High content imaging-based assay to classify estrogen receptor-α ligands based on defined mechanistic outcomes.
Estrogen receptor-α (ER) is an important target both for therapeutic compounds and endocrine disrupting chemicals (EDCs); however, the mechanisms involved in chemical modulation of regulating ER transcriptional activity are inadequately understood. Here, we report the development of a high content analysis-based assay to describe ER activity that uniquely exploits a microscopically visible multi-copy integration of an ER-regulated promoter. Through automated single-cell analyses, we simultaneously quantified promoter occupancy, recruitment of transcriptional cofactors and large-scale chromatin changes in response to a panel of ER ligands and EDCs. Image-derived multi-parametric data was used to classify a panel of ligand responses at high resolution. We propose this system as a novel technology providing new mechanistic insights into EDC activities in a manner useful for both basic mechanistic studies and drug testing.
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
F J
FJ
Ashcroft
J Y
JY
Newberg
E D
ED
Jones
I
I
Mikic
M A
MA
Mancini
D001559:Benzhydryl Compounds; D015153:Blotting, Western; D045744:Cell Line, Tumor; D016000:Cluster Analysis; D052244:Endocrine Disruptors / Q000494:pharmacology; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism*; D000077267:Fulvestrant; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D010636:Phenols / Q000494:pharmacology; D025341:Principal Component Analysis; D011401:Promoter Regions, Genetic / Q000235:genetics; D020849:Raloxifene Hydrochloride / Q000494:pharmacology; D015203:Reproducibility of Results; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology; D000098826:Bisphenol A Compounds
D001559:Benzhydryl Compounds; D052244:Endocrine Disruptors; D004958:Estradiol; D004965:Estrogen Antagonists; D047628:Estrogen Receptor alpha; D000077267:Fulvestrant; D049452:Green Fluorescent Proteins; D010636:Phenols; D020849:Raloxifene Hydrochloride; D013629:Tamoxifen; C016601:afimoxifene; C006780:bisphenol A; D000098826:Bisphenol A Compounds
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