Mapping chromatin secondary structure by sequencing correlated DNA strand breaks

通过对相关 DNA 链断裂进行测序来绘制染色质二级结构

• 批准号: 8683896
• 负责人: William James Greenleaf
• 金额: $ 20.06万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-15 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8683896
• 关键词: Aneuploidy; Arbitration; Architecture; B-Lymphocytes; Base Pairing; Binding; Biochemical; Biological; Biological Models; Biological Process; Caliber; Cell Line; Cell Nucleus; Cells; Chromatin; Chromatin Fiber; Chromatin Structure; Chromosome Structures; Chromosomes; Cleaved cell; Complex; Conflict (Psychology); DNA; DNA Folding; DNA Repair; DNA biosynthesis; DNA strand break; Data; Data Set; Dialysis procedure; Disease; Elements; Event; Exposure to; Fiber; Fibroblasts; Fingerprint; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genomics; Health; High-Throughput Nucleotide Sequencing; Histones; Human; Human Development; Human Genome; Hydroxyl Radical; In Vitro; Individual; Investigation; Ionizing radiation; Length; Libraries; Light; Location; Malignant Neoplasms; Maps; Measures; Mechanics; Methodology; Methods; Modeling; Molecular; Nucleosomes; Pattern; Pilot Projects; Play; Positioning Attribute; Post-Translational Protein Processing; Process; Proteins; Refractory; Regulation; Role; Saccharomyces cerevisiae; Single-Stranded DNA; Sodium Chloride; Stereotyping; Structure; System; Testing; Validation; Vertebral column; Work; Yeasts; base; chromatin modification; epigenome; epigenomics; genome-wide; in vivo; insight; meter; nuclease; physical model; public health relevance; transcription factor

DESCRIPTION (provided by applicant): Project summary In a single human cell, two meters of DNA is carefully packaged within a five-micron nucleus in such a way that allows complex biological necessities such as genome replication, DNA repair, and regulated gene expression. This spectacular organizational challenge is overcome through the hierarchical folding of DNA into chromatin. Our understanding of the structure of chromatin, from the level of individual nucleosomes at the ~100 bp level to higher-order, long-range interactions of chromosomes at the megabase level, is undergoing a profound expansion due to high-throughput methods of mapping structural elements to specific locations on the genome, allowing correlation with biological state. We expect the structure of chromatin at an intermediate length scale of ~2 kilobases to play a crucial role in regulating transcription, DNA replication, and DNA repair, but our structural understanding of this "secondary structure" of chromatin organization continues to lag behind our rapidly developing understanding of both the level of nucleosomes and higher-order, long-range interactions. After decades of work on the intermediate level of chromatin, the topology of this structure - or indeed the very existence of a well-stereotyped structure in vivo - is still holy debated, and almost nothing is known about the variability of these putative structures as a function of genome position. This pilot project builds methods that will develop a clearer picture of this scale of chromatin organization in order to integrate both the physical and biochemical views of the nucleus. We will study chromatin folding both in vivo and in vitro by applying ionizing radiation, which is known to generate correlated nicks to the DNA backbone at spatially proximal locations. The resulting single-stranded DNA fragments, which have ends that were within ~3 nm of each other in the folded chromatin structure, will be analyzed with high-throughput sequencing in order to map these fragments to the genome. This analysis will generate genome-wide pairwise distance constraints on the folded DNA. These data will provide an entirely new window into chromatin compaction and structure at the 30- nm length scale. Our investigations will begin with chromatin fibers assembled in vitro in order to troubleshoot and validate our methodology. Next we will investigate chromatin structure in S. cerevisiae, a model system with a small genome and extremely well characterized, well-positioned nucleosomes. Finally, we will pilot our chromatin structure mapping methodology in primary human fibroblasts and immortalized B-cells. This structural information will be combined with and compared to existing data sets that describe chromatin modifications and nuclease accessibility, laying the groundwork for an integrated physical model of chromatin structure by bridging the gap between our crystallographic understanding of the mononucleosome and our emerging understanding of the higher-order interactions at the megabase scale.

描述（由申请人提供）： 在单个人类细胞中的项目摘要，将两个米的DNA仔细包装在五微米核中，以允许复杂的生物学必需品，例如基因组复制，DNA修复和调节基因表达。通过将DNA分层折叠为染色质，克服了这一壮观的组织挑战。我们对染色质结构的理解，从〜100 bp水平的单个核小体的水平到巨大的染色体在巨大的高阶，长期相互作用，由于将结构元素映射到基因组的特定位置，使其与生物学状态相关，因此正在经历高通量的扩展。我们期望在〜2千倍酶的中间长度尺度上的染色质结构在调节转录，DNA复制和DNA修复方面起着至关重要的作用，但是我们对染色质组织的这种“次要结构”的结构性理解继续落后于我们对核体和高级相互作用的迅速发展的理解。经过数十年的染色质中间水平的工作，这种结构的拓扑 - 或实际上是体内良好的结构的存在 - 仍然是圣洁的辩论，几乎一无所知，这些假定结构的可变性是基因组位置的函数。该试点项目构建了方法，该方法将更清晰地了解这种染色质组织的规模，以便整合细胞核的物理和生化视图。我们将通过应用电离辐射研究染色质折叠，并在体外折叠，这是在空间近端位置在DNA主链中产生相关的迹线的。所得的单链DNA片段将使用高通量测序分析，其末端彼此之间的末端彼此之间彼此之间相互〜3 nm之内，以将这些片段映射到基因组。该分析将在折叠的DNA上产生全基因组的成对距离约束。这些数据将为30 nm长度尺度上的染色质压实和结构提供一个全新的窗口。我们的研究将从在体外组装的染色质纤维开始，以解决和验证我们的方法。接下来，我们将研究酿酒酵母中的染色质结构，这是一种模型系统，具有较小的基因组且表征良好的核小体。最后，我们将在原代人成纤维细胞和永生的B细胞中试用染色质结构映射方法。这些结构信息将与描述染色质修饰和核酸酶访问性的现有数据集结合并进行比较，通过弥合我们对单核体的晶体学理解与我们对巨型尺度上高级相互作用的新兴理解之间的差距，从而为染色质结构的综合物理模型奠定了基础。