Replication Domain Organization during hESC Differentiation

hESC 分化期间的复制域组织

• 批准号: 8382720
• 负责人: David M Gilbert
• 金额: $ 31.01万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8382720
• 关键词: Address; Affect; Biochemical; Biological; Biological Models; Cell Lineage; Cell Nucleus; Cells; Characteristics; Chromatin; Chromosome Structures; Chromosomes; DNA; DNA Sequence; Data Set; Ectoderm; Endoderm; Epiblast; Epigenetic Process; Fiber; Genes; Genetic; 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; Stretching; Structure; Syntenic Conservation; System; Testing; Time; base; cell type; dalton; genome wide association study; genome-wide; human embryonic stem cell; monolayer; novel; programs; research study; self-renewal; somatic cell nuclear transfer; stem cell differentiation

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分化过程中的复制结构域组织道尔顿，Stephen A.具体目标。 许多细胞学、遗传学和生物化学的高级染色体结构域的定义已经提出， 第四，但没有一个提供精确地划定域边界的属性，并且以一种 可以应用于全基因组。因此，虽然“域”一词经常用于描述 由于染色体结构域是一个大的，通常是多基因的染色体单元，因此对染色体结构域没有全面的定义。 我们证明，“复制域”是一个可定义的染色体单位，揭示了复制， 可能是唯一的染色体特性，提供了一个全面的分割整个基因组， 兆位水平。重要的是，我们发现这些领域的边界是戏剧性的， 在mESC和hESC分化过程中重组，以产生更大的时间整合结构域，其中 复制时间与染色体的序列性质更密切相关。因此，复制 胚胎干细胞中染色体的结构相对不受DNA序列的限制， 多能细胞的新特性。我们建议进一步研究结构域的生物学意义 巩固，其与细胞核中染色质的3D组织的关系以及 使用hESC作为模型系统进行整合。具体目标如下： 目的1：在血统承诺期间复制域边界的编排。 在这个目标中，我们将测试复制域边界是特定细胞特征的假设， 类型更具体地说，我们提出，较小的时间上不同的复制域是一个特点， 干细胞，分化将伴随着一个渐进的更严格的关系， 等容线序列组成和复制时间。为了确定边界是否是谱系特定的， 我们将研究hESC分化为独立胚细胞过程中复制域边界的变化， 层、外胚层和中内胚层。要区分复制域边界的更改是否发生在 一个单一的步骤，例如在失去多元性，或是否有一个连续的重组， 细胞谱系的选择变得更加有限，我们将检查下游谱系定形内胚层 和中胚层，以及分化为平滑肌的中胚层。最后，了解功能 结构域巩固的意义，我们将确定哪些基因受到巩固以及如何影响 它们与细胞谱系选择有关。 目的2：复制谱作为表征hESC的新手段。 复制分析提供了一种方便的全面的全基因组鉴定方法， 阐明细胞类型和多能细胞状态之间的重要关系， 在文学中争论。在这个目标中，我们将确定区分hESC与它们的结构域。 不同的对手。我们将研究小鼠和人类之间保守的同线性区域， 复制域结构的进化保守性。最后，我们将使用复制分析来解决 关于hESC是否更类似于mESC或小鼠外胚层样细胞的争论。 目的3：分化过程中染色质的空间整合。 在这里，我们将确定如何巩固的复制域定义的分子在Aim1对应 细胞核中这些区域的三维重组。使用原位杂交技术， 在分化的不同阶段，我们将追踪复制结构域的亚核定位， 特定的亚核区室，以询问暂时合并的结构域是否也合并 在空间上，以及它们是否获得更均匀的整体压实水平。 目标4：在域合并期间协调复制分叉。在这篇文章中，我们将分析 复制叉的极性、复制子的大小和单个伸展DNA上的复制起点活性区域 纤维从巩固域，以确定复制子集群和它们的边界相对的位置 到分子定义的复制域边界。这些实验将开始定义 域合并发生的分子机制。