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-like structures formed by heterochromatic region aggregation. However, insufficient data exist on how histone posttranslational modifications (PTMs), including acetylation, affect genome organization. In Neurospora, the HCHC complex [composed of the proteins HDA-1, CDP-2 (Chromodomain Protein-2), Heterochromatin Protein-1, and CHAP (CDP-2 and HDA-1 Associated Protein)] deacetylates heterochromatic nucleosomes, as loss of individual HCHC members increases centromeric acetylation, and alters the methylation of cytosines in DNA. Here, we assess whether 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 interchromosomal heterochromatic region interactions, while loss of CHAP decreases heterochromatic region compaction. Individual HCHC mutants exhibit different patterns of histone PTMs genome-wide, as CDP-2 deletion increases heterochromatic H4K16 acetylation, yet smaller heterochromatic regions lose H3K9 trimethylation and gain interheterochromatic 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.

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.

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

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 2 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 intercompartment 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.

Green Methodologies for Copper(I)-Catalyzed Azide-Alkyne Cycloadditions: A Comparative Study.

Successful copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reactions may be achieved by several methods. In this paper, four synthetic protocols were performed for direct comparison of time required for the synthesis, yield, and purity of the 1H-1,2,3-triazole products. The methods with Cu(I) catalysts were conventional, microwave heating, solvent-free, and a method using glycerol solvent. The compounds synthesized in this paper were known non-fluorinated triazoles and new fluorinated triazoles. The results lead to the conclusion that the microwave method should be strongly considered for CuAAC syntheses.