation or external perturbation and monitors DNA-DNA contact frequencies in live cells. The methodology exploits the transposition mechanism of bacteriophage Mu, and has been applied successfully to interrogate the 3D conformation of the E. coli genome. In contrast to the dominance of short-range contacts seen with 3C/HiC, the Mu methodology captured all genomic contacts, revealing that the genome was well-mixed. The methodology revealed widespread clustering of genetic loci in 3D space, many of the clusters consisting of co-regulated genes, which we subsequently validated using fluorescence-based measurements. These key features of the E. coli genome – generalized mixing with specific robust long-range contacts -- has not been detectable in studies using proximity ligation based methods. Our measurements using Mu also revealed that proteins that compact DNA (condensin and a histone-like protein) are responsible for the extensive long-range genomic contacts. In short, our Mu-based measurements changed the static, short-range contact view of the E. coli genome generated by 3C/HiC techniques to that of a dynamic chromosome anchored by specific contacts between biologically important regions. We propose to extend the Mu methodology to bacteria that are not a natural host for Mu in order to assess the universality of our findings among Bacteria, as well as to eukaryotes (first yeast and eventually mammalian cell lines) by designing Mu vectors that will function in cells of each target species. Given that chromatin folding is a major feature of gene regulation, and changes dynamically in development and disease, it is imperative that we assess genome architecture in live cells. Our Mu transposition based methods provide a new opportunity to unveil chromosome conformations without relying on the assumptions of proximity ligation experiments. Our ability to accurately track chromosomal conformations will open new avenues for disease diagnostics, disease target discovery and identification of structure-dependent gene regulation.

噬菌体 Mu 作为研究细菌和真核生物基因组组织的工具基因组的 3D 结构是复杂的、动态的，对于基因调控至关重要。最近对基因组构象的见解是使用基于染色体的邻近连接获得的构象捕获方法（例如 3C 和 HiC）和荧光原位杂交 (FISH) 技术。这两种方法的缺点是它们需要化学固定，并且在许多情况下需要规范邻近连接方法选择性地探测基因组上的少数目标位点。仅 DNA-蛋白质介导的相互作用，检测具有不同空间的接触的效率不同同一染色体内或不同染色体之间的距离，并且需要多个我们开发了化学交联后用于获取和处理数据的额外体外步骤。一种无需化学固定或外部扰动并监测 DNA-DNA 接触的新方法该方法利用噬菌体 Mu 的转座机制，并具有已成功应用于询问大肠杆菌基因组的 3D 构象。 3C/HiC 中短程接触占主导地位，Mu 方法捕获了所有基因组接触，该方法揭示了基因座的广泛聚集。 3D 空间，许多簇由共同调控的基因组成，我们随后使用它们进行了验证基于荧光的测量。大肠杆菌基因组的这些关键特征 - 广义混合特定的强远程接触——在使用基于邻近连接的研究中尚未检测到我们使用 Mu 进行的测量还揭示了压缩 DNA 的蛋白质（凝缩蛋白和 a）。组蛋白样蛋白）负责广泛的远程基因组接触。测量改变了 3C/HiC 生成的大肠杆菌基因组的静态、短程接触视图动态染色体的技术由生物学上重要的之间的特定接触锚定我们建议将 Mu 方法扩展到非 Mu 天然宿主的细菌，以便评估我们的研究结果在细菌以及真核生物（首先是酵母，最终是哺乳动物细胞系），通过设计将在每个目标物种的细胞中发挥作用的 Mu 载体。染色质折叠是基因调控的一个主要特征，在发育和疾病中动态变化，我们必须评估活细胞中的基因组结构，并提供基于 Mu 转座的方法。在不依赖邻近连接假设的情况下揭示染色体构象的新机会我们准确追踪染色体构象的能力将为疾病开辟新途径。诊断、疾病靶点发现和结构依赖性基因调控的鉴定。