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Quantification of transcription factor-DNA binding affinity in a living cell
Karolinska Inst, Dept Cell & Mol Biol, SE-17177 Stockholm, Sweden.;Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, SE-10691 Stockholm, Sweden..
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
Karolinska Inst, Dept Cell & Mol Biol, SE-17177 Stockholm, Sweden..
2016 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 7, 3045-3058 p.Article in journal (Refereed) PublishedText
Abstract [en]

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 [H-3]-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 similar to 1 mu M and dramatically stimulated DNA binding by AR with an apparent K-d of similar to 0.13 mu M 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.

Place, publisher, year, edition, pages
2016. Vol. 44, no 7, 3045-3058 p.
National Category
Cell and Molecular Biology
URN: urn:nbn:se:uu:diva-297901DOI: 10.1093/nar/gkv1350ISI: 000375800200017PubMedID: 26657626OAI: diva2:944361
Swedish Cancer Society, 14 0792 140792
Available from: 2016-06-29 Created: 2016-06-28 Last updated: 2016-06-29Bibliographically approved

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