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

项目总结 目前的3D基因组图谱技术(包括Chia-PET和Hi-C)每种都需要数百万个细胞 检测和主要依赖于邻近连接、连接产物的PCR扩增和基于Illumina的高 吞吐量排序。虽然这些方法揭示了染色质相互作用的许多原理和有用的 基于信号的3D基因组组织图平均覆盖数百万个细胞，它们仅提供二进制 (配对)染色质联系信息，因此无法阐明 在单个细胞中发生复杂的染色质相互作用。单细胞三维基因组图谱的研究进展 因此，技术引起了研究界的极大兴趣。然而，目前的单电池 这些策略很大程度上仍然基于前面提到的用于DNA操作的传统分子方法 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，我们计划开发一种全新的下一代染色质技术系统 基于SeqLL单分子蛋白质检测和DNA测序平台的相互作用分析。一个 原型单分子平台已被证明可同时检测组蛋白修饰 和个体核小体的基因组位置(Shema等人，2016)。我们将调整此平台以实现 单核单分子染色质相互作用分析。提出的smCHIA方法 与目前的方法相比，具有几个重要的优点，包括单分子在 最重要的是消除了邻近连接步骤。至 积极推动这项新一代染色质相互作用分析技术的发展，我们 组建了一支具有跨学科和互补知识和技能的综合团队，以确保 成功地落实了提案中的具体目标。我们还建立了一个学术产业 建立伙伴关系，向更大的研究界迅速传播这项技术。我们的预赛 研究结果为SMCHIA的战略提供了初步的概念验证。我们相信smChIA有潜力 彻底改变了从数据获取到解释的整个3D基因组生物学领域。一旦完全开发， 我们预计，smChIA数据将在3D的组织中产生令人兴奋的和新颖的生物学见解 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.