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开发用于映射和建模基因组的新实验和计算方法 结构，然后验证这些方法，以针对分子细胞遗传学中的建立基准测试。作为 这些技术出现了，项目4着重于建立研究核模型 体系结构和评估这种建筑在发育，疾病或回应期间如何演变 环境压力。我们将使用分化人类胚胎干细胞的发达系统 （HESC）并诱导多能干细胞（HIPSC）进入心肌细胞和内皮细胞。这个系统 已成功用作编码项目的一部分，以识别标记的动态染色质特征 心血管发育调节剂。最近的研究表明，核结构的差异是 在开发初期建立，但我们对这些差异有多稳定或是否了解 结构相互作用的域可以识别新的调节途径。同样，我们几乎一无所知 疾病期间核建筑的演变是如何演变 疾病发病机理。我们的目标是执行一系列关键的实验，这些实验将由DNase Hi-启用。 C和我们程序中的其他映射方法。在AIM 1中，我们将定义核架构的动力 在幼稚的hESC分化为心肌细胞和内皮细胞中。这项研究利用了 新得出的ELF1细胞系，在最早开发阶段是幼稚的hESC，并追踪了动态 使用批量和单细胞DNase Hi-C进行核架构。我们将研究分化为Primed HESC， 中胚层，心血管祖细胞，明确的心肌细胞和内皮。与 已建立的转录因子和表观遗传网络将确定协调激活的空间簇 并反映了调节心脏发育的基因。在AIM 2中，我们将检验以下假设。 心肌病诱导的核脚手架蛋白层粘连蛋白A/C（LMNA）的突变与 心肌细胞核结构的进化。我们将研究来自LMNA诱导的患者的HIPSC 扩张的心肌病和基因修复的同源控制，以确定LMNA突变是否不利 改变心肌细胞中的核结构。此外，我们将检验以下假设 机械应力加剧了这种混乱的体系结构。在AIM 3中，我们将确定核的变化 由21（唐氏综合症）引起的建筑。唐氏综合症是最常见的原因 先天性心脏病，我们假设额外的染色体21导致引起疾病 核结构的改变。我们将研究hipsc的等源性线，有和没有三体性的散装和三体性。 在单细胞水平上，以确定核构建方式如何受到额外的染色体21的干扰。 获得或丢失的相互作用将确定候选人局部引起先天性心脏病。