Name: yeast as a chassis for developing functional assays to study human p53
Uploaded: 2019-08-04T13:00:00
Description: Watch this Scientific Journal Video about Yeast As a Chassis for Developing Functional Assays to Study Human P53 at JoVE.com

We thank the European Union (FEDER funds POCI/01/0145/FEDER/007728 through Programa Operacional Factores de Competitividade - COMPETE) and National Funds (FCT/MEC, Funda&#231;&#227;o para a Ci&#234;ncia e Tecnologia and Minist&#233;rio da Educa&#231;&#227;o e Ci&#234;ncia) under the Partnership Agreement PT2020 UID/QUI/50006/2019&#160;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 L&#243;pez-Arias Montenegro (University of Trento, experimental sciences teaching laboratories) for assistance with video recording.

1. Construction of ADE2 or LUC1 reporter yeast strains containing a specific RE (yAFM-RE or yLFM-RE)
<ol>
	<li>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 &#176;C on a YPDA agar plate (Table 2). Let...

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...

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-&#945;&#160;(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.

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			<td height="21" style="height:21px;width:486px;">L-Aspartic acid</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">11189</td>
			<td style="width:63px;"></td>
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			<td style="width:123px;">28104</td>
			<td></td>
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			<td height="21" style="height:21px;">L-Phenylalanine</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">78019</td>
			<td></td>
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		<tr height="22">
			<td height="22" style="height:23px;width:486px;">Peptone</td>
			<td style="width:119px;">BD Bacto</td>
			<td>211677</td>
			<td></td>
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			<td height="25" style="height:25px;width:486px;">Yeast ex+A2:C26tract</td>
			<td style="width:119px;">BD Bacto</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Difco Yeast Nitrogen Base w/o Amino Acids and Ammonium Sulfate</td>
			<td style="width:119px;">BDTM</td>
			<td style="width:123px;">233520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lithium Acetate Dihydrate</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">517992</td>
			<td></td>
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			<td height="42" style="height:42px;width:486px;">Bacteriological Agar Type A</td>
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			<td height="21" style="height:21px;width:486px;">G418 disulfate salt</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">A1720</td>
			<td></td>
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			<td height="21" style="height:21px;">Ammonium Sulfate</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">A2939</td>
			<td></td>
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			<td height="21" style="height:21px;">L-Arginine Monohydro-chloride</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">A5131</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:486px;">Adenine Hemisulfate Salt</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">A9126</td>
			<td></td>
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			<td height="21" style="height:21px;width:486px;">Passive Lysis Buffer 5x</td>
			<td style="width:119px;">PROMEGA</td>
			<td style="width:123px;">E1941</td>
			<td></td>
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			<td height="21" style="height:21px;width:486px;">Bright-Glo Luciferase Assay System&#160;</td>
			<td style="width:119px;">PROMEGA</td>
			<td style="width:123px;">E2620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:486px;">5-FOA</td>
			<td style="width:119px;">Zymo Research</td>
			<td style="width:123px;">F9001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:486px;">D-(+)-Galactose</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">G0750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamic acid</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">G1251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:486px;">Dextrose&#160;</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">G7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Histidine</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">H8125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Isoleucine</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">I2752</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Lysine</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">L1262</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:486px;">L-Leucine</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">L8000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Methionine</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">M2893</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PEG</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">P3640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:486px;">D-(+)-Raffinose Pentahydrate</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">R0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Serine</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">S4500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Tryptophan</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">T0271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Threonine</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">T8625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Uracil</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">U0750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Valine</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">V0500</td>
			<td></td>
		</tr>
	</tbody>
</table>

yeast as a chassis for developing functional assays to study human p53

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.

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...

Watch this Scientific Journal Video about Yeast As a Chassis for Developing Functional Assays to Study Human P53 at JoVE.com

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

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.

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 ...

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