Dynamical remodeling of the transcriptome during short-term anaerobiosis in Saccharomyces cerevisiae: differential response and role of Msn2 and/or Msn4 and other factors in galactose and glucose media

Abstract

In contrast to previous steady-state analyses of the O(2)-responsive transcriptome, here we examined the dynamics of the response to short-term anaerobiosis (2 generations) in both catabolite-repressed (glucose) and derepressed (galactose) cells, assessed the specific role that Msn2 and Msn4 play in mediating the response, and identified gene networks using a novel clustering approach. Upon shifting cells to anaerobic conditions in galactose medium, there was an acute ( approximately 10 min) yet transient (<45 min) induction of Msn2- and/or Msn4-regulated genes associated with the remodeling of reserve energy and catabolic pathways during the switch from mixed respiro-fermentative to strictly fermentative growth. Concomitantly, MCB- and SCB-regulated networks associated with the G(1)/S transition of the cell cycle were transiently down-regulated along with rRNA processing genes containing PAC and RRPE motifs. Remarkably, none of these gene networks were differentially expressed when cells were shifted in glucose, suggesting that a metabolically derived signal arising from the abrupt cessation of respiration, rather than O(2) deprivation per se, elicits this "stress response." By approximately 0.2 generation of anaerobiosis in both media, more chronic, heme-dependent effects were observed, including the down-regulation of Hap1-regulated networks, derepression of Rox1-regulated networks, and activation of Upc2-regulated ones. Changes in these networks result in the functional remodeling of the cell wall, sterol and sphingolipid metabolism, and dissimilatory pathways required for long-term anaerobiosis. Overall, this study reveals that the acute withdrawal of oxygen can invoke a metabolic state-dependent "stress response" but that acclimatization to oxygen deprivation is a relatively slow process involving complex changes primarily in heme-regulated gene networks.

FIG. 1.

Change in O₂ concentration over the first 10 min of the shift to anaerobiosis. The change in dissolved O₂ concentration (μM) in the fermentor is plotted as a function of time over the first ten minutes after switching the sparge gas from air to 2.5% CO₂ in O₂-free N₂. The O₂ concentration was calculated from the dissolved oxygen level measured with a 12-mm Ingold polarographic O₂ sensor and is based upon the solubility of O₂ in the media at 28°C and ambient barometric pressure.

FIG. 2.

Dynamics of oxygen-responsive gene induction and repression during short-term anaerobiosis in galactose medium. The number of genes that responded significantly (P < 0.01) to the shift in O₂ concentration in the wild-type strain (JM43) grown in galactose medium (SSG-TEA) is plotted as a function of both time and relative number of doublings in cell mass (generations) after the shift to anaerobiosis. Genes are divided into those that were significantly up-regulated and those that were significantly down-regulated. Black bars indicate the number of genes that were identified for the first time at that time point to exhibit a significant change in expression from that of the aerobic control (time zero). Gray bars indicate the number of genes that were differentially expressed relative to the aerobic control yet had already been identified at an earlier time point to have responded significantly to the shift in O₂ concentration. The combined height of the black and gray bars indicates the total number of genes at each time point that showed a significant change in expression relative to the aerobic control.

FIG. 3.

Comparison of the performance of different clustering algorithms in terms of consensus share and the motif configuration statistic as a function of cluster number (K). Three clustering algorithms were evaluated in terms of the resulting gene-to-cluster consensus (consensus share) and the motif configuration statistic (MCS) as a function of cluster numbers (K = 2 to 50) using Pearson correlation as the distance metric: SOM (Kohonen map) algorithm with 1D ring topology (solid line), K-means (dashed line), and K-medoids (dotted line). Consensus share (upper panel) is the percentage of genes that were consistently grouped together over ten runs of the algorithm. An average MCS P value (lower panel) for 1,813 transcription factor consensus binding motifs (TFMs) was calculated using a compiled list of both experimentally verified and putative motifs (see Table S1 in the supplemental material) as described in Materials and Methods. Lower MCS values indicate more nonrandom configurations of TFMs among gene clusters. The clustered data are individual replicate expression levels of all genes that significantly (P < 0.01) responded to the shift to anaerobiosis in SSG-TEA (galactose) medium.

FIG. 4.

Heat maps and statistical comparisons of oxygen-responsive genes in a wild-type strain grown in both galactose and glucose media and in an msn2/4 strain grown in galactose. The temporal profiles of genes whose transcript levels responded significantly (P < 0.01) to the shift to anaerobiosis (wild-type strain, SSG-TEA) were clustered using an SOM algorithm with 1D ring topology (K = 17). Panel D is a heat map of the temporal changes in gene expression relative to the aerobic control (time zero) plotted as a function of the number of generations of anaerobiosis (0, 0.04 0.08, 0.19, and 2). Green indicates down-regulated expression and red indicates up-regulated expression. Genes are sorted into 17 primary clusters as indicated in panel E. Cluster 0 contains genes that exhibited unstable cluster membership. Panel B shows the response of the same genes shown in panel D but in glucose (SSD-TEA) as opposed to galactose medium (wild-type strain). The black bars in panel A indicate genes that responded significantly (P < 0.01) to the shift in O₂ concentration in glucose medium. Black bars in panel C indicate genes that showed a differential response (P < 0.01) in the two media (galactose versus glucose) as assessed by ANOVA. The purple bars in panel F identify putative Msn2/4-regulated genes, that is, genes that were anaerobically induced (P < 0.01) in the wild type but expressed at significantly (P < 0.01) lower levels in the msn2/4 strain and contained one or more STRE sites. Panel G is an expansion of the heat map shown in panel D for Msn2/4-regulated genes in the wild-type strain. Panel H is the expression profiles of the Msn2/4-regulated genes in the mutant strain, with yellow boxes indicating samples for which the transcript level was significantly (P < 0.01) lower in the msn2/4 strain compared to its wild-type parent.

FIG. 5.

Dynamics of oxygen-responsive gene induction and repression during short-term anaerobiosis in glucose medium. The number of genes that responded significantly (P < 0.01) to the change in O₂ concentration in the wild-type strain (JM43) grown in glucose medium (SSD-TEA) is plotted as a function of the relative number of cell doublings (generations) after the shift to anaerobiosis. Genes are divided into those that were significantly up-regulated and those that were significantly down-regulated. Black bars indicate the number of genes that were identified for the first time at that time point to exhibit a significant change in expression from that of the aerobic control (time zero). Gray bars indicate the number of genes that were differentially expressed relative to the aerobic control yet had already been identified at an earlier time point to have responded significantly to the shift in O₂ concentration. The combined height of the black and gray bars indicates the total number of genes at each time point that showed a significant change in expression relative to the aerobic control.

FIG. 6.

Clustering quality assessment (A) and heat maps (B) for genes that significantly responded to the shift to anaerobiosis in glucose medium. The temporal profiles of genes whose expression responded significantly (P < 0.01) to the shift to anaerobiosis in glucose medium (SSD-TEA) were clustered using an SOM algorithm with 1D ring topology. Panel A shows the motif configuration statistic (solid line, left ordinate) and consensus share (dotted line, right ordinate) as a function of cluster number. Panel B is the heat map of the temporal changes in gene expression relative to the aerobic control (time zero) partitioned with 11 clusters. Green indicates down-regulated expression and red indicates up-regulated expression. Bars to the left of the heat map indicate genes that failed to respond (P > 0.01) to the O₂ shift in galactose medium, and bars to the right indicate genes that exhibited a differential response (P < 0.01) in glucose versus galactose medium.