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.