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

摘要-项目4：UW-CNOF生物模型开发和数据生成 项目1-3开发了新的实验和计算方法，用于基因组作图和建模 架构，然后验证这些方法对分子细胞遗传学中建立的基准。作为 这些技术到位后，项目4的重点是建立研究核武器的生物模型， 结构，并评估这种结构在发育、疾病或对疾病的反应中如何演变。 环境压力我们将使用我们成熟的分化人类胚胎干细胞的系统 在一些实施方案中，本发明的方法包括将人胚胎干细胞（hESC）和诱导多能干细胞（hiPSC）转化为心肌细胞和内皮细胞。该系统 已成功地用作ENCODE项目的一部分，以识别标记 心血管发育调节剂。最近的研究表明，核结构的差异是 这些差异在发育的早期就已经建立，但我们对这些差异的稳定性以及它们是否 结构上相互作用的结构域可以识别新的调节途径。同样地，我们对 核结构在疾病期间如何演变，或者这种变化是疾病的驱动者还是乘客， 发病机理我们的目标是进行一系列关键的实验，这些实验将由DNase Hi- C和其他映射方法。在目标1中，我们将定义核结构的动力学 在幼稚hESC分化为心肌细胞和内皮细胞的过程中。这项研究利用了 新衍生的ELF 1细胞系，一个幼稚的hESC在发展的最早阶段，并跟踪动态， 使用本体和单细胞DNA酶Hi-C的核结构。我们将研究分化成致敏的hESC， 中胚层、心血管祖细胞和定形心肌细胞和内皮细胞。相比 已建立的转录因子和表观遗传网络将识别协同激活的 以及抑制心脏发育的基因在目标2中，我们将检验以下假设： 核支架蛋白核纤层蛋白A/C（LMNA）的心肌病诱导突变与 心肌细胞核结构紊乱。我们将研究来自LMNA诱导的HiPSC患者的hiPSC。 扩张型心肌病和基因修复的同基因对照，以确定LMNA突变是否不利 改变心肌细胞的核结构。此外，我们将测试亚致死性 机械应力加剧了这种混乱的结构。在目标3中，我们将确定核武器的变化， 由21三体（唐氏综合征）诱导的结构。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21号染色体导致致病 核结构的变化。我们将大量研究具有和不具有21三体的hiPSC的等基因系， 在单细胞水平，以确定核结构是如何被额外的21号染色体干扰的。 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座。

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.