Hello, my name is Bartolomeo Bosco, and I'm a PhD student at the Laboratory of Transcriptional Network at the Centre for Integrative Biology of the University of Trento in Italy. As you know, transcription is an extremely complex process involving spatial and temporal organization of transcription factor and cofactor. Most of them, including the human P53, recognize specific cis-acting elements, called response elements, and the binding on this DNA-sequencing is required for the modulation of the transcription of target genes.
In this work, we would like to show you the protocol to study P53 sequence specific in transactivation functions using yeast as a model system as protein in a color reporter gene or the luciferase or on the quantification of cell growth to highlight the main experimental steps and the versatility of these approaches. Protocol one, construction of reporter yeast strains that contain a specific P53 response element upstream of the adenine-2 or the firefly luciferase gene. To construct a reporter strain, first follow steps 1.1 through 1.15 to transform yeast cells with oligonucleotides targeting the desired promoter region.
The transformation is based on the standard lithium acetate-based protocol. Once transformants appear on 5-FOA plates, usually after three days of incubation at 30 degrees, replica plate them onto non-selective YPDA plates and also on YPDA plates containing G418, marking each plate to facilitate their subsequent comparison. Incubate the plates overnight at 30 degrees.
The next day, identify the candidate reporter strains from colonies that are G418-sensitive but grew on YPDA plates. Streak the identified colonies on a new YPDA plate to obtain single colony isolates, and let them grow for two days at 30 degrees. Check the correct integration of the desired P53 response element, performing a PCR reaction with conditions mentioned in step 1.20.
Load an aliquot of the PCR product on agarose gel to check the correct amplicon length prior to Sanger sequencing. Protocol two, we present an example of how to evaluate P53 protein transactivation ability using the qualitative color-based adenine-2 yeast assay. Transform yeast cells with P53 expression vectors following the same lithium acetate-based protocol described in section one.
Streak single yeast transformant colonies, up to six per plate, on a new selective plate, and let them grow overnight at 30 degrees. The day after, using sterile velvets, replica plate the streaks on new selective plates that allow for the assessment of the color phenotype, i.e. selective plates containing limiting amount of adenine.
Incubate at 30 degrees for three days. The same streaks can be replica plated multiple times. Protocol three, we now present an example of the evaluation of P53 protein transactivation ability using the quantitative luminescence-based LUC1 yeast assay.
First, transform yeast cells with P53 expression vectors following, again, the lithium acetate-based protocol described in section two. Patch single transformants on new selective plates containing glucose as carbon source, and let them grow at 30 degrees overnight. For each transformation type, make five to seven different patches.
After the overnight growth, resuspend a small amount of cells by using a sterile toothpick or a pipette tip in synthetic selective medium containing raffinose as carbon source. These cell suspensions should have an optical density at 600 nanometers around 0.4 and can be then split in multiple 96-well plates for incubation at different temperatures, treatments, or to expose cells to varying amount of galactose to activate P53 expression. To measure the firefly reporter activity, transfer 10 to 20 microliters of cell suspension from the transparent 96-well plate into a white 384-well plate, and mix the cells with an equal volume of lysis buffer, incubating for 10, 15 minutes at room temperature on a shaker.
Add 10, 20 microliters of firefly luciferase substrate. And measure light units using a multilabel plate reader. Measure also the optical density of the cultures in the 96-well plate.
Protocol four, we now present an example of how to exploit the inhibition of the growth of yeast induced by P53. Transform yeast cells with P53 expression vectors, or cotransform them with expression vectors to coexpress P53 and one of its cofactors, such as MDM2, following, again, the lithium acetate-based protocol described in section one. Grow transformant cells in selective medium to approximately one optical density.
And dilute them to 0.05, and add a chosen molecule to the appropriate concentration. Incubate cells at 30 degrees on a shaker for 42 hours. Then, seed 100 microliters of yeast cell culture in minimal selective medium plates.
Incubate for two days at 30 degrees. The correct RE integration in place of the ICORE cassette can be check by colony PCR, starting from a tiny amount of cells from candidate yeast clones and using primers described in table three to amplify the targeted locus. PCR products, a band of about 500 nucleotides, can then be checked by Sanger sequencing to confirm the sequence of the edited genomic location for the presence of the correct P53 response element sequence.
The reporter strain is ready to be used in functional assays. To evaluate the P53 protein transactivation ability, check the color of yeast colonies. For example, we present a comparison between wild-type P53 and the mutants R175H and R282W using two different reporters, P21-5-prime and PUMA, and two different temperatures, 30 degrees and 37 degrees.
Interestingly, while R175H is a loss-of-function P53 allele, red colonies in all conditions, R282W shows temperature sensitivity with residual transactivation activity at 30 degrees, resulting in white colonies, but loss of activity at 37 degrees, resulting in red or pink colonies. An extended data set is presented in figure two B.Wild-type P53 in four mutant alleles were tested for transactivation using 10 different P53 response element reporter strains, three temperatures, and constitutive expression levels. Results are summarized in a color code that reminds the yeast colony color phenotype.
The P53 K139E mutant allele retains partial activity and is cold-sensitive. For example, colonies are red in the GADD45 reporter strain at 24 and 30 degrees, but they are white at 37 degrees. Here, we present an example of results obtained with this method, comparing the transactivation specificity of human and C.elegans P53 yeast.
Results in the bar graph are expressed as average relative light units with standard error of four replicates. Five different P53 response elements were compared. Three different levels of P53 protein expression were tested by varying the concentration of galactose in the medium.
The two P53 orthologs show markedly different transactivation specificity. This is exemplified by the results with ced13 and V1 response elements that share the same sequence, except for the presence or absence of a 28-nucleotide spacer. Human P53 exhibits much higher transactivation with the V1 binding site, while C.elegans P53 shows the opposite.
The results are independent from the level of P53 expression under the galactose-inducible promoter. Measure yeast growth by counting the number of colonies obtained in the 100 microliters culture drops. 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 represent zero level of reactivation.
In this example, Nutlin-3a relieves the inhibition of wild-type P53 caused by the coexpression of MDM2, while PhiKan083 partially reactivates the Y220C P53 mutant. In both cases, this results in a P53-dependent reduction of yeast growth. Yeast basal cells have proven useful to investigate values, aspects of P53 protein functions.
The protocol described in this work are particularly sensitive for evaluating transactivation potential of wild-type or cancer-associated mutant P53 to variants of target sites. Further, the approach can be used to identify small molecules affecting P53 functions and to study P53 interaction with cofactor. The use of the color reporters, the miniaturization of the luciferase, as well the growth inhibition test result in cost-effective and scalable assays potentially amenable to chemical library screening.
Finally, the system described in this work can be easily adopted with the study of other sequence-specific transcription factors.
