In the course of this ERC project, we gained important novel insights into epigenetic regulation of gene expression. The main results achieved can be grouped into five categories:
1) Transgenerational inheritance of epigenetic information
This project was based on our previous discovery of a counter-acting mechanism that impedes small RNA-directed formation of heterochromatin and epigenetic gene silencing. With this project we have further investigated this phenomenon. Intriguingly, working with cells in which counter-acting activity was turned off, we observed that newly established heterochromatin can be passed on to many subsequent generations, even in the absence of the primary siRNAs that initiated heterochromatin assembly. This complies with the classical definition of epigenetics, i.e. it is heritable even in the absence of the initiating signal.
2) Inheritance of a phenotypically neutral epimutation can evoke gene silencing in later generations
RNA-directed epimutagenesis is commonly associated with persistent gene repression. Within this project we found that RNA-induced epimutations are still inherited even when the silenced gene is reactivated, and descendants can reinstate the silencing phenotype that only occurred in their ancestors. Thus, yeast cells can acquire a new trait that is plastic and can be passed down to its offspring.
3) Genetic predisposition for transgenerational, RNAi-mediated gene silencing
This ERC grant enabled us to perform comprehensive forward genetic screens that has led to the identification of more than twenty novel mutant alleles that enable the RNA interference pathway to trigger de novo formation of heterochromatin in S. pombe. This leads us to speculate that fission yeast’s natural ecology may lead to the acquisition of silencing enabling mutations as part of a biological bet-hedging strategy. In other words, we propose that epigenetic gene silencing is always preceded by a genetic change in this organism.
4) Cdk1-dependent phosphorylation of Crl4Suv39H controls an H3 methylation switch that is essential for gametogenesis
To further dissect mechanisms of transgenerational epigenetic inheritance, we implemented protocols enabling us to perform biochemical and genomics experiments in meiotic cells. Thereby we found that constitutive heterochromatin temporarily loses H3K9me2 and becomes H3K9me3 when cells commit to meiosis. Cells lacking the ability to tri-methylate H3K9 exhibit meiotic chromosome segregation defects, which does not occur in mitotic cells. Intriguingly, we also found that the H3K9 methylation switch is accompanied by differential phosphorylation of Clr4 by the cyclin-dependent kinase (CDK) Cdk1, suggesting that Clr4 activity could be regulated by Cdk1-dependent phosphorylation.
5) RNAi-mediated chromatin silencing in mammals
Whereas well established in fission yeast, plants, and worms, RNAi-mediated chromatin regulation in mammalian cells is controversial. The counteracting mechanisms that are operational in yeast made us speculate that RNAi could be similarly controlled also in mammals. To study potential conservation of the pathways that we have been elucidating in fission yeast, we have been employing mouse embryonic stem cells as a mammalian model system. We have been using cutting-edge genome engineering and next-generation sequencing to test a possible conserved function of Paf1C in suppressing RNAi-directed epigenetic gene silencing also in mammalian cells. Whereas we have been making good progress from a technical perspective, we have not found evidence for conservation of RNAi-mediated epigenetic silencing in this system.
We have published our findings in original research publications, and we presented our work at international research conferences or at seminars I was invited to held at European research institutions. We have also published a protocol along with a deep learning pipeline for colony segmentation and classification. Finally, we published a Review Article for Trends in Genetics about transgenerational inheritance of epigenetic information by small RNAs, which was greatly inspired by the work we performed throughout this project.
