Project 4: UW-CNOF Biological Model Development and Data Generation

项目 4：UW-CNOF 生物模型开发和数据生成

• 批准号: 9021415
• 负责人: Charles E Murry
• 金额: $ 51.16万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9021415
• 关键词: Address; Affect; Alleles; Architecture; Benchmarking; Biological Models; Breathing; Cardiac; Cardiac Myocytes; Cardiac development; Cardiomyopathies; Cardiovascular system; Cell Line; Cell Nucleus; Cells; Cessation of life; Chromatin; Chromosomes; Chromosomes, Human, Pair 21; Clustered Regularly Interspaced Short Palindromic Repeats; Computing Methodologies; Cues; Data; Deoxyribonucleases; Development; Dilated Cardiomyopathy; Disease; Disease model; Down Syndrome; Endothelial Cells; Endothelium; Epigenetic Process; Functional disorder; Generations; Genes; Genome; Germ Layers; Goals; Heart; Hereditary Disease; Human; Human Development; Human Engineering; Induced Mutation; Lamin Type A; Maps; Mechanical Stress; Mesoderm; Methods; Modeling; Molecular Cytogenetics; Mus; Mutation; Nuclear; Nuclear Envelope; Nuclear Matrix-Associated Proteins; Nuclear Structure; Pathogenesis; Patients; Phenotype; Pluripotent Stem Cells; Regulatory Pathway; Role; Series; Staging; Stress; System; Techniques; Technology; Testing; Tissues; Universities; Ventricular Septal Defects; Washington; abstracting; atrioventricular septal defect; base; cardiogenesis; cell type; congenital heart disorder; developmental disease; human disease; human embryonic stem cell; in vivo; induced pluripotent stem cell; insight; member; model development; progenitor; programs; repaired; research study; response; tool; transcription factor

ABSTRACT – PROJECT 4: UW-CNOF BIOLOGICAL MODEL DEVELOPMENT AND DATA GENERATION Projects 1-3 develop new experimental and computational methods for mapping and modeling genome architecture and then validate these methods against established benchmarks in molecular cytogenetics. As these technologies come into place, Project 4 focuses on establishing biological models for studying nuclear architecture and assessing how this architecture evolves during development, disease, or in response to environmental stress. We will use our well-developed system of differentiating human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) into cardiomyocytes and endothelial cells. This system has been used successfully as part of the ENCODE project to identify dynamic chromatin signatures that mark cardiovascular developmental regulators. Recent studies suggest that differences in nuclear architecture are established early in development, but we know little about how stable these differences are or whether structurally interacting domains can identify new regulatory pathways. Similarly, we know almost nothing about how nuclear architecture evolves during disease or whether such changes are drivers or passengers in disease pathogenesis. Our goals are to perform a key series of experiments that will be enabled by DNase Hi- C and other mapping approaches in our program. In Aim 1, we will define the dynamics of nuclear architecture during the differentiation of naïve hESCs into cardiomyocytes and endothelial cells. This study utilizes the newly derived ELF1 cell line, a naïve hESC at the earliest stage of development, and tracks the dynamics of nuclear architecture using bulk and single-cell DNase Hi-C. We will study differentiation into primed hESCs, mesoderm, cardiovascular progenitors, and definitive cardiomyocytes and endothelium. Comparison with established transcription factor and epigenetic networks will identify spatial clusters of coordinately activated and repressed genes that regulate heart development. In Aim 2, we will test the hypothesis that cardiomyopathy-inducing mutations in the nuclear scaffolding protein, lamin A/C (LMNA), are associated with derangements in cardiomyocyte nuclear architecture. We will study hiPSCs from patients with LMNA-induced dilated cardiomyopathy and genetically repaired, isogenic controls to determine if LMNA mutations unfavorably change nuclear architecture in cardiomyocytes. Additionally, we will test the hypothesis that sub-lethal mechanical stress exacerbates this deranged architecture. In Aim 3, we will determine the changes in nuclear architecture induced by trisomy 21 (Down Syndrome). Down Syndrome is the most common cause of congenital heart disease, and we hypothesize that the additional chromosome 21 results in disease-causing alterations in nuclear structure. We will study isogenic lines of hiPSCs with and without trisomy 21 in bulk and at the single-cell level to determine how nuclear architecture is perturbed by an additional chromosome 21. Interactions that are gained or lost will identify candidate loci for causing congenital heart disease.

摘要-项目4：UW-CNOF生物模型开发和数据生成 项目1-3开发绘制和模拟基因组图的新的实验和计算方法 架构，然后对照分子细胞遗传学中已建立的基准来验证这些方法。AS 这些技术已经到位，项目4的重点是建立研究核的生物模型 体系结构，并评估该体系结构在开发、疾病或响应过程中如何演变 环境压力。我们将使用我们开发的分化人类胚胎干细胞的系统 (HESCs)，并诱导多能干细胞(HiPSCs)分化为心肌细胞和内皮细胞。这个系统 已经被成功地用作ENCODE项目的一部分，以识别标记为 心血管发育调节剂。最近的研究表明，核架构的差异是 建立在早期发展阶段，但我们对这些差异的稳定性或是否 结构上相互作用的结构域可以识别新的调控途径。同样，我们几乎对此一无所知 核结构在疾病期间是如何演变的，或者这种变化是驱动因素还是乘客 疾病发病机制。我们的目标是执行一系列关键的实验，这些实验将由DNase Hi- C和我们程序中的其他映射方法。在目标1中，我们将定义核体系结构的动态 在幼稚胚胎干细胞分化为心肌细胞和内皮细胞的过程中。这项研究利用了 新获得的Elf1细胞系，一个幼稚的hESC在最早的发展阶段，并跟踪动态 核结构，采用块状和单细胞DNase Hi-C。我们将研究分化为预置的HESCs， 中胚层，心血管祖细胞，以及明确的心肌细胞和内皮细胞。与 已建立的转录因子和表观遗传网络将识别协同激活的空间簇 并抑制了调节心脏发育的基因。在目标2中，我们将检验假设 核支架蛋白LMNA(LMNA)的突变与心肌病有关 心肌细胞核结构紊乱。我们将研究LMNA诱导的患者的HiPSCs 扩张型心肌病和基因修复的等基因对照以确定LMNA突变是否不利 改变心肌细胞的核结构。此外，我们将测试亚致命性的假设 机械压力加剧了这种错乱的建筑。在目标3中，我们将确定核变化 21三体引起的建筑(唐氏综合征)。唐氏综合症是导致 先天性心脏病，我们假设额外的21号染色体导致疾病 核结构的改变。我们将大量研究带有和不带有21三体的hPSCs等基因系和 在单细胞水平上，以确定额外的21号染色体是如何干扰核结构的。 获得或丢失的相互作用将确定导致先天性心脏病的候选基因。