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Nucleosome Positioning by an Evolutionarily Conserved Chromatin Remodeler Prevents Aberrant DNA Methylation in Neurospora.

ABSTRACT: Aberrant DNA methylation is often associated with cancers and DNA-demethylating agents such as 5-azacytidine (5-azaC) are often used for anti-tumor therapy. Although it is clinically effective at inhibiting DNA methylation, the mechanistic effect that 5-azaC elicits on eukaryotic cells is controversial. It has been proposed that incorporation of 5-azaC into DNA during replication irreversibly tethers cytosine methyltransferases (MTases) to DNA. This DNA-MTase adduct is targeted by the DNA repair machinery and removed to restore normal DNA functionality; as such some DNA repair mutants are sensitive to 5-azaC. However, double mutants lacking both the cytosine MTase and DNA repair enzymes are still 5-azaC susceptible. Here, we characterize a defective in methylation (dim) mutant of Neurospora crassa, dim-1, that is resistant to 5-azaC when DNA repair is abolished. The causative mutation in a dim-1 strain is in an AAA-ATPase chromatin remodeler conserved from yeast to humans, and indeed a Δdim-1 strain has atypically-spaced nucleosomes within heterochromatic, intergenic, and genic regions, suggesting that DIM-1 influences nucleosome positioning genome-wide. A dim-1 mutant has an ~40-50% reduction in total cytosine methylation but surprisingly gains new cytosine methylation peaks at intergenic regions that are often the promoters of highly expressed genes; nucleosome disorder and DIM-1 localization are also observed at these regions. We propose aberrant nucleosome density not only signals for the catalysis of cytosine methylation in Neurospora, but plays a role in 5-azaC resistance upon loss of DNA repair. Our data shed light on a new relationship between aberrant DNA methylation, DNA repair, an anti-tumor DNA demethylating agent, and nucleosome positioning.

ORGANISM(S): Neurospora crassa

PROVIDER: GSE98911 | GEO | 2019/01/16

REPOSITORIES: GEO

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Project description:Cytosine methylation, a fundamental form of epigenetic regulation, is found in many eukaryotes and plays a significant role in cancer and other diseases. Using the genetically tractable model organism Neurospora crassa, we have identified genes that when mutated, cause the strains to be defective in methylation (dim). The process of DNA methylation in Neurospora has been shown to be dependent on DCDC, a five-member complex that directs the histone methyltransferase DIM-5 to tri-methylate Lysine 9 on histone H3 (H3K9me3). This mark is recognized by HP1, which directs DIM-2 to methylate DNA. In contrast, the HCHC complex employs HDA-1, CDP-2, HP1, and CHAP to deacetylate that same residue on H3. While we know a good deal about DNA methylation, it is still unclear whether we have identified all the genes involved in the process. Thus, we continued our search for dim mutants, using a selection for reactivation of silenced drug resistance genes. Interestingly, we predominantly identified known dim genes, including dim-5, dim-7, dim-8, dim-9, hpo, chap, cdp-2, and hda-1. Using a Sanger sequencing-based approach, we identified mutations in these known dim genes, presumably responsible for the Dim- phenotype; many mutations were unique point mutants that could compromise protein activity or structure, or impact protein-protein interactions. For mutants in which the gene was not placed into a complementation group, we employed a bulk segregant analysis and whole genome sequencing approach to identify additional mutations in known DNA methylation genes, including hH3 and dim-1, as well as a novel dim mutant: dim-10, a fungal-specific protein that may work with DCDC for H3K9me3 catalysis. Not only will this dim mutant collection be a useful resource to investigate the roles of these dim genes and their protein products in DNA methylation, but the isolation of a novel dim gene by a forward genetics approach provides an exciting avenue of research into how incipient heterochromatin formation is achieved.

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Nucleosome Positioning by an Evolutionarily Conserved Chromatin Remodeler Prevents Aberrant DNA Methylation in Neurospora.

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