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中关键的未解决问题是染色质折叠形状的动力学基因表达模式。我们对染色质高阶3-D折叠动力学的了解非常严重有限，很大程度上是由于缺乏精确图像的技术，工程师和监视器循环精确地循环整个细胞种群的时空方式。在这里，我们建议通过开发工具以在细胞种群中同步改变染色质折叠的工具作为以及单个细胞，并测量染色质循环及其与高空间的转录的关系单个细胞的分辨率。在特定目标1中，我们将设计工具以控制循环动力学。我们将通过融合到一个部分，修改各种水平折叠染色质的因素，例如LDB1和CTCF 稳定性可以通过扩散的配体控制。与HI分辨率5C和单分子结合使用成像这些工具有望产生对核架构关系的基本见解和基因表达机制。在特定目的2中，我们计划设计可诱导的系统循环动力学的精确控制。使用可在与设计器DNA结合蛋白的结合，我们试图设计用于快速促进或在各种尺度上破坏染色质循环。这项技术不仅应该在人群中，而且可以使也位于单细胞级。在特定目标3中：我们将开发试剂以研究转录与单个单元格循环有关的动力学。我们将将RNA鱼与超分辨率相结合成像以开发一种探索新生转录的空间和时间结构的方法高分辨率。结合高通量图像采集，我们将区分时间动态通过测量由转录本不同部分产生的相对强度来进行转录。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。这些实验有望揭示强制染色质循环在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.