Sequence Variation in the Response Element Determines Binding by

Article pubs.acs.org/biochemistry

Sequence Variation in the Response Element Determines Binding by the Transcription Factor p73 Ana Ramos, Pui-Wah Tse, Jessie Wang, Abdul S. Ethayathulla, and Hector Viadiu* Instituto de Química, Universidad Nacional Autónoma de México (UNAM), Circuito Exterior, Ciudad Universitaria, Mexico City, D.F. 04510, Mexico ABSTRACT: How the sequence of a response element affects the binding of a transcription factor and, ultimately, the differential rate of transcription of genes under its control is not well-understood. In the case of the p73 transcription factor, it binds to >200 response elements to trigger developmental, cell arrest, and apoptotic pathways. The p73 response elements match the 20 bp consensus sequence of the p53 response elements that are formed by two 10 bp half-sites, where each half-site is an inverted repeat of two 5 bp quarter-sites. Using sedimentation velocity and fluorescence anisotropy experiments, we studied how systematic variations in the sequence of a half-site response element modify the DNA binding affinity of the p73 DNA-binding domain. We observed that each nucleotide position in the response element has a different influence in determining the binding of the p73 DNA-binding domain. The cytosine in the fourth position of each quarter-site is the largest determinant of DNA binding, followed by the nucleotide in the fifth position, and last, the first three positions show a slight regulatory preference for purines. Together with previous structural and functional results, our data suggest a hierarchical model of binding in which some nucleotide positions in the response element are more important than others in determining the binding of the transcription factor.

T

where each 10 bp half-site matches the 5′-PuPuPuC(A/T)(A/ T)GPyPyPy-3′ consensus sequence. Each 20 bp full-site response element can be divided into two 10 bp half-sites, and each 10 bp half-site is formed by two 5 bp inverted repeats called quarter-sites. Some response elements are longer because they include insertions of one to three nucleotides between the half-sites.11,25 To understand the first step of transcription activation, the binding of the specific transcription factor to the response element, we used the DNA-binding domain (DBD) of the p73 transcription factor as a model. The transcription factor p73 regulates neuronal and epidermal differentiation.26 Moreover, together with the other two members of the p53 transcription factor family, p63 and p53, p73 regulates apoptotic and DNA repair pathways.27 To regulate such a large network of genes, p73 binds to more than 200 response elements using the central DBD of the protein.28 The activation of transcription by the p73, p63, or p53 tetramers requires the binding of two DBD dimers to a 20 bp full-site response element. The structural studies on complexes between the DBD tetramers of the members of the p53 family with response elements have established that the spatial arrangement of the complexes follows the symmetry determined by the DNA sequence.29−31 As the full-site response element is a repeat of two half-sites, for each dimer

he differential expression of genes is a fundamental biological phenomenon that underlies cell differentiation and development.1 The existence of proteins (transcription factors) that bind to specific DNA sequences (response elements) and regulate the differential expression of genes was described decades ago.2 The first step for the activation of gene transcription is the binding of a specific transcription factor to the response element that controls that gene.3 Disregarding chromatin packing effects, the simplest model of transcription activation postulates that the response element sequence encodes gene activation and its transcriptional level;4 the transactivation induced by the transcription factor p53 is one example of such response element regulation.5 Nonetheless, the subtle relationship between variations in the sequence of the response elements and how such sequence variation results in differential gene expression remains a mystery.6 The first experiments to define the binding site of the transcription factor p53 found that p53 activated genes that contained two consecutive consensus 5′-PuPuPuC(A/T)(A/ T)GPyPyPy-3′ sequences.7,8 Many individual gene studies verified >200 genes that contain similar response elements that promote transcription.9−11 More recently, a large number of genome-wide studies, mainly using ChIP-seq technology, have mapped thousands of p53-binding sites that do not necessarily induce transactivation.12−24 Experiments that aimed to locate the response elements for family members p73 and p63 have resulted in binding sites that match the same p53 consensus. As it was first described for the p53 response elements, the p73 and p63 response elements are also DNA sequences of 20 bp © 2015 American Chemical Society

Received: February 14, 2015 Revised: October 15, 2015 Published: November 3, 2015 6961

DOI: 10.1021/acs.biochem.5b00152 Biochemistry 2015, 54, 6961−6972

Article

Biochemistry

chloride, 10 mM sodium citrate (pH 6.1), 5 mM dithiothreitol, and 5 μM zinc chloride. The different steps in the purification protocol were monitored by 15% Coomassie-stained sodium dodecyl sulfate−polyacrylamide gel electrophoresis and Western blots using an anti-His antibody. For future experiments, the pure p73DBD protein was concentrated with centrifugal filter concentrators to a final concentration of 10 mg/mL. Fluorescence Anisotropy. Fluorescence anisotropy experiments were performed in a buffer with 100 mM sodium chloride, 10 mM sodium citrate (pH 6.1), 5 mM DTT, and 5 μM zinc chloride. The concentrated pure p73DBD protein was serially diluted in 17 samples with a final volume of 500 μL with a protein concentration that ranged from 1 nM to 160 μM. The 5′-fluorescein-labeled double-stranded oligonucleotides were added to a constant final concentration of 50 nM. The reference half-site response element sequence was the palindromic 12 bp 5′-tGGGCATGCCCa-3′ double-stranded DNA (half-site in capital letters). For the experiments with shorter oligonucleotides, the length was shortened successively by 2 bp each time by removing 1 bp from each end to produce DNA lengths of 10, 8, and 6 bp. For the experiments with variations from the reference sequence, the three possible nucleotide substitutions were made in each of the five positions of each quarter-site. Tubes were incubated on ice for 45 min before the anisotropy measurements were taken at 25 °C in a Hitachi F-2000 fluorescence spectrophotometer with polarization filters at excitation and emission wavelengths of 494 and 521 nm, respectively. We did not observe any change in the excitation and emission of the fluorescein upon formation of the complex. Each experiment was repeated three times. The anisotropy, rn, for each binding experiment was calculated using the following equation:

to bind to one half-site response element, the two dimers in the tetramer are translated 10 nucleotides or 34 Å with respect to each other. As each quarter-site in the half-site is an invertedrepeat sequence of the other quarter-site, each monomer in the dimer is rotated 180° with respect to each other. Overall, each of the four monomers in the tetramer binds to one of the four quarter-sites in the full-site response element. This symmetry is conserved in every DBD tetramer of the p53 protein family. The full-site response element is the most relevant sequence for biological activity,11 and in previous work by our laboratory,25,29 we performed a structural−functional characterization of how the p73 DBD recognizes some full-site response elements. To facilitate the identification of the molecular determinants of the p73 DBD affinity for its response elements, we decided to systematically study the basic DNA-binding unit observed in the sedimentation experiments, a p73DBD dimer bound to a half-site response element. To further simplify our analysis, we used palindromic half-sites in which the inverted quarter-site sequences were identical. In this manner, to elucidate the contribution to binding of each nucleotide position, we measured the affinity of the p73 DBD toward half-site response elements with all the possible variations in every position of the quarter-site response element. We support a view in which small sequence variations in the response element result in different amounts of time that a transcription factor spends bound to its response element.

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MATERIALS AND METHODS Cloning. Using the human p73 gene as a template, the DNA sequence encoding amino acids 115−312 of the p73 DNAbinding domain was amplified by polymerase chain reaction (PCR). The PCR product was cloned into the pET28a bacterial expression vector (Novagen) using the EcoRI and HindIII restriction sites. Protein Expression and Purification. The pET28a plasmid with the p73 DBD sequence inserted with a His tag at the N-terminus was used to transform Escherichia coli BL21/ DE3 cells. In this work, we call the expression product of this construct p73DBD protein. Cells carrying the p73DBDpET28a plasmid were cultured overnight in 5 mL of LuriaBertani medium with 30 μg/mL kanamycin at 37 °C and used to incubate 1 L of Luria-Bertani/kanamycin media. When the optical density of the culture, measured at 600 nm, reached 0.6−0.8 absorbance units, protein overexpression was induced for 4 h at 25 °C with a final isopropyl β-D-thiogalactopyranoside concentration of 0.5 mM. Cells were harvested by centrifugation at 2500g, and the pelleted cells were resuspended in a lysis buffer of 500 mM sodium chloride, 20 mM sodium citrate (pH 6.1), and 10 μM zinc chloride. After phenylmethanesulfonyl fluoride had been added to a final concentration of 1 mM to inhibit proteases, cells were lysed in a French press. The cell lysate was centrifuged at 70000g for 30 min at 4 °C. The supernatant was incubated for 30 min at 4 °C with 1 mL of nickel-nitrilotriacetic resin. After the incubation step, the supernatant/resin solution was transferred to a gravity column. The resin was first washed with 100 mL of lysis buffer, and then 50 mL of the same buffer with 20 mM imidazole added to remove proteins bound nonspecifically to the resin. The p73DBD protein was eluted from the affinity resin in lysis buffer with 300 mM imidazole. The protein was further purified by gel filtration chromatography using a Superdex-200 column equilibrated with 100 mM sodium

I

rn =

I⊥ I I⊥

−1 +2

where I∥ is the intensity obtained when the emission polarizer is oriented parallel to the direction of the polarized excitation and I⊥ describes the intensity measured when the polarizer is perpendicular to the polarized excitation.32 The calculated anisotropy values were converted into the fraction bound with the equation r − rF Fbound = n rB − rF where rn is the anisotropy value corresponding to the nth measurement of a certain protein concentration and rF and rB are the anisotropies of the free and bound species, respectively. Using the software Prism, the anisotropy data were fitted to the one-site binding model with cooperativity described by the equation to determine the dissociation constant, Kdapp: Fbound =

rmaxX h Kdapp + X h

where rmax is the maximal anisotropy value, X the p73DBD concentration, and h the Hill cooperative coefficient. We treated the p73DBD dimer as a one-site binding species, and our fitting did not account for the existence of more than one binding species. Analytical Ultracentrifugation. Sedimentation velocity experiments were performed in a Beckman Optima XL-I 6962


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Table 1. Sedimentation Coefficients and Apparent Binding Constants of the p73DBD Protein in Complex with DNAs of Different Lengthsa

p73DBD p73DBD with 8 bp DNA p73DBD with 10 bp DNA p73DBD with 12 bp DNA

experimental sedimentation coefficient (S)

theoretical S assuming a p73DBD monomer

theoretical S assuming a p73DBD dimer

Kdapp (μM)

2.10 3.88 4.03 4.29

2.36 3.01 3.10 3.28

3.49 4.08 4.29 4.35

12.0 6.0 2.8

a

The bold data represent the calculated sedimentation coefficients that are closer to the experimental sedimentation coefficients. As the models used for the theoretical values are based on the p73DBD crystal structure with or without DNA of different lengths using HYDROPRO,35 the bold values represent the model most likely to explain the experimental results.

Figure 1. Minimum DNA length required for p73 DNA binding. (A) Sedimentation coefficient distributions of p73DBD bound to DNA molecules of 12, 10, and 8 bp. As a reference, we include the experiments with p73DBD in the absence of DNA that remains a monomer, and p73DBD in the presence of a full-site response element that binds as a tetramer (last peak) and as a dimer (not shown for the sake of clarity). The c(S) distribution graph resulted from fitting the data from sedimentation velocity experiments to the Lamm equation using SEDFIT.34 (B−D) Binding affinity constants of p73DBD for fluorescein-labeled double-stranded DNA molecules of 12, 10, 8, and 6 bp. The Kd estimation resulted from fitting the fluorescence anisotropy binding data to a one-site binding equation.

The data were fitted to the Lamm equation using the SEDFIT software to calculate c(s) distributions.34 To obtain the theoretical sedimentation coefficient, we used the p73DBD crystal structure alone and in complex with dsDNAs of 12, 10, and 8 bp. Using the crystal structure of p73DBD, a primary hydrodynamic model was used as implemented in the HYDROPRO software to calculate the theoretical sedimentation coefficients listed in Table 1.35

analytical ultracentrifuge with an An-60 Ti rotor at 50000 rpm and 20 °C. The 400 μL samples were loaded in a double-sector centerpiece with a buffer of 100 mM sodium chloride, 10 mM sodium citrate (pH 6.1), 5 mM DTT, and 5 μM zinc chloride as a reference. The same buffer was used to prepare the final dilution of all the samples. The radial scans for the protein samples were collected at 280 nm with a p73DBD protein concentration of 64.5 μM. The data for the protein−DNA samples were collected at the fluorescein absorbance wavelength of 488 nm, using the same p73DBD protein concentration of 64.5 μM, and a 5′-fluorescein-labeled double-stranded DNA concentration of 3−3.4 μM. The software SEDNTERP was used to calculate the partial specific volume, buffer viscosity, and buffer density of the samples.33

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RESULTS Oligomerization State of the p73DBD Protein in Complex with DNAs of Different Lengths. The p53 family of transcription factors binds to response elements with a consensus 5′-PuPuPuC(A/T)(A/T)GPyPyPy-3′ half-site re6963


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Figure 2. Sedimentation coefficient values for the complexes of p73DBD with half-site response elements. Graphs of sedimentation coefficient distributions for the complexes of p73DBD bound to fluorescein-labeled 12 bp double-stranded DNA molecules containing a half-site response element. Only the protein−DNA complexes and free DNA were detected because only the fluorescein signal at 488 nm was followed in the sedimentation velocity experiments. The c(S) distribution graph resulted from fitting the data from sedimentation velocity experiments to the Lamm equation using SEDFIT.34 As a reference, all the graphs include the c(S) distribution of p73DBD in the absence of DNA that remains a monomer (in this experiment, the signal at 280 nm was measured). (A) c(S) distribution of p73DBD in complex with the reference 5′-GGGCATGCCC-3′ response element sequence. All the graphs contain this c(S) distribution as a reference. (B) c(S) distribution of p73DBD in complex with response element sequences with purine and pyrimidine changes in the first position of the quarter-site. (C) c(S) distribution of p73DBD in complex with response element sequences with purine and pyrimidine changes in the second position of the quarter-site. (D) c(S) distribution of p73DBD in complex with response element sequences with purine and pyrimidine changes in the third position of the quarter-site. (E) c(S) distribution of p73DBD in complex with response element sequences with purine and pyrimidine changes in the fourth position of the quarter-site. (F) c(S) distribution of p73DBD in complex with response element sequences with purine and pyrimidine changes in the fifth position of the quarter-site. 6964


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Besides the oligomerization state analysis with oligonucleotides of different sizes, we studied the oligomerization state of the complexes between the p73DBD protein with half-site response elements that had single-site changes in each position of the two quarter-sites. Specifically, we have conducted a characterization of the sedimentation profile of the p73DBD dimer in complex with 11 half-site response elements. The majority of the measured sedimentation coefficients of the complexes between the p73DBD dimer and the 11 tested 12 bp oligonucleotides varied slightly depending of the position that was modified. We analyzed variations of the 12 bp 5′aGGGCATGCCCt-3′ oligonucleotide as a reference sequence for the consensus p53 half-site response element (Figure 2A). The sedimentation velocity experiments can be grouped into three classes. The first class corresponds to sequences with modifications at position 1, 2, or 3 of the reference quarter-site response element. Their S values were high and close to the 4.29 S sedimentation coefficient of the reference half-site sequence 5′-GGGCATGCCC-3′ (Figure 2A). In the first class of sequences, those with purines in the first three positions of the quarter-site (5′-AGGCATGCCT-3′, 5′-GAGCATGCTC3′, and 5′-GGACATGTCC-3′) had the highest S value with sedimentation coefficients between 4.12 and 4.36 S (Figure 2B−D). In the same group, the p73DBD complexes with a pyrimidine in one of the first three positions of the quarter-site (5′-TGGCATGCCA-3′, 5′-GTGCATGCAC-3′, and 5′-GGTCATGACC-3′) had slightly lower sedimentation coefficients, between 3.98 and 4.26 S, that reflected its slightly lower affinity (Figure 2B−D). The S values for sequences with modifications in the first three positions of each quarter-site are identical or very close to the value of 4.29 S observed for the reference sequence and the theoretical value of 4.35 S predicted for a p73DBD dimer bound to the 12-mer containing a half-site response element (Table 1); their values range between 3.98 and 4.36 S, indicating that a p73DBD dimer binds to these sequences (Figure 2B−D). The second class of sequences corresponds to those in which the conserved cytosine in the fourth position of each quarter-site response element was replaced (5′-GGGAATTCCC-3′, and 5′-GGGGATCCCC-3′); the S values for these complexes diminish significantly to 3.38 and 3.50 S (Figure 2E). Such lower values approach the theoretical value of 3.28 S calculated for the p73DBD monomer bound to a 12-mer oligonucleotide (Table 1), pointing to the possibility that a p73DBD monomer is able to bind to sequences that lack the most conserved feature of the p53 halfsite response element, the central 5′-CNNG-3′ sequence. Finally, a third class is formed by those sequences with variations in the fifth base of the quarter-site response element (5′-GGGCGCGCCC-3′, and 5′-GGGCTAGCCC-3′). These complexes had a sedimentation coefficient similar to, although lower than, those of the complexes with modifications in the first three positions but higher than when the fourth position was modified. Their S values ranged between 3.82 and 3.94 S, showing that a p73DBD dimer also binds to these sequences (Figure 2F). Overall, the p73DBD complexes bound to response elements with changes in the first three positions had sedimentation coefficients higher than those in cases in which variations were introduced in the fourth or fifth positions of the quarter-site response element (Figure 2). The sedimentation velocity experiments demonstrate that the p73DBD dimer is the oligomer that preferentially binds to the p53 half-site response

sponse element sequence. Exhaustive crystallization and biochemical experiments with the p73DBD protein have shown that the 5′-GGGCATGCCC-3′ half-site response element sequence favors the stability of the DNA−protein complex.25,29 For this reason, we decided to use this half-site sequence as a reference to define the role of each nucleotide position in determining p73DBD binding to half-site response elements. Our goal with these experiments was to pinpoint the contribution to p73DBD binding of each nucleotide position in the half-site response element. To understand the binding of p73DBD to response element sequences, we first determined the oligomerization state of the complexes between the p73DBD protein and half-site response elements. We measured the sedimentation velocity of the p73DBD−DNA complexes by following the absorption signal of the fluorescein-labeled oligonucleotides. From previous work, we knew that, in the absence of DNA, the p73DBD protein (23 kDa) is a monomer in solution that shows a sedimentation coefficient of 2.10 S.25 This S value is close to the theoretical value of 2.36 S calculated for the p73DBD monomer crystal structure, and far from the theoretical value of 3.49 S for the p73DBD dimer (Table 1).35 Gel filtration chromatography experiments confirm this observation because, in the absence of DNA, we never observed a p73DBD dimer. Instead, in the presence of DNA, our experiments showed a p73DBD dimer as the oligomeric form bound to half-site response elements in which p73DBD dimerizes and forms a DNA−protein complex of ∼54 kDa [p73DBD dimer (2 × 23 kDa) + 12 bp dsDNA (8 kDa)]. When oligonucleotides of different sizes were used, the sedimentation coefficient of the p73DBD dimer in complex with DNA varies depending on DNA length. The sedimentation velocity experiments with DNA of different lengths confirmed that a p73DBD dimer binds to 12, 10, and 8 bp double-stranded oligonucleotides (4.29, 4.03, and 3.88 S, respectively) (Table 1 and Figure 1A). The experimental sedimentation coefficient values for the p73DBD complexes with oligonucleotides of different sizes that contain the half-site response element are close to the theoretical values calculated using models based on the p73DBD crystal structure (4.35, 4.29, and 4.08 S) (Table 1).35 To corroborate the importance of the central base pairs for binding of p73DBD to the half-site response element, we determined the DNA affinity of p73DBD for half-site response elements of decreasing length. We measured that having 12 or 10 bp oligonucleotides containing the reference 5′-GGGCATGCCC-3′ half-site sequence yielded very similar apparent binding constants: 2.8 μM versus 6.0 μM (Figure 1B,C). Once the half-site length was reduced to only the central 8 bp 5′GGCATGCC-3′ sequence, DNA affinity decreased, but not drastically, to 12.1 μM (Figure 1D). In conclusion, these series of experiments indicate that p73DBD binds as a dimer to half-site response elements and that the flanking first and tenth base pairs in the half-site response element contribute little toward p73DBD binding. The observed differences in the sedimentation coefficient are due to the different sizes of the oligonucleotide, and the affinity decreases as the DNA length decreases. Together, the binding and sedimentation experiments conducted with DNA of different lengths confirm that the most important determinants of DNA binding by p73DBD lie within the central 8 bp of the half-site response element. Oligomerization State of the p73DBD Protein in Complex with Half-Site Response Element Variants. 6965


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Table 2. Apparent Binding Constants and Cooperativities of the p73DBD Protein for Half-Site Response Element Variants

The Hill coefficient was obtained from fitting the data to a one-site binding model with cooperativity (Materials and Methods and Figure 3). bFrom ref 36. a

shows the fitting and Table 2 the Kdapp and the Hill coefficient values for all the data. The DNA binding affinity results displayed in Table 2 can be divided into three classes. The first class corresponds to sequences with a single mutation in any of the first three positions of each quarter-site (Table 2 and Figure 3B−D). Among these sequences, there were differences in the observed DNA binding affinity when the introduced mutation was a purine or when it was a pyrimidine. When any of the guanines at positions 1−3 of the reference sequence were replaced with an adenine, the DNA affinity of the p73 DBD did not change. The three sequences with an adenine in any of the three first positions had basically identical apparent binding constants with respect to the standard sequence. The Kdapp values were 3.2 ± 0.6 μM for the 5′-AGGCA-3′ quarter-site, 5.0 ± 0.9 μM for 5′-GAGCA-3′, and 4.9 ± 0.4 μM for 5′-GGACA-3′ (Table 2 and Figure 3B−D). On the other hand, whenever any of the guanines at positions 1−3 were replaced with a pyrimidine, the DNA affinity of the p73 DBD slightly decreased. When the new base was a thymine, a slight decrease in the level of DNA binding was seen: 13.2 ± 0.7 μM for the 5′-TGGCA-3′ quartersite, 6.4 ± 0.5 μM for 5′-GTGCA-3′, and 8.6 ± 0.8 μM for 5′GGTCA-3′ (Table 2 and Figure 3B−D). The same small effect was observed when the new base was a cytosine: Kdapp values of 12.6 ± 0.5 μM for the 5′-CGGCA-3′ quarter-site, 8.0 ± 0.1 μM for 5′-GCGCA-3′, and 12.2 ± 0.5 μM for 5′-GGCCA-3′ (Table 2 and Figure 3B−D). In general, the nine variants in the first three positions of the quarter-site follow a pattern in which sequences with three purines show a higher affinity (3−5 μM) than the affinities of those sequences in which a pyrimidine replaced the guanine (6−13 μM).

element, except when the conserved 5′-CNNG-3′ sequence is modified. Binding Affinity of the p73DBD Protein for Half-Site Response Element Variants. To understand how variations from a high-affinity consensus response element sequence are tolerated to maintain p73DBD binding, we took the 5′GGGCATGCCC-3′ consensus response element sequence as a reference, and we introduced the three alternative nucleotides into each of the five positions of the quarter-site response element (5′-GGGCA-3′). The systematic single-change variations of our reference sequence allowed us to study the role of each position in the binding of the p73DBD dimer to a half-site response element. All the studied oligonucleotides were 12-mers with a half-site response element. We created a collection of 16 half-site response elements: the reference sequence and 15 variants, three for each of the five positions in the quarter-site. Each of the 15 variant oligonucleotides had a mutation in each of the two quarter-sites; thus, each variant half-site had two identically mutated quarter-sites. To evaluate the effect of response element mutations on the binding affinity of p73DBD for DNA, we used the 5′GGGCATGCCC-3′ half-site response element as a standard, which has a Kdapp for DNA equal to 3.2 ± 0.4 μM (Table 2 and Figure 3A). For all the studied sequences, the raw anisotropy value reached at saturation was similar, pointing to the presence of identical oligomeric species. For each response element variant, we calculated an apparent DNA binding constant by measuring the fraction of protein bound to the DNA at different p73DBD concentrations in fluorescence anisotropy experiments. The results were fitted to a one-site binding equation with cooperativity (Materials and Methods). Figure 3 6966


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Figure 3. Kd values of the p73 DBD for half-site response elements. Binding affinity curves of p73DBD for fluorescein-labeled 12 bp double-stranded DNA molecules containing a half-site response element. After the changes in fluorescence intensity had been measured at different axes of polarization when different p73DBD concentrations bind to a constant DNA concentration of 50 nM, all the Kdapp estimations resulted from fitting the calculated fluorescence anisotropy binding data to a one-site binding equation with cooperativity. Each curve was measured three times, and the apparent affinity constants and Hill coefficients with their respective estimated errors are listed in Table 2. (A) Binding affinity curve of p73DBD toward the reference 5′-GGGCATGCCC-3′ half-site response element sequence. (B) Binding affinity curve of p73DBD toward the response element sequences with the three possible changes in the first and tenth positions of the half-site response element. (C) Binding affinity curve of p73DBD toward the response element sequences with the three possible changes in the second and ninth positions of the half-site response element. (D) Binding affinity curve of p73DBD toward the response element sequences with the three possible changes in the third and eighth positions of the half-site response element. (E) Binding affinity curve of p73DBD toward the response element sequences with the three possible changes in the fourth and seventh positions of the half-site response element. (F) Binding affinity curve of p73DBD toward the response element sequences with the three possible changes in the fifth and sixth positions of the half-site response element.

The second class of sequences corresponds to those in which the cytosine in the fourth position was modified (Table 2 and Figure 3E). As this fourth position is the most conserved nucleotide in the described consensus sequence of the quarter-

site response element, it was expected that these modifications had the most deleterious effect on DNA binding. For example, the 5′-GGGGA-3′ sequence had a Kdapp of 27.6 ± 9.7 μM and the 5′-GGGAA-3′ sequence had a Kdapp of 27.2 ± 6.2 μM. In 6967


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Figure 4. Hierarchy of contacts for the molecular recognition of the 5′-GGGCA-3′ quarter-site response element by a monomer of p73DBD. The color code shows the importance of the nucleotide positions for p73DBD binding. The most important contact for response element recognition is the hydrogen bond between Arg300 and the guanine at position 4′ (dark gray), followed by the DNA deformation observed at position 5 (gray), and finally the positions that are the least affected by nucleotide changes are the first three nucleotides of the quarter-site response element (light gray). The scheme is based on PDB entry 3vd0.25

fluorescence anisotropy data in classifying the tested variations in three groups, which demonstrates the hierarchical importance of the different positions in the half-site response element.

comparison, the substitution for a thymine, 5′-GGGTA-3′, did not have produce a detrimental drop in DNA binding with a Kdapp of 7.4 ± 0.3 μM (Table 2 and Figure 3E). In the third class of sequences, we grouped those sequences in which mutations at the fifth position of the quarter-site response element were introduced (Table 2 and Figure 3F). Each of these mutations resulted in a significant decrease in the level of binding, but generally not as drastic as when the fourth position was modified. We found that the sequence 5′GGGCC-3′ had a Kdapp of 10.0 ± 0.6 μM, the sequence 5′GGGCG-3′ had a Kdapp of 12.9 ± 2.2 μM, and the sequence 5′GGGCT-3′ had a Kdapp of 12.8 ± 1.4 μM (Table 2 and Figure 3F). With regard to the cooperativity of the p73DBD dimer upon DNA binding, we found that all of the half-site response element sequences, except one, gave a Hill cooperativity coefficient close to 1, indicating low or no cooperativity (Table 2). Besides a slight tendency of sequences with a pyrimidine in the first three positions to show cooperativity, the only exception with high cooperativity was the sequence 5′GCGCATGCGC-3′ that had a Hill cooefficient of 1.8, a value closer to what we had previously observed for the longer ΔNp73δ isoform that includes the oligomerization domain of p73 (Table 2).36 Overall, modifications in the fourth and fifth positions of the quarter-site response element have a stronger effect on the DNA binding affinity of the p73 DBD than modifications in the first three positions of the quarter-site response element. Moreover, the sedimentation velocity data coincide with the

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DISCUSSION The ability to transcribe some genes more than others is a quintessential property of living organisms that underlies cell differentiation and development. The study of the molecular mechanisms that lead transcription factors to express some genes and not others is an active area of biomedical research.37 In this study, we have taken the DNA-binding domain of the transcription factor p73 as a model to examine how the different base pairs in a response element determine the binding of a transcription factor to DNA. Our main conclusion is to postulate a hierarchical model of p73 response element recognition in which some base pairs in the response element are more important than others. From biochemical, structural, and cellular data, a scheme for how to think about the role of the different nucleotides in the response element of the transcription factor p73 emerges.25,29,38 This model is likely to be conserved in all the members of the p53 family of transcription factors.10,39 As a consequence of our general conclusion that p73 uses a hierarchical mode of response element recognition, our data suggest three steps that the p73 DBD dimer follows to recognize its response elements. The existing structural evidence for p53 also supports a scheme of tripartite response element recognition.31,40−42 First, the conservation of cytosine 6968


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Biochemistry

pairs result in a DNA minor groove narrower than that of 5′NGCN-3′ tetranucleotides.43 Interestingly, all the determined p53−DNA tetrameric structures have also shown a narrower minor groove exactly at the center of each half-site response element.31,40−42 Moreover, the p53 DBD induces a narrow DNA minor groove in the middle of the half-site of all the determined p53 DBD−DNA crystal structures that might facilitate the switch to a Hoogsteen conformation.31,40 As an insightful observation, the highest-resolution structure of p53 in complex with a full-site response element has the purine ring of the adenine in each fifth position flipped 180°.40 The importance of these conformational changes remains to be understood, but the binding preference that we have observed for a sequence with a narrower minor groove like 5′-CATG-3′ supports the importance of a noncanonical DNA conformation for response element recognition by the members of the p53 protein family. As it has been observed in in vivo transactivation assays,10 presumably, a sequence like 5′-CATG-3′ that permits adenine to adopt a Hoogsteen conformation is important for activation. The third and least determining factor for p73 to recognize a response element is the sequence of the first three nucleotides in each quarter-site. From the structural data, we know that only two DBD residues in each monomer recognize the DNA bases in this region of the response element (Figure 4).25,29 First, a reduced Cys297 recognizes a pyrimidine complementary to the third position of the quarter-site. Second, Lys138 in loop L1 is able to form hydrogen bonds to O6 or N7 in the purine ring of the guanines at positions 2 and 3. Being in flexible loop L1, Lys138 in p73 might also recognize O6 in guanine 1. We have postulated that loop L1 flexibility and Lys138 acetylation act as a switch to activate apoptotic response elements.29 Interestingly, acetylation of the equivalent position in p53, Lys120, has been found to regulate p53 switching between DNA repair and apoptotic pathways.44,45 Loop L1 is flexible in all the tetrameric p53 protein family structures.31,40−42 Interestingly, we found that the presence of a pyrimidine at any of the first three positions of the quarter-site, particularly cytosine in the second position, resulted in an increase in the cooperativity of binding. The dimer of p73DBD is formed by one α-helix from each monomer contacting each other by hydrophobic contacts of residues Pro195 and Asn196.25 We have not determined a crystal structure of p73DBD bound to response elements containing pyrimidines in the first three positions; the determined structure closest to a nonconsensus response element (PDB entry 4g83) shows subtle conformational rotations around the dimerization helices with respect to the conformation observed when p73DBD binds to consensus response elements (for example, PDB entry 4g82).29 Moreover, we also observed conformational arrangements of the dimerization helix when the response element had insertions in the middle of the full-site response element.25 The ability of p53 to rearrange the dimerization interface has been previously linked to cooperativity,46 and the acetylation of Lys120 in p53, the residue homologous to Lys138 in p73, triggers the expression of apoptotic pathways.47,48 In the data presented here, we document the ability of p73DBD to modify its cooperativity depending on the sequence of the response element, but we still do not understand the physiological consequences of an increased cooperativity toward response elements with pyrimidines in the first three positions. There is no obvious explanation for why p73DBD shows an increased cooperativity when pyrimidines are in the first three positions,

and guanine in the 5′-CNNG-3′ sequence at the center of each half-site response element is the most determining feature for any DNA sequence to be recognized as a response element by the members of the p53 protein family. Second, the presence of flexible sequences at the center of each half-site response element that allow a narrow DNA minor groove is the second determining factor recognizing the p73/p63/p53 response element. Third, the first three positions for each quarter-site response element tolerate variations, and it appears that the purine/pyrimidine ratio in the first three positions of the quarter-site response element might have a regulatory role in transactivation.10 First, the most critical chemical recognition signature for the p73 dimer to identify response elements is the existence of a cytosine separated by two bases from a guanine (5′-CNNG-3′). We also know from sedimentation velocity experiments that the p73DBD dimer is the basic oligomer required for DNA recognition.25 At the same time, in every description of the p53 response element, the fourth base of each quarter-site is the most conserved.7−11 Our data show that this fourth position of the quarter-site is the most important position for DNA binding (Table 2). Structurally, we know that two Arg300 residues in the dimer, one from each p73DBD monomer, recognize two guanines in the center of the half-site response element (5′-CNNG-3′) (Figure 4).25 One of the recognized guanines is complementary to the first cytosine in position 4 of the half-site, and the second recognized guanine is separated by two nucleotides at position 7. The same double guaninearginine recognition motif is also found in the p53 and p63 dimers.31,40−42 The double arginine-guanine recognition motif always occurs at the center of each half-site, and it repeats twice in the tetramer of every p53 protein family member to recognize the full-site response element. Interestingly, the only sequences in which we did not find the p73DBD dimer as the main binding species in the sedimentation experiments was when the conserved cytosine in the fourth position was mutated. We considered this observation as a confirmation of the need to have the 5′-CNNG-3′ sequence as a requisite for response element recognition. In conclusion, the most important binding step is that in which the p73 tetramer uses the four Arg300 residues from the four DBD monomers to recognize the four fourth positions in each of the four quartersites of the full-site response element. The second factor determining the response element recognition by p73 lies in the nucleotide occupying the fifth position in each quarter-site response element. The preferred two nucleotides in the central fifth positions of the two quartersites in the half-site are adenine and thymine, as in the sequence 5′-CATG-3′. Any other substitution that we tested (5′-CGCG3′, 5′-CCGG-3′, or 5′-CTAG-3′) resulted in a lower affinity. Again, previous structural work helps us explain this preference. In all the determined p73DBD structures, although there are no direct protein contacts to the central bases (Figure 4), the DNA conformation at the center of each half-site shows a minor groove narrower than that of canonical B-form DNA.25,29 In an ideal B-DNA conformation, the distance between the two C1 atoms from each ribose in the central AT base pair is 10.7 Å. Instead, in the determined p73 DBD structures in complex with DNA, we have observed a narrower minor groove with C1−C1 distances between 10.5 and 9.8 Å. An exhaustive analysis of DNA conformation in tetranucleotide segments in the crystal structures of protein−DNA complexes in the Protein Data Bank has determined that 5′-NATN-3′ tetranucleotide base 6969


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specially cytosine in the second position; we think that it might be related to our previous observation that links the DNA recognition by Lys138 with rotation of the dimerization interface of the p73DBD dimer.29 We have proposed a structural explanation in which once p73 binds to the central nucleotides in the half-site response element, the purine/ pyrimidine ratio in the first three positions of each quarter-site read by Lys138 appears to regulate the level of activation of transcription.29 The stoichiometry of binding for the p73 DBD to its response element has been defined by sedimentation velocity experiments.25 When the tested DNA had a full-site response element, p73 DBD dimers or tetramers were bound to the DNA. When the DNA just had a half-site response element, as all the oligonucleotides tested in this work, only the p73 DBD dimer was bound. We observed p73DBD monomers bound when the conserved fourth position was mutated. Although structurally a single p73 DBD recognizes a single quarter-site response element, we have consistently found that the p73DBD dimer is the most stable oligomer that binds DNA. Interestingly, functional studies with p53 have shown that the p53 dimer bound to the half-site response element is able to promote transactivation, although at a level considerably lower than that of the p53 tetramer.20 Nonetheless, the 20 bp full-site response element is the most physiologically relevant sequence. We have previously studied some 20 bp full-site response elements that we characterized structurally.29 For those few examples of full-site response elements, we encountered a DNA binding affinity in the micromolar range as in the high-affinity consensus-like half-sites of this study. We also found low or no cooperativity among the dimers of the p73 DBD tetramer. Instead, we found that the ΔNp73δ isoform that contains the oligomerization domain binds to the full-site response element in a cooperative manner with an affinity much higher than that of the p73DBD tetramer.36 Our data suggest that the response element uses different nucleotide positions to sequentially achieve its dual function: (1) the binding of p73 and (2) the regulation of transactivation. Our work defines the central four nucleotides of each half-site response element as the crucial residues for binding and identifies the three flanking nucleotides in each side of the halfsite response element as positions that tolerate modification without strong effects on binding. We postulated a model in which positions 4 and 5 of each quarter-site response element determine binding, while positions 1−3 of each quarter-site modulate transactivation (Figure 4). Throughout the structural work of many research groups, the molecular understanding of the specificity of the DNA-binding domain of the members of the p53 family of transcription factors has converged to a model that allows prediction of which DNA sequences will be bound by the p73, p63, and p53 DBDs and indicates that the DBDs in this protein family act in a similar manner. Nonetheless, these structural studies of only the DBDs have not yet explained the diverse transcriptional profiles of the p53 protein family. There are already some efforts to understand how the C-termini that are unique for each member of the family might affect target binding specificity.42,49−53 In the coming years, we must direct our attention to understand how the C-termini of p73, p63, and p53 influence the transcriptional specificity of this fascinating family of transcription factors.

Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

A.R. and P.-W.T. contributed equally to this work. Funding

Research funded by Dirección General Asuntos del Personal Académico, Universidad Nacional Autónoma de México (IA202215). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS DBD, DNA-binding domain; dsDNA, double-stranded DNA; DTT, dithiothreitol.

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REFERENCES

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