Mechanisms Regulating DNA Replication in the Developing Vertebrate Embryo

项目总结/摘要 在显示个体复制叉的启动是如何被调节的方面不断取得重大进展， 下一个前沿是了解DNA复制是如何与转录和染色质结构协调的。 特别是脊椎动物DNA复制控制的机制和功能， 当转录和染色质结构是高度动态的时，发育。这种知识上的差距是 这是一个重要的问题，因为在它被填补之前，DNA复制在发育障碍和 与表观遗传或DNA复制失调有关的癌症在很大程度上是不可理解的。每一个细胞 类型复制其基因组在一个独特的时空模式，随着转录和表观遗传的变化， 修改.申请人已经建立了易于处理的斑马鱼模型，其允许容易的测量和 在发育中的胚胎中操纵复制时间，因为添加了表观遗传标记，如转录 开始，并随着细胞分化。这项赠款的总体目标是确定推广机制 斑马鱼胚胎的时间变化。研究计划旨在达到三个具体目标：1） 确定Rif1在复制时间和染色质结构的发育控制中的功能; 2） 确定单个基因是否驱动全域复制时序变化;以及3）确定如何驱动 复制起始因子的表观遗传靶向驱动复制定时模式。在目标1中，申请人将 他们的数据表明，从早到晚的复制时序变化需要Rif1， 基因组片段（Chr4q）。申请人将使用其已建立的复制计时测定以及RNaseq 和ChipSeq来测试Chr4q的异染色质化是否需要Rif1依赖性定时开关。 目标2的实验将测试单个基因是否可以顺式作用以驱动全域复制 变化申请人将诱导遗传和表观遗传修饰，以测试模型复制是否- 定时转换基因（nr2f2）对于跨越1.6Mb基因组结构域的定时改变是必要的和足够的。 目标3的工作将测试乙酰化染色质的早期复制是否依赖于物理相互作用， 申请人发现，在关键复制起始蛋白（TICRR）和组蛋白乙酰化之间， "读者"。复制时间将在人类细胞和斑马鱼中进行分析，其中TICRR突变以防止 这种互动。这项研究是创新的，因为它将是第一个使用真正的体内脊椎动物 胚胎模型，以研究DNA复制如何与动态转录和表观遗传协调 变化这项工作意义重大，因为它将回答关于如何和为什么的基本问题 时空DNA复制模式在发育过程中发生变化。鉴于之间的功能相互作用 DNA复制和表观遗传变化，这些研究将最终提高对广泛的 与表观遗传失调有关的疾病。

PROJECT SUMMARY/ABSTRACT Great strides are continually made in showing how the initiation of individual replication forks is regulated, and the next frontier is to understand how DNA replication is coordinated with transcription and chromatin structure. In particular, little is known about the mechanisms and functions of DNA replication control during vertebrate development, when transcription and chromatin structure are highly dynamic. This gap in knowledge is an important problem because, until it is filled, the roles for DNA replication in developmental disorders and cancers associated with epigenetic or DNA replication deregulation will be largely incomprehensible. Every cell type replicates its genome in a unique spatiotemporal pattern that changes with transcription and epigenetic modifications. The applicant has established a tractable zebrafish model that allows easy measurement and manipulation of replication timing in the developing embryo as epigenetic marks are added, as transcription begins, and as cells differentiate. The overall objective of this grant is to define the mechanisms of replication timing changes in the zebrafish embryo. The research proposal seeks to complete three specific aims: 1) Determine the function of Rif1 in the developmental control of replication timing and chromatin structure; 2) Determine whether individual genes drive domain-wide replication timing changes; and 3) Determine how epigenetic targeting of replication initiation factors drives replication timing patterns. In Aim 1, the applicant will build on their data showing that Rif1 is required for an early-to-late replication timing change of a nearly 50 Mb genomic segment (Chr4q). The applicant will use their established replication timing assays as well as RNAseq and ChipSeq to test whether heterochromatinization of Chr4q requires the Rif1-dependent timing switch. Experiments in Aim 2 will test whether an individual gene can act in cis to drive domain-wide replication changes. The applicant will induce genetic and epigenetic modifications to test whether a model replication- timing switching gene (nr2f2) is necessary and sufficient for a timing change across a 1.6 Mb genomic domain. Work in Aim 3 will test whether early replication of acetylated chromatin depends on a physical interaction, which the applicant discovered, between a key replication initiation protein (TICRR) and a histone acetylation “reader”. Replication timing will be profiled in human cells and zebrafish in which TICRR is mutated to prevent that interaction. The proposed research is innovative because it will be the first using a true in vivo vertebrate embryo model to investigate how DNA replication is coordinated with dynamic transcriptional and epigenetic changes. This work will be significant because it will answer fundamental questions about how and why spatiotemporal DNA replication patterns change during development. Given the functional interplay between DNA replication and epigenetic changes, these studies will ultimately improve understanding of a wide-range of diseases associated with epigenetic deregulation.