Mechanisms Regulating DNA Replication in the Developing Vertebrate Embryo

脊椎动物胚胎发育中 DNA 复制的调节机制

基本信息

批准号：
9216837
负责人：
CHRISTOPHER L SANSAM
金额：
$ 36.26万
依托单位：
OKLAHOMA MEDICAL RESEARCH FOUNDATION
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-03-01 至 2022-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9216837
关键词：
Acetylation Affect Base Pairing Binding Proteins Biological Assay Bromodomain Cancer Etiology Cell Culture Techniques Cell Differentiation process Cells Characteristics Chromatin Chromatin Structure Chromosomes DNA biosynthesis DNA replication fork Data Deacetylation Defect Development Disease Down-Regulation Embryo Embryonic Development Epigenetic Process Euchromatin Feedback Gene Expression Genes Genetic Genetic Transcription Genome Genomic Instability Genomic Segment Genomics Goals Grant Health Heterochromatin Histone Acetylation Histone Deacetylation Histones Human Individual Knowledge Life Link Maintenance Malignant Neoplasms Measurement Mediating Medical Mission Modeling Modification Mutate Mutation Nature Organism Pattern Peptide Initiation Factors Phosphoric Monoester Hydrolases Play Proteins Public Health Reader Recruitment Activity Regulatory Element Replication Initiation Research Research Proposals Role S Phase Shapes Switch Genes System Testing Time United States National Institutes of Health Work Zebrafish apoAI regulatory protein-1 cell type developmental disease disability experimental study frontier gastrulation improved in vivo in vivo Model innovation insight novel prevent programs spatiotemporal transcriptome sequencing vertebrate embryos

项目摘要

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.

项目概要/摘要在展示如何调节个体复制叉的启动方面不断取得巨大进步，并且下一个前沿领域是了解 DNA 复制如何与转录和染色质结构协调。特别是，人们对脊椎动物DNA复制控制的机制和功能知之甚少。当转录和染色质结构高度动态时，这种知识差距是一个问题。这是一个重要的问题，因为在这个问题得到解决之前，DNA 复制在发育障碍和与表观遗传或 DNA 复制失调相关的癌症在很大程度上是难以理解的。类型以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申请人已经建立了一个易于处理的斑马鱼模型，可以轻松测量和分析。当添加表观遗传标记时，控制发育中胚胎的复制时间，如转录随着细胞的分化，这项资助的总体目标是定义复制机制。该研究计划旨在实现三个具体目标：1）确定Rif1在复制时间和染色质结构发育控制中的功能；确定单个基因是否驱动全域复制时间变化；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计时模式在目标 1 中，申请人将。他们的数据显示，从早期到晚期复制时间变化近 50 Mb 需要 Rif1 申请人将使用他们已建立的复制计时分析以及 RNAseq。和 ChipSeq 来测试 Chr4q 的异染色质化是否需要依赖 Rif1 的定时开关。目标 2 中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申请人将诱导遗传和表观遗传修饰来测试模型是否可复制。时序转换基因 (nr2f2) 对于跨 1.6 Mb 基因组结构域的时序变化来说是必要且充分的。 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，关键复制起始蛋白（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 发生突变以防止复制。这种相互作用是创新的，因为它将是第一个使用真正的体内脊椎动物的研究。胚胎模型研究 DNA 复制如何与动态转录和表观遗传协调这项工作将意义重大，因为它将回答有关如何以及为何发生变化的基本问题。考虑到DNA之间的功能相互作用，时空DNA复制模式会在发育过程中发生变化。 DNA 复制和表观遗传变化，这些研究最终将增进对广泛领域的理解与表观遗传失调相关的疾病。