An Isogenic Yeast Model Reveals the Influence of Native Promoter G‑Quadruplex Forming Sequences on P53 and NF-κB Transactivation.

G-quadruplexes (G4s) are noncanonical, four-stranded nucleic acid structures formed in guanine-rich regions that influence many biological processes, including transcription. We previously developed a yeast-based system to study the transcriptional modulation of p53 family proteins by G4-forming sequences (G4FS) using artificial constructs. Here, motivated by the observation of genome-wide enrichment of G4FS in proximity to p53 and NF-κB response elements (REs), we examined how native combinations of G4FS and REs derived from human promoters influence the activity of p53 or NF-κB family proteins. To this end, five pairs of isogenic reporter strains were developed, and the propensity of G4FS to adopt G4 structures was confirmed by in vitro assays. Results in yeast showed that the presence of G4FS enhanced the transcriptional output of partial-function p53 mutants, p63, and p73 relative to wild-type p53, suggesting a general boost in the activity of weaker transcription factor proteins. Conversely, the effect of G4FS on transactivation by NF-κB proteins was context-dependent and mainly inhibitory. In conclusion, our findings highlight the importance of DNA topology in transcriptional regulation by both p53 and NF-κB proteins and demonstrate that the yeast-based system serves as a valuable tool for isolating the contribution of G4FS within a controlled genomic context.