Name: yeast as a chassis for developing functional assays to study human p53
Uploaded: 2019-08-04T13:00:00.000+00:00
Duration: 14 min 57 s
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 it grow for 2-3 days at 30 &#176;C.</li>
	<li>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 &#176;C overnight, shaking at 150-200 rpm.</li>
	<li>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).</li>
	<li>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 &#176;C, shaking at 150-200 rpm (necessary for the induction of I-SceI).</li>
	<li>Centrifuge the cells for 2 min at 3,000 x g and discard the supernatant by inversion of the tube.</li>
	<li>Resuspend the cell pellet in 30-50 mL of sterile water. Repeat step 1.5.</li>
	<li>Resuspend the cell pellet in 10 mL of sterile water. Repeat step 1.5.</li>
	<li>Resuspend the cell pellet in 5 mL of sterile LiAcTE (Table 3), an ionic solution that favors DNA uptake. Repeat step 1.5.</li>
	<li>Resuspend the cell pellet in 250 &#181;L 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 &#181;L of sterile LiAcTE.</li>
	<li>During the washes, denature a 10 mg/mL solution of salmon sperm DNA carrier for 10 min at 100 &#176;C and chill immediately on ice to maintain it as single-stranded DNA.</li>
	<li>In a separate 1.5 mL tube, add 500 picomoles of the desired oligonucleotide (Table 3), 5 &#181;L of the boiled salmon sperm carrier DNA, 300 &#181;L of sterile LiAcTE PEG (Table 3), and 50 &#181;L of the yeast cell suspension (from step 1.9).</li>
	<li>Vortex the tubes for 10 s to mix and incubate for 30 min at 30 &#176;C, shaking at 150-200 rpm. Place the 1.5 mL tubes on the side to favor shaking.</li>
	<li>Heat shock the yeast cells for 15 min at 42 &#176;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.</li>
	<li>Spread 100 &#181;L of the cell suspension on YPDA agar plate and incubate (upside down) for 1 day at 30 &#176;C. To ensure well-separated colonies are obtained, also spread 100 &#181;L of a 1:10 dilution.</li>
	<li>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).</li>
	<li>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 &#176;C.</li>
	<li>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 &#176;C.</li>
	<li>Patch single yeast colonies on a YPDA plate to isolate the colonies for further analyses. After 24 h at 30 &#176;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.</li>
	<li>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 &#956;L of 10x PCR buffer (1.5 mM MgCl2), 2 &#956;L of 10 picomoles/&#956;L primers (Table 3), 4 &#956;L of 2.5 mM dNTPs, 0.25 &#181;L of 5 U/&#956;L Taq polymerase, and water to a final volume of 50 &#956;L. Multiply the reaction mix for the number of yeast colonies that need to be screened and aliquot 50 &#956;L 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.</li>
	<li>Perform the PCR reaction with the following program: 94 &#176;C for 8 min followed by 35 cycles of denaturation for 1 min at 94 &#176;C, primers annealing for 1 min at 55 &#176;C, and extension for 2 min at 72 &#176;C.</li>
	<li>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) .</li>
	<li>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.</li>
	<li>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 &#176;C.</li>
</ol>
2. Evaluation of P53 protein transactivation ability using the qualitative color-based ADE2 yeast assay
<ol>
	<li>Repeat steps 1.1 and 1.2 using the yAFM-RE strain (Table 1).</li>
	<li>The day after, dilute the cell culture (1:10) in 30-50 mL of pre-warmed YPDA and continue to incubate at 30 &#176;C by shaking until the OD600nm reaches 0.8-1.0 (~2 h).</li>
	<li>Repeat steps 1.5-1.10.</li>
	<li>In a separate 1.5 mL tube, add 300-500 ng of yeast P53 (or control) expression vector (Table 4), 5 &#181;L of the boiled salmon sperm DNA carrier, 300 &#181;L of sterile LiAcTE PEG, and 50 &#181;L of yeast cell suspension.</li>
	<li>Repeat steps 1.12 and 1.13 but resuspend the cell pellet in 300 &#181;L of sterile water.</li>
	<li>Spread 100 &#181;L 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 &#176;C for 2-3 days.</li>
	<li>Streak single yeast transformant colonies (2-6 streaks per plate) on a new selective plate and let them grow overnight at 30 &#176;C.</li>
	<li>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 &#176;C for 3 days. Optionally, to evaluate temperature sensitivity of P53 protein, incubate at three different temperatures for 3 days: 24 &#176;C, 30 &#176;C, and 37 &#176;C. 
	NOTE: The same streak can be replica plated multiple times.</li>
	<li>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.</li>
</ol>
3. Evaluation of P53 protein transactivation ability using the quantitative luminescence based LUC1 yeast assay
<ol>
	<li>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).</li>
	<li>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 &#176;C overnight. For each transformation type, make 5-7 different patches.</li>
	<li>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 &#181;L 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.</li>
	<li>Measure the absorbance of each well at OD600nm after inducible P53 expression (4-8 h at 30&#176;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.</li>
	<li>Transfer 10-20 &#181;L 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 &#181;L) 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.</li>
	<li>Add 10-20 &#181;L of Firefly luciferase substrate and measure the light units (LU) by a multi-label plate reader.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
4. Evaluation of P53 protein growth inhibition using the yeast phenotypic assay
<ol>
	<li>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).</li>
	<li>Grow transformed cells in minimal selective medium (Table 2) to approximately 1 OD600nm.</li>
	<li>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.</li>
	<li>Incubate cells at 30 &#176;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).</li>
	<li>Spot 100 &#181;L aliquots of yeast cell cultures on minimal selective plates (Table 2).</li>
	<li>Incubate for 2 days at 30 &#176;C.</li>
	<li>Measure yeast growth by counting the number of colonies obtained in the 100 &#181;L 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.</li>
</ol>

The authors declare no conflict of interest.

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.

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

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

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

9.5K Views.  Ospedale Policlinico San Martino. 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.

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

Mating-based Overexpression Library Screening in Yeast

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

Genetics

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Immunology and Infection

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Biology

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

Growth Assays to Assess Polyglutamine Toxicity in Yeast

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's disease, and Huntington's disease 1. Huntington's disease (HD) is caused by the abnormal expansion of a polyglutamine (polyQ) region within the protein huntingtin. The polyQ-expanded huntingtin protein attains an aberrant conformation (i.e. it misfolds) and causes cellular toxicity 2. At least eight further neurodegenerative diseases are caused by polyQ-expansions, including the Spinocerebellar Ataxias and Kennedy&#x2019;s disease 3.
The model organism yeast has facilitated significant insights into the cellular and molecular basis of polyQ-toxicity, including the impact of intra- and inter-molecular factors of polyQ-toxicity, and the identification of cellular pathways that are impaired in cells expressing polyQ-expansion proteins 3-8. Importantly, many aspects of polyQ-toxicity that were found in yeast were reproduced in other experimental systems and to some extent in samples from HD patients, thus demonstrating the significance of the yeast model for the discovery of basic mechanisms underpinning polyQ-toxicity. 
A direct and relatively simple way to determine polyQ-toxicity in yeast is to measure growth defects of yeast cells expressing polyQ-expansion proteins. This manuscript describes three complementary experimental approaches to determine polyQ-toxicity in yeast by measuring the growth of yeast cells expressing polyQ-expansion proteins. The first two experimental approaches monitor yeast growth on plates, the third approach monitors the growth of liquid yeast cultures using the BioscreenC instrument. 
Furthermore, this manuscript describes experimental difficulties that can occur when handling yeast polyQ models and outlines strategies that will help to avoid or minimize these difficulties. The protocols described here can be used to identify and to characterize genetic pathways and small molecules that modulate polyQ-toxicity. Moreover, the described assays may serve as templates for accurate analyses of the toxicity caused by other disease-associated misfolded proteins in yeast models.

This manuscript describes three complementary protocols for assessing the toxicity of polyglutamine (polyQ)-expansion proteins in the yeast Saccharomyces cerevisiae. These protocols can easily be modified to monitor the toxicity of other misfolded proteins in yeast.

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's ...

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and &#945;-synuclein, implicated in Parkinson&#8217;s disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders. Thus, yeast are an ideal platform for isolating toxicity suppressors from libraries of protein variants. We are interested in applying protein disaggregases to eliminate misfolded toxic protein conformers. Specifically, we are engineering Hsp104, a hexameric AAA+ protein from yeast that is uniquely capable of solubilizing both disordered aggregates and amyloid and returning the proteins to their native conformations. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. Hsp104 has only limited ability to eliminate disordered aggregates and amyloid fibers implicated in human disease. Thus, we aim to engineer Hsp104 variants to reverse the protein misfolding implicated in neurodegenerative disorders. We have developed methods to screen large libraries of Hsp104 variants for suppression of proteotoxicity in yeast. As yeast are prone to spontaneous nonspecific suppression of toxicity, a two-step screening process has been developed to eliminate false positives. Using these methods, we have identified a series of potentiated Hsp104 variants that potently suppress the toxicity and aggregation of TDP-43, FUS, and &#945;-synuclein. Here, we describe this optimized protocol, which could be adapted to screen libraries constructed using any protein backbone for suppression of toxicity of any protein that is toxic in yeast.

Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

A Method to Study &#945;-Synuclein Toxicity and Aggregation Using a Humanized Yeast Model

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by progressive cell death caused by the formation of Lewy bodies containing misfolded and aggregated &#945;-synuclein. &#945;-synuclein is an abundant presynaptic protein that regulates synaptic vesicle trafficking, but the accumulation of its proteinaceous inclusions results in neurotoxicity. Recent studies have revealed that various genetic factors, including bacterial chaperones, could reduce the formation of &#945;-synuclein aggregates in vitro. However, it is also important to monitor the anti-aggregation effect in the cell to apply this as a potential treatment for the patients. It would be ideal to use neuronal cells, but these cells are difficult to handle and take a long time to exhibit the anti-aggregation phenotype. Therefore, a quick and effective in vivo tool is required for the further evaluation of in vivo anti-aggregation activity. The method described here was used to monitor and analyze the anti-aggregation phenotype in the humanized yeast Saccharomyces cerevisiae, which expressed human &#945;-synuclein. This protocol demonstrates in vivo tools that could be used for monitoring &#945;-synuclein-induced cellular toxicity, as well as the formation of &#945;-synuclein aggregates in cells.

An in vivo physiological model of &#945;-synuclein is required to study and understand the pathogenesis of Parkinson's disease. We describe a method to monitor the cytotoxicity and aggregate formation of &#945;-synuclein using a humanized yeast model.

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by progressive cell death caused by the formation of Lewy ...

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

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Chapters in this video

Mating-based Overexpression Library Screening in Yeast

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Growth Assays to Assess Polyglutamine Toxicity in Yeast

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

A Method to Study α-Synuclein Toxicity and Aggregation Using a Humanized Yeast Model