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Bisulfite-seq from Neurospora crassa a wild type (WT) strain grown in minimum medium, a dim-3 strain grown in minimum medium, and a dim-3 strain grown with supplemented histidine

ABSTRACT: We report the placement of cytosine methylation from the filamentous fungus Neurospora crassa in wild type and dim-3 strains by bisulfite-sequencing. Compared to a wild type strain, the dim-3 strain has a global reduction in cytosine methylation, and this reduction in cytosine methylation is exacerbated by the supplementation of histidine to the growth medium. This global reduction in cytosine methylation results from a causative mutation in importin alpha (NUP-6), a component of the nuclear transport machinery, which severely reduces the level of the heterochromatic mark H3K9me3. NUP-6(dim-3) compromises the heterochromatic localization of several components of the DCDC, a histone H3K9 methyltransferase complex, which in turn reduces the level of Heterochromatin Protein-1 (HP1) found at heterochromatin and compromises the direct interaction between HP1 and the DNA methyltransferase dim-2. To determine whether the reduced level of cytosine methylation, as assayed by Southern blotting heterochromatic regions following digestion of genomic DNA with methylation-sensitive restriction endonucleases, is globally reduced at the A:T rich, repetitive DNA comprising heterochromatin, we performed Bisulfite-seq on a dim-3 strain (grown either in minimal medium or medium supplemented with histidine) and compared that to a wild type strain (grown in minimum medium; histidine does not reduce the levels of cytosine methylation in a wild type strain, as assayed by Southern blotting).

ORGANISM(S): Neurospora crassa

PROVIDER: GSE61174 | GEO | 2015/03/22

SECONDARY ACCESSION(S): PRJNA260402

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.

Dataset Information

Bisulfite-seq from Neurospora crassa a wild type (WT) strain grown in minimum medium, a dim-3 strain grown in minimum medium, and a dim-3 strain grown with supplemented histidine

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