The genome organization of Neurospora crassa at high-resolution uncovers principles of fungal chromosome topology

For a eukaryotic genome to properly function, its chromatin must be precisely organized, as genome topology impacts transcriptional regulation, cell division, DNA replication, and DNA repair, among other essential processes. Disruption of human genome topology can lead to disease states, such as cancer. The advent of chromosome conformation capture with high-throughput sequencing (Hi-C) technologies to assess genome organization has revolutionized our understanding of the arrangement of chromosomes within the nuclear genome. Critical developments include chromosomal territories, active/silent chromatin compartments, Topologically Associated Domains (TADs), and chromatin loops. However, to fully elucidate folding principles at the gene level, Hi-C datasets at an extremely high resolution are required: while low resolution (e.g., 40 kb bin) heatmaps can provide important insights into chromosomal level contacts, lower resolution datasets cannot provide information about individual gene or promoter contacts. Thus, high-resolution Hi-C datasets can elucidate principles of folding, including those of model organisms whose chromosome conformation can elucidate the folding of the human genome. Here, we have examined the high-resolution genome topology of the model fungal organism Neurospora crassa: our composite Hi-C dataset, generated using two restriction enzymes to monitor euchromatin (DpnII) and heterochromatin (MseI), provides exquisite detail for both larger chromosomal structures and individual gene contacts, including how repressed euchromatic genes associate with constitutive heterochromatic regions. Our high-resolution Neurospora Hi-C datasets should be an outstanding resource to the fungal community and provide valuable insights to the folding of higher organism genomes. Overall design: We analyzed genome organization of two wild type strains of filamentous fungus Neurospora crassa by chromosome conformation capture by paired-end high-throughput sequencing (Hi-C); Hi-C libraries were generated with the four-base cutters DpnII and MseI, which monitor euchromatic and heterochromatic regions of the Neurospora genome, respectively, due to the location of the recognition sequences of these restriction enzymes. Four DpnII replicates (three techincal replicates and a biological replicate) and three MseI replicates (two technical replicates, one biological) were generated using eight PCR cycles; replicates were merged into DpnII and MseI datasets, and those two datasets were merged into a comprehensive interaction matrix.