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.

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.

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.

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.

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.

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.

Dissection of androgen receptor-promoter interactions: steroid receptors partition their interaction energetics in parallel with their phylogenetic divergence.

Steroid receptors comprise a homologous family of ligand-activated transcription factors. The members include androgen receptor (AR), estrogen receptor (ER), glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and progesterone receptor (PR). Phylogenetic studies demonstrate that AR, GR, MR, and PR are most closely related, falling into subgroup 3C. ER is more distantly related, falling into subgroup 3A. To determine the quantitative basis by which receptors generate their unique transcriptional responses, we are systematically dissecting the promoter-binding energetics of all receptors under a single "standard state" condition. Here, we examine the self-assembly and promoter-binding energetics of full-length AR and a mutant associated with prostate cancer, T877A. We first demonstrate that both proteins exist only as monomers, showing no evidence of dimerization. Although this result contradicts the traditional understanding that steroid receptors dimerize in the absence of DNA, it is fully consistent with our previous work demonstrating that GR and two PR isoforms either do not dimerize or dimerize only weakly. Moreover, both AR proteins exhibit substantial cooperativity between binding sites, again as seen for GR and PR. In sharp contrast, the more distantly related ER-α dimerizes so strongly that energetics can only be measured indirectly, yet cooperativity is negligible. Thus, homologous receptors partition their promoter-binding energetics quite differently. Moreover, since receptors most closely related by phylogeny partition their energetics similarly, such partitioning appears to be evolutionarily conserved. We speculate that such differences in energetics, coupled with different promoter architectures, serve as the basis for generating receptor-specific promoter occupancy and thus function.

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.

Thermodynamic dissection of progesterone receptor interactions at the mouse mammary tumor virus promoter: monomer binding and strong cooperativity dominate the assembly reaction.

Progesterone receptors (PRs) play critical roles in eukaryotic gene regulation, yet the mechanisms by which they assemble at their promoters are poorly understood. One of the few promoters amenable to analysis is the mouse mammary tumor virus gene regulatory sequence. Embedded within this sequence are four progesterone response elements (PREs) corresponding to a palindromic PRE and three half-site PREs. Early mutational studies indicated that the presence of all four sites generated a synergistic and strong transcriptional response. However, DNA binding analyses suggested that receptor assembly at the promoter occurred in the absence of significant cooperativity. Taken together, the results indicated that cooperative interactions among PREs could not account for the observed functional synergy. More broadly, the studies raised the question of whether cooperativity was a common feature of PR-mediated gene regulation. As a step toward obtaining a quantitative and, thus, predictive understanding of receptor function, we have carried out a thermodynamic dissection of PR A-isoform interactions at the mouse mammary tumor virus promoter. Utilizing analytical ultracentrifugation and quantitative footprinting, we have resolved the microscopic energetics of PR A-isoform binding, including cooperativity terms. Our results reveal a model contrary to that inferred from previous biochemical investigations. Specifically, the binding unit at a half-site is not a receptor dimer but is instead a monomer; monomers bound at half-sites are capable of significant pairwise cooperative interactions; occupancy of all three half-sites is required to cooperatively engage the palindromic-bound dimer; and finally, large unfavorable forces accompany assembly. Overall, monomer binding accounts for the majority of the intrinsic binding energetics and cooperativity contributes an approximately 1000-fold increase in receptor-promoter stability. Finally, the partitioning of cooperativity suggests a framework for interpreting in vivo transcriptional synergy. These results highlight the insight available from rigorous analysis and demonstrate that receptor-promoter interactions are considerably more complex than typically envisioned.

Coactivator assembly at the promoter: efficient recruitment of SRC2 is coupled to cooperative DNA binding by the progesterone receptor.

A largely unsolved problem in eukaryotic gene regulation focuses on the mechanisms by which DNA-bound transcription factors recruit coactivators to a promoter. Recent work has suggested that promoter DNA acts as an allosteric ligand, serving not only to bind and localize transcription factors but also to trigger structural changes within the proteins in order to elicit coactivator recruitment. Unfortunately, a quantitative and molecular understanding of this phenomenon remains unclear. We have previously resolved the microstate interaction energetics of progesterone receptor A-isoform (PR-A) assembly at multiple promoters; here we extend this work to the role of PR-A in mediating promoter-dependent recruitment of the coactivator, SRC2. Quantitative footprinting and statistical thermodynamic modeling of PR-A:promoter interactions in the presence and absence of coactivator demonstrate that receptor binding to a single response element is maximally coupled to a 2-fold enhancement in SRC2 binding. By contrast, PR-A assembly at multiple response elements is linked to an additional 6- to 10-fold increase in SRC2 affinity. This effect arises due to a coupled reaction between SRC2 uptake and enhanced cooperative interactions between adjacently bound PR-A dimers. Put another way, increased coactivator levels stabilize a higher-order receptor-promoter complex. These results may thus not only offer a mechanism for explaining the weak transcriptional activity seen for promoters containing a single binding site and the synergistically strong activity seen for multisite promoters but also suggest that in vivo fluctuations of coactivator levels might serve as a physiological regulator of assembly for PR-A (and for other nuclear receptors) at the promoter.

Thermodynamic analysis of progesterone receptor-promoter interactions reveals a molecular model for isoform-specific function.

Human progesterone receptors (PR) exist as two functionally distinct isoforms, PR-A and PR-B. The proteins are identical except for an additional 164 residues located at the N terminus of PR-B. To determine the mechanisms responsible for isoform-specific functional differences, we present here a thermodynamic dissection of PR-A-promoter interactions and compare the results to our previous work on PR-B. This analysis has generated a number of results inconsistent with the traditional, biochemically based model of receptor function. Specifically, statistical models invoking preformed PR-A dimers as the active binding species demonstrate that intrinsic binding energetics are over an order of magnitude greater than is apparent. High-affinity binding is opposed, however, by a large energetic penalty. The consequences of this penalty are 2-fold: Successive monomer binding to a palindromic response element is thermodynamically favored over preformed dimer binding, and DNA-induced dimerization of the monomers is largely abolished. Furthermore, PR-A binding to multiple PREs is only weakly cooperative, as judged by a 5-fold increase in overall stability. Comparison of these results to our work on PR-B demonstrates that whereas both isoforms appear to have similar DNA binding affinities, PR-B in fact has a greatly increased intrinsic binding affinity and cooperative binding ability relative to PR-A. These differences thus suggest that residues unique to PR-B allosterically regulate the energetics of cooperative promoter assembly. From a functional perspective, the differences in microscopic affinities predict receptor-promoter occupancies that accurately correlate with the transcriptional activation profiles seen for each isoform.

Nuclear receptor structure: implications for function.

Small lipophilic molecules such as steroidal hormones, retinoids, and free fatty acids control many of the reproductive, developmental, and metabolic processes in eukaryotes. The mediators of these effects are nuclear receptor proteins, ligand-activated transcription factors capable of regulating the expression of complex gene networks. This review addresses the structure and structural properties of nuclear receptors, focusing on the well-studied ligand-binding and DNA-binding domains as well as our still-emerging understanding of the largely unstructured N-terminal regions. To emphasize the allosteric interdependence among these subunits, a more detailed inspection of the structural properties of the human progesterone receptor is presented. Finally, this work is placed in the context of developing a quantitative and mechanistic understanding of nuclear receptor function.

Hydrodynamic analysis of the human progesterone receptor A-isoform reveals that self-association occurs in the micromolar range.

Human progesterone receptors exist as two functionally distinct isoforms, an 83 kDa A-receptor (PR-A) and a 99 kDa B-receptor (PR-B). The isoforms are identical except that PR-B has an additional 164 amino acids at its N-terminus. We have previously characterized the hydrodynamics and solution assembly energetics of PR-B [Heneghan, A. F., et al. (2005) Biochemistry 44, 9528-9537], and here we present an analysis of PR-A. At micromolar concentrations of the receptor, sedimentation velocity studies demonstrate that PR-A undergoes a concentration-dependent change in its sedimentation coefficient distribution, indicative of a self-associating system. Global analysis of data sets collected at multiple PR-A concentrations supports the presence of a hydrodynamically homogeneous 3.50 S monomer species in equilibrium with a 7.15 S dimer species. Sedimentation equilibrium analysis demonstrates that self-association can be rigorously described by a monomer-dimer assembly reaction and a dimerization free energy of -7.6 +/- 0.6 kcal/mol. Both the PR-A monomer and dimer are structurally asymmetric, although the extent of asymmetry is significantly decreased for the dimer, indicative of quaternary-induced hydrodynamic compaction. Limited proteolysis studies suggest that PR-A asymmetry arises from an ensemble of partially folded conformations within the N-terminal half of the molecule. Finally, comparison to our previous work on PR-B self-association energetics demonstrates that it dimerizes, under identical solution conditions, with an affinity at least 8-fold weaker than that of PR-A. Thus, residues unique to the B-isoform destabilize receptor assembly energetics. Importantly, the physical and chemical driving forces underlying isoform-specific dimerization suggest that B-unique amino acids modulate the energetics through an allosteric mechanism.

Cooperative DNA binding by the B-isoform of human progesterone receptor: thermodynamic analysis reveals strongly favorable and unfavorable contributions to assembly.

Progesterone receptors (PR) play critical roles in eukaryotic gene regulation, yet the mechanisms by which they assemble at multisite promoters are poorly understood. Here we present a thermodynamic analysis of the interactions of the PR B-isoform (PR-B) with promoters containing either one or two progesterone response elements (PREs). Utilizing quantitative footprinting, we have resolved the microscopic energetics of PR-B binding, including cooperativity terms. The results of this analysis challenge a number of assumptions found in traditional models of receptor function. First, PR-B interactions at a single PRE can be equally well described by mechanisms invoking either the receptor monomer or the dimer as the active DNA binding species. If, as is commonly accepted, PR-B interacts with response elements only as a preformed dimer, then its intrinsic binding affinity is not the typically observed nanomolar but is rather picomolar. This high affinity binding is opposed, however, by a large energetic penalty. The penalty presumably pays for costly structural rearrangements of the receptor dimer and/or response element that are needed to form the protein-DNA complex. If PR-B assembles at a single response element via successive monomer binding reactions, then this penalty minimizes cooperative interactions between adjacent monomers. When binding to two response elements, the receptor exhibits strong intersite cooperativity. Although this phenomenon has been observed before, the present work demonstrates that the energetics reach levels seen in highly cooperative systems such as lambda cI repressor. This first quantitative dissection of cooperative receptor-promoter interactions suggests that PR-B function is more complex than traditionally envisioned.

Self-association energetics of an intact, full-length nuclear receptor: the B-isoform of human progesterone receptor dimerizes in the micromolar range.

We are focused on understanding the mechanisms underlying eukaryotic gene regulation, using the human progesterone receptor (PR) and its interactions with its DNA response elements as a model system. An understanding of PR function is complicated by the presence of two transcriptionally distinct isoforms, an 83 kDa A-receptor (PR-A) and a 99 kDa B-receptor (PR-B). The two isoforms are identical except the B-receptor contains an additional 164 residues at its N-terminus. As a first step toward understanding the principles by which the two isoforms assemble at complex promoters, we examined the energetics of PR-B self-association using sedimentation velocity and sedimentation equilibrium methods. Full-length human PR-B was purified to 95% homogeneity from baculovirus-infected insect cells. Using a fluorescence hormone binding assay, we determined the purified protein to be highly active in its ability to bind ligand. Sedimentation velocity studies of hormone-bound PR-B at pH 8.0, 4 degrees C, and 50 mM NaCl demonstrate that it undergoes a concentration-dependent change in its sedimentation coefficient, existing as a 4.0S species at submicromolar concentrations but forming a 5.7S species at higher concentrations. These results strongly suggest that PR-B undergoes self-association in the micromolar range. This hypothesis was examined rigorously using sedimentation equilibrium. Global analysis of the sedimentation equilibrium data demonstrated that PR-B self-association was well described by a monomer-dimer model with a dimerization free energy of -7.2 +/- 0.7 kcal/mol. The role of NaCl in regulating PR-B dimerization was examined by carrying out sedimentation velocity and equilibrium studies under high salt conditions. At 300 mM NaCl, PR-B is exclusively monomeric in the micromolar range, thus revealing a significant ionic contribution to the assembly energetics. Further, the monomer sediments as a structurally homogeneous, but highly asymmetric, 4.0S species. Limited proteolysis of PR-B demonstrated that the hydrodynamic asymmetry is due in part to an extended, nonglobular conformation localized to the N-terminal region of PR-B. In contrast, the DNA binding domain (DBD) and hormone binding domain (HBD) exist as independent structural units, and the activation function N-terminal to the DBD (AF-1) shows moderate structure. These results represent the first rigorous analysis of the self-assembly energetics of an intact nuclear receptor and suggest that PR function is more complex than envisioned by traditional models.