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：
10001247
负责人：
Gerd A Blobel
金额：
$ 27.1万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2020-07-31
项目状态：
已结题

项目摘要

Abstract 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 develop 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.

抽象的基因组生物学领域尚未解答的关键问题是染色质折叠形状的动力学如何基因表达模式。我们对染色质高阶 3D 折叠动力学的了解非常严重有限，主要是由于缺乏精确成像、设计和监控循环的技术跨越细胞群的时空方式。在这里，我们建议通过以下方式解决这些限制开发工具以同步方式动态改变细胞群中的染色质折叠以及单个细胞，并在高空间下测量染色质环及其与转录的关系单细胞分辨率。在具体目标 1 中，我们将设计工具来控制循环动态。我们将通过融合到一个部分来修改在不同水平上折叠染色质的因子，例如 Ldb1 和 CTCF 稳定性可以通过扩散配体来控制。结合高分辨率 5C 和单分子成像这些工具有望产生对核结构关系的基本见解和基因表达机制。在具体目标 2 中，我们计划设计光诱导系统精确控制循环动态。使用可用于与设计DNA结合蛋白相结合，我们尝试设计用于快速促进或在不同尺度上破坏染色质环。这项技术不仅能够在人群中进行研究，而且能够在人群中进行研究也在单细胞水平上。具体目标3：我们将开发试剂来研究转录与单细胞水平循环相关的动力学。我们将RNA FISH与超分辨率结合起来成像开发一种方法来探索新生转录的空间和时间结构高分辨率。结合高通量图像采集，我们将判别时间动态通过测量转录本不同部分产生的相对强度来分析转录。我们将采用超分辨率成像（STORM）来测量转录位点的空间结构。这些实验有望揭示强制染色质循环对不同阶段的影响转录周期并阐明转录爆发动力学与物理基因之间的关系结构。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Comparative analysis of three-dimensional chromosomal architecture identifies a novel fetal hemoglobin regulatory element.

DOI：
10.1101/gad.303461.117
发表时间：
2017-08-15
期刊：
Genes & development
影响因子：
10.5
作者：
Huang P;Keller CA;Giardine B;Grevet JD;Davies JOJ;Hughes JR;Kurita R;Nakamura Y;Hardison RC;Blobel GA
通讯作者：
Blobel GA

Crossed wires: 3D genome misfolding in human disease.