Background Selective gene silencing is key to development. It is generally accepted that H3K27me3-enriched heterochromatin maintains transcriptional repression established during early development and regulates cell fate. Conversely, H3K9me3-enriched heterochromatin prevents differentiation but constitutes protection against transposable elements. We exploited the fungus Podospora anserina, a valuable alternative to higher eukaryote models, to question the biological relevance and functional interplay of these two distinct heterochromatin conformations. Results We established genome-wide patterns of H3K27me3 and H3K9me3 modifications, and found these marks mutually exclusive within gene-rich regions but not within repeats. We generated the corresponding histone methyltransferase null mutants and showed an interdependence of H3K9me3 and H3K27me3 marks. Indeed, removal of the PaKmt6 EZH2-like enzyme resulted not only in loss of H3K27me3 but also in significant H3K9me3 reduction. Similarly, removal of PaKmt1 SU(VAR)3–9-like enzyme caused loss of H3K9me3 and substantial decrease of H3K27me3. Removal of the H3K9me binding protein PaHP1 provided further support to the notion that each type of heterochromatin requires the presence of the other. We also established that P. anserina developmental programs require H3K27me3-mediated silencing, since loss of the PaKmt6 EZH2-like enzyme caused severe defects in most aspects of the life cycle including growth, differentiation processes and sexual reproduction, whereas loss of the PaKmt1 SU(VAR)3–9-like enzyme resulted only in marginal defects, similar to loss of PaHP1. Conclusions Our findings support a conserved function of the PRC2 complex in fungal development. However, we uncovered an intriguing evolutionary fluidity in the repressive histone deposition machinery, which challenges canonical definitions of constitutive and facultative heterochromatin.

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 Δ PaRid defective mutants. Crosses involving P . anserina Δ PaRid mutants are sterile. Our results show that, although gametes are readily formed and fertilization occurs in a Δ PaRid background, sexual development is blocked just before the individualization of the dikaryotic cells leading to meiocytes. Complementation of Δ PaRid 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.