The long-term goal of this research is to determine the molecular mechanisms responsible for higher eukaryotic gene regulation. The model system employed is the human progesterone receptor (PR), a member of the nuclear receptor superfamily of ligand-activated transcription factors. PR co-exist naturally as two isoforms: an 83 kD A-receptor (PR-A) and a 99 kD B-receptor (PR-B). The isoforms are identical except that PR-B has an additional 164 residue B-unique sequence (BUS) at its N- terminus. Despite their near sequence identity, the two receptors generate distinctly different molecular, physiological and clinical outcomes. The molecular origins of these differences are unknown. Studies by this laboratory have determined that the extent of cooperative DNA binding seen for each isoform is modulated by BUS. It has also been determined that isoform-specific cooperativity can be regulated by promoter architecture. As a consequence, the distributions of cooperative binding energetics predict PR-A and PR-B promoter occupancies well correlated with their transcriptional activation profiles. Since only cooperative receptor binding is coupled to efficient coactivator recruitment (thus leading to transcriptional activation), it is hypothesized that the extent and type of cooperativity may be the key physiological regulator of isoform-specific gene control. In support of this, this laboratory has discovered that Na+ and K+ are positive and negative regulators of isoform-specific cooperativity, respectively. Since PR-A and PR-B differentially regulate the promoter encoding the Na+, K+-ATPase beta1 gene, the linkage between ion binding and BUS-modulated cooperativity may define a molecular mechanism for isoform-specific beta1 gene regulation. The thermodynamic and kinetic mechanisms of cooperative isoform binding at this promoter will be determined as a test of the stated hypothesis.
Aim 1 The energetics and driving forces responsible for cation-dependent PR-A and PR-B self-assembly will be determined using analytical ultracentrifugation. The cation binding affinities and stoichiometries will additionally be determined.
Aim 2 The thermodynamic and kinetic mechanisms of isoform-specific assembly at the Na+, K+- ATPase promoter will be determined using quantitative footprinting and statistical thermodynamic modeling. These studies will be carried out as a function of cation type in order to assess the role of these ions in regulating assembly.
Aim 3 - The energetics, stoichiometry and driving forces responsible for isoform-specific recruitment of the SRC3 coactivator to the Na+, K+-ATPase promoter will be determined using quantitative footprinting, and as a function of cation-type.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Macromolecular Structure and Function B Study Section (MSFB)
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Margolis, Ronald N
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University of Colorado Denver
Schools of Pharmacy
United States
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Connaghan, Keith D; Miura, Michael T; Maluf, Nasib K et al. (2013) Analysis of a glucocorticoid-estrogen receptor chimera reveals that dimerization energetics are under ionic control. Biophys Chem 172:8-17
Moody, Amie D; Robblee, James P; Bain, David L (2012) Dissecting the linkage between transcription factor self-assembly and site-specific DNA binding: the role of the analytical ultracentrifuge. Methods Mol Biol 796:187-204
Bain, David L; Yang, Qin; Connaghan, Keith D et al. (2012) Glucocorticoid receptor-DNA interactions: binding energetics are the primary determinant of sequence-specific transcriptional activity. J Mol Biol 422:18-32
Robblee, James P; Miura, Michael T; Bain, David L (2012) Glucocorticoid receptor-promoter interactions: energetic dissection suggests a framework for the specificity of steroid receptor-mediated gene regulation. Biochemistry 51:4463-72
Moody, Amie D; Miura, Michael T; Connaghan, Keith D et al. (2012) Thermodynamic dissection of estrogen receptor-promoter interactions reveals that steroid receptors differentially partition their self-association and promoter binding energetics. Biochemistry 51:739-49
Connaghan, Keith D; Moody, Amie D; Robblee, James P et al. (2011) From steroid receptors to cytokines: the thermodynamics of self-associating systems. Biophys Chem 159:24-32
Connaghan, Keith D; Heneghan, Aaron F; Miura, Michael T et al. (2010) Na(+) and K(+) allosterically regulate cooperative DNA binding by the human progesterone receptor. Biochemistry 49:422-31
Connaghan-Jones, Keith D; Bain, David L (2009) Using thermodynamics to understand progesterone receptor function: method and theory. Methods Enzymol 455:41-70
Connaghan-Jones, Keith D; Heneghan, Aaron F; Miura, Michael T et al. (2008) Thermodynamic dissection of progesterone receptor interactions at the mouse mammary tumor virus promoter: monomer binding and strong cooperativity dominate the assembly reaction. J Mol Biol 377:1144-60
Connaghan-Jones, Keith D; Moody, Amie D; Bain, David L (2008) Quantitative DNase footprint titration: a tool for analyzing the energetics of protein-DNA interactions. Nat Protoc 3:900-14

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