Center for 3D Structure and Physics of the Genome

项目总结-生物学确认 来自组件2和3的参考相互作用图的有效性，其主要依赖于基于连接的 邻近作图，将通过独立的方法测试基因组拓扑结构及其动态进行评估 在细胞周期和细胞分化中。此外，拓扑特征和其他特征之间的相关性也得到了进一步的研究。 过程（例如，局部转录速率、组蛋白修饰等）将暗示这些功能的功能角色 需要进行评估。我们将使用强大的成像技术来测试和进一步阐述基因组 我们细胞系统中的结构和动力学。此外，特定元素的直接扰动 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的工具和试剂，并对选定的小规模 深度上的TAD数量。将选择来自分化中的hESC和来自分裂中的成纤维细胞的TADs， 这些分析基于它们的动态拓扑行为和嵌入基因的存在， 动态表达模式通过验证和扰动人类胚胎干细胞和 成纤维细胞，我们将能够比较和对比这些细胞行为的相似性和差异性， 系统.这些实验应该有助于定义基本的语法基础的形成，维护 和拓扑相互作用的分解。 在目标1中，我们将建立克隆的hESC和成纤维细胞“成像”系，其来源于相同的系， 正在由联盟绘制和分析，其中包含集成成像工具（例如核酸酶死亡的Cas9 [dCas 9]变体与荧光蛋白连接）。这些细胞系将用于（i）对动态TAD进行成像 使用多种独立的方法来测试它们的可见行为是否与结构一致， 从测序方法推断的转换，和（ii）测量TAD特异性转录 动态拓扑行为的后果，以及动态拓扑行为的后果 转录行为在目标2中，我们将在边界和内部拓扑中设计突变， 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细胞系的作用。 在形成或维持基因组拓扑结构中改变序列。在Aim 3中，我们将使用融合到 组蛋白修饰酶来定义“写入”或“擦除”特异性的拓扑和功能效应 单个TAD内和周围的染色质标记。此外，我们还将探讨创建 通过dCas 9相互作用结构域生成人工环相互作用，获得新的拓扑特征。 通过这些研究生成的数据集将允许更准确地参数化 用于定量表示和解释参考相互作用图的计算模型，以及 提供关键的洞察力的生物功能的拓扑特征包含在地图。

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.