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组织的工具 基因组的3D构型是复杂的、动态的，对基因调控至关重要。大多数人 最近对基因组构象的洞察是使用基于邻近连接的染色体 构象捕捉方法(如3C和HIC)和荧光原位杂交(FISH)技术。 这两种方法的缺点都是它们需要化学固定，而且在许多情况下需要指定 要追踪的基因组上的一小部分靶点。近距离结扎方法选择性探查 只有DNA-蛋白质介导的相互作用，才有不同的效率来检测不同空间的接触 在同一染色体内或不同染色体之间的距离，并且需要几个 化学交联后的附加体外步骤以获得和处理数据。我们已经开发出 一种不需要化学固定或外部干扰并监测DNA-DNA接触的新方法 活细胞的频率。该方法利用了噬菌体Mu的转座机制，并具有 已成功应用于查询大肠杆菌基因组的3D构象。与之相对的是 在3C/HIC中看到的短程接触的主导地位，Mu方法捕获了所有基因组接触， 揭示出基因组是混合良好的。方法论揭示了遗传基因座的广泛聚集性 3D空间，许多簇由共同调控的基因组成，我们随后使用 基于荧光的测量。大肠杆菌基因组的这些关键特征--广泛地与 特定的强健的远距离接触--在使用邻近结扎的研究中没有检测到 方法：研究方法。我们使用Mu进行的测量还显示，紧凑DNA的蛋白质(凝集素和a 组蛋白样蛋白)是造成广泛的远距离基因组接触的原因。简而言之，我们的木本 测量改变了由3C/HIC产生的大肠杆菌基因组的静态、短距离接触视角 通过生物上重要的特定接触而锚定的动态染色体的技术 地区。我们建议将Mu方法扩展到Mu的非自然宿主细菌，以便 评估我们的发现在细菌以及真核生物(首先是酵母，最后是酵母)中的普遍性 通过设计在每个目标物种的细胞中起作用的Mu载体。考虑到 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的基因组结构。我们基于Mu转位的方法提供了 揭示染色体构象的新机会，而不依赖邻近连接的假设 实验。我们准确追踪染色体构象的能力将为疾病开辟新的途径 诊断、疾病靶点发现和结构依赖基因调控的识别。