A molecular mechanism for the "digital" response of p53 to stress

Abstract

The tumor suppressor protein p53 accumulates in response to cellular stress and consequently orchestrates the expression of multiple genes in a p53-level and time-dependent manner to overcome stress consequences, for which a molecular mechanism is currently unknown. Previously, we reported that DNA torsional flexibility distinguishes among p53 response elements (REs) and that transactivation at basal p53 levels is correlated with p53 REs flexibility. Here, we calculated the flexibility of ~200 p53 REs. By connecting functional outcomes of p53-target genes' activation to the calculated flexibility of their REs, we show that genes known to belong to pathways that are activated rapidly upon stress contain REs that are significantly more flexible relative to REs of genes known to be involved in pathways that are activated later in the response to stress. The global structural properties of several p53 REs belonging to different pathways were experimentally validated. Additionally, reporter-gene expression driven by flexible p53 REs occurred at lower p53 levels and with faster rates than expression from rigid REs. Furthermore, analysis of published endogenous mRNA levels of p53-target genes as a function of REs' flexibility showed that early versus late genes differ significantly in their flexibility properties of their REs and that highly flexible p53 REs enable high-activation level exclusively to early-response genes. Overall, we demonstrate that DNA flexibility of p53 REs contributes significantly to functional selectivity in the p53 system by facilitating the initial steps of p53-dependent target-genes expression, thereby contributing to survival versus death decisions in the p53 system.

Keywords: DNA twist flexibility; gene-expression regulation; p53; protein/DNA interactions.

Fig. 1.

p53 REs belonging to genes in functional groups that are needed early in the response to stress are more deformable than REs belonging to genes needed later in the response to stress. Shown are functional groups with at least five genes each. All data points are shown as dots. The line across all groups marks the mean of means of the deformability of all analyzed groups. Center lines show the medians of each group. The figure was created by BoxPlotR (49). Box limits indicate the 25th and 75th percentiles, as determined by R software. Whiskers extend to maximum and minimum values. Boxes are arranged by decreasing group average values from left to right. Blue boxes denote the “flexible REs category,” and red boxes signify the “rigid REs category.” For significant differences between the groups, see SI Appendix, Table S2. DDR = DNA-damage response.

Fig. 2.

Sequence logos and information content of the studied REs. (A) All 210 p53 REs without spacers belonging to genes with the known functional outcome of p53 activation studied here. (B) REs belonging to the flexible REs category. (C) REs belonging to genes with intermediate flexible REs. (D) REs belonging to the rigid REs category. All included REs are those belonging to functional groups with at least five genes each, and the RE sequences were aligned by transcription direction before generating the sequence logos. Sequence logos were generated by Weblogo (57). (E) Information content per position (Iseq_l) on the mononucleotide level for the REs belonging to the flexible category (blue squares), rigid category (red circles), or REs with intermediate flexibility (green triangles).

Fig. 3.

Luciferase activity from p53 REs as a function of the p53 expression level and of time. (A and B), Transactivation in cells transfected with the indicated p53-plasmid amounts measured 48 h post-transfection. (C and D) Fold increase in transactivation levels as a function of time, using 25 ng p53 plasmid (C) or 2 ng p53 plasmid (D). Kinetics of p53 accumulation (0 to 24 h) in cells transfected with 25 ng p53 plasmid are shown in panel C Inset. Luminescence values were normalized to the transfection efficiency of co-transfected, constitutively expressed Gaussia luciferase (Gluc). Results were further normalized first to the empty Cypridina secreted luciferase (CLuc) vector and then to results obtained without p53. Error bars represent the mean ± SD of three to eight independent experiments, each containing two technical replicas. The order of the REs in the legend within (A) is according to the measured torsional flexibility, decreasing from top to bottom.

Fig. 4.

Comparison of ectopic p53 levels to its induced levels under stress. (A), p53 null H1299 cells were transfected with the indicated amounts of pC53-SN3 vector. p53 was immunodetected in whole cell lysates collected 48 h post transfection. (B and C) p53 levels in MCF-7 cells treated with 2 µM or 10 µM CPT for the indicated time points. Actin serves as a loading control. Representative gels are shown, UD = untreated. (D) Quantitative analysis for ectopic p53 protein levels shown in (A). (E) Quantitative analysis for p53 levels induced by CPT stress in MCF-7 cells shown in (B), white, and (C), gray. Intensity of bands was quantitated using Cliqs software. Error bars are the result of two to four independent experiments.

Fig. 5.

Endogenous mRNA levels from p53-dependent genes are grouped by their RE deformability. The data shown are based on the RNA-seq results from Hafner et al. (75). Fragments Per Kilobase Million (FPKM) values of each gene (i), at each time point (j) were normalized to their FPKM value at T = 0, arriving at fold-change values. Then, these values were further normalized to the average fold-change of all genes at each particular time point (j). That is, each point is calculated from the equation: (FPKM_iT_j/FPKM_iT₀)/$\frac{1}{n}\sum _{i=1}^n$ (FPKM_iT_j/FPKM_iT₀). Deformability of genes’ REs was normalized in the same manner. Plotted are the normalized fold-change values as a function of normalized deformability values at even numbers post irradiation. The lines dissecting each axis at 1 unit is the normalized average of each parameter, for visualization of values that are above/below the average value of these parameters.