Mutant p53 is a transcriptional co-factor that binds to G-rich regulatory regions of active genes and generates transcriptional plasticity

Abstract

The molecular mechanisms underlying mutant p53 (mutp53) "gain-of-function" (GOF) are still insufficiently understood, but there is evidence that mutp53 is a transcriptional regulator that is recruited by specialized transcription factors. Here we analyzed the binding sites of mutp53 and the epigenetic status of mutp53-regulated genes that had been identified by global expression profiling upon depletion of endogenous mutp53 (R273H) expression in U251 glioblastoma cells. We found that mutp53 preferentially and autonomously binds to G/C-rich DNA around transcription start sites (TSS) of many genes characterized by active chromatin marks (H3K4me3) and frequently associated with transcription-competent RNA polymerase II. Mutp53-bound regions overlap predominantly with CpG islands and are enriched in G4-motifs that are prone to form G-quadruplex structures. In line, mutp53 binds and stabilizes a well-characterized G-quadruplex structure in vitro. Hence, we assume that binding of mutp53 to G/C-rich DNA regions associated with a large set of cancer-relevant genes is an initial step in their regulation by mutp53. Using GAS1 and HTR2A as model genes, we show that mutp53 affects several parameters of active transcription. Finally, we discuss a dual mode model of mutp53 GOF, which includes both stochastic and deterministic components.

Figure 1. Array-wide analysis of mutp53 binding sites. (A) Consensus signals for the whole genome and for chromosome 5 are shown. The EGR1 gene is shown as an example of a known mutp53 target gene. The lower panel displays the detected peaks in the EGR1 gene dependent on the selected p value threshold during peak calling. CpG-islands, DNaseI-HS regions and G4 motif locations extracted from the public databases are plotted. (B) Distribution of mutp53-BR around TSS. The median length of mutp53-BR varied from 1270 bp to 990 bp. (C) Motifs overrepresented in mutp53 binding regions (p value 0.001) identified by MEME-ChIP analysis.

Figure 2. Analysis of the interaction of mutant p53 proteins and of wtp53 with the MYC-PU52 quadruplex structure by EMSA and CD spectroscopy. (A-C) Comparison of the binding affinity of mutant p53 and of wtp53 for the MYC-Pu52 intramolecular quadruplex structure (A), double-stranded (ds) oligonucleotide (Py52/Pu52) (B) and p53CON ds sequence (C). Twenty-five, 50 and 100 ng of wtp53, G245S, R248W and R273H mutant p53 were incubated under binding conditions (see M&M) with ³²P-radiolabeled DNA oligonucleotides and 30 ng competitor DNA. (D) CD spectra of the MYC-Pu52 oligonucleotide (0.5 µM) in 5 mM TRIS-HCl, supplemented with 24 mM KCl, supplemented with mutp53R273H (3 µM) and 24 mM KCl. CD spectra were recorded after 24 h when equilibrium was attained. The dotted line corresponds to the CD spectrum of mutp53 alone (0.5 µM). Strong absorption of protein shifts measurement to shorter wavelength.

Figure 3. Array-wide analysis of mutp53, SP1 and ETS1 binding sites. (A) Distribution of the scores calculated for the overlap of mutp53, ETS1 and SP1 binding regions with CpG-islands and DNaseI-HS regions. The distribution was used to identify a significance threshold, where peaks with a score > = 20 are assumed to overlap significantly. (B) Venn-diagram displaying the number of overlapping mutp53, ETS1 and SP1 binding regions.

Figure 4. Expression analysis in U251 cells after mutp53 depletion. (A) U251 cells were transfected with p53- and scr-siRNA in three biological replicates and mutp53 protein was detected 96 h post-transfection by WB. HSC70 was used as a loading control. (B) Total RNA from three replicate transfections with p53- and scr-siRNA was subjected to microarray gene expression analysis. The Venn diagram shows the number of probes regulated in three experiments. (C) Transcript levels of GAS1 and HTR2A genes after mutp53-depletion were measured relative to the scr-control (scr1) in the same samples that had been subjected to microarray analysis. (D) Transcript levels of GAS1 and HTR2A genes were measured in a time-course experiment after mutp53-depletion until re-expression of mutp53 occurred (for WB, see Fig. S6B). (E and F) Transcript levels of GAS1 and HTR2A genes were measured 96 h after mutp53 depletion in two additional experiments including mock transfections and a different scr-siRNA (scr2; E) as well as two different p53-siRNAs (s606, s607) (F). WB analysis of mutp53 expression in transfected cells is shown below the graphs. Tubulin was used as a loading control.

Figure 5. Stability of GAS1 and HTR2A transcripts after mutp53-depletion. (A) Mutp53 protein levels at the start and at the end of ActD treatment. U251 cells were transfected with p53- and scr-siRNA. At the time of maximal mutp53 depletion (96 h), transcription was stopped by addition of ActD (1 µg/ml final concentration) and RNA was isolated every 30 min for 6 h. Tubulin was used as a loading control. (B) GAS1 and HTR2A transcript levels in scr- and sip53-transfected U251 cells were measured by qPCR for every time point after ActD treatment, normalized to GAPDH and calculated as %-mRNA remaining. The mRNA half-life was calculated using an exponential regression curve derived from the data points.

Figure 6. Histograms displaying the frequency of H3K4me3, H3K9ac, Pol II-S2P and Pol II-S5P peaks in the vicinity of mutp53 binding regions (mutp53-BR). The distance is displayed relative to the mutp53-BR center positions. The profiles were determined for control siRNA (blue) and p53 siRNA (red) treated samples and superimposed (combined color: black).

Figure 7. (A, B) ChIP-chip profiles of H3K4me3, H3K9ac, Pol II S2-P and Pol II S5-P occupancy in the GAS1 (A) and the HTR2A (B) genes in scr- and sip53-transfected U251 cells. Mean log2-values of the signal ratio “ChIP-sample/input” for every probe on the tiling array from two biological replicates are plotted against the probe position. The transcription start site (TSS) is marked by an arrow and exons are highlighted. (C, D) A qPCR-based analysis of a representative experiment for the promoter regions of GAS1 (C) and HTR2A (D). Data for scr- and p53-siRNA-transfected cells are presented as percent recovery of input DNA by the specific antibodies. A control without addition of antibodies is shown. The results of the three replicate experiments are summarized as fold change of mutp53-depleted cells vs. control cells.