This study has revealed novel insight into the transcriptomic response referred to as the "common environmental response" (CER) [4
] or "environmental stress response" (ESR) [2
] and differentiates between networks that respond to respiratory inhibition and those that respond to oxygen deprivation. First, we show that the acute inhibition of respiration by either oxygen deprivation (anoxia) or chemical means (antimycin A) in catabolite non-repressed conditions evokes a transient ESR/CER-like response. Gene network analyses suggest that changes in the activity of Fhl1 and PAC/RRPE-associated factors result in the transient down-regulation of genes involved in energetically costly programs of ribosomal biogenesis, protein synthesis, and rRNA transcription/processing. Simultaneously, transient changes in the activity of the SBF (Swi4-Swi6) and MBF complexes (Mbp1-Swi6) result in the down-regulation of genes involved in late G1 and the G1/S transition of the cell cycle and a predicted delay in its progression as mass and energy are assessed before committing to another round. At the same time Msn2/4-regulated networks involved in carbohydrate import/utilization and reserve energy metabolism are transiently activated.
When viewed together it seems clear that the transient changes in the activity of these gene networks may be required for balancing energy supply and demand and regulating entrance into the cell cycle. Functional gene network analyses suggest this program includes simultaneously bolstering catabolic potential, regulating reserve energy supplies (trehalose and glycogen), and sparing energetic demand by down-regulating early steps in the biogenesis of the cytoplasmic ribosomes. Given that changes in the activity of these gene networks are not observed when respiration is inhibited well before (3 generations) the aerobic-to-anaerobic transition, we hypothesize these networks may respond to the acute decrease in energy status that accompanies respiratory inhibition as it is clear they do not respond directly to the change in oxygen availability. This conclusion is further supported by previous genomic comparisons of the anaerobic shift in catabolite-repressed and non-repressed cells [5
], which showed that these networks also fail to respond during the aerobic-to-anaerobic transition when respiration is inhibited by the presence of glucose and, thus, when the transition precedes with no change in growth rate or energetic status. During the review of this manuscript, an additional genomic study examining the switch to fermentation also concluded that the mere depletion of oxygen does not evoke a stress response [11
After a substantive delay (≥ 2 generations), more chronic changes in gene network activity were observed under anaerobiosis that were not observed under aerobiosis in the presence of antimycin A. Network analyses show these included the chronic down-regulation of Hap1- and Hap2/3/4/5-regulated ones involved in mitochondrial functions as well as Rox1-regulated ones involved in redox regulation and carbohydrate usage. The last networks to respond (≥ 1 generation) were Upc2-regulated ones involved in cell wall functions and sterol homeostasis. These same networks responded with similar kinetics to anoxia in the presence of antimycin A. An extensive body of previous research (reviewed in [12
]) has shown that changes in the activity of these trans
-acting factors result from decreased heme levels as a result of the loss of oxygen for its synthesis and, thus, it is not surprising that these networks fail to respond to antimycin A treatment under normoxic conditions. Others include those involved in mitochondrial ribosomal biogenesis that have apparently lost their 5' ancestral cis
-regulatory sites and appear to be regulated post-transcriptionally by Puf3 [15
]. Although some genes were found to respond in a unique manner to each treatment, clustering analyses reveal the same or similar gene networks respond to oxygen deprivation both the presence and absence of inhibitor.
Given that both oxygen deprivation and antimycin A poisoning require metabolic retooling of mitochondrial functions, it is reasonable to postulate that many of the observed changes in gene expression result from retrograde signaling [16
]. The retrograde response signals mitochondrial dysfunction to the nucleus and causes changes in the expression of genes associated with peroxisomal activities and anaplerotic pathways that mitigate the loss of the tricarboxylic acid cycle activity [17
]. This response also correlates metabolism with stress responses, chromatin-dependent gene activation, and genome stability in yeast aging [18
]. Not surprisingly, many of the known RTG-dependent genes responded to antimycin A treatment in air [19
]. However, there was surprisingly little overlap (21%) between the genes identified here to respond to antimycin A treatment and those from Epstein et. al's study [17
], differences that most likely can be attributed to the time points that were sampled in each study, array platforms and differences in experimental conditions. Rather, it is clear that the gene networks that respond to anoxia as well as antimycin A treatment are those involved in the general cellular response to stress.
Is the acute response to respiratory inhibition an "environmental stress response"? While it is common practice to look for congruence in lists of differentially expressed genes among various treatments to answer this question, such comparisons are more meaningful and robust when comparing functional groups and transcriptional units defined by common cis
-regulatory sequences statistically enriched in the clustered genes. Even when identical experimental treatments are compared, differences in strain backgrounds, media, time courses, expression platforms, statistical criteria, and experimental variation can result in frighteningly little congruence among gene sets. For example, when we compare our transiently responding genes to those that comprise the ESR [2
], there is only modest (40%) overlap. However, after performing SOM clustering of the ESR data and calculating FCS and CS as we did for our data sets, we recovered eight clusters of transiently up-regulated genes and four clusters of transiently down-regulated genes in which the predominant cis
-regulatory sequences and functional categories were nearly the same as in our study. These include rapidly repressed networks involved in aminoacyl-tRNA synthesis and protein synthesis that are enriched for SCB, Swi6 and Abf1 binding sites, and several clusters of transiently repressed genes involved in ribosomal biogenesis, rRNA transcription/processing and/or protein synthesis that are significantly enriched for Fhl1, Rap1, PAC, RRPE, and/or Abf1 sites. Those that were transiently induced are predominated by Msn2/4 and/or Nrg1 sites and enriched for categories of rescue and defense, the stress response, metabolism of energy reserves and peroxisome function. Thus, from a network comparison it is clear that the same or similar gene networks respond to respiratory inhibition as they do to a large number of other environmental challenges.
Functional Attributes of the General Stress Response
The general stress response was first postulated to explain the phenomenon of cross-protection, wherein exposure to a non-lethal dose of one stress protects against a potentially lethal dose of another, often seemingly unrelated, stress [20
]. It is now clear that this cross protection is afforded by the transcriptional up-regulation of a common set of general stress-responsive genes involved in diverse cellular functions, including reserve energy regulation, carbohydrate metabolism, protein folding and degradation, oxidative stress defense, autophagy, cytoskeletal reorganization, DNA-damage repair, and other processes [2
]. However, the degree of cross-protection varies depending on the stresses and it is not always reciprocal, indicating that stress-specific responses are required for full protection from a specific stressor. Stress resistance has also been shown to occur following nutrient deprivation and stationary phase [2
]. A number of different signaling pathways acting in response to specific stressors have been shown to control these stress-responsive genes. Signaling pathways implicated in coordinating the response include the protein kinase C (PKC)-mitogen-activated protein (MAP) kinase pathway following secretion defects and cell wall damage [29
], the MEC1 pathway following DNA damage [33
], and the Ssk1/Ste11-dependent pathways and high osmolarity glycerol (HOG)-MAP kinase cascade following osmotic stress [1
]. Pathways involved in suppressing the response include the target of rapamycin (TOR) pathway [35
], the Snf1 protein kinase pathway [36
], and the PKA-MAP kinase pathway [37
]. Although seemingly complex, such a multiplex of signaling cascades is likely required for dictating specificity in the cellular response to an environment in which a multitude of different parameters (e.g., temperature, osmolarity, pH, O2
and other nutrients) can change simultaneously.
From functional analyses of the response, it is clear that a rapid change in steady-state conditions (environmental or physiological) prepares the cell to meet a myriad of unforeseen challenges. From a systems level viewpoint, the cell must coordinate energy metabolism with growth rate, the cell cycle, and any specific requirements dictated by the environmental conditions. Given the gene networks that respond, it would appear that at its core is the sparing of energetic demand by rapidly down-regulating the expression of a large set of genes (~600) involved in energy consuming pathways. Simultaneously, supplies may be bolstered by up-regulating key genes involved in import/utilization of primary and secondary carbon substrates and presumably recycling cellular components through autophagy. More complex changes are observed for genes involved in reserve energy stores (trehalose and glycogen), which presumably reflect appropriate parsing of supplies for cell-cycle function and stress mitigation while preserving a modicum of reserve capacity. In addition to changes in gene expression, many of these latter genes are regulated post-transcriptionally.
In terms of potential energetic sparing measures, more than 70% of the characterized genes [40
] whose expression is down-regulated in the ESR are involved in protein synthesis, including genes required for ribosome synthesis and processing, RNA polymerase I- and III-dependent transcription, and protein translation. Thus, it should be of no surprise that these include some of the most abundant and shortest-lived mRNAs, namely genes for ribosomal proteins (RP genes) and rRNA transcription. RP genes alone account for about 60% of all transcription-initiation events in rapidly growing yeast cells [42
] and a remarkable 90% of all mRNA splicing events. Temporal analyses suggest rapid transcriptional deactivation or silencing of these genes followed by mRNA decay given that the decline in their transcript levels tracks well with their estimated half-lives. Predictable yet less dramatic differences in the expression of these, as well as most other genes that comprise the common stress response, have been shown to result from simply varying the growth rate [7
], providing further evidence that their expression is in step with the physiological status of the cell.
In nearly all conditions examined except nutrient starvation [2
], the stress response has shown to be transitory, with maximal transcriptional changes frequently observed between 15 and 30 min and a diminishing response after 60 min. Although, multiple interpretations have been provided (e.g., see discussions in [2
]), a simple hypothesis that fits with the transcriptomic data is that all of these environmental challenges, whether perturbations that temporally disrupt catabolic pathways or ones that require rapidly up-regulating costly programs for combating a specific stressor, require the immediate expenditure of sufficient energy as to require the suspension of other cellular operations, such as general protein synthesis. Obviously, such a hypothesis needs to be corroborated with careful measurements of cellular energetics, measurements that are the focus of ongoing studies in the laboratory. After the initial energetic drain, it would appear that a new balance is quickly achieved and cells resume their normal activities given these genes do not continue to be differentially expressed except under nutrient starvation conditions. Moreover, such an interpretation suggests the response is graded according to the degree to which the energetic status of the cell is perturbed, a hypothesis that is directly testable and the subject of additional ongoing experiments in our laboratory.
Finally, several recent studies have suggested that many of the genes that have been previously characterized as "stress responsive" are perhaps more appropriately labeled "growth-rate responsive" [7
]. In a serious of elegant, nutrient-limited chemostat studies, these authors have shown the expression of most of the genes comprising the ESR is in-step with steady-state growth rate. Thus, rather than responding to the stress directly, perhaps they are responding to a change in growth rate secondary to the stress [8
]. To determine if the genes we identified are correlated with growth rate, we divide the clustered expression profiles of the anaerobic dataset into groups of transiently and chronically responding ones and then further into repressed and induced sets. Using the online utility http://growthrate.princeton.edu
developed by Brauer et al.
] we plotted the distribution of growth-rate slopes for these sets. As shown in Additional file 7
, those that are transiently expressed are highly correlated with growth rate whereas those that were more chronically expressed are not. Given this, it is also possible to infer the instantaneous growth rate using these transiently expressed genes [9
], which in our datasets suggests a maximal slowing of growth between 0.13 and 0.19 generations (ca. 30 min) in response to N2
and antimycin A treatment in air and complete recovery by about 0.5 generations (ca. 2 h). If changes in the expression of these genes are a direct result of differences in growth rate, what is puzzling here is the fact they are not differentially expressed between the strictly fermentative phase, in which the mass doubling time is 4 h, and the respiro-fermentative phase, in which the mass doubling time is 2.4 h. Rather the response is strictly transitory. What's more is that inhibiting galactose-dependent respiration limits the rate at which ATP can be generated (QATP
) and, thus, it is hard to see how acute changes in cellular energetics cannot be at the core of this response. In other words, that it is the acute change in cellular energetics that initiates the response resulting in the change in growth rate, a hypothesis that is being tested with on-going experiments in our laboratory.
In summary, all eukaryotic cells have necessarily evolved mechanisms for effectively dealing with fluctuations in physical, chemical, and physiological parameters. Even within the relatively protected confines of multicellular organisms, cells experience fluctuations in osmolarity, oxygen and reactive oxygen species as well as various nutrients and noxious substances. Comparative studies have shown that many of the factors important for regulating the stress response, including both signaling pathways (e.g., MAPK) and transcription factors (e.g., AP-1), are conserved from yeast to humans. Understanding the factors that influence this response has important implications for pathophysiological conditions, such as heart disease, tumorigenesis, various metabolic syndromes, and aging, all of which contribute to stress at the cellular level. Here we set forth a hypothesis that at its core is the regulation of cellular energetics via an as yet unidentified metabolic signal. Such internal signals are being actively studied in bacteria [44
] but comparatively little work has been done in eukaryotes. Two, partially redundant, serine/threonine kinases containing PAS domains, Psk1 andPsk2, which coordinately regulate protein synthesis and carbohydrate metabolism in response to nutritional status of the cell, have been implicated in signaling in yeast. Although a small molecule/metabolite is presumed to trigger this response by interacting with the PAS domain, it has not been identified [45
]. Variants of this system are found from yeast to mammals [47
]. Thus, future studies with such factors will be of great interest to unravel both the sensing and signaling aspects of this conserved stress response program.