Histone deacetylation and cytosine methylation compartmentalize heterochromatic regions in the genome organization of Neurospora crassa

Chromosomes must correctly fold in eukaryotic nuclei for proper genome function. Eukaryotic organisms hierarchically organize their genomes, including in the fungus Neurospora crassa, where chromatin fiber loops compact into Topologically Associated Domain (TAD)-like structures formed by heterochromatic region aggregation. However, insufficient data exists on how histone post-translational modifications, including acetylation, affect genome organization. In Neurospora, the HCHC complex (comprised of the proteins HDA-1, CDP-2, HP1, and CHAP) deacetylates heterochromatic nucleosomes, as loss of individual HCHC members increases centromeric acetylation and alters the methylation of cytosines in DNA. Here, we assess if the HCHC complex affects genome organization by performing Hi-C in strains deleted of the cdp-2 or chap genes. CDP-2 loss increases intra- and inter-chromosomal heterochromatic region interactions, while loss of CHAP decreases heterochromatic region compaction. Individual HCHC mutants exhibit different patterns of histone post-translational modifications genome-wide: without CDP-2, heterochromatic H4K16 acetylation is increased, yet smaller heterochromatic regions lose H3K9 trimethylation and gain inter-heterochromatic region interactions; CHAP loss produces minimal acetylation changes but increases heterochromatic H3K9me3 enrichment. Loss of both CDP-2 and the DIM-2 DNA methyltransferase causes extensive genome disorder, as heterochromatic-euchromatic contacts increase despite additional H3K9me3 enrichment. Our results highlight how the increased cytosine methylation in HCHC mutants ensures genome compartmentalization when heterochromatic regions become hyperacetylated without HDAC activity.