Final Report Summary - RNAIGENREG (RNAi-mediated genome regulation)
RNA interference (RNAi) 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 diseases. A surprising link between epigenetic genome regulation and the RNAi pathway was discovered in fission yeast and plants, and our group was involved in this new and exciting field of research from the beginning. Despite intensive research, our insight into certain aspects of nuclear RNAi is rudimentary at best and it remains largely unknown to what extent the mechanisms we have been studying in yeast are conserved in other eukaryotes. The goal of this ERC-funded project was to go beyond the state of the art and make substantial advances in our understanding of nuclear functions of RNAi pathways. We were combining yeast genetics with innovative imaging and genomics approaches to address fundamental questions that have remained open. During the project we have successfully implemented a microscopy protocol (CLEM) that combines light and electron microscopy by imaging a specially prepared ultrathin section with two microscopy modalities. This protocol allows us to integrate intracellular localization of a protein using a fluorescent tag with high resolution imaging of the same cell, which also provides information about the cellular context the protein is located in. We have been using CLEM to study subcellular localization of RNAi factors in fission yeast and have found that Dicer is localized to electron-dense membrane-free particles in the cytoplasm, whose formation is heat dependent. This has led to the discovery of an important feedback loop that regulates the RNAi pathway in yeast.
Besides employing cutting edge microscopy techniques, we have been using a special chromatin profiling technique referred to as DamID, which led to the identification of novel physiological targets of RNAi. We discovered a class of genes that are bound by the stress response transcription factor Atf1 and which we refer to as BANCs. We found that BANC genes are associated with nuclear pore complexes (NPCs) and RNAi proteins, which are all required for their tight repression. Because RNA polymerase II occupancy is not affected in RNAi mutants at the majority of BANCs, we conclude that RNAi functions on a truly co-transcriptional level to tightly repress this class of genes under non-stressful conditions.
Finally, through a forward genetic screen we uncovered fission yeast mutants that are highly susceptible to de novo formation of heterochromatin and stable gene silencing by siRNAs. Beyond basic research, these findings are highly relevant for biotechnology and biomedicine. Specifically, inducing stable epigenetic-mediated repression of a disease-causing gene by transient delivery of siRNAs would constitute a big step forward in developing successful RNAi-based therapeutics. In the course of a follow-up project that is funded by an ERC Consolidator Grant, we will dissect the details of this repressive mechanism and test whether similar activities exist in mammalian cells.
Besides employing cutting edge microscopy techniques, we have been using a special chromatin profiling technique referred to as DamID, which led to the identification of novel physiological targets of RNAi. We discovered a class of genes that are bound by the stress response transcription factor Atf1 and which we refer to as BANCs. We found that BANC genes are associated with nuclear pore complexes (NPCs) and RNAi proteins, which are all required for their tight repression. Because RNA polymerase II occupancy is not affected in RNAi mutants at the majority of BANCs, we conclude that RNAi functions on a truly co-transcriptional level to tightly repress this class of genes under non-stressful conditions.
Finally, through a forward genetic screen we uncovered fission yeast mutants that are highly susceptible to de novo formation of heterochromatin and stable gene silencing by siRNAs. Beyond basic research, these findings are highly relevant for biotechnology and biomedicine. Specifically, inducing stable epigenetic-mediated repression of a disease-causing gene by transient delivery of siRNAs would constitute a big step forward in developing successful RNAi-based therapeutics. In the course of a follow-up project that is funded by an ERC Consolidator Grant, we will dissect the details of this repressive mechanism and test whether similar activities exist in mammalian cells.