Genetic Specification and Evolution of 3D Genome Organization

项目摘要 染色体构象捕获技术的发展，如Hi-C，使我们有可能 在整个基因组中以高分辨率识别核内相互作用的染色质区域。这些 结构域称为拓扑关联结构域(TADS)，它们的边界起到绝缘体的作用 元素。虽然现在很容易确定这样的领域，但在知识方面存在差距，因为 TAD边界强度和位置的遗传控制尚不完全清楚，也不清楚 这些边界属性会影响基因表达等分子表型。这样做的长期目标是 申请人的实验室是了解导致基因组组织变化的进化过程 在物种内部和物种之间。该项目的总体目标是确定并确定功能特征 果蝇TAD边界强度的遗传决定因素，以及 黑腹鱼与近缘种的边界得与失。初步数据产生于 申请人的实验室表明，在黑腹黄鼠之间，TAD边界强度存在差异。 果蝇遗传资源小组(DGRP)的个体和新的TAD界限存在 甚至在关系密切的黑腹夜蛾和雅库巴夜蛾之间也是如此。建议的理由是 研究表明，了解TAD边界的遗传基础将提供对3D变化的洞察 基因组组织与新的表型和疾病状态的出现有关。目标是 将通过追求三个具体目标来实现该项目的目标：1)识别影响TAD的序列变体 使用关联映射的边界强度；2)确定TAD边界如何在 基因组；以及3)使用CRISPR对黑腹隐翅虫的候选变异体进行功能表征。这个 DGRP是专门作为数量性状作图资源开发的，将用于识别 与TAD边界强度相关的序列变体，而来自 黑素胃泌素基因组将被用来确定与黑色素瘤形成有关的谱系特异性序列替换 新奇的TAD边界。候选因果序列将使用CRISPR基因组编辑进行验证，以 确定它们对TAD边界强度和位置的影响。这项拟议的研究具有创新性，因为它 代表着与现状的实质性背离：不是测试特定基因组的浓缩 TAD范围内的特征，本项目建议利用TAD范围内和 在果蝇的物种之间。该领域的主要目标之一是最终预测TAD边界 仅从DNA序列就可以确定其强度和位置。拟议的研究具有重要意义，因为它将为我们带来 通过增加我们对TAD边界的起源和贡献的理解，更接近这一目标 基因表达水平在物种内和物种间的差异。

Project Summary The development of chromosome conformation capture techniques such as Hi-C have made it possible to identify domains of interacting chromatin within the nucleus at high resolution across the entire genome. These domains are known as Topologically Associating Domains (TADs) and their boundaries act as insulator elements. While it is now straightforward to identify such domains, there is a gap in knowledge because the genetic control of TAD boundary strength and location is not fully understood, nor is it clear how variation in these boundary properties affects molecular phenotypes such as gene expression. The long-term goal of this applicant’s laboratory is to understand the evolutionary processes that lead to changes in genome organization within and between species. The overall objective of this project is to identify and functionally characterize the genetic determinants of TAD boundary strength within Drosophila melanogaster, as well as the determinants of boundary gain and loss between D. melanogaster and closely related species. Preliminary data produced in the applicant’s laboratory suggests that there is variation in TAD boundary strength among D. melanogaster individuals from the Drosophila Genetic Resource Panel (DGRP) and that novel TAD boundaries are present even between the closely related species of D. melanogaster and D. yakuba. The rationale for the proposed research is that understanding the genetic basis of TAD boundaries will provide insight into how variation in 3D genome organization is related to the emergence of novel phenotypes as well as disease states. The objective of this project will be achieved by pursuing three specific aims: 1) Identify sequence variants that affect TAD boundary strength using association mapping; 2) Determine how TAD boundaries evolve at novel locations in the genome; and 3) Functionally characterize candidate variants using CRISPR in D. melanogaster. The DGRP, which was specifically developed as a resource for mapping quantitative traits, will be used to identify sequence variants associated with TAD boundary strength, whereas 13 Drosophila species from the melanogaster group will be used to identify lineage-specific sequence substitutions involved in the formation of novel TAD boundaries. Candidate causal sequences will be validated using CRISPR genome editing to determine their effect on TAD boundary strength and location. The proposed research is innovative because it represents a substantial departure from the status quo: instead of testing for enrichment of specific genomic features within TAD boundaries, this project proposes to take advantage of natural variation within and between species of Drosophila. One of the major goals of the field is to ultimately predict TAD boundary strength and location from DNA sequence alone. The proposed research is significant because it will bring us closer to this goal by increasing our understanding of how TAD boundaries originate and how they contribute to variation in gene expression levels both within and between species.