Discovery and Analysis of Network Components via High Throughput Sequencing

项目3：“通过高吞吐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，并利用这些信号来传递过渡信号。 从早期到晚期的营养生长阶段，直到完成无性发育。这可能发生 在一天的过程中，响应两个突出的环境线索-光和暴露于 空气/干燥-协同作用，引起细胞转录组的全面变化。这 生物学是在一个模型系统中观察到的，脉孢菌，具有小的基因组，易处理的遗传学，（现在） 易于重组。通过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%那样容易地被检测到。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测序以鉴定和表征N.染色质内的粗糙DNA和蛋白质元件， 在基因组水平上影响基因表达。我们寻求全系统对监管的理解， 潜在的全球变化的转录组和表观基因组引起的，并伴随着， 环境因素触发发育基于Solexa的测序将与染色质免疫沉淀相结合 （ChIP-Seq）以鉴定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。胞嘧啶甲基化位点与核小体 将评估染色质中的定相。这些分析将有助于了解这些组成部分， 控制这种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）收集N。在随后的一天内，在14个时间段内对厚皮组织进行处理， 暴露于引发发育的线索（光和空气），（ii）每次对全转录组进行编目，（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 （iv）通过ChIP-seq绘制表观基因组的地理图， 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 将在整个时间序列的选定点进行跟踪，（v）绘制所有核小体和 在实验开始时以及在稍后确定的一个或多个额外时间的DNA甲基化 基于何时观察到最显著的表观基因组转变。

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.