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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • 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 logos<....

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.......

Representative 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.......

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 M....

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.....

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