Using thermodynamics to understand progesterone receptor function: method and theory.

Progesterone receptors (PRs) are members of the nuclear receptor superfamily of ligand-activated transcription factors. The mechanisms by which receptors such as PR assemble at a promoter and recruit coactivators are well understood at the biochemical level. However, a rigorous and thus quantitatively predictive understanding of function is entirely lacking. This is so in part because the study of receptor function has largely been carried out using semiquantitative or qualitative approaches. These types of analyses are limited in their ability to resolve thermodynamically valid and physically meaningful microscopic interaction parameters. This includes resolution of intrinsic binding constants and cooperativity terms, as well as the mathematical framework for integrating these values into a larger molecular code for function. Here we present our experimental and theoretical approach for dissecting the linked reactions associated with PR and coactivator assembly at complex promoter sequences. We discuss the use of analytical ultracentrifugation and quantitative DNase footprint titration and their coupling to exact theoretical treatments. We then highlight the major findings of these studies and their implications for understanding and reevaluating receptor function.

Quantitative DNase footprint titration: a tool for analyzing the energetics of protein-DNA interactions.

A major goal in biomedical research is to determine the mechanisms responsible for gene regulation. However, the promoters and operators that control transcription are often complex in nature, containing multiple-binding sites with which DNA-binding proteins can interact cooperatively. Quantitative DNase footprint titration is one of the few techniques capable of resolving the microscopic binding affinities responsible for the macroscopic assembly process. Here, we present a step-by-step protocol for carrying out a footprint titration experiment. We then describe how to quantify the resultant images to generate individual-site binding curves. Finally, we derive basic equations for binding at each site and present an overview of the fitting process, applying it to the anticipated results. Users should anticipate that the footprinting experiment will take 3-5 d starting from DNA template isolation to image acquisition and quantitation.

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.

High-risk surgical stage 1 endometrial cancer: outcomes with vault brachytherapy alone.

OBJECTIVE: Prior to 1995, in our center, patients with surgically staged endometrial cancer with greater than 50% myoinvasion (FIGO 1C) were treated with vault brachytherapy and whole pelvis (WP) radiotherapy despite negative nodes. After October 1, 1995, these patients were treated with vault brachytherapy alone. The aim of this study was to ensure that the survival and recurrence rate had not changed. METHODS: A retrospective review of Cancer Care Manitoba charts was undertaken. All patients diagnosed with endometrioid adenocarcinoma between October 1, 1995, and March 1, 2001, were reviewed. Data for all FIGO surgical stage 1 patients, and a subset of stage 1C patients, were analyzed and compared with those of a historical control group, composed of patient data previously collected in our center (1978 to 1990) [Gynecol. Oncol. 55 (1994), 51]. RESULTS: A total of 172 patients had negative selective pelvic lymphadenectomy and FIGO stage 1 disease. Fifty-three stage 1C patients were spared WP radiotherapy. Median follow-up was 32 months. Recurrence rate in FIGO stage 1 disease was 2.3% (4/172) and for the subset 1C was 5.7% (3/53). The recurrence rate was not statistically significantly different from that of the historical control group, 3.6% for stage 1 (P = 0.562) and 7.2% for stage 1C (P = 0.51). Two- and five-year survival rates for stage 1 patients in this study were 97 and 95%, respectively. In the historical group, 2- and 5-year survival rates were 97 and 94%. CONCLUSION: Whole pelvis radiotherapy can be safely omitted in patients with FIGO stage 1C endometrial cancer if nodal status is known.