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: 9003449
• 负责人: Gerd A Blobel
• 金额: $ 76.76万
• 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 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9003449
• 关键词: 3-Dimensional; ATAC-seq; Address; Affect; Architecture; Biological Models; Biology; Cells; Chromatids; Chromatin; Chromatin Loop; Chromatin Structure; Chromosomes; Color; Communities; DNA; DNA-Binding Proteins; Dimerization; Dissociation; Engineering; Enhancers; Epigenetic Process; Erythroid Cells; Foundations; Frequencies; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Structure; Genes; Genetic Transcription; Genome; Genome Mappings; Globin; Heterodimerization; Higher Order Chromatin Structure; Image; Imagery; 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; Relative (related person); Resolution; Shapes; Site; Specific qualifier value; Staging; Structure; System; Technology; Testing; Time; Tissues; Transcript; Transcription Elongation; Transcription Initiation; Transcriptional Activation; Work; Zinc Fingers; cell type; design; embryonic stem cell; fluorophore; gene repression; insight; interest; promoter; public health relevance; research study; 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.

 描述（适用提供）：基因组生物学领域中的关键未解决问题是染色质折叠形状基因表达模式的动力学。我们对染色质高阶3-D折叠动力学的了解受到严重限制，这主要是由于缺乏精确图像，工程和监视器循环的技术，以精确的时空方式跨细胞群体进行。在这里，我们建议通过开发工具来解决这些局限性，以在单个细胞和单个细胞中以同步方式动态改变染色质折叠，并测量染色质循环及其与单个细胞中高空间分辨率下转录的关系。在特定目标1中，我们将 设计循环动力学的工具。我们将通过融合到一个可以通过扩散的配体控制稳定性的部分来修改各种水平折叠染色质的因素，例如LDB1和CTCF。结合HI分辨率5C和单分子成像，这些工具有望产生对核结构与基因表达机制关系的基本见解。在特定目标2中，我们计划设计可诱导的系统，以精确控制循环动力学。使用可与设计器DNA结合蛋白结合使用的光激活二聚域，我们试图设计用于快速促进或破坏各种尺度上的染色质循环的因子。这项技术不仅应该在人群中，而且可以在单细胞水平上进行研究。在特定的目标3中：我们将开发试剂以研究与单细胞水平循环相关的转录动力学。我们将将RNA鱼与超分辨率成像相结合，以开发一种方法，用于探索高分辨率下新生转录的空间和临时结构。结合高通量图像采集，我们将通过测量转录不同部分产生的相对强度来区分转录的临时动力学。我们将采用超分辨率成像（风暴）来测量转录位点的空间结构。预计这些实验将揭示强制染色质循环在转录周期的不同阶段的影响，并阐明转录爆发动力学与物理基因结构之间的关系。