cis-Acting Elements Regulating Developmental Control of Replication Timing

描述(由申请人提供)： 项目摘要/摘要在许多疾病中观察到了复制的异常时间控制，但因果联系尚不清楚。在了解正常发育过程中调控复制时间的机制之前，这种差距仍将是不可理解的。长期目标是了解复制时机与细胞表观遗传状态和疾病的关系。目前的目标是确定顺式作用的DNA/染色质元件，这些元件调节小鼠胚胎干细胞(ESCs)分化过程中复制时间的变化。小鼠胚胎干细胞是一个理想的实验系统，因为有染色体工程工具、定向细胞分化系统和全面的全基因组复制计时和转录图谱。这些图谱已经确定了复制计时中发生的程序性变化的分子坐标，这些变化以400-800kb的单位发生，称为“复制域”。中心假设是离散的可识别的染色质或DNA序列特征决定了复制域的边界和发育引起的复制时间的变化。这一建议的基本原理是，确定调控复制时机的DNA/染色质元件是阐明调控复制时机及其与疾病的关系的机制的关键下一步。AIM1将检验这样一个假设，即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。来自发育调节复制域的大片段克隆的基因组DNA将被引入到构成复制计时的区域。在分化过程中，将监测插入DNA和侧翼DNA的复制时间，以确定构成调控单位的最小序列。AIM2将区分两种模型，一种是特定边界元素点缀在时间上不同的区域，另一种是边界模型，即主动编程区域之间被动复制的染色质。嵌套缺失将在发育控制的复制计时过渡区中设计，并将确定缺失对复制计时调节的影响。AIM3将测试这一假设，即沉默的晚期复制结构域中的转录启动向早期复制的切换。在发育调节的复制结构域内控制转录的启动子和调控元件将被删除，代之以可诱导的启动子，并将分析这种操作对复制时机调节的影响。这里描述的研究将确定调控复制时间的发育控制的顺式作用元件。这一贡献是重要的，因为确定复制定时控制的调节元件是理解复制定时在基于染色体的疾病中的作用的先决条件。这里提出的工作是创新的，它提出了一种染色体工程和定向胚胎干细胞(ESC)分化的新组合，以解决在复制时间引起发育程序性变化的机制。

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.