The eukaryotic genome must be precisely organized for its proper function, as genome topology impacts transcriptional regulation, cell division, replication, and repair, among other essential processes. Disruptions to human genome topology can lead to diseases, including cancer. The advent of chromosome conformation capture with high-throughput sequencing (Hi-C) to assess genome organization has revolutionized the study of nuclear genome topology; Hi-C has elucidated numerous genomic structures, including chromosomal territories, active/silent chromatin compartments, Topologically Associated Domains, and chromatin loops. While low-resolution heatmaps can provide important insights into chromosomal level contacts, high resolution Hi-C datasets are required to reveal folding principles of individual genes. Of particular interest are high-resolution chromosome conformation datasets of organisms modeling the human genome. Here, we report the genome topology of the fungal model organism Neurospora crassa at a high resolution. Our composite Hi-C dataset, which merges two independent datasets generated with restriction enzymes that monitor euchromatin (DpnII) and heterochromatin (MseI), along with our DpnII/MseI double digest dataset, provide exquisite detail for both the conformation of entire chromosomes and the folding of chromatin at the resolution of individual genes. Within constitutive heterochromatin, we observe strong yet stochastic internal contacts, while euchromatin enriched with either activating or repressive histone post-translational modifications associates with constitutive heterochromatic regions, suggesting inter-compartment contacts form to regulate transcription. Consistent with this, a strain with compromised heterochromatin experiences numerous changes in gene expression. Our high-resolution Neurospora Hi-C datasets are outstanding resources to the fungal community and provide valuable insights into higher organism genome topology.

Comparative genomics has recently provided unprecedented insights into the biology and evolution of the fungal lineage. In the post-genomics era, a major research interest focuses now on detailing the functions of fungal genomes, i.e. how genomic information manifests into complex phenotypes. Emerging evidence across diverse eukaryotes has revealed that the organization of DNA within the nucleus is critically important. Here, we discuss the current knowledge on the fungal genome organization, from the association of chromosomes within the nucleus to topological structures at individual genes and the genetic factors required for this hierarchical organization. Chromosome conformation capture followed by high-throughput sequencing (Hi-C) has elucidated how fungal genomes are globally organized in Rabl configuration, in which centromere or telomere bundles are associated with opposite faces of the nuclear envelope. Further, fungal genomes are regionally organized into Topologically Associated Domain-like (TAD-like) chromatin structures. We discuss how chromatin organization impacts the proper function of DNA-templated processes across the fungal genome. Nevertheless, this view is limited to a few fungal taxa given the paucity of fungal Hi-C experiments. We advocate for exploring genome organization across diverse fungal lineages to ensure the future understanding of the impact of nuclear organization on fungal genome function.