Journal of Molecular Biology
Thermodynamic Dissection of Progesterone Receptor Interactions at the Mouse Mammary Tumor Virus Promoter: Monomer Binding and Strong Cooperativity Dominate the Assembly Reaction☆
Introduction
Progesterone receptors (PRs) are members of the nuclear receptor superfamily of ligand-activated transcription factors.1 The traditional understanding of PR function is that the receptors dimerize in solution, bind to progesterone response elements (PREs) at upstream promoter sites, and recruit an array of coactivating proteins in order to remodel chromatin and activate transcription. This model is based upon an enormous number of biochemical and molecular biological studies, and it has lent great insight into the qualitative and semiquantitative aspects of receptor-mediated gene regulation. However, for PR and all other nuclear receptors, a quantitative and, thus, truly predictive understanding of function is still lacking. Even the seemingly straightforward basis by which receptors assemble at multisite promoter sequences is not understood from any physicochemical perspective. This absence of knowledge has limited our insight into the basic principles responsible for higher eukaryotic gene regulation and likely hindered the development of new drugs and therapeutics.
One of the very few (and perhaps only) PR-regulated promoters amenable to detailed analysis is the mouse mammary tumor virus (MMTV) gene regulatory sequence. This promoter has long been known to be regulated by PR and its closely related homolog, the glucocorticoid receptor (GR).2 As shown in Fig. 1a, the sequence contains a number of cis-acting elements including a TATA box, two Oct-1 sites, an NF-1 site, and multiple response elements for PR or GR binding. More specifically, there are at least four clearly recognizable receptor binding sequences—a palindromic PRE (site 1) and three identical half-site PREs (sites 2–4). Semiquantitative footprinting and filter binding studies indicated that receptors bound at all four of the PREs,2, 3, 4 and glycerol gradient centrifugation studies4 suggested that dimers assembled at each site.5 Finally, mutational studies indicated that loss of any one site decreased the level of transcriptional activity up to 10-fold,6 demonstrating that transcriptional activation was synergistic in character and, thus, suggestive of communication among the sites.
It had been reasonably hypothesized that MMTV promoter functional synergy could arise through receptor-mediated cooperative interactions.3 However, the results of the footprinting studies noted above suggested that receptor assembly at the promoter was accompanied by little to no cooperativity. This conclusion was based on the observation that mutation of individual response elements changed receptor half-saturation values at the remaining sites by only two- to threefold.3, 4 This was despite the fact that, at least for GR, the binding transitions at each response element were extremely steep,4 indicating that additional reactions were somehow coupled to DNA binding (e.g., cooperative assembly or solution dimerization). Furthermore, the results were in contrast to concurrent studies of a synthetic PR-regulated promoter, which indicated the presence of highly cooperative interactions between adjacently bound PR dimers.7 This discordance thus left unanswered the mechanism of receptor-mediated transcriptional synergy and raised the broader question of whether cooperativity was a common feature of PR (and GR) function. These elementary and essential issues have never been resolved for any nuclear receptor, even as current research has now moved toward addressing the even more complex phenomena of coactivator recruitment and chromatin remodeling.
Any attempt to quantitatively understand PR function must first take into account the presence of two distinct isoforms: an 83-kDa PR A-isoform (PR-A) and a 99-kDa PR B-isoform (PR-B) (Fig. 1b).8 The two proteins are identical in primary structure except for the addition of 164 amino acids located at the N-terminus of PR-B. These residues define the B-unique sequence or BUS. Despite their high degree of sequence identity, the two isoforms exhibit a number of unique functional properties, including differences in transcriptional activity,9, 10 ligand response,11 gene regulation,12 and tissue-specific physiological effects.13, 14 We have previously analyzed the thermodynamics of PR-A and PR-B binding to synthetic promoters containing either one or two palindromic response elements and have found that a role for BUS is to allosterically enhance the cooperative binding energetics of PR-B relative to PR-A.15, 16 As a functional consequence, the increased affinity seen for the B-isoform predicts promoter occupancies that accurately correlate with its increased transcriptional activation properties relative to PR-A. Since this difference in activation is also maintained on the natural, nonsynthetic MMTV promoter,17 we hypothesized that PR-A should be capable of assembling at the promoter via high-affinity binding but with moderate cooperative interactions. Furthermore, since our work on the self-association energetics of PR-A found that there are few receptor dimers present upon initiation of DNA binding and that dimer binding is penalized relative to monomer binding,15, 16, 18, 19 we anticipated that monomers rather than dimers should assemble at individual half-sites. This issue of cooperative assembly is of particular interest in light of our work on steroid receptor coactivator-2 (SRC2), which demonstrated that efficient coactivator recruitment to the promoter is dependent upon cooperativity between tandemly linked PREs.20
As a step toward a quantitative understanding of PR function, we present here a thermodynamic dissection of PR-A interactions with the MMTV promoter sequence. Using analytical ultracentrifugation and quantitative footprint titrations, we have resolved the stoichiometries of binding, the intrinsic DNA binding energetics, and the microstate cooperativity terms for PR-A assembly at this promoter. Our analysis of the data reveals that, in contrast to previous reports, the binding unit at a half-site is not a receptor dimer but is instead a monomer. Moreover, monomers bound at half-sites are capable of significant pairwise cooperative interactions, and occupancy of all three half-sites is required to cooperatively engage the palindromic-bound dimer. Each of these cooperative interactions is of moderate free energy, but as a whole, they translate into an approximately 1000-fold increase in receptor promoter stability. Somewhat unexpectedly, the large amount of cooperative free energy is balanced by unfavorable forces that penalize both monomer and dimer assembly reactions at the promoter. Finally, the distribution of the cooperative binding energetics may lend insight into the functional synergy observed in early mutational analyses. This study represents the first rigorous dissection of the interactions between a full-length nuclear receptor and any natural promoter and may serve as a template for dissecting and understanding other newly identified PR-regulated promoter sequences.
Section snippets
Results
Shown in Fig. 2a is a quantitative DNase footprint titration of the wild-type MMTV promoter. It is evident that PR-A binding is specific for five regions, labeled here as sites 1–5. Dideoxy sequencing analysis reveals that the first four sites correspond to the palindromic site 1 and half-sites 2–4, respectively. Close inspection of the sequencing results indicates that only one or two additional nucleotides flanking each of the four PREs are also protected (data not shown). We also identified
Discussion
As noted in the Introduction, the long-accepted picture for PR interactions at the MMTV promoter is that only preformed dimers are capable of binding, that binding appears to be coupled to weak or nonexistent cooperative interactions, and that the functional synergy observed for this promoter cannot be accounted for by cooperative assembly. This understanding was based upon a number of pioneering investigations that focused on elucidating the molecular origins of receptor–promoter interactions.2
Purification and hydrodynamic characterization of PR-A
An expression vector encoding full-length human PR-A (residues 165–933) fused to an N-terminal hexahistidine sequence was a generous gift from Dr. Dean Edwards (Baylor College of Medicine, Houston, TX). A detailed description of the PR-A purification process and a quantitative analysis of its hydrodynamic solution properties were published previously.18 Briefly summarized, PR-A expressed in Sf9 insect cells can be purified to at least 95% homogeneity. As judged by sedimentation velocity
Acknowledgements
This work was supported by National Institutes of Health Grant R01-DK061933 to D.L.B. We thank Drs. Dean Edwards and Steven Nordeen for constructs. We thank Dr. N. Karl Maluf for insightful discussions.
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Cited by (20)
Dissecting Steroid Receptor Function by Analytical Ultracentrifugation
2015, Methods in EnzymologyGlucocorticoid receptor-DNA interactions: Binding energetics are the primary determinant of sequence-specific transcriptional activity
2012, Journal of Molecular BiologyStructural and functional analysis of domains of the progesterone receptor
2012, Molecular and Cellular EndocrinologyCitation Excerpt :PR binding sites on natural target genes are generally composites of near palindromic PREs with adjacent PRE half-sites. The mouse mammary tumor virus (MMTV) promoter, comprised of a single PRE with three tandemly arranged PRE half-sites and a cryptic PRE half-site, was used to examine the binding of PR-A (Connaghan-Jones et al., 2008). In contrast to the weak inter-site cooperativity observed in the PRE2 system, the apparent binding affinity for all MMTV sites has multiple contributions including the modest monomer intrinsic binding affinity, the strong inter-site cooperativity for the tandem half-sites, and additional anti-cooperativity associated with complete saturation of the PRE half-sites.
From steroid receptors to cytokines: The thermodynamics of self-associating systems
2011, Biophysical ChemistryCitation Excerpt :Because PR-A is less soluble at low salt concentrations, we were unable to estimate Na+ binding affinity for this isoform. Millimolar ion affinities may seem weak when compared to the micromolar or nanomolar affinities associated with PR dimerization and DNA binding [16,17,21,22,26]. However, this is comparable to intracellular M+ concentration.
Chapter 2 Using Thermodynamics to Understand Progesterone Receptor function. Method and Theory
2009, Methods in EnzymologyCitation Excerpt :In order to confirm such stoichiometry, we used sedimentation equilibrium to measure the reduced molecular weight of an MMTV promoter containing only the three half‐sites when in the presence of increasing concentrations of PR‐A. The resolved reduced molecular weights (σ) are shown in Fig. 2.7 (Connaghan‐Jones et al., 2008a). Comparison of the resolved σ values with the predicted σ for saturation of monomers at the three half‐sites (short dashed line) is clearly consistent with a binding stoichiometry of one monomer per site.
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K.D.C.-J. and A.F.H. contributed equally to this work.
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K.D.C.-J. and A.F.H. contributed equally to this work.