A major challenge facing biomedical research is to determine how the network of interactions involving gene regulatory proteins is controlled in the context of the natural environment inside living cells, and to understand how disease processes affect these activities. The expression of the prolactin (PRL) gene in anterior pituitary lactotrope cells is a proven model system to define the molecular mechanisms that contribute to the control of cell type-specific gene regulation. In the pituitary cell nucleus, the homeodomain (HD) transcription factor Pit-1 orchestrates the activities of a network of regulatory proteins that control PRL gene expression. The broad objective of this proposal is to use emerging concepts in nuclear architecture and chromatin remodeling to determine how Pit-1 coordinates the activities of multi-protein complexes at specific gene enhancers and promoters. Pit-1 regulates PRL transcription through its interactions with other coregulatory proteins including the CCAAT/enhancer-binding protein alpha (C/EBP1). C/EBP1 in turn associates with the heterochromatin binding protein 1 alpha (HP11) in regions of centromeric heterochromatin, and Pit-1 can recruit C/EBP1 from the regions of compact chromatin. Disease-causing mutations in Pit-1 disrupt this network of protein interactions, and these results have broad implications for many human diseases linked to mutations in the HD proteins. The studies in this proposal use the combination of biochemical analysis and live-cell imaging to test the hypothesis that Pit-1 interactions with the C/EBP1-HP11 complex function to remodel densely packaged chromatin, allowing the access of pituitary-specific transcription factors to target genes.
The first aim i s to define the interactions of C/EBP1 and HP11 in regions of heterochromatin in pituitary cells, and then to determine the role of Pit-1 in regulating this network of protein interactions.
The second aim i s to determine how these interactions function to control local chromatin remodeling.
The third aim will take advantage of newly developed transgenic mouse models that allow the unambiguous identification of living lactotrope or somatotropes cells to map the network of Pit-1 interactions in the normal mature mouse pituitary cells. Relevance: The anterior pituitary hormone PRL has many diverse physiological roles, and the failure to regulate PRL synthesis leads to reproductive disturbances in both men and women, and can lead to prolactinomas, the most common intracranial primary tumor. If we are to understand disease processes and design therapeutic strategies, it is important to define how specific gene regulatory complexes are assembled in the intact cell nucleus. Discovering how nuclear architecture controls gene expression will be the cornerstone for understanding how genomes work.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK043701-18
Application #
8230671
Study Section
Molecular and Cellular Endocrinology Study Section (MCE)
Program Officer
Malozowski, Saul N
Project Start
1994-07-11
Project End
2014-02-28
Budget Start
2012-03-01
Budget End
2014-02-28
Support Year
18
Fiscal Year
2012
Total Cost
$289,790
Indirect Cost
$99,228
Name
Indiana University-Purdue University at Indianapolis
Department
Physiology
Type
Schools of Medicine
DUNS #
603007902
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
Day, Richard N (2014) Measuring protein interactions using Forster resonance energy transfer and fluorescence lifetime imaging microscopy. Methods 66:200-7
Siegel, Amanda P; Hays, Nicole M; Day, Richard N (2013) Unraveling transcription factor interactions with heterochromatin protein 1 using fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. J Biomed Opt 18:25002
Sun, Yuansheng; Hays, Nicole M; Periasamy, Ammasi et al. (2012) Monitoring protein interactions in living cells with fluorescence lifetime imaging microscopy. Methods Enzymol 504:371-91
Day, Richard N; Davidson, Michael W (2012) Fluorescent proteins for FRET microscopy: monitoring protein interactions in living cells. Bioessays 34:341-50
Sun, Yuansheng; Day, Richard N; Periasamy, Ammasi (2011) Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy. Nat Protoc 6:1324-40
Day, Richard N; Davidson, Michael W (2009) The fluorescent protein palette: tools for cellular imaging. Chem Soc Rev 38:2887-921
Day, Richard N; Booker, Cynthia F; Periasamy, Ammasi (2008) Characterization of an improved donor fluorescent protein for Forster resonance energy transfer microscopy. J Biomed Opt 13:031203
Voss, Ty C; Demarco, Ignacio A; Booker, Cynthia F et al. (2005) Quantitative methods to analyze subnuclear protein organization in cell populations with varying degrees of protein expression. J Biomed Opt 10:024011
Voss, Ty C; Demarco, Ignacio A; Booker, Cynthia F et al. (2005) Corepressor subnuclear organization is regulated by estrogen receptor via a mechanism that requires the DNA-binding domain. Mol Cell Endocrinol 231:33-47
Voss, Ty C; Demarco, Ignacio A; Day, Richard N (2005) Quantitative imaging of protein interactions in the cell nucleus. Biotechniques 38:413-24

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