Project 1: Defining the Logic of Genome Organization In Pluripotent Cells

项目 1：定义多能细胞基因组组织的逻辑

• 批准号: 8710263
• 负责人: Kathrin Plath
• 金额: $ 32.83万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8710263
• 关键词: Address; Affect; Animal Experimentation; Architecture; Automobile Driving; Binding; Bioinformatics; Biology; Cell Maintenance; Cell Nucleus; Cells; Chromatin; Chromatin Structure; Chromosome Structures; Collaborations; DNA; DNA Replication Timing; DNA biosynthesis; Data; ES Cell Line; Engineering; Enhancers; Enzymes; Epigenetic Process; Gene Cluster; Gene Expression; Gene Targeting; Genes; Genetic; Genome; Genomics; Germ Layers; Goals; Human; Human Experimentation; Laboratories; Link; Logic; Methods; Modeling; Molecular; Mus; Nuclear; Nuclear Structure; Nucleic Acid Regulatory Sequences; Pattern; Polycomb; Positioning Attribute; Process; Proteins; Regulation; Staging; Structure; System; Testing; Time; Work; base; chromatin modification; chromatin protein; embryonic stem cell; fascinate; human embryonic stem cell; improved; induced pluripotent stem cell; loss of function; member; mutant; pluripotency; three dimensional structure; transcription factor

Description The goal of this proposal is to reveal the mechanisms that govern the three-dimensional (3D) architecture of the genome in pluripotent cells and during reprogramming to the IPS cell state. The spatial organization of chromosomes is a fascinating problem of metazoan biology, but leaves many unanswered questions, particularly the challenge to decipher the mechanisms driving the co-localization of genetic loci. Based on differentiated cell data, the 3D network of chromosomal interactions in the nucleus is thought to be important for the maintenance of cell identity and affect gene expression by spatial clustering of genes and their regulatory regions and by congregating groups of genes at the same sub-nuclear structure allowing their coordinated expression and modulation of epigenetic states. The 3D structure of the pluripotent cell genome is basically unstudied. However, a recent study of DNA replication timing, to which my lab contributed, suggested that a large-scale reorganization of the genome coincides with the commitment of pluripotent cells to differentiation, prior to germ layer specification. The reversal of this process appears to be one of the final steps of reprogramming, linked to the binding of the reprogramming factors to pluripotency gene targets and changes in global chromatin structure and DNA replication patterns. Based on these findings, we hypothesize that a true understanding of genome regulation in pluripotent cells and during reprogramming can only be obtained by revealing the structural and functional relationships between the spatial organization of the genome and linear genomic features such as chromatin and expression states and association with transcription factors; and that the establishment of the pluripotent nuclear architecture represents a road block to reprogramming. In an effort to begin to address 3D genomic interactions in pluripotent cells, my laboratory has successfully established the 4C-seq method to identify regions throughout the genome that are physically close to the Oct4 locus, which revealed that this locus interacts with early replicating, highly expressed genes that are bound by pluripotency transcription factors that themselves are enriched at the Oct4 locus. Based on this extensive work, our expertise in reprogramming and pluripotent cell chromatin, along with specific collaborations within the P01 and strong ties to the P01 Bioinformatics Core, we are well positioned to unveil molecular mechanisms governing the 3D genomic interactions in human and mouse embryonic stem (ES) cells and to study how the differentiated cell genome is reorganized during human cell reprogramming; with these Aims: Aims: Aim 1. We will assess and compare genomic interactions of key loci in mouse and human ES cells; discern the relationship with linear genomic features; and unveil mechanisms that control 3D organization. Aim 2. We will systematically test how expression level, pluripotency transcription factors, and chromatin proteins regulate 4C interactions at an isolated locus in mouse ES cells. Aim 3. We will define how genomic interactions are reorganized during human cell reprogramming and discover how chromatin limits this process. Each of the aims will be performed in conjunction with other members of the P01 proposal and build on the work of the P01 Bioinformatics Core. We do not propose human and animal experimentation.

描述 该提案的目标是揭示控制三维 (3D) 的机制 多能细胞中以及重编程为 IPS 细胞状态期间的基因组结构。染色体的空间组织是后生动物生物学中一个令人着迷的问题，但仍有许多悬而未决的问题，特别是破译驱动遗传位点共定位的机制的挑战。 基于分化的细胞数据，细胞核中染色体相互作用的 3D 网络被认为对于维持细胞身份非常重要，并通过基因及其调控区域的空间聚类以及通过将基因组聚集在同一子区域来影响基因表达。核结构允许它们协调表达和表观遗传状态的调节。多能细胞基因组的 3D 结构基本上尚未被研究。然而，我的实验室最近参与的一项关于 DNA 复制时间的研究表明，基因组的大规模重组与多能细胞的承诺同时发生。 分化，在胚层规范之前。这一过程的逆转似乎是重编程的最后步骤之一，与重编程因子与多能性基因靶标的结合以及整体染色质结构和 DNA 复制模式的变化有关。基于这些发现，我们假设只有通过揭示多能细胞的空间组织之间的结构和功能关系才能真正理解多能细胞和重编程过程中的基因组调控。 基因组和线性基因组特征，例如染色质和表达状态以及与转录因子的关联；多能核结构的建立是重编程的障碍。为了开始解决多能细胞中的 3D 基因组相互作用，我的实验室成功建立了 4C-seq 方法来识别整个基因组中物理上接近 Oct4 基因座的区域，这表明该基因座与早期复制、高度复制相互作用。表达的基因与多能性转录因子结合，这些转录因子本身在 Oct4 基因座上富集。基于这项广泛的工作、我们在重编程和多能细胞染色质方面的专业知识，以及 P01 内部的具体合作以及与 P01 生物信息学核心的紧密联系，我们有能力揭示控制人类和小鼠胚胎干中 3D 基因组相互作用的分子机制(ES) 细胞并研究分化的细胞基因组在人类细胞重编程过程中如何重组；目标如下： 目标： 目标 1. 我们将评估和比较小鼠和人类 ES 细胞中关键位点的基因组相互作用；辨别与线性基因组特征的关系；并揭示控制 3D 组织的机制。 目标 2. 我们将系统地测试表达水平、多能性转录因子和染色质蛋白如何调节小鼠 ES 细胞中孤立位点的 4C 相互作用。 目标 3。我们将定义人类细胞重编程过程中基因组相互作用如何重组，并发现染色质如何限制这一过程。 每个目标都将与 P01 提案的其他成员一起实现，并以 P01 生物信息学核心的工作为基础。我们不建议进行人类和动物实验。