Tra1 controls the transcriptional landscape of the aging cell

Abstract

Gene expression undergoes considerable changes during the aging process. The mechanisms regulating the transcriptional response to cellular aging remain poorly understood. Here, we employ the budding yeast Saccharomyces cerevisiae to better understand how organisms adapt their transcriptome to promote longevity. Chronological lifespan assays in yeast measure the survival of nondividing cells at stationary phase over time, providing insights into the aging process of postmitotic cells. Tra1 is an essential component of both the yeast Spt-Ada-Gcn5 acetyltransferase/Spt-Ada-Gcn5 acetyltransferase-like and nucleosome acetyltransferase of H4 complexes, where it recruits these complexes to acetylate histones at targeted promoters. Importantly, Tra1 regulates the transcriptional response to multiple stresses. To evaluate the role of Tra1 in chronological aging, we took advantage of a previously characterized mutant allele that carries mutations in the TRA1 PI3K domain (tra1Q3). We found that loss of functions associated with tra1Q3 sensitizes cells to growth media acidification and shortens lifespan. Transcriptional profiling reveals that genes differentially regulated by Tra1 during the aging process are enriched for components of the response to stress. Notably, expression of catalases (CTA1, CTT1) involved in hydrogen peroxide detoxification decreases in chronologically aged tra1Q3 cells. Consequently, they display increased sensitivity to oxidative stress. tra1Q3 cells are unable to grow on glycerol indicating a defect in mitochondria function. Aged tra1Q3 cells also display reduced expression of peroxisomal genes, exhibit decreased numbers of peroxisomes, and cannot grow on media containing oleate. Thus, Tra1 emerges as an important regulator of longevity in yeast via multiple mechanisms.

Keywords: SAGA complex; Tra1; chronological aging; peroxisomes; yeast.

Fig. 1.

Functional Tra1 is required for chronological aging. a) CLS is defined as the amount of time yeast cells survive at stationary phase. Experimentally, longevity is assessed by labeling cells with a viability dye such as propidium iodide at various time points during the aging process. Adapted from Chadwick et al. 2016. b) tra1_Q3 cells display reduced CLS. TRA1 and tra1_Q3 cells were grown for the indicated times in standard synthetic complete medium and stained with propidium iodide to measure cell survival. Stained cells were imaged in a 96-well plate. For each time point, a negative control (unstained cells), a positive control (boiled cells), and 5 replicates were analyzed. c) Normalized survival rates over the aging process and d) survival integral are shown in the bar graph. Significance was assessed using an unpaired Student T-test.

Fig. 2.

Tra1 function is dispensable for lifespan extension by caloric restriction. a) TRA1 and tra1_Q3 cells were grown for the indicated time in standard synthetic complete medium containing either 2% glucose or 0.1% glucose (CR) and stained with propidium iodide to measure cell survival. Stained cells were imaged in a 96-well plate. For each time point, a negative control (unstained cells), a positive control (boiled cells), and 5 replicates were analyzed. b) Normalized survival rates over the aging process and calculated survival integral are shown in graphs. Significance was assessed using a 1-way ANOVA followed by a Tukey’s multiple comparison test. **P < 0.01 and ****P < 0.0001.

Fig. 3.

Increased sensitivity of tra1_Q3 cells to media acidification is linked to shortened lifespan. a) tra1_Q3 cells display increased sensitivity to acetic acid. TRA1 and tra1_Q3 cells were grown for 4 days and subsequently treated with the indicated concentrations of acetic acid for 200 min and stained with propidium iodide to measure cell survival. b) Normalized survival rates upon acetic acid treatment were calculated and survival integrals are shown. c) TRA1 and tra1_Q3 cells were grown for the indicated time in standard synthetic complete medium with or without 0.1 M MES and stained with propidium iodide to measure cell survival. Stained cells were imaged in a 96-well plate. For each time point, a negative control (unstained cells), a positive control (boiled cells) and 5 replicates were analyzed. d) Normalized survival rates over the aging process and calculated survival integrals are shown. Significance was assessed using a 1-way ANOVA followed by a Tukey’s multiple comparison test. ***P < 0.005 and ****P < 0.0001.

Fig. 4.

The tra1_Q3 mutation alters the transcriptional landscape of aging cells. a) Principal component analysis of centered log ratio of normalized reads from TRA1 and tra1_Q3 cells at day 0 and day 3. Each point represents a single biological replicate (n = 3). b) Volcano plot of genes that respond differently to the aging process in the tra1_Q3 cells compared to wild-type TRA1 (coloured for dark points represent P < 0.05, log₂ fold change >1). c) Significantly enriched GO biological processes were determined for genes with both positive and negative age–genotype interaction with log₂ interaction score >2 (P < 0.05) in tra1_Q3 cells compared to wild type. d) Examples of genes with positive (SIP18, MLS1) and negative (RGL1, BTN2) age–genotype interaction. Normalized RNA sequencing read counts are shown for TRA1 and tra1_Q3 cells at day 0 and day 3.

Fig. 5.

Aged tra1_Q3 cells display increased TRA1 mRNA abundance. a) Normalized RNA sequencing read counts are shown for TRA1 mRNA in wild-type TRA1 and tra1_Q3 cells at day 0 and day 3. b) Log₂ fold change (tra1_Q3/TRA1) for the mRNA of genes encoding SAGA and NuA4 components at day 0 and day 3.

Fig. 6.

tra1_Q3 cells are sensitive to oxidative stress. a) Log₂ fold change (TRA1/tra1_Q3) for mRNA of genes associated with oxidoreductase activity in wild-type TRA1 and tra1_Q3 cells at day 0 and day 3. b) Normalized RNA sequencing read counts are shown for CTT1 and CTA1 in TRA1 and tra1_Q3 cells at day 0 and day 3. c) tra1_Q3 cells are sensitive to diamide. TRA1 and tra1_Q3 cells were spotted onto agar plates without (untreated) or with 1 mM diamide.

Fig. 7.

tra1_Q3 results in defective peroxisomes. a) Log₂ fold change (TRA1/tra1_Q3) for beta-oxidation genes at day 0 and day 3. b) tra1_Q3 cells show decreased growth in the presence of oleic acid. TRA1 and tra1_Q3 cells were spotted on agar plates containing either glucose (YPD), oleate, myristate, or glycerol as the carbon source. Growth of tra1_Q3 cells relative to TRA1 cells was quantified and is shown in the bar graph. c) Normalized RNA sequencing read counts are shown for PEX34 and PEX21 in wild-type TRA1 and tra1_Q3 cells at day 0 and day 3. d) TRA1 and tra1_Q3 cells expressing yemRFP-SKL were imaged using fluorescence microscopy at day 0 and day 3 of the aging process. The number of peroxisomes/cell is shown in bar graphs. Bar: 10 µm.