Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - REpiReg (RNAi-mediated Epigenetic Gene Regulation)

Teaser

RNA interference (RNAi) refers to the ability of small RNAs to silence expression of homologous sequences. A surprising link between epigenetics and RNAi was discovered a while ago. It is now well established that endogenous small RNAs have a direct impact on the genome in...

Summary

RNA interference (RNAi) refers to the ability of small RNAs to silence expression of homologous sequences. A surprising link between epigenetics and RNAi was discovered a while ago. It is now well established that endogenous small RNAs have a direct impact on the genome in various organisms. Yet, the initiation of chromatin modifications in trans by exogenously introduced small RNAs has been inherently difficult to achieve in all eukaryotic cells. This has sparked controversy about the importance and conservation of RNAi-mediated epigenome regulation and hampered systematic mechanistic dissection of this phenomenon.
This has recently changed when we discovered mutations in the polymerase associated factor 1 complex (Paf1C), which enable de novo formation of heterochromatin triggered by ectopic expression of synthetic siRNAs. The goal of this research project is to identify additional factors that impede small RNA-directed formation of heterochromatin, and to dissect their mode of action at the molecular level. We are aiming at closing several knowledge gaps and testing the intriguing possibility that the suppressive mechanisms that we are elucidating in fission yeast are conserved in mammalian cells.

Work performed

Experiments performed in the course of this ERC CoG so far have led to original research papers that we published in Molecular Cell. In one paper, we report the isolation of Mst2 as a novel trans-acting factor that is specifically involved in preventing the initiation of heterochromatin assembly but not maintenance. This is achieved by H3K36me3-dependent sequestration of Mst2 on actively transcribed genes, which is mediated by its interaction partner Pdp3. By restricting Mst2 activity to transcribed protein coding genes, H3K36me3 maintains those in a euchromatic state. Surprisingly, we discovered that Mst2 acetylates a specific lysine in Brl1, a component of the histone H2B ubiquitin ligase complex (HULC), revealing insights into the mechanism by which Mst2 antagonizes the assembly of ectopic heterochromatin and secures epigenome integrity (Flury et al, Molecular Cell, 2017).
In another paper, we describe the discovery of a novel epigenetic phenomenon. Phenotypic effects caused by epimutations rather than changes in DNA sequence have been described in various organisms. Prominent examples of heritable phenotypic changes caused by epimutations are paramutation in plants and RNA-induced epigenetic silencing (RNAe) in nematodes, in which small RNAs trigger the formation of epialleles that are stably silenced across generations. Consequently, RNA-directed epimutagenesis is associated with persistent gene repression. In the course of this ERC 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. Specifically, we found that yeast experiencing an RNAi-mediated gene silencing response acquire an epigenetic imprint that is inherited by subsequent generations even when silencing of the target gene is lost. We have dissected this phenotypically neutral imprint and could show that it is comprised of H3K9me3 marks and secondary siRNA production, and its transgenerational inheritance is mediated by the coupling of H3K9 methylation and the RNAi machinery (Duempelmann et al, Molecular Cell, 2019).
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 so far.

Final results

Our results provide proof of concept that an organism is able to adapt to a change in the environment by inheriting this “knowledge” from its ancestors, even when it hasn’t experienced the environmental trigger directly itself. Because the epigenetic information is inherited with no phenotypic consequences explains why such “adaptive” epigenetic processes have remained obscure. It also means that our finding might have an impact on our understanding of natural variation, and may provide us with a tractable model system to study a potential role of epigenetics in evolution. Thus, besides answering a longstanding question, this work will stimulate future research on transgenerational inheritance in my lab and others.

Website & more info

More info: https://buehlerlab.org/awards-honors/.