Mechanisms Regulating DNA Replication in the Developing Vertebrate Embryo

项目摘要/摘要 在显示个体复制分叉的启动是如何被调节的方面不断取得巨大进展，并且 下一个前沿是了解DNA复制是如何与转录和染色质结构相协调的。 特别是，对脊椎动物DNA复制控制的机制和功能知之甚少。 发育，当转录和染色质结构高度动态时。这种知识上的差距是一种 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复制放松管制相关的癌症将在很大程度上令人费解。每一个细胞 TYPE以一种独特的时空模式复制其基因组，该模式随转录和表观遗传而变化 修改。申请人已经建立了一种易于驯服的斑马鱼模型，该模型允许轻松测量和 随着表观遗传标记的加入，在发育中的胚胎中复制时间的操纵，如转录 开始，随着细胞的分化。这笔赠款的总体目标是定义复制机制 斑马鱼胚胎的时间变化。该研究提案寻求完成三个具体目标：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间的变化；以及3)确定如何 复制起始因子的表观遗传靶向驱动复制计时模式。在目标1中，申请者将 根据他们的数据显示，需要Rif1进行从早到晚的近50 Mb的复制时间更改 基因组片段(Chr4q)。申请者将使用他们建立的复制时间测定以及RNAseq 和ChipSeq来测试是否需要Rif1依赖的定时开关才能实现Chr4q的异染色化。 Aim 2的实验将测试单个基因是否可以在顺式结构中起作用，以驱动全域复制。 改变。申请人将诱导遗传和表观遗传学修改，以测试模型复制是否- 时序转换基因(Nr2f2)对于跨越1.6Mb基因组结构域的时序变化是必要的，也是充分的。 AIM 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，在关键的复制起始蛋白(TICRR)和组蛋白乙酰化之间 “读者”。将在人类细胞和斑马鱼中分析复制时间，其中TICRR发生突变以防止 这种互动。这项拟议的研究具有创新性，因为它将是第一次使用真正的活体脊椎动物 研究DNA复制如何与动态转录和表观遗传协调的胚胎模型 改变。这项工作将具有重要意义，因为它将回答有关如何以及为什么的基本问题 时空DNA复制模式在发育过程中会发生变化。鉴于两者之间的功能相互作用 DNA复制和表观遗传学的变化，这些研究最终将提高对大范围的认识 与表观遗传去调控相关的疾病。

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.