Abstract

The long-term goal of this research is to elucidate the molecular mechanisms underlying eukaryotic gene regulation. Focus is centered on the mechanisms by which human progesterone receptors (PR) cooperatively bind to complex promoters, and the role of hormone agonists and antagonists in regulating these reactions. A further goat is to determine the principles by which the associated structural transitions are propagated to neighboring domains. PR co-exist as two functionally distinct isoforms: an 83 kD A-receptor and a 99 kD B-receptor. The two isoforms are identical except that the B-receptor has an additional 164 amino acids at its N-terminus. The B-receptor often functions as a strong transcriptional activator while the A-receptor generally acts as a weak activator. It is hypothesized that this difference arises through the ability of the B-receptor to bind cooperatively at PR-regulated promoters. Mechanistically, the B-unique residues impose a hormone-dependent conformational constraint upon the remainder of the receptor. This constraint causes changes in PR structure and stability- changes that can include dramatic disorder-order transitions - resulting in cooperative DNA binding. It is proposed that a role of antagonists is to decouple these linkages by stabilizing ineffective conformations within the PR hormone-binding domain. This hypothesis and the underlying mechanism will be examined by carrying out the following studies:
Aim 1 - The energetics of self-assembly for both isoforms in the presence and absence of progestin agonists and antagonists will be determined using analytical ultracentrifugation.
Aim 2 - A rigorous thermodynamic analysis of the interactions of each PR isoform with the multi-site mouse mammary tumor virus promoter will be determined using quantitative DNAse footprinting.
Aim 3 - The changes in isoform structure and stability associated with ligand and DNA-binding will be mapped using hydroxyl radical proteolytic footprinting, CD spectroscopy and microcalorimetry.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK061933-05
Application #
7176169
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Margolis, Ronald N
Project Start
2003-03-15
Project End
2008-01-31
Budget Start
2007-02-01
Budget End
2008-01-31
Support Year
5
Fiscal Year
2007
Total Cost
$219,030
Indirect Cost

  Institution

Name
University of Colorado Denver
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
041096314
City
Aurora
State
CO
Country
United States
Zip Code
80045

  Publications

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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