NSF-ANR: Detailed and Mechanistic Characterization of TAD Boundaries Using Complementary Single-Molecule Sequencing and Super-Resolution Imaging Approaches

真核生物基因组以其线性序列编码遗传信息，但基因的适当表达需要染色体折叠成复杂且空间上不同的三维结构。尽管这些组织特征保存得很好，但人们对它们如何使染色体发挥功能的理解有限。将基因组结构与功能联系起来的一个重大飞跃是拓扑相关结构域（TADs）的发现，TADs是哺乳动物基因组中涉及基因调控以及DNA复制、修复和重组的区域单位。tad之间的边界被认为可以防止参与各种过程的分子机制的“扩散”。本研究项目旨在阐明TAD边界的复杂性，并更好地了解它们如何使TAD结构和功能成为可能。该项目还将为研究生和本科生提供跨学科的培训机会，以补充实验和生物信息学方法。目前TAD形成的实验和计算机模型包括简化的TAD边界，由间断和稳定的CTCF蛋白结合组成。相比之下，最近的证据表明，边界是扩展的，动态的，并表现出细胞间的可变性。该项目将采用新颖的单分子基因组学方法与超分辨率成像相结合，以识别和量化在单个细胞中隔离邻近TADs的遗传元件。研究结果有助于更新TAD形成模型，提高对边界如何影响TAD结构、动力学和功能的理解和预测。这一新知识反过来可以帮助确定机制，即未表征的遗传元件（例如，在GWAS研究中发现的）导致基因调控、DNA复制、修复和重组的微妙变化。它也可能有助于解释TAD结构如何在遗传或获得性非编码基因组变化（例如，结构变异，多态性）时进行重组。这个美国/法国合作项目由美国国家科学基金会和法国国家研究机构支持，其中美国国家科学基金会资助美国研究者，法国国家研究机构资助法国合作伙伴。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Eukaryotic genomes encode genetic information in their linear sequence, but appropriate expression of genes requires chromosomes to fold into complex and spatially distinct three-dimensional structures. Despite the remarkable conservation of these organizational features, there is limited understanding of how they enable chromosome function. A major leap forward in linking genome structure to function has been the discovery of Topologically Associating Domains (TADs), which are regional units of mammalian genomes implicated in gene regulation as well as DNA replication, repair and recombination. The boundaries between TADs are thought to prevent the “spreading” of molecular machineries involved in various processes. This research project aims to clarify the complex nature of TAD boundaries and better understand how they enable TAD structure and function. The project will also offer cross-disciplinary training opportunities for graduate and undergraduate students in complementary experimental and bioinformatic methods.Current experimental and in-silico models for TAD formation include simplified TAD boundaries consisting of punctuated and stable binding of CTCF protein. In contrast, recent evidence indicates that the boundaries are extended, dynamic and exhibit cell-to-cell variability. This project will employ novel single-molecule genomics approaches coupled with super-resolution imaging to identify and quantify the genetic elements that insulate neighboring TADs in single cells. The outcomes can help update models of TAD formation and improve understanding and prediction of how the boundaries contribute to TAD structure, dynamics and function. This new knowledge could in turn help identify mechanisms whereby uncharacterized genetic elements (e.g., identified in GWAS studies) cause subtle changes to gene regulation, DNA replication, repair and recombination. It may also help explain how TAD structure can be reorganized upon inherited or acquired changes to the non-coding genome (e.g., structural variation, polymorphisms).This collaborative US/France project is supported by the US National Science Foundation and the French Agence Nationale de la Recherche, where NSF funds the US investigator and ANR funds the partners in France.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.