UC San Diego

Sir2 is the founding sirtuin, a protein class of nicotinamide adenine dinucleotide (NAD) dependent histone deacetylases, that plays a prominent role in the aging of budding yeast by silencing ribosomal DNA. Previous single-cell analysis of Sir2 activity has revealed dynamic, multigenerational oscillations in ribosomal DNA silencing important to longevity. Time lapse fluorescence microscopy and microfluidic technologies are combined with a reporter expressed by a promoter regulated by a non-catalytic, NAD(+) sensitive activator to better characterize Sir2 silencing dynamics. Results reveal that multigenerational NAD(+) temporal patterns exist in synchrony with Sir2-mediated ribosomal DNA silencing. NAD(+) levels were also shown to often decrease across life span in a death phenotype. Decay of both Sir2-silencing and NAD(+) metabolism at similar timepoints suggest that both dynamics are related but that Sir2 dysregulation is not likely caused by NAD(+) oscillations. Additional experiments suggest that regulation of NAM by the nicotinamidase, Pnc1, is a driving factor in rDNA silencing dynamics. These findings strengthen the connection between metabolic and silencing dynamics as key factors in cellular aging and could provide groundwork for future studies into possible pathways between mitochondrial and nuclear regulation of aging.