Project 3: UW-CNOF Biological Validation Development

项目 3：UW-CNOF 生物验证开发

• 批准号: 9021414
• 负责人: Christine M. Disteche
• 金额: $ 49.94万
• 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/9021414
• 关键词: Address; Affect; Alleles; Architecture; Biological; Biological Assay; Biological Models; Cell Cycle; Cell Line; Cell Nucleus; Cell physiology; Cells; Chromatin; Chromosomes; Clone Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Computing Methodologies; DNA; Data; Data Analyses; Deoxyribonucleases; Detection; Development; Diploid Cells; Effectiveness; Embryo; Engineering; Enhancers; Epigenetic Process; Female; Gene Expression; Generations; Genes; Genetic Polymorphism; Genome; Genomic Segment; Guide RNA; Haploidy; Human; In Vitro; K-562; Knowledge; Link; Mammals; Maps; Measures; Mediating; Mediator of activation protein; Methods; Modeling; Molecular Conformation; Mus; Myoblasts; Nuclear; Nuclear Structure; Population; Process; Promoter Regions; Protocols documentation; Publishing; RNA; Regulation; Resolution; Site; Skeletal Myoblasts; Staging; Structure; System; Testing; Tissues; Transcriptional Regulation; Universities; Validation; Washington; Work; X Chromosome; X Inactivation; abstracting; autosome; base; biological systems; cell type; cohesin; combinatorial; data modeling; design; embryonic stem cell; genome editing; imprint; in vivo; internal control; model development; novel strategies; promoter; stem; stem cell differentiation; technology development; three dimensional structure

ABSTRACT – PROJECT 3: UW-CNOF BIOLOGICAL VALIDATION DEVELOPMENT The complexity of hierarchical interactions within the nucleus demands that easy-to-use and accurate methods for mapping and modeling both close-range and long-range interactions be developed. In this project, the experimental and computational methods developed in Projects 1 and 2 will be put to the test to evaluate their ability to detect interactions at high resolution, as well as for the prediction of the sequence determinants of specific aspects of genome architecture. In Aim 1, we will apply newly developed methods to well-defined mouse and human biological systems in which the 4D structure of specific genomic regions and chromosomes can be anticipated. Our validation strategy will employ systems in which alleles can be identified to facilitate studies of diploid cells, i.e. tissues and cell lines from a mouse interspecific cross. Interactions between loci will be tested at enhancer/promoter regions, while interactions at topologically associated domains (TADs) will be tested by comparing the active and inactive X chromosomes in female cells. This functional validation approach will be complemented by high resolution DNA-FISH analyses to verify specific interactions. In Aim 2, to validate our approaches for generating a 4D view of dynamic changes in nuclear structure, we will measure interactions in single cells during the cell cycle and during mouse myoblast and embryonic stem (ES) cell differentiation. By focusing on relatively well understood aspects of these systems, we will achieve validation by linking dynamic changes in the nucleome to other layers of regulation. Analysis of mouse ESC differentiation will exploit the wealth of knowledge that surrounds X inactivation, a process central to nuclear remodeling in mammals, while skeletal myoblast differentiation is well characterized with respect to its transcriptional regulation. In Aim 3, we will validate our ability to predict the sequence determinants of genome architecture. Specifically, we will perform controlled manipulations of defined genomic regions, either by allele- specific heterozygous CRISPR/Cas9 targeting to generate cells with engineered deletions at promoter/enhancer interacting regions and at regions between TADs, or by Xist-mediated silencing of full autosomes. The biological validation work performed in all aims of this project will facilitate the progressive optimization of both bulk and single cell DNase Hi-C protocols (Project 1) and new approaches to modeling the 4D nucleome (Project 2), while also paving the way for biological model development and data generation (Project 4).

摘要-项目3：UW-CNOF生物学验证开发 核内层次交互的复杂性要求易于使用和准确的方法 为绘制近距离和远距离相互作用的地图和建立模型，应开发。本工程 在项目1和2中开发的实验和计算方法将进行测试，以评估其 能够高分辨率检测相互作用，以及预测序列决定因素 基因组结构的特定方面。在目标1中，我们将应用新开发的方法来定义良好的 小鼠和人类生物系统，其中特定基因组区域和染色体的4D结构 可以预期。我们的验证策略将采用可以识别等位基因的系统， 二倍体细胞的研究，即来自小鼠种间杂交的组织和细胞系。基因座之间的相互作用将 在增强子/启动子区域进行测试，而在拓扑相关结构域（TAD）的相互作用将被测试。 通过比较女性细胞中活跃和不活跃的X染色体进行测试。此功能验证 高分辨率DNA-FISH分析将补充这种方法，以验证特定的相互作用。在目标2中， 为了验证我们生成核结构动态变化4D视图的方法，我们将测量 在细胞周期和小鼠成肌细胞和胚胎干细胞（ES） 分化通过关注这些系统中相对较好理解的方面，我们将实现验证 通过将核组的动态变化与其他调控层联系起来。小鼠ESC分析 分化将利用围绕X失活的丰富知识，这是一个核心过程， 哺乳动物的重塑，而骨骼成肌细胞的分化在其方面得到了很好的表征 转录调控在目标3中，我们将验证我们预测基因组序列决定簇的能力 架构具体地说，我们将对定义的基因组区域进行受控操作，或者通过等位基因- 特异性杂合CRISPR/Cas9靶向产生具有工程化缺失的细胞， 启动子/增强子相互作用区域和TADs之间的区域，或通过Xist介导的完全沉默， 常染色体在本项目的所有目标中进行的生物学验证工作将促进 优化散装和单细胞DNase Hi-C协议（项目1）和新的方法来建模 4D核组（项目2），同时也为生物模型开发和数据生成铺平了道路 （项目4）。