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 核内层次相互作用的复杂性要求易于使用和准确的方法 为了映射和建模近距离和远程相互作用。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 能够以高分辨率检测相互作用，以及预测的序列决定符 基因组体系结构的特定方面。在AIM 1中，我们将应用新开发的方法来定义明确 小鼠和人类生物学系统，其中特定基因组区域和染色体的4D结构 我们的验证策略将采用可以识别等位基因的系统以促进 研究二倍体细胞的研究，即来自小鼠间特定交叉的组织和细胞系。基因座之间的相互作用将 在增强子/启动子区域进行测试，而拓扑相关的域（TAD）的相互作用将为 通过比较雌性细胞中的活性和非活性X染色体进行测试。此功能验证 高分辨率DNA-FISH分析将完成方法，以验证特定的相互作用。在AIM 2中， 为了验证我们为产生核结构动态变化的4D观点的方法，我们将测量 在细胞周期和小鼠成肌细胞和胚胎茎（ES）细胞中，单个细胞中的相互作用 分化。通过专注于相对了解这些系统的方面，我们将实现验证 通过将核心的动态变化与其他调节层联系起来。小鼠ESC分析 差异化将利用周围环境X失活的知识，这是核核心的一个过程 在哺乳动物中进行重塑，而骨骼肌细胞的分化相对于它的特征很好 转录调节。在AIM 3中，我们将验证我们预测基因组序列的能力 架构。特别是，我们将对定义的基因组区域进行受控操纵，要么由等位基因 - 特定的杂合CRISPR/cas9靶向以产生具有工程删除的细胞 启动子/增强子相互作用区域以及TAD之间的区域，或通过XIST介导的完整沉默 常染色体。在该项目的所有目标中进行的生物验证工作将有助于进步 优化批量和单细胞DNase HI-C协议（项目1）以及建模的新方法 4D核心（项目2），同时也为生物模型开发和数据生成覆盖了道路 （项目4）。