Genome topology in the filamentous fungus Neurospora crassa: organizing factors and impact on genome function - Project Summary/Abstract Eukaryotic chromosomes compact into the nucleus while maintaining a precise, non-stochastic organization that allows genome function, including gene expression and chromatin formation. In humans, genome disorder can cause to aberrant gene expression and uncontrolled cellular growth, leading to oncogenic tumor formation, as observed in neuroblastoma and pancreatic cancers. However, a complete understanding of the mechanisms necessary to organize eukaryotic genomes is not known in any species, which is a critical gap in our collective knowledge. Specifically, many (epi)genetic factors have not been examined for roles in organizing genomic DNA. The long-term goal of this project is to elucidate how genome organization is formed in eukaryotic nuclei and dissect the role of genome organization on transcriptional control. To this end, we will use the innovative fungal organism Neurospora crassa as a model for human systems, given the similarities of its epigenetic regulation and extent of DNA compaction with humans; the Neurospora genome is subdivided into active euchromatin and silent heterochromatin catalyzed by similar proteins. However, the smaller fungal genome allows cost-efficient genomic studies, and the genetically tractable Neurospora employs simple epigenetic machinery often involving single protein complexes to allow direct study of individual histone marks. Further, study of a model fungal system will allow direct comparisons to fungal pathogens of humans, as both have similar genome folding patterns. The proposed research, building upon previous results, will examine fungal genome organization by chromosome conformation capture with high-throughput sequencing (Hi-C) in different genetic backgrounds to understand the (epi)genetic factors organizing the fungal genome and the resulting effects on genome function. Previously, we showed loss of the HCHC histone deacetylase complex caused hyperacetylation of heterochromatin which influenced genome organization. First, we will characterize the histone lysines targeted by the HCHC, examine HCHC deacetylation over the cell cycle, and identify the histone acetyltransferase acetylating heterochromatic regions. Secondly, given how heterochromatic region bundling is one of the main features of fungal genome organization, we will determine if the de novo formation H3K9me3 or a protein binding to the underlying AT-rich DNA is necessary for genome organization; we will also assess if changes to heterochromatin region boundaries influence genome topology. Lastly, since subtelomere clusters are one of the main features of organizing fungal chromosomes, we will characterize a telomere-binding protein complex for role(s) in genome organization. The research for these Specific Aims will be primarily led by undergraduate students, who will directly build strains, perform genomics experiments (e.g., Hi-C and Chromatin Immunoprecipitation-sequencing), analyze resulting datasets, and present results in conference presentations and peer-reviewed manuscripts, thereby creating an outstanding training environment for UCCS students for careers in scientific research or medicine. Together, our multi-faceted approach will advance our understanding of eukaryotic genome organization and function.
