Abstract
Aging, defined as the loss of cellular function over time, affects all eukaryotic organisms. The phenotypes seen in cellular aging have been well categorized into the “Hallmarks of Aging”. Although these hallmarks are well defined, many of the molecular mechanisms that drive these phenotypes remain unknown. Furthermore, it is difficult to determine which of these aging phenotypes are a driver of aging or simply a byproduct of the aging process. Using budding yeast replicative aging as a model for aging in actively dividing cells, we investigate two major Hallmarks of Aging: loss of proteostasis and genomic instability.
Using proteomics, we identified a set of proteins that are depleted from the nucleus in the earliest stages of aging, as well as several that are increased in the nucleus with replicative age. We found that many of the age-depleted nuclear proteins were involved in ribosome biogenesis (ribosome processing) or in maintenance of chromatin stability. Focusing on the topoisomerase, Top1, we found that its depletion in the early stages of aging was not a result of transcriptional changes or changes in protein turnover, but likely resulted from a decrease in translation. Despite the importance of Top1 in maintaining genome stability, we found that a rescue of this age-dependent depletion via mild TOP1 overexpression decreased replicative lifespan. We uncovered a possible mechanism for this RLS decrease in which Top1 overexpression disrupts the stoichiometry of the RENT complex by pulling Sir2 away from the rDNA, a phenotype that is further enhanced when Top1 is mutated to be catalytically inactive. Additionally, we found that overexpression of the catalytically dead TOP1 mutant protein decreased RNA pol II silencing of a reporter gene integrated at the rDNA locus. The Top1 mutant protein was also unable to complement the rDNA silencing defect of a top1∆ strain, suggesting that Top1 plays an important role in the establishment of rDNA silencing, and leading us to hypothesize that DNA topology is important for the unidirectional spreading of rDNA silencing.
We next focused on identifying genomic hotspots for double-strand DNA breaks, a lethal form of genomic instability known to play a role in cancer formation and progression, and how those DSB hotspots change in the context of replicative aging. Using progressively aged populations of yeast mother cells, we generated genome wide DSB maps for early and late stages of replicative aging. We found that the amount of DSBs in the rDNA increases with replicative age, specifically at the end of A/T tracts in the IGS1 spacer region. We also discovered that DSBs at the centromeres decrease. Furthermore, in all age groups, DSBs were enriched at TSS and TTS. The accumulation of breaks in TSS and TTS increased with age compared to the young control. Additional identification of A/T rich DSB hotspots revealed that many of the A/T rich DSB peaks are mapped to mitochondrial DNA.
Taken together, my thesis work uses yeast RLS as a model to identify molecular events that contribute to two key Hallmarks of Aging, loss of proteostasis and genomic instability. We demonstrate that nuclear depletion of proteins begins in early replicative aging. We found that translational changes responsible for age-associated Top1 protein depletion may impact rDNA silencing and lifespan via a role for Top1 (both structurally and catalytically) in establishing rDNA silencing. Furthermore, we uncover age-dependent changes in levels of double-strand DNA breaks, particularly at the rDNA locus, as well as transcriptional start and stop sites throughout the genome, suggesting altered genome maintenance mechanisms during aging. These insights open many unanswered questions about the intersections of a disrupted proteome with genomic instability in aging and provide avenues for further exploration of the molecular mechanisms of aging.
