Replication Domain Organization during hESC Differentiation

hESC 分化期间的复制域组织

• 批准号: 7538033
• 负责人: David M Gilbert
• 金额: $ 34.38万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7538033
• 关键词: Address; Affect; Biochemical Genetics; Biological; Biological Models; Cell Differentiation process; Cell Lineage; Cell Nucleus; Cells; Characteristics; Chromatin; Chromosome Structures; Chromosomes; DNA; DNA Sequence; Data Set; Ectoderm; Endoderm; Epiblast; Epigenetic Process; Fiber; Fire - disasters; Genes; Genetic Transcription; Genie; Genome; Germ Layers; Human; In Situ Hybridization; Individual; Isochores; Light; Literature; Location; Maps; Mesoderm; Methods; Microscopic; Molecular; Mus; Nuclear; Nucleosomes; Positioning Attribute; Process; Property; Relative (related person); Replication Origin; Replicon; Role; Smooth Muscle; Staging; Stem cells; Stretching; Structure; Syntenic Conservation; System; Testing; Time; base; cell type; dalton; human embryonic stem cell; monolayer; novel; programs; research study; size; somatic cell nuclear transfer

Project 2: Replication Domain Organization During hESC Differentiation Dalton, Stephen A. Specific Aims. Many cytological, genetic and biochemical definitions of higher-order chromosomal domains have been put forth, but none provides a property that delineates boundaries of domains with precision and in a manner that can be applied genome-wide. Hence, while the word "domain" is frequently used to describe properties of large, often multi-genie, units of chromosomes, there is no comprehensive definition of a chromosome domain. We demonstrate that the "replication domain" is a definable chromosomal unit, revealing replication as potentially the only chromosomal property that affords a comprehensive segmentation of the entire genome at the megabase level. Importantly, we have discovered that the boundaries of these domains are dramatically re-organized during mESC and hESC differentiation to create larger temporally consolidated domains in which replication timing correlates more closely with sequence properties of chromosomes. Hence, replication structure of chromosomes in ESCs is relatively free from constraints imposed by DNA sequences, defining a novel property of pluripotent cells. We propose to further investigate the biological significance of domain consolidation, its relationship to the 3D organization of chromatin in the nucleus and the mechanisms by which consolidation occurs using hESCs as a model system. The specific aims are as follows: Aim 1: Choreography of Replication Domain Boundaries During Lineage Commitment. In this aim, we will test the hypothesis that replication domain boundaries are characteristic of particular cell types. More specifically, we propose that smaller temporally distinct replication domains is a characteristic of stem cells, and that differentiation will be accompanied by a progressively more rigid relationship between isochore sequence composition and replication timing. To determine whether boundaries are lineage specific, we will examine changes in replication domain boundaries during differentiation of hESCs to independent germ layers, ectoderm and mesendoderm. To distinguish whether changes in replication domain boundaries occur in a single-step, for example during loss of pluripotence, or whether there is a continuum of re-organization as cell lineage choices become more restricted, we will examine the downstream lineages definitive endoderm and mesoderm, as well as mesoderm differentiated to smooth muscle. Finally, to understand the functional significance of domain consolidation, we will determine which genes are affected by consolidation and how they relate to cell lineage choices. Aim 2: Replication Profiling as a Novel Means to Characterize hESCs. Replication profiling provides a convenient comprehensive genome-wide identification method that may shed light on important relationships between cell types and pluripotent cellular states that are currently being debated in the literature. In this Aim, we will identify the domains that distinguish hESCs from their differentiated counterparts. We will examine regions of conserved synteny between mouse and human for evolutionary conservation of replication domain structure. Finally, we will use replication profiling to address the debate as to whether hESCs are more similar to mESCs or mouse epiblast-like cells. Aim 3: Spatial consolidation of chromatin during differentiation. Here we will determine how the consolidation of replication domains defined molecularly in Aim1 corresponds to the three-dimensional re-organization of these domains in the cell nucleus. Using in situ hybridization at different stages of differentiation, we will trace the sub-nuclear localization of replication domains relative to specific sub-nuclear compartments to ask whether domains that consolidate temporally also consolidate spatially and whether they acquire a more uniform overall level of compaction. Aim 4: Coordination of replication forks during domain consolidation. In this Aim, we will analyze the polarity of replication forks, replicon sizes and regions of replication origin activity on individual stretched DNA fibers from the consolidating domains to identify replicon clusters and the locations of their boundaries relative to the boundaries of replication domains defined molecularly. These experiments will begin to define the molecular mechanisms by which domain consolidation occurs.

项目2：hESC分化期间的复制域组织Dalton，Stephen A.具体目标。 已经提出了许多高阶染色体结构域的细胞学，遗传和生化定义 第四，但没有人提供以精确和方式描述域边界的属性 可以应用全基因组。因此，而“域”一词经常用于描述 染色体的大型，通常是多机构的单位，没有染色体域的全面定义。 我们证明“复制域”是一个可定义的染色体单位，将复制揭示为 可能是唯一对整个基因组进行全面分割的染色体特性 巨型银行级别。重要的是，我们发现这些领域的边界是巨大的 在MESC和HESC分化过程中重新组织，以创建更大的时间合并域，其中 复制时机与染色体的序列特性更紧密相关。因此，复制 ESC中染色体的结构相对不受DNA序列施加的约束，定义A 多能细胞的新型特性。我们建议进一步研究域的生物学意义 巩固，其与核中染色质3D组织的关系以及通过的机制 使用hESC作为模型系统发生合并。具体目的如下： 目标1：谱系承诺期间复制域边界的编排。 在此目标中，我们将检验以下假设：复制域边界是特定细胞的特征 类型。更具体地说，我们建议较小的时间上不同的复制域是 干细胞，分化将伴随 等距序列组成和复制时机。要确定边界是否特定于谱系， 我们将检查hESC与独立细菌分化过程中复制域边界的变化 层，外胚层和中胚层。区分复制域边界的变化是否发生在 一个步骤，例如在多能丧失期间，或者是否有一个连续的重组 细胞谱系选择变得更加受限制，我们将检查下游谱系确定的内胚层 和中胚层以及中胚层与平滑肌区分开。最后，了解功能 域合并的重要性，我们将确定哪些基因受巩固的影响以及如何影响 它们与细胞谱系选择有关。 AIM 2：复制分析作为一种新颖的手段来表征HESC。 复制分析提供了一种方便的全面基因组识别方法，可能会脱落 阐明当前正在的细胞类型和多能细胞状态之间的重要关系 文学辩论。在此目标中，我们将确定将hESC与他们的hESC区分开的领域 差异化对应物。我们将检查小鼠与人之间保守同义区域的区域 复制域结构的进化保护。最后，我们将使用复制分析来解决 关于hESC是否与MESC或小鼠表皮样细胞更相似的争论。 AIM 3：分化过程中染色质的空间巩固。 在这里，我们将确定如何在AIM1中定义的复制域的巩固如何对应 这些结构域在细胞核中的三维重组。在原位杂交处 分化的不同阶段，我们将追踪相对于复制域的亚核定位 特定的亚核隔室，询问巩固时间合并的域是否也合并 在空间上以及他们是否获得了更均匀的总体压实水平。 目标4：域合并过程中复制叉的协调。在此目标中，我们将分析 复制叉的极性，复制子大小和复制起源活性在单个拉伸DNA上 来自合并域的纤维以识别复制子簇及其边界相对的位置 到复制域的边界，定义了分子。这些实验将开始定义 发生结构域合并的分子机制。