Engineering and visualizing genome folding at high spatiotemporal resolution

 描述(由申请人提供)：基因组生物学领域中尚未回答的关键问题是染色质折叠的动态如何塑造基因表达模式。我们对染色质高阶三维折叠动力学的了解严重有限，这在很大程度上是因为缺乏技术来精确地成像、设计和监测跨细胞群体的精确时空循环。在这里，我们建议通过开发工具来解决这些限制，以跨细胞群体以及单个细胞以同步的方式动态改变染色质折叠，并在单个细胞中以高空间分辨率测量染色质循环及其与转录的关系。在具体目标1中，我们将 设计控制环路动态的工具。我们将通过融合来修饰在不同水平折叠染色质的因子，如LDB1和CTCF，使其部分稳定，其稳定性可以由可扩散的配体控制。结合高分辨率5C和单分子成像，这些工具有望对核结构和基因表达机制的关系产生基本的见解。在具体目标2中，我们计划设计用于精确控制环路动力学的光感应系统。利用光激活的二聚化结构域，可以与设计的DNA结合蛋白一起使用，我们试图设计用于快速促进或破坏不同规模的染色质环路的因子。这项技术不仅应该能够在群体中进行研究，而且还应该能够在单细胞水平上进行研究。在特定目标3：我们将开发试剂来研究与单细胞水平上的环有关的转录动力学。我们将结合RNA FISH和超分辨率成像来开发一种以高分辨率探索新生转录的时空结构的方法。结合高通量图像采集，我们将通过测量转录记录不同部分产生的相对强度来区分转录的时间动力学。我们将使用超分辨率成像(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转录周期不同阶段的影响，并阐明转录猝发动力学与物理基因结构之间的关系。

 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.