Thermodynamic dissection of estrogen receptor-promoter interactions reveals that steroid receptors differentially partition their self-association and promoter binding energetics

Abstract

Steroid receptors define a family of ligand-activated transcription factors. Recent work has demonstrated that the receptors regulate distinct but overlapping gene networks, yet the mechanisms by which they do so remain unclear. We previously determined the microscopic binding energetics for progesterone receptor (PR) isoform assembly at promoters containing multiple response elements. We found that the two isoforms (PR-A and PR-B) share nearly identical dimerization and intrinsic DNA binding free energies but maintain large differences in cooperative free energy. Moreover, cooperativity can be modulated by monovalent ion binding and promoter layout, suggesting that differences in cooperativity might control isoform-specific promoter occupancy and thus receptor function. To determine whether cooperative binding energetics are common to other members of the steroid receptor family, we dissected the thermodynamics of estrogen receptor-α (ER-α):promoter interactions. We find that the ER-α intrinsic DNA binding free energy is identical to that of the PR isoforms. This was expected, noting that receptor DNA binding domains are highly conserved. Unexpectedly, ER-α generates negligible cooperativity-orders of magnitude less than predicted based on our studies of the PR isoforms. However, analysis of the cooperativity term suggests that it reflects a balance between highly favorable cooperative stabilization and unfavorable promoter bending. Moreover, ER-α cooperative free energy is compensated for by a large increase in dimerization free energy. Collectively, the results demonstrate that steroid receptors differentially partition not only cooperative energetics but also dimerization energetics. We speculate that this ability serves as a framework for regulating receptor-specific promoter occupancy and thus receptor-specific gene regulation.

Figure 1

Estrogen receptor-α domain structure and promoter assembly states. (A) Schematic of ER-α primary sequence. Functional domains as labeled: DBD, DNA binding domain; LBD, ligand binding domain; AF, activation functions are present in both the N-terminus and LBD. (B) Schematic describing the dimer binding pathway for ER-α assembling onto the ERE₂ promoter. The circle represents an ER-α monomer. Squares represent ER-α dimers either free in solution (k_di) or bound to an ERE (k_int). Binding at both promoter sites can be accompanied by intersite cooperative interactions (k_c). Events potentially associated with cooperativity are illustrated through protein-protein contacts and bending of promoter DNA. The arrow on the promoter indicates transcriptional start site. (C) Sequence of the ERE₂ promoter in the vicinity of the two binding sites, ERE 1 and ERE 2. Underlined sequences indicate the half-sites of each palindromic response element.

Figure 2

Purification of full-length, human ER-α and analysis by sedimentation velocity. (A) ER-α (5 μg) purified from baculovirus-infected Sf9 cells, resolved by 4–12% gradient SDS-PAGE and Coomassie stained. Molecular weight markers are indicated to left. (B) Sedimentation velocity data of His-ER-α (0.5 μM) collected at 300 mM NaCl, pH 8.0, 4°C. Circles represent scans collected at 50,000 rpm, plotted as a function of time and radial position. Solid lines represent direct fitting to the Lamm equation as implemented in Sedfit. For clarity, only every sixth scan is shown. (C) The residuals associated with the fit to the data presented in Panel B. (D) Overlaid c(s) distributions determined for three concentrations of His-ER-α: 1.7 μM (dashed grey line), 1.0 μM (solid black line) and 0.3 μM (solid grey line).

Figure 3

Sedimentation equilibrium analysis of His-ER-α at 300 mM NaCl, pH 8.0 and 4°C, plotted as r²/2. (A) Initial ER-α loading concentration was 0.5 μM. Circles represent absorbance at 10,800 rpm. Solid line represents best fit using a single species model. Standard deviation of the fit was 0.0054 absorbance units. (B) Residuals of fit using single species model.

Figure 4

Quantitative DNase footprint titrations and individual-site binding isotherms determined from global analysis of the ERE_1- and ERE₂ promoters. (A) Representative DNase footprint titration of an ERE₂ promoter carried out in 100 mM NaCl. ER-α concentration increases from left to right. Schematic of ERE₂ structure shown to the right. Arrows at left highlight hypersensitive sites associated with ERE₂ and ERE_1- promoters (thin arrows), and site seen only with ERE₂ promoter (thick arrow). (B) Solid red circles represent binding to site 1 and open red circles represent binding to site 2 of the ERE₂ promoter (three independent footprint titrations); open blue squares represent binding to the ERE_1- promoter (three independent footprint titrations). Red and blue lines represent best fit from global analysis of nine isotherms. Binding energetics for each site of the ERE₂ promoter are identical, therefore the lines for each site overlay. Lines represent best fit assuming a dimerization dissociation constant of 1 nM; however, a visually identical result is obtained regardless of assumed k_di.

Figure 5

Resolved ER-α binding parameters and associated error surface determined as a function of dimerization constant. (A) Resolved intrinsic binding affinity, k_int, versus dimerization constant, k_di. Both parameters are plotted as dissociation constants in molar units. Error determined by Monte Carlo analysis. (B) Resolved cooperativity, k_c, versus dimerization constant, k_di. Error determined by Monte Carlo analysis. (C) Standard deviation of global fit plotted as a function of assumed k_di. Shaded area represents binding parameters eliminated from consideration as a result of the sedimentation studies.

Figure 6

The distinctive hypersensitive region between the binding sites shown in Figure 4A was quantified and fit globally with the binding data from the ERE_1- promoter to resolve k_c from the hypersensitivity. (A) Open squares represent the data from three ERE_1- footprint titrations. The line through the data represents the best fit from the global analysis with the hypersensitivity data. (B) Open triangles represent the quantification of the hypersensitive region. The line represents the best fit through the data from the global analysis. Data were arbitrarily rescaled from 0 to −1 after fitting to make the distinction between the decreasing band intensity of the footprint titrations and the increasing band intensity associated with the hypersensitive site.

Figure 7

Comparative Monte Carlo analysis of errors associated with global analyses of ERE₂ and ERE_1- isotherms versus hypersensitive region and ERE_1- isotherm. (A) Overlaid histograms reporting the error distribution of k_int from the ERE₂ and ERE_1- (black bars) and hypersensitivity (grey bars). (B) Overlaid histograms reporting the error distribution of k_c from the ERE₂ and ERE_1- (black bars) and hypersensitivity (grey bars). Solid and dashed horizontal bars represent 68% confidence limits for ERE₂/ERE_1- and ERE_1-/hypersensitivity binding parameters, respectively.

Figure 8

Individual-site binding isotherms for PR-B assembly at the PRE₂ promoter. Solid red circles represent binding to site 1 and open red circles represent binding to site 2 the PRE₂ promoter. Open blue squares represent data from the PRE_1- promoter. Lines through the data represent best fit from the global analysis. PRE₂, solid red line; PRE_1-, dashed blue line. Binding energetics for each site of the PRE₂ promoter are identical, therefore the lines for each site overlay. For comparative purposes, the isotherms determined from analysis of the ER-α footprint titration data are overlaid. ERE₂, solid black line; ERE_1-, dashed black line.

Figure 9

Predicted ligation states for PR-B and ER-α assembly at respective two-site promoters. (A) PR-B:PRE₂ probabilities as determined from the experimentally determined interaction energetics. (B) Same as (A) but ER-α:ERE₂. Unligated HRE₂ promoter is represented by the dashed black lines, singly ligated promoter by the solid black lines and the fully ligated HRE₂ by the red dotted lines. The proportion of dimer in solution is represented by the open-dashed black lines.