Dissecting Steroid Receptor Function by Analytical Ultracentrifugation.

Steroid receptors comprise a family of ligand-activated transcription factors. The members include the androgen receptor (AR), estrogen receptor (ER), glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and progesterone receptor (PR). Each receptor controls distinct sets of genes associated with development, metabolism, and homeostasis. Although a qualitative understanding of how individual receptors mediate gene expression has come into focus, quantitative insight remains less clear. As a step toward delineating the physical mechanisms by which individual receptors activate their target genes, we have carried out a systematic dissection of receptor interaction energetics with their multisite regulatory elements. Analytical ultracentrifugation (AUC) has proved indispensable in these studies, in part by revealing the energetics of receptor self-association and its thermodynamic coupling to DNA binding. Here, we discuss these findings in the context of understanding specificity of receptor-mediated gene control. We first highlight the role of sedimentation velocity and sedimentation equilibrium in addressing receptor assembly state, and present a comparative analysis across the receptor family. We then use these results for understanding how receptors assemble at multisite regulatory elements, and hypothesize how these findings might play a role in receptor-specific gene regulation. Finally, we examine receptor behavior in a cellular context, with a view toward linking our in vitro studies with in vivo function.

A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation.

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

Homologous steroid receptors assemble at identical promoter architectures with unique energetics of cooperativity.

Steroid receptors comprise a homologous family of ligand-activated transcription factors. The receptors bind largely identical response elements in vitro, yet regulate distinct gene networks in vivo. This paradox raises the issue of how transcriptional specificity is achieved, particularly if multiple receptor populations are competing for identical sites. Noting that receptor-DNA energetics are a primary force in driving transcriptional activity, differences in interaction energetics among the receptors might underlie receptor-specific transcriptional control. Thermodynamic dissections support this premise-upon assembling at an identical promoter architecture, individual receptors exhibit vast differences in cooperative and self-association energetics. More intriguingly, these parameters distribute in a way that mirrors the evolutionary divergence of the steroid receptor family. For example, the closely related progesterone and glucocorticoid receptors (PR and GR) display little or no self-association but strong intersite cooperativity, whereas the more distantly related estrogen receptor (ER-α) shows inverse behavior. These findings suggest that receptors view genomic promoter architectures as a collection of affinity landscapes; receptors select from this landscape via their unique interaction energetics. To test this idea, we analyzed the cooperative binding energetics of the above three receptors using an array of promoters. We find that cooperativity is not only receptor-specific but also highly promoter-specific. Thus PR shows maximal cooperativity at promoters with closely spaced and in phase binding sites. GR cooperativity is maintained over greater distances, is larger energetically, and shows markedly different phase dependency. Finally, ER-α appears incapable of cooperativity regardless of promoter architecture, consistent with its more distant phylogeny.

Steroid receptor-DNA interactions: toward a quantitative connection between energetics and transcriptional regulation.

Steroid receptors comprise an evolutionarily conserved family of transcription factors. Although the qualitative aspects by which individual receptors regulate transcription are well understood, a quantitative perspective is less clear. This is primarily because receptor function is considerably more complex than that of classical regulatory factors such as phage or bacterial repressors. Here we discuss recent advances in placing receptor-specific transcriptional regulation on a more quantitative footing, specifically focusing on the role of macromolecular interaction energetics. We first highlight limitations and challenges associated with traditional approaches for assessing the role of energetics (more specifically, binding affinity) with functional outcomes such as transcriptional activation. We next demonstrate how rigorous in vitro measurements and straightforward interaction models quantitatively relate energetics to transcriptional activity within the cell, and follow by discussing why such an approach is unexpectedly effective in explaining complex functional behavior. Finally, we examine the implications of these findings for considering the unique gene regulatory properties of the individual receptors.

Analysis of a glucocorticoid-estrogen receptor chimera reveals that dimerization energetics are under ionic control.

Steroid receptors assemble at DNA response elements as dimers, resulting in coactivator recruitment and transcriptional activation. Our work has focused on dissecting the energetics associated with these events and quantitatively correlating the results with function. A recent finding is that different receptors dimerize with large differences in energetics. For example, estrogen receptor-α (ER-α) dimerizes with a ΔG=-12.0 kcal/mol under conditions in which the glucocorticoid receptor (GR) dimerizes with a ΔG≤-5.1 kcal/mol. To determine the molecular forces responsible for such differences, we created a GR/ER chimera, replacing the hormone-binding domain (HBD) of GR with that of ER-α. Cellular and biophysical analyses demonstrate that the chimera is functionally active. However, GR/ER dimerization energetics are intermediate between the parent proteins and coupled to a strong ionic linkage. Since the ER-α HBD is the primary contributor to dimerization, we suggest that GR residues constrain an ion-regulated HBD assembly reaction.

Glucocorticoid receptor-DNA interactions: binding energetics are the primary determinant of sequence-specific transcriptional activity.

The glucocorticoid receptor (GR) is a member of the steroid receptor family of ligand-activated transcription factors. A long-standing question has focused on how GR and other receptors precisely control gene expression. One difficulty in addressing this is that GR function is influenced by multiple factors including ligand and coactivator levels, chromatin state, and allosteric coupling. Moreover, the receptor recognizes an array of DNA sequences that generate a range of transcriptional activities. Such complexity suggests that any single parameter-DNA binding affinity, for example-is unlikely to be a dominant contributor to function. Indeed, a number of studies have suggested that for GR and other receptors, binding affinity toward different DNA sequences is poorly correlated with transcriptional activity. As a step toward determining the factors most predictive of GR function, we rigorously examined the relationship between in vitro GR-DNA binding energetics and in vivo transcriptional activity. We first demonstrate that previous approaches for assessing affinity-function relationships are problematic due to issues of data transformation and linearization. Thus, the conclusion that binding energetics and transcriptional activity are poorly correlated is premature. Using more appropriate analyses, we find that energetics and activity are in fact highly correlated. Furthermore, this correlation can be quantitatively accounted for using simple binding models. Finally, we show that the strong relationship between energetics and transcriptional activity is recapitulated in multiple promoter contexts, cell lines, and chromatin environments. Thus, despite the complexity of GR function, DNA binding energetics are the primary determinant of sequence-specific transcriptional activity.

Progestin regulated miRNAs that mediate progesterone receptor action in breast cancer.

Progesterone receptors (PRs) mediate response to progestins in the normal breast and breast cancer. To determine if liganded PR regulate microRNAs (miRNAs) as a component of their action, we profiled mature miRNA levels following progestin treatment. Indeed, 28 miRNAs are significantly altered by 6h of progestin treatment. Many progestin-responsive genes are putative targets of progestin-regulated miRNAs; for example, progestin treatment decreases miR-29, thereby relieving repression of one of its direct targets, the gene encoding ATPase, Na(+)/K(+) transporting, beta 1 polypeptide (ATP1B1). Thus, liganded PR regulates ATP1B1 through sites in the promoter and the 3'UTR, to achieve maximal tight hormonal regulation of ATP1B1 protein via both transcriptional and translational control. We find that ATP1B1 serves to limit migration and invasion in breast cancer cells. Lastly, we demonstrate that PR itself is regulated by a progestin-upregulated miRNA, miR-513a-5p, providing a novel mechanism for tight control of PR protein expression.

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

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.

From steroid receptors to cytokines: the thermodynamics of self-associating systems.

Since 1987, the Gibbs Conference on Biothermodynamics has maintained a focus on understanding the quantitative aspects of gene regulatory systems. These studies coupled rigorous techniques with exact theory to dissect the linked reactions associated with bacterial and lower eukaryotic gene regulation. However, only in the last ten years has it become possible to apply this approach to clinically relevant, human gene regulatory systems. Here we summarize our work on the thermodynamics of human steroid receptors and their interactions with multi-site promoter sequences, highlighting results not available from more traditional biochemical and structural approaches. Noting that the Gibbs Conference has also served as a vehicle to promote the broader use of thermodynamics in understanding biology, we then discuss collaborative work on the hydrodynamics of a cytokine implicated in tumor suppression, prostate derived factor (PDF).

Na(+) and K(+) allosterically regulate cooperative DNA binding by the human progesterone receptor.

Cooperativity is a common mechanism used by transcription factors to generate highly responsive yet stable gene regulation. For the two isoforms of human progesterone receptor (PR-A and PR-B), differences in cooperative DNA binding energetics may account for their differing transcriptional activation properties. Here we report on the molecular origins responsible for cooperativity, finding that it can be activated or repressed with Na(+) and K(+), respectively. We demonstrate that PR self-association and DNA-dependent cooperativity are linked to a monovalent cation binding event and that this binding is coupled to modulation of receptor structure. K(+) and Na(+) are therefore allosteric effectors of PR function. Noting that the apparent binding affinities of Na(+) and K(+) are comparable to their intracellular concentrations and that PR isoforms directly regulate the genes of a number of ion pumps and channels, these results suggest that Na(+) and K(+) may additionally function as physiological regulators of PR action.