Unusual Characteristics of the DNA Binding Domain of Epigenetic Regulatory Protein MeCP2 Determine Its Binding Specificity

Protein, DNA, and Other Reagents

The DNA binding domain of MeCP2 (MBD, residues 76–167; Uniprot entry P51608) was inserted into a PET21 derivative by the Einstein Protein Production Facility to yield pMCSG7. Following transformation into Rosetta (DE3) cells, the protein was expressed following standard procedures in media containing 0.4% sorbitol. The MBD was purified as previously described²⁸ except that HEPES buffer was used throughout and the His tag was cleaved by recombinant Tev protease as described by the manufacturer (Invitrogen). A molar extinction coefficient (ε_M) of 11460 M⁻¹cm⁻¹ was used to determine the MBD concentration from their 280 nm absorption spectra.

All buffer solution solutes were purchased from Sigma. Labeled and unlabeled DNA oligonucleotides were purchased from Integrated DNA Technologies, Inc. (Coralville, IA), and their concentration was optically determined using extinction coefficients calculated from sequence. The extinction was corrected for the fluorescein absorption at 260 nm when appropriate. The 20 bp oligonucleotide 5′-TCTGGAACGGAATTCTTCTA-3′, with C methylated or unmethylated, was used in our study. This sequence is taken from promoter III of the mouse BDNF gene and is the DNA present in the MBD cocrystal structure.⁸ A random sequence 20 bp oligonucleotide not bearing the CpG site (5′-TCTGGTATGAACTTCTA-3′) was also analyzed. The top strands of each duplex were 5′-labeled with fluorescein or unlabeled. The duplexes used in our studies have stabilities of −35.9 and −26.5 kcal/mol corresponding to melting temperatures of 40.7 and 32.5 °C, respectively, under our experimental conditions, resulting in an undetectable single-strand oligonucleotide at the lowest concentration used in binding assays (5 nM).

Fluorescence, Absorption, and Light Scattering Measurements

All experiments were performed in buffer containing 25 mM Tris-HCl, 6% glycerol, 0.1 mM EDTA, 0.1 mM TCEP, and either 25 or 150 mM KCI (pH 7.6) at 22 °C except for the salt-induced dissociation experiments for which the KCl concentration is specified. The presence or absence of 10 μg/mL nonspecific competitor poly(dA-dT) is noted in the figure and table legends. Absorption measurements were taken with a NanoDrop 2000 UV–vis spectrophotometer. Fluorescence measurements were taken with a Jobin Yvon (Edison, NJ) Fluoromax-3 spectrofluorometer. The intensity of the Raman scattering band of water was used as the internal standard of fluorometer sensitivity. Elastic light scattering (ELS) was also recorded using the Fluoromax-3 spectrofluorometer as a control for protein aggregation. The scattered 350 nm light was collected at an angle of 90° to the incident illumination.

Analytical Ultracentrifuge

Sedimentation equilibrium experiments were performed using the absorption optics of a Beckman XL-I analytical ultracentrifuge with six-channel centerpieces in the Ti-60 rotor. Three concentrations of the MBD in buffer were equilibrated at 20 °C for 24 h each at 10000, 20000 and 30000 rpm. The absorbance scans obtained at 280 nm were globally analyzed using HeteroAnalysis version 1.0.114 (J. L. Cole and J. W. Lary, Analytical Ultracentrifugation Facility, Biotechnology Services Center, University of Connecticut, Storrs, CT) for the weight-average molecular weight. The resolved molecular weight and the 95% joint confidence intervals are reported. The values of ν̅ (from the amino acid composition), density, and viscosity were calculated using Sednterp version 1.06 (B. Hayes, T. Laue, and J. Philo, Sedimentation Interpretation Program, 2003, University of New Hampshire, Durham, NH).

Equilibrium Binding

MBD binding isotherms were calculated and analyzed as described below. Fractional saturation (Y̅) is calculated for equilibrium titrations by

1

where A_obs, A_min, and A_max are the observed, minimum, and maximum values of the measured fluorescence anisotropy, respectively. For the equilibrium P_f^n_H + O_f ↔ PO^n_H, where P_f and O_f are the free concentrations of protein and DNA, respectively, PO is the protein–DNA complex, and n_H is the Hill coefficient, fractional saturation is related to the binding polynomial by

2

where PO and O_tot are the protein–DNA complex and total DNA concentrations, respectively. Substitution and transformation²⁹ yield

3

where k_d is the equilibrium dissociation constant and P_tot is the total protein concentration. Equation 3 can be transformed into a convenient form in which P_tot is substituted with the ratio P_tot^n_H/O_tot as the independent variable to yield

4

Equation 4 reduces to the single-site (Langmuir) binding model with modifications explicitly considering the total DNA concentration where n_H = 1. Fitting eq 4 against Y̅ obtained from the experimental data accurately yields k_d even when the concentration of the DNA is comparable to k_d.

Two-Site Binding Model

If a molecule has two binding sites with different affinities for the same ligand, then eq 2 can be rewritten as

5

where Y̅ is the total saturation of the two sites (Y̅₁ and Y̅₂) and PO₁, PO₂, and O_tot are the protein–DNA complex and total DNA concentrations, respectively. At low concentrations of the DNA target where P_tot ≈ P_free, eq 5 can be transformed into the sum of two isotherms

5a

where k_d1 and k_d2 are the dissociation constants for the first and second binding sites, respectively, and P_tot is the total protein concentration. Because the total anisotropy change is the sum of the change for the two binding events (A_obs = A_obs1 + A_obs2), eq 5a can be transformed into

6

where A_obs, A_obs1, A_obs2, A_min, and A_max are defined for two binding sites as in eq 1. A₀ is the initial value of A_obs. If the measured parameter A_obs is a relative quantity, then A₀ is equal to 0 and eq 6 is transformed into

7

We used the relative value of anisotropy A_rel = (A_obs – A_DNA)/A_DNA as a binding parameter, where A_obs is as in eqs 6 and 7 and A_DNA is the value of the anisotropy of the DNA in the absence of the MBD. Nonlinear least-squares fitting of eq 7 yields values of k_d1, k_d2, A_max1, and A_max2.

We have used oligonucleotides end labeled with fluorescein as in many other studies of protein–DNA complexes.^11,30,31 In those studies and this study, we measure no significant change in the fluorescence intensity of the fluorescein probe upon protein binding under a given solution condition. In this study, we conducted control experiments in which labeled DNA was present in “tracer amounts” to which was added unlabeled DNA. Isotherms identical to those obtained using only the labeled DNA were obtained (data not shown).

Salt-Induced Dissociation Titrations

The electrostatic contribution to formation of the MBD–DNA complex was determined from the net number of ions (cations, m⁺, and anions, x^-) released

8

where k_d is the experimentally determined equilibrium dissociation constant, (k_d)₀ is the value of k_d extrapolated to 1 M salt, and m⁺ is the cation concentration.³² Usually, m⁺ ≫ x^– because of the polyanionic nature of DNA and the polyampholyte nature of proteins. Because the influence of water is negligible at high salt concentrations, SK reflects the net cation release or uptake upon formation of a protein–DNA complex.

SK can be conveniently obtained from salt displacement isotherms in which a protein–DNA complex is dissociated by titration with increasing salt concentrations.³³ In these experiments

9

where

10

with (k_d)₀ transformed into mp, the salt concentration at which half of the initial protein–DNA complex is dissociated. The nonelectrostatic portion of ΔG is calculated from (k_d)₀ (eq 8) by ΔG_nel = −RT ln[1/(K_d)₀].³³

Because the affinity of the protein for the different sequences analyzed differs, Y̅ for the complexes differs under the initial condition of the experiment. An impact of different Y̅ values on the resolved values of SK and mp is circumvented by setting the initial condition of the salt dissociation experiments at P_t = O_t. As was shown previously,³³ there is no dependence on the concentrations of the reactants, P_t and O_t, under that condition. Thus, eq 10 can be used to measure relative rather than absolute values of Y̅.

Equimolar concentrations of the MBD and the unlabeled duplex (0.5–1.0 uM) along with the labeled oligonucleotide at 15 nM were mixed and incubated for 1 h. KCl was then added at the specified concentration and the solution incubated for 1 h prior to measurements. Because the fluorescence of fluorescein is sensitive to high salt concentrations, dissociation of the MBD from DNA was followed by measurement of the anisotropy of the intrinsic protein fluorescence at 330 nm and excitation of 280 nm. A_norml = (A_obs – A₀)/(A_max – A₀), where A_obs, A₀, and A_max are the observed and fitted initial and final values of anisotropy, respectively, and A_norml is a measured parameter. The relative values of Y̅ analyzed range from unity to zero for each salt displacement isotherm (Figure 9B). Nonlinear least-squares fitting of salt displacement isotherms to eqs 8–10 yields values of (k_d)₀, mp, and SK at any macromolecule concentration.³³

Comparison of the Goodness of Fit of Alternative Binding Models

Comparison of the performance of models is typically characterized by the root-mean-square deviations (rmsds) between the theoretical and experimental estimates of the data

11

where X_i and Y_i are the theoretical and experimental estimates, i, respectively, in N experimental points. The normalized rmsd (nrmsd) or error (nrmse) is the rmsd divided by the range of observed values of a variable being predicted

12

The quality of the fit of a model to the data is reflected in the rmsd and nrmsd values. Smaller values indicate a closer relationship between theory and experiment.

MBD Binding Specificity

Key characteristics of a protein–DNA interaction that drive its biological function are its specificity for its target site and its stoichiometry. Our studies compare the binding of the MBD to the DNA duplex present in the MBD cocrystal structure⁸ bearing the CpG site with C either methylated or unmethylated and a “random” sequence oligonucleotide lacking any known determinant for MBD affinity. Figures 1 and 2 summarize the dramatic effect of salt concentration on the discrimination of mCpG from CpG. As noted previously,¹¹ the MBD lacks specificity for mCpG at low salt concentrations. Consistent with this finding, we find that the MBD binds in a low-salt solution mCpG, CpG, and random sequence with comparable affinity within experimental error (Figure 1A and Table 1). Increasing the salt concentration to an approximately physiological concentration (150 mM KCl) slightly increases the affinity of the MBD for mCpG while dramatically weakening its binding to CpG and the random sequence (Figures 1B and Table 1). The observed increase in binding affinity for mCpG with an increasing salt concentration is very unusual for a protein–DNA interaction. In contrast, the suppression of CpG and random sequence binding by salt is canonical protein–DNA binding behavior.³⁴

The 150 mM KCl binding isotherms shown in Figure 1B were extended to higher MBD concentrations in an attempt to define their upper plateaus (Figure 2). Even so, an upper plateau could not be determined, allowing calculation of only lower limits for these binding affinities, ≥10.3 and 17.7 μM, respectively (Table 1). The mCpG isotherm encompasses both specific and nonspecific binding, an observation consistent with the literature.³⁵ Under these conditions, the MBD displays ≥86-fold specificity for mCpG over CpG and ≥147-fold specificity over random sequence DNA (Table 1).

MBD Binding Stoichiometry

We routinely conduct titrations at DNA concentrations greater than the K_D of the reaction to determine the stoichiometry of a binding reaction.^28,33 In light of the structures of the 1:1 MBD–mCpG complexes determined by NMR and crystallography from equimolar mixtures of protein and DNA,^8,9 we were quite surprised to measure a 2:1 stoichiometry for the MBD–mCpG complex (Figure 3). This result was confirmed using analytical ultracentrifugation by monitoring the dye absorption of the fluorescein-labeled mCpG oligonucleotide (Figure 4). The sedimentation equilibrium analysis yielded M_w values for the free DNA, and complexes assembled from 1:1 and 2:1 molar ratios of the MBD and DNA are within error of the values calculated from sequence (square brackets): 12.6 (12.1, 13.0), [12.8]; 24.3 (23.1, 25.4), [23.2]; and 33.1 (31.1, 35.3), [33.6] kDa, respectively. Each of the data sets is described as a single species with no evidence of heterogeneity or active self-association. The homogeneity of the 1:1 and 2:1 complexes was further confirmed by sedimentation velocity analysis (data not shown). Together, stoichiometric titration and analytical ultracentrifugation results make an unassailable case that the MBD binds as a dimer to mCpG-bearing DNA.

A Two-Site “Sequential” Binding Mechanism

How can the 2:1 stoichiometry be reconciled with the published monomeric structures and our binding analyses? To answer this question, we conducted data-dense titrations at 150 mM KCl so that the shape of the isotherms could be precisely determined. It is readily apparent in these isotherms that the Langmuir binding model inadequately describes the binding of the MBD to mCpG (Figure 5, dotted line). Without sufficient data density and careful analysis, this poor fit is easy to either miss or ascribe to experimental error (e.g., Figure 1B; analysis not shown). Indeed, the initial portion of the isotherm (<1 μM) is perfectly described by the Langmuir binding model (Figure 5, inset). [In retrospect, a clue that the binding was more complex than Langmuir was that fits of the Hill equation (eq 4) to isotherms such as that shown in Figure 1 were typically described with coefficients below unity. Such behavior can indicate anticooperativity or heterogeneity.] Thus, the complexity of MBD–mCpG binding is not evident if either the number of data points is too small or the MBD concentration range is too narrow.

The simplest binding model that describes these isotherms is a noninteracting two-site model (Figure 5, solid line). The MBD binds tightly to the first site (K_D1 = 75 ± 10 nM) and 50-fold less so (K_D2 = 3.7 ± 2.6 μM) to the second site (Table 1). Random residuals and lower nrmsd values demonstrate the superiority of the two-site model for formation of the MBD–mCpG complex (Figure 5 and Table 1). Because K_D2 is 1 order of magnitude tighter than nonspecific binding, the second binding event is a distinct and unique molecular interaction.

Because of the wide separation of the resolved affinities, the two binding events occur in sequence. Only after the first site is almost fully occupied does the second site become bound. This conclusion is supported by three independent observations. First, we observe that the MBD is monomeric in solution [M_w = 10.6 (9.8, 11.2), [10.4] kDa] as assayed by sedimentation equilibrium and velocity analysis consistent with literature observations.³⁶ Second, each of the two binding events is separately well described by the Langmuir polynomial. Third, the 1:1 complex is unequivocally monodisperse as assayed by sedimentation equilibrium and velocity analysis. If the binding were at all cooperative or the two binding events were closer in affinity, a mixture of 1:1 and 2:1 complexes would be observed at substoichiometric protein concentrations. It is the last observation that rationalizes the existence of the published 1:1 MBD–mCpG structures.^8,9 A 1:1 protein:DNA ratio was used to form the complexes that were structurally analyzed. Because binding of the second monomer follows complete occupancy of the first, the 1:1 complex is observed to be homogeneous (Figure 4, middle panel).

To test whether dimeric binding of the MBD was unique to modified DNA, we determined “data-dense” isotherms at 25 mM KCl, conditions under which the binding affinity of the MBD for mCpG, CpG, and random sequence DNA is comparably high. The two-site binding model better fits all three isotherms (Figure 6). Again, the values of K_D1 and K_D2 determined are readily distinguishable (Table 1). From these results, we conclude that the assembly of MBD monomers to form a discrete DNA-bound dimer is not unique to its interaction with modified DNA.

A Complementary Probe of mCpG Binding Specificity

The addition of competitor DNA to “soak up” nonspecifically bound protein is also used to distinguish specific binding.³⁷ When we added the poly(dA-dT) oligonucleotide to 25 mM KCl MBD binding titrations, the affinity of the MBD for mCpG increases several-fold and dramatically decreases for CpG and random sequence oligonucleotides to undetectable (Figure 7A and Table 1). This behavior is comparable to that observed at 150 mM KCl (Figure 1B and Table 1). The presence of poly(dA-dT) has little effect at 150 mM KCl on either binding affinity or dimer formation (Figure 7 and Table 1).

The dependence of mCpG binding specificity on either salt or the presence of competitor shows that suppression of nonspecific binding underlies the ability of the MBD to discriminate sites of modified nucleotides at physiological salt concentrations. The fact that the poly(dA-dT) competitor obliterates binding of the MBD to CpG and random DNA at 25 mM KCl shows that these reactions are entirely electrostatic in nature. However, one aspect of the binding of the MBD to mCpG suggests additional complexity to its binding mechanism. That aspect is the increase in binding affinity when the KCl concentration is increased from 25 to 150 mM. The canonical behavior of DNA binding proteins is for the affinity to decrease with an increasing counterion concentration; this is true for both specific and nonspecific interactions, albeit with different magnitudes for the dependencies.³² We will return to this issue following the presentation of two other unexpected binding behaviors.

The MBD Binds Tightly to Single-Stranded DNA (ssDNA)

The MBD binds with high affinity to single strands of the duplexes discussed above, methylated, unmethylated, and random (Figure 7B and Table 2). The binding of the MBD to ssDNA is mostly electrostatic and can be detected only at low salt concentrations and independent of CpG methylation. Remarkably, the MBD binds ssDNA more tightly than the corresponding duplex DNA sequence (Tables 1 and 2). The binding isotherms are perfectly fit to the Langmuir equation, suggesting that these reactions also lack cooperativity.

The MBD Binds Poorly to Hydroxymethylated CpG

The discovery of high levels of 5-hydroxymethylcytosine (hmC) in neuronal genomes suggests that this epigenetic mark plays a cell-type-specific role in gene regulation.³⁸ Published in vitro studies of MeCP2 and the binding of the MBD to hmCpG provide contradictory results; both high-affinity binding³⁹ and low-affinity binding^40,41 to hmCpG have been reported. In light of the salt dependence of the specificity of the MBD for mCpG discussed above, we determined isotherms for the binding of the MBD to both symmetrically and hemimodified hydroxymethylated oligonucleotides under both conditions.

At 150 mM KCl, the MBD binds 5hmCpG-containing DNA 20-fold less tightly than mCpG and only several-fold tighter than CpG (Table 1). Because the affinity of the MBD for hmCpG is so low at high salt concentrations, it was convenient to explore the binding of the MBD to symmetric and hemimodified targets at 100 mM KCl. The relative affinity of the MBD for mCpG and hmCpG is comparable to that at 150 mM KCl (Figure 8). The MBD binds hmCpG much less tightly than mCpG; there is a slight preference for the binding of symmetric hmCpG over CpG (0.5-fold) and random sequence (2-fold). The binding of the MBD to symmetric hmCpG and hemimodified (hmCpG/CpG) is the same within experimental error. Interestingly, the MBD binds the asymmetrically modified hmCpG/mCpG with greater affinity, although still weaker than symmetric mCpG. These data suggest that the asymmetrically modified hmCpG/mCpG may represent a distinct epigenetic state with regard to MeCP2 binding.

Quantitation of MBD Binding Electrostatics

Although nonelectrostatic interactions typically dominate DNA sequence-specific binding, electrostatics also plays an essential role in specific protein–DNA interactions.^34,42,43 Thus, it is unsurprising that cations are required to neutralize some of this charge during the formation of a specific complex. A standard tool for partitioning a reaction into its electrostatic and nonelectrostatic components is the measurement of binding affinity as a function of salt concentration (linkage analysis). A typical protein–DNA interaction displays linear log–log dependence with the steeper slope of nonspecific binding resulting in intersection with the specific binding curve at low salt concentrations.⁴²

The MBD is atypical of protein–DNA interactions in that it displays nonlinear linkage (Figure 9A). Formation of the MBD–mCpG complex is minimally salt-dependent from 25 to 150 mM. We interpret this observation to reflect a net cation uptake that offsets the displacement of condensed counterions typically observed when proteins bind to DNA. At ∼150 mM, the uptake reaction saturates in mCpG binding and the canonical cation displacement is observed. The binding of the MBD to CpG- and random sequence-containing DNA shows a similar salt dependence except that the inflection points are shifted to lower KCl concentrations (Figure 9A). Thus, cation uptake is not restricted to mCpG binding.

A complementary approach to partitioning the electrostatic and nonelectrostatic contributions of protein–DNA interactions is the salt displacement isotherm.³³ Salt displacement isotherms for MBD complexes with mCpG-, CpG-, and random sequence-containing DNA are shown in Figure 9B. Because this assay is conducted at stoichiometric concentrations of the protein and DNA, it is minimally sensitive to the enhanced protein binding at low cation concentrations where the rate of dissociation of the complex is low. A slight plateau in the isotherm with an increased salt concentration is observed at low KCl concentrations for mCpG. Plateaus for CpG and random DNA could not be resolved in these salt displacement isotherms.

The energetic portioning calculated from these data is summarized in Table 3. The binding of the MBD to mCpG DNA displaces an average of six cations with an unusually large electrostatic component of binding of 73%. As expected, the binding of the MBD to random sequence DNA is completely electrostatic and accompanied by displacement of a slightly larger number of cations. Although the binding of the MBD to CpG is slightly favored over that to random sequence DNA, a nonelectrostatic component to its binding was not resolved. The energetics of the binding of the MBD to mCpG DNA is minimally affected by the presence of poly(dA-dT).

What Are the Biological Implications of These Results?

The studies presented here demonstrate that the MBD alone is necessary and sufficient for MeCP2 to discriminate mCpG sites in genomic DNA. Because MeCP2 is strongly expressed in neuronal tissues, its binding to modified, unmodified, and random sequence DNA may all contribute to biological function, perhaps in different ways. Salt plays a critical and unprecedented role in mediating binding. A striking aspect of the salt dependence of MBD specificity for binding to mCpG is its invariance up to ion concentrations that are considered physiological. Thus, the salt independence of binding of MeCP2 to DNA at low salt concentrations may reflect a buffer against these fluctuations or provide a specific response to them. In addition, the MBD binds to DNA as a dimer. Thus, the published structural studies do not provide a complete description of this protein–DNA interaction. Because MeCP2 is present at high concentrations in neuronal tissues⁴ and the full-length protein is reported to cooperatively bind DNA in chromatin as a dimer,¹¹ it is possible that some of the structurally or thermodynamically unannotated Rett syndrome-linked mutations impact the dimer interface.