High Resolution Screening of Plant Natural Product Extracts for Estrogen Receptor α and β Binding Activity Using an Online HPLC-MS Biochemical Detection System

A new screening technology that combines biochemical analysis with the resolution power of high-performance liquid chromatography (HPLC), referred to here as high-resolution screening (HRS) technique, is described. The capability of the HRS technology to analyze biologically active compounds in complex mixtures is demonstrated by screening a plant natural product extract library for estrogen receptor (ER) a and fi binding activity. The simultaneous structure elucidation of biologically active components in crude extracts was achieved by operating the HRS system in combination with mass spectrometry (MS). In contrast to conventional microtiter-type bioassays, the interactions of the extracts with the ER and the employed label, coumestrol, proceeded at high speed in a closed, continuous-flow reaction detection system, which was coupled directly to the outlet of a HPLC separation column. The reaction products of this homogeneous fluorescence enhancement-type assay were detected online using a flow-through fluorescence detector. Primary screening of the extract library was performed in the fast-flow injection analysis mode (FlowScreening) wherein the chromatographic separation system was bypassed. The library was screened at high speed, using two assay lines in parallel. A total of 98% of the identified hits were confirmed in a traditional 96-well microplate-based fluorescence polarization assay, indicating the reliability of the FlowScreening process. Active extracts were reassayed in a transcriptional activation assay in order to assess the functional activity of the bioactive extracts. Only functional active extracts were processed in the more time-consuming HRS mode, which was operated in combination with MS. Information on the number of active compounds, their retention times, the molecular masses, and the MS/MS-fingerprints as a function of their biological activity was obtained from 50% of the functional active extracts in real time. This dramatically enhances the speed of biologically active compound characterization in natural product extracts compared to traditional fractionation approaches.

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Estrogen Receptor-Independent Catechol Estrogen Binding Activity: Protein Binding Studies in Wild-Type, Estrogen Receptor-α KO, and Aromatase KO Mice Tissues†

Biochemistry ◽

10.1021/bi036154j ◽

2004 ◽

Vol 43 (21) ◽

pp. 6698-6708 ◽

Cited By ~ 17

Author(s):

Brian J. Philips ◽

Pete J. Ansell ◽

Leslie G. Newton ◽

Nobuhiro Harada ◽

Shin-Ichiro Honda ◽

...

Keyword(s):

Estrogen Receptor ◽

Protein Binding ◽

Estrogen Receptor Α ◽

Binding Activity ◽

Wild Type ◽

Binding Studies ◽

Catechol Estrogen ◽

Estrogen Binding

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The Phosphorylated Estrogen Receptor α (ER) Cistrome Identifies a Subset of Active Enhancers Enriched for Direct ER-DNA Binding and the Transcription Factor GRHL2

Molecular and Cellular Biology ◽

10.1128/mcb.00417-18 ◽

2018 ◽

Vol 39 (3) ◽

Cited By ~ 5

Author(s):

Kyle T. Helzer ◽

Mary Szatkowski Ozers ◽

Mark B. Meyer ◽

Nancy A. Benkusky ◽

Natalia Solodin ◽

...

Keyword(s):

Estrogen Receptor ◽

Transcription Factor ◽

Dna Binding ◽

Protein Interactions ◽

Posttranslational Modifications ◽

Binding Sites ◽

Protein Function ◽

Estrogen Receptor Α ◽

Binding Activity

ABSTRACT Posttranslational modifications are key regulators of protein function, providing cues that can alter protein interactions and cellular location. Phosphorylation of estrogen receptor α (ER) at serine 118 (pS118-ER) occurs in response to multiple stimuli and is involved in modulating ER-dependent gene transcription. While the cistrome of ER is well established, surprisingly little is understood about how phosphorylation impacts ER-DNA binding activity. To define the pS118-ER cistrome, chromatin immunoprecipitation sequencing was performed on pS118-ER and ER in MCF-7 cells treated with estrogen. pS118-ER occupied a subset of ER binding sites which were associated with an active enhancer mark, acetylated H3K27. Unlike ER, pS118-ER sites were enriched in GRHL2 DNA binding motifs, and estrogen treatment increased GRHL2 recruitment to sites occupied by pS118-ER. Additionally, pS118-ER occupancy sites showed greater enrichment of full-length estrogen response elements relative to ER sites. In an in vitro DNA binding array of genomic binding sites, pS118-ER was more commonly associated with direct DNA binding events than indirect binding events. These results indicate that phosphorylation of ER at serine 118 promotes direct DNA binding at active enhancers and is a distinguishing mark for associated transcription factor complexes on chromatin.

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Involvement of estrogen receptors α and β in the regulation of cervical permeability

AJP Cell Physiology ◽

10.1152/ajpcell.2000.278.4.c689 ◽

2000 ◽

Vol 278 (4) ◽

pp. C689-C696 ◽

Cited By ~ 20

Author(s):

George I. Gorodeski ◽

Dipika Pal

Keyword(s):

Estrogen Receptor ◽

Binding Sites ◽

Estrogen Receptor Α ◽

Binding Activity ◽

Scatchard Analysis ◽

Single Class ◽

Estrogen Receptor Modulators ◽

Transcription Inhibitor ◽

Estrogen Binding ◽

In Cells

Estrogen increases the permeability of cultured human cervical epithelia (Gorodeski, GI. Am J Physiol Cell Physiol 275: C888–C899, 1998), and the effect is blocked by the estrogen receptor modulators ICI-182780 and tamoxifen. The objective of the study was to determine involvement of estrogen receptor(s) in mediating the effects on permeability. In cultured human cervical epithelial cells estradiol binds to high-affinity, low-capacity sites, in a specific and saturable manner. Scatchard analysis revealed a single class of binding sites with a dissociation constant of 1.3 nM and binding activity of ∼0.5 pmol/mg DNA. Estradiol increased the density of estrogen-binding sites in a time- and dose-related manner (half time ≈ 4 h, and EC50≈ 1 nM). RT-PCR assays revealed the expression of mRNA for the estrogen receptor α (αER) and estrogen receptor β (βER). Removal of estrogen from the culture medium decreased and treatment with estrogen increased the expression of αER and βER mRNA. In cells not treated with estrogen, ICI-182780 and tamoxifen increased βER mRNA. In cells treated with estrogen, neither ICI-182780 nor tamoxifen had modulated significantly the increase in αER or βER mRNA. The transcription inhibitor actinomycin D blocked the estrogen-induced increase in permeability, and it abrogated the estradiol-induced increase in estrogen binding sites. These results suggest that the estrogen-dependent increase in cervical permeability is mediated by an αER-dependent increase in transcription.

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Loss of ERE binding activity by estrogen receptor-α alters basal and estrogen-stimulated bone-related gene expression by osteoblastic cells

Journal of Cellular Biochemistry ◽

10.1002/jcb.21459 ◽

2008 ◽

Vol 103 (3) ◽

pp. 896-907 ◽

Cited By ~ 16

Author(s):

Volha Rudnik ◽

Arunik Sanyal ◽

Farhan A. Syed ◽

David G. Monroe ◽

Thomas C. Spelsberg ◽

...

Keyword(s):

Gene Expression ◽

Estrogen Receptor ◽

Estrogen Receptor Α ◽

Binding Activity ◽

Osteoblastic Cells ◽

Related Gene

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Determining the Transrepression Activity of Xenoestrogen on Nuclear Factor-κB in Cos-1 Cells by Estrogen Receptor-α

International Journal of Toxicology ◽

10.1080/10915810701620317 ◽

2007 ◽

Vol 26 (5) ◽

pp. 441-449 ◽

Cited By ~ 1

Author(s):

R. A. Ansari ◽

J. Gandy

Keyword(s):

Estrogen Receptor ◽

Internal Control ◽

Firefly Luciferase ◽

Nuclear Factor Κb ◽

Estrogen Receptor Α ◽

Binding Activity ◽

Renilla Luciferase ◽

Nuclear Factor ◽

Sv40 Promoter ◽

Human Estrogen Receptor

Functional assays have been used to define the estrogenicity of xenoestrogens in cotransfection studies employing estrogen receptors in various cell lines. It is known that estrogen is able to affect transcription from other nuclear transcription factors, especially the nuclear factor- κB (NF- κB). The ability of selected xenoestrogens (methoxychlor [MXC], dieldrin, and o′, p′-DDT) to transrepress the NF- κB–mediated transcription in Cos-1 cells was evaluated by cotransfection of human estrogen receptor- α (hER α). These xenoestrogens have been described as comparably potent xenoestrogens, whereas their relative binding activity (RBA) has been relegated to a lower order as compare to estrogen. The two NF- κB response element–containing SV40 promoter and −242/+54 cytomegalovirus (CMV)–expressing firefly luciferase (2 × NRE-PV-Luc and 2 × NRE-CMV-Luc, respectively) were transfected into Cos-1 cells with pRL-tk, expressing the renilla luciferase as internal control. The estrogen receptor was expressed from cytomegalovirus major immediate early promoter (CMV-MIEP) (CMV5-hER α). Treatment with 1 nM estrogen (E2) (26.2%), 5 nM E2 (41.4%; p < .05), and xenoestrogens (methoxychlor [1 nM: 29.6%, p < .05; 10 nM: 22.6%), dieldrin [1 nM: 10.3%; 10 nM: 36.06%, p < .05], and o′, p′-DDT [1 nM: 17.0%; 10 nM: 7.15%]) repressed transcription from 2 × NREX-PV-Luc. The antiestrogen, ICI 182,780, failed to antagonize the effects of xenoestrogens. The effects of xenoestrogens in transrepression of NF- κB by ER α were similar when 2 × NRE-CMV-Luc was employed as reporter. Statistically significant ( p < .01) repression by 1 nM E2 (69.2%), 5 nM E2 (69.1%), 1 nM o′, p′-DDT (51.4%), 1 nM dieldrin (47.3%), and 1 nM MXC (73.3%) were observed. The effect of these xenoestrogens without ER α cotransfection on 2 × NRE-PV-Luc- and 2 × NRE-CMV-Luc-mediated NF- κB transcription was not affected by the treatment alone. It is concluded that xenoestrogens, like estrogens, are capable of producing transrepression of NF- κB by hER α.

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Identification of Compounds by High-Content Screening That Induce Cytoplasmic to Nuclear Localization of a Fluorescent Estrogen Receptor α Chimera and Exhibit Agonist or Antagonist Activity In Vitro

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057113504136 ◽

2013 ◽

Vol 19 (2) ◽

pp. 242-252 ◽

Cited By ~ 5

Author(s):

Angie B. Dull ◽

Anuja A. George ◽

Ekaterina I. Goncharova ◽

Jason R. Evans ◽

Antony Wamiru ◽

...

Keyword(s):

Estrogen Receptor ◽

Natural Product ◽

Fluorescent Protein ◽

Nuclear Translocation ◽

Imaging System ◽

Estrogen Receptor Α ◽

Significant Activity ◽

Antagonist Activity ◽

Green Fluorescent

We have completed a robust high-content imaging screen for novel estrogen receptor α (ERα) agonists and antagonists by quantitation of cytoplasmic to nuclear translocation of an estrogen receptor chimera in 384-well plates. The screen was very robust, with Z′ values >0.7 and coefficients of variation (CV) <5%. The screen utilized a stably transfected green fluorescent protein–tagged glucocorticoid/estrogen receptor (GFP-GRER) chimera, which consisted of the N-terminus of the glucocorticoid receptor fused to the human ERα ligand binding domain. The GFP-GRER exhibited cytoplasmic localization in the absence of ERα ligands and translocated to the nucleus in response to stimulation with ERα agonists and antagonists. The BD Pathway 435 imaging system was used for image acquisition, analysis of translocation dynamics, and cytotoxicity measurements. We screened 224,891 samples from our synthetic, pure natural product libraries, prefractionated natural product extracts library, and crude natural product extracts library, which produced a 0.003% hit rate. In addition to identifying several known ER ligands, five compounds were discovered that elicited significant activity in the screen. Transactivation potential studies demonstrated that two hit compounds behave as agonists, while three compounds elicited antagonist activity in MCF-7 cells.

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Acetylation of Estrogen Receptor α by p300 at Lysines 266 and 268 Enhances the Deoxyribonucleic Acid Binding and Transactivation Activities of the Receptor

Molecular Endocrinology ◽

10.1210/me.2005-0531 ◽

2006 ◽

Vol 20 (7) ◽

pp. 1479-1493 ◽

Cited By ~ 128

Author(s):

Mi Young Kim ◽

Eileen M. Woo ◽

Yee Ting Esther Chong ◽

Daria R. Homenko ◽

W. Lee Kraus

Keyword(s):

Estrogen Receptor ◽

Trichostatin A ◽

Estrogen Receptor Α ◽

Binding Activity ◽

Sirtuin 1 ◽

Dependent Manner ◽

Class Iii ◽

Dna Binding Activity ◽

Gene Assay ◽

Lysine Residues

Abstract Using a variety of biochemical and cell-based approaches, we show that estrogen receptor α (ERα) is acetylated by the p300 acetylase in a ligand- and steroid receptor coactivator-dependent manner. Using mutagenesis and mass spectrometry, we identified two conserved lysine residues in ERα (Lys266 and Lys268) that are the primary targets of p300-mediated acetylation. These residues are acetylated in cells, as determined by immunoprecipitation-Western blotting experiments using an antibody that specifically recognizes ERα acetylated at Lys266 and Lys268. The acetylation of ERα by p300 is reversed by native cellular deacetylases, including trichostatin A-sensitive enzymes (i.e. class I and II deacetylases) and nicotinamide adenine dinucleotide-dependent/nicotinamide-sensitive enzymes (i.e. class III deacetylases, such as sirtuin 1). Acetylation at Lys266 and Lys268, or substitution of the same residues with glutamine (i.e. K266/268Q), a residue that mimics acetylated lysine, enhances the DNA binding activity of ERα in EMSAs. Likewise, substitution of Lys266 and Lys268 with glutamine enhances the ligand-dependent activity of ERα in a cell-based reporter gene assay. Collectively, our results implicate acetylation as a modulator of the ligand-dependent gene regulatory activity of ERα. Such regulation is likely to play a role in estrogen-dependent signaling outcomes in a variety of estrogen target tissues in both normal and pathological states.

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