Several lines of our research show that the organizational status of regulatory elements in the eukaryotic nucleus can impact strongly on their utilization. We have developed a reagent that will be very useful in testing models related to the domain structure of chromatin and intranuclear organization. We have constructed chimeras between the green fluorescent protein (GFP) and several of the major nuclear receptors, including the glucocorticoid receptor, progesterone receptor, estrogen receptor, AhR receptor, PPAR receptor, RAR receptor, and the thyroid receptor. All of the receptor chimeras have been shown to be efficiently expressed in cultured cells, and to be functional in terms of promoter transactivation and ligand binding. GFP-GR has been characterized in particular detail. Using a cell line, 3134, that harbors a 200-copy, head-to-tail tandem repeat of an MMTV LTR-ras-BPV fusion, we have been able to demonstrate direct binding of the receptor to its cognate response element in living cells. Using these cells, a bright region of GFP-GR localization is observed that represents binding to the tandem array. This technology allows us, for the first time, to characterize the interactions of nuclear receptors with regulatory elements in living cells in real time.

Agency
National Institute of Health (NIH)
Institute
Division of Basic Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010027-09
Application #
6950637
Study Section
(LRBG)
Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
2003
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
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