The glucocorticoid receptor (GR) dynamically interacts with response elements in the 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 GFP-GR fusion protein to the MMTV regulatory elements can be observed in living cells. Following ligand treatment, MMTV-dependent transcription in individual cells was detected by RNA 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. While dexamethasone (Dex) increased the mean RNA FISH signal 10 fold, RU486 (RU) produced only 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 ChIP 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 non-linear 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 RU treatment compared with Dex 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.