Yeast As a Chassis for Developing Functional Assays to Study Human P53

Summary

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

Abstract

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Introduction

Transcription is an extremely complex process involving dynamic, spatial, and temporal organization of transcription factors (TFs) and cofactors for the recruitment and modulation of RNA polymerases on chromatin regions in response to specific stimuli¹. Most TFs, including the human P53 tumor suppressor, recognize specific cis-acting elements in the form of DNA sequences called response elements (REs), which consist of single (or multiple) unique motifs ~6-10 nucleotides long. Within these motifs, individual positions may show various degrees of variability², usually summarized by position weight matrices (PWM) or logos^3,4.

The yeast S. cerevisiae is a suitable model system for studying different aspects of human proteins through complementation assays, ectopic expression, and functional assays, even when an orthologous yeast gene is not present^5,6,7. Due to the evolutionary conservation of basal components of the transcriptional system⁸, many human TFs (when ectopically expressed in yeast cells) can modulate the expression of a reporter gene by acting through promoters engineered to contain appropriate REs. The transcription model system presented here for human P53 is characterized by three major variables whose effects can be modulated: 1) the modality of expression and type of P53, 2) the RE sequence controlling P53-dependent transcription, and 3) the type of reporter gene (Figure 1A).

Concerning the modality of P53 expression, S. cerevisiae allows the choice of inducible, repressible, or constitutive promoters^9,10,11. In particular, the inducible GAL1 promoter allows basal (using raffinose as a carbon source) or variable (by changing the amount of galactose in the media) expression of a TF in yeast. In fact, the finely tunable expression represents a critical development for studying not only P53 itself but also other P53 family proteins^12,13.

Regarding the type of REs controlling the P53-dependent expression, S. cerevisiae allows the construction of different reporter strains possessing unique differences in the RE of interest in an otherwise isogenic background. This goal is reached using an adaptation of a particularly versatile genome editing approach developed in S. cerevisiae, called delitto perfetto^12,14,15,16.

Furthermore, different reporter genes (i.e., URA3, HIS3, and ADE2) can be used to qualitatively and quantitatively evaluate transcriptional activities of human TFs in S. cerevisiae, each with specific features that can be tailored to experimental needs^{17,18,19,20,21}. The expression of these reporter genes confers uracil, histidine, and adenine prototrophy, respectively. The URA3 reporter does not allow the growth of cells in the presence of 5-FOA as well, and thus it can be counterselected. The ADE2 reporter system has the advantage that, besides nutritional selection, it allows the identification of yeast cells that express wild-type (i.e., functional on ADE2 expression) or mutant (i.e.,not functional on ADE2) P53 from the colony color.

For example, yeast cells expressing the ADE2 gene generate normally sized white colonies on plates containing limiting amounts of adenine (2.5-5.0 mg/L), while those that poorly or do not transcribe it appear on the same plate as smaller red (or pink) colonies. This is due to accumulation of an intermediate in the adenine biosynthetic pathway (i.e., P-ribosylamino-imidazole, which has been previously called amino-imidazole ribotide or AIR), which is converted to form a red pigment. The qualitative color based ADE2 reporter gene has since been replaced with the quantitative Firefly Photinus pyralis (LUC1)^12,22. More recently, the ADE2 reporter has been combined with the lacZ reporter in an easy-to-score, semi-quantitative, double reporter assay that can be exploited to sub-classify P53 mutants according to their residual level of functionality²³.

Fluorescent reporters such as EGFP (enhanced green fluorescent protein) or DsRed (Discosoma sp. red fluorescent protein) have also been used for the quantitative evaluation of transactivation activity associated with all possible missense mutations in the TP53 coding sequence²⁴. Lastly, the chance of combining tunable promoters for P53 allele expression with isogenic yeast strains differing for the RE and/or reporter gene has led to the development of a data matrix that generates a refined classification of cancer-associated and germline mutant P53 alleles^25,26,27.

The approaches described above are used to measure the transcriptional activity of the P53 protein. However, the expression of wild-type P53 in the yeast S. cerevisiae²⁸ and Schizosaccharomyces pombe²⁹ can cause growth retardation, which has been associated with cell cycle arrest^28,30 or cell death³¹. In both cases, yeast growth inhibition is triggered by high P53 expression and has been correlated with potential transcriptional modulation of endogenous yeast genes involved in cell growth. Supporting this hypothesis, the loss-of-function mutant P53 R273H did not interfere with yeast cell growth when expressed at similar levels as wild-type P53³². Conversely, the expression in yeast of the toxic mutant P53 V122A (known for higher transcriptional activity compared to wild-type P53) caused a stronger growth inhibitory effect than wild-type P53³².

Additionally, it was demonstrated that human MDM2 was able to inhibit the human P53 transcriptional activity in yeast, promoting its ubiquitination and subsequent degradation³³. Accordingly, the ability of human MDM2 and MDMX to inhibit P53-induced yeast growth inhibition was demonstrated^32,34. In an additional study, a correlation between P53 transcriptional activity and actin expression levels was established, with the identification of a putative P53 RE upstream on the ACT1 gene in yeast³². Consistently, actin expression was enhanced by wild-type P53 and even more so by P53 V122A, but not by mutant P53 R273H. Conversely, actin expression by P53 decreased in the co-presence of P53 inhibitors MDM2, MDMX, or pifithrin-α (a small-molecule inhibitor of P53 transcriptional activity), consistent with results based on the yeast-growth assay. Importantly, these results established a correlation between P53-induced growth inhibition and degree of its activity in yeast, which has been also exploited to identify and study small molecules modulating P53 functions^28,34,35.

Protocol

1. Construction of ADE2 or LUC1 reporter yeast strains containing a specific RE (yAFM-RE or yLFM-RE)

1. Streak a yAFM-ICORE or yLFM-ICORE strain^12,14 (ICORE = I, ISce-I endonuclease under GAL1 promoter; CO = counter selectable URA3; RE = reporter KanMX4 conferring kanamycin resistance; Table 1) from a 15% glycerol stock stored at -80 °C on a YPDA agar plate (Table 2). Let it grow for 2-3 days at 30 °C.
2. Take one yeast colony from the fresh plate (no more than 3 weeks old) and place it in a small flask containing 5 mL of YPDA (Table 2). Incubate at 30 °C overnight, shaking at 150-200 rpm.
3. The next day, to remove all traces of dextrose, pellet the cells for 2 min at 3,000 x g and discard the supernatant by inversion of the tube.
   NOTE: Perform all centrifugations in this protocol at room temperature (RT).
4. Resuspend the cell pellet in 30-50 mL of pre-warmed CM (complete media) containing galactose (Table 2) and incubate for 4 h at 30 °C, shaking at 150-200 rpm (necessary for the induction of I-SceI).
5. Centrifuge the cells for 2 min at 3,000 x g and discard the supernatant by inversion of the tube.
6. Resuspend the cell pellet in 30-50 mL of sterile water. Repeat step 1.5.
7. Resuspend the cell pellet in 10 mL of sterile water. Repeat step 1.5.
8. Resuspend the cell pellet in 5 mL of sterile LiAcTE (Table 3), an ionic solution that favors DNA uptake. Repeat step 1.5.
9. Resuspend the cell pellet in 250 µL of sterile LiAcTE and transfer the cells to a 1.5 mL tube. Repeat step 1.5 and resuspend the cell pellet in 300-500 µL of sterile LiAcTE.
10. During the washes, denature a 10 mg/mL solution of salmon sperm DNA carrier for 10 min at 100 °C and chill immediately on ice to maintain it as single-stranded DNA.
11. In a separate 1.5 mL tube, add 500 picomoles of the desired oligonucleotide (Table 3), 5 µL of the boiled salmon sperm carrier DNA, 300 µL of sterile LiAcTE PEG (Table 3), and 50 µL of the yeast cell suspension (from step 1.9).
12. Vortex the tubes for 10 s to mix and incubate for 30 min at 30 °C, shaking at 150-200 rpm. Place the 1.5 mL tubes on the side to favor shaking.
13. Heat shock the yeast cells for 15 min at 42 °C in a heating block, then centrifuge the cells for 20 s at 10,000 x g. Remove the supernatant and resuspend the cells in 1 mL of sterile water.
14. Spread 100 µL of the cell suspension on YPDA agar plate and incubate (upside down) for 1 day at 30 °C. To ensure well-separated colonies are obtained, also spread 100 µL of a 1:10 dilution.
15. The next day, replica plate using sterile velvets onto CM agar plate containing dextrose and 5-FOA (Table 2). Consider a second replica plate on a new plate if many cells are transferred (i.e., some growth of URA3 cells).
16. Three days later, replica plate on non-selective YPDA and YPDA containing G418 (an aminoglycoside antibiotic similar to kanamycin) agar plates (Table 2), marking each plate to facilitate their subsequent comparison. Incubate the plates overnight at 30 °C.
17. The next day, identify the candidate reporter strains from colonies that are G418-sensitive but grow on YPDA plates (e.g., yLFM- or yAFM-RE colonies). Streak the identified colonies (3-6 colonies) on a new YPDA plate to obtain single colony isolates and let them grow for 2 days at 30 °C.
18. Patch single yeast colonies on a YPDA plate to isolate the colonies for further analyses. After 24 h at 30 °C, test them by replica plating on a YPGA agar plate (Table 2) that prevent the growth of petite mutants (i.e., respiratory-deficient mutants). At the same time, replica plate on a new YPDA agar plate.
19. Test the correct patches (i.e., growth on YPDA and YPGA agar plates; 1-3 colonies) from step 1.18 for the presence of correct oligonucleotide integration by colony PCR. Assemble a reaction mix by adding 5 μL of 10x PCR buffer (1.5 mM MgCl₂), 2 μL of 10 picomoles/μL primers (Table 3), 4 μL of 2.5 mM dNTPs, 0.25 µL of 5 U/μL Taq polymerase, and water to a final volume of 50 μL. Multiply the reaction mix for the number of yeast colonies that need to be screened and aliquot 50 μL into each PCR tube. Using a pipette, add a very small amount of yeast cells from the YPDA agar plate into a single PCR reaction mix.
20. Perform the PCR reaction with the following program: 94 °C for 8 min followed by 35 cycles of denaturation for 1 min at 94 °C, primers annealing for 1 min at 55 °C, and extension for 2 min at 72 °C.
21. After the reaction is completed, load an aliquot of the PCR reaction (about one-tenth the volume) on an agarose gel to check the correct size (~500 bp) .
22. Sequence the PCR product after purification with a commercial kit to confirm the integration of the desired RE sequence using the same primers of step 1.19.
23. After the validation of the correct sequence, make a 15% glycerol stock of yAFM-RE or yLFM-RE strain culture (in YPDA) and store it at -80 °C.

2. Evaluation of P53 protein transactivation ability using the qualitative color-based ADE2 yeast assay

1. Repeat steps 1.1 and 1.2 using the yAFM-RE strain (Table 1).
2. The day after, dilute the cell culture (1:10) in 30-50 mL of pre-warmed YPDA and continue to incubate at 30 °C by shaking until the OD_600nm reaches 0.8-1.0 (~2 h).
3. Repeat steps 1.5-1.10.
4. In a separate 1.5 mL tube, add 300-500 ng of yeast P53 (or control) expression vector (Table 4), 5 µL of the boiled salmon sperm DNA carrier, 300 µL of sterile LiAcTE PEG, and 50 µL of yeast cell suspension.
5. Repeat steps 1.12 and 1.13 but resuspend the cell pellet in 300 µL of sterile water.
6. Spread 100 µL of the cell suspension on synthetic selective (for P53 expression or control vector) plates containing dextrose as a carbon source and high amount of adenine (Table 2), then incubate (upside down) at 30 °C for 2-3 days.
7. Streak single yeast transformant colonies (2-6 streaks per plate) on a new selective plate and let them grow overnight at 30 °C.
8. The day after, using sterile velvets replica plate onto new selective plates that allow for the assessment of the color phenotype (i.e., plates containing dextrose as carbon source but limiting the amount of adenine ; Table 2). Incubate the plates upside down at 30 °C for 3 days. Optionally, to evaluate temperature sensitivity of P53 protein, incubate at three different temperatures for 3 days: 24 °C, 30 °C, and 37 °C.
   NOTE: The same streak can be replica plated multiple times.
9. To evaluate the P53 protein transactivation ability, check the color-based phenotype of yeast colonies and compare the P53 protein phenotype with respect to P53 wild-type and empty vector phenotypes.

3. Evaluation of P53 protein transactivation ability using the quantitative luminescence based LUC1 yeast assay

1. Transform yeast cells with P53 (or control) expression vectors (Table 4) using the LiAc based method described in protocol 2. Use yLFM-RE strain (Table 1).
2. Patch single transformants on a new selective plate with the high amount of adenine containing glucose as the carbon source and let them grow at 30 °C overnight. For each transformation type, make 5-7 different patches.
3. After overnight growth, resuspend a small amount of yeast cells using a sterile toothpick or pipette tip from the plate in synthetic selective medium containing dextrose or raffinose as the carbon source (200 µL final volume in a transparent 96 well plate, with a round or flat bottom). If the experiment requires inducible P53 expression, add galactose to the raffinose medium to modulate the level of induction (Table 2).
   NOTE: These cell suspensions should have an OD_600nm of about 0.4 and no higher than 1.
4. Measure the absorbance of each well at OD_600nm after inducible P53 expression (4-8 h at 30°C with 150-200 rpm shaking) using a multilabel plate reader. Make sure the cell suspensions are homogeneous by mixing each well with a multichannel pipette.
5. Transfer 10-20 µL of cell suspension from the transparent 96 well plate into a white 384 (or 96) well plate and mix with an equal volume (10-20 µL) of lysis buffer. Incubate for 10-15 min at RT on a shaker (150-200 rpm) to achieve permeabilization of the cell to the luciferase substrate.
6. Add 10-20 µL of Firefly luciferase substrate and measure the light units (LU) by a multi-label plate reader.
7. To determine P53 protein transactivation ability, normalize the LUs of each well to the corresponding OD_600nm (relative light unit, RLU). Calculate average RLU and standard deviation from 3-4 patches of yeast transformant colonies.
8. Compare the P53 protein transactivation data with respect to P53 wild-type and empty vector, either by subtracting the values obtained with the empty vector or by dividing by the values obtained with the empty vector (i.e., computing fold of induction).
   NOTE: The P53 transactivation activity can be also evaluated using the same experimental set-up in the presence of P53-interacting proteins (i.e., MDM2 and MDMX) and/or including drug treatment.

4. Evaluation of P53 protein growth inhibition using the yeast phenotypic assay

1. Transform yeast cells with P53/MDM2/MDMX (or control) expression vectors (Table 4) using the LiAc-based method described in section 2. Use the CG379 strain (Table 1) and spread yeast transformants on minimal selective plates (Table 2).
2. Grow transformed cells in minimal selective medium (Table 2) to approximately 1 OD_600nm.
3. Dilute yeast cells to 0.05 OD_600nm in the selective induction medium (Table 2), and optionally, add a chosen small molecule to the appropriate concentration (or solvent only) to test its efficacy in reactivating mutant P53 or in inhibiting MDM2-/MDMX-P53 interactions.
4. Incubate cells at 30 °C under continuous orbital shaking (200 rpm) for approximately 42 h (time required by negative control yeast to reach mid-log phase, about 0.45 OD_600nm).
5. Spot 100 µL aliquots of yeast cell cultures on minimal selective plates (Table 2).
6. Incubate for 2 days at 30 °C.
7. Measure yeast growth by counting the number of colonies obtained in the 100 µL culture drops (colony forming unit, CFU counts). For example, calculate the mutant reactivating effect of compounds considering the growth of wild-type P53 expressing yeast as the maximal possible effect (set to 100%), while the growth of cells expressing mutant P53 (but exposed to solvent control) represents the zero level of reactivation.

Results

Construction of ADE2 or LUC1 reporter yeast strains

Thedelitto perfettoapproach^12,14,15,16 has been adapted to enable the construction of P53 reporter yeast strains (Figu...

Discussion

Yeast-based assays have proven useful to investigate various aspects of P53 protein functions. These assays are particularly sensitive for evaluating P53 transactivation potential towards variants of RE target sites, including the evaluation of functional polymorphisms. The use of color reporters as well as miniaturization of the luciferase assay result in cost-effective and relatively scalable assays. Also, the growth inhibition test is potentially amenable to being used in chemical library screening, automating the qua...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
L-Aspartic acid	SIGMA	11189
QIAquick PCR Purification Kit	QIAGEN	28104
L-Phenylalanine	SIGMA	78019
Peptone	BD Bacto	211677
Yeast ex+A2:C26tract	BD Bacto	212750
Difco Yeast Nitrogen Base w/o Amino Acids and Ammonium Sulfate	BDTM	233520
Lithium Acetate Dihydrate	SIGMA	517992
Bacteriological Agar Type A	Biokar Diagnostics	A1010 HA
G418 disulfate salt	SIGMA	A1720
Ammonium Sulfate	SIGMA	A2939
L-Arginine Monohydro-chloride	SIGMA	A5131
Adenine Hemisulfate Salt	SIGMA	A9126
Passive Lysis Buffer 5x	PROMEGA	E1941
Bright-Glo Luciferase Assay System	PROMEGA	E2620
5-FOA	Zymo Research	F9001
D-(+)-Galactose	SIGMA	G0750
L-Glutamic acid	SIGMA	G1251
Dextrose	SIGMA	G7021
L-Histidine	SIGMA	H8125
L-Isoleucine	SIGMA	I2752
L-Lysine	SIGMA	L1262
L-Leucine	SIGMA	L8000
L-Methionine	SIGMA	M2893
PEG	SIGMA	P3640
D-(+)-Raffinose Pentahydrate	SIGMA	R0250
L-Serine	SIGMA	S4500
L-Tryptophan	SIGMA	T0271
L-Threonine	SIGMA	T8625
Uracil	SIGMA	U0750
L-Valine	SIGMA	V0500