Regulatory elements of replication timing and 3D genome organization

项目摘要 越来越多的证据表明，需要三维（3D）基因组组织来调节 基因功能及其改变与许多疾病有关。基因组被组织成 与DNA复制的时间顺序（复制定时- RT）对齐的区室。然而，在这方面， 关于RT控制和3D基因组组织的机制知之甚少。最近我们 鉴定了一类新的控制RT和3D基因组组织的顺式调控元件： 复制控制元件。我们的长期目标是研究什么是监管要素 人类分化细胞类型的基因组组织，研究反式作用因子如何控制 这些元素，并定义了3D基因组组织在发育过程中如何重塑， 进化为了实现这些目标，我们将在人类基因组中删除和插入候选ERCE， 分化的细胞类型，并测试它们对RT、3D基因组组织和基因表达的影响。我们 将使用高度同步的人类胚胎干细胞分化来跟踪ERCE激活 系统.最后，我们将分析3D基因组组织进化使用原代细胞来自 不同的物种我们将使用最近开发的基于芯片的细胞纯化技术- 封闭的微流控流式细胞术以分离活的单细胞以建立携带 ERCE缺失和插入。此外，我们将利用这项技术进行精确识别， 分离特定细胞周期阶段的细胞群，以系统地研究 RT、基因组结构和基因表达之间的关系。总的来说，我们预计， 微流控流式细胞术将使我们能够优化我们的细胞系开发，以及我们的样品 纯化用于下游全基因组分析。我们期望我们的工作将大大有助于 了解基因组组织的基本原则及其与基因功能的关系。

PROJECT SUMMARY Increasing evidence indicates that three-dimensional (3D) genome organization is required to regulate gene function and its alterations are associated with many diseases. The genome is organized into compartments that align with the temporal order of DNA replication (replication timing – RT). However, little is known about the mechanisms underlying RT control and 3D genome organization. Recently, we identified a novel class of cis regulatory elements that control RT and 3D genome organization: early replicating control elements – ERCEs. Our long-term goals are to study what are the regulatory elements of genome organization in human differentiated cell types, investigate how trans-acting factors control these elements, and define how 3D genome organization is remodeled during development and evolution. To achieve these goals, we will delete and insert candidate ERCEs into the genome of human differentiated cell types and test their effect on RT, 3D genome organization and gene expression. We will track ERCE activation using highly-synchronous human embryonic stem cells differentiation systems. Finally, we will analyze 3D genome organization evolution using primary cells derived from different species. We will use the recently developed technologies for cell purification based on chip- enclosed microfluidic flow cytometry to isolate live, single cells to establish clonal cell lines carrying ERCE deletions and insertions. Moreover, we will use this technology for precise identification and isolation of cell populations in specific cell cycle stages to systematically investigate the connections between RT, genome architecture and gene expression. Overall, we anticipate that the application of microfluidic flow cytometry will allow us to optimize our cell line development, as well as our sample purification for downstream genome-wide analyses. We expect that our work will contribute significantly to understand the fundamental principles of genome organization and its relationship to gene function.