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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
5P01GM068087-09
Application #
8375312
Study Section
Special Emphasis Panel (ZRG1-GGG-M)
Project Start
Project End
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
9
Fiscal Year
2012
Total Cost
$483,428
Indirect Cost
$14,570
Name
Dartmouth College
Department
Type
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755
Wang, Zheng; Wang, Junrui; Li, Ning et al. (2017) Light sensing by opsins and fungal ecology: NOP-1 modulates entry into sexual reproduction in response to environmental cues. Mol Ecol :
Decker, Logan M; Xiao, Hua; Boone, Erin C et al. (2017) The Nuclear Cap-Binding Complex Mediates Meiotic Silencing by Unpaired DNA. G3 (Bethesda) 7:1149-1155
Ivanov, Ivaylo P; Wei, Jiajie; Caster, Stephen Z et al. (2017) Translation Initiation from Conserved Non-AUG Codons Provides Additional Layers of Regulation and Coding Capacity. MBio 8:
Havlik, David; Brandt, Ulrike; Bohle, Kathrin et al. (2017) Establishment of Neurospora crassa as a host for heterologous protein production using a human antibody fragment as a model product. Microb Cell Fact 16:128
Dekhang, Rigzin; Wu, Cheng; Smith, Kristina M et al. (2017) The Neurospora Transcription Factor ADV-1 Transduces Light Signals and Temporal Information to Control Rhythmic Expression of Genes Involved in Cell Fusion. G3 (Bethesda) 7:129-142
Nguyen, Tu Anh; Cissé, Ousmane H; Yun Wong, Jie et al. (2017) Innovation and constraint leading to complex multicellularity in the Ascomycota. Nat Commun 8:14444
De Souza, Colin P; Hashmi, Shahr B; Hage, Natalie et al. (2017) Location and functional analysis of the Aspergillus nidulans Aurora kinase confirm mitotic functions and suggest non-mitotic roles. Fungal Genet Biol 103:1-15
Samarajeewa, Dilini A; Manitchotpisit, Pennapa; Henderson, Miranda et al. (2017) An RNA Recognition Motif-Containing Protein Functions in Meiotic Silencing by Unpaired DNA. G3 (Bethesda) 7:2871-2882
Carrillo, Alexander J; Schacht, Patrick; Cabrera, Ilva E et al. (2017) Functional Profiling of Transcription Factor Genes in Neurospora crassa. G3 (Bethesda) 7:2945-2956
Weichert, Martin; Lichius, Alexander; Priegnitz, Bert-Ewald et al. (2016) Accumulation of specific sterol precursors targets a MAP kinase cascade mediating cell-cell recognition and fusion. Proc Natl Acad Sci U S A 113:11877-11882

Showing the most recent 10 out of 149 publications