Variation in the modality of a yeast signaling pathway is mediated by a single regulator
- Department of Systems Biology, Harvard Medical School, United States
- Department of Chemical Engineering, University of Washington, United States
Figures
Figure 1 with 5 supplements
Genetic and environmental factors change galactose-utilization (GAL) modality.
(A) Experimental workflow. Natural isolates of yeast tagged with a fluorescent reporter of GAL1 (GAL1pr-YFP) expression were first grown in synthetic (S) medium with a pre-induction carbon source for 16 hr, then switched to S medium with mixtures of glucose and galactose. After 8 hr, GAL1 expression was analyzed by flow cytometry. (B–C) GAL induction of two natural isolates (DBVPG1106 and BC187) or a lab strain (S288C) in mixtures of glucose and galactose after pre-induction growth in raffinose or mannose. Glucose concentration was titrated in twofold steps from 0.0039% to 1% while galactose concentration was kept constant at 0.25%. Top: Induction profiles of two natural isolates. Each plot is composed of nine histograms with color intensities corresponding to the density of cells with a given YFP abundance. Galactose concentration was titrated in twofold steps from 1% to 0.0039%. Bottom: Blow out of two histograms of YFP level normalized by side scatter (SSC) at a single concentration of glucose and galactose (see Figure 1—figure supplement 4 for histograms of all conditions). Since the GAL pathway responds to the ratio of galactose and glucose (Escalante-Chong et al., 2015), increasing the glucose concentration while keeping the galactose concentration constant simultaneously decreases GAL activation and increases glucose repression. All measurements are representative examples of at least two independent repeats. Repeat measurements are plotted in Figure 1—figure supplement 5. (D) GAL induction of two natural isolates in different galactose concentrations after pre-induction growth in raffinose.
Figure 1—figure supplement 1
Galactose-utilization (GAL) induction of natural isolates in different glucose and galactose concentrations.
Each plot is composed of nine histograms with color intensities corresponding to the density of cells with a given YFP abundance (normalized by side scatter [SSC]). Glucose concentration is titrated in twofold steps from 1% to 0.0039%, galactose concentration is constant at 0.25%. Orange isolate names indicate unimodal induction, blue isolate names indicate bimodal induction.
Figure 1—figure supplement 2
Galactose-utilization (GAL) induction profiles of 14 natural isolates (panel titles) with unimodal induction behavior after 8 or 24 hr in the same environment.
Figure 1—figure supplement 3
Galactose-utilization (GAL) induction of natural isolates in different galactose concentrations.
Each plot is composed of nine histograms with color intensities corresponding to the density of cells with a given YFP abundance (normalized by side scatter [SSC]). Galactose concentration is titrated in twofold steps from 1% to 0.0039%.
Figure 1—figure supplement 4
Histograms of the glucose gradient heatmaps plotted in Figure 1B and C.
Figure 1—figure supplement 5
Reproducibility of induced fraction (blue) and induced mean (green) measurements of different 30 natural isolates (panel titles).
Each line represents an independent repeat. Glucose concentration was titrated in twofold steps from 0.0039% to 1%. Galactose concentration was kept constant at 0.25%.
Figure 2 with 2 supplements
Phenomenological modeling of galactose-utilization (GAL) induction.
(A) Schematic description of the GAL pathway with indirect or direct inhibition by glucose. (B–C) Modeling results for varying expression level regulation with constant induced fraction regulation (B) or varying induced fraction regulation with constant expression level regulation (C). The induced fraction and mean induced level curves were chosen to be Hill functions based on empirical data (e.g. Figure 1—figure supplement 5). To create a population distribution, normal distributions were defined around the mean (log) induced level and a constant uninduced expression level, with standard deviations determined by fitting to observed distributions (Figure 2—figure supplement 1). The overall expression distribution is the induced-fraction-weighted sum of induced and uninduced distributions. For the uninduced subpopulation, the relative expression level was set to 10−3. Model results are represented by histograms at nine different glucose and galactose combinations. The intensity of the color on the plot corresponds to the density of cells with a given induction value. This analysis can be extended to a continuous range of induced fraction and induced level behaviors (Figure 2—figure supplement 2).
Figure 2—figure supplement 1
Fit of the standard deviation to the mean induction level.
Points represent the population mean of unimodal strains in nine different combinations of glucose and galactose (see Figure 1—figure supplement 1). The orange line represents a manual fit to the data (standard deviation = −0.2 * (log10(population mean + 1.75)2 + 0.4)).
Figure 2—figure supplement 2
Comparison of a wide range of induced fraction and induced level behaviors.
Simulations were performed as described in Figure 2. The color of the panel describes the modality of the induction profile (see Materials and methods).
Figure 3 with 8 supplements
Experimental validation of model predictions.
(A) Expression level metric (E10). GAL induction is measured in 2% galactose to determine the maximal expression level. The E10 is the glucose concentration at which the expression level of the induced subpopulation reaches 10% of the expression level in 2% galactose in the absence of glucose. (B) Induced fraction metric (F90). The induced fraction for each strain is calculated as the fraction of cells with an expression level that is outside the range of an uninduced population grown in 2% glucose (Figure 3—figure supplement 1). The F90 is the glucose concentration where the induced fraction reaches 90%. (C–D) Modality prediction based on induction metrics. In the phenomenological model, the position of the induced fraction and induced level functions are determined by the F90 and the E10, respectively. (E) F90 and E10 values of a panel of 30 natural isolates. Values correspond to the mean of two to five replicates. Modality was determined by comparing the fit of the data to a single or double Gaussian model (See Materials and methods). Background colors correspond to predicted regimes for unimodal and bimodal strains. Simulations with diverse combinations of F90 and E10 values as well as different slopes for the induced fraction and induced level curves delineate regimes of unimodal and bimodal behaviors. The overlap represents an ambiguous regime where both unimodal and bimodal behaviors are possible. Standard deviations of the F90 and E10 measurements are plotted in Figure 3—figure supplement 7. (F) Effect of mig1Δ on GAL1 induction profiles.
Figure 3—figure supplement 1
Identification of the induced subpopulation.
A reference distribution from 2% glucose (black histogram) is subtracted from the population distribution (orange histogram) to yield the induced subpopulation (orange shading).
Figure 3—figure supplement 2
Experimental and simulated galactose-utilization (GAL) induction profiles of bimodal strains.
Simulations were performed as shown in Figure 3. Glucose concentration is titrated in twofold steps from 1% to 0.0039%, galactose concentration is constant at 0.25%.
Figure 3—figure supplement 3
Experimental and simulated galactose-utilization (GAL) induction profiles of unimodal strains.
Simulations were performed as shown in Figure 3. Glucose concentration is titrated in twofold steps from 1% to 0.0039%, galactose concentration is constant at 0.25%.
Figure 3—figure supplement 4
Experimental and simulated galactose-utilization (GAL) induction profiles of unimodal strains with higher F90.
Simulations were identical to those in Figure 3—figure supplement 3, but the experimentally determined F90 was increased twofold for simulation purposes.
Figure 3—figure supplement 5
Experimental and simulated galactose-utilization (GAL) induction profiles of unimodal strains with varying steepness.
Simulations were identical to those in Figure 3—figure supplement 3, but the n parameter of the functions (see Figure 3 and Materials and methods) was increased to the highest value that was observed when the functions were fitted to experimental data (induced level curve: 1.72, induced fraction curve: 3.10, see Figure 3—figure supplement 6).
Figure 3—figure supplement 6
Fitted values for the n parameter.
The functions described in Figure 3 were fitted to (A) the induced level curves of all strains or (B) the induced fraction curves of bimodal strains and the n parameter of the fit was extracted. Shown values represent the mean of fits to replicates. Blue lines indicate the mean of all means.
Figure 3—figure supplement 7
Effect of the threshold for the size of the smaller subpopulation on the modality metric.
Left: Modality calls of natural isolate induction profiles after pre-induction growth in raffinose (Figure S1) when the smaller population size threshold is varied from 0.15. Right: Induction profiles that are always called as unimodal (‘BC187’, ‘YJM653’), always called as bimodal (‘273614N’, ‘DBVPG6040’, ‘YJM981’), for which the modality metric changes to bimodal when the threshold is decreased from 0.15 (‘S288C’, ‘YPS163’) or for which the modality metric changes the unimodal when the threshold is increased from 0.15 (‘DBVPG1788’, ‘Y12-SGRP’). Each plot is composed of nine histograms with color intensities corresponding to the density of cells with a given YFP abundance (normalized by side scatter [SSC]). Glucose concentration is titrated in twofold steps from 1% to 0.0039%, galactose concentration is constant at 0.25%.
Figure 3—figure supplement 8
F90 and E10 values of a panel of the 30 natural isolates in Figure 3.
Values correspond to the mean of two to five replicates. Error bars correspond to the standard deviation.
Figure 4 with 4 supplements
Metabolic history changes modality.
(A) History dependence of induction profiles. Induction profiles of S288C in mixtures of glucose and galactose after pre-induction growth in different carbon sources for 16 hr. (B) F90 and E10 values for isolates that are unimodal after growth in raffinose and bimodal after growth in mannose. (C) Induction profiles of Y12-WashU after growth in different carbon sources for 16 hr. (D) F90 and E10 values for isolates that are bimodal after growth in raffinose and unimodal after growth in either acetate or glycerol. (E) Induction profiles of YPS1009 after growth in different carbon sources for 16 hr. (F) F90 and E10 values for isolates that are always unimodal or bimodal. All measurements are representative examples of two independent repeats (compared in Figure 4—figure supplements 3–4).
Figure 4—figure supplement 1
Galactose-utilization (GAL) induction of natural isolates in different glucose and galactose concentrations after growth in different carbon sources.
Each plot is composed of nine histograms with color intensities corresponding to the density of cells with a given YFP abundance (normalized by side scatter [SSC]). Glucose concentration is titrated in twofold steps from 1% to 0.0039%, galactose concentration is constant at 0.25%. Orange titles indicate unimodal induction, blue titles indicate bimodal induction.
Figure 4—figure supplement 2
Fold change between the highest and lowest E10 and F90 values after growth in different pre-induction conditions for all isolates shown in Figure 4—figure supplement 1.
Gray bars indicate the means of the maximal fold changes.
Figure 4—figure supplement 3
Reproducibility of induced fraction (blue) and induced mean (green) measurements of 14 natural isolates (left titles) after growth in different pre-induction carbon sources (top titles).
Each line represents an independent repeat. Glucose concentration was titrated in twofold steps from 0.0039% to 1%. Galactose concentration was kept constant at 0.25%.
Figure 4—figure supplement 4
Reproducibility of F90 and E10 measurements.
(A+B) Comparison of two independent repeats of (A) F90 and (B) E10 measurements after growth in different pre-induction conditions (Figure 4).
Figure 5
Pre-induction GAL3 levels determine the modality of induction profiles.
(A) Expression of galactose-utilization (GAL) genes in different pre-induction carbon sources in S288C as determined by fluorescence of GAL promoter-YFP protein transcriptional reporter strains. (B) Effect of pre-induction growth in different carbon sources on the expression of a GAL3S288C reporter in S288C and nine natural isolates. Colors correspond to the modality of the induction profile of these strains in the given pre-induction carbon source. (C) Left: Effect of GAL3 overexpression during pre-induction growth in mannose. Connected points correspond to a doxycycline titration series for a S288C TetO7pr-GAL3 strain. Right: Complete induction profiles of S288C in mannose (I) or raffinose (II) and S288C TetO7pr-GAL3 in mannose at two concentrations of doxycycline that lead to GAL3 expression levels that bracket the expression level of GAL3 from a raffinose pre-induction culture (III, IV). (D) Left: Effect of synthetic GAL3 expression in a Δgal3 during pre-induction growth in different carbon sources. Connected points correspond to a doxycycline titration series in different carbon sources for a S288C Δgal3 TetO7pr-GAL3-mScarlet strain. Right: Complete induction profiles of S288C Δgal3 TetO7pr-GAL3-mScarlet after pre-induction growth in different carbon sources with constant GAL3 expression.
Figure 6 with 5 supplements
Allele swaps of GAL3 change modality.
(A) F90 and E10 values for a panel of allele swaps (10 GAL3 alleles in three different genetic backgrounds). Strains that never reach an induced fraction of 90% are not shown here. Black outlines denote the wild-type strains. All measurements are representative examples of two independent repeats (compared in Figure 6—figure supplements 4–5). (B–E) Effect of GAL3 allele swaps on modality. (Top) Induction profiles of (B–D) wild-type isolates or (E) S288C GAL3YPS606. (Middle) Induction profiles of (B–D) GAL3 allele swaps and (E) GAL3 promoter or GAL3 coding regions (CDS) swaps. (Bottom) Effect of the perturbation on F90 and E10. Arrows start at the values of the wild-type or S288C GAL3YPS606 strains and end at the values of the perturbed strain.
Figure 6—figure supplement 1
Effect of GAL1 and GAL4 allele swaps.
Top titles indicate the genotype of the strain, right titles indicate the source of the swapped allele. All swaps are in the S288C background. Each plot is composed of nine histograms with color intensities corresponding to the density of cells with a given YFP abundance (normalized by side scatter [SSC]). Glucose concentration is titrated in twofold steps from 1% to 0.0039%, galactose concentration is constant at 0.25%.
Figure 6—figure supplement 2
Effect of GAL1 and GAL4 allele swaps in the GAL3 allele swap background.
Top titles indicate the genotype of the strain, right titles indicate the source of the swapped allele(s). All swaps are in the S288C background. Each plot is composed of nine histograms with color intensities corresponding to the density of cells with a given YFP abundance (normalized by side scatter [SSC]). Glucose concentration is titrated in twofold steps from 1% to 0.0039%, galactose concentration is constant at 0.25%.
Figure 6—figure supplement 3
Reproducibility of induced fraction (blue) and induced mean (green) measurements of 10 GAL3 alleles (left titles) in three different strain backgrounds (top titles).
Each line represents an independent repeat. Glucose concentration was titrated in twofold steps from 0.0039% to 1%. Galactose concentration was kept constant at 0.25%.
Figure 6—figure supplement 4
Galactose-utilization (GAL) induction of allele swap strains in different glucose and galactose concentrations.
Each plot is composed of nine histograms with color intensities corresponding to the density of cells with a given YFP abundance (normalized by side scatter [SSC]). Glucose concentration is titrated in twofold steps from 1% to 0.0039%, galactose concentration is constant at 0.25%. Plot titles indicate the source of the GAL3 allele. Orange titles indicate unimodal induction, blue titles indicate bimodal induction.
Figure 6—figure supplement 5
Reproducibility of F90 and E10 measurements.
(A+B) Comparison of two independent repeats of (A) F90 and (B) E10 measurements of GAL3 allele swap strains (Figure 6).
Additional files
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Variation in the modality of a yeast signaling pathway is mediated by a single regulator
eLife 10:e69974.
https://doi.org/10.7554/eLife.69974
