Ligand-induced large-scale chromatin dynamics as a biosensor for the detection of estrogen receptor subtype selective ligands.

Estrogen receptors (ER), members of the nuclear steroid receptor superfamily, act to activate transcription through ligand-dependent recruitment of coregulators and chromatin modifications. A series of synthetic A-ring reduced 19-nortestosterone-derived progestins has the capacity to selectively bind ERalpha for activated transcription, and to recruit coregulatory factors. In this study, we have analyzed the ability of synthetic 19-nortestosterone derivatives to visibly alter the configuration of ER-target gene chromatin using a novel mammalian promoter transcriptional biosensor (PRL-array) stably transfected into the genome of HeLa cells (PRL-HeLa cells). Results from synthetic steroid-treated cells expressing functional GFP-ERalpha or YFP-ERbeta chimeras were compared to those obtained with estradiol (E(2)) and the antiestrogen tamoxifen. In the presence of synthetic ligands or E(2) a concentration-dependent increase in area of the biosensor array was observed in GFP-ERalpha-expressing PRL-HeLa cells. No significant differences were found between the effects obtained with natural and synthetic steroids. Similarly, E(2) or synthetic steroids-treated PRL-HeLa cells also resulted in similar colocalization of SRC-1- and RNAPII-immunofluorescence at the array. YFP-ERbeta-expressing PRL-HeLa cells treated with E(2) showed increases in array area that were similar to ERalpha; however, treatment of YFP-ERbeta-expressing cells with synthetic ligands was indistinguishable from vehicle controls. These data indicate that A-ring reduced 19-nortestosterone derivatives have an estrogen-like effect on chromatin, including recruitment of transcription factors through selective interactions with ERalpha.