Small ncRNAs play critical roles in endogenous chromatin-mediated processes in plants, C. elegans, D. melanogaster, ciliates, and fungi. For example, siRNAs are indispensable for the maintenance of functional heterochromatin at fission yeast centromeres. Yet, efforts to initiate chromatin modifications in trans using synthetic siRNAs have proved inherently difficult in all eukaryotic cells.
We hypothesized that siRNA-directed formation of heterochromatin is strictly controlled by an unknown mechanism and therefore we designed a forward genetic screen in fission yeast. EMS mutagenesis of a reporter strain uncovered five mutants that are highly susceptible to de novo formation of heterochromatin and stable gene silencing by siRNAs in trans. Using whole-genome next generation sequencing, we mapped causative missense or nonsense mutations in genes coding for subunits of the conserved RNA Polymerase-Associated Factor 1 complex (Paf1C).
Importantly, the Paf1C mutations enabled siRNA-directed heterochromatin formation in trans at all euchromatic loci tested. Our results clearly demonstrate that siRNAs can initiate the formation of heterochromatin and gene silencing, but that this is under strict negative control by Paf1C. This explains previous unsuccessful attempts to induce stable heterochromatin formation in trans using synthetic siRNAs and might also explain why similar efforts in mammalian cells were not very successful.
Remarkably, we observed that newly established heterochromatin was passed on to the next generation in Paf1C mutant cells even in the absence of the primary siRNAs that triggered the assembly of heterochromatin. This phenomenon complies with the classical definition of epigenetics (i.e., that it is heritable even in the absence of the initiating signal) and highlights fundamental roles of Paf1C and the RNAi machinery in building up epigenetic memory. Elucidating the mechanism and potential evolutionary conservation of this stunning phenomenon is the subject of current studies.
This ERC funded project aims at a systematic investigation of counter-acting mechanisms that prevent siRNA-directed epigenetic repression of protein-coding genes in yeast and mammalian cells.
Zaratiegui, Nature, 2015