Engineering and visualizing genome folding at high spatiotemporal resolution

以高时空分辨率对基因组折叠进行工程设计和可视化

• 批准号: 9762161
• 负责人: Gerd A Blobel
• 金额: $ 71.78万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-15 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9762161
• 关键词: 3-Dimensional; ATAC-seq; Address; Affect; Architecture; Biological Models; Biology; Cell Communication; Cells; Chemicals; Chromatids; Chromatin; Chromatin Loop; Chromatin Structure; Chromosomes; Color; Communities; DNA; DNA-Binding Proteins; Diffuse; Dimerization; Dissociation; Engineering; Enhancers; Epigenetic Process; Erythroid Cells; Foundations; Frequencies; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Structure; Genes; Genetic Transcription; Genome; Heterodimerization; Higher Order Chromatin Structure; Image; Imaging Device; Individual; Interphase; Kinetics; Knowledge; Label; Length; Ligands; Light; Link; Location; Maps; Measures; Mediating; Memory; Methodology; Modeling; Molecular Conformation; Monitor; Nuclear; Pattern; Physiologic pulse; Plant Roots; Population; Process; Proteins; RNA; Reagent; Regulation; Resolution; Shapes; Site; Specific qualifier value; Structural Genes; Structure; System; Technology; Testing; Time; Tissues; Transcript; Transcription Elongation; Transcription Initiation; Transcriptional Activation; Visualization software; Work; Zinc Fingers; beta Globin; cell type; design; embryonic stem cell; experimental study; fluorophore; gene repression; high resolution imaging; insight; interest; molecular imaging; promoter; public health relevance; response; single molecule; spatiotemporal; tool; transcriptome sequencing

 DESCRIPTION (provided by applicant): Critical unanswered questions in the field of genome biology are how the dynamics of chromatin folding shape gene expression patterns. Our knowledge of the dynamics of higher-order 3-D folding of chromatin is severely limited, largely due to the lack of technologies to precisely image, engineer and monitor looping in a precise spatiotemporal manner across a population of cells. Here we propose to address these limitations by developing tools to dynamically alter chromatin folding in a synchronous manner across populations of cells as well as individual cells, and measure chromatin looping and its relationship to transcription at high spatial resolution in single cells. In Specific Aim 1 we will design tools to control looping dynamics. We will modify factors that fold chromatin at various levels, such as Ldb1 and CTCF by fusion to a moiety whose stability can be controlled by diffusible ligands. In combination with hi resolution 5C and single molecule imaging these tools are expected to generate fundamental insights into the relationship of nuclear architecture and gene expression mechanisms. In Specific Aim 2 we plan to engineer light-inducible systems for the precise control of looping dynamics. Using light activated dimerization domains that can be used in conjunction with designer DNA binding proteins we attempt to engineer factors used to rapidly promote or disrupt chromatin looping at various scales. This technology should enable studies not only in populations but also at the single cell level. In Specific Aim 3: we will develp reagents to study the transcriptional dynamics in relation to looping at the single cell level. We will combine RNA FISH with super-resolution imaging to develop a methodology for exploring the spatial and temporal structure of nascent transcription at high resolution. Combined with high-throughput image acquisition, we will discriminate the temporal dynamics of transcription by measuring the relative intensities arising from the different parts of the transcript. We will employ super-resolution imaging (STORM) to measure the spatial structure of transcription sites. These experiments are expected to reveal the impact of forced chromatin looping on distinct stages of the transcription cycle and elucidate the relationship between transcriptional burst kinetics and physical gene structure.