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 该提案的目的是揭示支配三维（3D）的机制 基因组在多能细胞中的结构以及重新编程到IPS细胞状态。染色体的空间组织是后生生物学的一个有趣的问题，但留下了许多未解决的问题，尤其是破译促进遗传基因座共定位机制的挑战。 基于分化的细胞数据，认为核中染色体相互作用的3D网络被认为对于维持细胞身份和通过空间聚类的基因及其调节区域以及通过在相同的亚核结构上聚集基因组来影响基因表达至关重要，从而允许其表观遗传状态的协调表达和调节。多能细胞基因组的3D结构基本上是未研究的。然而，我的实验室对DNA复制时机的最新研究表明，基因组的大规模重组与多能细胞对多能细胞的承诺相吻合 分化，在细菌层规范之前。该过程的逆转似乎是重编程的最后一步之一，与重编程因子与多能基因靶标的结合以及全局染色质结构和DNA复制模式的变化有关。基于这些发现，我们假设对多能细胞中的基因组调节以及在重编程过程中的真正理解只能通过揭示该空间组织之间的结构和功能关系才能获得。 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；并且建立多能核结构代表了重新编程的路障。为了开始解决多元细胞中的3D基因组相互作用，我的实验室成功建立了4C-Seq方法，以识别整个基因组的区域，这些区域在物理上接近OCT4基因座，该基因座与早期复制的，高度表达的基因相互作用，这些基因受到Pluripotency转录因素的束缚，该基因本身是10月4日在44个locus plocus poct poct poct poc poc poc poc poc pericus ever poc ect4 locus ever poc ect4 locus。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中的特定协作以及与P01生物信息学核心的紧密联系，我们在人类和小鼠胚胎（ES）细胞中的3D基因组相互作用在人类和小鼠基因组中的研究中，我们有好处公布分子机制，以揭示3D基因组的相互作用，在差异化的情况下，我们是相同的基因组合。以这些目的： 目标： 目标1。我们将评估和比较小鼠和人ES细胞中关键基因座的基因组相互作用；辨别与线性基因组特征的关系；以及控制3D组织的机制。 AIM 2。我们将系统地测试表达水平，多能转录因子和染色质蛋白如何调节小鼠ES细胞中分离的基因座的4C相互作用。 目标3。我们将定义在人类细胞重编程过程中如何重组基因组相互作用，并发现染色质如何限制该过程。 每个目标都将与P01提案的其他成员一起执行，并以P01生物信息学核心的工作为基础。我们不提出人类和动物实验。