Development of Single Molecule Chromatin Interaction Assays (smChIA) in Single Nuclei

项目摘要 目前的3D基因组图谱技术（包括ChIA-PET和Hi-C）需要数百万个细胞才能完成每个基因组。 该方法主要依赖于邻位连接、连接产物的PCR扩增和基于Illumina的高通量测序。 吞吐量排序。虽然这样的方法已经揭示了染色质相互作用的许多原理， 基于数百万个细胞平均信号的3D基因组组织图，他们只提供了二进制 （配对的）染色质接触信息，因此不能阐明细胞凋亡的精确分子机制。 在单个细胞中发生的复杂的染色质相互作用。单细胞3D基因组作图的研究进展 因此，技术引起了研究界的极大兴趣。目前，单细胞 这些策略仍然主要基于上述用于DNA操作的常规分子方法 这些技术障碍阻碍了真正的单分子和单细胞全基因组分析。 在这个项目中，我们提出了一个全新的系统，下一代技术的染色质 基于SeqLL单分子蛋白质检测和DNA测序平台的相互作用测定。一 原型单分子平台已被证明用于同时检测组蛋白修饰 和单个核小体的基因组位置（Shema等人，2016）。我们将调整这个平台， 单核中的单分子染色质相互作用分析（smChIA）。提出的smChIA方法 具有几个重要的优势，目前的方法，包括单分子分辨率在单一的 核，不需要PCR或任何扩增，并且最显著地消除了邻近连接步骤。到 积极推进下一代染色质相互作用分析技术的发展，我们 我组建了一个具有跨学科和互补知识和技能的综合团队，以确保 成功实现提案中的具体目标。我们还建立了一个学术产业 合作伙伴关系，使技术迅速传播到更大的研究界。我们的初步 结果为smChIA策略提供了初步的概念验证。我们相信smChIA有潜力 彻底改变整个3D基因组生物学领域，从数据采集到解释。一旦发育完全， 我们预计smChIA数据将在3D组织中产生令人兴奋的新颖生物学见解， 基因组及其动态调控。

PROJECT SUMMARY The current 3D genome mapping technologies (including ChIA-PET and Hi-C) require millions of cells for each assay and primarily rely on proximity ligation, PCR amplification of the ligation products, and Illumina-based high throughput sequencing. While such methods have revealed many principles of chromatin interaction and useful 3D genome organizational maps based on signals averaged over millions of cells, they provided only binary (paired) chromatin contact information, and thus do not enable elucidating the precise molecular mechanisms of complex chromatin interactions occurring in individual cells. The development of single-cell 3D genome mapping technologies has thus attracted significant interest in the research community. However, current single cell strategies are still largely based on the conventional molecular approaches for DNA manipulation mentioned above, which present technical barriers that prevent true single-molecule and single cell analyses genome-wide. In this project, we propose to develop a completely new system of next generation technology for chromatin interaction assays based on the SeqLL single-molecule protein-detection and DNA-sequencing platform. A prototype single-molecule platform has been demonstrated for simultaneous detection of histone modifications and genomic positions of individual nucleosomes (Shema et al., 2016). We will adapt this platform to enable single-molecule Chromatin Interaction Analysis (smChIA) in single nuclei. The proposed smChIA method possesses several important advantages over the current methods, including single-molecule resolution in single nuclei, no need of PCR or any amplification, and most significantly the elimination of proximity ligation steps. To aggressively advance the development of this next generation chromatin interaction analysis technology, we have assembled an integrative team with interdisciplinary and complementary knowledge and skills to ensure successful implementation of the specific aims in the proposal. We have also established an academic-industry partnership to make quick dissemination of the technology to the larger research community. Our preliminary results have provided initial proof-of-concept for the strategy of smChIA. We believe smChIA has the potential to revolutionize the entire field of 3D genome biology from data acquisition to interpretation. Once fully developed, we expect that the smChIA data will generate exciting and novel biological insights in the organization of the 3D genome and its dynamic modulation.