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

• 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
• 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

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结构的工具 基因组的3D构型是复杂的、动态的，对于基因调控至关重要。大多数 最近，利用基于邻近连接的染色体 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格说明。 基因组上的少量目标位点被追踪。邻位连接选择性地接近探针 只有DNA-蛋白质介导的相互作用，具有不同的效率检测接触与不同的空间 在同一染色体内或不同染色体之间的距离，并需要几个 在化学交联之后用于获得和处理数据的附加体外步骤。我们已经开发 一种新的方法，不需要化学固定或外部干扰，并监测DNA-DNA接触 活细胞中的频率。该方法利用噬菌体Mu的转座机制， 成功地应用于E.大肠杆菌基因组。相对于 在3C/HiC中观察到的短程接触占主导地位，Mu方法捕获了所有基因组接触， 揭示了基因组是混合的。该方法揭示了广泛的遗传位点聚类， 在3D空间中，许多簇由共调控基因组成，我们随后使用 基于荧光的测量E.大肠杆菌基因组-广义混合 特异性强的长距离接触-在使用基于邻位连接的研究中尚未检测到 方法.我们使用Mu的测量还显示，压缩DNA的蛋白质（凝聚素和蛋白质）， 组蛋白样蛋白）负责广泛的长距离基因组接触。简而言之， 测量改变了E. 3C/HiC产生的大肠杆菌基因组 通过生物学上重要的染色体之间的特定接触锚定的动态染色体的技术 地区我们建议将Mu方法扩展到不是Mu天然宿主的细菌，以便 评估我们的研究结果在细菌中的普遍性，以及真核生物（首先是酵母，最终 哺乳动物细胞系），通过设计将在每种靶物种的细胞中发挥功能的Mu载体。鉴于 染色质折叠是基因调控的主要特征，并且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。我们的基于Mu转座的方法提供 一个新的机会，揭示染色体构象，而不依赖于邻近连接的假设， 实验我们精确追踪染色体构象的能力将为疾病的研究开辟新的途径 诊断、疾病靶点发现和结构依赖性基因调控的鉴定。