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3D基因组组织的基本机制知之甚少。最近，我们发现 RT和3D基因组组织控制元件（早期复制控制元件-ERCE）， 小鼠胚胎干细胞ERCE富含增强子表观遗传标记，形成强染色质 在一些实施方案中，多能性蛋白与多能性特异性转录因子相互作用并被多能性特异性转录因子结合。此外，我开发了一个 预测细胞类型特异性共占据基因调控网络的整合模型 转录因子调控RT，在此，我们将研究基因组的调控元件 组织在人类分化的细胞类型，调查如何反式作用因子控制这些 元素，并定义了3D基因组组织在发育和进化过程中如何重塑。 我们的中心假设是，ERCE中细胞类型特异性转录因子的共同占据 是调节3D基因组结构所必需的。为了验证这一假设，我们将删除和插入 将候选ERCE插入人分化细胞类型的基因组中，并测试它们对RT、3D 基因组组织和基因表达。我们将使用高度同步的 人胚胎干细胞分化系统。最后，我们将分析3D基因组组织 利用来自不同物种的原代细胞进行进化。我的实验室有着独特的优势 在实验设计和3D分析方面具有广泛的专业知识， 基因组组织在人类细胞命运的承诺。此外， 明尼苏达大学将促进该项目的成功，包括最先进的 基因组编辑技术（基因组工程共享资源），下一代测序 （基因组学中心），干细胞（干细胞研究所），成像（成像中心）和生物信息学工具 明尼苏达超级计算研究所（Minnesota Supercomputing Institute）我们希望我们的工作将有助于理解 基因组组织的基本原则及其与基因功能的关系。拟议 研究结合了几个创新方面，如综合计算模型，以预测 大规模染色质组织的调控元件，基因组工程技术和 优化人类胚胎干细胞的分化方案，以剖析 控制发育和进化过程中的3D基因组组织。

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 3D genome organization. Recently, we identified cis elements of RT and 3D genome organization control (early replicating control elements – ERCEs) in murine embryonic stem cells. ERCEs are enriched in enhancer epigenetic marks, form strong chromatin interactions and are bound by pluripotency-specific transcription factors. Moreover, I developed an integrative model of gene regulatory networks that predicts that co-occupancy of cell type-specific transcription factors regulate RT. Here, we will 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. Our central hypothesis is that co-occupancy of cell type-specific transcription factors at ERCEs is required to regulate the 3D genome organization. To test this hypothesis, 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. My laboratory is uniquely positioned to perform the proposed research with broad expertise in the experimental design and analysis of 3D genome organization during human cell fate commitment. Moreover, numerous resources available at the University of Minnesota will facilitate the success of this project, including state-of-the-art technologies for genome editing (Genome Engineering Shared Resource), next-generation sequencing (Genomics Center), stem cells (Stem Cell Institute), imaging (Imaging Center) and bioinformatic tools (Minnesota Supercomputing Institute). We expect that our work will contribute significantly to understand the fundamental principles of genome organization and its relationship to gene function. The proposed research combines several innovative aspects such as integrative computational models to predict regulatory elements of large-scale chromatin organization, genome engineering technologies and optimized differentiation protocols of human embryonic stem cells to dissect the mechanisms that control 3D genome organization during development and evolution.