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: 8912084
• 负责人: Monica P Gutierrez
• 金额: $ 3.36万
• 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/8912084
• 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; Histones; Immunoprecipitation; In Vitro; Inherited; Institutes; Label; Laboratories; Lead; Location; Maps; 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; epigenome; genome-wide; helicase; histone modification; human disease; in vivo; innovation; insight; mutant; novel; novel strategies; nucleoside analog; nucleotide analog; origin recognition complex; programs; protein complex; public health relevance; research study; spatiotemporal; transcription factor; tumorigenesis

 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（由申请人提供）：DNA 复制是涉及遗传和表观遗传的重要过程。在每个细胞周期中，整个基因组必须在 S 期进行复制，DNA 复制从多个染色体位置开始，称为起点，由起点识别复合物 (ORC) 标记。此外，表观遗传状态（例如核小体定位、转录因子结合和翻译后组蛋白修饰）也必须在复制叉后面重新建立。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，染色质结构是起点选择和激活的主要决定因素，并且染色质组装与复制体和 DNA 复制叉的通道紧密耦合。 DNA 复制程序或染色质组装的失调可能会导致基因组不稳定和表观遗传状态的丧失——这两者都是肿瘤发生的标志。 我建议在核苷酸分辨率下评估果蝇复制起点的染色质结构。我将应用最近开发的基于微球菌核酸酶 (MNase) 的测定法来“足迹”蛋白质-DNA 在整个果蝇基因组中的占据情况。该测定能够以独立于染色质免疫沉淀的因素不可知的方式以核苷酸分辨率全面解析核小体、转录因子、复制因子和染色质重塑因子。我希望能够识别出特定的染色质特征，这些特征可以定义 ORC 结合和起源激活的细胞类型特定模式。我将使用突变型 ATP 依赖性染色质重塑剂来检验以下假设：主动染色质重塑对于定义 DNA 复制起始位点很重要。此外，我将开发一种新颖的方法来表征 通过将基于 MNase 的染色质测定与掺入的核苷酸类似物的免疫沉淀相结合，研究复制叉后面染色质成熟的时空动态，这将使我能够区分新生和成熟染色质。我期望识别染色质成熟中的位点和因子特异性差异，为表观遗传状态如何在每个细胞周期遗传提供新的机制见解。