Discovery and Analysis of Network Components via High Throughput Sequencing

项目3：“通过高吞吐量排序发现和分析网络组件” 丝状真菌对环境线索非常敏感，并利用这些线索发出过渡的信号。 从营养生长早期到后期，直至完成无性发育。这是有可能发生的 在一天的过程中对两个突出的环境线索做出反应-光和暴露在 空气/干燥--它们协同作用，引起细胞转录组的全面变化。这 生物学是在一个模型系统中观察到的，脉孢子虫，具有小的基因组，易处理的遗传学，和(现在) 便捷的重组工程。通过负担得起的超高通量测序；脉孢子虫的整个基因组 可以很容易地检测到人类基因组的1%。因此，它是一种非常好的测试系统 描述、模拟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测序鉴定和鉴定粗面核菌DNA和染色质中的蛋白质元件 在基因组水平上影响基因表达。我们寻求对监管机构的全系统理解 在转录组和表观基因组中的全球变化的基础上，由 引发发展的环境线索。基于Solexa的测序将与染色免疫沉淀相结合 (ChIP-Seq)以确定与不同转录因子相关的染色质位点和 核小体含有各种组蛋白修饰。胞嘧啶甲基化和核小体的位置 将评估染色质的阶段性。这些分析将导致理解组件和 控制这种有机体对光和空气的反应的调控网络的相互作用，以及对 发展。具体目标如下：(I)在接下来的一天中分14次收集粗毛夜蛾组织 暴露在诱导发育的线索(光和空气)中，(Ii)每次编目完整的转录组，(Iii) 利用表位标记菌株，用芯片序列技术检测50多个相关转录因子与DNA的结合 在项目#1中开发，在时间序列上的点上，(Iv)通过芯片序列绘制表观基因组的地理位置， 追踪带有特定修饰的组蛋白和组蛋白变体结合的区域；50个不同物种 将在时间序列中的选定点处跟踪，(V)绘制所有核小体的位置和 DNA甲基化在实验开始时，以及稍后确定的一个或多个额外时间 基于观察到最显著的表观基因组转换的时间。

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.