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The structure formed by inverted repeats in p53 response elements determines the transactivation activity of p53 protein
Biochemical and Biophysical Research Communications xxx (2016) 1e6
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
The structure formed by inverted repeats in p53 response elements determines the transactivation activity of p53 protein clav Bra zda a, *, Jana Cechov a, Michele Battistin b, Jan Coufal a, Eva B. Jagelska a, Va a Ivan Raimondi b, Alberto Inga b a b
lovopolska 135, 61265, Brno, Czechia Institute of Biophysics, Academy of Sciences of the Czech Republic v.v.i., Kra Laboratory of Transcriptional Networks, Center for Integrative Biology, CIBIO, University of Trento, via Sommarive 9, 38123, Trento, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 November 2016 Accepted 17 December 2016 Available online xxx
The TP53 gene is the most frequently mutated gene in human cancer and p53 protein plays a crucial role in gene expression and cancer protection. Its role is manifested by interactions with other proteins and DNA. p53 is a transcription factor that binds to DNA response elements (REs). Due to the palindromic nature of the consensus binding site, several p53-REs have the potential to form cruciform structures. However, the influence of cruciform formation on the activity of p53-REs has not been evaluated. Therefore, we prepared sets of p53-REs with identical theoretical binding affinity in their linear state, but different probabilities to form extra helical structures, for in vitro and in vivo analyses. Then we evaluated the presence of cruciform structures when inserted into plasmid DNA and employed a yeast-based assay to measure transactivation potential of these p53-REs cloned at a chromosomal locus in isogenic strains. We show that transactivation in vivo correlated more with relative propensity of an RE to form cruciforms than to its predicted in vitro DNA binding affinity for wild type p53. Structural features of p53-REs could therefore be an important determinant of p53 transactivation function. © 2016 Elsevier Inc. All rights reserved.
Keywords: Inverted repeat p53 protein Protein-DNA interaction Cruciform structure
1. Introduction The ability of p53 to accomplish its biological functions is mainly attributed to its action as a transcription factor that binds to specific DNA sequences to regulate gene expression in processes including apoptosis, DNA repair and cell cycle control [1]. TP53 mutations result in altered or loss of p53-binding activity in approximately 50% of human tumors, and p53 activity is compromised by other mechanisms in most tumors with wild type p53 [2]. Human p53 is a nuclear phosphoprotein. The N-terminal domain contains the transactivation domains and also regulates stability [3]. The central part allows the specific recognition of target sequences and ~80% of mutations associated with cancer are located in the central DNA binding domain [3]. The C-terminal part contains a thermodynamically stable tetramerization domain and a basic regulatory domain [4]. Sequence-specific binding of p53 to DNA may require specific combinations of cofactors and post-translational modifications [5,6]. p53 recognizes and binds to a target sequence consisting of
* Corresponding author. zda). E-mail address: [email protected] (V. Bra
two decamers (RRRCWWGYYY - R ¼ A or G; W ¼ A or T; Y ¼ C or T) that can be separated by a spacer. p53 binds as a dimer of dimers and each subunit contacts three nucleotides of the quarter site formed by the RRRCW or WGYYY pentamer [7]. In vitro studies of RE sequence variations and their influence on p53 binding have provided an overview of the importance of each base within the canonical consensus sequence. These data led to a binding predictor matrix that provides the theoretical affinity of wild type p53 to any target sequence [8]. The primary sequence of DNA is not the only feature that determines recognition and interaction with proteins. Epigenetic modifications and alternative DNA structures contribute significantly to the regulation of biological processes. The threedimensional structure of DNA is often crucial for DNA-protein interactions [9,10]. Local DNA structures, like cruciforms, triplexes and quadruplexes, were originally described in vitro, but are now known to play important roles in vivo. The formation of cruciforms is closely related to DNA sequence, requiring inverted repeats of at least 6 nucleotides in length. Cruciform structures consist of a branch point, a stem and a loop whose size depends on the sequence that separates inverted repeats [11]. The presence of cruciforms was first found in plasmids with negative supercoils that stabilize their formation both in vitro and in vivo [12].
http://dx.doi.org/10.1016/j.bbrc.2016.12.113 0006-291X/© 2016 Elsevier Inc. All rights reserved.
zda, et al., The structure formed by inverted repeats in p53 response elements determines the Please cite this article in press as: V. Bra transactivation activity of p53 protein, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.12.113
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Cruciforms can influence DNA winding, the formation of other secondary structures and direct interaction with proteins. Monoclonal antibodies that recognize and stabilize cruciforms, but not antibodies recognizing B-DNA and Z-DNA, cause increased replication rates in vivo, suggesting that cruciforms contribute to activation of DNA replication origins [13]. Negative supercoiling in DNA during transcription can mediate cruciform formation in vivo and cruciform formation is related to increased transcriptional activity of the promoter [14]. Some DNA-binding proteins show only a weak sequence specificity but preferentially bind cruciform structures, while other DNA-binding proteins induce cruciform formation after DNA binding [15]. A high proportion of p53 target sequences have internal symmetry and adopt non-B-DNA conformation [16]. Notably, p53 target sites are not only direct repeats of two half-sites, but in some REs there are inverted repeats also in individual half-sites [17]. Binding of p53 to its target sequence in vitro is highly dependent on the presence of inverted repeats and there is a strong correlation between propensity to form cruciform and p53 specific binding [18e20], although whether non-B DNA structures independently influence transcriptional activity has not been demonstrated. Therefore, we analyzed transcriptional activity of several p53 target sequences presented within chromatin in isogenic yeast reporter strains. Comparing p53-REs with the same predicted p53 binding affinity but different propensities to adopt non-B DNA structures, our results suggest that cruciform structures with longer loops in the center of p53 target sites stimulate p53-dependent transactivation. 2. Materials and methods 2.1. DNA Supercoiled plasmid DNAs of pBluescriptIISK() and derived plasmids pCFNO [38], pB-XA, pB-TT, pB-XG, pB-GCG, pB-XT and pBWC were isolated from DH5a as described in the QIAGEN protocol. pB-series was constructed by cloning the oligonucleotide sequences into HindIII site of pBluescript. 2.2. Detection of non-B DNA structures in plasmids by S1 nuclease cleavage 2 mg of plasmid DNA was digested by S1 nuclease for 2 h at 37 C in S1 nuclease buffer, precipitated in ethanol, dissolved in water and digested by ScaI for 1 h at 37 C before separation by electrophoresis on 1% agarose gels. 2.3. Yeast strains and luciferase assay p53-REs of interest were cloned following the Delitto Perfetto technique, starting from the yLFM ICORE strain [21]. yLFM isogenic derivative strains were transformed with pTSG-empty (control vector) or pTSG-hp53 (for the expression of p53). Purified transformant colonies were inoculated on 96-well plates in 100 ml of selective media containing 2% raffinose-SRtA-as carbon source or 2% raffinose supplemented with different concentrations of galactose to induce p53 expression [21]. Luciferase was measured as described [22]. 2.4. Statistical analysis Transactivation data are plotted as fold luciferase induction relative to reporter activity measured with cells that do not contain p53 plasmid, cultured under the same conditions. Mean and standard deviation of at least three biological replicates are presented.
Statistical significance was evaluated using Student's t-test. 3. Results and discussion 3.1. Selection and in silico analyses of p53-REs for the potential to form cruciform structures To elucidate the role of inverted repeats and cruciform structure in p53-REs we analyzed six different p53 target sequences (Table 1). The REs were designed as pairs of sequences with the same DlogKd (a measure of reduced binding affinity relative to an optimal p53 consensus sequence [8]), but with different propensities to form cruciform structures [23], and/or with similar dG but different locations of the inverted repeat involved in forming the predicted cruciform stem. This latter feature was analyzed using “Palindrome analyser” (bioinformatics.ibp.cz, [24]). The energy required for destabilization of the cruciform structure is presented in Table 1 (dG column) next to the inverted repeat signature (CF rank). Every inverted repeat is characterized by three basic properties in the CF rank: L-S-M, where L is length of repeat, S is length of spacer and M is amount of mismatches in the sequence. The results section of the program shows the position of repeats in DNA, dG, and highlights inverted repeats, which can be visualized in their predicted cruciform structure (Fig. 1). We cloned the chosen p53-RE sequences into pBluescript to measure cruciform formation and into yeast at a chosen chromosomal location upstream of the luciferase reporter gene, to measure the transactivation potential of wild type p53 protein. The predicted best targets for p53 binding affinity are the XA and TT REs. XA contains a triplet of adenine at the beginning of the first decamer, but still respects the p53 consensus sequence and has a low DlogKd penalty score. The inverted repeat in this sequence is localized in the central part of the sequence (Table 1, bold). The TT RE has the same predicted DlogKd but a lower predicted dG as this sequence has more regular Watson-Crick pairs in cruciform and the predicted cruciform is larger with a spacer in the inverted repeat in the center of the sequence. The dG is the lowest of all tested sequences, suggesting higher stability of the cruciform. A second pair of REs, XG and GCG, have lower predicted p53 binding affinity. XG contains a triplet of adenines at the beginning of the second decamer, which complies with the p53 consensus sequence, but results in lower affinity. However the structure of the cruciform is almost identical to TT sequence e only with one additional imperfect base pairing. The high affinity of the GCG RE is reduced by a single mismatch from consensus in the second
Table 1 p53 target sequences and their characterization according to potential p53 binding affinity [8], energy required for cruciform formation [24] and CF rank by Palindrome analyser (inverted repeats in bold, mismatches from p53 consensus RE are in lowercase; for the p53 REs, all nucleotide changes in comparison to the TT RE sequence are highlighted in gray). Name
Sequence
XA: TT: XG:
GGGCATGTCT GGGCATGCCC
GCG: XT: WC: CFNO:
cAtgATgTga tcACATgaTg
DlogKd
dGa
CF rank
0.08
10.48
7-0-0
0.08 0.18
18.01 17.75
9-2-1 7-6-0
0.18 0.32
16.91 10.00
10-0-1 7-0-0
0.32
16.82
10-0-1
2.60
17.32
10-0-0
a
5 nt upstream and downstream flanking the cloned p53-RE in the yeast reporter locus are included in the dG calculations.
zda, et al., The structure formed by inverted repeats in p53 response elements determines the Please cite this article in press as: V. Bra transactivation activity of p53 protein, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.12.113
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chosen sequences embedded in plasmid DNA by S1 nuclease cleavage. As controls, we also analyzed pBluescript and pCFNO, which is derived from pBluescript by insertion of a 20bp long ideal inverted repeat without any mismatch [25]. We confirmed that this sequence forms cruciform at natural superhelical density (Fig. 2A, lane 4). Densitometry of five independent experiments showed that 74% of plasmids contain cruciform structure in this sequence (Fig. 2B). However, pBluescript at natural superhelical density also
Fig. 1. Models of cruciform structures within tested p53-REs generated with “Palindrome Finder”. Cruciform structures can be sorted into three groups. XA and XT each have a 7bp long inverted repeat in the center of the RE sequence. TT and XG also have a 7bp long inverted repeat (TT has an additional base pairing after one mismatch) but this reaches the edge of the sequence. The CF ranks of these sequences are 9-2-1 and 7-6-0, respectively (Table 1). CGC and WC have relatively strong theoretical CF propensity, however the inverted repeat is interrupted by one mismatch and also reaches the edge of the sequence. Without considering the base pairing before the mismatch, the structure of the CGC sequence will be similar to XA and XT sequences.
position (where C is in place of a purine). This change also disrupts the inverted repeat at the beginning of the sequence, however due to the overall potential base pairing, the dG of the predicted cruciform is similar to that of the XG sequence. The third pair of sequences, XT and WC, have even lower p53 affinity according to DlogKd score. XT contains a triplet of thymines at the beginning of the first decamer. Thymine at these positions does not respect the p53 consensus. The predicted cruciform is virtually identical to that of the XA sequence, with a 7 nucleotide inverted repeat located in the center of the sequence. WC contains a single mismatch at position 6, which decreases the predicted p53 binding affinity. The mismatch also interrupts the inverted repeat of this sequence. While the stability of the predicted cruciform remains relatively high within our panel, WC, as well as XA, XT and GCG rely on adjacent inverted repeat sequences, hence the extrahelical Watson-Crick pairing is probably overestimated.
3.2. Formation of cruciform structure in p53-REs cloned in plasmid DNA We analyzed the formation of cruciform structures in the
Fig. 2. Evidence for p53-REs adopting non-B DNA structures by nuclease cleavage assay in plasmid DNA. The p53-REs or an ideal 20bp palindrome (pCFNO) were cloned into pBluescript. A) S1 nuclease cleavage with subsequent linearization by ScaI restriction endonuclease was performed at natural superhelical density (lanes 4e9). Lane 1 contains the 500 bp DNA ladder. Supercoiled (lane 2) and nuclease S1 þ ScaI digested pBluescript (lane 3) were used as controls. S1/ScaI cleavage bands of pCFNO plasmid are 1837 and 1124 bp. S1/Sca I cleavage bands of empty pBluescript are about 2179 and 782, indicating an S1 cleavage site at the ori site. The digestion patterns indicate that the two extra-helical structures do not coexist. One representative image is shown. B) Bars plot the average proportion S1/Sca I cleavage bands from the densitometry of five experiments. The shades of gray indicate the relative proportion of cleavage site at ori (light) or at the HindIII site, where the control palindrome or the p53-REs are cloned. C) Map of pBluescript highlighting positions of the unique ScaI site, the cloning site, and the ori site. The linear fragments obtained after S1/ScaI cleavage are shown.
zda, et al., The structure formed by inverted repeats in p53 response elements determines the Please cite this article in press as: V. Bra transactivation activity of p53 protein, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.12.113
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zda et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6 V. Bra
showed the presence of local DNA structure by S1 cleavage (Fig. 2, lane 3). In silico analyses of pBluescript showed a long inverted repeat at position 1745 in ori sequence capable of forming a cruciform structure. Notably, cloning of the 20bp inverted repeats apparently removes the local DNA structure at the ori sequence. The p53-REs inserted in pBluescript led to the appearance of S1 cleavage patterns consistent with cruciform formation but with lower efficiency compared to pCFNO. Plasmids isolated in natural superhelical density showed the presence of the cruciform in either the ori site or the inserted RE sequences. Plasmids pB-TT and pB-XG showed preferential cruciform formation in the p53 target sequence (43 and 47% of plasmids with cruciform extrusion in p53 target site), while this was less apparent for pB-XA, pB-GCG, pB-XT and pB-WC, which showed preferential cruciform formation at the ori site (Fig. 2). Densitometry did not show significant differences among pB-XA, pB-GCG, pB-XT and pB-WC plasmids, where cruciform was formed at the p53 target sequence in 11e18% plasmid molecules (Fig. 2B). Cruciform formation was significantly more frequent in the pB-TT and pB-XG plasmids that contain an inverted repeat sequence separated by a spacer that could accommodate the disruption caused by the extrusion. It seems that disruption of the inverted repeat in the middle or at the edge of the sequence decreases cruciform structure formation, even if the total number of bases in the inverted repeat is identical. 3.3. p53 transactivation differs in yeast cells not only according to theoretical binding affinity, but also according to predicted RE structure We analyzed the level of p53-dependent transactivation from a luciferase reporter gene placed in a specific chromatin context in yeast. The assay is based on the ability to generate isogenic strains that differ only in the p53 RE sequence that is being tested and enables p53 protein expression at different levels. This sensitive assay identifies subtle changes in transactivation potential [2,21,26]. Specifically, the p53 target sequences were cloned upstream of the luciferase gene at the ADE2 locus and we analyzed transactivation induced by three different levels of p53 protein 1.5, 3 and 4.5 h after transferring cells to galactose-containing media, resulting in transcription of the TP53 cDNA that is controlled by the GAL1 promoter. We analyzed the level of transcription without galactose or with 0.008% and 0.032% of galactose, leading to basal,
moderate or high p53 levels [26]. Induction over time was gradual and constant in all strains (not shown) with peak activity at 4.5 h. The isogenic yLFM derivative containing the CFNO sequence inserted upstream of the luciferase reporter did not show any significant change in transactivation, even when p53 was expressed at high levels (fold induction equal to ~1, Fig. 3). We observed two broad groups of REs in terms of responsiveness to p53 level, with constructs XA, TT and XG showing significantly higher transactivation compared to GCG, XT and WC (Fig. 3). The last three strains showed only slight, non-significant differences between each other, even after long induction. Although the predicted p53 binding affinity for XA and TT was identical, the TT RE was more responsive in all tested culture conditions and particularly in galactose-containing media. Furthermore, the XG RE has a lower predicted Kd compared to XA, yet showed higher responsiveness, particularly to high levels of p53 (0.032% galactose). 3.4. Changes in p53-dependent transactivation due to mutated RE sequences To support our results we performed additional mutagenesis. We made mutants (a) in the central part of the sequence to change bases in the loop part of the predicted cruciform structure, (b) in the stem part of the cruciform to destroy or (c) recover the possibility of cruciform formation (Table 2). TT-8,10 mutation leads to an RE with ideal palindromic sequence. This sequence has higher cruciform propensity (see arrows in Fig. 4 and Table 2) and is moreover the best p53 RE in terms of p53 binding affinity according to DlogKd estimation. Due to the combination of these two factors, TT-8,10mut transactivation by wild type p53 was slightly higher than that for TT (Fig. 4A). However even if the cruciform structure is formed by adjacent inverted repeat sequence stretches, there has to be a small loop in the middle of the structure due to steric hindrance, so the difference of transactivation is not statistically significant compared to TT construct. On the other hand, comparing XA, XT and TT-8,10mut (identical sequences except for the first three residues) shows significant improvement of p53 transactivation for TT-8,10mut that can form a cruciform from the peripheral areas of the sequence. In the TT-2mut RE, the inverted repeat of the TT construct is destroyed by mutation of the second base pair (only a 5bp inverted repeat remains, which is insufficient for CF formation). p53 transactivation for TT-2mut is significantly lower than the original TT construct (Fig. 4A). However, the nucleotide change at position 2 is a p53 nonconsensus base that also leads to the lowest theoretical p53 DNA binding affinity. Therefore, we constructed another TT variant RE -TT-19mut. As expected, this construct is significantly less responsive to p53 compared to TT (Fig. 4A), but slightly more than TT-2, which can be dependent both on the predicted
Table 2 Mutagenesis of the target sequences.
Fig. 3. p53-dependent transactivation potential in yeast. Wild type p53 is expressed under an inducible GAL1 promoter. Histogram plots average fold induction over empty vector and standard deviations of three biological replicates. For each strain, the results with three levels of p53 induction obtained after 4.5 h of culture in inducing media are presented. Asterisks indicate that, except for the CFNO, all REs led to a significant induction of p53-dependent transactivation at each galactose level.
Name
Sequence
DlogKd
CF rank
TT (a)TT-8,10muta (b)TT-2mut (b)TT-19mut GCG (c)GCG-2muta (c)GCG-19mut
GGGCATGTCT GGGCATGCCC GGGCATGCCC GGGCATGCCC GcGCATGTCT GGGCATGCCC GGGCATGTCT GGGCATGCtC GcGCATGCCC GGGCATGCCC GGGCATGCCC GGGCATGCCC GcGCATGCCC GGGCATGCgC
0.08 0.00 0.26 0.11 0.18 0.00 0.36
9-2-1 10-0-0 7-2-1 7-2-1 10-0-1 10-0-0 10-0-0
New sequences are in bold. a Identical sequences, ideal p53 target and also ideal inverted repeat e alternative name TT-8,10mut (identical sequence as TT construct with two mutated bases at position 8 and 10) or GCG-2mut (identical to GCG with one nucleotide change in position 2).
zda, et al., The structure formed by inverted repeats in p53 response elements determines the Please cite this article in press as: V. Bra transactivation activity of p53 protein, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.12.113
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respective cruciform structures [27]. As expected, p53-dependent transactivation of GCG-2mut is higher than the original GCG construct (Fig. 4B). This difference is partly influenced by the improvement in p53 binding affinity conferred by the change at position 2. The same change also improves the cruciform propensity (Fig. 4B, arrows). We also tested a second mutation of the original GCG RE, GCG-19mut that has a very low predicted p53 binding affinity but enhanced potential for cruciform formation by the introduction of a second p53 nonconsensus base at position 19. This mutated RE has similar transactivation to the original GCG RE, supporting the proposal that both DNA sequence and DNA structure are important determinants for effective p53 transactivation. 3.5. Discussion
Fig. 4. Changes in p53-dependent transactivation due to mutated RE sequences. A) The TT RE was mutated to extend (TT-8,10 mut) or reduce (TT-2mut, TT-19mut) the inverted repeat sequence (arrows), affecting palindrome propensity. Some of the changes affect also DNA binding affinity (see Table 2) B) The GCG RE was mutated to extend the inverted repeat sequence (arrows). In the case of GCG-19mut the mutation also reduces the predicted DNA binding affinity. * denotes altered p53-dependent transactivation. C) Schematic model of p53 binding to a target RE in linear and cruciform structure. Transcriptionally active DNA is dynamic and can adopt cruciform structure according to its sequence. p53-RE is highlighted in red. A p53 tetramer (dimer of dimers) can bind the RE both in linear and cruciform DNA. The transition from linear to non-B DNA could drive mutual allosteric changes to p53 quaternary structure and potentially affect off rates by inhibiting protein sliding [28]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
difference in binding affinity and the relative higher stability of a G:T compared to C:C mismatch in the predicted stem of the
The transactivation assay of p53-REs highlighted that luminescence did not simply correlate with p53 binding affinity predicted on the basis of nucleotide sequence [8]. A trend for weaker transactivation with the lowest Kd REs was observed, particularly at low p53 levels (raffinose, Fig. 3). However, pairs of sequences with the same predicted value of DlogKd have very different levels of transactivation, which are related to the propensity to form cruciform structures. However, an ideal cruciform sequence that is not a p53 RE was not active in the functional assay. XA and TT are REs with the highest affinity among those analyzed and should give the highest luminescence. Instead, XA showed lower transactivation than TT, especially 4.5 h after induction by galactose. This finding can be explained by the different structures that these sequences can form. Based on S1 cleavage of plasmid DNA, XA forms a cruciform less favorably, while TT forms a more stable structure. Even with a lower Kd, the XG RE shows p53dependent luminescence values similar to TT. These two REs are expected to form very similar cruciform structures, with a long stem without mismatches and non-complementary loop. The very low values of luminescence for the sequence GCG are not explicable in terms of affinity, which is equal to XG, but can be related to the instability of the secondary structure, which could compromise recognition and/or binding by p53. The sequences XT and WC have similar values of luminescence and also the same (low) affinity for p53. Overall, these results suggest that p53 transactivation is determined not only by the target sequence but also by local DNA structure. DNA structure is an important determinant for binding of proteins [10,11]. The importance of the DNA-conformation for p53 binding was proposed previously by in vitro analyses and pointed to the importance of inverted repeats in p53-REs [16,25]. It was also demonstrated that DNA topology influences p53 sequence-specific DNA binding through structural transitions within target sites [19]. Specific mutations introduced in the TT or GCG REs showed that changes in the loop part of the inverted repeat did not weaken p53dependent transactivation, but changes at the edge or in the middle of the inverted repeat significantly weakened p53 activity. Such extra-helical structures could favor the recognition/binding of p53, and/or have an allosteric effect on p53 tetramer structure that results in stronger stimulation of transcription (Fig. 4C). This does not negate the role of sequence-specific DNA recognition by p53 and the contribution of binding affinity due to direct protein:DNA contacts. For example, XA and XT have very similar predicted propensity to form cruciform and showed overlapping results in the S1 nuclease assay, but their transactivation potential is correlated to DNA binding affinity. However, our results established that primary sequence of DNA is not the only determining factor in the recognition, binding and transactivation by p53 from a target site in a chromatin context.
zda, et al., The structure formed by inverted repeats in p53 response elements determines the Please cite this article in press as: V. Bra transactivation activity of p53 protein, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.12.113
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Acknowledgements We thank Dr. Philip J Coates for proof-reading and editing the manuscript. This work was supported by the Grant Agency of the Czech Republic (15-21855S). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.12.113. References [1] A.J. Levine, M. Oren, The first 30 years of p53: growing ever more complex, Nat. Rev. Cancer 9 (2009) 749e758. [2] D. Menendez, A. Inga, M.A. Resnick, The expanding universe of p53 targets, Nat. Rev. Cancer 9 (2009) 724e737. [3] R. Beckerman, C. Prives, Transcriptional regulation by p53, Cold Spring Harb. Perspect. Biol. 2 (2010) a000935. [4] T. Gohler, M. Reimann, D. Cherny, K. Walter, G. Warnecke, E. Kim, W. Deppert, Specific interaction of p53 with target binding sites is determined by DNA conformation and is regulated by the C-terminal domain, J. Biol. Chem. 277 (2002) 41192e41203. [5] J.P. Kruse, W. Gu, Modes of p53 regulation, Cell 137 (2009) 609e622. [6] D.W. Meek, Mechanisms of switching on p53: a role for covalent modification? Oncogene 18 (1999) 7666e7675. [7] K.G. McLure, P.W. Lee, How p53 binds DNA as a tetramer, EMBO J. 17 (1998) 3342e3350. [8] D.B. Veprintsev, A.R. Fersht, Algorithm for prediction of tumour suppressor p53 affinity for binding sites in DNA, Nucleic Acids Res. 36 (2008) 1589e1598. [9] Z. Shakked, D. Rabinovich, The effect of the base sequence on the fine structure of the DNA double helix, Prog. Biophys. Mol. Biol. 47 (1986) 159e195. [10] V. Brazda, L. Haronikova, J.C. Liao, M. Fojta, DNA and RNA quadruplex-binding proteins, Int. J. Mol. Sci. 15 (2014) 17493e17517. [11] V. Brazda, R.C. Laister, E.B. Jagelska, C. Arrowsmith, Cruciform structures are a common DNA feature important for regulating biological processes, BMC Mol. Biol. 12 (2011) 33. [12] N. Panayotatos, A. Fontaine, A native cruciform DNA structure probed in bacteria by recombinant T7 endonuclease, J. Biol. Chem. 262 (1987) 11364e11368. [13] M. Zannis-Hadjopoulos, L. Frappier, M. Khoury, G.B. Price, Effect of anticruciform DNA monoclonal antibodies on DNA replication, EMBO J. 7 (1988) 1837e1844.
[14] A. Dayn, S. Malkhosyan, S.M. Mirkin, Transcriptionally driven cruciform formation in vivo, Nucleic Acids Res. 20 (1992) 5991e5997. [15] S. Chasovskikh, A. Dimtchev, M. Smulson, A. Dritschilo, DNA transitions induced by binding of PARP-1 to cruciform structures in supercoiled plasmids, Cytom. A 68 (2005) 21e27. [16] E. Kim, N. Albrechtsen, W. Deppert, DNA-conformation is an important determinant of sequence-specific DNA binding by tumor suppressor p53, Oncogene 15 (1997) 857e869. [17] B. Ma, Y. Pan, J. Zheng, A.J. Levine, R. Nussinov, Sequence analysis of p53 response-elements suggests multiple binding modes of the p53 tetramer to DNA targets, Nucleic Acids Res. 35 (2007) 2986e3001. [18] J. Coufal, E.B. Jagelska, J.C. Liao, V. Brazda, Preferential binding of p53 tumor suppressor to p21 promoter sites that contain inverted repeats capable of forming cruciform structure, Biochem. Biophys. Res. Commun. 441 (2013) 83e88. [19] E.B. Jagelska, V. Brazda, P. Pecinka, E. Palecek, M. Fojta, DNA topology influences p53 sequence-specific DNA binding through structural transitions within the target sites, Biochem. J. 412 (2008) 57e63. [20] E. Palecek, V. Brazda, E. Jagelska, P. Pecinka, L. Karlovska, M. Brazdova, Enhancement of p53 sequence-specific binding by DNA supercoiling, Oncogene 23 (2004) 2119e2127. [21] M. Lion, I. Raimondi, S. Donati, O. Jousson, Y. Ciribilli, A. Inga, Evolution of p53 transactivation specificity through the lens of a yeast-based functional assay, PLoS One 10 (2015) e0116177. [22] J.J. Jordan, D. Menendez, A. Inga, M. Noureddine, D.A. Bell, M.A. Resnick, Noncanonical DNA motifs as transactivation targets by wild type and mutant p53, PLoS Genet. 4 (2008) e1000104. [23] M. Zuker, Mfold web server for nucleic acid folding and hybridization prediction, Nucleic Acids Res. 31 (2003) 3406e3415. [24] V. Brazda, J. Kolomaznik, J. Lysek, L. Haronikova, J. Coufal, J. St'astny, Palindrome analyser - a new web-based server for predicting and evaluating inverted repeats in nucleotide sequences, Biochem. Biophys. Res. Commun. 478 (2016) 1739e1745. [25] E.B. Jagelska, H. Pivonkova, M. Fojta, V. Brazda, The potential of the cruciform structure formation as an important factor influencing p53 sequence-specific binding to natural DNA targets, Biochem. Biophys. Res. Commun. 391 (2010) 1409e1414. [26] A. Inga, F. Storici, T.A. Darden, M.A. Resnick, Differential transactivation by the p53 transcription factor is highly dependent on p53 level and promoter target sequence, Mol. Cell. Biol. 22 (2002) 8612e8625. [27] S. Pan, X.J. Sun, J.K. Lee, Stability of complementary and mismatched DNA duplexes: comparison and contrast in gas versus solution phases, Int. J. Mass Spectrom. 253 (2006) 238e248. [28] J.S. Leith, A. Tafvizi, F. Huang, W.E. Uspal, P.S. Doyle, A.R. Fersht, L.A. Mirny, A.M. van Oijen, Sequence-dependent sliding kinetics of p53, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 16552e16557.
zda, et al., The structure formed by inverted repeats in p53 response elements determines the Please cite this article in press as: V. Bra transactivation activity of p53 protein, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.12.113
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The structure formed by inverted repeats in p53 response elements determines the transactivation activity of p53 protein clav Bra zda a, *, Jana Cechov a, Michele Battistin b, Jan Coufal a, Eva B. Jagelska a, Va a Ivan Raimondi b, Alberto Inga b a b
lovopolska 135, 61265, Brno, Czechia Institute of Biophysics, Academy of Sciences of the Czech Republic v.v.i., Kra Laboratory of Transcriptional Networks, Center for Integrative Biology, CIBIO, University of Trento, via Sommarive 9, 38123, Trento, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 November 2016 Accepted 17 December 2016 Available online xxx
The TP53 gene is the most frequently mutated gene in human cancer and p53 protein plays a crucial role in gene expression and cancer protection. Its role is manifested by interactions with other proteins and DNA. p53 is a transcription factor that binds to DNA response elements (REs). Due to the palindromic nature of the consensus binding site, several p53-REs have the potential to form cruciform structures. However, the influence of cruciform formation on the activity of p53-REs has not been evaluated. Therefore, we prepared sets of p53-REs with identical theoretical binding affinity in their linear state, but different probabilities to form extra helical structures, for in vitro and in vivo analyses. Then we evaluated the presence of cruciform structures when inserted into plasmid DNA and employed a yeast-based assay to measure transactivation potential of these p53-REs cloned at a chromosomal locus in isogenic strains. We show that transactivation in vivo correlated more with relative propensity of an RE to form cruciforms than to its predicted in vitro DNA binding affinity for wild type p53. Structural features of p53-REs could therefore be an important determinant of p53 transactivation function. © 2016 Elsevier Inc. All rights reserved.
Keywords: Inverted repeat p53 protein Protein-DNA interaction Cruciform structure
1. Introduction The ability of p53 to accomplish its biological functions is mainly attributed to its action as a transcription factor that binds to specific DNA sequences to regulate gene expression in processes including apoptosis, DNA repair and cell cycle control [1]. TP53 mutations result in altered or loss of p53-binding activity in approximately 50% of human tumors, and p53 activity is compromised by other mechanisms in most tumors with wild type p53 [2]. Human p53 is a nuclear phosphoprotein. The N-terminal domain contains the transactivation domains and also regulates stability [3]. The central part allows the specific recognition of target sequences and ~80% of mutations associated with cancer are located in the central DNA binding domain [3]. The C-terminal part contains a thermodynamically stable tetramerization domain and a basic regulatory domain [4]. Sequence-specific binding of p53 to DNA may require specific combinations of cofactors and post-translational modifications [5,6]. p53 recognizes and binds to a target sequence consisting of
* Corresponding author. zda). E-mail address: [email protected] (V. Bra
two decamers (RRRCWWGYYY - R ¼ A or G; W ¼ A or T; Y ¼ C or T) that can be separated by a spacer. p53 binds as a dimer of dimers and each subunit contacts three nucleotides of the quarter site formed by the RRRCW or WGYYY pentamer [7]. In vitro studies of RE sequence variations and their influence on p53 binding have provided an overview of the importance of each base within the canonical consensus sequence. These data led to a binding predictor matrix that provides the theoretical affinity of wild type p53 to any target sequence [8]. The primary sequence of DNA is not the only feature that determines recognition and interaction with proteins. Epigenetic modifications and alternative DNA structures contribute significantly to the regulation of biological processes. The threedimensional structure of DNA is often crucial for DNA-protein interactions [9,10]. Local DNA structures, like cruciforms, triplexes and quadruplexes, were originally described in vitro, but are now known to play important roles in vivo. The formation of cruciforms is closely related to DNA sequence, requiring inverted repeats of at least 6 nucleotides in length. Cruciform structures consist of a branch point, a stem and a loop whose size depends on the sequence that separates inverted repeats [11]. The presence of cruciforms was first found in plasmids with negative supercoils that stabilize their formation both in vitro and in vivo [12].
http://dx.doi.org/10.1016/j.bbrc.2016.12.113 0006-291X/© 2016 Elsevier Inc. All rights reserved.
zda, et al., The structure formed by inverted repeats in p53 response elements determines the Please cite this article in press as: V. Bra transactivation activity of p53 protein, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.12.113
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Cruciforms can influence DNA winding, the formation of other secondary structures and direct interaction with proteins. Monoclonal antibodies that recognize and stabilize cruciforms, but not antibodies recognizing B-DNA and Z-DNA, cause increased replication rates in vivo, suggesting that cruciforms contribute to activation of DNA replication origins [13]. Negative supercoiling in DNA during transcription can mediate cruciform formation in vivo and cruciform formation is related to increased transcriptional activity of the promoter [14]. Some DNA-binding proteins show only a weak sequence specificity but preferentially bind cruciform structures, while other DNA-binding proteins induce cruciform formation after DNA binding [15]. A high proportion of p53 target sequences have internal symmetry and adopt non-B-DNA conformation [16]. Notably, p53 target sites are not only direct repeats of two half-sites, but in some REs there are inverted repeats also in individual half-sites [17]. Binding of p53 to its target sequence in vitro is highly dependent on the presence of inverted repeats and there is a strong correlation between propensity to form cruciform and p53 specific binding [18e20], although whether non-B DNA structures independently influence transcriptional activity has not been demonstrated. Therefore, we analyzed transcriptional activity of several p53 target sequences presented within chromatin in isogenic yeast reporter strains. Comparing p53-REs with the same predicted p53 binding affinity but different propensities to adopt non-B DNA structures, our results suggest that cruciform structures with longer loops in the center of p53 target sites stimulate p53-dependent transactivation. 2. Materials and methods 2.1. DNA Supercoiled plasmid DNAs of pBluescriptIISK() and derived plasmids pCFNO [38], pB-XA, pB-TT, pB-XG, pB-GCG, pB-XT and pBWC were isolated from DH5a as described in the QIAGEN protocol. pB-series was constructed by cloning the oligonucleotide sequences into HindIII site of pBluescript. 2.2. Detection of non-B DNA structures in plasmids by S1 nuclease cleavage 2 mg of plasmid DNA was digested by S1 nuclease for 2 h at 37 C in S1 nuclease buffer, precipitated in ethanol, dissolved in water and digested by ScaI for 1 h at 37 C before separation by electrophoresis on 1% agarose gels. 2.3. Yeast strains and luciferase assay p53-REs of interest were cloned following the Delitto Perfetto technique, starting from the yLFM ICORE strain [21]. yLFM isogenic derivative strains were transformed with pTSG-empty (control vector) or pTSG-hp53 (for the expression of p53). Purified transformant colonies were inoculated on 96-well plates in 100 ml of selective media containing 2% raffinose-SRtA-as carbon source or 2% raffinose supplemented with different concentrations of galactose to induce p53 expression [21]. Luciferase was measured as described [22]. 2.4. Statistical analysis Transactivation data are plotted as fold luciferase induction relative to reporter activity measured with cells that do not contain p53 plasmid, cultured under the same conditions. Mean and standard deviation of at least three biological replicates are presented.
Statistical significance was evaluated using Student's t-test. 3. Results and discussion 3.1. Selection and in silico analyses of p53-REs for the potential to form cruciform structures To elucidate the role of inverted repeats and cruciform structure in p53-REs we analyzed six different p53 target sequences (Table 1). The REs were designed as pairs of sequences with the same DlogKd (a measure of reduced binding affinity relative to an optimal p53 consensus sequence [8]), but with different propensities to form cruciform structures [23], and/or with similar dG but different locations of the inverted repeat involved in forming the predicted cruciform stem. This latter feature was analyzed using “Palindrome analyser” (bioinformatics.ibp.cz, [24]). The energy required for destabilization of the cruciform structure is presented in Table 1 (dG column) next to the inverted repeat signature (CF rank). Every inverted repeat is characterized by three basic properties in the CF rank: L-S-M, where L is length of repeat, S is length of spacer and M is amount of mismatches in the sequence. The results section of the program shows the position of repeats in DNA, dG, and highlights inverted repeats, which can be visualized in their predicted cruciform structure (Fig. 1). We cloned the chosen p53-RE sequences into pBluescript to measure cruciform formation and into yeast at a chosen chromosomal location upstream of the luciferase reporter gene, to measure the transactivation potential of wild type p53 protein. The predicted best targets for p53 binding affinity are the XA and TT REs. XA contains a triplet of adenine at the beginning of the first decamer, but still respects the p53 consensus sequence and has a low DlogKd penalty score. The inverted repeat in this sequence is localized in the central part of the sequence (Table 1, bold). The TT RE has the same predicted DlogKd but a lower predicted dG as this sequence has more regular Watson-Crick pairs in cruciform and the predicted cruciform is larger with a spacer in the inverted repeat in the center of the sequence. The dG is the lowest of all tested sequences, suggesting higher stability of the cruciform. A second pair of REs, XG and GCG, have lower predicted p53 binding affinity. XG contains a triplet of adenines at the beginning of the second decamer, which complies with the p53 consensus sequence, but results in lower affinity. However the structure of the cruciform is almost identical to TT sequence e only with one additional imperfect base pairing. The high affinity of the GCG RE is reduced by a single mismatch from consensus in the second
Table 1 p53 target sequences and their characterization according to potential p53 binding affinity [8], energy required for cruciform formation [24] and CF rank by Palindrome analyser (inverted repeats in bold, mismatches from p53 consensus RE are in lowercase; for the p53 REs, all nucleotide changes in comparison to the TT RE sequence are highlighted in gray). Name
Sequence
XA: TT: XG:
GGGCATGTCT GGGCATGCCC
GCG: XT: WC: CFNO:
cAtgATgTga tcACATgaTg
DlogKd
dGa
CF rank
0.08
10.48
7-0-0
0.08 0.18
18.01 17.75
9-2-1 7-6-0
0.18 0.32
16.91 10.00
10-0-1 7-0-0
0.32
16.82
10-0-1
2.60
17.32
10-0-0
a
5 nt upstream and downstream flanking the cloned p53-RE in the yeast reporter locus are included in the dG calculations.
zda, et al., The structure formed by inverted repeats in p53 response elements determines the Please cite this article in press as: V. Bra transactivation activity of p53 protein, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.12.113
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chosen sequences embedded in plasmid DNA by S1 nuclease cleavage. As controls, we also analyzed pBluescript and pCFNO, which is derived from pBluescript by insertion of a 20bp long ideal inverted repeat without any mismatch [25]. We confirmed that this sequence forms cruciform at natural superhelical density (Fig. 2A, lane 4). Densitometry of five independent experiments showed that 74% of plasmids contain cruciform structure in this sequence (Fig. 2B). However, pBluescript at natural superhelical density also
Fig. 1. Models of cruciform structures within tested p53-REs generated with “Palindrome Finder”. Cruciform structures can be sorted into three groups. XA and XT each have a 7bp long inverted repeat in the center of the RE sequence. TT and XG also have a 7bp long inverted repeat (TT has an additional base pairing after one mismatch) but this reaches the edge of the sequence. The CF ranks of these sequences are 9-2-1 and 7-6-0, respectively (Table 1). CGC and WC have relatively strong theoretical CF propensity, however the inverted repeat is interrupted by one mismatch and also reaches the edge of the sequence. Without considering the base pairing before the mismatch, the structure of the CGC sequence will be similar to XA and XT sequences.
position (where C is in place of a purine). This change also disrupts the inverted repeat at the beginning of the sequence, however due to the overall potential base pairing, the dG of the predicted cruciform is similar to that of the XG sequence. The third pair of sequences, XT and WC, have even lower p53 affinity according to DlogKd score. XT contains a triplet of thymines at the beginning of the first decamer. Thymine at these positions does not respect the p53 consensus. The predicted cruciform is virtually identical to that of the XA sequence, with a 7 nucleotide inverted repeat located in the center of the sequence. WC contains a single mismatch at position 6, which decreases the predicted p53 binding affinity. The mismatch also interrupts the inverted repeat of this sequence. While the stability of the predicted cruciform remains relatively high within our panel, WC, as well as XA, XT and GCG rely on adjacent inverted repeat sequences, hence the extrahelical Watson-Crick pairing is probably overestimated.
3.2. Formation of cruciform structure in p53-REs cloned in plasmid DNA We analyzed the formation of cruciform structures in the
Fig. 2. Evidence for p53-REs adopting non-B DNA structures by nuclease cleavage assay in plasmid DNA. The p53-REs or an ideal 20bp palindrome (pCFNO) were cloned into pBluescript. A) S1 nuclease cleavage with subsequent linearization by ScaI restriction endonuclease was performed at natural superhelical density (lanes 4e9). Lane 1 contains the 500 bp DNA ladder. Supercoiled (lane 2) and nuclease S1 þ ScaI digested pBluescript (lane 3) were used as controls. S1/ScaI cleavage bands of pCFNO plasmid are 1837 and 1124 bp. S1/Sca I cleavage bands of empty pBluescript are about 2179 and 782, indicating an S1 cleavage site at the ori site. The digestion patterns indicate that the two extra-helical structures do not coexist. One representative image is shown. B) Bars plot the average proportion S1/Sca I cleavage bands from the densitometry of five experiments. The shades of gray indicate the relative proportion of cleavage site at ori (light) or at the HindIII site, where the control palindrome or the p53-REs are cloned. C) Map of pBluescript highlighting positions of the unique ScaI site, the cloning site, and the ori site. The linear fragments obtained after S1/ScaI cleavage are shown.
zda, et al., The structure formed by inverted repeats in p53 response elements determines the Please cite this article in press as: V. Bra transactivation activity of p53 protein, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.12.113
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zda et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6 V. Bra
showed the presence of local DNA structure by S1 cleavage (Fig. 2, lane 3). In silico analyses of pBluescript showed a long inverted repeat at position 1745 in ori sequence capable of forming a cruciform structure. Notably, cloning of the 20bp inverted repeats apparently removes the local DNA structure at the ori sequence. The p53-REs inserted in pBluescript led to the appearance of S1 cleavage patterns consistent with cruciform formation but with lower efficiency compared to pCFNO. Plasmids isolated in natural superhelical density showed the presence of the cruciform in either the ori site or the inserted RE sequences. Plasmids pB-TT and pB-XG showed preferential cruciform formation in the p53 target sequence (43 and 47% of plasmids with cruciform extrusion in p53 target site), while this was less apparent for pB-XA, pB-GCG, pB-XT and pB-WC, which showed preferential cruciform formation at the ori site (Fig. 2). Densitometry did not show significant differences among pB-XA, pB-GCG, pB-XT and pB-WC plasmids, where cruciform was formed at the p53 target sequence in 11e18% plasmid molecules (Fig. 2B). Cruciform formation was significantly more frequent in the pB-TT and pB-XG plasmids that contain an inverted repeat sequence separated by a spacer that could accommodate the disruption caused by the extrusion. It seems that disruption of the inverted repeat in the middle or at the edge of the sequence decreases cruciform structure formation, even if the total number of bases in the inverted repeat is identical. 3.3. p53 transactivation differs in yeast cells not only according to theoretical binding affinity, but also according to predicted RE structure We analyzed the level of p53-dependent transactivation from a luciferase reporter gene placed in a specific chromatin context in yeast. The assay is based on the ability to generate isogenic strains that differ only in the p53 RE sequence that is being tested and enables p53 protein expression at different levels. This sensitive assay identifies subtle changes in transactivation potential [2,21,26]. Specifically, the p53 target sequences were cloned upstream of the luciferase gene at the ADE2 locus and we analyzed transactivation induced by three different levels of p53 protein 1.5, 3 and 4.5 h after transferring cells to galactose-containing media, resulting in transcription of the TP53 cDNA that is controlled by the GAL1 promoter. We analyzed the level of transcription without galactose or with 0.008% and 0.032% of galactose, leading to basal,
moderate or high p53 levels [26]. Induction over time was gradual and constant in all strains (not shown) with peak activity at 4.5 h. The isogenic yLFM derivative containing the CFNO sequence inserted upstream of the luciferase reporter did not show any significant change in transactivation, even when p53 was expressed at high levels (fold induction equal to ~1, Fig. 3). We observed two broad groups of REs in terms of responsiveness to p53 level, with constructs XA, TT and XG showing significantly higher transactivation compared to GCG, XT and WC (Fig. 3). The last three strains showed only slight, non-significant differences between each other, even after long induction. Although the predicted p53 binding affinity for XA and TT was identical, the TT RE was more responsive in all tested culture conditions and particularly in galactose-containing media. Furthermore, the XG RE has a lower predicted Kd compared to XA, yet showed higher responsiveness, particularly to high levels of p53 (0.032% galactose). 3.4. Changes in p53-dependent transactivation due to mutated RE sequences To support our results we performed additional mutagenesis. We made mutants (a) in the central part of the sequence to change bases in the loop part of the predicted cruciform structure, (b) in the stem part of the cruciform to destroy or (c) recover the possibility of cruciform formation (Table 2). TT-8,10 mutation leads to an RE with ideal palindromic sequence. This sequence has higher cruciform propensity (see arrows in Fig. 4 and Table 2) and is moreover the best p53 RE in terms of p53 binding affinity according to DlogKd estimation. Due to the combination of these two factors, TT-8,10mut transactivation by wild type p53 was slightly higher than that for TT (Fig. 4A). However even if the cruciform structure is formed by adjacent inverted repeat sequence stretches, there has to be a small loop in the middle of the structure due to steric hindrance, so the difference of transactivation is not statistically significant compared to TT construct. On the other hand, comparing XA, XT and TT-8,10mut (identical sequences except for the first three residues) shows significant improvement of p53 transactivation for TT-8,10mut that can form a cruciform from the peripheral areas of the sequence. In the TT-2mut RE, the inverted repeat of the TT construct is destroyed by mutation of the second base pair (only a 5bp inverted repeat remains, which is insufficient for CF formation). p53 transactivation for TT-2mut is significantly lower than the original TT construct (Fig. 4A). However, the nucleotide change at position 2 is a p53 nonconsensus base that also leads to the lowest theoretical p53 DNA binding affinity. Therefore, we constructed another TT variant RE -TT-19mut. As expected, this construct is significantly less responsive to p53 compared to TT (Fig. 4A), but slightly more than TT-2, which can be dependent both on the predicted
Table 2 Mutagenesis of the target sequences.
Fig. 3. p53-dependent transactivation potential in yeast. Wild type p53 is expressed under an inducible GAL1 promoter. Histogram plots average fold induction over empty vector and standard deviations of three biological replicates. For each strain, the results with three levels of p53 induction obtained after 4.5 h of culture in inducing media are presented. Asterisks indicate that, except for the CFNO, all REs led to a significant induction of p53-dependent transactivation at each galactose level.
Name
Sequence
DlogKd
CF rank
TT (a)TT-8,10muta (b)TT-2mut (b)TT-19mut GCG (c)GCG-2muta (c)GCG-19mut
GGGCATGTCT GGGCATGCCC GGGCATGCCC GGGCATGCCC GcGCATGTCT GGGCATGCCC GGGCATGTCT GGGCATGCtC GcGCATGCCC GGGCATGCCC GGGCATGCCC GGGCATGCCC GcGCATGCCC GGGCATGCgC
0.08 0.00 0.26 0.11 0.18 0.00 0.36
9-2-1 10-0-0 7-2-1 7-2-1 10-0-1 10-0-0 10-0-0
New sequences are in bold. a Identical sequences, ideal p53 target and also ideal inverted repeat e alternative name TT-8,10mut (identical sequence as TT construct with two mutated bases at position 8 and 10) or GCG-2mut (identical to GCG with one nucleotide change in position 2).
zda, et al., The structure formed by inverted repeats in p53 response elements determines the Please cite this article in press as: V. Bra transactivation activity of p53 protein, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.12.113
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respective cruciform structures [27]. As expected, p53-dependent transactivation of GCG-2mut is higher than the original GCG construct (Fig. 4B). This difference is partly influenced by the improvement in p53 binding affinity conferred by the change at position 2. The same change also improves the cruciform propensity (Fig. 4B, arrows). We also tested a second mutation of the original GCG RE, GCG-19mut that has a very low predicted p53 binding affinity but enhanced potential for cruciform formation by the introduction of a second p53 nonconsensus base at position 19. This mutated RE has similar transactivation to the original GCG RE, supporting the proposal that both DNA sequence and DNA structure are important determinants for effective p53 transactivation. 3.5. Discussion
Fig. 4. Changes in p53-dependent transactivation due to mutated RE sequences. A) The TT RE was mutated to extend (TT-8,10 mut) or reduce (TT-2mut, TT-19mut) the inverted repeat sequence (arrows), affecting palindrome propensity. Some of the changes affect also DNA binding affinity (see Table 2) B) The GCG RE was mutated to extend the inverted repeat sequence (arrows). In the case of GCG-19mut the mutation also reduces the predicted DNA binding affinity. * denotes altered p53-dependent transactivation. C) Schematic model of p53 binding to a target RE in linear and cruciform structure. Transcriptionally active DNA is dynamic and can adopt cruciform structure according to its sequence. p53-RE is highlighted in red. A p53 tetramer (dimer of dimers) can bind the RE both in linear and cruciform DNA. The transition from linear to non-B DNA could drive mutual allosteric changes to p53 quaternary structure and potentially affect off rates by inhibiting protein sliding [28]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
difference in binding affinity and the relative higher stability of a G:T compared to C:C mismatch in the predicted stem of the
The transactivation assay of p53-REs highlighted that luminescence did not simply correlate with p53 binding affinity predicted on the basis of nucleotide sequence [8]. A trend for weaker transactivation with the lowest Kd REs was observed, particularly at low p53 levels (raffinose, Fig. 3). However, pairs of sequences with the same predicted value of DlogKd have very different levels of transactivation, which are related to the propensity to form cruciform structures. However, an ideal cruciform sequence that is not a p53 RE was not active in the functional assay. XA and TT are REs with the highest affinity among those analyzed and should give the highest luminescence. Instead, XA showed lower transactivation than TT, especially 4.5 h after induction by galactose. This finding can be explained by the different structures that these sequences can form. Based on S1 cleavage of plasmid DNA, XA forms a cruciform less favorably, while TT forms a more stable structure. Even with a lower Kd, the XG RE shows p53dependent luminescence values similar to TT. These two REs are expected to form very similar cruciform structures, with a long stem without mismatches and non-complementary loop. The very low values of luminescence for the sequence GCG are not explicable in terms of affinity, which is equal to XG, but can be related to the instability of the secondary structure, which could compromise recognition and/or binding by p53. The sequences XT and WC have similar values of luminescence and also the same (low) affinity for p53. Overall, these results suggest that p53 transactivation is determined not only by the target sequence but also by local DNA structure. DNA structure is an important determinant for binding of proteins [10,11]. The importance of the DNA-conformation for p53 binding was proposed previously by in vitro analyses and pointed to the importance of inverted repeats in p53-REs [16,25]. It was also demonstrated that DNA topology influences p53 sequence-specific DNA binding through structural transitions within target sites [19]. Specific mutations introduced in the TT or GCG REs showed that changes in the loop part of the inverted repeat did not weaken p53dependent transactivation, but changes at the edge or in the middle of the inverted repeat significantly weakened p53 activity. Such extra-helical structures could favor the recognition/binding of p53, and/or have an allosteric effect on p53 tetramer structure that results in stronger stimulation of transcription (Fig. 4C). This does not negate the role of sequence-specific DNA recognition by p53 and the contribution of binding affinity due to direct protein:DNA contacts. For example, XA and XT have very similar predicted propensity to form cruciform and showed overlapping results in the S1 nuclease assay, but their transactivation potential is correlated to DNA binding affinity. However, our results established that primary sequence of DNA is not the only determining factor in the recognition, binding and transactivation by p53 from a target site in a chromatin context.
zda, et al., The structure formed by inverted repeats in p53 response elements determines the Please cite this article in press as: V. Bra transactivation activity of p53 protein, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.12.113
zda et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6 V. Bra
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