cis-Acting Elements Regulating Developmental Control of Replication Timing

调节复制时间发育控制的顺式作用元件

• 批准号: 8238959
• 负责人: David M Gilbert
• 金额: $ 29.69万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-30 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8238959
• 关键词: Address; Boundary Elements; Cations; Cell Differentiation process; Cell Nucleus; Cell division; Cells; Characteristics; Chromatin; Chromosome Structures; Chromosomes; DNA; DNA Sequence; DNA biosynthesis; Development; Disease; Dissection; Eating; Elements; Engineering; Epigenetic Process; Euchromatin; Eukaryotic Cell; Genes; Genetic; Genetic Recombination; Genetic Transcription; Genome; Genomics; Goals; Growth and Development function; Heterochromatin; Human; Human Chromosomes; Integrase; Knowledge; Link; Location; Malignant Neoplasms; Mammals; Maps; Mediating; Mission; Modeling; Molecular; Monitor; Mus; Nuclear; Pathogenesis; Public Health; Regulation; Regulatory Element; Replicon; Role; Shipping; Ships; Site; Specific qualifier value; System; Testing; Time; Tissues; Transcriptional Regulation; Work; abstracting; base; cell type; cis acting element; disability; embryonic stem cell; genome-wide; human disease; innovation; insight; novel; programs; promoter; research study; stem cell differentiation; tool

DESCRIPTION (provided by applicant): PROJECT SUMMARY / ABSTRACT Abnormal temporal control of replication is observed in many diseases but causal linkages are unknown. This gap will remain incomprehensible until the mechanisms regulating replication timing during normal development are understood. The long-term goal is to understand the relationship of replication timing to cellular epigenetic states and disease. The immediate goal is to identify cis-acting DNA/chromatin elements that regulate changes in replication timing during differentiation of mouse embryonic stem cells (ESCs). Mouse ESCs are an ideal experimental system due to the availability of chromosome engineering tools, directed cell differentiation systems, and comprehensive genome-wide maps of replication timing and transcription. These maps have identified the molecular coordinates of programmed changes in replication timing that occur in 400-800kb units termed "replication domains". The central hypothesis is that discrete identifiable chromatin or DNA sequence features dictate the boundaries of replication domains and the developmentally induced changes in their replication time. The rationale for this proposal is that identifying DNA/chromatin elements regulating replication timing is the essential next step in elucidating mechanisms regulating replication timing and its relationship to disease. Aim1 will test the hypothesis that replication domains are fundamental units of chromosome structure and function that can be transferred to an ectopic location. Large pieces of cloned genomic DNA from a developmentally regulated replication domain will be introduced into a region of constitutive replication timing. Repli- cation timing of the insert and flanking DNA will be monitored during differentiation to identify the minimal sequences constituting a unit of regulation. Aim2 will distinguish between models in which specific boundary elements punctuate temporally distinct domains vs. models of boundaries as passively replicated chromatin between actively programmed domains. Nested deletions will be engineered in developmentally controlled replication timing transition regions and the effects of deletions on the regulation of replication timing will be determined. Aim3 will test the hypothesis that transcription within a silent late replicating domain initiates a switch to early replication. Promoter and regulatory elements controlling transcription within a developmentally regulated replication domain will be deleted, replaced with an inducible promoter, and the effects of such manipulations on the regulation of replication timing will be analyzed. Studies described here will identify cis-acting elements regulating the developmental control of replication timing. This contribution is significant because identifying regulatory elements of replication timing control is a pre-requisite to understanding the role of replication timing in chromosome-based diseases. The work proposed here is innovative in that it proposes a novel combination of chromosome engineering and directed embryonic stem cell (ESC) differentiation to address the mechanisms eliciting developmentally programmed changes in replication timing. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE Accurate duplication of chromosomes during each cell division is essential to normal growth and development. The proposed project is important for public health because abnormalities in the temporal order in which chromosome segments are duplicated have been detected in many diseases and are expected to reflect the origins of these diseases, yet we have a poor understanding of how replication timing is regulated and why it is disrupted in disease states. Thus, the proposed experiments are relevant to the part of NIH's mission that pertains to developing fundamental knowledge that will increase our understanding of the pathogenesis of disease, suggest novel treatments, and reduce the burdens of human disability.

描述（由申请人提供）： 项目摘要/摘要 在许多疾病中观察到复制的时间异常控制，但因果关系尚不清楚。在理解正常发育期间调节复制时间的机制之前，这一差距将仍然难以理解。长期目标是了解复制时间与细胞表观遗传状态和疾病的关系。近期目标是鉴定顺式作用 DNA/染色质元件，调节小鼠胚胎干细胞 (ESC) 分化过程中复制时间的变化。由于染色体工程工具、定向细胞分化系统以及全面的全基因组复制时间和转录图谱的可用性，小鼠 ESC 是理想的实验系统。这些图谱已经确定了复制时间程序化变化的分子坐标，这些变化发生在称为“复制域”的 400-800kb 单元中。中心假设是离散的可识别染色质或 DNA 序列特征决定了复制域的边界以及发育诱导的复制时间变化。该提议的基本原理是，识别调节复制时间的 DNA/染色质元件是阐明调节复制时间的机制及其与疾病关系的重要下一步。 Aim1 将检验以下假设：复制域是染色体结构和功能的基本单位，可以转移到异位位置。来自发育调控复制域的大片段克隆基因组 DNA 将被引入组成型复制计时区域。在分化过程中将监测插入片段和侧翼DNA的复制时间，以识别构成调控单元的最小序列。 Aim2 将区分特定边界元素在时间上打断不同域的模型与作为主动编程域之间被动复制染色质的边界模型。将在发育控制的复制时间过渡区域中设计嵌套删除，并确定删除对复制时间调节的影响。 Aim3 将测试以下假设：沉默的晚期复制域内的转录启动向早期复制的切换。发育调控的复制域内控制转录的启动子和调控元件将被删除，并用诱导型启动子取代，并且将分析此类操作对复制计时调控的影响。这里描述的研究将确定调节复制时间发育控制的顺式作用元件。这一贡献意义重大，因为识别复制计时控制的调控元件是了解复制计时在染色体疾病中的作用的先决条件。这里提出的工作具有创新性，因为它提出了染色体工程和定向胚胎干细胞（ESC）分化的新颖组合，以解决引起复制时间发育编程变化的机制。 公共卫生相关性： 项目叙述 每次细胞分裂期间染色体的精确复制对于正常生长和发育至关重要。该项目对公共卫生很重要，因为在许多疾病中都检测到了染色体片段复制时间顺序的异常，并有望反映这些疾病的起源，但我们对复制时间是如何调节的以及为什么它在疾病状态下被破坏知之甚少。因此，拟议的实验与 NIH 使命的一部分相关，即发展基础知识，这将增加我们对疾病发病机制的理解，提出新的治疗方法，并减轻人类残疾的负担。