High content imaging-based assay to classify estrogen receptor-α ligands based on defined mechanistic outcomes

Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Gene (Impact Factor: 2.14). 05/2011; 477(1-2):42-52. DOI: 10.1016/j.gene.2011.01.009
Source: PubMed


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.

Download full-text


Available from: Michael A Mancini
  • Source
    • "Z-stacks were imaged at 1 µM intervals at 1 × 1 binning. Nuclear array segmentation and signal quantization were performed using PipelinePilot image analysis software as previously described (20). Briefly, maximum intensity projections were created for the GFP-ERα and antibody channels followed by background subtraction to all images. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Nuclear receptors (NRs) are central regulators of pathophysiological processes; however, how their responses intertwine is still not fully understood. The aim of this study was to determine whether and how steroid NRs can influence each other’s activity under co-agonist treatment. We used a unique system consisting of a multicopy integration of an estrogen receptor responsive unit that allows direct visualization and quantification of estrogen receptor alpha (ERα) DNA binding, co-regulator recruitment and transcriptional readout. We find that ERα DNA loading is required for other type I nuclear receptors to be co-recruited after dual agonist treatment. We focused on ERα/glucocorticoid receptor interplay and demonstrated that it requires steroid receptor coactivators (SRC-2, SRC-3) and the mediator component MED14. We then validated this cooperative interplay on endogenous target genes in breast cancer cells. Taken together, this work highlights another layer of mechanistic complexity through which NRs cross-talk with each other on chromatin under multiple hormonal stimuli.
    Full-text · Article · Feb 2013 · Nucleic Acids Research
  • [Show abstract] [Hide abstract]
    ABSTRACT: The nuclear receptors (NR) are a critical superfamily of gene regulatory proteins involved in all of human physiology. Classic biochemical techniques have provided deep insights into NR function but, due to their nature as single endpoint assays derived from pooled cell populations and tissues, these approaches are intrinsically unable to address important mechanistic considerations linked to cellular heterogeneity. Androgen receptor (AR) and estrogen receptor-α (ER) are members of the Type 1 steroid receptor subfamily of NRs that contribute to sex determination. Numerous recent advances have permitted direct, quantitative visualization of AR and ER functions within a cellular context that can be combined with conventional molecular and biochemical approaches. This new approach, essentially single cell-based systems biology, now enables researchers to follow NR shuttling through subcellular compartments in response to physiologic, pathologic or pharmacologic stimuli, to quantify transcriptional activity, and to identify interacting molecules leading to induction, or repression, of transcription. Here, we discuss recent reports that have applied advanced imaging systems [high throughput microscopy (HTM), high content analysis (HCA), FRET, FRAP] to dissect the biology of ER and AR at the single cell level. First, we discuss the use of HCA to examine AR functions in genital skin fibroblasts derived from normally virilized individuals and patients with androgen insensitivity syndrome (AIS), and in prostate cancer cell lines. Second, we highlight how these imaging techniques have been applied to define compound-specific mechanisms of ER gene regulation and coregulator interactions. These methods reveal new findings about the actions of AR and ER that are overlooked by conventional methods. © 2012 Springer Science+Business Media, LLC. All rights reserved.
    No preview · Article · Aug 2011
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cofactors for estrogen receptor α (ERα) can modulate gene activity by posttranslationally modifying histone tails at target promoters. Here, we found that stimulation of ERα-positive cells with 17β-estradiol (E2) promotes global citrullination of histone H3 arginine 26 (H3R26) on chromatin. Additionally, we found that the H3 citrulline 26 (H3Cit26) modification colocalizes with ERα at decondensed chromatin loci surrounding the estrogen-response elements of target promoters. Surprisingly, we also found that citrullination of H3R26 is catalyzed by peptidylarginine deiminase (PAD) 2 and not by PAD4 (which citrullinates H4R3). Further, we showed that PAD2 interacts with ERα after E2 stimulation and that inhibition of either PAD2 or ERα strongly suppresses E2-induced H3R26 citrullination and ERα recruitment at target gene promoters. Collectively, our data suggest that E2 stimulation induces the recruitment of PAD2 to target promoters by ERα, whereby PAD2 then citrullinates H3R26, which leads to local chromatin decondensation and transcriptional activation.
    Full-text · Article · Aug 2012 · Proceedings of the National Academy of Sciences
Show more