Differential recruitment of co-regulatory proteins to the human estrogen receptor 1 in response to xenoestrogens

Abstract

The diverse biological effects of xenoestrogens may be explained by their ability to differentially recruit co-regulatory proteins to the estrogen receptor (ER). We employed high-throughput receptor affinity binding and co-regulatory protein recruitment screening assays based on fluorescence polarization and time resolved florescence resonance energy transfer (TR-FRET), respectively, to assess xenoestrogen-specific binding and co-regulatory protein recruitment to the ER. Then we used a functional proteomic assay based on co-immunoprecipitation of ER-bound proteins to isolate and identify intact co-regulatory proteins recruited to a ligand-activated ER. Through these approaches, we revealed differential binding affinity of bisphenol-A (BPA) and genistein (GEN) to the human ERα (ESR1) and ligand-dependent recruitment of SRC-1 and SRC-3 peptides. Recruitment profiles were variable for each ligand and in some cases were distinct compared to 17β-estradiol (E2). For example, E2 and GEN recruited both SRC-1 and -3 peptides whereas BPA recruited only SRC-1 peptides. Results of the functional proteomic assay showed differential recruitment between ligands where E2 recruited the greatest number of proteins followed by BPA then GEN. A number of proteins share previously identified relationships with ESR1 as determined by STRING analysis. Although there was limited overlap in proteins identified between treatments, all ligands recruited proteins involved in cell growth as determined by subnetwork enrichment analysis (p < 0.05). A comparative, in silico analysis revealed that fewer interactions exist between zebrafish (Danio rerio) esr1 and zebrafish orthologs of proteins identified in our functional proteomic analysis. Taken together these results identify recruitment of known and previously unknown co-regulatory proteins to ESR1 and highlight new methods to assay recruitment of low abundant and intact, endogenous co-regulatory proteins to ESR1 or other nuclear receptors, in both human and aquatic species.

ERα mediated gene state switching regulates the extent of the single-cell estrogen response

“…Our study establishes a correlation between the fraction of cells actively transcribing TFF1 and GREB1, and the recruitment of ERα and FOXA1 at the gene loci. This result, in the context of studies which profiled the protein-protein interactions formed by ERα in the presence of E2 and BPA [20, 21], suggest that activation of GREB1 does not depend on the same cofactors as TFF1 . Supporting this speculation, different subsets of cofactors are necessary for maximal activation of E2 responsive genes [34].…”

“…Notably, the efficiency of ERα interactions with several cofactors including MED1 and SRC-3 is altered when the receptor is bound by estrogen-mimicking chemicals known as endocrine-disrupting chemicals (EDCs) [20, 21]. Humans are continuously exposed to a complex mixture of EDCs including genistein (Gen) from soy products, Bisphenol S (BPS), and Bisphenol A (BPA) from plastics [22, 23].…”

Thrap3 promotes R-loop resolution via interaction with methylated DDX5

“…It has been reported that treatment of human breast cancer cells with 17β-estradiol (E2) dramatically increases the accumulation of R-loops, particularly in regions of the genome containing estrogen-induced genes 35 . Interestingly, estrogen receptor α (ERα) specifically interacts with Thrap3 under conditions of E2 exposure 36 . Because E2-dependent R-loop generation and rearrangement in breast cancer cells are mainly enriched at E2-responsive genomic loci, it is possible that Thrap3 might be localized to E2-responsive genomic loci with ERα and that this localization is followed by DDX5 recruitment for precise removal of cotranscriptional R-loops in breast cancer cells, which is critical for cancer progression.…”

Influence of the Gastrointestinal Environment on the Bioavailability of Ethinyl Estradiol Sorbed to Single-Walled Carbon Nanotubes

“…To determine the impact of adsorption of EE2 to SWCNTs on estrogen receptor (ER) binding by EE2, a fluorescence polarization (FP)-based ER binding assay was utilized. The advantages of this assay over traditional radioligand binding assays and detailed methods have been described previously . Briefly, the recombinant human estrogen receptor 1 ligand binding domain (ESR1-LBD) was produced in bacterial cells and purified for use in the assays.…”