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

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. Overall design: MNase-seq: We analyzed nucleosome positioning by micrococcal nuclease (MNase) digestion followed by paired end sequencing of the mono- and di-nucleosome fractions (as well as the linker DNA) from the 8 and 10 minute timepoints from a MNase timecourse digestion of uncrosslinked, isolated nuclei. ChIP-seq: We analyzed a dim-1 complete deletion strain (N5801) of Neurospora crassa by Chromatin Immunoprecipitation for the histone marks H3K9me3, H3K4me3, and H3K27me2me3. Strains were grown, crosslinked, lysed, the histone marks were immunopurified, and associated DNA was sequenced. RNA-seq: We analyzed a dim-1 complete deletion strain of Neurospora crassa by poly-A+ mRNA-sequencing for gene expression changes by monitoring poly-adenylated messenger RNA levels in duplicate. A wild type strain (N3752) serves as the reference strain (deposited in GSE82222). Bisulfite-seq: Examination of cytosine methylation, as determined by bisulfite treatment, of two dim-1 strains (a "natural" strain that was the outcome of a mutant selection, and a dim-1 [NCU06484] complete deletion strain). Wild type Bisulfite-seq, deposited in GSE61175, was used as a positive control for comparison