Project Summary. Heterochromatin consists of large domains of repetitive DNA such as centromeres, telomeres, rDNA, and retrotransposons that remain organized into compact chromatin structures throughout interphase, when other portions of the chromosomes (euchromatin) usually decondense. Heterochromatin is generally repressive to transcription and plays important roles in maintaining genome stability, whether it is through facilitating centromere function, telomere protection, or suppressing recombination between the underlying DNA repeats to help maintain genomic integrity. Such is the case with the rDNA locus, a relatively understudied and unusual form of heterochromatin consisting of tandemly repeated rRNA genes. Paradoxically, the rDNA is heavily transcribed by RNA polymerase I (Pol I) to synthesize ribosomal RNA, yet retains several key heterochromatin characteristics such as suppression of recombination and “silencing” of RNA polymerase II (Pol II)-dependent transcription. In budding yeast rDNA, transcription of non-coding RNAs from the intergenic spacers must be silenced by the conserved NAD+-dependent histone deacetylase Sir2 to maintain rDNA stability and support replicative lifespan. Remarkably, silencing of these non-coding RNAs by Sir2 actually requires Pol I-dependent transcription of the large rRNA coding genes. Therefore, the nucleolus has a rather complex and dynamic chromatin environment designed to optimize rRNA synthesis while maintaining integrity of the tandem array. During replicative aging of budding yeast, the Sir2 protein, along with the cohesin complex, and other nuclear proteins, are progressively depleted as the cells get older. This results in deterioration of the rDNA heterochromatin and instability of the array. Interestingly, the replicatively aging cells also have a chromosome instability (CIN) phenotype that is driven by the rDNA instability. Overexpression of the Mcd1 subunit of cohesin suppresses the age-induced rDNA and CIN phenotypes, and strongly extends lifespan. The experiments in this project are designed to identify determine how stabilization of the rDNA array by SIR2 and cohesin leads to improved fidelity of chromosome segregation. We hypothesize that rDNA interactions with the genome, including centromeres, play a significant role in the age-induced CIN phenotype. Therefore, we will develop a method to define genome-wide rDNA contacts, and test how these contacts change with age or when rRNA synthesis is compromised. Mechanistic experiments will also address why certain nuclear proteins are depleted or destabilized with aging, and identify additional dose dependent longevity factors. We anticipate these studies in yeast will provide a paradigm for future structural-function studies of rDNA heterochromatin during aging in mammalian cells.