Inducible, tightly regulated and growth condition-independent transcription factor in Saccharomyces cerevisiae

Abstract

The precise control of gene expression is essential in basic biological research as well as in biotechnological applications. Most regulated systems available in yeast enable only the overexpression of the target gene, excluding the possibility of intermediate or weak expression. Moreover, these systems are frequently toxic or depend on growth conditions. We constructed a heterologous transcription factor that overcomes these limitations. Our system is a fusion of the bacterial LexA DNA-binding protein, the human estrogen receptor (ER) and an activation domain (AD). The activity of this chimera, called LexA-ER-AD, is tightly regulated by the hormone β-estradiol. The selection of the AD proved to be crucial to avoid toxic effects and to define the range of activity that can be precisely tuned with β-estradiol. As our system is based on a heterologous DNA-binding domain, induction in different metabolic contexts is possible. Additionally, by controlling the number of LexA-binding sites in the target promoter, one can scale the expression levels up or down. Overall, our LexA-ER-AD system is a valuable tool to precisely control gene expression in different experimental contexts without toxic side effects.

Figure 1.

The mechanism of action of the LexA-ER-AD system. ER: hormone-binding domain of the human estrogen receptor; AD: activation domain; Ptarget: target promoter; ORF: open reading frame.

Figure 2.

Time course of the titration of LexA-ER-AD activity. Strains containing LexA-ER-B42, LexA-ER-B112, LexA-ER-Gal4AD or LexA-ER-VP16 and a target promoter with four lexA boxes driving Citrine expression (FRY418, FRY666, FRY667, FRY743) were incubated in a concentration series of β-estradiol in SDC. At each time point, the induction levels were measured by flow cytometry. Experimental (symbols, mean ± standard deviation) and simulated (gray surfaces and contour lines) time-dependent dose-response are plotted against the logarithmic concentration of inducer.

Figure 3.

Induction levels and tight regulation of LexA-ER-AD. (A) The expression levels of constitutive promoters were plotted on top of the LexA-ER-AD 24-h titration curves obtained by flow cytometry shown in Figure 2. We only considered the concentration ranges of β-estradiol in which our expression system reached a steady state. The x-axis is logarithmic. Symbols represent the median and error bars the 25th and the 75th percentiles of the fluorescence signal (area) distribution measured by the cytometer. Each dashed horizontal line represents the median of the yellow fluorescence signal distributions obtained by expressing Citrine from the constitutive promoters indicated on the right side of the graph (FRY744, FRY745, FRY746, FRY748 and FRY757). (B) Western blots to determine protein induction fold by LexA-ER-B42 (left) and LexA-ER-B112 (right) upon incubation with 2000 nM β-estradiol in SDC for 24 h (strains: FRY418 and FRY667). Citrine levels were assayed using an anti-GFP antibody. As loading control, we detected the β-actin with an anti-β-actin antibody. The induced samples were diluted as indicated. As controls, we loaded the un-induced strains (FRY418 and FRY667), a strain bearing only the target gene (Citrine under the control of four lexA boxes, FRY484), a strain bearing only the transcription factor (FRY312) and an ‘empty’ strain (FRY11). (C) Flow cytometry of the basal activity of LexA-ER-AD. Cells were cultivated in SDC lacking β-estradiol. The target gene strain contained only the target gene with four lexA boxes in its promoter (FRY484); the LexA-ER-AD + target gene strains contained both transcription factor and target gene (FRY418, FRY666, FRY667 and FRY743). The LexA-ER-AD variant is indicated under each boxplot, which summarizes the distribution of the fluorescence signal (height) measured.

Figure 4.

Effects of LexA-ER-AD activation on cell growth. (A) Strains containing a LexA-ER-AD variant and no target gene (FRY312, FRY460, FRY544 and FRY758) were induced with variable amounts of β-estradiol in SDC. In each panel, we plotted two growth curves of a LexA-ER-AD variant incubated with two different β-estradiol concentrations. As control, we plotted an ‘empty’ strain (FRY11) grown in 2500 nM β-estradiol. For each curve, we plotted the mean of triplicates (in full color), and ± standard deviation (in semi-transparent color). (B) Experimental (symbols; exponential growth rate with mean and ± standard deviation) and predicted (lines; 5 h after induction by β-estradiol) dose-response curves for the specific cellular growth rate, representing dose-dependent toxicity of the individual constructs.

Figure 5.

Induction of LexA-ER-B42 in different growth conditions. The induction levels of a prototroph strain containing LexA-ER-B42 and target gene with four lexA boxes in its promoter (FRY865) grown in SDC, SGlyC and SDP were measured by flow cytometry. Symbols represent the median and error bars the 25th and the 75th percentiles of the fluorescence signal (area) distribution measured. (A) Timing of the induction with 2000 nM β-estradiol. (B) Titration using a concentration series of β-estradiol. Cells were induced for 24 h. The x-axis is logarithmic.

Figure 6.

Characterization of the synthetic target promoter by flow cytometry. (A) Structure of the synthetic target promoter. Insulator: DEG1 terminator sequence; PminCYC1: minimal CYC1 promoter; ORF: open reading frame. (B) Experimental (filled symbols, mean ± standard deviation) and simulated (open symbols and dashed lines) dependency of fluorescence output on the number of lexA boxes after 28 h induction with 2000 nM β-estradiol; all computational results, except for four lexA boxes are independent predictions. The strains used for this experiment are FRY400, FRY401, FRY403, FRY417 and FRY418. (C) Titration of the strains bearing LexA-ER-B42 and the target gene (Citrine) with one, two, three, four or eight lexA boxes in the promoter (FRY400, FRY401, FRY403, FRY417 and FRY418), as indicated on the right. Cells were induced for 28 h in SDC with a concentration series of β-estradiol. The x-axis is logarithmic. Symbols represent the median of the fluorescence signal (area) distribution measured. For clarity, we only plotted the error bars (the 25th and the 75th percentiles of the distributions) of the induction of the strain containing four lexA boxes (FRY418). Similar error bars were observed for the other strains. (D) Normalized induction of strains bearing LexA-ER-B42 and one, two, three, four or eight lexA boxes in the target promoter driving Citrine expression (FRY400, FRY401, FRY403, FRY417 and FRY418). The strains were induced with 2000 nM β-estradiol. The gray trajectories represent the normalized median of the induction levels of each strain; the black trajectory is the mean of the normalized medians. (E) Basal activity of the system incubated in SDC without β-estradiol. The target gene strains only contained the target Citrine (FRY482, FRY484, FRY485, FRY486 and FRY487); the LexA-ER-B42 + target gene strains contained both constructs (FRY400, FRY401, FRY403, FRY417 and FRY418). The copy number of lexA boxes in the target promoter is indicated under the boxplots, which summarize the distribution of the fluorescence signal (height) measured. (F) Predicted alternative configurations for the example of LexA-ER-B42. Relative fluorescence output after 24-h induction for four lexA boxes with modified binding affinities (dissociation constant K_D) to the transcription factor. Values are normalized to the reference operator with 2000 nM β-estradiol induction.