Project Summary/Abstract Genome instability increases the rates of mutations, chromosomal rearrangements, and aneuploidy and drives many age-related human diseases, including cancer. The challenges of replicating our genome and epigenome with each cell division requires molecular pathways that ensure the propagation of stable genomes to daughter cells. To date, the roles of mammalian noncoding RNAs (ncRNAs) in the maintenance of genome stability is incompletely understood. We propose that filling this gap is essential to expand our knowledge of human development and homeostasis and to identify novel risk factors that contribute to human disease. In fission yeast, the RNA interference (RNAi) pathway acts during DNA synthesis at the repeats of pericentromeres via locally produced ncRNAs to establish the heterochromatin needed for genome stability. It is unclear if similar cell cycle-specific RNAi mechanisms of genome stability are present in mammals. In preliminary studies, we used mouse stem cell systems to map high-confidence interactions of the RNAi effector Argonaute (Ago) proteins with ncRNAs. We determined that Ago binds directly to pericentromeric ncRNAs. Furthermore, we found that pericentromeric ncRNAs are overexpressed during DNA synthesis in Ago-deficient cells and observed multiple signatures of genomic instability caused by full Ago depletion. In parallel, previous studies in mouse models have established that overexpression of pericentromeric ncRNA suffices to cause genomic instability and tumorigenesis. Based on these intriguing findings, we hypothesize that pericentromeric ncRNA expression, established via cell cycle-specific Ago regulation, is critical for the maintenance of genomic stability in mammalian cells, analogous to fission yeast. In this RO1 research project, we examine the potential roles of RNAi in preventing genomic instability caused by accumulating pericentromeric ncRNA and/or disruption of local heterochromatin structure. We propose to define cell cycle- specific Ago activities to elucidate the molecular triggers for pericentromere regulation and to determine how pericentromeric ncRNAs contribute to genome stability. In Aim 1, we will determine how and when Ago functions at pericentromeres for the establishment of heterochromatin. In Aim 2, we determine the direct molecular consequence of Ago recruitment to chromatin. Finally, in Aim 3, we will elucidate how ncRNAs control Ago activity at pericentromeres during cell cycle progression. These innovative studies will answer the long-standing question of whether the mammalian RNAi pathway contributes to the maintenance of pericentromere heterochromatin and genome stability and will break new ground in our understanding of RNA- mediated gene regulation. We predict these studies will drive and yield a framework for future strategies to modulate pericentromeric ncRNA in disease settings.