University of Washington Center for Nuclear Organization and Function

华盛顿大学核组织与功能中心(UW-CNOF) 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3D DNA在体内的构象，然后将构象变化与细胞等动态过程联系起来 周期、分化和疾病。在这里，我们建议创建华盛顿大学核能中心 组织和职能，汇聚了一个跨学科的调查团队，他们的不同领域 专业知识--技术开发、计算建模、老鼠和人类生物学--使它们 非常适合这项挑战。我们的总体假设是，刻画和理解 随着时间的推移，基因组结构(4D基因组)将导致对人类生物学和 疾病。我们将通过开发实验和计算相结合的方法来解决这一假设 方法开发，加上它们的系统生物学验证和应用于开发--和 与疾病相关的系统。在实验方面，我们将进一步优化我们最近开发的DNA酶Hi- C检测，包括单细胞的组合方法，最终目标是同时检测核 许多单个细胞中的每一个细胞的结构和基因表达。在计算方面，我们将扩展 我们现有的3D建模算法考虑了二倍体、细胞间的可变性、细胞的层次性 基因组结构，并显式模拟细胞周期和细胞分化时间的结构变化 比例。然后，我们将使用几种互补的计算方法将我们的4D核基因组模型连接到 现有的一维基因组数据集。这些新的实验和计算技术的成果将 在几个众所周知的模型系统中进行正交验证：人体细胞系，活体 来自种间杂交F1小鼠、小鼠胚胎干细胞(ESCs)和骨骼肌母细胞的组织。我们会 也测试模型的特定预测，以响应定向(基因组编辑)或大规模 (染色体沉默)扰动。在初始验证之后并且与进一步的方法开发并行， 我们将应用我们的新工具来分析三个生物系统：我们将表征 人胚胎干细胞定向分化为心肌细胞过程中的核构筑 内皮细胞；我们将测试心肌病在核支架中诱导突变的假设 蛋白质，层蛋白A，与心肌细胞核结构的紊乱有关；我们将 检测21三体对人心肌细胞核结构的影响。建议数 该中心将制定新的实验方案，以确定4D核组结构，两个新软件 用于模拟4D核组并将核组的特征与其他类型的基因组数据相关联的工具包，a 在老鼠和人类系统中各种公开可用的大规模4D核基因组数据集，以及 对人类生物学和疾病的基本见解。在所有这些工作中，我们将密切和公开地与 NOFIC和4DN网络，以最大限度地发挥我们中心和整体方案的影响。

ABSTRACT – UNIVERSITY OF WASHINGTON CENTER FOR NUCLEAR ORGANIZATION AND FUNCTION (UW-CNOF) 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 variability, 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.