Glucocorticoid Receptor–Promoter Interactions: Energetic Dissection Suggests a Framework for the Specificity of Steroid Receptor-Mediated Gene Regulation

The glucocorticoid receptor (GR) is a member of the steroid receptor family of ligand-activated transcription factors. A number of studies have shown that steroid receptors regulate distinct but overlapping sets of genes; however, the molecular basis for such specificity remains unclear. Previous work from our laboratory has demonstrated that under identical solution conditions, three other steroid receptors [the progesterone receptor A isoform (PR-A), the progesterone receptor B isoform (PR-B), and estrogen receptor α (ER-α)] differentially partition their self-association and promoter binding energetics. For example, PR-A and PR-B generate similar dimerization free energies but differ significantly in their extents of intersite cooperativity. Conversely, ER-α maintains an intersite cooperativity most comparable to that of PR-A yet dimerizes with an affinity orders of magnitude greater than that of either of the PR isoforms. We have speculated that these differences serve to generate receptor-specific promoter occupancies, and thus receptor-specific gene regulation. Noting that GR regulates a unique subset of genes relative to the other receptors, we hypothesized that the receptor should maintain a unique set of interaction energetics. We rigorously determined the self-association and promoter binding energetics of full-length, human GR under conditions identical to those used in our earlier studies. We find that unlike all other receptors, GR shows no evidence of reversible self-association. Moreover, GR assembles with strong intersite cooperativity comparable to that seen only for PR-B. Finally, simulations show that such partitioning of interaction energetics allows for receptor-specific promoter occupancies, even under conditions where multiple receptors are competing for binding at identical sites

Examples of transient changes in the console temperature reading during the SV experiment, as saved in the scan file data.

<p>For comparison, the maximum adiabatic cooling of -0.3°C would be expected after approximately 300 sec, recovering to the equilibrium temperature after approximately 1,200 s (see Fig 3 in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126420#pone.0126420.ref033" target="_blank">33</a>]).</p

Histogram and box-and-whisker plot of <i>s</i>-values of the BSA monomer after different corrections: Raw experimental <i>s</i>-values (black, with grey histogram), scan time corrected <i>s</i>_<i>t</i>-values (blue), rotor temperature corrected <i>s</i>_<i>20T</i>-values (green), or radial magnification corrected <i>s</i>_<i>r</i>-values (cyan), and fully corrected <i>s</i>_{<i>20T</i>,<i>t</i>,<i>r</i>,<i>v</i>}-values (red with red histogram).

<p>The box-and-whisker plots indicate the central 50% of the data as solid line and draw the smaller and larger 25% percentiles as individual circles. The median for each group is displayed as a vertical line.</p

Corrected best-fit apparent monomer molecular mass from integration of the <i>c</i>(<i>s</i>) peak when scanned with the absorbance system (green) and the interference system (magenta).

<p>Only data with rmsd less than 0.01 OD or 0.01 fringes were included. The box-and-whisker plot indicates the central 50% of the data as solid line and draws the smaller and larger 25% percentiles as individual circles. The median is displayed as a vertical line.</p

Absence of a long-term trend in <i>s</i>_{<i>20T</i>,<i>t</i>,<i>r</i>,<i>v</i>}-values of the BSA monomer with time of experiment for the three kits (blue, green, and magenta).

<p>Highlighted as bold solid line is the overall average, and the grey area indicates one standard deviation.</p

Observed fraction of dimer (as a ratio of dimer peak area to the sum of monomer plus dimer peak areas).

<p>The box-and-whisker plot indicates the central 50% of the data as solid line and draws the smaller and larger 25% percentiles as individual circles. The median displayed as vertical line. The mean and standard deviations are 18.5% ± 1.1% for the absorbance system, and 19.0% ± 2.1% for the interference system.</p

Example for the analysis of absorbance data from the sedimentation velocity experiment of BSA.

<p>(A) Absorbance scans (symbols) and best-fit <i>c</i>(<i>s</i>) model at different points in time indicated by color temperature. (B and C) Bitmap and overlay of the residuals of the fit. (D) <i>c</i>(<i>s</i>) sedimentation coefficient distribution showing peaks for monomer, dimer, trimer, and traces of higher oligomers.</p

Magnitude of the radial magnification correction obtained with the absorbance system (green) and the interference system (magenta).

<p>The box-and-whisker plot above the histogram indicates the central 50% of the data as solid horizontal line and draws data in the smallest and highest 25% percentiles as individual circles. The median is displayed as vertical line. The mean and standard deviations are -0.43% ±1.36% for the absorbance system, and -0.75% ± 0.82% for the interference system (once the three outliers are excluded).</p