Chromatin accessibility profiling in Neurospora crassa reveals molecular features associated with accessible and inaccessible chromatin.

BACKGROUND: Regulation of chromatin accessibility and transcription are tightly coordinated processes. Studies in yeast and higher eukaryotes have described accessible chromatin regions, but little work has been done in filamentous fungi. RESULTS: Here we present a genome-scale characterization of accessible chromatin regions in Neurospora crassa, which revealed characteristic molecular features of accessible and inaccessible chromatin. We present experimental evidence of inaccessibility within heterochromatin regions in Neurospora, and we examine features of both accessible and inaccessible chromatin, including the presence of histone modifications, types of transcription, transcription factor binding, and relative nucleosome turnover rates. Chromatin accessibility is not strictly correlated with expression level. Accessible chromatin regions in the model filamentous fungus Neurospora are characterized the presence of H3K27 acetylation and commonly associated with pervasive non-coding transcription. Conversely, methylation of H3 lysine-36 catalyzed by ASH1 is correlated with inaccessible chromatin within promoter regions. CONCLUSIONS: In N. crassa, H3K27 acetylation is the most predictive histone modification for open chromatin. Conversely, our data show that H3K36 methylation is a key marker of inaccessible chromatin in gene-rich regions of the genome. Our data are consistent with an expanded role for H3K36 methylation in intergenic regions of filamentous fungi compared to the model yeasts, S. cerevisiae and S. pombe, which lack homologs of the ASH1 methyltransferase.

IMITATION SWITCH is required for normal chromatin structure and gene repression in PRC2 target domains.

Polycomb Group (PcG) proteins are part of an epigenetic cell memory system that plays essential roles in multicellular development, stem cell biology, X chromosome inactivation, and cancer. In animals, plants, and many fungi, Polycomb Repressive Complex 2 (PRC2) catalyzes trimethylation of histone H3 lysine 27 (H3K27me3) to assemble transcriptionally repressed facultative heterochromatin. PRC2 is structurally and functionally conserved in the model fungus Neurospora crassa, and recent work in this organism has generated insights into PRC2 control and function. To identify components of the facultative heterochromatin pathway, we performed a targeted screen of Neurospora deletion strains lacking individual ATP-dependent chromatin remodeling enzymes. We found the Neurospora homolog of IMITATION SWITCH (ISW) is critical for normal transcriptional repression, nucleosome organization, and establishment of typical histone methylation patterns in facultative heterochromatin domains. We also found that stable interaction between PRC2 and chromatin depends on ISW. A functional ISW ATPase domain is required for gene repression and normal H3K27 methylation. ISW homologs interact with accessory proteins to form multiple complexes with distinct functions. Using proteomics and molecular approaches, we identified three distinct Neurospora ISW-containing complexes. A triple mutant lacking three ISW accessory factors and disrupting multiple ISW complexes led to widespread up-regulation of PRC2 target genes and altered H3K27 methylation patterns, similar to an ISW-deficient strain. Taken together, our data show that ISW is a key component of the facultative heterochromatin pathway in Neurospora, and that distinct ISW complexes perform an apparently overlapping role to regulate chromatin structure and gene repression at PRC2 target domains.

Normal Patterns of Histone H3K27 Methylation Require the Histone Variant H2A.Z 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 trimethyl groups on lysine 27 of histone H3, and trimethyl 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 colocalized 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 ΔhH2Az;Δeed background restored most H3K27 methylation. Reduced eed expression leads to region-specific losses of H3K27 methylation, suggesting that differential dependence on EED concentration is critical for normal H3K27 methylation at certain regions in the genome.

Involvement of MAK-1 and MAK-2 MAP kinases in cell wall integrity in Neurospora crassa.

Among three MAPK disruptants of Neurospora crassa, Δmak-1 was sensitive and Δmak-2 was hypersensitive to micafungin, a beta-1,3-glucan synthase inhibitor, than the wild-type or Δos-2 strains. We identified six micafungin-inducible genes that are involved in cell wall integrity (CWI) and found that MAK-1 regulated the transcription of non-anchored cell wall protein gene, ncw-1, and the beta-1,3-endoglucanase gene, bgt-2, whereas MAK-2 controlled the expression of the glycosylhydrolase-like protein gene, gh76-5, and the C4-dicarboxylate transporter gene, tdt-1. Western blotting analysis revealed that, in the wild-type strain, MAK-1 was constitutively phosphorylated from conidial germination to hyphal development. In contrast, the phosphorylation of MAK-2 was growth phase-dependent, and micafungin induced the phosphorylation of unphosphorylated MAK-2. It should be noted that the phosphorylation of MAK-1 was virtually abolished in the Δmak-2 strain, but was significantly induced by micafungin, suggesting functional cross talk between MAK-1 and MAK-2 signalling pathway in CWI.

Histone H1 Limits DNA Methylation in Neurospora crassa.

Histone H1 variants, known as linker histones, are essential chromatin components in higher eukaryotes, yet compared to the core histones relatively little is known about their in vivo functions. The filamentous fungus Neurospora crassa encodes a single H1 protein that is not essential for viability. To investigate the role of N. crassa H1, we constructed a functional FLAG-tagged H1 fusion protein and performed genomic and molecular analyses. Cell fractionation experiments showed that H1-3XFLAG is a chromatin binding protein. Chromatin-immunoprecipitation combined with sequencing (ChIP-seq) revealed that H1-3XFLAG is globally enriched throughout the genome with a subtle preference for promoters of expressed genes. In mammals, the stoichiometry of H1 impacts nucleosome repeat length. To determine if H1 impacts nucleosome occupancy or nucleosome positioning in N. crassa, we performed micrococcal nuclease digestion in the wild-type and the [Formula: see text]hH1 strain followed by sequencing (MNase-seq). Deletion of hH1 did not significantly impact nucleosome positioning or nucleosome occupancy. Analysis of DNA methylation by whole-genome bisulfite sequencing (MethylC-seq) revealed a modest but global increase in DNA methylation in the [Formula: see text]hH1 mutant. Together, these data suggest that H1 acts as a nonspecific chromatin binding protein that can limit accessibility of the DNA methylation machinery in N. crassa.

The LSH/DDM1 Homolog MUS-30 Is Required for Genome Stability, but Not for DNA Methylation in Neurospora crassa.

LSH/DDM1 enzymes are required for DNA methylation in higher eukaryotes and have poorly defined roles in genome maintenance in yeast, plants, and animals. The filamentous fungus Neurospora crassa is a tractable system that encodes a single LSH/DDM1 homolog (NCU06306). We report that the Neurospora LSH/DDM1 enzyme is encoded by mutagen sensitive-30 (mus-30), a locus identified in a genetic screen over 25 years ago. We show that MUS-30-deficient cells have normal DNA methylation, but are hypersensitive to DNA damaging agents. MUS-30 is a nuclear protein, consistent with its predicted role as a chromatin remodeling enzyme, and levels of MUS-30 are increased following DNA damage. MUS-30 co-purifies with Neurospora WDR76, a homolog of yeast Changed Mutation Rate-1 and mammalian WD40 repeat domain 76. Deletion of wdr76 rescued DNA damage-hypersensitivity of Δmus-30 strains, demonstrating that the MUS-30-WDR76 interaction is functionally important. DNA damage-sensitivity of Δmus-30 is partially suppressed by deletion of methyl adenine glycosylase-1, a component of the base excision repair machinery (BER); however, the rate of BER is not affected in Δmus-30 strains. We found that MUS-30-deficient cells are not defective for DSB repair, and we observed a negative genetic interaction between Δmus-30 and Δmei-3, the Neurospora RAD51 homolog required for homologous recombination. Together, our findings suggest that MUS-30, an LSH/DDM1 homolog, is required to prevent DNA damage arising from toxic base excision repair intermediates. Overall, our study provides important new information about the functions of the LSH/DDM1 family of enzymes.

Deletion and expression analysis of beta-(1,3)-glucanosyltransferase genes in Neurospora crassa.

GPI(glycosylphosphatidylinositol)-anchored beta-(1,3)-glucanosyltransferases play an active role in cell wall biosynthesis in fungi. Neurospora crassa has 5 putative beta-(1,3)-glucanosyltransferase genes, namely, gel-1, gel-2, gel-3, gel-4, and gel-5, in its genome. Among them, the gel-3 gene is constitutively expressed at the highest level in growing hyphae, whereas gel-1 is expressed at the lowest level. The gel-3 deletion mutant displayed slow growth, while other gel gene disruptants exhibited normal growth. Although no gel gene disruption affected pH sensitivity and fertility, all Δgel mutants were resistant to cell wall degradation enzymes. Micafungin, a beta-(1,3)-glucan synthase inhibitor, induced gel-4 expression in the wild-type and 2 MAP kinase mutants mak-1 and mak-2 strains. In contrast, fludioxonil, an activator of OS-2 MAP kinase, strongly induced the gel-1 gene in the wild-type strain. Its induction was nearly abolished in the os-2 and in the atf-1/asl-1 mutant. These suggested that GEL-3 is a major factor in mycelial growth, while GEL-1 and GEL-4 may play important roles in cell wall remodeling in response to stress conditions or cell wall damage, respectively.