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Updated: Dec 10, 2025
Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe
Published on: September 13, 2022
Selection and Characterization of Mutants Defective in DNA Methylation in Neurospora crassa
Andrew D Klocko1, Calvin A Summers1, Marissa L Glover1
1Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403.
Abstract:
DNA methylation, a prototypical epigenetic modification implicated in gene silencing, occurs in many eukaryotes and plays a significant role in the etiology of diseases such as cancer. The filamentous fungus Neurospora crassa places DNA methylation at regions of constitutive heterochromatin such as in centromeres and in other A:T-rich regions of the genome, but this modification is dispensable for normal growth and development. This and other features render N. crassa an excellent model to genetically dissect elements of the DNA methylation pathway. We implemented a forward genetic selection on a massive scale, utilizing two engineered antibiotic-resistance genes silenced by DNA methylation, to isolate mutants efective n ethylation (dim). Hundreds of potential mutants were characterized, yielding a rich collection of informative alleles of 11 genes important for DNA methylation, most of which were already reported. In parallel, we characterized the pairwise interactions in nuclei of the DCDC, the only histone H3 lysine 9 methyltransferase complex in Neurospora, including those between the DIM-5 catalytic subunit and other complex members. We also dissected the N- and C-termini of the key protein DIM-7, required for DIM-5 histone methyltransferase localization and activation. Lastly, we identified two alleles of a novel gene, dim-10 - a homolog of Clr5 in Schizosaccharomyces pombe - that is not essential for DNA methylation, but is necessary for repression of the antibiotic-resistance genes used in the selection, which suggests that both DIM-10 and DNA methylation promote silencing of constitutive heterochromatin.
Insights
This study identifies novel genes involved in DNA methylation in Neurospora crassa, a key epigenetic process. Findings reveal new insights into gene silencing mechanisms and their role in heterochromatin regulation.
Area of Science:
- Epigenetics
- Molecular Biology
- Genetics
Background:
- DNA methylation is a crucial epigenetic modification involved in gene silencing and disease etiology, including cancer.
- Neurospora crassa serves as an ideal model organism for studying DNA methylation due to its unique genomic features and dispensable methylation for growth.
Purpose of the Study:
- To genetically dissect the DNA methylation pathway in Neurospora crassa using large-scale forward genetic selection.
- To characterize interactions within the DIM-5 histone methyltransferase complex and identify novel factors involved in heterochromatin silencing.
Main Methods:
- Large-scale forward genetic screen using engineered antibiotic-resistance genes silenced by DNA methylation.
- Characterization of mutant alleles affecting DNA methylation and heterochromatin function.
- Analysis of protein interactions within the DIM-5 complex and functional dissection of DIM-7.
Main Results:
- Isolation and characterization of numerous alleles across 11 genes critical for DNA methylation.
- Detailed analysis of interactions within the DIM-5 histone methyltransferase complex.
- Identification of a novel gene, DIM-10, essential for silencing antibiotic-resistance genes, suggesting a role alongside DNA methylation in constitutive heterochromatin repression.
Conclusions:
- The study successfully identified key genetic components of the DNA methylation pathway in Neurospora crassa.
- DIM-10 represents a novel factor contributing to heterochromatin-mediated gene silencing, working in concert with DNA methylation.
- Neurospora crassa continues to be a valuable model for understanding fundamental epigenetic mechanisms.
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