Genomic Regulation of Chromatin Accessibility during Drosophila Development

果蝇发育过程中染色质可及性的基因组调控

• 批准号: 7808971
• 负责人: Daniel J McKay
• 金额: $ 4.76万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2011-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7808971
• 关键词: Adult; Affect; Belief; Biology; Cell Count; Cell Nucleus; Cell division; Cells; Chromatin; Chromatin Structure; Collection; Communities; DNA; Data; Development; Developmental Gene Expression Regulation; Digestion; Disease; Double-Stranded RNA; Drosophila genus; Drosophila inturned protein; Embryo; Euchromatin; Formaldehyde; Gene Expression Regulation; Genes; Genome; Genomics; Heterochromatin; Knowledge; Malignant Neoplasms; Maps; Measures; Mediating; Microscopy; Modeling; Molecular; Molecular Genetics; Organism; Pathway interactions; Play; Polycomb; Process; Property; Proteins; Public Health; RNA Interference; Regulation; Regulatory Element; Research; Resistance; Resolution; Resources; Role; Specific qualifier value; Staging; System; Techniques; Time; Tissues; Validation; Work; cell determination; cell type; follow-up; genome-wide; human disease; in vivo; knock-down; novel; nuclease; programs

DESCRIPTION (provided by applicant): The organization of the genome into chromatin plays a central role in the regulation of developmental gene expression programs. One widely held belief to explain this regulation is the coordinated control of chromatin accessibility. However, there is little high resolution data measuring chromatin accessibility in vivo, the regulators of large scale chromatin accessibility are largely unknown, and the molecular mechanisms by which chromatin accessibility affects gene activity are unclear. This proposal describes a genomics approach to directly measure chromatin accessibility at high resolution in order to develop a system in the experimentally tractable Drosophila model that will examine the relationship between chromatin accessibility and gene activity. To accomplish this objective, we will utilize a technique developed by the Lieb lab termed FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements). We present data here not only demonstrating the ability of FAIRE to reproducibly isolate discrete regions of 'open' chromatin, but also to identify broad chromosomal regions that share chromatin accessibility properties, even using small numbers of cells. Specifically, we will first perform a genomics screen to identify novel regulators of chromatin accessibility by using RNAi-mediated knockdown in Drosophila S2 cells, followed by analysis of FAIRE-enriched DNA on microarrays. Second, we will generate genomic chromatin accessibility maps for different tissues and stages of Drosophila development by performing FAIRE followed by high-throughput sequencing, providing a framework to assess the relationship between chromatin accessibility and gene activity during development and identifying cis-regulatory modules that control developmental expression programs. Finally, we will begin to characterize potential regulators of chromatin accessibility in vivo through mutational analyses. Relevance to Public Health: Many developmental pathways and mechanisms, when misregulated, contribute to human disease. Therefore, the knowledge gained by these efforts will have applications in understanding mechanisms that underlie disease states such as cancer.

描述(由申请人提供)：基因组到染色质的组织在发育基因表达程序的调节中起着核心作用。解释这一规律的一个普遍看法是染色质可及性的协调控制。然而，目前在体内测量染色质可及性的高分辨率数据很少，大规模染色质可及性的调节因素在很大程度上是未知的，染色质可及性影响基因活性的分子机制也不清楚。这项建议描述了一种基因组学方法，以高分辨率直接测量染色质可及性，以便在实验上可驯化的果蝇模型中开发一种系统，该系统将检查染色质可及性与基因活性之间的关系。为了实现这一目标，我们将利用LIEB实验室开发的一种名为FIRE(甲醛辅助分离调节元件)的技术。我们在这里提供的数据不仅证明了FIRE能够重复地分离“开放”染色质的离散区域，而且还能够识别广泛的染色体区域，这些区域共享染色质可及性特性，即使使用少量细胞也是如此。具体地说，我们将首先进行基因组学筛选，通过在果蝇S2细胞中使用RNAi介导的击倒来确定染色质可及性的新调节因子，然后在微阵列上分析富含FIRE的DNA。其次，我们将通过FIRE和高通量测序为果蝇发育的不同组织和阶段生成基因组染色质可及性图，提供一个框架来评估染色质可及性和发育过程中基因活性之间的关系，并识别控制发育表达程序的顺式调控模块。最后，我们将开始通过突变分析来表征体内染色质可及性的潜在调节因子。与公共卫生的相关性：许多发展途径和机制在受到不当管理时，会导致人类疾病。因此，通过这些努力获得的知识将在理解癌症等疾病状态的基础机制方面有所应用。