Estrogen-receptor-α exchange and chromatin dynamics are ligand- and domain-dependent

We report a mammalian-based promoter chromosomal array system developed for single-cell studies of transcription-factor function. Designed after the prolactin promoter-enhancer, it allows for the direct visualization of estrogen receptor alpha (ER alpha) and/or Pit-1 interactions at a physiologically regulated transcription locus. ER alpha-and ligand-dependent cofactor recruitment, large-scale chromatin modifications and transcriptional activity identified a distinct fingerprint of responses for each condition. Ligand-dependent transcription ( more than threefold activation compared with vehicle, or complete repression by mRNA fluorescent in situ hybridization) at the array correlated with its state of condensation, which was assayed using a novel high throughput microscopy approach. In support of the nuclear receptor hit-and-run model, photobleaching studies provided direct evidence of very transient ER-array interactions, and revealed ligand-dependent changes in k(off). ER alpha-truncation mutants indicated that helix-12 and interactions with co-regulators influenced both large-scale chromatin modeling and photobleaching recovery times. These data also showed that the ER alpha DNA-binding domain was insufficient for array targeting. Collectively, quantitative observations from this physiologically relevant biosensor suggest stochastic-based dynamics influence gene regulation at the promoter level.