Fast-acting and nearly gratuitous induction of gene expression and protein depletion in Saccharomyces cerevisiae - PubMed (original) (raw)

Fast-acting and nearly gratuitous induction of gene expression and protein depletion in Saccharomyces cerevisiae

R Scott McIsaac et al. Mol Biol Cell. 2011 Nov.

Abstract

We describe the development and characterization of a system that allows the rapid and specific induction of individual genes in the yeast Saccharomyces cerevisiae without changes in nutrients or temperature. The system is based on the chimeric transcriptional activator Gal4dbd.ER.VP16 (GEV). Upon addition of the hormone β-estradiol, cytoplasmic GEV localizes to the nucleus and binds to promoters containing Gal4p consensus binding sequences to activate transcription. With galactokinase Gal1p and transcriptional activator Gal4p absent, the system is fast-acting, resulting in readily detectable transcription within 5 min after addition of the inducer. β-Estradiol is nearly a gratuitous inducer, as indicated by genome-wide profiling that shows unintended induction (by GEV) of only a few dozen genes. Response to inducer is graded: intermediate concentrations of inducer result in production of intermediate levels of product protein in all cells. We present data illustrating several applications of this system, including a modification of the regulated degron method, which allows rapid and specific degradation of a specific protein upon addition of β-estradiol. These gene induction and protein degradation systems provide important tools for studying the dynamics and functional relationships of genes and their respective regulatory networks.

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Figures

FIGURE 1:

FIGURE 1:

Schematic of the GEV overexpression system. GEV is constitutively expressed from an ACT1 promoter. Before activation by β-estradiol, GEV is inactive in the cytoplasm, associating with the Hsp90 chaperone complex. β-Estradiol diffuses through the cell membrane and binds to the GEV estrogen receptor domain, resulting in release of GEV from the Hsp90 chaperone complex. GEV then localizes to the nucleus, binds to the UASGAL consensus sequences, and strongly activates transcription through its VP16 domain.

FIGURE 2:

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 min. The input medium was then switched to medium lacking β-estradiol, and the kinetics of delocalization were quantified. (C) Images of single cells from the washout experiment in (B). For each cell, the localization score is defined as the mean of the top 5% of pixel intensities minus the mean pixel intensity over the entire cell.

FIGURE 3:

FIGURE 3:

The kinetics of GEV-mediated genetic switch. (A) We followed transcription of P_GAL1_-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 DBY12040 8 min after β-estradiol addition to the medium (FISH). Active transcription sites of P_GAL1_-CBF1 can be seen in single cells. (C) GEV activation of a P_GAL1_-GFP reporter in DBY12039 as measured by flow cytometry.

FIGURE 4:

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) The mean GFP intensity from histograms in (A) as a function of β-estradiol dose. The Hill coefficient (nH) is 0.95 (95% CI, nH = 0.72 to 1.18). The strain used in this experiment is DBY12039.

FIGURE 5:

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 SD of three replicates. (B) The heat sensitivity of tps2Δ is repaired by GEV-mediated induction of TPS2 with 10 nM β-estradiol. (WT = DBY12001; GEV = DBY12021; GEV + PGAL1-TPS2 = DBY12086).

FIGURE 6:

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 genes from (A). (C) Transcriptional response of YPL066W and YPL067C in (A). (D) The 10 most strongly repressed genes in (A). Clusters in (B) and (D) were hierarchically clustered.

FIGURE 7:

FIGURE 7:

Quantifying the kinetics of MET4's downstream targets. DBY12027 (GEV, P_GAL1_-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 β-estradiol. Left, hierarchical clustering of the sulfur metabolic genes with three clearly identifiable categories based on kinetics. Right, the mean expression of genes in each category is plotted (color) along with individual traces (gray). Error bars represent ±1 SD of the mean.

FIGURE 8:

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 P_GAL1_-driven TEV. (B) The target gene of interest (YFG) is regulated by its native promoter. TEV protease binding to its cleavage site in NDeg-modified YFG, facilitated by p14-SF3b binding, results in cleavage, exposing the destabilizing amino acid phenylalanine at the N-terminus. Proteasome-mediated digestion of YFG is the result of N-end rule degradation pathway.

FIGURE 9:

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) and DBY12235 (Met31p-13Myc) before (0′) and after (10′, 20′, and 40′) 1 μM β-estradiol addition to the culture. (C) Normalized protein levels from experiments in (A) and (B) with DBY12055 and DBY12199. Protein levels were quantified in ImageJ and then fitted to a power law for the 10′, 20′, and 40′ time points.

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