Defining chromatin architecture and maturation at sites of DNA replication in the Drosophila melanogaster genome

定义果蝇基因组 DNA 复制位点的染色质结构和成熟

• 批准号: 9242656
• 负责人: Monica P Gutierrez
• 金额: $ 3.45万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9242656
• 关键词: Address; Architecture; Binding; Biochemical; Biological Assay; Biological Models; Biological Process; Cell Cycle; Cell Line; Cell division; Cells; Characteristics; Chromatin; Chromatin Disassembly; Chromatin Modeling; Chromatin Structure; Chromosomes; Complex; Coupled; Coupling; DNA; DNA Footprint; DNA biosynthesis; DNA replication fork; DNA replication origin; Data; Development; Drosophila genome; Drosophila genus; Drosophila melanogaster; Environment; Epigenetic Process; Euchromatin; Eukaryota; Event; Exhibits; Genetic; Genetic Transcription; Genome; Genomic Instability; Heterochromatin; Immunoprecipitation; In Vitro; Inherited; Institutes; Label; Laboratories; Lead; Location; Medicine; Micrococcal Nuclease; Mission; Monitor; Nucleosomes; Nucleotides; Pattern; Physiologic pulse; Positioning Attribute; Post-Translational Protein Processing; Process; Protein Footprinting; Proteins; Replication Origin; Resolution; Role; S Phase; Saccharomyces cerevisiae; Series; Site; Specific qualifier value; Structure; System; Testing; Work; base; cell type; chromatin immunoprecipitation; chromatin remodeling; chromosomal location; comparative; epigenome; experimental study; genome-wide; helicase; histone modification; human disease; in vivo; innovation; insight; mutant; novel; novel strategies; nucleoside analog; nucleotide analog; origin recognition complex; programs; protein E; protein complex; public health relevance; spatiotemporal; transcription factor; tumorigenesis; whole genome

 DESCRIPTION (provided by applicant): DNA replication is an essential process involved in both genetic and epigenetic inheritance. In every cell cycle, the entire genome must be duplicated in S-phase with DNA replication starting at multiple chromosomal locations, termed origins, marked by the origin recognition complex (ORC). In addition, the epigenetic state (e.g. nucleosome positioning, transcription factor binding, and post-translational histone modifications) must also be re-established behind the replication fork. Not surprisingly, DNA replication and the chromatin environment are mutually dependent. Increasing data suggests that chromatin structure is a primary determinant for origin selection and activation, and that chromatin assembly is tightly coupled to the replisome and passage of the DNA replication fork. Deregulation of either the DNA replication program or chromatin assembly may result in genomic instability and loss of the epigenetic state -- both of which are hallmarks of tumorigenesis. I propose to assess the chromatin architecture of Drosophila replication origins at nucleotide resolution. I will apply a recently developed micrococcal nuclease (MNase) based assay to 'footprint' protein-DNA occupancy throughout the Drosophila genome. This assay is capable of comprehensively resolving nucleosomes, transcription factors, replication factors, and chromatin remodelers at nucleotide resolution in factor agnostic manner that is independent of chromatin immunoprecipitation. I expect to identify specific chromatin signatures that define cell type specific patterns of ORC binding and origin activation. I will use mutant ATP-dependent chromatin remodelers to test the hypothesis that active chromatin remodeling is important to define start sites of DNA replication. In addition, I will develop a novel approach to characterize the spatiotemporal dynamics of chromatin maturation behind the replication fork by coupling the MNase based chromatin assay with the immunoprecipitation of an incorporated nucleotide analog, which will allow me to discriminate between nascent and mature chromatin. I expect to identify locus and factor specific differences in chromatin maturation providing new mechanistic insights into how the epigenetic state is inherited every cell cycle.

 描述（通过应用程序提供）：DNA复制是遗传和表观遗传涉及的重要过程。在每个细胞周期中，必须在S期中重复整个基因组，并在多个染色体位置开始，以原始识别复合物（ORC）为标志。此外，还必须在复制叉后面重新建立表观遗传态（例如核小体定位，转录因子结合和翻译后组蛋白修饰）。毫不奇怪，DNA复制和染色质环境是相互依赖的。越来越多的数据表明，染色质结构是原点选择和激活的主要确定剂，并且染色质组装紧密耦合到复制体和DNA复制叉的通过。 DNA复制程序或染色质组装的放松管制可能会导致基因组不稳定性和表观遗传状态的丧失 - 这两者都是肿瘤发生的标志。我建议评估果蝇复制起源的染色质结构。我将在整个果蝇基因组中应用最近开发的基于微球菌核酸酶（MNase）评估，以“足迹”蛋白-DNA占用率。该评估能够以核苷酸分辨率的因素不可知的方式全面解决核苷酸，转录因子，复制因子和染色质重塑剂，而核苷酸分辨率与染色质免疫沉淀无关。我希望确定特定的染色质特征，以定义细胞类型的特定于ORC结合和原点激活的模式。我将使用突变体ATP依赖性染色质重塑剂来测试以下假设：活动染色质重塑对于定义DNA复制的起始位点很重要。此外，我将开发一种新颖的方法来表征 复制叉背后染色质成熟的时空动力学通过将基于MNase的染色质测定与掺入的核毒性类似物的免疫沉淀耦合，这将使我能够区分新生和成熟的染色质。我期望确定染色质成熟的基因座和因素特定的差异，从而为表观遗传状态如何遗传每个细胞周期提供了新的机械见解。