Expressing the Cryptic Genome: Identification of Feedstocks After Removal of Histone Modifications
EMSL Project ID
48817
Abstract
Responses to external (environmental) or internal (cellular) stimuli require space- and time-dependent transcription of genes in the context of chromatin, an assembly of DNA and proteins that compacts chromosomes. Thus, changes in post-translational histone modifications are essential to coordinate eukaryotic transcriptional networks. We study these changes as consequences of alterations in nutritional status of fungi. Transcriptionally silent genes are often enriched with H3 lysine 27 trimethylation (H3K27me3). We use genetically, biochemically and cytologically tractable filamentous soil fungi in the genus Fusarium as models to understand this chromatin-mediated gene silencing. We recently showed that Fusarium contains a "Polycomb Repressive Complex 2" (PRC2) that catalyzes formation of H3K27me3. Mutation of three Fusarium PRC2 subunits (KMT6, EED, SUZ12) results in severe defects during development and altered morphology. We also found that >2,500 genes are overexpressed in these mutants under laboratory growth conditions whereas they are silent or barely expressed in the wild type. This results in de-repression of dozens of novel metabolic pathways that remain to be studied in detail. Working hypotheses for this work are that (1) PRC2 core complex components can be readily identified by forward genetics, (2) removal of H3K27me3 results in widespread changes of additional histone modifications on H3 and other core histones, and (3) “subtractive metabolic profiling” of different secondary metabolism gene cluster mutants will aid in sorting metabolites to genes responsible for specific metabolic pathways. To address our hypothesis, we aim to (1) understand how H3K27me3 controls expression of gene clusters by UV mutagenesis of strains with reporter genes inserted at usually silent loci to identify novel components of the silencing machinery, (2) determine the spectrum of differential histone modifications in the presence or absence of PRC2 components to study “crosstalk” between histone modifications, and (3) discover which specific secondary metabolite gene clusters are required to produce novel metabolites in novel PRC2 mutants. Our project is designed as a broad collaboration between laboratories at the Oregon State University main and Cascades campuses and several scientists at EMSL. Here we combine four state-of-the-art capabilities at EMSL to (1) improve our yield of high-throughput sequencing by strand-specific total RNA-seq (via SOLiD cDNA sequencing), (2) identify combinatorial core histone modifications by comparing chromatin enriched with or lacking H3K27me3 (by "top down" FT-ICR MS), and (3) identify and characterize novel metabolites or enzymes produced in PRC2 mutants (by MS, GC-MS and –if applicable– NMR). This combination of capabilities is not currently available at Oregon State University. Our experiments will provide key knowledge on chromatin-mediated gene silencing in industrially important fungi and improve strategies to overexpress enzymes or novel compounds. Taken together, we will gain insights into the nature of chromatin silencing complexes and associated proteins, the composition of H3K27me3 chromatin, and the interplay between epigenetic modifications and chromatin proteins that are required for gene silencing in secondary metabolite gene clusters. Innovation also lies in our combination of established methods and materials for the immediate discovery and sorting of new metabolites to specific secondary metabolite gene clusters.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2015-10-01
End Date
2017-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Zhou M., S. Wu, D.L. Stenoien, Z. Zhang, L. Connolly, M. Freitag, and L. Pasa-Tolic. 2016. "Profiling Changes in Histone Post-translational Modifications by Top-Down Mass Spectrometry." In Methods in Molecular Biology: Eukaryotic transcriptional and post-transcriptional gene expression regulation, edited by N Wajapeyee and R Gupta. 153-168. New York, New York:Springer. PNNL-SA-113153. doi:10.1007/978-1-4939-6518-2_12