Document Detail

Single-cell analysis of glucocorticoid receptor action reveals that stochastic post-chromatin association mechanisms regulate ligand-specific transcription.
MedLine Citation:
PMID:  16873444     Owner:  NLM     Status:  MEDLINE    
The glucocorticoid receptor (GR) dynamically interacts with response elements in the mouse mammary tumor virus (MMTV) promoter to regulate steroid-dependent transcription. In a clonal mammary carcinoma cell line containing a tandem array of MMTV promoter-reporter gene cassettes integrated at a single genomic locus, direct binding of a green fluorescent protein (GFP)-GR fusion protein to the MMTV regulatory elements can be observed in living cells. After ligand treatment, MMTV-dependent transcription in individual cells was detected by RNA fluorescence in situ hybridization (FISH). High-resolution fluorescence images were acquired from large numbers of randomly selected cells. Images were analyzed with a novel automated computer algorithm, measuring the RNA FISH signal and the relative GFP-GR fluorescence intensity at the MMTV array for each cell. Although dexamethasone increased the mean RNA FISH signal approximately 10-fold, RU486 produced only about a 2-fold induction, as expected for this mixed antagonist. For all treatment conditions, the relative GFP-GR fluorescence at the array for the averaged cells paralleled the RNA FISH measurements, suggesting that image analysis accurately detected an increase in steady-state GR association with the MMTV array that was responsible for the increase in transcriptional activity. The antagonist-dependent decreases in GR association with the MMTV promoter were confirmed by chromatin immunoprecipitation experiments, supporting the image analysis results. A pronounced cell-to-cell variability was observed in RNA FISH signal and GR-MMTV association within treatment groups. We observed a nonlinear relationship between GR-MMTV association and RNA FISH in individual cells, indicating that differences in GR-MMTV interaction account for some, but not all, of the transcriptional heterogeneity between individual cells. In selected cell subpopulations with equal levels of GR-MMTV association, there was a decrease in RNA FISH signal with RU486 treatment compared with dexamethasone treatment. These results indicate that stochastic events occurring after GR-promoter association, such as the actions of chromatin remodeling complexes or other cofactors, change in a ligand-dependent manner and regulate heterogeneous transcription in individual cells.
Ty C Voss; Sam John; Gordon L Hager
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Publication Detail:
Type:  Journal Article     Date:  2006-07-27
Journal Detail:
Title:  Molecular endocrinology (Baltimore, Md.)     Volume:  20     ISSN:  0888-8809     ISO Abbreviation:  Mol. Endocrinol.     Publication Date:  2006 Nov 
Date Detail:
Created Date:  2006-10-26     Completed Date:  2007-02-12     Revised Date:  2008-11-21    
Medline Journal Info:
Nlm Unique ID:  8801431     Medline TA:  Mol Endocrinol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  2641-55     Citation Subset:  IM    
Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-5055, USA.
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MeSH Terms
Chromatin / metabolism*
DNA-Binding Proteins / physiology
Mammary Tumor Virus, Mouse / genetics
Models, Biological
Promoter Regions, Genetic
Receptors, Glucocorticoid / physiology*
Stochastic Processes
Tissue Array Analysis / methods*
Transcription, Genetic / physiology*
Transcriptional Activation
Tumor Cells, Cultured
Reg. No./Substance:
0/Chromatin; 0/DNA-Binding Proteins; 0/Ligands; 0/Receptors, Glucocorticoid

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