A toolkit for visualizing and manipulating chromosomal interactions in living cells.

用于可视化和操纵活细胞中染色体相互作用的工具包。

• 批准号: 9168109
• 负责人: Orion D Weiner
• 金额: $ 23.78万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9168109
• 关键词: 3-Dimensional; Address; Alleles; Bacteriophages; Biological Assay; CRISPR/Cas technology; Cells; Chimeric Proteins; Chromosome Structures; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Color; Communities; Coupling; DNA; Disease; Distal; Elements; Enhancers; Fluorescent in Situ Hybridization; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Genetic Enhancer Element; Genetic Recombination; Genetic Transcription; Genome; Genomics; Heterodimerization; Human; Image; Image Analysis; Imagery; Individual; Label; Life; Link; Location; Logic; Measures; Modeling; Molecular; Monitor; Mus; Nature; Pharmaceutical Preparations; Physiological; Play; Population; Protein Array; Proteins; Protocols documentation; RNA; Reading; Real-Time Systems; Regulation; Reporter; Role; Specimen; Structure; System; Techniques; Testing; Time; Tissues; Transcriptional Regulation; base; biomedical resource; chromosome conformation capture; combinatorial; embryonic stem cell; gene interaction; genome editing; imaging platform; interest; mouse genome; pluripotency; portability; promoter; protein expression; protein protein interaction; temporal measurement; three dimensional structure; tool; transcription factor; 3-Dimensional; Address; Alleles; Bacteriophages; Biological Assay; CRISPR/Cas technology; Cells; Chimeric Proteins; Chromosome Structures; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Color; Communities; Coupling; DNA; Disease; Distal; Elements; Enhancers; Fluorescent in Situ Hybridization; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Genetic Enhancer Element; Genetic Recombination; Genetic Transcription; Genome; Genomics; Heterodimerization; Human; Image; Image Analysis; Imagery; Individual; Label; Life; Link; Location; Logic; Measures; Modeling; Molecular; Monitor; Mus; Nature; Pharmaceutical Preparations; Physiological; Play; Population; Protein Array; Proteins; Protocols documentation; RNA; Reading; Real-Time Systems; Regulation; Reporter; Role; Specimen; Structure; System; Techniques; Testing; Time; Tissues; Transcriptional Regulation; base; biomedical resource; chromosome conformation capture; combinatorial; embryonic stem cell; gene interaction; genome editing; imaging platform; interest; mouse genome; pluripotency; portability; promoter; protein expression; protein protein interaction; temporal measurement; three dimensional structure; tool; transcription factor

PROJECT SUMMARY/ABSTRACT Eukaryotic genome structure fluctuates across both 3-dimensional space and time and must be precisely orchestrated to achieve regulated gene expression. This structure underlies regulatory interactions between distal control elements (i.e. enhancers) and the genes that they target. While many thousands of enhancers have now been annotated and their interacting genic partners identified, we have a poor understanding of the molecular nature of these interactions, their regulation, and their significance in the cell. Chromosome conformation capture (3C) techniques have accelerated study of chromosomal organization but provide only population-averaged snapshots with poor temporal resolution and so fail to describe chromosomal interactions in their relevant context. Elucidating the molecular basis of enhancer-gene interactions in living, single cells is paramount to understanding transcriptional control. To address this need we are building new tools to visualize and manipulate chromosomal interactions in living cells. This proposal aims to dissect enhancer function by probing the dynamics and control of a model chromosomal interaction: the Sox2 gene and its distal, essential Sox2 Control Region (SCR). Sox2 encodes a tissue-specific transcription factor (TF) involved in pluripotency and reprogramming, making its transcriptional control of broad interest. In mouse embryonic stem cells (ESCs), Sox2 expression is established through the function of the SCR, located >100 kb away. To probe the dynamics of Sox2's interaction with its enhancer in living ESCs, we are combining techniques for marking DNA with advances in genome editing. To determine how these dynamics inform gene expression, we are developing tools to acutely and specifically manipulate the stability of the Sox2/SCR interaction and assay the consequences on Sox2 gene expression in individual living cells. These studies should broadly inform our understanding of transcriptional control by enhancer elements and should be generalizable to a wide range of other genomic contexts.

项目摘要/摘要 真核基因组结构在三维空间和时间上都波动，必须精确 精心策划以实现受调节的基因表达。这种结构是调节性相互作用的基础 远端控制元素（即增强子）及其靶向的基因。而成千上万的增强剂 现在已经被注释了，他们的相互作用的基因伴侣确定了，我们对 这些相互作用的分子性质，它们的调节及其在细胞中的意义。染色体 构象捕获（3C）技术已经加速了染色体组织的研究，但仅提供 人口平均的快照，时间分辨率差，因此无法描述染色体相互作用 在他们的相关背景下。阐明在活着的单个细胞中增强子基因相互作用的分子基础 了解转录控制的至高无上。为了满足这一需求，我们正在构建新工具来可视化 并操纵活细胞中的染色体相互作用。 该建议旨在通过探测模型的动力学和控制来剖析增强子功能 染色体相互作用：SOX2基因及其远端Sox2控制区（SCR）。 Sox2编码a 涉及多能性和重编程的组织特异性转录因子（TF），使其转录 控制广泛的兴趣。在小鼠胚胎干细胞（ESC）中，SOX2表达是通过 SCR的功能，位于> 100 kb之外。探测Sox2与增强器相互作用的动态 生活ESC，我们正在将标记DNA的技术与基因组编辑的进步相结合。确定 这些动力学如何使基因表达信息，我们正在开发敏锐，专门操纵的工具 SOX2/SCR相互作用的稳定性，并分析对个体中SOX2基因表达的后果 活细胞。这些研究应该广泛地告知我们通过增强器对转录控制的理解 元素，应推广到各种其他基因组环境。