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

基本信息

批准号：
9323543
负责人：
Gerd A Blobel
金额：
$ 72.87万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
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/9323543
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 Structure System Technology Testing Time Tissues Transcript Transcription Elongation Transcription Initiation Transcriptional Activation Visualization software Work Zinc Fingers beta Globin cell type design dimer 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

项目摘要

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（由申请人提供）：基因组生物学领域中尚未解答的关键问题是染色质折叠的动力学如何塑造基因表达模式，我们对染色质高阶 3-D 折叠动力学的了解严重有限，这主要是由于。缺乏以精确的时空方式对细胞群中的循环进行精确成像、设计和监控的技术。在此，我们建议通过开发动态改变染色质折叠的工具来解决这些限制。在特定目标 1 中，我们将在细胞群以及单个细胞之间以同步方式测量染色质循环及其与高空间分辨率转录的关系。我们将修改在不同水平上折叠染色质的因子，例如通过与可扩散配体控制稳定性的部分融合，将这些工具与高分辨率 5C 和单分子成像相结合。期望对核结构和基因表达机制的关系产生基本的见解，在特定目标 2 中，我们计划设计光诱导系统，以使用光激活的二聚化结构域来精确控制循环动力学。与设计DNA结合蛋白结合使用，我们尝试设计用于在不同规模上快速促进或破坏染色质循环的因子，该技术不仅可以在群体中进行研究，而且还可以在单细胞水平上进行研究：我们将在特定目标3中进行研究。我们将开发一种在单细胞水平上研究与循环相关的转录动力学的试剂，以开发一种在高分辨率下探索新生转录的空间和时间结构的方法。高通量图像采集，我们将通过测量转录本不同部分产生的相对强度来区分转录的时间动态。我们将采用超分辨率成像（STORM）来测量转录位点的空间结构。有望揭示强制染色质循环对转录周期不同阶段的影响，并阐明转录爆发动力学与物理基因结构之间的关系。