cis-Acting Elements Regulating Developmental Control of Replication Timing

描述（由申请人提供）： 在许多疾病中观察到复制的异常时间控制，但因果关系尚不清楚。在正常发育过程中调节复制时间的机制被理解之前，这种差距仍然是不可理解的。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。目前的目标是确定顺式作用的DNA/染色质元件，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，由于可用的染色体工程工具，定向细胞分化系统，和全面的全基因组复制定时和转录图谱。这些图谱已经确定了复制时间程序性变化的分子坐标，这些变化发生在称为“复制结构域”的400- 800 kb单元中。核心假设是，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诱导的复制时间的变化。这一建议的基本原理是，确定DNA/染色质元件调节复制时间是阐明机制调节复制时间及其与疾病的关系的重要下一步。目的1将测试的假设，复制域是染色体结构和功能的基本单位，可以转移到一个异位位置。将来自发育调节复制结构域的克隆基因组DNA的大片段引入组成性复制时序区域。在分化过程中监测插入片段和侧翼DNA的复制时间，以鉴定构成调控单元的最小序列。Aim 2将区分特定边界元件在时间上不同的结构域之间标点的模型与作为主动编程结构域之间的被动复制染色质的边界模型。巢式缺失将在发育控制的复制时序过渡区进行工程化，并将确定缺失对复制时序调节的影响。aim 3将测试沉默的晚期复制结构域内的转录启动向早期复制的转换的假设。在发育调控的复制结构域内控制转录的启动子和调控元件将被删除，用诱导型启动子取代，并将分析此类操作对复制时机调控的影响。这里描述的研究将确定顺式作用元件调节复制时间的发育控制。这一贡献是重要的，因为确定复制时间控制的调控元件是理解复制时间在染色体疾病中的作用的先决条件。这里提出的工作是创新的，因为它提出了一种新的染色体工程和定向胚胎干细胞（ESC）分化的组合，以解决机制引起发育编程的复制时间的变化。

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.