Investigating the role of RNAi and epigenetic modifications in the basidiomycetes yeast Cryptococcus deneoformans

The basidiomycete yeasts from the Cryptococcus neoformans/gattii species complex are major fungal pathogens and are particularly prevalent in the developing world. The species within the complex are rapidly evolving, with several species loosing genes encoding proteins required for silencing by the RNA interference (RNAi) pathway which correlate with an increase virulence. The species C. deneoformans, however, has retained all five of the core RNAi components (Rdp1, Ago1, Ago2, Dcr1, Dcr2), and has been shown to have a functional RNAi pathway involved in the silencing of transposable elements (TEs). Centromeric TEs have also been shown to coincide with DNA methylation in C. deneoformans, and also with H3K9 methylation in neighbouring species C. neoformans. Here I look at the relationship between these three potential mechanisms of silencing in C. deneoformans, RNAi, DNA methylation and H3K9 methylation, focussing on how RNAi interacts with both methylation marks in TE regulation. Identification of the H3K9 methyltransferase Clr4 and H3K9me2-ChIP confirmed the presence of H3K9 methylation at the centromeres in C. deneoformans. Analysis of strains with deletions of core RNAi components revealed wild-type levels of centromeric H3K9 methylation, confirming that RNAi is not required for maintenance of this heterochromatin mark. Analysis of transcript levels at RNAi target sites showed no difference between wild-type and RNAi deficient strains. This suggests that RNAi silences targets through a post-transcriptional gene silencing (PTGS) method out with RNA degradation. To investigate the role of RNAi in suppressing transposon activity, mutation rate assays were carried out by screening for spontaneous 5-FOA resistance that results from disruption of the URA3 or URA5 genes. Strains lacking both H3K9 methylation and DNA methylation (clr4Δdnmt5Δ) had the highest drug resistance rates. PCR screening determined if 5-FOA resistance was due to transposon insertion into URA3 or URA5, and both T1 and T2 DNA transposon insertions were identified. The rate of inserts identified within rdp1Δ and clr4Δdnmt5Δ strains was significantly higher than in WT, showing increased transposon mobility in both strains. Analysis of DNA transposable element expression showed large variance between replicate cultures but suggested that T3 may be regulated by an RNAi-independent mechanism, unlike T1 and T2 where suppression appears dependent on Rdp1. Analysis of retrotransposon copy numbers showed no significant increase in any strains tested when compared to WT. Overall this shows a potential role for H3K9 and/or DNA methylation in controlling transposon mobility alongside RNAi. Finally, analysis was carried out into the roles of both Argonaute proteins within C. deneoformans, as Ago2 is frequently lost within the species complex, and is not present within neighbouring species C. neoformans. Mass spectrometry of tagged proteins showed that each Ago binds to a different subset of proteins, suggesting a different role for each protein within the RNAi pathway. Deletion of Gwo1, the main Ago1 interactor, increases the interaction of Ago1 with Ago2. The work undertaken here contributes to the further understanding of the interaction between RNAi and the DNA and H3K9 methylation silencing pathway in C. deneoformans and shed lights on the different roles of the two Argonaute proteins in this species.