title: Single-cell analysis of glucocorticoid receptor action reveals that stochastic post-chromatin association mechanisms regulate ligand-specific transcription.
abstract: 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.affiliation: Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-5055, USA.
authors.forename: Ty C
authors.initials: TC
authors.lastname: Voss
authors.forename: Sam
authors.initials: S
authors.lastname: John
authors.forename: Gordon L
authors.initials: GL
authors.lastname: Hager
mesh_terms: D000818:Animals; D002843:Chromatin / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000502:physiology; D008024:Ligands; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D051379:Mice; D008954:Models, Biological; D011401:Promoter Regions, Genetic; D011965:Receptors, Glucocorticoid / Q000502:physiology*; D013269:Stochastic Processes; D046888:Tissue Array Analysis / Q000379:methods*; D014158:Transcription, Genetic / Q000502:physiology*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
chemical_list: D002843:Chromatin; D004268:DNA-Binding Proteins; D008024:Ligands; D011965:Receptors, Glucocorticoid