Replication domain organization during hESC differentiation

hESC 分化过程中的复制域组织

• 批准号: 8641824
• 负责人: David M Gilbert
• 金额: $ 33万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8641824
• 关键词: Address; Affect; Architecture; Biological; Cell Cycle; Cell Cycle Regulation; Cell Nucleus; Cells; Chromatin; Chromosome Structures; Chromosomes; Data; Dependence; Development; Disease; Dissection; Event; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Germ Layers; Goals; Human; Human Development; Investigation; Knowledge; Life; Link; Location; Malignant Neoplasms; Mediating; Methods; Molecular; Pathogenesis; Pathway interactions; Phase; Positioning Attribute; Program Research Project Grants; Property; Proteins; Public Health; Regulation; Replicon; Research; Role; S Phase; Signal Transduction; Structure; System; Testing; Time; Work; cellular imaging; chromatin remodeling; chromosome replication; computerized data processing; design; gene therapy; genome-wide; histone modification; human disease; human embryonic stem cell; imaging modality; innovation; insight; programs; response; self-renewal; stem cell differentiation

Abnormal large-scale chromosome structure is a hallmark of cancer and many other human diseases, but the mechanisms linking chromosome structure to function remain unresolved. The temporal order of replication is developmentally regulated at the level of large (400-800kb) "replication domains" that correspond to stable units of chromosome structure. Our long-term goal is to understand the role of largescale chromosome architecture in cell fate transitions. The objective of this application is to determine the causal interdependency of changes in replication timing and its correlated chromosome properties during human embryonic stem cell (hESC) differentiation. Our central hypothesis is that differentiation signals directiy modify replication timing to alter chromatin composition, which will in turn influence 3D folding in the next cell cycle, contributing to the robustness of transcription networks. Our rationale is that knowledge of causal relationships is an essential first step of mechanistic studies linking large-scale chromosome structure to cell fate transitions. Aimi will determine the order in which changes in replication timing, histone modifications, 3P chromatin interactions and transcription occur in response to differentiation and their dependence upon completion of prior events. Preliminary data describe newly developed hESC differentiation and cell cycle synchronization methods that can achieve this goal. Aim2 will test the hypothesis that human Rifl protein, which we recentiy identified as essential to maintain replication timing, is redistributed during differentiation to regulate replication timing. Gene disruption, genome wide ChlP, and single cell methods will localize Rifl and determine its role in regulating replication and transcription. These Aims are significant because identifying causal relationships and molecular players involved will remove a major obstacle in the field, paving the way to investigate mechanisms linking large-scale chromosome structure to cell fate commitment and, ultimately, human disease. The work is innovative in developing a system to study cell cycle regulated events in response to differentiation and in pioneering investigations into the newly identified role of Rifl in replication timing during early human development.

异常的大规模染色体结构是癌症和许多其他人类疾病的标志，但将染色体结构与功能联系起来的机制仍然没有得到解决。复制的时间顺序在发育过程中在大的（400- 800 kb）“复制域”水平上受到调节，这些复制域对应于染色体结构的稳定单位。我们的长期目标是了解大规模染色体结构在细胞命运转变中的作用。本申请的目的是确定人类胚胎干细胞（hESC）分化过程中复制时间及其相关染色体特性变化的因果关系。我们的中心假设是，分化信号直接修改复制时机以改变染色质组成，这反过来又会影响下一个细胞周期中的3D折叠，从而有助于转录网络的鲁棒性。我们的基本原理是，因果关系的知识是连接大规模染色体结构的机制研究的必要的第一步 细胞命运的转变。艾米将决定复制时间、组蛋白 修饰、3 P染色质相互作用和转录响应于分化及其对先前事件完成的依赖性而发生。初步数据描述了新开发的hESC 分化和细胞周期同步的方法，可以实现这一目标。aim 2将测试 假设我们最近鉴定为维持复制定时所必需的人Rifl蛋白在分化期间重新分布以调节复制定时。基因破坏、全基因组ChIP和单细胞方法将定位Rifl并确定其在调节复制和转录中的作用。这些目标是重要的，因为确定因果关系和分子参与者将消除该领域的主要障碍，为研究大规模染色体结构与细胞命运承诺以及最终人类疾病之间的联系机制铺平道路。这项工作在开发一个系统来研究细胞周期调节事件响应分化和开拓性的调查到新确定的作用Rifl在复制时间在早期人类发育过程中是创新的。