Authors
Contact Us

Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 logos3,4.

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 present5,6,7. 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 promoters9,10,11. 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 proteins12,13.

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 perfetto12,14,15,16.

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 needs17,18,19,20,21. 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 alleles25,26,27.

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 arrest28,30 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 demonstrated32,34. 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 functions28,34,35.

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 strain12,14 (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 OD600nm 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 OD600nm of about 0.4 and no higher than 1.
  4. Measure the absorbance of each well at OD600nm 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 OD600nm (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 OD600nm.
  3. Dilute yeast cells to 0.05 OD600nm 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 OD600nm).
  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 perfettoapproach12,14,15,16 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...

Disclosures

The authors declare no conflict of interest.

Acknowledgements

We thank the European Union (FEDER funds POCI/01/0145/FEDER/007728 through Programa Operacional Factores de Competitividade - COMPETE) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020 UID/QUI/50006/2019 and the projects (3599-PPCDT) PTDC/DTP-FTO/1981/2014 - POCI-01-0145-FEDER-016581. FCT fellowships: SFRH/BD/96189/2013 (S. Gomes). This work was supported by the Compagnia S. Paolo, Turin, Italy (Project 2017.0526) and Ministry of Health, (Project 5x1000, 2015 and 2016; current research 2016). We deeply thank Dr. Teresa López-Arias Montenegro (University of Trento, experimental sciences teaching laboratories) for assistance with video recording.

Materials

NameCompanyCatalog NumberComments
L-Aspartic acidSIGMA11189
QIAquick PCR Purification KitQIAGEN28104
L-PhenylalanineSIGMA78019
PeptoneBD Bacto211677
Yeast ex+A2:C26tractBD Bacto212750
Difco Yeast Nitrogen Base w/o Amino Acids and Ammonium SulfateBDTM233520
Lithium Acetate DihydrateSIGMA517992
Bacteriological Agar Type ABiokar DiagnosticsA1010 HA
G418 disulfate saltSIGMAA1720
Ammonium SulfateSIGMAA2939
L-Arginine Monohydro-chlorideSIGMAA5131
Adenine Hemisulfate SaltSIGMAA9126
Passive Lysis Buffer 5xPROMEGAE1941
Bright-Glo Luciferase Assay System PROMEGAE2620
5-FOAZymo ResearchF9001
D-(+)-GalactoseSIGMAG0750
L-Glutamic acidSIGMAG1251
Dextrose SIGMAG7021
L-HistidineSIGMAH8125
L-IsoleucineSIGMAI2752
L-LysineSIGMAL1262
L-LeucineSIGMAL8000
L-MethionineSIGMAM2893
PEGSIGMAP3640
D-(+)-Raffinose PentahydrateSIGMAR0250
L-SerineSIGMAS4500
L-TryptophanSIGMAT0271
L-ThreonineSIGMAT8625
UracilSIGMAU0750
L-ValineSIGMAV0500

References

  1. Spitz, F., Furlong, E. E. Transcription factors: from enhancer binding to developmental control. Nature Reviews Genetics. 13 (9), 613-626 (2012).
  2. Pan, Y., Tsai, C. J., Ma, B., Nussinov, R. Mechanisms of transcription factor selectivity. Trends in Genetics. 26 (2), 75-83 (2010).
  3. Das, M. K., Dai, H. K. A survey of DNA motif finding algorithms. BMC Bioinformatics. 8 Suppl 7, S21 (2007).
  4. Sebastian, A., Contreras-Moreira, B. The twilight zone of cis element alignments. Nucleic Acids Resesarch. 41 (3), 1438-1449 (2013).
  5. Iggo, R., et al. Validation of a yeast functional assay for p53 mutations using clonal sequencing. Journal of Pathology. 231 (4), 441-448 (2013).
  6. Kaeberlein, M., Burtner, C. R., Kennedy, B. K. Recent developments in yeast aging. PLoS Genetics. 3 (5), e84 (2007).
  7. Scharer, E., Iggo, R. Mammalian p53 can function as a transcription factor in yeast. Nucleic Acids Research. 20 (7), 1539-1545 (1992).
  8. Kennedy, B. K. Mammalian transcription factors in yeast: strangers in a familiar land. Nature Reviews Molecular Cellular Biology. 3 (1), 41-49 (2002).
  9. Belli, G., Gari, E., Piedrafita, L., Aldea, M., Herrero, E. An activator/repressor dual system allows tight tetracycline-regulated gene expression in budding yeast. Nucleic Acids Research. 26 (4), 942-947 (1998).
  10. Mumberg, D., Muller, R., Funk, M. Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. Nucleic Acids Research. 22 (25), 5767-5768 (1994).
  11. Weinhandl, K., Winkler, M., Glieder, A., Camattari, A. Carbon source dependent promoters in yeasts. Microbial Cell Factories. 13, 5 (2014).
  12. Inga, A., Storici, F., Darden, T. A., Resnick, M. A. Differential transactivation by the p53 transcription factor is highly dependent on p53 level and promoter target sequence. Molelcular and Cellular Biology. 22 (24), 8612-8625 (2002).
  13. Resnick, M. A., Inga, A. Functional mutants of the sequence-specific transcription factor p53 and implications for master genes of diversity. Proceeding of the National Academy of Science USA. 100 (17), 9934-9939 (2003).
  14. Tomso, D. J., et al. Functionally distinct polymorphic sequences in the human genome that are targets for p53 transactivation. Proceeding of the National Academy of Science USA. 102 (18), 6431-6436 (2005).
  15. Storici, F., Lewis, L. K., Resnick, M. A. In vivo site-directed mutagenesis using oligonucleotides. Nature Biotechnology. 19 (8), 773-776 (2001).
  16. Storici, F., Resnick, M. A. The delitto perfetto approach to in vivo site-directed mutagenesis and chromosome rearrangements with synthetic oligonucleotides in yeast. Methods in Enzymology. 409, 329-345 (2006).
  17. Campomenosi, P., et al. p53 mutants can often transactivate promoters containing a p21 but not Bax or PIG3 responsive elements. Oncogene. 20 (27), 3573-3579 (2001).
  18. Flaman, J. M., et al. A simple p53 functional assay for screening cell lines, blood, and tumors. Proceedings of the National Academy of Science USA. 92 (9), 3963-3967 (1995).
  19. Kovvali, G. K., Mehta, B., Epstein, C. B., Lutzker, S. G. Identification of partial loss of function p53 gene mutations utilizing a yeast-based functional assay. Nucleic Acids Research. 29 (5), E28 (2001).
  20. Shimada, A., et al. The transcriptional activities of p53 and its homologue p51/p63: similarities and differences. Cancer Research. 59 (12), 2781-2786 (1999).
  21. Sunahara, M., et al. Mutational analysis of p51A/TAp63gamma, a p53 homolog, in non-small cell lung cancer and breast. Oncogene. 18 (25), 3761-3765 (1999).
  22. Andreotti, V., et al. p53 transactivation and the impact of mutations, cofactors and small molecules using a simplified yeast-based screening system. PLoS ONE. 6 (6), e20643 (2011).
  23. Hekmat-Scafe, D. S., et al. Using yeast to determine the functional consequences of mutations in the human p53 tumor suppressor gene: An introductory course-based undergraduate research experience in molecular and cell biology. Biochemistry Molecular Biology Educaction. 45 (2), 161-178 (2017).
  24. Kato, S., et al. Understanding the function-structure and function-mutation relationships of p53 tumor suppressor protein by high-resolution missense mutation analysis. Proceedings of the National Academy of Science USA. 100 (14), 8424-8429 (2003).
  25. Monti, P., et al. Transcriptional functionality of germ line p53 mutants influences cancer phenotype. Clinical Cancer Research. 13 (13), 3789-3795 (2007).
  26. Monti, P., et al. Dominant-negative features of mutant TP53 in germline carriers have limited impact on cancer outcomes. Molecular Cancer Research. 9 (3), 271-279 (2011).
  27. Leroy, B., et al. The TP53 website: an integrative resource centre for the TP53 mutation database and TP53 mutant analysis. Nucleic Acids Research. 41 (Database issue), D962-D969 (2013).
  28. Nigro, J. M., Sikorski, R., Reed, S. I., Vogelstein, B. Human p53 and CDC2Hs genes combine to inhibit the proliferation of Saccharomyces cerevisiae. Molecular and Cellular Biology. 12 (3), 1357-1365 (1992).
  29. Bureik, M., Jungbluth, A., Drescher, R., Wagner, P. Human p53 restores DNA synthesis control in fission yeast. Biological Chemistry. 378 (11), 1361-1371 (1997).
  30. Leao, M., et al. Alpha-mangostin and gambogic acid as potential inhibitors of the p53-MDM2 interaction revealed by a yeast approach. Journal of Natural Products. 76 (4), 774-778 (2013).
  31. Hadj Amor, I. Y., et al. Human p53 induces cell death and downregulates thioredoxin expression in Saccharomyces cerevisiae. FEMS Yeast Research. 8 (8), 1254-1262 (2008).
  32. Leao, M., et al. Novel simplified yeast-based assays of regulators of p53-MDMX interaction and p53 transcriptional activity. FEBS J. 280 (24), 6498-6507 (2013).
  33. Di Ventura, B., Funaya, C., Antony, C., Knop, M., Serrano, L. Reconstitution of Mdm2-dependent post-translational modifications of p53 in yeast. PLoS ONE. 3 (1), e1507 (2008).
  34. Leao, M., et al. Discovery of a new small-molecule inhibitor of p53-MDM2 interaction using a yeast-based approach. Biochemichal Pharmacology. 85 (9), 1234-1245 (2013).
  35. Billant, O., Blondel, M., Voisset, C. p53, p63 and p73 in the wonderland of S. cerevisiae. Oncotarget. 8 (34), 57855-57869 (2017).
  36. Cho, Y., Gorina, S., Jeffrey, P. D., Pavletich, N. P. Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations. Science. 265 (5170), 346-355 (1994).
  37. Muller, P. A., Vousden, K. H., Norman, J. C. p53 and its mutants in tumor cell migration and invasion. Journal of Cellular Biology. 192 (2), 209-218 (2011).
  38. Oren, M., Rotter, V. Mutant p53 gain-of-function in cancer. Cold Spring Harbor Perspectives in Biology. 2 (2), a001107 (2010).
  39. Walerych, D., Napoli, M., Collavin, L., Del Sal, G. The rebel angel: mutant p53 as the driving oncogene in breast cancer. Carcinogenesis. 33 (11), 2007-2017 (2012).
  40. Ciribilli, Y., et al. The coordinated p53 and estrogen receptor cis-regulation at an FLT1 promoter SNP is specific to genotoxic stress and estrogenic compound. PLoS ONE. 5 (4), e10236 (2010).
  41. Menendez, D., Inga, A., Resnick, M. A. Potentiating the p53 network. Discovery Medicine. 10 (50), 94-100 (2010).
  42. Monti, P., et al. Characterization of the p53 mutants ability to inhibit p73 beta transactivation using a yeast-based functional assay. Oncogene. 22 (34), 5252-5260 (2003).
  43. Monti, P., et al. Tumour p53 mutations exhibit promoter selective dominance over wild-type p53. Oncogene. 21 (11), 1641-1648 (2002).
  44. Shiraishi, K., et al. Isolation of temperature-sensitive p53 mutations from a comprehensive missense mutation library. Journal of Biological Chemistry. 279 (1), 348-355 (2004).
  45. Soussi, T. TP53 mutations in human cancer: database reassessment and prospects for the next decade. Advances in Cancer Research. 110, 107-139 (2011).
  46. Malkin, D. Li-Fraumeni Syndrome. Genes and Cancer. 2, 474-484 (2001).
  47. el-Deiry, W. S., Kern, S. E., Pietenpol, J. A., Kinzler, K. W., Vogelstein, B. Definition of a consensus binding site for p53. Nature Genetics. 1 (1), 45-49 (1992).
  48. Menendez, D., Inga, A., Resnick, M. A. The expanding universe of p53 targets. Nature Reviews in Cancer. 9, 724-737 (2009).
  49. Ciribilli, Y., et al. Transactivation specificity is conserved among p53 family proteins and depends on a response element sequence code. Nucleic Acids Research. 41 (18), 8637-8653 (2013).
  50. Smeenk, L., et al. Characterization of genome-wide p53-binding sites upon stress response. Nucleic Acids Research. 36, 3639-3654 (2008).
  51. Vyas, P., et al. Diverse p53/DNA binding modes expand the repertoire of p53 response elements. Proceeding of the National Academy of Science USA. 114 (40), 10624-10629 (2017).
  52. Tebaldi, T., et al. Whole-genome cartography of p53 response elements ranked on transactivation potential. BMC Genomics. 16, 464 (2015).
  53. Wang, T., et al. Species-specific endogenous retroviruses shape the transcriptional network of the human tumor suppressor protein p53. Proceedings of the National Academy of Science USA. 104, 18613-18618 (2007).
  54. Bond, G. L., et al. A single nucleotide polymorphism in the MDM2 promoter attenuates the p53 tumor suppressor pathway and accelerates tumor formation in humans. Cell. 119 (5), 591-602 (2004).
  55. Zeron-Medina, J., et al. A polymorphic p53 response element in KIT ligand influences cancer risk and has undergone natural selection. Cell. 155 (2), 410-422 (2013).
  56. Lion, M., et al. Evolution of p53 transactivation specificity through the lens of a yeast-based functional assay. PLoS ONE. 10 (2), e0116177 (2015).
  57. Schumacher, B., et al. C. elegans ced-13 can promote apoptosis and is induced in response to DNA damage. Cell Death and Differentiation. 12 (2), 153-161 (2005).
  58. Jordan, J. J., et al. Low-level p53 expression changes transactivation rules and reveals superactivating sequences. Proceeding of the National Academy of Science USA. 109 (36), 14387-14392 (2012).
  59. Soares, J., et al. Oxazoloisoindolinones with in vitro antitumor activity selectively activate a p53-pathway through potential inhibition of the p53-MDM2 interaction. European Journal of Pharmaceutical Sciences. 66, 138-147 (2015).
  60. Leao, M., et al. Enhanced cytotoxicity of prenylated chalcone against tumour cells via disruption of the p53-MDM2 interaction. Life Sciences. 142, 60-65 (2015).
  61. Soares, J., et al. A tryptophanol-derived oxazolopiperidone lactam is cytotoxic against tumors via inhibition of p53 interaction with murine double minute proteins. Pharmacol Research. 95-96, 42-52 (2015).
  62. Soares, J., et al. DIMP53-1: a novel small-molecule dual inhibitor of p53-MDM2/X interactions with multifunctional p53-dependent anticancer properties. Molecular Oncology. 11 (6), 612-627 (2017).
  63. Soares, J., et al. Reactivation of wild-type and mutant p53 by tryptophanolderived oxazoloisoindolinone SLMP53-1, a novel anticancer small-molecule. Oncotarget. 7 (4), 4326-4343 (2016).
  64. Sharma, V., Monti, P., Fronza, G., Inga, A. Human transcription factors in yeast: the fruitful examples of P53 and NF-small ka, CyrillicB. FEMS Yeast Research. 16 (7), (2016).
  65. Stepanov, A., Nitiss, K. C., Neale, G., Nitiss, J. L. Enhancing drug accumulation in Saccharomyces cerevisiae by repression of pleiotropic drug resistance genes with chimeric transcription repressors. Molecular Pharmacology. 74 (2), 423-431 (2008).
  66. Sharma, V., et al. Quantitative Analysis of NF-kappaB Transactivation Specificity Using a Yeast-Based Functional Assay. PLoS ONE. 10 (7), e0130170 (2015).
  67. Epinat, J. C., et al. Reconstitution of the NF-kappa B system in Saccharomyces cerevisiae. Yeast. 13 (7), 599-612 (1997).
  68. Inga, A., Reamon-Buettner, S. M., Borlak, J., Resnick, M. A. Functional dissection of sequence-specific NKX2-5 DNA binding domain mutations associated with human heart septation defects using a yeast-based system. Human Molecular Genetics. 14 (14), 1965-1975 (2005).
  69. Reamon-Buettner, S. M., Ciribilli, Y., Inga, A., Borlak, J. A loss-of-function mutation in the binding domain of HAND1 predicts hypoplasia of the human hearts. Human Molecular Genetics. 17 (10), 1397-1405 (2008).
  70. Balmelli-Gallacchi, P., et al. A yeast-based bioassay for the determination of functional and non-functional estrogen receptors. Nucleic Acids Research. 27 (8), 1875-1881 (1999).

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Explore More Articles

P53Yeast ModelTranscriptional NetworkFunctional AssaysResponse ElementsYeast TransformationReporter StrainsAdenine 2Firefly LuciferasePCR ReactionAgarose GelSanger SequencingTransactivation AbilityColor based AssayProtein Expression Vectors

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Contact Us

Recommend to library

JoVE NEWSLETTERS

Research

Education

Authors

Librarians

ABOUT JoVE

Copyright © 2025 MyJoVE Corporation. All rights reserved