A RID-like putative cytosine methyltransferase homologue controls sexual development in the fungus Podospora anserina.

DNA methyltransferases are ubiquitous enzymes conserved in bacteria, plants and opisthokonta. These enzymes, which methylate cytosines, are involved in numerous biological processes, notably development. In mammals and higher plants, methylation patterns established and maintained by the cytosine DNA methyltransferases (DMTs) are essential to zygotic development. In fungi, some members of an extensively conserved fungal-specific DNA methyltransferase class are both mediators of the Repeat Induced Point mutation (RIP) genome defense system and key players of sexual reproduction. Yet, no DNA methyltransferase activity of these purified RID (RIP deficient) proteins could be detected in vitro. These observations led us to explore how RID-like DNA methyltransferase encoding genes would play a role during sexual development of fungi showing very little genomic DNA methylation, if any. To do so, we used the model ascomycete fungus Podospora anserina. We identified the PaRid gene, encoding a RID-like DNA methyltransferase and constructed knocked-out ΔPaRid defective mutants. Crosses involving P. anserina ΔPaRid mutants are sterile. Our results show that, although gametes are readily formed and fertilization occurs in a ΔPaRid background, sexual development is blocked just before the individualization of the dikaryotic cells leading to meiocytes. Complementation of ΔPaRid mutants with ectopic alleles of PaRid, including GFP-tagged, point-mutated and chimeric alleles, demonstrated that the catalytic motif of the putative PaRid methyltransferase is essential to ensure proper sexual development and that the expression of PaRid is spatially and temporally restricted. A transcriptomic analysis performed on mutant crosses revealed an overlap of the PaRid-controlled genetic network with the well-known mating-types gene developmental pathway common to an important group of fungi, the Pezizomycotina.

Modulation of C-to-T mutation by recombination-independent pairing of closely positioned DNA repeats.

Repeat-induced point mutation is a genetic process that creates cytosine-to-thymine (C-to-T) transitions in duplicated genomic sequences in fungi. Repeat-induced point mutation detects duplications (irrespective of their origin, specific sequence, coding capacity, and genomic positions) by a recombination-independent mechanism that likely matches intact DNA double helices directly, without relying on the annealing of complementary single strands. In the fungus Neurospora crassa, closely positioned repeats can induce mutation of the adjoining nonrepetitive regions. This process is related to heterochromatin assembly and requires the cytosine methyltransferase DIM-2. Using DIM-2-dependent mutation as a readout of homologous pairing, we find that GC-rich repeats produce a much stronger response than AT-rich repeats, independently of their intrinsic propensity to become mutated. We also report that direct repeats trigger much stronger DIM-2-dependent mutation than inverted repeats. These results can be rationalized in the light of a recently proposed model of homologous DNA pairing, in which DNA double helices associate by forming sequence-specific quadruplex-based contacts with a concomitant release of supercoiling. A similar process featuring pairing-induced supercoiling may initiate epigenetic silencing of repetitive DNA in other organisms, including humans.