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.