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

基本信息

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
项目类别：
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

项目摘要

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 开发用于基因组绘图和建模的新实验和计算方法架构，然后根据分子细胞遗传学的既定基准验证这些方法。这些技术到位后，项目4专注于建立研究核的生物模型架构并评估该架构在发育、疾病或响应过程中如何演变我们将使用我们成熟的分化人类胚胎干细胞系统。 (hESC) 和诱导多能干细胞 (hiPSC) 转化为心肌细胞和内皮细胞。已成功用作 ENCODE 项目的一部分，用于识别标记的动态染色质特征最近的研究表明核结构的差异。在开发早期就已确定，但我们对这些差异的稳定性或是否存在知之甚少同样，我们对结构相互作用的域可以识别新的调控途径几乎一无所知。疾病期间核结构如何演变，或者这种变化是疾病的驱动者还是乘客我们的目标是进行一系列由 DNase Hi- 实现的关键实验。在我们的程序中，C 和其他映射方法将定义核架构的动态。本研究利用了幼稚 hESC 分化为心肌细胞和内皮细胞的过程。新衍生的 ELF1 细胞系是处于发育最早阶段的幼稚 hESC，并跟踪使用批量和单细胞 DNase Hi-C 的核结构我们将研究分化为引发的 hESC，中胚层、心血管祖细胞、定形心肌细胞和内皮细胞的比较。建立的转录因子和表观遗传网络将识别协调激活的空间簇在目标 2 中，我们将检验以下假设：核支架蛋白核纤层蛋白 A/C (LMNA) 中诱发心肌病的突变与我们将研究 LMNA 诱导的患者的 hiPSC。扩张型心肌病和基因修复、同基因对照以确定 LMNA 突变是否不利此外，我们将检验亚致死的假设。机械应力加剧了这种混乱的结构。在目标 3 中，我们将确定核的变化。 21 三体（唐氏综合症）引起的结构是唐氏综合症的最常见原因。先天性心脏病，我们发现额外的 21 号染色体会导致疾病我们将大量研究具有和不具有 21 三体性的 hiPSC 的同基因系。在单细胞水平上确定额外的 21 号染色体如何扰乱核结构。获得或失去的相互作用将确定导致先天性心脏病的候选位点。