The long-term goal of this research is to determine the mechanisms responsible for higher eukaryotic gene regulation. As a model system we are studying the five members of the steroid receptor family of ligand-activated transcription factors. These proteins - the estrogen receptor, progesterone receptor, glucocorticoid receptor, androgen receptor and mineralocorticoid receptor - are structurally similar. However, the basis by which each receptor executes its unique gene regulatory functions is unclear, particularly so when in the presence of its remaining family members. Our research indicates that a) different steroid receptors exhibit variable energetics of inter-site cooperativity when binding to an identical promoter;and b) the energetics of cooperativity exhibited by a steroid receptor can vary at different promoters. We therefore hypothesize that the extent to which a gene is activated by a particular steroid receptor correlates with the energetics of receptor cooperativity when assembling at the promoter of that gene. Differences in cooperative binding energetics will thus dictate the degree of successful promoter occupancy by each steroid receptor, and thus the extent of receptor-specific gene regulation.
In Aim 1, we will determine the microscopic binding energetics for steroid receptor assembly at the mouse mammary tumor virus (MMTV) promoter. We will resolve the DNA- independent self-association properties of each receptor using analytical ultracentrifugation, and we will determine the intrinsic and cooperative binding free energies for receptor assembly at the promoter using quantitative DNase footprint titration.
In Aim 2, we will characterize androgen receptor transcriptional activation mutants identified in prostate carcinomas. We will examine their self-association properties using analytical ultracentrifugation, and we will dissect the microstate binding energetics of these mutants to the MMTV promoter using footprint titrations.
In Aim 3, we will correlate the binding energetics of the receptors with their promoter occupancy and transcriptional activation ability within the cell. Cellular occupancy at the promoter will be determined by chromatin immunoprecipitation (ChIP) assays, and the extent of mRNA production will be determined by quantitative RT-PCR.

Public Health Relevance

The long-term goal of this research is to determine the mechanisms responsible for human gene regulation. As a model system we are studying steroid receptors and their interactions with complex promoter sequences. The results of this work should reveal a new and more quantitative way to understand receptor-mediated gene regulation. As a result, these studies should open up a number of new avenues for drug design.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK088843-03
Application #
8293234
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Margolis, Ronald N
Project Start
2010-07-01
Project End
2015-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
3
Fiscal Year
2012
Total Cost
$308,813
Indirect Cost
$103,388
Name
University of Colorado Denver
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
041096314
City
Aurora
State
CO
Country
United States
Zip Code
80045
Bain, David L; Connaghan, Keith D; Maluf, Nasib K et al. (2014) Steroid receptor-DNA interactions: toward a quantitative connection between energetics and transcriptional regulation. Nucleic Acids Res 42:691-700
Connaghan, Keith D; Yang, Qin; Miura, Michael T et al. (2014) Homologous steroid receptors assemble at identical promoter architectures with unique energetics of cooperativity. Proteins 82:2078-87
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
De Angelis, Rolando W; Yang, Qin; Miura, Michael T et al. (2013) Dissection of androgen receptor-promoter interactions: steroid receptors partition their interaction energetics in parallel with their phylogenetic divergence. J Mol Biol 425:4223-35
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