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Quantification of transcription factor-DNA binding affinity in a living cell

The apparent dissociation constant (K(d)) for specific binding of glucocorticoid receptor (GR) and androgen receptor (AR) to DNA was determined in vivo in Xenopus oocytes. The total nuclear receptor concentration was quantified as specifically retained [(3)H]-hormone in manually isolated oocyte nucl...

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Detalles Bibliográficos
Autores principales: Belikov, Sergey, Berg, Otto G., Wrange, Örjan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838337/
https://www.ncbi.nlm.nih.gov/pubmed/26657626
http://dx.doi.org/10.1093/nar/gkv1350
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author Belikov, Sergey
Berg, Otto G.
Wrange, Örjan
author_facet Belikov, Sergey
Berg, Otto G.
Wrange, Örjan
author_sort Belikov, Sergey
collection PubMed
description The apparent dissociation constant (K(d)) for specific binding of glucocorticoid receptor (GR) and androgen receptor (AR) to DNA was determined in vivo in Xenopus oocytes. The total nuclear receptor concentration was quantified as specifically retained [(3)H]-hormone in manually isolated oocyte nuclei. DNA was introduced by nuclear microinjection of single stranded phagemid DNA, chromatin is then formed during second strand synthesis. The fraction of DNA sites occupied by the expressed receptor was determined by dimethylsulphate in vivo footprinting and used for calculation of the receptor-DNA binding affinity. The forkhead transcription factor FoxA1 enhanced the DNA binding by GR with an apparent K(d) of ∼1 μM and dramatically stimulated DNA binding by AR with an apparent K(d) of ∼0.13 μM at a composite androgen responsive DNA element containing one FoxA1 binding site and one palindromic hormone receptor binding site known to bind one receptor homodimer. FoxA1 exerted a weak constitutive- and strongly cooperative DNA binding together with AR but had a less prominent effect with GR, the difference reflecting the licensing function of FoxA1 at this androgen responsive DNA element.
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spelling pubmed-48383372016-04-21 Quantification of transcription factor-DNA binding affinity in a living cell Belikov, Sergey Berg, Otto G. Wrange, Örjan Nucleic Acids Res Gene regulation, Chromatin and Epigenetics The apparent dissociation constant (K(d)) for specific binding of glucocorticoid receptor (GR) and androgen receptor (AR) to DNA was determined in vivo in Xenopus oocytes. The total nuclear receptor concentration was quantified as specifically retained [(3)H]-hormone in manually isolated oocyte nuclei. DNA was introduced by nuclear microinjection of single stranded phagemid DNA, chromatin is then formed during second strand synthesis. The fraction of DNA sites occupied by the expressed receptor was determined by dimethylsulphate in vivo footprinting and used for calculation of the receptor-DNA binding affinity. The forkhead transcription factor FoxA1 enhanced the DNA binding by GR with an apparent K(d) of ∼1 μM and dramatically stimulated DNA binding by AR with an apparent K(d) of ∼0.13 μM at a composite androgen responsive DNA element containing one FoxA1 binding site and one palindromic hormone receptor binding site known to bind one receptor homodimer. FoxA1 exerted a weak constitutive- and strongly cooperative DNA binding together with AR but had a less prominent effect with GR, the difference reflecting the licensing function of FoxA1 at this androgen responsive DNA element. Oxford University Press 2016-04-20 2015-12-10 /pmc/articles/PMC4838337/ /pubmed/26657626 http://dx.doi.org/10.1093/nar/gkv1350 Text en © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Gene regulation, Chromatin and Epigenetics
Belikov, Sergey
Berg, Otto G.
Wrange, Örjan
Quantification of transcription factor-DNA binding affinity in a living cell
title Quantification of transcription factor-DNA binding affinity in a living cell
title_full Quantification of transcription factor-DNA binding affinity in a living cell
title_fullStr Quantification of transcription factor-DNA binding affinity in a living cell
title_full_unstemmed Quantification of transcription factor-DNA binding affinity in a living cell
title_short Quantification of transcription factor-DNA binding affinity in a living cell
title_sort quantification of transcription factor-dna binding affinity in a living cell
topic Gene regulation, Chromatin and Epigenetics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838337/
https://www.ncbi.nlm.nih.gov/pubmed/26657626
http://dx.doi.org/10.1093/nar/gkv1350
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