RNA interference (RNAi) mediates chromatin modifications in fungi, plants, drosophila and vertebrates. In the fission yeast Schizosaccharomyces pombe, where the molecular mechanisms are the most detailed, it is believed that small interfering RNAs (siRNAs) guide the RNA-Induced Transcriptional Silencing (RITS) complex to specific chromatin regions to trigger formation of heterochromatin. Although it is clear that siRNAs are required for RITS localization to chromatin, important events of RITS siRNA-dependent recruitment remain to be elucidated. Additionally, critical aspects of the following RITS-driven heterochromatin formation mechanism need to be clarified. Genetic data indicate that among proteins required for assembly of heterochromatin some act very early in this process to the point that they should interact directly or indirectly with RITS. However, no physical connection between RITS and these enzymes has yet been found. This project proposes to address these fundamental aspects of RNAi-mediated heterochromatin assembly in S.pombe, by coupling interdisciplinary and innovative approaches to classical protein affinity purifications, yeast molecular genetics and chromatin techniques. The first part of the project will concentrate on the characterization of an unprecedented physical connection between RITS and a chromatin-modifying activity. In parallel, we propose to develop two complementary approaches to identify new proteins that physically and/or genetically interact with RITS. Finally, thanks to a UV light-induced crosslinking technology, we intend to study RITS recruitment to chromatin by analyzing its direct interaction with nucleic acids both in vitro and in vivo. As the control of chromatin structure by RNAi is evolutionary conserved, our studies have the potential to bring general insights into how RNAi-based chromatin modifications take place and are regulated in the kingdom of eukaryotes.