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: 8254481
• 负责人: MATTHEW Steven SACHS
• 金额: $ 45.62万
• 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
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8254481
• 关键词: Advocate; Air; Base Sequence; Binding; Biological Models; Biology; Cataloging; Catalogs; Cells; 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; 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 (ChlP-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 ChlP-seq, using epitope-tagged strains developed in Project #1, at points across the time series, (iv) Map by ChlP-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” 丝状真菌对环境信号非常敏感，并利用这些信号来发出转变信号 从营养生长的早期到晚期，直至无性发育完成。这可能会发生 在一天的过程中对两个突出的环境线索做出反应——光线和暴露 空气/干燥 - 协同作用引起细胞转录组的全面变化。这 生物学是在模型系统脉孢菌中观察到的，该系统具有较小的基因组、易于处理的遗传学，并且（现在） 轻松重组。具有经济实惠的超高通量测序；整个脉孢菌基因组 可以像人类基因组的 1% 一样轻松地进行检查。因此，它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 中生成的资源和项目 #2 中描述的计算资源 Solexa 测序可识别和表征粗糙链孢霉 DNA 和染色质内的蛋白质元件， 在基因组规模上影响基因表达。我们寻求全系统对监管的理解 转录组和表观基因组的整体变化是由 触发发育的环境线索。基于 Solexa 的测序将与染色质免疫沉淀相结合 (ChlP-Seq) 识别与不同转录因子相关的染色质位点 核小体含有各种组蛋白修饰。胞嘧啶甲基化位点和核小体 将评估染色质的分相。这些分析将有助于理解组件和 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。具体目标如下： (i) 在接下来的一天内收集 14 次粗糙猪笼草组织 暴露于引发发育的线索（光和空气），(ii) 每次对完整转录组进行编目，(iii) 使用表位标记的菌株，通过 ChlP-seq 检查 50 多种相关转录因子与 DNA 的结合 在项目 #1 中开发，在整个时间序列的点上，(iv) 通过 ChlP-seq 绘制表观基因组地理图， 跟踪由具有特定修饰的组蛋白和组蛋白变体结合的区域； 50 个不同的物种 将在整个时间序列中的选定点进行跟踪，（v）绘制所有核小体和 实验开始时以及稍后确定的一个或多个额外时间的 DNA 甲基化 基于观察到最显着的表观基因组转变的时间。