| Single-cell analysis of glucocorticoid receptor action reveals that stochastic post-chromatin association mechanisms regulate ligand-specific transcription. | |
MedLine Citation:
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PMID: 16873444 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
Authors:
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Ty C Voss; Sam John; Gordon L Hager |
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Publication Detail:
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Type: Journal Article Date: 2006-07-27 |
Journal Detail:
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Title: Molecular endocrinology (Baltimore, Md.) Volume: 20 ISSN: 0888-8809 ISO Abbreviation: Mol. Endocrinol. Publication Date: 2006 Nov |
Date Detail:
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Created Date: 2006-10-26 Completed Date: 2007-02-12 Revised Date: 2008-11-21 |
Medline Journal Info:
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Nlm Unique ID: 8801431 Medline TA: Mol Endocrinol Country: United States |
Other Details:
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Languages: eng Pagination: 2641-55 Citation Subset: IM |
Affiliation:
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Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-5055, USA. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Animals Chromatin / metabolism* DNA-Binding Proteins / physiology Ligands* Mammary Tumor Virus, Mouse / genetics Mice Models, Biological Promoter Regions, Genetic Receptors, Glucocorticoid / physiology* Stochastic Processes Tissue Array Analysis / methods* Transcription, Genetic / physiology* Transcriptional Activation Tumor Cells, Cultured |
| Chemical | |
Reg. No./Substance:
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0/Chromatin; 0/DNA-Binding Proteins; 0/Ligands; 0/Receptors, Glucocorticoid |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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