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.
The proposed work will study at an unprecedented systems level the transcriptomic and epigenomic changes that lead to spore formation in a non-yeast fungus. Such spores are a major dispersal mechanism for pathogenic fungi. The proposed studies will provide the basis for a greater understanding of fundamental regulatory processes relevant to the study of many organisms important to human health and welfare.
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