Mapping chromatin secondary structure by sequencing correlated DNA strand breaks

描述（由申请人提供）： 在一个单一的人类细胞中，两米长的DNA被精心包装在一个五微米的细胞核中，以这种方式允许复杂的生物必需品，如基因组复制，DNA修复和调控基因表达。这种壮观的组织挑战是通过DNA分层折叠成染色质来克服的。我们对染色质结构的理解，从~100 bp水平的单个核小体水平到兆碱基水平的染色体的更高阶、远程相互作用，由于将结构元件映射到基因组上特定位置的高通量方法，允许与生物状态相关，正在经历深刻的扩展。我们期望染色质的结构在一个中等长度的尺度上发挥至关重要的作用，在调节转录，DNA复制和DNA修复，但我们的结构的理解，这种“二级结构”的染色质组织继续落后于我们迅速发展的理解水平的核小体和高阶，远程相互作用。在对染色质的中间水平进行了数十年的研究之后，这种结构的拓扑结构--或者说在体内是否存在一个良好的定型结构--仍然存在着激烈的争论，而且几乎没有人知道这些假定结构的变异性是基因组位置的函数。这个试点项目建立的方法，将开发一个更清晰的图片，这种规模的染色质组织，以整合细胞核的物理和生物化学的观点。我们将研究染色质折叠在体内和体外通过应用电离辐射，这是已知的产生相关的缺口的DNA骨架在空间上接近的位置。将使用高通量测序分析所得单链DNA片段（其在折叠染色质结构中具有彼此在约3 nm内的末端），以将这些片段映射到基因组。该分析将在折叠的DNA上产生全基因组成对距离约束。这些数据将提供一个全新的窗口，染色质压缩和结构在30纳米的长度尺度。我们的研究将从体外组装的染色质纤维开始开始，以排除故障并验证我们的方法。接下来我们将研究S.酿酒酵母，一个模型系统，具有一个小的基因组和非常好的特点，定位良好的核小体。最后，我们将在原代人成纤维细胞和永生化B细胞中试验我们的染色质结构作图方法。这些结构信息将与描述染色质修饰和核酸酶可及性的现有数据集相结合并进行比较，通过弥合我们对单胞体的晶体学理解与我们对兆尺度上的高阶相互作用的新兴理解之间的差距，为染色质结构的综合物理模型奠定基础。

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.