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中基因组组织的工具 基因组的3D构型对于基因调节是复杂的，动态的和关键的。大多数 使用基于接近诱因的染色体进行了对基因组会议的最新见解 构象捕获方法（例如3C和HIC）和荧光原位杂交（FISH）技术。 两种方法的缺点是它们需要化学固定，在许多情况下需要规格 要跟踪的基因组上的少数目标位点。接近连接方法有选择地证明 仅DNA蛋白介导的相互作用，在检测与空间不同的接触中具有不同的影响 在同一染色体或不同染色体之间的距离，需要几个 化学交联后的其他体外步骤，以获取和处理数据。我们已经发展了 一种不需要化学固定或外部扰动的新方法，并监视DNA-DNA接触 活细胞中的频率。该方法利用了噬菌体MU的传输机制，并具有 我们成功地应用了大肠杆菌基因组的3D构象。与 3C/HIC看到的短程接触的优势，MU方法捕获了所有基因组触点， 揭示了基因组的混杂性。该方法揭示了遗传基因座的宽度聚类 3D空间，许多由共同调节基因组成的簇，随后我们使用它们验证 基于荧光的测量。大肠杆菌基因组的这些关键特征 - 与 特定的健壮的远程接触 - 在使用基于接近的研究中尚不可检测 方法。我们使用MU的测量还表明，紧凑DNA的蛋白质（冷凝蛋白和A Hisstone类蛋白）负责广泛的远程基因组接触。简而言之 测量改变了由3C/HIC产生的大肠杆菌基因组的静态，短距离接触视图 由生物学上重要的特定接触锚定的动态染色体技术的技术 地区。我们建议将MU方法扩展到不是MU天然宿主的细菌 在细菌和真核生物中评估我们发现的宇宙（第一酵母，有时 哺乳动物细胞系）通过设计将在每个目标物种的细胞中起作用的MU载体。鉴于 染色质折叠是基因调节的主要特征，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换位的方法提供 一个新的机会来揭露染色体会议，而无需依靠接近结扎的假设 实验。我们准确跟踪染色体构象的能力将为疾病打开新的途径 诊断，疾病靶向发现和结构依赖性基因调控的鉴定。