Evaluating the Influence of a G-Quadruplex Prone Sequence on the Transactivation Potential by Wild-Type and/or Mutant P53 Family Proteins through a Yeast-Based Functional Assay

Abstract

P53, P63, and P73 proteins belong to the P53 family of transcription factors, sharing a common gene organization that, from the P1 and P2 promoters, produces two groups of mRNAs encoding proteins with different N-terminal regions; moreover, alternative splicing events at C-terminus further contribute to the generation of multiple isoforms. P53 family proteins can influence a plethora of cellular pathways mainly through the direct binding to specific DNA sequences known as response elements (REs), and the transactivation of the corresponding target genes. However, the transcriptional activation by P53 family members can be regulated at multiple levels, including the DNA topology at responsive promoters. Here, by using a yeast-based functional assay, we evaluated the influence that a G-quadruplex (G4) prone sequence adjacent to the p53 RE derived from the apoptotic PUMA target gene can exert on the transactivation potential of full-length and N-terminal truncated P53 family α isoforms (wild-type and mutant). Our results show that the presence of a G4 prone sequence upstream or downstream of the P53 RE leads to significant changes in the relative activity of P53 family proteins, emphasizing the potential role of structural DNA features as modifiers of P53 family functions at target promoter sites.

Keywords: G-quadruplex (G4) prone sequence; P53 family; transactivation potential; wild-type and mutant P53/P63 proteins; yeast.

Figure 1

Fold change transactivation by wild-type P53 family proteins. Yeast cells expressing different P53 family isoforms by an inducible GAL1,10 promoter were grown for 8 h in Galactose 1% to evaluate their transactivation ability. (A) Evaluation of wild-type P53, P63, and P73 (ΔN variants: ΔN40P53α, ΔNP63α, and ΔNP73α; TA variants: full-length P53, TAP63α, and TAP73α) transcriptional activity as fold change over the empty vector in presence of the P53 response element (RE) from the PUMA target gene (yLFM-PUMA) or a G4 prone sequence (yLFM-KSHV). (B) Evaluation of transcriptional activity as in panel A in the presence of the G4 prone sequence upstream (yLFM-KSHV-PUMA) or downstream (yLFM-PUMA-KSHV) of the P53 RE from the PUMA target gene.

Figure 2

Relative activity of wild-type P53 family α isoforms. (A) Comparison of P53 (full-length P53) or P63 (TAP63α) TA variant activity with respect to the corresponding ΔN variant (ΔN40P53α and ΔNP63α, respectively) in yLFM-PUMA, yLFM-KSHV-PUMA, and yLFM-PUMA-KSHV strains. (B) Comparison of ΔNP63α, TAP63α, or TAP73α variant activity with respect to TAP53 (full-length P53) variant in yLFM-PUMA, yLFM-KSHV-PUMA, and yLFM-PUMA-KSHV strains. ns: not significant, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 3

Fold change transactivation by mutant P53 family proteins. Yeast cells expressing wild-type and mutant TAP53 or ΔNP63 proteins by an inducible GAL1,10 promoter were grown for 8 h in Galactose 1% to evaluate the transactivation ability. (A) Evaluation of wild-type (TAP53: full-length P53; ΔNP63: ΔNP63α) or mutant (TAP53: R175H and R282W; ΔNP63: G134V and R204W) P53 and P63 proteins transcriptional activity as fold change over the empty vector in presence of the P53 RE from the PUMA target gene (yLFM-PUMA) or a G4 prone sequence (yLFM-KSHV). (B) Evaluation of transcriptional activity as in panel (A) in presence of the G4 prone sequence upstream (yLFM-KSHV-PUMA strain) or downstream (yLFM-PUMA-KSHV) of the P53 RE from the PUMA target gene. WT, wild-type.

Figure 4

Relative activity of co-expressed wild-type and mutant TAP53 or ΔNP63 proteins compared to the corresponding single protein expression. (A) Comparison of wild-type and mutant TAP53 co-expression activity (i.e., constitutive pADH1-wild-type TAP53 + inducible pGAL1,10 TAP53 R175H or R282W) with that of wild-type TAP53 expressed alone (i.e., constitutive pADH1-wild-type TAP53) in presence of the P53 RE from the PUMA target gene (yLFM-PUMA) or the same p53 RE along with a G4 prone sequence upstream (yLFM-KSHV-PUMA) or downstream (yLFM-PUMA-KSHV). (B) Comparison of wild-type and mutant ΔNP63 co-expression activity (i.e., constitutive pADH1-wild-type ΔNP63 + inducible pGAL1,10 ΔNP63 G134V or R204W) with that of wild-type ΔNP63 expressed alone (i.e., constitutive pADH1 wild-type ΔNP63) in presence of the P53 RE from the PUMA target gene (yLFM-PUMA) or the same P53 RE along with a G4 prone sequence upstream (yLFM-KSHV-PUMA) or downstream (yLFM-PUMA-KSHV). WT, wild-type. ns: not significant, ** p < 0.01; *** p < 0.001; **** p < 0.0001.