Decoding chromosome structure with multiplexed super-resolution microscopy

总结 几十年的研究表明，基因组的组织是非随机的，并严重影响许多核 包括转录调节、DNA复制和DNA修复等过程， 表明染色体采用的三维结构对发育至关重要， 经常被疾病困扰。我们目前的大部分理解来自于生物化学技术， 在大量的细胞上，导致我们对建立和 保持组织状态，特别是在单个细胞的上下文中。我们建议引进一套新的 基于单分子超分辨率成像方法DNA-PAINT的单细胞技术， 用一套同时具有高多重性和空间分辨率的工具来弥补这一差距。具体到 目的1我们将开发一种多路复用（>20色）超分辨率染色体成像策略， 基因组靶点范围从内切酶到长度为数百万个碱基，这将使我们能够研究 单细胞中染色质纤维在一定长度范围内的折叠特性。在目标2中，我们将开发 多重分析，以共同定位蛋白质，RNA分子，和特定的基因组位点在个别细胞在 纳米级的然后，我们将研究在一个大染色质模型中心的结构蛋白的组织 在人类不活跃的X染色体上的环。在目标3中，我们将开发一种依赖于邻近度的超分辨率 一种探测蛋白质和DNA靶标之间特异性相互作用的方法， 在拥挤的环境中检测分子间的相互作用。我们将部署此技术来查询 在单个细胞中，上述染色质成环枢纽的组成和表观遗传状态。总的来说， 我们的方法将使许多问题的定位，组成，和表观遗传状态的具体 研究人员第一次接触到单个细胞中的基因组位点，并有望影响不同的领域 超越了染色体生物学

Summary Decades of study have revealed that genome organization is non-random and critically impacts many nuclear processes including the regulation of transcription, DNA replication, and DNA repair, and increasing evidence suggests that the three-dimensional structures adopted by chromosomes are critical for development and are often perturbed in disease. Much of our current understanding comes from biochemical techniques performed on large populations of cells, leading to many gaps in our understanding of the mechanisms that establish and maintain organizational states, particularly in the context of individual cells. We propose to introduce a new set of single-cell technologies based on the single-molecule super-resolution imaging method DNA-PAINT to bridge this gap with a suite of tools possessing both high multiplexibility and spatial resolution. Specifically, in Aim 1 we will develop a multiplexed (>20 color) super-resolution chromosomal imaging strategy to image genomic targets ranging from kilobases to multiple megabases in Iength, which will enable us to investigate the folding properties of the chromatin fiber in single cells over a range of length-scales. In Aim 2, we will develop multiplexed assays to co-localize proteins, RNA molecules, and specific genomic sites in individual cells at the nanoscale. We will then investigate organization of architectural proteins at a model hub of large chromatin loops on the human inactive X-chromosome. In Aim 3, we will develop a proximity-dependent super-resolution method to probe specific interactions between protein and DNA targets that will allow for the sensitive detection of molecular interactions in crowded environments. We will deploy this technology to query the composition and epigenetic states of the aforementioned chromatin looping hub in individual cells. Collectively, our methods will make many questions about the positioning, composition, and epigenetic states of specific genomic loci in individual cells accessible to researchers for the first time, and promise to impact diverse fields beyond chromosome biology.