Biology subjects

Lewis, Z. A.

Publications and source records attributed to Lewis, Z. A..

10 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↗

bioRxiv2026

Epigenetic maintenance of PRC2-repressed chromatin requires RTT109 but not H3K56 acetylation

In animals, plants, and some fungi, Polycomb Repressive Complex 2 (PRC2) catalyzes trimethylation of histone H3 lysine 27 (H3K27me3) to establish transcriptionally repressed chromatin. Here, we identify the histone acetyltransferase RTT109 as a key regulator of PRC2-repressed domains in the model fungus Neurospora crassa. Although RTT109 interacts with the VPS75 homolog Nucleosome Assembly Factor 2 (NAF-2), we show that proper structure and function of PRC2-methylated chromatin require RTT109 catalytic activity but are independent of NAF-2 and H3K56 acetylation. We further demonstrate that H3K27me3 can be stably propagated over multiple rounds of mitosis in the absence of sequence-specific PRC2 targeting, and that RTT109 is essential for maintenance of the repressed state. These findings uncover a replication-linked mechanism for epigenetic memory and establish RTT109 as a key regulator of Polycomb-mediated chromatin inheritance.

genetics↗

bioRxiv2025

Polycomb repressive complex 2 regulates sexual development in Neurospora crassa

In most branches of the fungal kingdom, sexual development marks a dramatic shift from simple multicellular hyphae to complex fruiting bodies composed of multiple tissues and cell types. It is essential to tightly regulate development to prevent unnecessary energy investment into building these structures. Polycomb Repressive Complex 2 (PRC2) is a highly conserved regulator of development in multicellular organisms. In plants, animals, and some fungi, PRC2 tri-methylates histone H3 lysine 27 in promoters and gene bodies to repress gene expression. In Neurospora crassa, H3K27me3-associated genes are poorly conserved and stably repressed in standard lab conditions. Through analysis of publicly available RNA-seq experiments, we found that PRC2-methylated genes are broadly and uniquely activated during sexual development. PRC2-methylated genes comprise a distinct subset of developmentally induced genes (DIGs) characterized by an exceptionally high degree of cell-type specificity. Loss of PRC2 activity results in the precocious formation of perithecia-like structures (dubbed false perithecia), even in the absence of a compatible mating-type partner. These structures show a unique gene expression profile with activation of both PRC2-methylated and unmethylated DIGs, showing evidence of a transcriptional reprogramming event. Together, these data suggest that PRC2 is part of a developmental checkpoint that restricts fruiting body development to the appropriate conditions.

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

Chromatin regulator HELLS mediates SSB repair and responses to DNA alkylation damage.

The SNF2 family chromatin remodeler HELLS has emerged as an important regulator of cell proliferation, genome stability, and several cancer pathways. Significant upregulation of HELLS has been reported in 33 human cancer types. While HELLS has been implicated in DNA damage response, its function in DNA repair is poorly understood. Here we report a new regulatory link between HELLS and single-strand break (SSB) repair in cellular responses to DNA alkylation damage. We found that loss of HELLS impairs SSB repair, and selectively sensitizes cells to DNA alkylating agents and PARP inhibitors (PARPi). Furthermore, we found that HELLS is co-expressed with PARP1 in cancer cells, and its loss is synthetic lethal with homologous recombination deficiency (HRD). This work unveils new functions of HELLS in modulating SSB repair and responses to clinically relevant DNA alkylation damage, thus offering new insights into the potential therapeutic value of targeting HELLS in cancer. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/629292v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@2bbd3eorg.highwire.dtl.DTLVardef@1956f14org.highwire.dtl.DTLVardef@1afcb7corg.highwire.dtl.DTLVardef@58504c_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2021

DectiSomes: Glycan Targeting of Liposomal Drugs Improves the Treatment of Disseminated Candidiasis

Candida albicans causes life-threatening disseminated candidiasis. Individuals at greatest risk have weakened immune systems. An outer cell wall, exopolysaccharide matrix, and biofilm rich in oligoglucans and oligomannans help Candida spp. evade host defenses. Even after antifungal drug treatment the one-year mortality rate exceeds 25%. Undoubtedly there is room to improve antifungal drug performance. The mammalian C-type lectin pathogen receptors Dectin-1 and Dectin-2 bind to fungal oligoglucans and oligomannans, respectively. We previously coated amphotericin B-loaded liposomes, AmB-LLs, pegylated analogs of AmBisome, with the ligand binding domains of these two Dectins. DectiSomes, DEC1-AmB-LLs and DEC2-AmB-LLs, showed two distinct patterns of binding to the exopolysaccharide matrix surrounding C. albicans hyphae grown in vitro, while untargeted AmB-LLs did not bind. DectiSomes were preferentially associated with fungal colonies in the kidneys. In a neutropenic mouse model of candidiasis, DEC1-AmB-LLs and DEC2-AmB-LLs delivering only one dose of 0.2 mg/kg AmB significantly reduced the kidney fungal burden several fold relative to AmB-LLs, based on either colony forming units (P= 0.013 to 8.8 x 10-5) or quantitative PCR of fungal rRNA ITS (P= 5.5x10-5 to 3.0x10-10). DEC1-AmB-LLs and DEC2-AmB-LLs significantly increased the percent of surviving mice relative to AmB-LLs. Dectin-2 targeted anidulafungin loaded liposomes and AmBisomes, DEC2-AFG-LLs and DEC2-AmBisome reduced fungal burden in the kidneys several fold over their untargeted counterparts (P=7.8x10-5 and 0.0020, respectively). The data herein suggest that targeting of a variety of antifungal drugs to fungal glycans may achieve lower safer effective doses and improve drug efficacy against a variety of invasive fungal infections.

microbiology↗

bioRxiv2021

Versatile Cell-Based Assay for Measuring Base Excision Repair of DNA Alkylation Damage

DNA alkylation damage induced by environmental carcinogens, chemotherapy drugs, or endogenous metabolites plays a central role in mutagenesis, carcinogenesis, and cancer therapy. Base excision repair (BER) is a conserved, front line DNA repair pathway that removes alkylation damage from DNA. The capacity of BER to repair DNA alkylation varies markedly between different cell types and tissues, which correlates with cancer risk and cellular responses to alkylation chemotherapy. The ability to measure cellular rates of alkylation damage repair by the BER pathway is critically important for better understanding of the fundamental processes involved in carcinogenesis, and also to advance development of new therapeutic strategies. Methods for assessing the rates of alkylation damage and repair, especially in human cells, are limited, prone to significant variability due to the unstable nature of some of the alkyl adducts, and often rely on indirect measurements of BER activity. Here, we report a highly reproducible and quantitative, cell-based assay, named alk-BER (alkylation Base Excision Repair) for measuring rates of BER following alkylation DNA damage. The alk-BER assay involves specific detection of methyl DNA adducts (7-methyl guanine and 3-methyl adenine) directly in genomic DNA. The assay has been developed and adapted to measure the activity of BER in fungal model systems and human cell lines. Considering the specificity and conserved nature of BER enzymes, the assay can be adapted to virtually any type of cultured cells. Alk-BER offers a cost efficient and reliable method that can effectively complement existing approaches to advance integrative research on mechanisms of alkylation DNA damage and repair.

molecular biology↗

bioRxiv2020

Shannon Entropy as a metric for conditional gene expression in Neurospora crassa

Neurospora crassa has been an important model organism for molecular biology and genetics for over 60 years. N. crassa has a complex life cycle, with over 28 distinct cell types and is capable of transcriptional responses to many environmental conditions including nutrient availability, temperature, and light. To quantify variation in N. crassa gene expression, we analyzed public expression data from 97 conditions and calculated the Shannon Entropy value for Neurosporas approximately 11,000 genes. Entropy values can be used to estimate the variability in expression for a single gene over a range of conditions and be used to classify individual genes as constitutive or condition-specific. Shannon entropy has previously been used measure the degree of tissue specificity of multicellular plant or animal genes. We use this metric here to measure variable gene expression in a microbe and provide this information as a resource for the N. crassa research community. Finally, we demonstrate the utility of this approach by using entropy values to identify genes with constitutive expression across a wide range of conditions and to identify genes that are activated exclusively during sexual development.

bioinformatics↗

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↗