The GEV system
We constructed two isogenic strains of opposite mating type (DBY12020, DBY12021) to use for studies of gene induction/overexpression in S. cerevisiae. These strains (see ) contain an integrated copy of PACT1-GEV, so that GEV is produced constitutively and at relatively high levels. To study dynamically the consequences of induction of a gene, its promoter is replaced by the GAL1 promoter (PGAL1) in either DBY12020 or DBY12021. If one wants a fluorescent reporter to monitor induction, one can use DBY12039. To study the consequences of the degradation of a protein, a peptide sequence recognized by TEV (i.e., N-degron sequence) can be appended to the N-terminus of the gene in DBY12132, as described in Materials and Methods.
Note that a gene whose promoter has been replaced with a GAL regulated promoter should be expressed only after addition of β-estradiol to the growth medium; this has been the case with the several dozen constructs we have made. Importantly, although GEV binds DNA via the Gal4p DNA binding domain, it is not subject to inhibition or repression by glucose, making feasible induction and overexpression experiments in standard glucose-containing media simply by the addition of the inducer β-estradiol.
Quantitation of GEV localization
Generally, hormone receptors localize to the nucleus and activate transcription after binding their cognate ligands (Pratt and Toft, 1997
). To our knowledge, this model has not been experimentally verified in the case of GEV. Therefore, to study GEV nuclear localization following hormone addition, we constructed a functional (Supplemental Figure 1), chromosomally integrated, C-terminus–tagged GEV-GFP (green fluorescent protein) reporter and measured its localization following pulses of β-estradiol ( and Supplemental Movie 1) in single cells. We observed a level of GEV localization that is proportional to the amount of added β-estradiol for a range of concentrations. At higher doses (100 nM to 10 μM), we can see the nuclear localization signal emerging from the background 6–8 min following hormone addition. The initial rate of localization achieved its maximum at concentrations at or above 1 μM β-estradiol.
FIGURE 2: Single-cell analyses of a GEV-GFP fusion protein. (A) Measuring nuclear localization of GEV-GFP in the presence of different amounts of β-estradiol in DBY11415. (B) DBY11415 was grown in the presence of 1 μM β-estradiol for 50 (more ...)
To determine the nuclear residence time of activated GEV after removal of β-estradiol, cells were grown in a flow chamber. For the first 50 min, medium containing 1 μM β-estradiol was flowed over the cells. Then the medium inflow was switched (, black line) to a medium lacking β-estradiol, resulting in an approximately exponential reduction in nuclear fluorescence with time. In this way we determined that the nuclear half-life of GEV is ~40 min ( and , and Supplemental Movie 2).
Kinetics of GEV-mediated gene induction
To investigate the kinetics of target gene induction by GEV, we inserted PGAL1
in front of various reporter genes (Liko et al., 2006
). First, we constructed a strain in which PGAL1
is driving transcription of the CBF1
gene at its native chromosomal locus. The level of CBF1
transcript, as determined by microarray analysis, reaches ~70% its maximum by 5 min following addition of 1 μM β-estradiol (). Using fluorescence in situ hybridization (FISH) to monitor transcription in single cells, we observed both the cytoplasmic transcripts and nuclear transcription sites of CBF1
, as shown in the image from , which was taken 8 min following addition of 1 μM β-estradiol to the culture. We used a PGAL1
-GFP reporter to find the point at which GEV becomes saturated by inducer. The percentage of induced cells is determined (see Materials and Methods
) over time, and we find that GEV is saturated between 100 nM and 1 μM β-estradiol, as the induction kinetics are similar between these two concentrations ().
FIGURE 3: The kinetics of GEV-mediated genetic switch. (A) We followed transcription of PGAL1-CBF1 induced by GEV by microarray (strain = DBY12040), with DBY12001 RNA as a reference. Values are zero-normalized. (B) Maximal projection image of CBF1 transcripts from (more ...)
GEV-mediated gene induction shows a graded response
The generally accepted model for the mechanism of nuclear receptors predicts that subsaturating concentrations of inducer result in a response that is proportional to the concentration of inducer (i.e., exhibit a graded as opposed to switch-like response). Indeed this behavior was reported for GEV by Takahashi and Pryciak (2008
). To verify a graded response for our system, we performed experiments in which the population of cells containing PGAL1
-GFP was induced by β-estradiol for 12 h and then quantified by flow cytometry.
At concentrations less than 1 nM β-estradiol, the system is ineffective: the average intensity of the reporter is at nearly the background level (). At 10 nM β-estradiol, GFP intensity is increased but is well below what is achieved at higher inducer concentrations (); the significant point is that all the cells exposed to 10 nM β-estradiol exhibit significant fluorescence, as shown by the shift of the entire distribution to the right (). At 100 nM, the average induction is near maximum and the distribution of cells closely resembles that found for higher, completely saturating concentrations of β-estradiol.
FIGURE 4: GEV activity shows a graded response in response to β-estradiol. (A) Flow cytometry data at different doses of β-estradiol. Cells were grown to mid-log phase and incubated with the indicated amount of β-estradiol for 12 h. (B) (more ...)
A trade-off between GEV activation and cell growth
We find that strong GEV activation achieved at high concentrations of β-estradiol results in a slowing of growth (). This is not due to β-estradiol toxicity because the slow growth does not occur in a strain lacking the GEV system. We speculate that this effect could be due to “squelching” (Gill and Ptashne, 1988
), whereby strong transcriptional activators repress off-target genes through titration of the RNA polymerase machinery, and thus reduce the cell's capacity for growth. Lang et al. (2009)
have provided evidence in yeast that there is indeed a fitness cost exacted by synthesis of proteins not required for asexual proliferation.
FIGURE 5: GEV is a gratuitous inducer at 10 nM β-estradiol without a growth defect. (A) DBY12021 grown in YPD liquid in the presence of β-estradiol. A600 was monitored over time, and growth rates were spline-fit (inset). Error bars represent ±1 (more ...)
At lower concentrations of β-estradiol (10 nM), the GEV-containing cells no longer show the growth defect (), but are able to induce the PGAL1-GFP reporter essentially in all the cells nonetheless (). Inducing GEV at 1 μM β-estradiol results in a greater than twofold reduction in growth rate (, inset).
To determine whether physiologically significant expression of target genes can be induced at intermediate (10 nM) levels of induction, where no growth consequence is observed, we designed an experiment in which a lethal defect might be complemented in a GEV-dependent manner. TPS2
encodes an enzyme that converts trehalose-6-phosphate to trehalose. Deletion of TPS2
causes a heat-sensitive growth phenotype, likely due to the buildup of trehalose-6-phosphate (Devirgilio et al., 1993
). By placing TPS2
downstream of PGAL1
, cells become heat sensitive in the absence of β-estradiol (). Growth is restored to wild-type levels in the presence of 10 nM β-estradiol, and the resulting growth is indistinguishable from wild type ().
This result suggests that maximal induction is not likely to be necessary to achieve physiological levels of protein in all experiments. Intermediate levels of inducer suffice to avoid the growth-inhibiting side effects and complement the deletion. In practice, the system is fast-acting at high (>100 nM β-estradiol) concentrations, but has increased growth rate inhibition. Even at the highest inducer concentrations, few unintended genes are expressed at early times after induction. Thus the user has a trade-off: if the goal of experiments is just to see which genes are induced first, high concentrations will yield this result; if the goal is longer-term specificity, where speed is not of the essence, low concentrations of inducer can be used. It is not clear that this problem is avoidable: using a less strong promoter than that of ACT1 to drive GEV produces a system with the mitigated growth effect but at the price of less speedy induction.
GEV induction in chemostats is suitable for dynamically studying the physiological consequences of gene action
Because yeast are extremely sensitive to slight changes in the extracellular environment, the ideal growth setting for induction experiments is in the chemostat. Briefly, a chemostat works by flowing fresh medium into a growth vessel, and culture medium is diluted from the vessel at a rate determined by the experimenter. With this experimental setup, we can grow to steady state a culture of cells containing the GEV construct and PGAL1 fused to any gene of interest. In the case in which a gene's native promoter is replaced with PGAL1 (in a haploid), the cells are effectively deletion mutants of that gene in the absence of β-estradiol due to the tight repression of PGAL1 in the presence of 2% glucose. By adding β-estradiol to such cultures, we can rapidly and strongly induce the gene of interest and follow the physiological consequences. For the particular case of genes encoding transcription factors, the consequence to be followed is the genome-wide transcriptional response of the cells. The goal is to infer a transcription factor's immediate targets and quantify the strength of their response. For those target genes that respond most quickly, the inference of causation is strengthened.
GEV induction in a steady-state culture is nearly gratuitous
For this approach to be effective, GEV needs to be nearly gratuitous, meaning that it does not have a large effect on the transcriptional landscape of the cell. Although this definition is slightly ambiguous, we can define a large effect as one that results in strong increases/decreases in many genes such that it becomes difficult to separate changes due to overexpressing the target gene from those due to background. By growing a strain (DBY12021) that contains GEV in a chemostat, we find that GEV can be nearly gratuitous (). In DBY12021, the GAL1
genes are deleted, as is the GAL10
promoter. Two hours following addition of saturating (1 μM) β-estradiol, 105 genes decreased twofold (Supplemental data set 1) and 94 genes increased twofold (Supplemental data set 2; Supplemental Figure 7). This finding accounts for ~3% of all S. cerevisiae
genes. Although this can be seen as modest compared with all yeast genes, it is nevertheless more than one sees in many deletion mutants, including transcription factors (Hughes et al., 2000
; Hu et al., 2007
). By using the gene ontology (GO) term finder to find enriched processes within these groups, we found that repressed genes are most enriched for glucose catabolic processes (corrected p-value = 8.49 × 10−9
) and activated genes are most enriched for oxidation-reduction processes (corrected p-value = 1.73 × 10−7
). Note that at 18 min following addition of 1 μM β-estradiol, a total of 57 genes have changed twofold (Supplemental Figure 7).
FIGURE 6: (A) Hierarchical clustering of gene expression of DBY12021 grown to steady state in a phosphate-limited chemostat with a doubling time of 4.3 h, and at t = 0 min, pulsed with 1 μM β-estradiol. (B) The transcriptional response of the GAL (more ...)
GEV induction results in strong activation of GAL2 and GAL7 and moderate induction of GAL3, GAL5, and GAL80 (). Surprisingly, despite the removal of the GAL10 promoter by loxP in DBY12021, we observe slight induction of GAL10 over the course of the experiment.
We found two genes (YPL067C and YPL066W) that are divergently transcribed in response to GEV induction (). These genes are reminiscent of GAL1 and GAL10, which are divergently transcribed in response to Gal4p activation. YPL066W and YPL067C are separated by a 385-nucleotide region. This region contains a single UASGAL sequence, which is conserved upstream of the YPL066W and YPL067C orthologues in Saccharomyces mikatae and Saccharomyces paradoxus. Despite the conservation of the Gal4p binding site, these genes have no known role in galactose metabolism and are of unknown function.
The most strongly repressed gene by 2 h following GEV activation is HSP30 (17.4-fold). The 10 genes most strongly repressed at 2 h following GEV activation are shown in . We could not find any significant correlation with annotated functions that these genes might hold in common.
Using GEV overexpression to probe kinetics of transcriptional regulatory networks
Large-scale ChIP-chip studies have defined extensive transcription factor–DNA binding maps, yielding a highly informative, although static, view of genetic regulation. Using the GEV system to induce rapid and high-level expression of a single transcription factor, we can look at the dynamics of target gene activation or repression.
As an example and proof of principle, we induced the MET4 gene, which encodes a well-studied, strong transcriptional activator of sulfur metabolic genes in S. cerevisiae. A strain containing PGAL1-MET4 was grown to steady state in a phosphate-limited chemostat containing high levels of extracellular methionine, a condition in which Met4p is supposed to be less active. By hierarchically clustering the transcriptional profile of the sulfur metabolic genes as a time series up to 90 min following MET4 induction, we were able to observe that the kinetics of MET4-target induction can vary from gene to gene ().
FIGURE 7: Quantifying the kinetics of MET4's downstream targets. DBY12027 (GEV, PGAL1-MET4) was grown to steady state in a phosphate-limited chemostat with excess methionine (200 mg/l) with a doubling time of 4.3 h. At t = 0 min, cells were pulsed with 1 μM (more ...)
We divide the targets into three categories based on their kinetic profiles: strongly induced (group 1), weakly induced (group 2), and uninduced (group 3) (G1, G2, and G3, respectively, in ). The MUP1 and MUP3 genes, which encode the high- and low-affinity methionine permeases, respectively, and SAM1 and SAM2, which encode enzymes that catalyze the synthesis of S-adenosylmethionine (AdoMet), are strongly induced group 1 genes. AdoMet is the primary source of methyl donor groups in the cell.
The MET31 and MET32 genes encode homologous zinc-finger proteins (46% identical), which aid in the recruitment of Met4p to many sulfur metabolic gene promoters. MET32 is weakly induced (group 2), and we do not detect any induction of MET31 (group 3). Whereas met31Δ and met32Δ mutant cells are able to produce their own methionine when grown on minimal medium, met31Δmet32Δ double-mutants are methionine auxotrophs. Despite the sequence and functional similarity of these proteins, the observation of differential regulation is suggestive of distinct roles for MET31 and MET32, at least under particular conditions, resulting in retention of both copies during evolution.
GEV-mediated induction of TEV results in rapid degradation of N-degron–tagged proteins
We chose the MET4
genes to use in a proof-of-principle experiment to test our version of the Taxis et al. (2009)
protein depletion method (). We constructed Ndeg-modified MET4
alleles in separate strains containing PACT1
(see Materials and Methods
). Cells were grown in rich medium (YPD) and treated with 1 μM β-estradiol at ~2 × 107
cells/ml. Western blots were performed on proteins extracted before the addition of β-estradiol, and at several time points after induction (). Treatment of cultures with 1 μM β-estradiol had no effect on levels of Met4p-13Myc or Met31p-13Myc lacking NDeg ( and ). However, 1 μM β-estradiol treatment of NDeg-MET4
cells showed rapid reduction of the NDeg-tagged proteins ( and ). By performing a best fit of the data from 10-, 20-, and 40-min time points to a power law, we find that the half-life of NDeg-Met4p-13Myc is 16 min and that of NDeg-Met31p-13Myc is 13.75 min ().
FIGURE 8: (A) GEV is expressed under the regulation of the constitutive ACT1 promoter. Treatment with β-estradiol induces nuclear entry of GEV and subsequent induction of PGAL1-driven TEV. (B) The target gene of interest (YFG) is regulated by its native (more ...)
FIGURE 9: (A) Western blot of DBY12055 (NDeg-Met4p-13Myc) and DBY11440 (Met4p-13Myc) before (0′) and after (10′, 20′, and 40′) 1 μM β-estradiol addition to the culture. (B) Western blot of DBY12234 (NDeg-Met31p-13Myc) (more ...)
null allele has a growth defect and produces small colonies on YPD plates (Hickman et al., 2011
). We therefore started with a diploid cell for construction of NDeg-MET4
. Sporulation of this diploid followed by dissection of the resulting tetrads showed that the spore colonies had the same wild-type size, confirming that the modified gene was functional.
Met4p is known to be ubiquitinated (Rouillon et al., 2000
) and is, as a result, unstable. We observed three clear bands in our Met4p Western blots, consistent with the presence of nonubiquitinated Met4p in addition to heavier ubiquitinated forms (). When MET4
is tagged with NDeg, both the ubiquitinated and nonmodified Met4p proteins are targeted for degradation by TEV (). This finding demonstrates that even heavily modified forms of a protein can be cleaved by TEV.
Finally, we assayed the phenotypic effect of β-estradiol treatment of NDeg-MET4–
containing cells. As expected, only cells treated with β-estradiol displayed a methionine auxotrophy (met–
) phenotype (Supplemental Figure 8), as Met4p is required for methionine synthesis (Masselot and Robichon-Szulmajster, 1975