University of Washington Center for Nuclear Organization and Function

 描述：基因组学目前的一个重大挑战是在所有相关的尺度上准确地分析体内DNA的3D构象，然后将构象变化与细胞周期、分化和疾病等动态过程联系起来。在这里，我们建议创建华盛顿大学核组织和功能中心，将一个跨学科的研究团队聚集在一起，他们的不同专业领域--技术开发、计算建模以及老鼠和人类生物学--使他们非常适合这一挑战。我们的总体假设是，表征和理解基因组结构随时间的变化(4D核组)将导致对人类生物学和疾病的基本见解。我们将通过开发实验和计算方法的组合开发，以及它们的系统生物学验证和应用于与发育和疾病相关的系统来解决这一假设。在实验方面，我们将进一步优化我们最近开发的DNase Hi-C分析，包括单细胞的组合方法，最终目标是同时分析多个单细胞中的每个细胞的核结构和基因表达。在计算方面，我们将扩展我们现有的3D建模算法，以考虑二倍体、细胞到细胞的可变性、基因组结构的分层性质，并显式地模拟细胞周期和细胞分化时间尺度上的结构变化。然后，我们将使用几种互补的计算方法将我们的4D基因组模型与现有的1D基因组数据集联系起来。这些新的实验和计算技术的输出将在几个众所周知的模型系统中进行正交验证：人类细胞系、种间杂交小鼠的体内组织、小鼠胚胎干细胞(ESCs)和骨骼肌母细胞。我们还将测试模型对定向(基因组编辑)或大规模(染色体沉默)扰动的特定预测。在初步验证之后，随着进一步的方法开发，我们将把我们的新工具应用于三个生物系统的分析：我们将表征原始的人ESCs定向分化为心肌细胞和内皮细胞过程中核结构的动态变化；我们将测试核支架蛋白lamin A中导致心肌病的突变与心肌细胞核结构紊乱相关的假设；以及我们将确定21三体引起的人心肌细胞核结构的变化。拟议的中心将产生新的实验方案，用于确定4D基因组结构，两个新的软件工具包，用于建模4D基因组并将基因组的特征与其他类型的基因组数据联系起来，各种公开可用的大规模4D基因组数据集，以及对人类生物学和疾病的基本见解。在所有这些工作中，我们将与NOFIC和4DN网络密切而公开地合作，以最大限度地发挥我们中心和整个项目的影响。

 DESCRIPTION: A current grand challenge in genomics involves accurately assaying, at all relevant scales, the 3D conformation of DNA in vivo and then linking conformational changes to dynamic processes such as the cell cycle, differentiation and disease. Here we propose to create the University of Washington Center for Nuclear Organization and Function, bringing together an interdisciplinary team of investigators whose diverse areas of expertise - technology development, computational modeling, and mouse and human biology - make them ideally suited to this challenge. Our overall hypothesis is that characterizing and understanding changes in genome architecture over time (the 4D nucleome) will lead to fundamental insights into human biology and disease. We will address this hypothesis by developing a combination of experimental and computational methods development, coupled with their systematic biological validation and application to development- and disease-relevant systems. On the experimental side, we will further optimize our recently developed DNase Hi- C assay, including combinatorial methods for single cells, ultimately aiming to concurrently assay nuclear architecture and gene expression within each of many single cells. On the computational side, we will extend our existing 3D modeling algorithms to account for diploidy, cell-to-cell variabilit, the hierarchical nature of genome architecture, and to explicitly model architectural changes over cell cycle and cell differentiation time scales. We will then employ several complementary computational methods to link our 4D nucleome models to existing, 1D genomics data sets. The outputs of these new experimental and computational technologies will be subjected to orthogonal validation in several well-understood model systems: human cell lines, in vivo tissues from interspecific F1 hybrid mice, mouse embryonic stem cells (ESCs) and skeletal myoblasts. We will also test specific predictions of the models in response to targeted (genome editing) or large-scale (chromosome silencing) perturbations. After initial validation and in parallel with further methods development, we will apply our new tools to the analysis of three biological systems: we will characterize the dynamics of nuclear architecture during the directed differentiation of naïve human ESCs into cardiomyocytes and endothelial cells; we will test the hypothesis that cardiomyopathy-inducing mutations in the nuclear scaffolding protein, lamin A, are associated with derangements in cardiomyocyte nuclear architecture; and we will determine the changes in human cardiomyocyte nuclear architecture induced by trisomy 21. The proposed center will produce new experimental protocols for ascertaining 4D nucleome architecture, two new software toolkits for modeling the 4D nucleome and linking features of the nucleome to other types of genomic data, a variety of publicly available, large-scale 4D nucleome data sets in mouse and human systems, and fundamental insights into human biology and disease. In all of this work, we will work closely and openly with NOFIC and the 4DN Network to maximize the impact of our center and the overall program.