A genome-scale yeast library with inducible expression of individual genes

Abstract

The ability to switch a gene from off to on and monitor dynamic changes provides a powerful approach for probing gene function and elucidating causal regulatory relationships. Here, we developed and characterized YETI (Yeast Estradiol strains with Titratable Induction), a collection in which > 5,600 yeast genes are engineered for transcriptional inducibility with single-gene precision at their native loci and without plasmids. Each strain contains SGA screening markers and a unique barcode, enabling high-throughput genetics. We characterized YETI using growth phenotyping and BAR-seq screens, and we used a YETI allele to identify the regulon of Rof1, showing that it acts to repress transcription. We observed that strains with inducible essential genes that have low native expression can often grow without inducer. Analysis of data from eukaryotic and prokaryotic systems shows that native expression is a variable that can bias promoter-perturbing screens, including CRISPRi. We engineered a second expression system, Z₃ EB42, that gives lower expression than Z₃ EV, a feature enabling conditional activation and repression of lowly expressed essential genes that grow without inducer in the YETI library.

Keywords: BAR-seq; CRISPRi; gene overexpression; yeast genomics; yeast mutant array.

Figure 1. The Z₃EV system

1. Outline of the β‐estradiol‐inducible gene expression system. Z₃EV is composed of a 3‐zinc finger DNA‐binding domain (Zif268), human estrogen receptor domain (hER), and transcription activation domain (VP16). β‐estradiol displaces Hsp90 from the estrogen receptor, allowing Z₃EV to translocate to the nucleus and induce gene expression. Zif268 binds preferentially to a sequence that is present in six copies in Z₃pr. In the strain collection, gene‐specific DNA barcodes are flanked by universal primer sequences: 5’‐GCACCAGGAACCATATA‐3’ and 5’‐GATCCGCTCGCACCG‐3’.

2. GFP intensity as a function of β‐estradiol concentration. Strains with an integrated Z₃pr driving GFP (Y15292; blue) and a control strain (Y15483; gray) were incubated with a concentration series of β‐estradiol in YNB for 6 h, and then cells were fixed. GFP intensity was measured by flow cytometry. Error bars represent standard deviation for three replicates.

3. Y15292 (blue) and Y15477 (gray) cultures were induced with 10 nM β‐estradiol for 6 h. Cells were washed, and β‐estradiol was removed from the medium at time = 0 h on the figure. Error bars represent ± 1 standard deviation for three biological replicates.

Figure EV1. Transcriptional induction of 18 YETI‐NE strains

1. Expression data of 18 strains during exponential growth in 1 ml batch culture of SC medium after 30 min of induction with varying concentrations of β‐estradiol in a deep‐well 96‐well plate. The y‐axis shows transcripts per million (TPM) values from RNA‐seq experiments, and the x‐axis shows concentrations of β‐estradiol (nM). The dotted line indicates the endogenous level of expression for each gene in this experiment, calculated as the average TPM value from all strains in the panel that contain a native copy of the allele.

2. Comparing Z₃EV inducibility to endogenous gene expression. The graph shows maximum TPM values after β‐estradiol induction for the 18 genes assayed in (A) (y‐axis) versus native expression (x‐axis). For genes with maximum endogenous expression level of < 250 TPM, a linear model was fit (y = 87.96 + 4.79x, R ² = 0.76).

Figure 2. Hierarchical clustering growth patterns of YETI‐E haploid strains

The YETI‐E diploid strains were sporulated, and haploids were selected on SC SGA selection medium with monosodium glutamate as a nitrogen source at 12 β‐estradiol concentrations (0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1,000 nM) and clustered by growth profile. Growth was measured as the area under the growth curve (AUGC); blue represents little growth and yellow represents more growth. AUGC values were quantified for sixteen separate colonies per genotype and dose (see Materials and Methods). Strains were clustered using a Chebyshev distance metric, resulting in five clusters (numbered 1–5). Representative strains for each of the five clusters and their growth patterns are plotted to the right of the clustergram. Error bars represent ± 1 standard deviation.

Figure EV2. Growth measurements for YETI alleles of ALG1, ULP2, CDC48, and ADE13

LOESS curve fits are shown (blue lines) from individual measurements of four separate colonies (gray dots).

Figure 3. Synthetic control of growth depends on native gene expression levels in yeast and in E. coli

1. A

   Yeast strains in which Z₃EV targets essential genes are more likely to grow in the absence of inducer if the Z₃EV‐controlled gene is lowly expressed. The boxplots show YETI‐E AUGC (area under the growth curve) values from 0 nM (gray) and 1,000 nM (blue) β‐estradiol experiments. The six bins (bins 1–6) are based on gene expression level of native genes (Lipson et al, 2009).

2. B–F

   Box plots showing native levels of gene expression binned by growth characteristics for other yeast strain collections. (B) The TET‐allele collection. Genes not repressible with Tet‐off (DOX‐non‐responsive) and genes that are repressible with Tet‐off (DOX‐responsive) are shown. (C) The DAmP allele collection. Box plots showing fitness of DAmP alleles as a function of native expression level. (D) TS‐degron strains. Levels of native gene expression for genes for which the addition of an N‐terminal degron does not affect growth versus those for which the degron confers temperature‐sensitive growth are plotted. (E) CRISPRi strains. Box plots showing fitness of CRISPRi alleles as a function of native expression level. (F) E. coli strains in which CRISPRi targets essential genes are more likely to grow if the targeted essential gene is lowly expressed. Boxplots show the distribution of gene expression levels for E. coli genes tested in a CRISPRi screening experiment (Wang et al, 2018). The genes are grouped into essential genes whose repression by CRISPRi inhibits growth (does not grow) or fails to inhibit growth (grows). Gene expression data from Gene Expression Omnibus (GSE67218). RPKM (Reads Per Kilobase of transcript, per Million reads) median values are 166 and 503 for the “Grows” and “Does not grow” classes, respectively. For all box plots, the central band represents the median value. The bottom and top hinges represent the 25 and 75% quantiles, respectively.

Figure 4. Comparing β‐estradiol‐dependent growth of strains expressing YETI‐NE genes on YNB and SC

Distribution of Aux scores of YETI‐NE haploid strains. The y‐axis shows the Aux score value (see main text and Materials and Methods) measured for each YETI‐NE strain displayed on the x‐axis. YETI‐NE strains are shown as gray dots with metabolic auxotroph panel members labeled in blue. The identities of the 41 YETI‐NE strains with the highest Aux scores are listed. The inset shows the distribution pattern for all strains tested.

Figure EV3. Analysis of genes that impair growth when overexpressed

1. Venn diagram of genes that are toxic upon overexpression from this study as compared to previous overexpression studies.

2. Barplot of Z₃EV toxic strains grouped by overlap with previous overexpression studies seen in (A). For each grouping the percentage of Z₃EV toxic strains that are toxic at only 100 nM β‐estradiol (solid color) versus the percentage that are toxic at β‐estradiol concentrations < 100 nM (white) is shown.

Figure 5. BAR‐seq of YETI‐NE haploid strains

1. Diagram illustrating the pooled culture growth and harvesting strategy for BAR‐seq experiments with YETI‐NE strains. The labels in the diagram are also used to indicate the relevant growth conditions in the corresponding clustergram (part B).

2. BAR‐seq analysis of YETI‐NE pools in SC and YNB medium in the absence or presence of 100 nM β‐estradiol. Data were hierarchically clustered and 11 clusters labeled based on conditions in which strains exhibited reduced growth: Cluster YNB +e (showing depletion in 100 nM β‐estradiol in YNB only), Cluster YNB (depletion in 0 nM β‐estradiol in YNB only), Clusters All 1 and All 2 (time‐dependent depletion in all conditions), Cluster YNB or SC (depletion in either YNB or SC at 0 nM β‐estradiol), Clusters “+e 1a” and “+e 1b” (fast depletion in YNB [12 h] and SC [9 h] at 100 nM β‐estradiol), Cluster “+e 2” (depletion by 24 h in YNB and 18 h in SC at 100 nM β‐estradiol), Clusters “SC+e 1” and “SC+e 2” (depletion in SC only at 100 nM β‐estradiol), Cluster SC (depletion in SC only at 0 nM β‐estradiol). Experiments were performed in biological triplicate.

Figure 6. Rof1 is a transcriptional repressor

Microarray expression data resulting from induction of Z₃pr‐ROF1 over a 90‐min time‐course. The Z₃pr‐ROF1 strain was grown in the presence of 1 µM β‐estradiol in a phosphate‐limited chemostat, and samples were harvested at the indicated times. Two independent time courses are shown. Gene expression data were clustered using a Pearson correlation distance metric. One regulon of Rof1‐repressed genes was identified, which is highlighted on the right. Motif enrichment was calculated using the cumulative binomial distribution in the HOMER software suite (Heinz et al, 2010).

Figure 7. Number of binding sites, URS1 presence, and ATF choice all affect mNeonGreen reporter induction

Strains with all combinations of ATFs and promoters driving mNeonGreen were grown to early log phase in YNB and mNeonGreen was induced with various levels of β‐estradiol. Strains were imaged every hour using a Phenix automated confocal microscope and average fluorescence/cell was calculated using Harmony software. Average of two replicates is shown at t = 8 h following induction with > 300 cells quantified per dose. Full time series are shown in Fig EV4.

Figure EV4. Number of binding sites, URS1 presence, and ATF choice all affect mNeonGreen reporter induction time series

Full time series collected for testing the relationship between ATF, URS1, and binding site copy number. Strains were imaged every hour using a Phenix automated confocal microscope, and average fluorescence/cell was calculated using Harmony software. Average of two replicates is shown following induction with > 300 cells quantified per dose per replicate.

Figure 8. Engineered gene expression system for more stringent regulation

1. A

   Diagrams of Z₃EV artificial transcription factor with Z₃pr promoter and Z₃EB42 artificial transcription factor with Z₃pr‐Zif2‐URS1 promoter.

2. B–D

   Serial spot dilutions onto medium containing various concentrations of β‐estradiol. Diploid strains containing the indicated regulatory systems (see (A)) controlling the target genes were incubated in sporulation medium for 5 days. Serial dilutions of cells were plated on SD‐his‐ura‐arg‐lys with canavanine, thialysine, and ClonNAT with indicated concentrations of β‐estradiol to obtain haploid cell growth.

3. E

   Distribution of growth phenotypes for strains carrying Z₃EB42 promoter alleles for 45 essential genes from Cluster 1 and 2 (see Fig 2).

4. F

   Distribution of growth phenotypes for strains carrying Z₃EB42 promoter alleles for 68 essential genes from Cluster 4 and 5 (see Fig 2).

5. G

   Distribution of Z₃EB42 promoter reversibility phenotype for 29 tested essential genes.

Data information: The color key for interpreting the figure is below panels (E–G).

Figure EV5. Expression (TPM) levels of Core Essential Genes (CEG2) Essential/Non‐Essential genes in K562 cells

The CEG2 essential list is from (Hart et al, 2017). RNA‐seq data can be found on the Gene Expression Omnibus with accession GSE88351. The median TPM of CEG2 non‐essential genes is 5 TPM. The median TPM of CEG2 essential genes is 80 TPM.