Supporting Information S1 - p53 Transactivation and the Impact of Mutations, Cofactors and Small Molecules Using a Simplified Yeast-Based Screening System

1. Small-volume yeast functional assay with constitutive expression of p53 proteins. Presented is the comparison of the relative transactivation capacity of wild type (WT) and the R282Q p53 towards four different response elements (REs) obtained with the traditional assay based on 2 ml liquid cultures in individual tubes (A, traditional assay) and with the permeabilized assay format based on 100 µl cultures prepared directly in 96-well plates (C, miniaturized assay). p53 proteins were expressed under the moderate, constitutive ADH1 promoter. Cells collected from the two different culture protocols were used for the measurement of luciferase activity as described in the Materials and Methods section. Presented are the average fold-induction of luciferase by p53 proteins relative to the activity obtained with an empty vector; included is the standard deviations of three replicates. In these experiments the light units per OD for WT p53 and the p21-5′ RE were 2.8×106 for the 2 ml cultures and 2.5×107 for the 100 µl cultures. 2. Impact of genetic modifications at the ABC transporter system on cell sensitivity to cycloheximide. Based on the experiments described by Stepanov et. al. [39] we used cycloheximide treatment to evaluate whether the disruption of PDR1 and replacement with the PDR1-repressor construct, the disruption of PDR5, or the combined modifications would result in enhanced toxicity in our reporter strain background. Cells from the indicated strains were resuspended in sterile water and transferred to a 96-well plate. Serial dilutions (1:5) were prepared and cells were transferred to plates containing synthetic medium (SD) with different concentrations of cycloheximide using a 48-pin replicator. A rich (YPDA) and an SD control plates were also spotted for comparison. Plates were incubated for two days at 30°C. 3. Phenotypic analysis of the impact of MDM2 on WT and mutant p53 transactivation. The ADE2-based red/white assay was used to examine p53 dependent transactivation and the impact of MDM2. p53 was expressed at low levels under the GAL1 promoter in media containing only raffinose (2%), or raffinose plus 0.002%, 0.004% or 0.016%, galactose. MDM2 was expressed from the constitutive PGK1 promoter. p53 transactivation was examined from three REs upstream of ADE2-based p53 reporter strains as indicated. The optimized consensus (CON) and P21-5′ p53 RE yield levels of high transactivation while the NOXA RE is weaker [38]. In the ADE2-based p53 functional assays, cells grown on plates containing a low-amount of adenine (5 mg/L) result in small red colonies when p53 is not present or not transcriptionally active. p53-dependent expression of ADE2 results in the appearance of colonies with a color ranging from light red to white, depending on the level of transactivation. To reveal the dependency of the phenotype on p53 expression levels, streaks are prepared on glucose plates containing high amount of adenine (200 mg/L) and the plates are incubated for two days at 30°C, resulting in the appearance of white colonies. These plates are then replica-plated to a stack of plates containing 2% raffinose plus various levels galactose along with the low-level of adenine. The replica plates are then incubated at 30°C for 2-3 days. Images of a section of the replicas are presented. For each image the upper section corresponds to colonies expressing p53 alone, while in the lower section the colonies also express MDM2. Various multiple mutants were tested, as indicated. 4. Relative transactivation capacity of p53 phosphorylation-site mutants. The activity of the p53 mutants described in Figure 4 is presented as relative light units in two p53 reporter strains using the Killer/DR5 or the p21-5′ REs upstream of a luciferase reporter. Results were obtained with the traditional assay format and are normalized to amount of soluble proteins. 4D refers to a quadruple mutant with the S15D, T18E, S20D, S33D changes in p53. 6A indicates a multiple mutant with alanine changes at S15, T18, S20, S33, S37, S46. 6D indicates a multiple mutant with aspartic acid changes at S15, S20, S33, S37, S46 and a glutamic acid change at T18. 5. Impact of small molecules Nutlin and RITA on the growth of WT yeast reporter strains or the isogenic derivatives with modified chemical uptake. Overnight cultures grown in synthetic glucose medium were washed and diluted to ∼0.1 OD600nm as measured by a plate reader. (A) The WT strain was treated with 40 µM or 80 µM Nutlin (indicated as nutlin 1 and nutlin 2 respectively) and 1 µM or 2 µM RITA (indicated as rita1 and rita2). (B) The indicated mutant ABC-transporter strains were treated with1 µM RITA (or DMSO solvent control). OD was measured at the following times: 2, 4, 8, 12 and 24 hrs. Error bars correspond to the standard deviations of three biological replicates. 6. Negative impact of RITA on the Firefly luciferase but not the Renilla luciferase. To confirm that the negative impact of RITA on the Firefly reporter was not dependent on modulation of p53 transactivation, an isogenic derivative strain was developed containing the PUMA p53 RE upstream of the Renilla luciferase. Wild-type p53-dependent transactivation was examined in cultures treated with DMSO control solvent or with 1 µM RITA. Presented are relative light units normalized to OD600nm of the cultures. The error bars correspond to standard deviations for three biological repeats. 7. Apparent lack of PRIMA-1 effects on yeast growth or p53-dependent transactivation. (A) The small molecule PRIMA-1 does not affect yeast growth. Overnight cultures grown in synthetic glucose medium were washed, diluted to ∼0.1 OD600nm, as measured by a plate reader, and treated with 200 µM PRIMA-1. Growth curves were compared for the wild type strain or the indicated ABC-transporter mutants. OD600nmwas measured at the 2-, 4-, 8-, 12- and 24-hr time points. Presented are standard deviations for three biological repeats. (B) The small molecule PRIMA-1 does not impact wild type p53 transactivation capacity. Cells were grown in glucose-containing media to keep p53 expression repressed and transferred to galactose-containing media followed by the addition of PRIMA-1. Dual luciferase assays were conducted 16 hrs after the treatment. Renilla luciferase was used as normalization factor. There was no significant effect of PRIMA-1 on WT p53 transactivation. The same result was obtained with a diploid yeast strain, in which both the p53-dependent reporter (Firefly) and the control luciferase (Renilla) were placed at the ADE2 chromosomal locus (i.e., heteroalleles), thus removing potential chromatin effects on reporter expression. The diploid strain was obtained starting from two isogenic isolates of our yLFM strain background that differ for the mating type locus. Presented is the fold-induction of the Firefly reporter over the Renilla reporter relative to strains that do not express p53, as they contain an empty expression vector. (C) The small molecule PRIMA-1 does not affect mutant p53 transactivation capacity. Different p53 alleles were expressed at moderate levels using medium containing 0.128% galactose. PRIMA-1 (200 µM) was added to the cultures at the time of the switch to galactose-containing medium. Presented are the average fold-induction by p53 proteins compared to empty vector and normalized using the Renilla control luciferase. Presented are standard deviations for three biological repeats.
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