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 defective in methylation (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.
| Original language | English |
|---|---|
| Pages (from-to) | 671-688 |
| Number of pages | 18 |
| Journal | Genetics |
| Volume | 216 |
| Issue number | 3 |
| DOIs | |
| State | Published - Nov 2020 |
| Externally published | Yes |
Funding
The authors would like to thank the undergraduate students enrolled in Eric Selker’s Advanced Molecular Genetics course at the University of Oregon for initiating some of the genetic experiments, as well as Alicia Seymour, Rohan Borkar, Christian Lindner, Anthony Shiver, and Hendrik Tegethof for help with the mutant hunt, high school students Lisa and Kevin Selker for early work on the mutant hunt, Josh Lowry (UO) for advice on SNP identification and filtering, Jordan Gessaman and Paula Grisafi for their initial efforts dissecting protein interactions within the DCDC, Yusuke Kazama and Tomoko Abe (RIKEN Nishina Center, Wako, Saitama, Japan) for help with carbon-and argon-ion beam mutagenesis, and all members of the Selker laboratory for helpful comments and discussions. We also thank Kevin McCluskey (Kansas State University) for making the sequence of the Mauriceville strain available prior to publication, as well as the three anonymous reviewers and associate editor Michael Freitag for their comments. Funding for this work was provided by grants from the National Institutes of Health (NIH) to E.U.S. (R01GM035690 and R35GM127142). A.D.K. was supported in part by an NIH postdoctoral fellowship (F32GM097821), and K.J.M. was supported in part by an NIH predoctoral training grant (T32HD007348). The authors would like to thank the undergraduate students enrolled in Eric Selker's Advanced Molecular Genetics course at the University of Oregon for initiating some of the genetic experiments, as well as Alicia Seymour, Rohan Borkar, Christian Lindner, Anthony Shiver, and Hendrik Tegethof for help with the mutant hunt, high school students Lisa and Kevin Selker for early work on the mutant hunt, Josh Lowry (UO) for advice on SNP identification and filtering, Jordan Gessaman and Paula Grisafi for their initial efforts dissecting protein interactions within the DCDC, Yusuke Kazama and Tomoko Abe (RIKEN Nishina Center, Wako, Saitama, Japan) for help with carbon- and argonion beam mutagenesis, and all members of the Selker laboratory for helpful comments and discussions. We also thank Kevin McCluskey (Kansas State University) for making the sequence of the Mauriceville strain available prior to publication, as well as the three anonymous reviewers and associate editor Michael Freitag for their comments. Funding for this work was provided by grants from the National Institutes of Health (NIH) to E.U.S. (R01GM035690 and R35GM127142). A.D.K. was supported in part by an NIH postdoctoral fellowship (F32GM097821), and K.J.M. was supported in part by an NIH predoctoral training grant (T32HD007348).
| Funders | Funder number |
|---|---|
| National Institutes of Health | T32HD007348, R01GM035690, R35GM127142 |
| National Institute of General Medical Sciences | F32GM097821 |
| Kansas State University |
Keywords
- Cytosine methylation
- Epigenetics
- H3K9me3
- Heterochromatin
- Neurospora
