Biology subjects

Klocko, A. D.

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

5 recordsLinked to original sources

bioRxiv2026

A Translocation within the Ogataea Species Complex Alters Local Subtelomeric Chromatin while Maintaining Overall Genome Organization

Eukaryotic genomic DNA is packaged in the nucleus as chromatin - a DNA-protein aggregate regulating genome function, including transcription. Chromatin is classified as either active euchromatin or silent heterochromatin, with each marked by distinct histone post-translational modifications (PTMs). Chromatin composition also mediates genome organization, including how heterochromatin aggregates at the nuclear periphery while euchromatin localizes to the nucleus center. In fungi, heterochromatic loci cluster, including independent centromere and telomere clusters that form the Rabl chromosome conformation. However, it is unknown if chromatin composition and genome organization are conserved in closely related fungi, and how these features are impacted by large-scale chromosomal rearrangements. Here, we examined differences in histone PTM deposition, gene expression, and genome organization in two yeast species from the order Pichiales, which diverged from the common ancestor shared with Saccharomyces cerevisiae more than 200 million years ago. We focused on Ogataea polymorpha, which is used for industrial protein production, and Ogataea haglerorum, an isolate of which harbors a translocation between chromosomes 1 and 6. We show that the enrichment of three activating PTMs - the trimethylation of lysine 4 of histone H3 (H3K4me3) and the acetylation of lysine 9 of histone H3 (H3K9ac) or lysine 16 of histone H4 (H4K16ac) - are similar genome-wide yet gene orthologs have distinct chromatin and expression patterns. While both Ogataea genomes organize into a Rabl conformation, the O. haglerorum translocation alters subtelomeric chromatin composition and expression of genes affected by the translocation. Our work highlights the genome function differences that occur on a microevolutionary scale. Article SummaryTo assess changes in chromatin - the DNA-protein aggregate controlling genome function - between closely related species of fungi, we compared histone modifications, gene expression, and DNA folding in two yeasts in the same species complex, Ogataea polymorpha and Ogataea haglerorum. Globally, we found similar patterns of chromatin composition, transcription, and genome folding, with distinct differences in individual genes. Further, a large genome rearrangement in O. haglerorum alters histone mark deposition and gene expression at affected chromosome ends. Our results provide insights into functional genome changes that occur over short evolutionary time scales and in response to large chromosomal changes.

genomics↗

bioRxiv2026

Centromeres are hotspots of cytosine methylation epimutations in a filamentous fungus

Epimutations are changes in chromatin modifications, such as DNA methylation or histone modifications. Some of these epigenetic changes can be inherited for several generations, and they potentially contribute to evolutionary processes. Estimates of epimutation rates now exists in a few species, but the presence and function of epigenetic marks are not conserved across different species. To understand the properties of epimutations in fungi, we performed a mutation accumulation experiment with the filamentous fungus Neurospora crassa and investigated spontaneous changes in DNA methylation and trimethylation of lysine 9 on histone H3 (H3K9me3) in the mutation accumulation lines. We observed that centromeric regions are hotspots of spontaneous DNA methylation changes in N. crassa. In these hotspot regions, DNA methylation changes were transmitted across mitoses, but changes occurring in euchromatin were not maintained. The rate of DNA methylation changes was around 30 000 fold faster than the genetic mutation rate. We did not observe spontaneous changes in H3K9me3 that were transmitted across mitoses. Our results show that while spontaneous epimutations occur in this species, they occur predominantly in gene poor heterochromatic regions, so their impact for evolutionary adaptation may be limited.

genetics↗

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↗