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

基本信息

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
项目类别：
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

项目摘要

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 (ChIP-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 ChIP-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 ChIP-seq 绘制表观基因组地理图，跟踪由带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。