Intersections of Nutrient Availability, Heterochromatic Silencing, Growth, Aging and DNA Damage Repair in Saccharomyces cerevisiae

AbstractIntersections of Nutrient Availability, Heterochromatic Silencing, Growth, Aging and DNA Damage Repair in Saccharomyces cerevisiaeByDavid Frankel McClearyDoctor of Philosophy in Molecular and Cell BiologyUniversity of California, BerkeleyProfessor Jasper D. Rine, ChairCalorie restriction extends lifespan in organisms as diverse as yeast and mammals through incompletely understood mechanism(s). The role of NAD+-dependent deacetylases, known as Sirtuins, in this process, particularly in the yeast S. cerevisiae is controversial. In Chapter 1, I measured chronological lifespan of wild-type and sir2Δ strains over a higher glucose range than typically used for studying yeast calorie restriction. sir2Δ extended lifespan in high-glucose synthetic complete medium, and had little effect in low-glucose medium, revealing a partial role for Sir2 in the calorie restriction response under these conditions. I measured growth, glucose consumption, and pH of these cultures and revealed a mild effect of sir2Δ that inhibited growth, lowered glucose consumption, and increased extracellular pH. Buffering of growth medium eliminated the sir2Δ-dependent extension of chronological lifespan, as did replacing stationary-phase media with water. Replacing medium with water at stationary phase actually led to a sir2Δ-dependent reduction in lifespan in high-glucose. Growth on high levels of other fermentable sugars still resulted in the sir2Δ-dependent extension of lifespan, whereas growth on high levels of glycerol, a non-fermentable carbon source, did not allow for sir2Δ-dependent lifespan extension. When cells were grown in high- or low-glucose yeast peptone medium, large amounts of stationary-phase adaptive regrowth were observed, especially in high-glucose cultures. This regrowth complicated analysis of chronological aging. Interestingly, high-glucose yeast peptone cultures had dramatically decreased initial viability as compared to low-glucose yeast peptone or any other media tested. Also of interest, adaptively regrown cells from high-glucose yeast peptone cultures displayed enhanced growth, a trait that was heritable.In Chapter 2, I measured chronological lifespan of wild-type and sir2Δ strains in rich yeast peptone medium with a newly-developed genetic strategy. This approach revealed that sir2Δ shortened lifespan in low glucose while having little effect in high glucose, revealing a partial role for Sir2 in calorie restriction-mediated lifespan extension in yeast peptone medium. In complete minimal media like synthetic complete, Sir2 shortened lifespan as glucose levels increased, whereas in rich media, Sir2 extended lifespan as glucose levels decreased. Both of these phenotypes are consistent with roll(s) for Sir2 in the calorie restriction response. Using a second genetic strategy to measure the strength of gene silencing at HML, I determined increasing glucose stabilized Sir2-based silencing during growth on complete minimal media. Conversely, increasing glucose destabilized Sir-based silencing during growth on rich media, specifically during late cell divisions. In rich medium, silencing was far less stable in high glucose than in low glucose during stationary phase. Therefore, Sir2 is involved in a response to nutrient cues including glucose that regulates chronological aging, possibly through Sir2-dependent modification of chromatin or deacetylation of a non-histone protein.In Chapter 3, I investigated the origins of a color change of the growth medium at stationary phase when sir2Δ cells were grown on 3% glycerol synthetic complete medium. sir2Δ cultures eventually turned a yellow-brown color while wild-type cultures remained near-white throughout stationary phase. To determine whether the color change was caused by wild-type and sir2Δ cells having different levels of anabolism or catabolism of any small molecules, I performed gas chromatography and mass spectroscopy on filtered medium from wild-type and sir2Δ stationary-phase cultures. Quinoline and several chemical derivatives were highly enriched in filtered sir2Δ medium relative to wild-type. These molecules in solution are reported to have a yellow-brown color, consistent with my observations. GC-MS analysis of fresh 3% glycerol synthetic complete medium revealed that these quinoline compounds were present before cultures were seeded. Thus, wild-type cultures catabolized these molecules at a higher rate than sir2Δ cultures, removing them from the extracellular environment. The quinoline compounds identified here are biosynthesized from tryptophan in some organisms, and are closely related to known UV-degradation products of tryptophan. Closely related compounds including kynurenine and quinolinic acid are also chemical intermediates of the yeast kynurenine pathway, which generates NAD+ from tryptophan. Since sirtuins including Sir2 are the only enzymes that consume NAD+ in yeast, loss of Sir2 could lead to less kynurenine pathway activity, limiting consumption of quinoline compounds that formed either through UV-degradation or catabolism of tryptophan.In Chapter 4, I exploited a microfluidics approach to investigate yeast replicative lifespan using the same calorie restriction paradigm used to investigate chronological lifespan in past chapters. I used a microfluidics chip, provided by Dr. Hao Li, which was designed to trap aging mother cells while washing away smaller, newborn daughter cells. By filming multiple positions on the chip over the course of a single experiment, I was able to count the number of offspring given off by dozens of cells at once. Surprisingly, wild-type cells had the same replicative lifespan in high- and low-glucose, defying the logic of calorie restriction and the results of past studies of the effects of calorie restriction on replicative lifespan. Since classical replicative lifespan experiments involve micromanipulating daughter cells away from a stationary mother cell growing on solid media, that mother cell is experiencing a changing environment as it metabolizes essential nutrients locally and generates waste. In the case of my microfluidics approach, mother cells are experiencing a static environment, since fresh media is constantly flowing through the chip. Therefore, a changing extracellular environment is likely necessary for calorie restriction to increase replicative lifespan. Additional experiments will be necessary to determine this with certainty.Finally, in Chapter 5, we investigate a report claiming Sir2 protein was recruited to dicentric chromosomes under tension, and such chromosomes are reported to be especially vulnerable to breakage in sir2Δ mutants. We found that the loss of viability in such mutants was an indirect effect of the repression of non-homologous end joining in Sir- mutants, and that the apparent recruitment of Sir2 protein to chromosomes under tension was likely due to methodological weakness in early chromatin immunoprecipitation studies