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.
Highlights
Introduction cis-regulatory elements (CREs) are defined as regions of DNA containing a set of transcription factor binding sites recognized by sequence-specific transcription factors (TFs)
We focused on the impact of response elements (REs) sequence, p53 protein level and temperature on transactivation using a yeast-based functional assay that minimizes the impact of variables such as chromatin state, promoter landscape and the influence of cofactors
Conserved enhancer elements that can be bound by TFs are thought to be functionally essential and are usually enriched in proximity of key target genes, such as those controlling body plan development
Summary
Ten isogenic yeast strains (yLFM), auxotrophic for tryptophan and containing different human p53 response element (RE) sequences cloned upstream the reporter Firefly luciferase gene were tested [26] [27]. The recombination process occurs in vivo in yeast cells through a gap repair transformation assay [33] using pTSG plasmids digested with BamHI HF and XhoI restriction enzymes (New England Biolabs). The recombination occurs in vivo in yeast cells through a Gap repair transformation assay using pTSG plasmids single digested with BamHI HF restriction enzyme (New England Biolabs). Luciferase quantitative assay yLFM yeast strains were transformed with the appropriate pTSG plasmid or the empty vector pRS314 (as vehicle control) and selected for 48 h at 30°C on SDtA plates [30]. The tanglegram algorithm, implemented in Dendroscope 3 [42] was used to compare the phylogenetic trees
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