Analysis of Self-Activating Bait and Prey Constructs by HT Flow Cytometry
Self-activating constructs refer to the bait or prey constructs in Y2H assays that activate Y2H reporters on their own without the occurrence of protein-protein interactions. This is observed more often for bait constructs and occasionally for prey constructs. For example, eukaryotic transcription factors, when fused with BD (bait constructs), often self-activate Y2H reporters. Other BD-fused proteins with features that can induce transcription in yeast also activate Y2H reporters (13). These self-activators in Y2H assay are often required to be determined before the HT screen and removed from the bait pools or prey arrays. We tested whether the flow cytometry based approach can be readily applied to determine the self-activators in a high throughput manner. Two self-activators, AH109-yEGFP bearing the BD-VP16 plasmid encoding the fusion protein of the Gal4BD and the VP16AD as well as Y187-yEGFP bearing the PCL1 plasmid were arrayed respectively in the bait and prey plate as shown in Figure 3A. The GFP signal of cells in each well of the bait and prey plates was measured by HT flow cytometry (Figs. 3B and 3C). Compared with the wells containing the nonself-activating baits, such as BD, BD-Lam, and BD-P53, all 24 wells in columns 10, 11, and 12 containing BD-VP16 showed clear GFP signal (mean GFP fluorescence = 635.4 ± 21.5) with 33.5% GFP positive cells (33.5 ± 0.6). Compared with the wells containing nonself-activating prey constructs, all 24 wells in row G and row H containing PCL1 triggered extremely high GFP expression (mean GFP fluorescence = 1,962.9 ± 93.2) with almost 100% GFP-positive cells (99.8 ± 0.1). This indicates that the GFP reporter in both strains is suitable for detection of self-activators in a high throughput manner. Therefore, this HT flow cytometry based Y2H system affords unique advantages for analysis of self-activators.
Figure 3. Determination of the self-activators by flow cytometry. (A) The plate map for AH109-yEGFP cells bearing various bait constructs (bait plate) are shown in the left panel. The plate map for the Y187-yEGFP cells bearing prey constructs (prey plate) are shown in the right panel. (B and C) Flow cytometry analysis of the GFP signal (FL1 Log) in each well of bait (B) and prey (C) plates. Two microliters cells from the stock bait and prey plates were transferred to 100 μl SD-T and SD-L media, respectively, to grow over night. PBS/BSA buffer was added to the each well and measured by HyperCyt flow cytometry row by row. The GFP signal (FL1 Log) of the whole plates (96 samples resolved by time) is shown.
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Evaluation of an Integrated Liquid Handing Array Approach for Analysis of PPIs
To eliminate the agar plating and the lacZ assay procedures in the current Y2H array approach, we evaluated an integrated liquid handling Y2H array approach carried out in a 96-well plate by employing HT flow cytometry analysis of the yEGFP reporter (Fig. 1B).
To mimic the standard Y2H array approach, we made frozen stocks of the bait and prey cells in 96-well plates. The bait plate was arrayed with the AH109-yEGFP cells bearing the bait constructs BD, BD-Lam, BD-T, and BD-VP16. The prey plate was arrayed with the Y187-yEGFP cells bearing the prey constructs AD, AD-T, and PCL1. Plate maps are shown in Figure 3. The cells in each well of the bait plate were mated with cells in the corresponding well of the prey plate (for example, bait A1 mated with prey A1). This mating strategy generated six to nine replicates of 12 bait/prey pairs including: (1) nine replicates of one positive control pair, P53/T; (2) nine replicates of five negative control pairs, BD/AD, BD/T, Lam/AD, Lam/T, and P53/AD and (3) six to nine replicates of the self-activators, BD-VP16 or PCL1 pairing with any other constructs (in rows G and H and columns 10–12).
Next, we optimized the mating conditions in 96-well deep well plates as follows: (1) the bait and prey cells were grown to 0.6 to 1.2 OD595 before mating; (2) 50 μl bait cells (∼105 cells) and 50 μl prey cells were mated in 100 μl 2× YPD media for 18 to 20 hr. Under these conditions, the cell density of the postmating culture is ∼107 cells/ml. The mating efficiency of 12 wells representing the 12 bait/prey pairs was determined as follows: (1) negative controls: 0.48% (BD/AD, well A2), 0.47% (Lam/AD, well A5), 0.26% (P53/AD, well A7), 0.44% (BD/T, well E2) and 0.62% (Lam/T, well, E5); (2) positive control: 0.23% (P53/T, well E7); (3) self-activators: 0.13% (BD-VP16/AD, well A11), 0.10% (BD-VP16/T, well, E11), 0.28% (BD-VP16/PCL1, well H11), 0.53% (BD/PCL1, H2), 0.55% (Lam/PCL1, well H5), and 0.25% (P53/PCL1, well H7). This indicates that the mating efficiency in each well is between 0.1 and 1%, and the mating efficiency of the positive control P53/T is equal or lower than the negative controls.
Then, we evaluated the GFP reporter and determined the optimal assay time for identification of P53/T interaction by measuring the GFP signal at 0, 24, 30, 48, 54, 72, and 96 hr postmating (Fig. 4). We first determined the appropriate region (gate) for data analysis by displaying the cell populations from the whole 96-well plate in a forward scatter versus side scatter dot plot at 0, 24, and 72 hr time points (Fig. 4A). At the zero time point, ∼95% cells were in the R1 region. A distinct yeast cell population in the R2 region with similar FSC but larger SSC values appeared after 24 hr. The percentage of cells in the R2 region reached ∼20% at 24 hr and increased to ∼50% at 72 hr and 96 hr (Fig. 4A and data not shown). To determine if the R2 region needs to be included for data analysis, we compared the GFP histogram of the cells in the R1, R2, and R1 plus R2 regions from the whole 96-well plate at 72 hr (Fig. 4B). Cells (45.4%) were GFP-positive in the R1 region whereas only 8.0% cells were GFP-positive in the R2 region. The overall percentage of GFP-positive cells decreased to 25.2%. These data indicate that cells in the R2 region contain a minor portion of GFP-positive cells. We also displayed the GFP histogram of the R1 and R2 regions in individual wells at 72 hr. For example, in well 43 containing the P53/T pair, the percentages of GFP-positive cells were 23%, 7%, and 16% in the R1, R2, and R1 plus R2 regions, respectively. In well 90 containing the Lam/PCL1 pair, the percentages of GFP-positive cells were 91%, 26%, and 60% in the R1, R2, and R1 plus R2 regions, respectively (data not shown). These results also showed that the R2 region contains much fewer GFP-positive cells than the R1 region. Cells in the R2 region were likely to be dying haploid or diploid cells that could not grow in the SD-T-L-H media. This agrees with the observation that cells in the R2 region showed similar FSC but larger SSC as well as higher background FL1 fluorescence compared with cells in the R1 region (Fig. 4A, 72hr). Therefore, only cells in the R1 region were used in the subsequent data analysis. We then evaluated GFP expression at different time points by analyzing the GFP histogram of the cells on the whole plate (Fig. 4C) and by displaying and quantifying the GFP signal (Log FL1) of cells in each well (Figs. 4D–4F). Because 51 wells would be anticipated to express GFP in the 96-well plate format, including 42 wells containing the self-activators and nine wells containing the P53/T positive control, the GFP histogram of the whole plate would define the optimal assay time point. At the zero time point, 18.2% cells from the whole plate were positive (Fig. 4C). These positive cells were contributed by the haploid cells bearing self-activators, AH109-GFP/BD-VP16 (columns 10–12) and Y187-GFP/PCL1 (rows G and H), as shown in Figure 4D (0 hr). The percentage of GFP-positive cells decreased to 11.2% at 24 hr, indicating that the percentage of GFP-positive haploid cells was decreased and that the diploid cells were growing. The percentage of GFP-positive cells increased to 19.3% at 48 hr and reached a plateau of 45.4% at 72 hr and remained 43.6% at 96 hr (Fig. 4C and data not shown). These data show that the diploid cells bearing the positive triggers (positive interactions or self-activators) started to overgrow after 48 hr and dominated after 72 hr incubation, suggesting that 72 hr is the optimal time to assay PPIs.
Figure 4. Evaluation of the flow cytometry based array approach by analysis of the GFP signal at different time points postmating. The bait plate and the prey plate were grown over night and mated with each other in YPD media. Positive PPIs were selected in SD-T-L-H media and cells were sampled at 0, 24, 30, 48, 54, 72, and 96 hr postmating to measure the GFP signal by HyperCyt/CyAn flow cytometer. (A) FSC/SSC dot plot analysis of two populations of cells in regions R1 and R2 from the whole 96-well plate at 0, 24, and 72 hr postmating. (B) Histogram analysis of GFP signals of the cells in regions R1, R2, and R1 plus R2 from the whole 96-well plate at 72 hr postmating. The percentage of GFP positive cells in each region is shown. (C) Histogram analysis of the GFP signal of the cells in the region R1 at 0, 24, 48, 72 hr postmating. The percentage of GFP-positive cells on the whole plate at each time point is shown. (D) The GFP signal of the cells in each well at 0, 24, 48, 72 hr postmating are shown in alignment. The nine wells containing the P53/T mating pairs at each time point are highlighted by horizontal lines. (E) The mean value of the percentage of GFP-positive cells (mean ± SD) in all nine wells containing the P53/T or Lam/T mating pairs are shown as a function of time. (F) The mean value of the GFP fluorescence of the cells (mean ± SD) in all nine wells containing the P53/T or Lam/T mating pairs are shown as a function of time. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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To evaluate the GFP signal in the wells containing positive control P53/T mating, we displayed the FL1 signal of the cells in each well and quantified the GFP signal to compare the P53/T pair and the negative control pairs using Lam/T as an example (Figs. 4D–4F). At 0 and 24 hr time points, only the wells containing self-activators (columns 10–12 as well as rows G and H) showed GFP signal due to the GFP-positive haploid cells whereas the wells containing all five negative controls and the positive control P53/T did not show any GFP-positive cells. A small population of GFP-positive cells in the P53/T wells appeared at 48 hr (Fig. 4D) while a larger population of GFP-positive cells was shown clearly in all the P53/T wells at the 72 hr time point and remained at the same level at 96 hr (Fig. 4D and data not shown). No GFP-positive cells were detected in the wells containing five negative control pairs (45 wells) at any time points (Fig. 4D). The percentage of GFP-positive cells in the wells containing the self-activators BD-VP16 (columns 10–12) or PCL1 (rows G and H) also increased significantly at 72 hr compared with that at 24 to 48hr (Fig. 4D). These data further confirm the whole-plate histogram analysis (Fig. 4C) and show that 72 hr postmating is an optimal time point to detect the P53/T interaction. The percentage of GFP-positive cells in the P53/T and Lam/T wells were quantified in Figure 4E, showing that the percentage of GFP-positive cells in the P53/T wells increased to 3 to 5% at 30 to 48 hr and reached a plateau (23.8 ± 3.1%) at 72 hr, which remained at the same level (23.5 ± 3.4%) at 96 hr. The mean GFP fluorescence in the P53/T wells also reached the peak at 72 hr (156.4 ± 16.2) and decreased slightly (116 ± 19.2) at 96 hr (Fig. 4F). Both the percentage of GFP-positive cells and the mean GFP fluorescence in the Lam/T wells and the other negative control wells remained at the same low level at different time points (Figs. 4E and 4F and data not shown). It is noteworthy that the mating efficiency of the negative control pairs was equal to or greater than the P53/T pair, ruling out the possibility that the negative results from these wells were due to the failure of mating. These data demonstrate that the interaction of the P53/T pair can be consistently detected in all nine wells by flow cytometry at 72 hr postmating and the longer incubation time (96 hr) still generated a significant GFP signal. They clearly show that GFP is a robust and reproducible reporter and that the flow cytometry array approach can be utilized for identification of PPIs in a high throughput manner.
Finally, we evaluated the His3 reporter gene in the liquid array approach. The cell growth in the selective media lacking histidine (SD-T-L-H) was scored by measuring OD595 at different time points. The wells containing PCL1 (0.91 OD) and the P53/T pair (0.83 OD) showed statistically significant faster cell growth than that containing the Lam/T pair (0.61 OD) at 72 hr postmating, suggesting that His3 could be used as a reporter gene in the array approach (Fig. 5). However, we observed a significant and continuous cell growth (from 0.2 OD to 0.6 OD) in wells containing the Lam/T pair and other negative controls after mating (Fig. 5 and data not shown). The background cell growth may be due to the leaky expression and moderate stringency of the His3 reporter genes since we removed the YPD media by washing the wells with SD-T-L-H media after mating. This is commonly observed and typically overcome through the use of the His3 competitor 3-aminotriazole (3-AT) in the medium in the plate-based array approach. Addition of an appropriate amount of 3-AT may increase selection stringency in our assay, but the His3 reporter in our approach is not necessary as we are measuring GFP expression in the diploid cells which are selected by the Trp and Leu markers.
Figure 5. Evaluation of the His3 Y2H reporter by measuring cell growth in the selective medium. The bait and prey cells were mated as described in Figure 4 and the mated cells were grown in SD-T-L-H media. Cells were sampled at 0, 24, 30, 48, 54, and 72 hr postmating to measure the optical density at 595 nm by plate reader. Statistical analysis of OD595 of Lam/T wells (n = 9), P53/T wells (n = 9), and BD/PCL1 wells (n = 6) was performed by the unpaired t-test using GraphPad Prism 5.
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