Biology subjects

Ferraro, A. R.

Publications and source records attributed to Ferraro, A. R..

4 recordsLinked to original sources

bioRxiv2026

Chromatin Assembly Factor 1 is required for normal structure and function of facultative heterochromatin in Neurospora crassa

Polycomb Repressive Complex 2 (PRC2) is a conserved epigenetic regulator that represses gene expression through methylation of histone H3 lysine 27 (H3K27me3). In animals, plants, and some fungi, PRC2-directed facultative heterochromatin plays essential roles in development and cellular differentiation. Here, we show that the replication-dependent histone chaperone Chromatin Assembly Factor 1 (CAF-1) is required for proper structure and function of facultative heterochromatin in the model fungus Neurospora crassa. Loss of CAF-1 causes widespread transcriptional misregulation, particularly within PRC2-repressed regions, and leads to redistribution of H3K27me3, reduced ASH1-dependent H3K36 methylation, and accumulation of chromatin marks associated with active transcription. CAF-1 was not required for repressive histone methylation within constitutive heterochromatin. A double mutant lacking both CAF-1 and PRC2 components displayed a synergistic silencing defect, suggesting these complexes make distinct contributions to facultative heterochromatin. Together, our findings indicate that CAF-1 works in concert with PRC2 to silence transcription within N. crassa facultative heterochromatin domains.

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↗

bioRxiv2020

The histone variant H2A.Z is required to establish normal patterns of H3K27 methylation in Neurospora crassa

Neurospora crassa contains a minimal Polycomb repression system, which provides rich opportunities to explore Polycomb-mediated repression across eukaryotes and enables genetic studies that can be difficult in plant and animal systems. Polycomb Repressive Complex 2 is a multi-subunit complex that deposits mono-, di-, and tri-methyl groups on lysine 27 of histone H3, and tri-methyl H3K27 is a molecular marker of transcriptionally repressed facultative heterochromatin. In mouse embryonic stem cells and multiple plant species, H2A.Z has been found to be co-localized with H3K27 methylation. H2A.Z is required for normal H3K27 methylation in these experimental systems, though the regulatory mechanisms are not well understood. We report here that Neurospora crassa mutants lacking H2A.Z or SWR-1, the ATP-dependent histone variant exchanger, exhibit a striking reduction in levels of H3K27 methylation. RNA-sequencing revealed downregulation of eed, encoding a subunit of PRC2, in an hH2Az mutant compared to wild type and overexpression of EED in a {Delta}hH2Az;{Delta}eed background restored most H3K27 methylation. Reduced eed expression leads to region-specific losses of H3K27 methylation suggesting that EED-dependent mechanisms are critical for normal H3K27 methylation at certain regions in the genome. AUTHOR SUMMARYEukaryotic DNA is packaged with histone proteins to form a DNA-protein complex called chromatin. Inside the nucleus, chromatin can be assembled into a variety of higher-order structures that profoundly impact gene expression. Polycomb Group proteins are important chromatin regulators that control assembly of a highly condensed form of chromatin. The functions of Polycomb Group proteins are critical for maintaining stable gene repression during development of multicellular organisms, and defects in Polycomb proteins are linked to disease. There is significant interest in elucidating the molecular mechanisms that regulate the activities of Polycomb Group proteins and the assembly of transcriptionally repressed chromatin domains. In this study, we used a model fungus to investigate the regulatory relationship between a histone variant, H2A.Z, and a conserved histone modifying enzyme complex, Polycomb Repressive Complex 2 (PRC2). We found that H2A.Z is required for normal expression of a PRC2 component. Mutants that lack H2A.Z have defects in chromatin structure at some parts of the genome, but not others. Identification of PRC2-target domains that are differentially dependent on EED provides insights into the diverse mechanisms that regulate assembly and maintenance of facultative heterochromatin in a simple model system. Data Reference NumbersGSE146611

genetics↗

bioRxiv2019

Targeted antifungal liposomes

Aspergillus species cause pulmonary invasive aspergillosis resulting in nearly a hundred thousand deaths each year. Patients at the greatest risk of developing life-threatening aspergillosis have weakened immune systems and/or various lung disorders. Patients are treated with antifungals such as amphotericin B (AmB), casofungin acetate, or triazoles (itraconazole, voriconazole etc.), but these antifungal agents have serious limitations due to lack of sufficient fungicidal effect and human toxicity. Liposomes with AmB intercalated into the lipid membrane (AmBisomes, AmB-LLs), have several-fold reduced toxicity compared to detergent solubilized drug. However, even with the current antifungal therapies, one-year survival among patients is only 25 to 60%. Hence, there is a critical need for improved antifungal therapeutics.\n\nDectin-1 is a mammalian innate immune receptor in the membrane of some leukocytes that binds as a dimer to beta-glucans found in fungal cell walls, signaling fungal infection. Using a novel protocol, we coated AmB-LLs with Dectin-1s beta-glucan binding domain to make DEC-AmB-LLs. DEC-AmB-LLs bound rapidly, efficiently, and with great strength Aspergillus fumigatus and to Candida albicans and Cryptococcus neoformans, highly divergent fungal pathogens of global importance. By contrast, un-targeted AmB-LLs and BSA-coated BSA-AmB-LLs showed 200-fold lower affinity for fungal cells. DEC-AmB-LLs reduced the growth and viability of A. fumigatus an order of magnitude more efficiently than untargeted control liposomes delivering the same concentrations of AmB, in essence increasing the effective dose of AmB. Future efforts will focus on examining pan-antifungal targeted liposomal drugs in animal models of disease.\n\nTweetWe coated anti-fungal drug loaded liposomes to fungal cell walls with a beta-glucan binding protein and thereby increased drug effectiveness by an order of magnitude.\n\nImportanceThe fungus Aspergillus fumigatus causes pulmonary invasive aspergillosis resulting in nearly a hundred thousand deaths each year. Patients are often treated with antifungal drugs such as amphotericin B loaded into liposomes, AmB-LLs, but all antifungal drugs including AmB-LLs have serious limitations due to human toxicity and insufficient fungal cell killing. Even with the best current therapies, one-year survival among patients with invasive aspergillosis is only 25 to 60%. Hence, there is a critical need for improved antifungal therapeutics.\n\nDectin-1 is a mammalian protein that binds to beta-glucan polysaccharides found in nearly all fungal cell walls. We coated AmB-LLs with Dectin-1 to make DEC-AmB-LLs. DEC-AmB-LLs bond strongly to fungal cells, while AmB-LLs had little affinity. DEC-AmB-LLs killed or inhibited A. fumigatus ten times more efficiently than untargeted lipsomes, increasing the effective dose of AmB. Dectin-1 coated liposomes targeting fungal pathogens have the potential to greatly enhance antifungal therapeutics.

molecular biology↗