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. 2015 Feb 10;10(2):e0116177.
doi: 10.1371/journal.pone.0116177. eCollection 2015.

Evolution of p53 transactivation specificity through the lens of a yeast-based functional assay

Mattia Lion  1 , Ivan Raimondi  1 , Stefano Donati  1 , Olivier Jousson  2 , Yari Ciribilli  1 , Alberto Inga  1
Affiliations

Evolution of p53 transactivation specificity through the lens of a yeast-based functional assay

Mattia Lion et al. PLoS One. .

Abstract

Co-evolution of transcription factors (TFs) with their respective cis-regulatory network enhances functional diversity in the course of evolution. We present a new approach to investigate transactivation capacity of sequence-specific TFs in evolutionary studies. Saccharomyces cerevisiae was used as an in vivo test tube and p53 proteins derived from human and five commonly used animal models were chosen as proof of concept. p53 is a highly conserved master regulator of environmental stress responses. Previous reports indicated conserved p53 DNA binding specificity in vitro, even for evolutionary distant species. We used isogenic yeast strains where p53-dependent transactivation was measured towards chromosomally integrated p53 response elements (REs). Ten REs were chosen to sample a wide range of DNA binding affinity and transactivation capacity for human p53 and proteins were expressed at two levels using an inducible expression system. We showed that the assay is amenable to study thermo-sensitivity of frog p53, and that chimeric constructs containing an ectopic transactivation domain could be rapidly developed to enhance the activity of proteins, such as fruit fly p53, that are poorly effective in engaging the yeast transcriptional machinery. Changes in the profile of relative transactivation towards the ten REs were measured for each p53 protein and compared to the profile obtained with human p53. These results, which are largely independent from relative p53 protein levels, revealed widespread evolutionary divergence of p53 transactivation specificity, even between human and mouse p53. Fruit fly and human p53 exhibited the largest discrimination among REs while zebrafish p53 was the least selective.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. p53 DNA binding domain sequence alignment of Homo sapiens, Mus musculus, Xenopus laevis, Danio rerio, Drosophila melanogaster and Caenorhabditis elegans.
Sequences were aligned using ClustalW tool [74] and the alignment was visualized using Jalview (http://www.jalview.org) [39]. The three shades of blue highlight different percentage agreement, respectively from darker to lighter in this order: >80%, >60%, >40%. A percentage agreement ≤40% is not highlighted. The alignment shows the conservation of human functional residues involved in DNA-protein interaction (solid box), zinc-binding (dashed box), DBD stability (asterisk, *), protein-protein interface (empty circle, #) and DBD thermostability (hash mark, #). Human zinc-binding and DNA-binding residue positions are also typed. On the top of the alignment, human p53 Loop, Sheet, Helix motifs (L, dashed lines; S, left-right double arrows; H, two-dimensional arrows) are also presented. Conservation and consensus graphs are shown. Conservation is visualized as a histogram with the relative score for each column. Conserved columns are indicated with an asterisk, and columns with mutations, where all properties are conserved, are marked with a plus. Consensus is displayed as the percentage of the modal residue per column. The plus symbol is used instead of displaying multiple characters in a single character space. A consensus logo is also generated and the scale of the letter is in agreement with the conservation of the residues.
Fig 2
Fig 2. Yeast-based transactivation analysis of p53 proteins towards a 10 RE panel.
Results were obtained as indicated in Material and Methods. Presented are the averages of relative light units (RLU), defined as the light unit normalized by the optical density at 600nm and after the subtraction of the empty values, i.e. the p53-independent expression of the reporter. Error bars plot the standard deviation of at least four independent replicates. The activity of p53 proteins from the indicated species was measured after 6 hrs incubation at 30°C in media containing two concentrations of galactose (0.008% and 0.064%), that regulates the expression of the p53 transgene. Results obtained with fruit fly p53 and worm p53 are plotted with a different scale due to the low induction of the reporter. The p53 REs used are indicated in the X-axis (see Table 3).
Fig 3
Fig 3. Evolutionary changes in p53 transactivation specificity.
Transactivation potentials for the ten REs tested are presented as radar plot graphs in Log10 scale, relative to the results obtained with CON1 (set to 1). The resulting images represent the transactivation specificity for the indicated p53 proteins and that of human p53 is also overlaid (gray line) in every panel to facilitate comparisons. The yeast-based transactivation results at 0.064% of galactose concentration were used. Results obtained at 0.008% galactose are presented in S1 File.
Fig 4
Fig 4. Influence of temperature on the transactivation potential of p53 proteins.
The ability of p53 proteins to transactivate from the five natural REs at different expression levels was measured culturing yeast cells at 24°C (white bars) or 37°C (black bars) in media containing the indicated amount of galactose. Data obtained at 30°C (Fig. 2) were re-plotted (gray bars) for comparison. The experiments were performed and data were analyzed and presented as for Fig. 2.
Fig 5
Fig 5. Chimeric Dm_p53 exhibited higher transactivation potential but no changes in transactivation specificity.
A) schematic view of the two types of chimeric constructs tested, as described in Material and Methods. B, C) Transactivation activity of Dm_p53, hN63-Dm_p53 and hN92-ΔNDm_p53, and Hs_p53 proteins on five human natural p53 binding sites (see Table 3). Two galactose concentrations (0.008% and 0.064%) were tested. D) Radar plot charts in Log10 scale of relative transactivation potential of chimeric Dm_p53 (black line) relative to the results with the p21 RE (set to 1). Results with non-chimeric Dm_p53 are overlaid (gray line).
Fig 6
Fig 6. Wild type Cep-1 is functional on a C. elegans natural RE.
Transactivation activity of Hs_p53 and Cep-1 proteins on the human natural p21, used as a positive control (see Table 3), and the worm natural ced-13 binding sites. Results were obtained as indicated for Fig. 2, except for a 4-hour incubation time in galactose-containing media. Two galactose concentrations (0.008% and 0.064%) were tested. Caenorhabditis elegans Cep-1 binding site of ced-13 is the following: AAACATGTTT(N)28AAACATGTTT.
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References

    1. Maeso I, Irimia M, Tena JJ, Casares F, Gomez-Skarmeta JL (2013) Deep conservation of cis-regulatory elements in metazoans. Philos Trans R Soc Lond B Biol Sci 368: 20130020 10.1098/rstb.2013.0020 - DOI - PMC - PubMed
    1. Rubinstein M, de Souza FS (2013) Evolution of transcriptional enhancers and animal diversity. Philos Trans R Soc Lond B Biol Sci 368: 20130017 10.1098/rstb.2013.0017 - DOI - PMC - PubMed
    1. Wittkopp PJ, Kalay G (2012) Cis-regulatory elements: molecular mechanisms and evolutionary processes underlying divergence. Nat Rev Genet 13: 59–69. 10.1038/nrg3095 - DOI - PubMed
    1. Wagner GP, Lynch VJ (2008) The gene regulatory logic of transcription factor evolution. Trends Ecol Evol 23: 377–385. 10.1016/j.tree.2008.03.006 - DOI - PubMed
    1. Bode AM, Dong Z (2004) Post-translational modification of p53 in tumorigenesis. Nat Rev Cancer 4: 793–805. - PubMed

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This work was partially supported by the Italian Association for Cancer Research, AIRC (IG # 12869 to AI) (www.airc.it/). IR is supported by a Pezcoller Foundation Fellowship (http://www.pezcoller.it/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.