Activation of Estrogen Receptor-α by E2 or EGF Induces Temporally Distinct Patterns of Large-Scale Chromatin Modification and mRNA Transcription

Abstract

Estrogen receptor-α (ER) transcription function is regulated in a ligand-dependent (e.g., estradiol, E2) or ligand-independent (e.g., growth factors) manner. Our laboratory seeks to understand these two modes of action. Using a cell line that contains a visible prolactin enhancer/promoter array (PRL-HeLa) regulated by ER, we analyzed ER response to E2 and EGF by quantifying image-based results. Data show differential recruitment of GFP-ER to the array, with the AF1 domain playing a vital role in EGF-mediated responsiveness. Temporal analyses of large-scale chromatin dynamics, and accumulation of array-localized reporter mRNA over 24 hours showed that the EGF response consists of a single pulse of reporter mRNA accumulation concomitant with transient increase in array decondensation. Estradiol induced a novel cyclical pattern of mRNA accumulation with a sustained increase in array decondensation. Collectively, our work shows that there is a stimuli-specific pattern of large-scale chromatin modification and transcript levels by ER.

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

“…3. reporter, based on ER-mediated colocalization of SRC-1 and RNAPII (Figs. 4 and 5), and colocalized reporter mRNA accumulation (Sharp et al, 2006;Berno et al, 2008). Addition of an ER agonist (E 2 ) to PRLHeLa cells is followed by a visible decondensation of the PRL-promoter array and colocalization of endogenous transcription factors.…”

“…The PRL-HeLa line used in this and other studies (Sharp et al, 2006;Amazit et al, 2007;Berno et al, 2008) was designed for singlecell studies of transcription factor function.…”

“…For this purpose we used a recently developed transcriptional biosensor constructed from mammalian promoter/ enhancer components to study ligand-dependent large-scale chromatin modifications at the single cell level, including recruitment of endogenous chromatin-remodeling complexes at the promoter level. In this assay, ER-mediated chromosomal dynamics can be assayed using an integrated responsive array of promoter/enhancer elements based on the mammalian prolactin (PRL) promoter/enhancer (Sharp et al, 2006;Berno et al, 2008). The PRL promoter contains both Pit-1 (a POU-class transcription factor) (Andersen and Rosenfeld, 2001) and ER binding sites (Day and Maurer, 1989;Day et al, 1990).…”

The myImageAnalysis Project: A Web-Based Application for High-Content Screening

“…We have validated that these cells allow us to characterize physiological responses to hormones and their effect upon ER DNA binding, promoter/chromatin remodeling, coregulator recruitment, and mRNA production. 14,26,27 We have also used these cell lines to characterize how environmental estrogens differentially affect the ER response. 13 The image analysis tools to perform these studies have been incorporated into the mIA algorithm editor, creating the predefined ''Nuclear Spot'' analysis pipeline.…”

Androgen Receptor Mutations Associated with Androgen Insensitivity Syndrome: A High Content Analysis Approach Leading to Personalized Medicine

“…1A shows examples of typical images obtained with the IC-100 microscope of normal GSFs (L7728, M7118, A4676) or GSFs from AIS patients [marked as GSF strain 571 (AR mutant F764L), 691 (AR mutant R840C) and 851(AR mutant P766S)], stained with DAPI, AR, and CellMask solution, at concentrations of Mibolerone of 0 and 100 nM. While our initial AR studies using HTM were based upon CytoShop (Beckman-Coulter) [14,15,33], these approaches were refined in the current work with the more robust server-client Pipeline Pilot image analysis platform (Basic and Advanced Imagine Collections, Pipeline Pilot 7.5, Accelrys, San Diego). Initially, the background signal was removed from all images using plate-and channel-specific correction images, generated by the sum projection of .600 randomly selected images for each channel from the image set.…”