Normal development relies on regulated gene expression, which in many organisms is achieved by protein complexes that alter the structure of chromatin to silence or activate genes. Chromatin consists of DNA packaged together with histone and other proteins. How Polycomb Repressive Complexes (PRCs) silence genes that are important for development is not completely understood in any organism. This project seeks to uncover how specific genes are targeted and inactivated by PRCs, using fungi as convenient model systems. Societal impacts will be via a combination of research on gene regulation, natural products, biofuels and the discovery of putative fungal pathogenicity factors. The broader impact of the work will also provide hands on laboratory education of undergraduates via intensive long-term research projects. Other outreach efforts include two-day genomics workshops for high school teachers and presentations to the general public.
This project addresses mechanisms of chromatin-based gene silencing by PRC2 and H3K27 methylation in the filamentous fungus, Fusarium graminearum. Using a forward genetics screen, two novel chromatin proteins that impact H3K27 methylation (DIS2 and DIS10) were discovered. The main objectives of this work are to uncover the mechanism for PRC2 targeting and the composition of PRC2 complexes. The main hypotheses for the current project are: (1) Fusarium DIS2 and DIS10 use their putative chromatin-binding motifs to interact with specific regions on histones or other chromatin proteins to target the core PRC2 to chromatin segments that will be enriched for H3K27 trimethylation; (2) targeting is based on portable, nucleic acid-based signals. Core PRC2 subunits are regulated by DIS2 and DIS10 and themselves regulate a large network of transcription factors and major regulators of signal transduction. Completion of this project will result in gaining insights into the global H3K27 methylation network. Methods used to achieve the aims are: (1) forward and reverse genetics by undirected selections and screens, gene deletions, and site-directed mutagenesis; (2) biochemistry by chromatin immunoprecipitation, genome-wide expression analyses and mass spectrometry; (3) biophysical studies to probe interactions between DIS2 or DIS10 and histone or other chromatin proteins, and (4) cytology by examination of "silencing bodies" (containing PRC2) in association with DNA. In combination, these techniques will allow identification and characterization of PRC2 signals and novel proteins involved in gene silencing. By studying mechanisms of PRC2 targeting, this research will provide key knowledge on chromatin-mediated silencing in eukaryotes.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
