The Effects of Physical Disruption on Genome Organization and Integrity

描述(申请人提供)：人类基因组在细胞核中的三维(3D)组织不仅对于将2米长的DNA放入2微米直径的细胞核非常重要，而且对于基因调控和其他重要的生物学功能也是重要的。利用最近开发的Hi-C技术来捕获细胞内整个基因组中的染色体折叠和循环，拟议的研究将调查这种基因组组织与细胞和DNA经历的物理破坏之间的关系。如果不正确的断裂修复导致的染色体易位扰乱了基因或基因与调控元件之间的关系，DNA双链断裂(DSB)的戏剧性物理破坏可能会导致癌症和其他疾病。因此，了解影响DSB后可能发生易位的因素是很重要的。测量小鼠B淋巴细胞基因组组织的HI-C实验将被用来确定先前存在的基因组区域的接近程度与在相同细胞中诱导DSB后观察到的易位位置的相关性。在物理接近的高概率不能解释反复易位的情况下，将通过将本研究中获得的相互作用和易位数据与先前发表的关于基因表达、蛋白质-DNA相互作用和染色体常见脆性部位等特征的数据进行计算整合，来检验其他因素对易位易感性的贡献。然后，将通过将实验观察到的易位与根据聚合物物理原理和实验Hi-C数据得出的基因组生物物理模型进行比较，来评估3D基因组结构的物理性质对非近端基因组区域之间易位形成的影响。这个模型将被用来预测在寻找修复伙伴的过程中，基因组结构的物理属性可能在多大程度上限制或增强基因组区域和DNA断端的可移动性。在这些模拟之后，将通过对受到物理力和约束的细胞进行Hi-C实验来直接实验测试基因组的变形性。这些力实验的结果将检验生物物理模型的预测，并使物理变形性和移位敏感性之间的比较成为可能。成功观察细胞内基因组对物理作用力的反应也将有助于深入了解组织和器官中的生物相关作用力是否会通过直接改变染色质结构来影响基因表达、细胞行为和细胞命运。这样的洞察力可以为未来做出贡献 力在组织工程细胞定向分化中的应用。通过整合新的实验数据、物理模型和以前发表的基因组数据，这项研究将有助于更全面地了解复杂的3D基因组对健康和疾病中的物理干扰的反应所涉及的因素。 公共卫生相关性：这个项目调查物理干扰对细胞和DNA的影响，这通常会影响人类健康，如何与细胞核内DNA的三维组织有关。了解基因组组织如何影响DNA断裂后可能发生的染色体重排，将有助于解释通常由这些重排引起或影响的癌症的起源和发展。测量细胞感受到的其他力和压力对基因组组织的影响将提供关于人类组织和器官中的细胞受到的力如何影响其正常发育和功能的信息。

DESCRIPTION (provided by applicant): The three-dimensional (3D) organization of the human genome in the nucleus is important not only for packing 2 meters of DNA inside a 2 micron diameter nucleus, but also for gene regulation and other important biological functions. Taking advantage of the Hi-C technology recently developed to capture chromosome folding and looping throughout the genome within a cell, the proposed research will investigate the relationship between this genome organization and physical disruptions experienced by the cell and DNA. The dramatic physical disruption of a DNA double strand break (DSB) can lead to cancer and other diseases if chromosomal translocations resulting from incorrect break repair disrupt genes or the relationship between genes and regulatory elements. Thus, it is important to understand the factors influencing which translocations are likely to occur after a DSB. Hi-C experiments measuring genome organization in mouse B lymphocytes will be used to determine the extent to which pre-existing proximity of genomic regions correlates with the locations of translocations observed after an induced DSB in the same cells. In cases where a high probability of physical proximity does not explain recurrent translocations, the contribution of other factors to translocation susceptibility will be examined by computationally integrating interaction and translocation data obtained in this research with previously published data on features such as gene expression, protein-DNA interactions, and chromosomal common fragile sites. The influence of the physical properties of the 3D genome structure on the formation of translocations between non-proximal genome regions will then be evaluated by comparing experimentally observed translocations with a biophysical model of the genome derived from principles of polymer physics and experimental Hi-C data. This model will be used to predict how much the physical properties of the genome structure might constrain or enhance the movability of genomic regions and broken ends of DNA in their search for repair partners. These simulations will be followed by direct experimental tests of the deformability of the genome by performing Hi-C experiments on cells subjected to physical forces and constraints. The results of these force experiments will test the biophysical model predictions and enable comparisons between physical deformability and translocation susceptibility. Successfully observing the response of the genome inside cells to physical forces will also provide insight into whether biologically relevant forces in tissues and organs might influence gene expression, cell behavior, and cell fate by direct changes to the chromatin structure. Such insight could contribute to future applications of force in the directed differentiation of cells for tissue engineering purposes. By integrating new experimental data, physical models, and previously published genomic data, this research will contribute to a more complete understanding of the factors involved in the response of the complex 3D genome to physical disruptions in health and disease. PUBLIC HEALTH RELEVANCE: This project investigates how the effects of physical disruptions to cells and DNA, which often affect human health, are related to the three dimensional organization of DNA within the cell nucleus. Understanding how genome organization affects chromosomal rearrangements that can occur after DNA breakage will help explain the origin and progression of cancers that are often caused or affected by these rearrangements. Measuring the effects of other forces and stresses felt by the cell on genome organization will provide information about how forces on cells in human tissues and organs can affect their proper development and function.