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HiC of Wild Type Neurospora crassa and mutants deficient in heterochromatin formation

ABSTRACT: Eukaryotic genomes are organized into chromatin domains with distinct three-dimensional arrangements resulting from nucleic acid and protein factor interactions within the physical constraints of the nucleus. It is of obvious interest to determine interactions between various chromosomal regions defined by these nuclear constraints, and to identify important factors that limit the interactions. We used chromosome conformation capture (3C) followed by high-throughput sequencing (HiC) to improve our understanding of Neurospora crassa genome organization and to examine if known components of heterochromatin machinery influence nuclear organization. In heterochromatin establishment, DIM-5 tri-methylates histone H3 on lysine 9 (H3K9me3), and this mark is subsequently bound by Heterochromatin Protein-1 (HP1). NUP-6 is required to target DIM-5 to incipient A:T rich DNA and the dim-3 mutant of NUP-6 is deficient in DIM-5 localization. We performed HiC on chromatin from wildtype, Δdim-5, Δhpo, and dim-3 strains. The genome configuration of wild type nuclei revealed strong intra- and inter-chromosomal associations between both constitutive and facultative heterochromatic domains, with the strongest interactions among the centromeres, telomeres and interspersed heterochromatin. We found that loss of the H3K9me3 mark, as well as loss of HP1, minimally altered the chromatin organization found at these strongly interacting heterochromatic regions. Surprisingly, dim-3 chromatin was highly disorganized suggesting NUP-6 plays key role(s) in genome structure. Thus, our datasets suggest that while heterochromatic regions are critical to defining the genome conformation in Neurospora, non-canonical protein factors may play a key role in maintaining this organization.

ORGANISM(S): Neurospora crassa

PROVIDER: GSE71024 | GEO | 2016/05/19

SECONDARY ACCESSION(S): PRJNA290116

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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HiC of Wild Type Neurospora crassa and mutants deficient in heterochromatin formation

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