Biology subjects

Klocko, A. D.

Publications and source records attributed to Klocko, A. D..

3 recordsLinked to original sources

bioRxiv2025

Histone deacetylase-1 is required for epigenome stability in Neurospora crassa

Polycomb group (PcG) proteins form chromatin modifying complexes that stably repress lineage- or context-specific genes in animals, plants, and some fungi. Polycomb Repressive Complex 2 (PRC2) catalyzes trimethylation of lysine 27 on histone H3 (H3K27me3) to assemble repressive chromatin. In the model fungus Neurospora crassa, H3K27me3 deposition is controlled by the H3K36 methyltransferase ASH1 and components of constitutive heterochromatin including the H3K9me3-binding protein HETEROCHROMATIN PROTEIN 1 (HP1). Hypoacetylated histones are a defining feature of both constitutive heterochromatin and PcG-repressed chromatin, but how histone deacetylases (HDACs) contribute to normal H3K27me3 and transcriptional repression within PcG-repressed chromatin is poorly understood. We performed a genetic screen to identify HDACs required for repression of PRC2-methylated genes. In the absence of HISTONE DEACETYLASE-1 (HDA-1), PRC2-methylated genes were activated and H3K27me3 was depleted from typical PRC2-targeted regions. At constitutive heterochromatin, HDA-1 deficient cells displayed reduced H3K9me3, hyperacetylation, and aberrant enrichment of H3K27me3 and H3K36me3. CHROMODOMAIN PROTEIN-2 (CDP-2) is required to target HDA-1 to constitutive heterochromatin and was also required for normal H3K27me3 patterns. Patterns of aberrant H3K27me3 were distinct in isogenic {Delta}hda-1 strains, suggesting that loss of HDA-1 causes stochastic or progressive epigenome dysfunction. To test this, we constructed a new{Delta} hda-1 strain and performed a laboratory evolution experiment. Deletion of hda-1 led to progressive epigenome decay over hundreds of nuclear divisions. Together, our data indicate that HDA-1 is a critical regulator of epigenome stability in N. crassa.

genetics↗

bioRxiv2024

A Constitutive Heterochromatic Region Shapes Genome Organization and Impacts Gene Expression in Neurospora crassa

BackgroundOrganization of the eukaryotic genome is essential for proper function, including gene expression. In metazoans, chromatin loops and Topologically Associated Domains (TADs) organize genes into transcription factories, while chromosomes occupy nuclear territories in which silent heterochromatin is compartmentalized at the nuclear periphery and active euchromatin localizes to the nucleus center. A similar hierarchical organization occurs in the fungus Neurospora crassa where its seven chromosomes form a Rabl conformation typified by heterochromatic centromeres and telomeres independently clustering at the nuclear membrane, while interspersed heterochromatic loci aggregate across Megabases of linear genomic distance to loop chromatin in TAD-like structures. However, the role of individual heterochromatic loci in normal genome organization and function is unknown. ResultsWe examined the genome organization of a Neurospora strain harboring a [~]47.4 kilobase deletion within a temporarily silent, facultative heterochromatic region, as well as the genome organization of a strain deleted of a 110.6 kilobase permanently silent constitutive heterochromatic region. While the facultative heterochromatin deletion minimally effects local chromatin structure or telomere clustering, the constitutive heterochromatin deletion alters local chromatin structure, the predicted three-dimensional chromosome conformation, and the expression of some genes, which are qualitatively repositioned into the nucleus center, while increasing Hi-C variability. ConclusionsOur work elucidates how an individual constitutive heterochromatic region impacts genome organization and function. Specifically, one silent region indirectly assists in the hierarchical folding of the entire Neurospora genome by aggregating into the "typical" heterochromatin bundle normally observed in wild type nuclei, which may promote normal gene expression by positioning euchromatin in the nucleus center.

genomics↗

bioRxiv2023

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. Significance StatementThe mechanisms driving chromosome organization in eukaryotic nuclei, including in the filamentous fungus Neurospora crassa, are currently unknown, but histone post-translational modifications may be involved. Histone proteins can be acetylated to form active euchromatin while histone deacetylases (HDACs) remove acetyl marks to form silent heterochromatin; these heterochromatic regions cluster, forming strong interactions, in Neurospora genome organization. Here, we show that mutants of a heterochromatin-specific HDAC, HCHC, increase heterochromatic histone acetylation genome-wide and contact probability between distant heterochromatic loci. HCHC loss also impacts cytosine methylation, and in strains lacking both the HCHC and cytosine methylation, heterochromatic regions interact more with euchromatin. Our results suggest cytosine methylation normally functions to segregate silent and active loci when heterochromatic acetylation increases.

genomics↗