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 生物验证开发 细胞核内层次相互作用的复杂性要求易于使用且准确的方法 为了绘制和建模，需要开发近距离和远程交互。在这个项目中， 项目 1 和 2 中开发的实验和计算方法将进行测试，以评估其效果 能够以高分辨率检测相互作用，以及预测序列决定因素 基因组结构的具体方面。在目标 1 中，我们将应用新开发的方法来明确定义 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 可以预见。我们的验证策略将采用可以识别等位基因的系统，以促进 二倍体细胞的研究，即来自小鼠种间杂交的组织和细胞系。位点之间的相互作用将 在增强子/启动子区域进行测试，而拓扑相关域（TAD）的相互作用将 通过比较女性细胞中活跃和不活跃的 X 染色体进行测试。本次功能验证 该方法将得到高分辨率 DNA-FISH 分析的补充，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 细胞周期期间以及小鼠成肌细胞和胚胎干 (ES) 细胞期间单细胞的相互作用 差异化。通过关注这些系统相对容易理解的方面，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，X失活是核的核心过程 哺乳动物的重塑，而骨骼肌成肌细胞分化的特征在于其 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因素的能力 建筑学。具体来说，我们将通过等位基因对定义的基因组区域进行受控操作 特异性杂合 CRISPR/Cas9 靶向生成具有工程缺失的细胞 启动子/增强子相互作用区域和 TAD 之间的区域，或通过 Xist 介导的完全沉默 常染色体。在该项目的所有目标中进行的生物验证工作将促进进步 批量和单细胞 DNase Hi-C 方案的优化（项目 1）以及建模的新方法 4D 核组（项目 2），同时也为生物模型开发和数据生成铺平道路 （项目 4）。