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<....

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 con....

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

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