Center for 3D Structure and Physics of the Genome

基因组 3D 结构和物理中心

基本信息

批准号：
9021492
负责人：
ERIK J. SONTHEIMER
金额：
$ 75.66万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
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/9021492
关键词：
Behavior Beryllium Biological Biological Process Cell Cycle Cell Differentiation process Cell Line Cell physiology Cells Chromatin Chromatin Structure Chromosome Structures Computer Simulation Data Data Analyses Data Set Development Elements Engineering Enzymes Epigenetic Process Fibroblasts Fluorescence Microscopy Generations Genes Genetic Transcription Genome Genomics Goals Higher Order Chromatin Structure Histones Human Image Imaging Device Imaging Techniques In Situ Indium Individual Lead Life Ligation MS2 coat protein Maintenance Maps Measures Methods Microscopy Mining Modeling Mutation Output Pattern Physics Play Process Proteins Reagent Regulation Reporter Reporter Genes Repression Resolution Role Shapes Site Structure System Techniques Testing Transcript Transplantation Untranslated Regions Validation Variant Writing base data modeling genome editing histone modification human embryonic stem cell insight molecular imaging nuclease photoactivation physical model predictive modeling promoter research study stem technology development three dimensional structure tool transcriptome sequencing

项目摘要

Project Summary – Biological Validation The validity of the Reference Interaction Map from Components 2 and 3, which rely primarily on ligation-based proximity mapping, will be assessed through independent methods testing genomic topology and its dynamics during cell cycle and cell differentiation. In addition, the correlations between topological features and other processes (e.g. local transcription rate, histone modifications, etc.) will imply functional roles for these features that need to be evaluated. We will use powerful imaging techniques to test and further elaborate the genomic structure and dynamics within our cellular systems. In addition, the direct perturbation of elements of specific topological features will define their biological roles in the cellular processes under study. To pursue these goals, we will develop a core set of tools and reagents and characterize a selected small number of TADs in depth. TADs from differentiating hESCs and from dividing fibroblasts will be selected for these analyses based on their dynamic topological behaviors and on the presence of embedded genes with dynamic expression patterns. By validating and perturbing topological features in both human ESCs and fibroblasts, we will be able to compare and contrast the similarities and differences in the behavior of these systems. These experiments should help define the basic grammar that underlies the formation, maintenance and dissolution of topological interactions. In Aim 1, we will establish clonal hESC and fibroblast “imaging” lines, derived from the same lines that are being mapped and analyzed by the consortium, that harbor integrated imaging tools (e.g. nuclease-dead Cas9 [dCas9] variants tethered to fluorescent proteins). These cell lines will be used to (i) image dynamic TADs using multiple, independent methods to test whether their visible behavior is consistent with the structures and transitions inferred from sequencing approaches, and to (ii) measure the TAD-specific transcriptional consequences of dynamic topological behavior, as well as the topological consequences of dynamic transcriptional behavior. In Aim 2, we will engineer mutations in TAD boundaries and intra-TAD topological elements within these imaging cell lines, and use both imaging and molecular analyses to test the roles of the altered sequences in forming or maintaining genome topology. In Aim 3, we will use dCas9 variants fused to histone modification enzymes to define the topological and functional effects of “writing” or “erasing” specific chromatin marks within and around individual TADs. In addition we will probe the requirements for creating new topological features through the generation of artificial looping interactions via dCas9-interaction domains. The dataset generated through these studies will allow the more accurate parameterization of the computational models used to quantitatively represent and interpret the Reference Interaction Map as well as provide critical insights into the biological functions of the topological features contained within the map.

项目摘要 – 生物学验证来自组件 2 和 3 的参考相互作用图的有效性主要依赖于基于连接的邻近映射，将通过测试基因组拓扑及其动态的独立方法进行评估此外，细胞周期和细胞分化之间的拓扑特征和其他相关性。过程（例如局部转录率、组蛋白修饰等）将暗示这些特征的功能作用我们将使用强大的成像技术来测试和进一步阐述基因组。此外，我们的细胞系统内的结构和动力学。拓扑特征将定义它们在所研究的细胞过程中的生物学作用。为了实现这些目标，我们将开发一套核心工具和试剂，并表征选定的小分子将选择来自分化 hESC 和分裂成纤维细胞的 TAD 数量。这些分析基于它们的动态拓扑行为和嵌入基因的存在通过验证和扰动人类 ESC 和成纤维细胞，我们将能够比较和对比这些行为的异同这些实验应该有助于定义构成和维护系统的基本语法。和拓扑相互作用的消解。在目标 1 中，我们将建立克隆 hESC 和成纤维细胞“成像”系，这些系源自与正在由该联盟进行绘图和分析，其中包含集成成像工具（例如核酸酶死亡的 Cas9 [dCas9] 变体与荧光蛋白相连）。这些细胞系将用于 (i) 动态 TAD 成像。使用多种独立的方法来测试它们的可见行为是否与结构和结构一致从测序方法推断的转换，以及 (ii) 测量 TAD 特异性转录动态拓扑行为的后果，以及动态拓扑后果在目标 2 中，我们将设计 TAD 边界和 TAD 内拓扑的突变。这些成像细胞系中的元素，并使用成像和分子分析来测试这些成像细胞系的作用在目标 3 中，我们将使用融合的 dCas9 变体来形成或维持基因组拓扑。组蛋白修饰酶来定义“写入”或“擦除”特定的拓扑和功能效应此外，我们将探讨创建单个 TAD 内部和周围的染色质标记。通过 dCas9 交互域生成人工循环交互来实现新的拓扑特征。通过这些研究生成的数据集将允许更准确的参数化用于定量表示和解释参考交互图的计算模型以及提供对地图中拓扑特征的生物学功能的重要见解。