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

Connaghan‐Jones, Keith D.; Heneghan, Aaron F.; Miura, Michael T.; Bain, David L.

doi:10.1073/pnas.0608848104

Cited by 33 publications

(70 citation statements)

References 41 publications

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“…Consistent with this, we also demonstrated that PR monomers assemble at isolated half-sites at natural promoters, with stabilization provided by longer-range cooperative interactions between sites (Connaghan-Jones, Heneghan, Miura, & Bain, 2008). Furthermore, simulations suggested that weak dimerization and cooperativity might play a role in specificity of receptor-promoter interactions (Connaghan-Jones et al, 2007;Robblee et al, 2012).…”

Section: Homologous Steroid Receptors Display a Vast Range Of Self-assupporting

confidence: 74%

“…In parallel with these studies, we had also dissected the energetics of receptor-promoter interactions, finding that steroid receptors also exhibit large difference in cooperative binding energetics (ΔG c in Fig. 7B) (Connaghan-Jones et al, 2007;De Angelis et al, 2013;Moody et al, 2012;Robblee et al, 2012). For example, ER-α displays negligible cooperativity (ΔG c ¼ À0.2 kcal/mol) on the two-site promoter in Fig.…”

Section: Integrating Auc Results Into Understanding Receptor-promotermentioning

confidence: 94%

“…As a step toward identifying mechanisms of receptor-specific gene control, we have carried out a systematic dissection of receptor-promoter interactions under a single "standard-state" condition , 2007De Angelis, Yang, Miura, & Bain, 2013;Heneghan, Berton, Miura, & Bain, 2005;Moody, Miura, Connaghan, & Bain, 2012;. Here, we present a summary of these studies, focusing on the role of analytical ultracentrifugation (AUC).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Dissecting Steroid Receptor Function by Analytical Ultracentrifugation

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Angelis

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et al. 2015

Methods in Enzymology

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Section: Homologous Steroid Receptors Display a Vast Range Of Self-assupporting

confidence: 74%

Section: Integrating Auc Results Into Understanding Receptor-promotermentioning

confidence: 94%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Dissecting Steroid Receptor Function by Analytical Ultracentrifugation

Bain

Angelis

Connaghan

et al. 2015

Methods in Enzymology

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“…Eleven GREs, seven of which had been previously characterized, 2 were generated in a pA3-Luc plasmid 27,28 by site-directed mutagenesis. A single GRE, corresponding to a sequence derived from the palindromic tyrosine amino transferase promoter (TAT 3 ; AGAACATCCTG-TACA), was successively mutated to generate a total of 11 GREs.…”

Section: Plasmid Construction For Cellular Assaysmentioning

confidence: 99%

Glucocorticoid Receptor–DNA Interactions: Binding Energetics Are the Primary Determinant of Sequence-Specific Transcriptional Activity

Bain

Yang

Connaghan

et al. 2012

Journal of Molecular Biology

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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.

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“…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 highaffinity binding but with moderate cooperative interactions.…”

Section: Introductionmentioning

confidence: 99%

Thermodynamic Dissection of Progesterone Receptor Interactions at the Mouse Mammary Tumor Virus Promoter: Monomer Binding and Strong Cooperativity Dominate the Assembly Reaction

Connaghan‐Jones

Heneghan

Miura

et al. 2008

Journal of Molecular Biology

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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.

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

Cited by 33 publications

References 41 publications

Dissecting Steroid Receptor Function by Analytical Ultracentrifugation

Dissecting Steroid Receptor Function by Analytical Ultracentrifugation

Glucocorticoid Receptor–DNA Interactions: 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

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