P53 binds preferentially to non-B DNA structures formed by the pyrimidine-rich strands of GaA·TTC trinucleotide repeats associated with Friedreich’s ataxia

Expansions of trinucleotide repeats (TNRs) are associated with genetic disorders such as Friedreich’s ataxia. The tumor suppressor p53 is a central regulator of cell fate in response to different types of insults. Sequence and structure-selective modes of DNA recognition are among the main attributes of p53 protein. The focus of this work was analysis of the p53 structure-selective recognition of TNRs associated with human neurodegenerative diseases. Here, we studied binding of full length p53 and several deletion variants to TNRs folded into DNA hairpins or loops. We demonstrate that p53 binds to all studied non-B DNA structures, with a preference for non-B DNA structures formed by pyrimidine (Py) rich strands. Using deletion mutants, we determined the C-terminal DNA binding domain of p53 to be crucial for recognition of such non-B DNA structures. We also observed that p53 in vitro prefers binding to the Py-rich strand over the purine (Pu) rich strand in non-B DNA substrates formed by sequence derived from the first intron of the frataxin gene. The binding of p53 to this region was confirmed using chromatin immunoprecipitation in human Friedreich’s ataxia fibroblast and adenocarcinoma cells. Altogether these observations provide further evidence that p53 binds to TNRs’ non-B DNA structures

p53 Specifically Binds Triplex DNA In Vitro and in Cells

Triplex DNA is implicated in a wide range of biological activities, including regulation of gene expression and genomic instability leading to cancer. The tumor suppressor p53 is a central regulator of cell fate in response to different type of insults. Sequence and structure specific modes of DNA recognition are core attributes of the p53 protein. The focus of this work is the structure-specific binding of p53 to DNA containing triplex-forming sequences in vitro and in cells and the effect on p53-driven transcription. This is the first DNA binding study of full-length p53 and its deletion variants to both intermolecular and intramolecular T.A.T triplexes. We demonstrate that the interaction of p53 with intermolecular T.A.T triplex is comparable to the recognition of CTG-hairpin non-B DNA structure. Using deletion mutants we determined the C-terminal DNA binding domain of p53 to be crucial for triplex recognition. Furthermore, strong p53 recognition of intramolecular T.A.T triplexes (H-DNA), stabilized by negative superhelicity in plasmid DNA, was detected by competition and immunoprecipitation experiments, and visualized by AFM. Moreover, chromatin immunoprecipitation revealed p53 binding T.A.T forming sequence in vivo. Enhanced reporter transactivation by p53 on insertion of triplex forming sequence into plasmid with p53 consensus sequence was observed by luciferase reporter assays. In-silico scan of human regulatory regions for the simultaneous presence of both consensus sequence and T.A.T motifs identified a set of candidate p53 target genes and p53-dependent activation of several of them (ABCG5, ENOX1, INSR, MCC, NFAT5) was confirmed by RT-qPCR. Our results show that T.A.T triplex comprises a new class of p53 binding sites targeted by p53 in a DNA structure-dependent mode in vitro and in cells. The contribution of p53 DNA structure-dependent binding to the regulation of transcription is discussed

Influence of DNA topology on mutp53 MSP/MST1 recognition in H1299 cells.

<p>A) Preferential binding of G245S to scMSP in sc/lin competition assay in H1299 cells by ChIP. pGL3-MSP (sc, 2 µg), pMSP (lin, 2 µg) and pCDNA3.1-G245S (2 µg) were co-transfected into H1299 cells. ChIP assay was performed after 20 h. DNAs (sc or lin) bound by mutp53 were detected by two (sc and lin) specific PCRs on 1.5% agarose gels. Left side shows detection of scDNA by PCR with GL2 and RV3 primers: control DNA for transfection (lane 1, scpGL3-MSP); 1/20 of ChIP input DNA (lane 5); as ChIP were marked all immunoprecipitation samples: DO1-Ab (lane 2, bound scDNA), whole mouse IgG-Ab (lane 3, negative control), IP without Ab (lane 4, negative control); negative control of PCR (lane 6). Right side shows detection of linDNA by PCR with BT3 and MSP primers: control DNA for transfection (lane 7, pMSP/SmaI); 1/20 of ChIP input DNA (lane 11); as ChIP were marked all immunoprecipitation samples: DO1-Ab (lane 8, bound linDNA), whole mouse IgG-Ab (lane 9), IP without Ab (lane 10). Results of PCR analysis of immunoprecipited DNA were detected on a 1.5% agarose gel. Samples for PCR on the gel are: plasmids (lane 1 (scpGL3MSP), lane 7 (linMSP)); 1/20 of input DNA (lanes 5, 11 marked as in); IP without Ab (lanes 4, 10); IP from DO1-Ab (lanes 2, 8); IP from whole mouse IgG-Ab (lanes 3, 9). (B) Preferential binding of R273H to scMSP in sc/rel competition assay in H1299 cells by ChIP. pMSP (sc, 2 µg), pGL3-MSP (rel, 2 µg) and pCDNA3.1-R273H (2 µg) were co-transfected into H1299 cells. Other condition was the same as in A). DNAs (sc or rel) bound by mutp53 were detected by two (sc and rel) specific PCRs. Left side shows detection of scDNA by PCR with BT7 and MSP primers: 1/20 of ChIP input DNA (lane 2); as ChIP were marked all immunoprecipitation samples: DO1-Ab (lane 3, bound scDNA), whole mouse IgG-Ab (lane 4), IP without Ab (lane 5); negative control of PCR (lane 1). Right side shows detection of relDNA by PCR with GL2 and RV3 primers: 1/20 of ChIP input DNA (lane 6); as ChIP were marked all immunoprecipitation samples: DO1-Ab (lane 7, bound relDNA), whole mouse IgG-Ab (lane 8), IP without Ab (lane 9).</p

Preferential Binding of Hot Spot Mutant p53 Proteins to Supercoiled DNA <i>In Vitro</i> and in Cells

<div><p>Hot spot mutant p53 (mutp53) proteins exert oncogenic gain-of-function activities. Binding of mutp53 to DNA is assumed to be involved in mutp53-mediated repression or activation of several mutp53 target genes. To investigate the importance of DNA topology on mutp53-DNA recognition <i>in vitro</i> and in cells, we analyzed the interaction of seven hot spot mutp53 proteins with topologically different DNA substrates (supercoiled, linear and relaxed) containing and/or lacking mutp53 binding sites (mutp53BS) using a variety of electrophoresis and immunoprecipitation based techniques. All seven hot spot mutp53 proteins (R175H, G245S, R248W, R249S, R273C, R273H and R282W) were found to have retained the ability of wild-type p53 to preferentially bind circular DNA at native negative superhelix density, while linear or relaxed circular DNA was a poor substrate. The preference of mutp53 proteins for supercoiled DNA (supercoil-selective binding) was further substantiated by competition experiments with linear DNA or relaxed DNA <i>in vitro</i> and <i>ex vivo</i>. Using chromatin immunoprecipitation, the preferential binding of mutp53 to a sc mutp53BS was detected also in cells. Furthermore, we have shown by luciferase reporter assay that the DNA topology influences p53 regulation of BAX and MSP/MST1 promoters. Possible modes of mutp53 binding to topologically constrained DNA substrates and their biological consequences are discussed.</p> </div

SCS-binding of p53 proteins by magnetic beads-based immunoprecipitation assay (MBIP) <i>in vitro and ex vivo</i>.

<p>A) Scheme of the procedure (from left to right): the cell lysate or purified proteins were mixed with antibody (Ab) and then with a DNA substrate and incubated to allow formation of the Ab-p53-DNA complex (a). Then, the complex is captured at DBG (magnetic beads-coated with protein G) (b). The bead suspension is washed (c), followed by dissociation of DNA from the complex with p53 (d) and DNA eluted from beads is detected by agarose electrophoresis (e, Et-Br staining). B) Control of specificity of MBIP assay for sc and lin DNAs. Binding of 100 ng of purified GST-p53CD protein to scBSK (lane 6), linBSK (lane 7), linPGM1 (lane 9) and mixtures of scBSK/linBSK (lane 8) and scBSK/linPGM1 (lane 10) by MBIP assay with anti-GST Ab are shown on the left. Lanes 1–5 (scBSK, linBSK, linPGM1 and sc+lin) were a control for input DNA (50 ng, 1/6 input DNA). After anti-GST immunoprecipitation, bound DNAs (lanes 6–15) were analyzed on 1% TAE gel (scDNA migrates faster than lin or oc forms). Binding of GST-Sp1 (construct without DNA binding domain) to different types of DNA (lanes 11–15) by MBIP assay with the same Ab was not observed (right panel). C) Binding of seven purified hot spots and wtp53 to scBSK, linBSK and a scBSK/linBSK mixture by MBIP assay with DO1. Lanes 1–3 and 16–18 are a control for input DNAs (50 ng). D) Control of p53 and actin levels in cell lysates from H1299 and H1299-R273H (induced by tetracyclin for 24 h) by WB with DO1 and anti-Actin antibody. E) Binding of R273H from H1299-R273H lysate to scMSP (lane 4), linMSP (lane 5) and a scMSP/linMSP mixture (lane 6) by MBIP assays. Lanes 1–3 are a control for input DNA (50 ng). Lysate from H1299 cells was used for control reaction for DO1 immunoprecipitation of sc, lin and scMSP/linMSP mixture (lanes 7–9). MBIP assay was performed also with recombinant purified protein R273H (100 ng) with scMSP (lane 10), linMSP (lane 11) or scMSP/linMSP mixture (lane 12). After DO1 immunoprecipitation, DNAs were analyzed on 1% 1x TAE gel (scDNA migrates faster than linDNA, ocDNA or d-sc (dimer of scDNA)).</p

Only full length mutp53 proteins exhibit strong SCS-binding in sc/lin competition assay for scBSK <i>in vitro</i>.

<p>A) All mutp53fl proteins selectively recognized scBSK by EMSA. P53 proteins (protein amount expressed as p53/total DNA ratio) were incubated with scBSK (200 ng, lanes 1, 3–25) and linBSK (200 ng, lanes 2–25) and separated on a 1.3% 0.33x TBE agarose gel at 4°C (linDNA migrated faster than scDNA). P53-DNA binding was detected by Et-Br staining of DNA. The conformation of p53 proteins is labeled: white (wtp53), grey (conformation mutants) and black (contact mutants). B) Binding of CΔ30-wtp53 (lanes 4–7), CΔ30-G245S (lanes 8–11), CΔ30-R248W (lanes 12–15) and CΔ30-R175H (lanes 16–19) to pBSK in sc/lin competition asssay was performed similarly to p53fl (A). CΔ30-R248W and CΔ30-R175H lose the ability for strong SCS-binding. Column graph below was plotted on the basis of Et-Br stained DNA on agarose gels (from three independent experiments), free DNA substrates labeled with arrows were measured by densitometry and % of bound DNAs (sc and lin) were calculated the same way as in Fig. 2. C) Preferential binding of CΔ30-wtp53 (lanes 2–4) to linPGM1 in sc/lin competition assay with scBSK, complex of p53-linDNA is bonded.</p

Hot spot mutp53 proteins and R248W preferential binding to scDNA.

<p>A) Distribution of hot spot mutations on full length p53 (conserved regions marked by cylinders) with a bar code representing their frequency; scheme of p53 molecules (p53 (aa 1–393), p53CΔ30 (aa 1–363)), DNA binding domains (shaded, central (CD) and C-terminal (CTDBS)) and positions of mAb epitope of DO1 (aa 21–25) are marked. The conformation of mutp53 proteins is labeled: grey (conformation mutants) and black (contact mutants). B) ScDNA binding of p53 R248W by agarose electrophoresis and DO1 immunoblotting. Increasing amounts of p53 protein (marked by the p53/DNA molar ratio) (lanes 2–5) were incubated with scDNA (pBSK, 200 ng) for 20 min and then separated in a 1% agarose gel at 4°C. DO1 immunodetection of R248W binding to scDNA showed that each retarded band of Et-Br visualized DNA on the agarose gel (lanes 2–5) corresponds to a p53 band on the DO1 immunoblot (lanes 7–10). Open circle DNA (oc) was not bound by p53.</p

Comparison of mutp53 binding to scDNA and linDNA by EMSA.

<p>Mutp53 proteins R175H, G245S, R248W, R249S, R273H, R273C, R282W and wtp53 were bound to scDNA (pBSK, 200 ng, A), linDNA (linBSK, 200 ng, B) and to linDNA with p53CON sequence (linPGM1, 200 ng, C) in p53/DNA molar ratios 1, 2, 3 and 5 (for R175H only) at 25°C; EMSA was performed at 4°C. Column graphs below (pictures A and C) were plotted on the basis of Et-Br stained agarose gels (A and C from three independent experiments); free DNA substrates labeled with arrows were measured by densitometry. Graphs show the evaluation of p53-DNA binding as the dependence of % of bound DNA (axis <i>y</i>) on the amount of input of p53 proteins in the reaction (expressed by molar ratio p53/DNA, axis <i>x</i>). Bound DNA (%) were calculated as % of decrease of free DNA after binding of p53 in comparison to input DNA (lane 1, 0% of bound DNA). The conformation of p53 proteins is labeled: white (wtp53), grey (conformation mutants) and black (contact mutants).</p

Mutant p53 recognition of MSP/MST1 sites in scDNA.

<p>A) Mutp53 proteins recognize MSP/MST1 site in scDNA differently. Mutp53s and wtp53 were bound to scMSP at a molar ratio p53/DNA 2, 4 and 6. B) R273H binding to scBSK (lanes 2–5), scMSP (lanes 7–10), linBSK (lanes 12–15) and linMSP (lanes 17–20) was compared at p53/DNA molar ratios of 0.5–4. P53-DNA binding and EMSA condition for A) and B) were the same as in Fig. 1B. Both graphs show the evaluation of p53-DNA binding (from three independent experiments) as the dependence of % of bound DNA (axis <i>y</i>) on the amount of input of p53 proteins in the reaction (expressed by molar ratio p53/DNA, axis <i>x</i>). C) Mutp53 R273H binds selectively to scMSP. In competition experiment R273H (lanes 2–4) were bound to a mixture of scMSP and linMSP, experimental condition was the same as in 3A). Observed p53-scDNA complexes and p53-linDNA complexes are marked. Evaluation of binding is shown on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059567#pone.0059567.s004" target="_blank">Fig. S4C</a>. D) Influence of N- and C-terminal antibodies on SCS-binding of R273H to a DNA mixture of scMSP and linMSP. DO1, Bp5310.1, PAb421 and ICA9 (50 ng, lanes 5–8) were preincubated with R273H (100 ng) at 25°C, sc/lin competition EMSA experiment was performed at RT. Complexes of Ab-p53-linDNA were observed mainly with Bp5310.1 and PAb421 (lanes 6 and 7); control DNAs (sc, lin, sc+lin; lanes 1, 2, 3).</p

Influence of DNA topology on mutp53-driven repression of BAX and MSP/MST1 promoters and mutp53 mediated down-regulation of <i>BAX</i> and <i>MSP/MST1</i> expression.

<p>A, C) Influence of DNA topology on mutp53-driven repression of BAX (A) and MSP/MST1 (C) promoters. Saos2 or H1299 cells were transiently transfected with plasmids expressing the p53 constructs (based on pCDNA3.1) or pCDNA3.1 vector alone (CMV) together with the reporter plasmids expressing the firefly luciferase gene under the transcriptional control of the indicated gene promoters (BAX, MSP/MST1) and a reference plasmid with the renilla gene under control of the SV40 promoter. Luciferase activity was analyzed 16–20 h after transfection as described in Material and methods. Transfections were carried out in triplicates and at least three independent times and standards deviations are indicated. Representative western blot analysis was performed using 50 µg of samples from the transfection to determine the expression status of p53. A) The BAX promoter in the pGL3-basic vector was repressed by mutp53 (R248W, G245S and R273H) more efficiently in scDNA form (left side) in comparison with relDNA form (right side) in H1299 cells. C) MSP/MST1 promoter (161 bp mutp53BS in pGL3-promoter vector) was transfected to Saos2 cells in sc (left side) or relaxed (right side) forms. Mutp53 proteins (R175H and R273H) repress MSP/MST1 promoter in scDNA form more efficiently than rel form in Saos2 cells. B, D) Analysis of down-regulation of BAX and MSP/MST1 mRNA levels in cells overexpressing mutant p53s. H1299 (B) and Saos2 (D) cells were transfected by p53 constructs or empty vector in the same conditions as A) and C). Samples marked DOX were additionally exposed 0.1 µM doxorubicin for 16 h. Total RNA was isolated, and mRNA levels of BAX (B) and MSP/MST1 (D) were determined by quantitative real-time reverse transcription PCR. BAX and MSP/MST1 values were normalized by GAPDH, HPRT1 or Actin. The values are the average of three biological independent experiments.</p