RNA interference (RNAi) is a highly conserved, sequence-specific gene regulatory mechanism among eukaryotes. It is critical for a variety of important biological functions and is being pursued as a promising new tool for the treatment of a variety of human maladies. A surprising link between heterochromatin and the RNAi pathway was discovered a few years ago in fission yeast and plants, and similar mechanisms have more recently been described in various eukaryotes. However, to what extent the mechanisms we have been studying in yeast are conserved up to humans remains unknown. The goal of this proposal is to further our understanding of RNAi-mediated heterochromatin assembly by using fission yeast as a model organism, but also to investigate to role of RNAi in the nucleus of human cells.My proposal consists of three major aims. In aim 1 I propose to combine light and electron microscopy to address important and largely unanswered questions such as subcellular localization and temporal regulation of the RNAi pathway. Aim 2 builds on our recent discovery that RNAi factors physically associate with chromatin to control genome activity also outside constitutive heterochromatin. I am proposing experiments in fission yeast that aim at understanding the biological role of this new mode of genome regulation and its mechanistic dissection . However, we will also extend our analysis to human cells which will shed new light on the role of the RNAi pathway in the nucleus of higher eukaryotes. Finally, we are aiming at identifying the features a target locus in the S. pombe genome must have to become susceptible to RNAi-mediated silencing at the level of chromatin. Thus, the outcome of these experiments may substantially influence the developments of siRNA-based therapeutics.