The presence of a G-quadruplex prone sequence upstream of a minimal promoter increases transcriptional activity in the yeast Saccharomyces cerevisiae

Abstract

Non-canonical secondary structures in DNA are increasingly being revealed as critical players in DNA metabolism, including modulating the accessibility and activity of promoters. These structures comprise the so-called G-quadruplexes (G4s) that are formed from sequences rich in guanine bases. Using a well-defined transcriptional reporter system, we sought to systematically investigate the impact of the presence of G4 structures on transcription in yeast Saccharomyces cerevisiae. To this aim, different G4 prone sequences were modeled to vary the chance of intramolecular G4 formation, analyzed in vitro by Thioflavin T binding test and circular dichroism and then placed at the yeast ADE2 locus on chromosome XV, downstream and adjacent to a P53 response element (RE) and upstream from a minimal CYC1 promoter and Luciferase 1 (LUC1) reporter gene in isogenic strains. While the minimal CYC1 promoter provides basal reporter activity, the P53 RE enables LUC1 transactivation under the control of P53 family proteins expressed under the inducible GAL1 promoter. Thus, the impact of the different G4 prone sequences on both basal and P53 family protein-dependent expression was measured after shifting cells onto galactose containing medium. The results showed that the presence of G4 prone sequences upstream of a yeast minimal promoter increased its basal activity proportionally to their potential to form intramolecular G4 structures; consequently, this feature, when present near the target binding site of P53 family transcription factors, can be exploited to regulate the transcriptional activity of P53, P63 and P73 proteins.

Keywords: G-quadruplex; p53; transcriptional activity; yeast.

Figure 1. Evaluation of G4 formation potential in vitro by ThT assay

Oligonucleotides were hybridized in 100 mM HCl (A) or in the same buffer with the addition of 100 mM KCl (B). The I_(KCl)/I₍₀₎ fold values are reported in Supplementary Table S1.

Figure 2. Evaluation of G4s formation potential in vitro by CD spectra analysis

CD spectra of oligonucleotides of (A) PUMA, (B) KSHV, (C) KSHV-Mut2.0, (D) KSHV-Mut1.5, (E) KSHV-1NO, (F) KSHV-2NO and (G) KSHV-3NO in medium without stabilizing potassium ions (dashed line) and in medium supplemented with 100 mM KCl (solid line). Wavelengths (210, 240, 260 and 295 nm) characteristic for the presence of G4 conformations in CD spectra are highlighted by gray dashed lines.

Figure 3. Effect of G4 prone sequences on basal reporter activity in the yeast S. cerevisiae

(A) RLU measurements for the indicated panel of yLFM reporter strains from empty plasmid (pRS314) yeast transformants at 0.016% galactose for 6 h. (B) RLU measurements as above at 1% galactose. Data are presented as mean ± standard deviation, (SD) of at least three biological replicates, and individual values are also plotted. The symbols * and **** indicate significant differences with P=0.0461 and P<0.0001, respectively between PUMA strain and those containing G4 regulatory elements. ns, not significant. Ordinary one-way ANOVA test.

Figure 4. Effect of G4 prone sequences on P53 family-dependent reporter activity in the yeast S. cerevisiae

(A–C): RLU measurements for the indicated panel of yLFM reporter strains expressing P53, P63 or P73 at 0.016% galactose for 6 h. (D–F) RLU measurements as above at 1% galactose for 6 h. Data are presented as mean ± standard deviation (SD) of at least three biological replicates, and individual values are also plotted. The symbols *, ** and **** indicate significant differences with P≤0.0406, P=0.0017 and P<0.0001, respectively between PUMA strain and those containing G4 regulatory elements. ns, not significant. Ordinary one-way ANOVA test.

Figure 5. Effect of G4 forming sequences on P53/P63 and P53/P73 relative activity plotted as a heat map

The upper heatmap presents the relative activity of P63 and P73 compared with P53 (shades of blue with the indicated color scale; average of three to six replicates) in yLFM reporter strains; the lower heatmap presents the results of multiple unpaired t-tests, assuming individual variance for each row and a two-stage step-up method to compare the relative activities measured in the control PUMA strain with strains containing the other G4 regulatory elements (shades of red, P-value <0.05; white = not significant.). Data with corresponding statistical analysis were obtained after 6 h of growth in media containing 0.016% or 1% galactose. Data are also presented as bar graphs in Supplementary Figure S4.

Figure 6. Scheme of G4 influence on yeast transcription

(A) Basal transcription (green arrows) correlates with the presence of G-quadruplex forming sequences and an increase in G4 hunter score (G4HS, gray) leads to higher activity of the luciferase-associated yeast minimal promoter. (B) P53 family proteins (P53:red arrows; P63/P73:blue arrows) are not equally influenced by the presence of the G-quadruplex forming sequence. The length and number of arrows correspond to the level of transcriptional activity.