Steady-state and dynamic gene expression programs in Saccharomyces cerevisiae in response to variation in environmental nitrogen

Abstract

Cell growth rate is regulated in response to the abundance and molecular form of essential nutrients. InSaccharomyces cerevisiae(budding yeast), the molecular form of environmental nitrogen is a major determinant of cell growth rate, supporting growth rates that vary at least threefold. Transcriptional control of nitrogen use is mediated in large part by nitrogen catabolite repression (NCR), which results in the repression of specific transcripts in the presence of a preferred nitrogen source that supports a fast growth rate, such as glutamine, that are otherwise expressed in the presence of a nonpreferred nitrogen source, such as proline, which supports a slower growth rate. Differential expression of the NCR regulon and additional nitrogen-responsive genes results in >500 transcripts that are differentially expressed in cells growing in the presence of different nitrogen sources in batch cultures. Here we find that in growth rate-controlled cultures using nitrogen-limited chemostats, gene expression programs are strikingly similar regardless of nitrogen source. NCR expression is derepressed in all nitrogen-limiting chemostat conditions regardless of nitrogen source, and in these conditions, only 34 transcripts exhibit nitrogen source-specific differential gene expression. Addition of either the preferred nitrogen source, glutamine, or the nonpreferred nitrogen source, proline, to cells growing in nitrogen-limited chemostats results in rapid, dose-dependent repression of the NCR regulon. Using a novel means of computational normalization to compare global gene expression programs in steady-state and dynamic conditions, we find evidence that the addition of nitrogen to nitrogen-limited cells results in the transient overproduction of transcripts required for protein translation. Simultaneously, we find that that accelerated mRNA degradation underlies the rapid clearing of a subset of transcripts, which is most pronounced for the highly expressed NCR-regulated permease genesGAP1,MEP2,DAL5,PUT4, andDIP5 Our results reveal novel aspects of nitrogen-regulated gene expression and highlight the need for a quantitative approach to study how the cell coordinates protein translation and nitrogen assimilation to optimize cell growth in different environments.

FIGURE 1:

Physiology of yeast cultures grown in nitrogen-limited batch and chemostat cultures. (A) When normalized for total nitrogen concentration, the biomass yield is the same regardless of nitrogen source. Values are determined by regression analysis of cell yield, measured using a Klett colorimeter, in batch cultures containing different concentrations of nitrogen. Error bars represent 95% confidence interval of the regression coefficient. (B) Approach to steady-state kinetics for glutamine- and proline-limited chemostats. Each chemostat culture was inoculated with 10⁶ cell/ml and monitored over an 80-h period. Experimental measurements for proline (black triangles) and glutamine (red circles) are in good agreement with a mathematical model of the chemostat (lines).

FIGURE 2:

Differential gene expression as a function of nitrogen source in nutrient-limited chemostats. (A) The nitrogen-responsive NCR-A, NCR-P, and SPS regulons respond specifically to nitrogen limitation, whereas the GAAC and UPR regulons are not differentially expressed in nitrogen-limited chemostats compared with carbon, sulfur, phosphorus, leucine, and uracil growth limitation. Gene expression values in Brauer et al. (2008) were relative to a common reference taken from a glucose-limited chemostat grown at a dilution rate of 0.25 h⁻¹. (B) A small number of nitrogen-regulated genes respond significantly to variation in nitrogen source in nitrogen-limited conditions. Regulon membership of genes is denoted by color corresponding to A (NCR-A, green; GAAC, orange; NCR-P, blue; UPR, red). GDH2 has been proposed to be a member of both NCR-A and GAAC regulons (Godard et al., 2007). Filled symbols represent significant effects as determined by permutation testing. Gene expression values are relative to gene expression in an ammonium-limited chemostat growing at a dilution rate of 0.12 h⁻¹.

FIGURE 3:

Kinetics of growth and predicted growth rate response in cultures transiently relieved from nitrogen limitation. Ammonium-limited chemostat cultures grown at 0.12 h⁻¹ were subjected to an instantaneous addition of (A) 400 μM glutamine, (B) 40 μM glutamine, (C) 800 μM proline, or (D) 80 μM proline. For each perturbation experiment, we modeled the change in growth rate as a continuous function of time (blue line). We measured culture density at discrete times points (blue points) and predicted the instantaneous growth rate on the basis of global gene expression (red points).

FIGURE 4:

Remodeling of the transcriptome in response to relief from nitrogen limitation. (A) We clustered global gene expression data for cultures limited for proline and glutamine grown at different growth rates with gene expression profiles for ammonium-limited cultures transiently perturbed by the addition of 80 and 800 μM proline or 40 and 400 μM glutamine or a mixture of 20 μM glutamine and 40 μM proline. Many transcripts that increase (red bar) or decrease (green bar) systematically with growth rate in steady-state chemostats are also altered in expression when transiently relieved from nitrogen limitation. Average response of the NCR-A, NCR-P, GAAC, SPS, and UPR regulons to a (B) large or (C) small pulse of glutamine or a (D) large or (E) small pulse of proline. Gene expression values are relative to gene expression in an ammonium-limited chemostat growing at a dilution rate of 0.12 h⁻¹.

FIGURE 5:

Growth rate response of gene expression in steady-state and dynamic conditions. (A) We estimated the growth rate response of each transcript in steady-state and dynamic conditions by linear regression. In dynamic conditions in which nitrogen limitation is transiently relieved, the mRNAs belonging to the RiBi and RP regulons exhibit a growth rate response that exceeds that observed in steady-state chemostats. High temporal resolution shows a reciprocal relationship between expression of the RiBi and RP regulons, which increase in expression, and the NCR-A and NCR-P regulons, which decrease in expression, in response to a (B) 40 μM glutamine pulse and a (C) 80 μM proline pulse. Time points (in seconds) at which samples were obtained and analyzed are indicated on the x-axis.

FIGURE 6:

Accelerated mRNA degradation contributes to gene expression remodeling. Upon addition of glutamine to NCR-derepressed cells, a subset of transcripts degrade more rapidly than their steady-state degradation rate both (A) in cells grown in ammonia-limited chemostats and (B) in cells growing in proline media in batch cultures. All points are genes that fit a model of exponential decrease in abundance (FDR < 0.05). Orange points are NCR genes that show significant accelerated degradation, blue points are NCR genes that are not significant, green points are non-NCR genes that show significantly accelerated degradation, and gray points are genes that are neither accelerated nor NCR. The dashed line denotes equal degradation rates in both conditions (i.e., slope equal to 1). Names of nitrogen transporter genes are displayed. We measured the transient changes in the degradation rates of (C) GAP1 and (D) DIP5 mRNA using a pulse-chase experiment. Cells were grown for 24 h in the presence of 4-thiouracil, which was chased at t = 0 min by the addition of excess uracil. At t = 13 min, we added either glutamine in water (orange) or equal volume of water (blue). We extracted and quantified the abundance of 4-thiouracil–labeled mRNA relative to a thiolated external spike-in using qPCR. We found significant acceleration of degradation for both GAP1 and DIP5 mRNAs (p < 0.001). Points are the mean of triplicate qPCR measurements, error bars are the propagated SD of transcript and spike-in measurements, and dotted lines are the log-linear model fit.

FIGURE 7:

Transcriptome allocation is dramatically altered in response to nitrogen availability. RP and RiBi transcripts represent 28.4% of the transcriptome and NCR transcripts comprise just 0.9% of the transcriptome in NCR-repressing conditions (growth in yeast extract/peptone/dextrose media; data from Waern and Snyder, 2013). In NCR-derepressing conditions (minimal media depleted of nitrogen), the RP and RiBi transcripts comprise 7.6% of the transcriptome and NCR mRNAs comprise 8.8%.