Discovery and Analysis of Network Components via High Throughput Sequencing

通过高通量测序发现和分析网络组件

• 批准号: 8466989
• 负责人: MATTHEW Steven SACHS
• 金额: $ 44.59万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8466989
• 关键词: Advocate; Air; Base Sequence; Binding; Biological Models; Biology; Cataloging; Catalogs; Cells; ChIP-seq; Chromatin; Computer Simulation; Coupled; Cues; Custom; Cytosine; DNA; DNA Methylation; Data; Data Analyses; Data Set; Desiccation; Development; Elements; Epitopes; Exposure to; Gene Expression; Gene Expression Profile; Genetic; Genome; Geography; Global Change; Growth; Health; Histones; Human; Human Genome; Immune Sera; Knock-out; Lead; Light; Location; Maps; Methylation; Modeling; Molds; Neurospora; Neurospora crassa; Nucleosomes; Organism; Pathway Analysis; Phase; Post-Translational Protein Processing; Process; Proteins; Reproduction spores; Resources; Series; Signal Transduction; Site; Social Welfare; Staging; System; Systems Biology; Technology; Testing; Time; Tissues; Transcriptional Regulation; Variant; Work; asexual; base; chromatin immunoprecipitation; computing resources; epigenome; epigenomics; fungus; histone modification; interest; research study; response; transcription factor; transcriptomics

Project 3: "Discovery and Analysis of Network Components via High Throughput Sequencing" Filamentous fungi are exquisitely sensitive to environmental cues, and use these to signal the transition from early to late stage vegetative growth and on to completion of asexual development. This can happen over the course of a day in response to two prominent environmental cues - light and exposure to air/desiccation - that act synergistically to elicit comprehensive changes in the cell's transcriptome. This biology is observed in a model system, Neurospora, with a small genome, tractable genetics, and (now) facile recombineering. With affordable ultra high through-put sequencing; the entire Neurospora genome can be examined as easily as 1% of the human genome. It is thus an exceptionally good test system for describing, modeling, and understanding the interplay between the transcriptome and the epigenome. We will leverage resources generated in Project #1 and the computational resources described in Project #2 with Solexa sequencing to identify and characterize N. crassa DNA and protein elements within chromatin that influence gene expression at the genome scale. We seek a system-wide understanding of the regulatory underlying the global changes in the transcriptome and epigenome elicited by, and accompanying, the environmental cues that trigger development. Solexa-based sequencing will be coupled to chromatinimmunoprecipitation (ChIP-Seq) to identify chromatin sites associated with different transcription factors and with nucleosomes containing various histone modifications. Sites of cytosine methylation and nucleosome phasing in chromatin will be assessed. These analyses will lead to understanding the components and interactions of the regulatory networks controlling this organism's responses to light and air, and to development. Specific aims are as follows: (i) Collect N. crassa tissue at 14 times spanning a day following exposure to cues (light and air) that elicit development, (ii) Catalog the full transcriptome at each time, (iii) Examine binding of over 50 relevant transcription factors to DNA by ChIP-seq, using epitope-tagged strains developed in Project #1, at points across the time series, (iv) Map by ChIP-seq the epigenome's geography, tracking regions bound by histones bearing specific modifications and by histone variants; 50 distinct species will be followed at selected points across the time series, (v) Map the locations of all the nucleosomes and of DNA methylation at the outset of the experiment, and at one or more additional times to be determined later based on when the most significant epigenomic transitions are observed.

项目3：“通过高通量测序发现和分析网络组件” 丝状真菌对环境提示非常敏感，并使用它们来发出过渡的信号 从早期到晚期的营养生长，再到无性发展的完成。这可能会发生 在一天的过程中，响应两个突出的环境线索 - 光线和暴露 空气/干燥 - 协同作用以引起细胞转录组的全面变化。这 在模型系统神经孢子中观察到生物学，具有较小的基因组，可处理的遗传学，并且（现在） 便捷的重新组合。负担得起的超高贯穿测序；整个Neurospora基因组 可以像人类基因组的1％一样容易研究。因此，这是一个非常好的测试系统 描述，建模和理解转录组和表观基因组之间的相互作用。我们 将利用项目＃1中生成的资源以及项目＃2中描述的计算资源 solexa测序以识别和表征染色质中的Crassa DNA和蛋白质元素， 在基因组量表上影响基因表达。我们寻求对监管的全系统理解 基本的转录组和表观基因组的全球变化是由 触发发展的环境提示。基于Solexa的测序将耦合到染色质原则 （chip-seq）确定与不同转录因子和不同的染色质位点和 含有各种组蛋白修饰的核小体。胞嘧啶甲基化和核小体部位 将评估染色质中的相位。这些分析将导致理解组件和 控制该生物体对光和空气的反应的调节网络的相互作用，以及 发展。具体目的如下：（i）在14次跨越后的一天中收集N. crassa组织 暴露于引起发展的提示（光和空气），（ii）每次分类完整的转录组，（iii） 使用表位标记的菌株，通过ChIP-Seq检查了50多个相关转录因子与DNA的结合 在项目＃1中开发的，在时间序列上的点，（iv）图中的地图，表观基因组的地理， 跟踪受组蛋白结合的区域，具有特定的修饰和组蛋白变体； 50种不同的物种 将在整个时间序列的选定点上遵循，（v）绘制所有核小体和的位置 实验开始时的DNA甲基化，并在一个或多个额外的时间待确定 基于观察到最显着的表观基因组过渡的何时。