Histone H1 Limits DNA Methylation in Neurospora crassa

Abstract

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 ΔhH1 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 ΔhH1 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.

Rapid and Inexpensive Preparation of Genome-Wide Nucleosome Footprints from Model and Non-Model Organisms

“…Similar to both S. cerevisiae and S. pombe cells, we were readily able to isolate nucleosome footprints from N. crassa using the rapid MNase protocol ( Figure 3D). Importantly, we verified that the genomic nucleosome positions that we obtained were similar to previously-published N. crassa data sets (SEYMOUR et al 2016;KLOCKO et al 2019) ( Figure 3E). Together these data validate the rapid MNase protocol as a reasonable, rapid strategy to map nucleosome positions in S. pombe and N. crassa, two widely-investigated model fungi.…”

“…pombe (THODBERG et al 2019). Previously-published S. cerevisiae data (SRX5086833, (DONOVAN et al 2019) ; SRX1176421, (MCKNIGHT et al 2016)), S. pombe data (SRX554384, (STEGLICH et al 2015) ; SRX331943, (DEGENNARO et al 2013)) and N. crassa data (SRX1596291, (SEYMOUR et al 2016) ;SRX2822417, (KLOCKO et al 2019)) were downloaded from the Sequence Read Archive (SRA) and analyzed using our computational pipeline to identify nucleosome dyad positions. Data were visualized using Integrated Genome Browser (FREESE et al 2016).…”

DNA Replication Is Required for Circadian Clock Function by Regulating Rhythmic Nucleosome Composition

“…Previous work show that WCC binding at the frq C-box region can be regulated by several chromatin modifiers that affect the nucleosome occupancy or positioning (Belden et al, 2011; Belden et al, 2007; Cha et al, 2013; Wang et al, 2014). We performed micrococcal nuclease digestion of Neurospora chromatin followed by deep sequencing (MNase-seq) as previously described (Seymour et al, 2016) to determine whether DNA replication affects nucleosome occupancy or positioning at the frq locus. MNase-seq data showed that although the nucleosome positioning at the frq locus in the WT strain was not affected by HU treatment, increased nucleosome occupancy was found at the frq promoter region, including the C-box, after HU treatment (Figure 4A), indicating that DNA replication affects nucleosome occupancy at the frq promoter.…”

“…Micrococcal nuclease (MNase) experiments was performed as previously described (Seymour et al, 2016). Neurospora was cultured in petri dishes in liquid medium for two days.…”

Production of a fungal furocoumarin by a polyketide synthase gene cluster confers the chemo‐resistance of Neurospora crassa to the predation by fungivorous arthropods

“…In filamentous fungi, prominent classes of secondary metabolites are synthesized by large multimodular enzymes, the polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) or hybrid NRPS-PKS/PKS-NRPS (Amnuaykanjanasin et al, 2009;Boettger et al, 2012). The genes encoding PKSs and NRPSs in filamentous fungi are often physically clustered with other functionally related genes (Bok et al, 2006) and tightly regulated through clusterspecific transcription factors (Netzker et al, 2015) or in some cases through the epigenetic regulators such as histone methyltransferases, histone deacetylases (Lee et al, 2009;Seymour et al, 2016). Among these, the epigenetic regulators such as histone methyltransferase 5 encoded by dim-5 in N. crassa are responsible for histone H3 lysine 9 trimethylation and play significant roles in heterochromatin formation (Lewis et al, 2010).…”