An estrogen receptor chimera senses ligands by nuclear translocation

Martínez, Elisabeth D.; Rayasam, Geetha Vani; Dull, Angie B.; Walker, Dawn A.; Hager, Gordon L.

doi:10.1016/j.jsbmb.2005.06.033

Cited by 22 publications

(18 citation statements)

References 60 publications

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“…However, unlike GR (Htun et al 1996; Ogawa et al 1995) or AR (Klokk et al 2007), the thyroid receptor isoforms are mostly nuclear even in the absence of hormone (Baumann et al 2001) and thus cannot be used directly in a nuclear translocation assay. To overcome this obstacle, we generated a fusion GFP-GR-TRβ construct, an approach used earlier to generate fluorescent chimeric molecules between the GR and the retinoic acid receptor (GFP-GR-RAR) and the GR and estrogen receptor (GFP-GR-ER) (Mackem et al 2001; Martinez et al 2005). The two major isoforms of the human thyroid receptor TRα and TRβ are encoded by two separate genes THRA and THRB located on chromosomes 3 and 17, respectively.…”

Section: Discussionmentioning

confidence: 99%

Novel cell-based assay for detection of thyroid receptor beta-interacting environmental contaminants

et al. 2016

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Even though the presence of endocrine disrupting chemicals (EDCs) with thyroid hormone (TH)-like activities in the environment is a major health concern, the methods for their efficient detection and monitoring are still limited. Here we describe a novel cell assay, based on the translocation of a green fluorescent protein (GFP) - tagged chimeric molecule of glucocorticoid receptor (GR) and the thyroid receptor beta (TRβ) from the cytoplasm to the nucleus in the presence of TR ligands. Unlike the constitutively nuclear TRβ, this GFP-GR-TRβ chimera is cytoplasmic in the absence of hormone while translocating to the nucleus in a time- and concentration-dependent manner upon stimulation with triiodothyronine (T3) and thyroid hormone analogue, TRIAC, while the reverse triiodothyronine (3,3′,5′-triiodothyronine, or rT3) was inactive. Moreover, GFP-GR-TRβ chimera does not show any cross-reactivity with the GR-activating hormones, thus providing a clean system for the screening of TR beta -interacting EDCs. Using this assay, we demonstrated that Bisphenol A (BPA) and 3,3′,5,5′-Tetrabromobisphenol (TBBPA) induced GFP-GR-TRβ translocation at micro molar concentrations. We screened over 100 concentrated water samples from different geographic locations in the United States and detected a low, but reproducible contamination in 53 % of the samples. This system provides a novel high-throughput approach for screening for endocrine disrupting chemicals (EDCs) interacting with TR beta.

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Section: Discussionmentioning

confidence: 99%

Novel cell-based assay for detection of thyroid receptor beta-interacting environmental contaminants

et al. 2016

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“…More recently, a number of fluorescent indicators have been designed for ligand-mediated responses of SRs. Some of these quantify ligand-induced activity by nuclear translocation or nuclear mobility of fluorescently labeled receptors or receptor chimeras (39)(40)(41)(42). Others make use of ligand-induced conformational changes in the SR-LBDs (43).…”

Section: Introductionmentioning

confidence: 99%

A multi‐parameter imaging assay identifies different stages of ligand‐induced androgen receptor activation

et al. 2013

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Androgens exert their key function in development and maintenance of the male phenotype via the androgen receptor (AR). Ligand-activated ARs also play a role in prostate cancer. Despite initial success of treatment by testosterone depletion or blocking of androgen binding to the AR using antiandrogens, eventually all tumors escape to a therapy resistant stage. Development of novel therapies by other antagonistic ligands or compounds that target events subsequent to ligand binding is very important. Here, we validate a fluorescence resonance energy transfer (FRET) based imaging assay for ligand-induced AR activity, based on the conformational change in the AR caused by interaction between the FQNLF motif in the N-terminal domain and the cofactor binding groove in the ligand-binding domain (N/C-interaction). We test the assay using known agonistic and antagonistic ligands on wild type AR and specific AR mutants. Our data show a strong correlation between the ligand-induced AR N/C-interaction and transcriptional activity in wild type AR, but also in AR mutants with broadened ligand responsiveness. Moreover, we explore additional readouts of this assay that contribute to the understanding of the working mechanism of the ligands. Together, we present a sensitive assay that can be used to quantitatively assess the activity of agonistic and antagonistic AR ligands. ' 2013 International Society for Advancement of Cytometry Key terms androgen receptor; recurrent prostate cancer; ligand; N/C-interaction; quantitative live cell imaging; FRET

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“…To temporarily control the induction of a DSB at the L-ISceI-T array in a single living cell, we took advantage of glucocorticoid-receptor chimeras that translocate from the cytoplasm to the nucleus on binding to the synthetic ligand triamcinolone acetonide (Fig. 1a) 15 . A chimera between the ISceI restriction endonuclease and the ligand-binding domain of the glucocorticoid receptor in frame with monomeric RFP (ISceI-GR) is cytoplasmic in the absence of triamcinolone acetonide and no DSBs were detected when cells were stained for phosphorylated H2AX (γ-H2AX; Fig.…”

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Positional stability of single double-strand breaks in mammalian cells

et al. 2007

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Formation of cancerous translocations requires the illegitimate joining of chromosomes containing double-strand breaks (DSBs). It is unknown how broken chromosome ends find their translocation partners within the cell nucleus. Here, we have visualized and quantitatively analysed the dynamics of single DSBs in living mammalian cells. We demonstrate that broken ends are positionally stable and unable to roam the cell nucleus. Immobilization of broken chromosome ends requires the DNAend binding protein Ku80, but is independent of DNA repair factors, H2AX, the MRN complex and the cohesion complex. DSBs preferentially undergo translocations with neighbouring chromosomes and loss of local positional constraint correlates with elevated genomic instability. These results support a contact-first model in which chromosome translocations predominantly form among spatially proximal DSBs.DSBs occur frequently in the genome through the action of DNA-damaging agents or during genome replication 1,2 . DSBs are hazardous to the cell because failure to properly repair them may lead to tumorigenic trans-locations 3 . How broken ends of different chromosomes meet in the cell nucleus to eventually form a translocation is poorly understood 4 . Two hypotheses have been put forth: the 'contact-first' model proposes that interactions between breaks on distinct chromosomes can only take place when the breaks are created in chromatid fibres that colocalize at the time of DNA damage 5 . In contrast, the 'breakage-first' hypothesis postulates that breaks formed at distant locations are able to scan the nuclear space for potential partners and come together to produce translocations 6 . The two models make divergent predictions as to the dynamic behaviour of broken chromosome ends. In the breakage-first model, single DSBs are required to undergo large-scale motions within the cell nucleus and must be able to roam the nuclear space in search of appropriate interaction partners. In the contact-first model, only limited local positional motion of DSBs is expected. The available experimental data is contradictory: in mammalian cells, induction of extensive chromosome damage using ultrasoft X-rays 7 , laser microirradiation 8 or γ-irradiation 8 indicates that damaged DNA is largely stationary. In contrast, α-particle irradiation leads to large-scale motion and clustering of 6Correspondence should be addressed to T.M. (e-mail: mistelit@mail.nih.gov). AUTHOR CONTRIBUTIONS E.S. and T.M. designed the study, E.S., J.F.D. and K.S. performed the experiments, J.M., A.N. and T.R. provided reagents and advice, and E.S. and T.M. wrote the manuscript. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.Note: Supplementary Information is available on the Nature Cell Biology website. NIH Public Access Author ManuscriptNat Cell Biol. Author manuscript; available in PMC 2008 July 3. Published in final edited form as:Nat Cell Biol. 2007 June ; 9(6): 675-682. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manus...

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An estrogen receptor chimera senses ligands by nuclear translocation

Cited by 22 publications

References 60 publications

Novel cell-based assay for detection of thyroid receptor beta-interacting environmental contaminants

Novel cell-based assay for detection of thyroid receptor beta-interacting environmental contaminants

A multi‐parameter imaging assay identifies different stages of ligand‐induced androgen receptor activation

Positional stability of single double-strand breaks in mammalian cells

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