title: Quantification of transcription factor-DNA binding affinity in a living cell.
abstract: The apparent dissociation constant (Kd) 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 Kd of ∼1 μM and dramatically stimulated DNA binding by AR with an apparent Kd 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.
authors.affiliation: Department of Cell and Molecular Biology, Karolinska Institutet, SE-17177 Stockholm, Sweden orjan.wrange@ki.se.
authors.forename: Sergey
authors.initials: S
authors.lastname: Belikov
authors.affiliation: Department of Cell and Molecular Biology, Uppsala University, BMC Box 596, SE-75124 Uppsala, Sweden.
authors.forename: Otto G
authors.initials: OG
authors.lastname: Berg
authors.affiliation: Department of Cell and Molecular Biology, Karolinska Institutet, SE-17177 Stockholm, Sweden orjan.wrange@ki.se.
authors.forename: Örjan
authors.initials: Ö
authors.lastname: Wrange
mesh_terms: D000818:Animals; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D051541:Hepatocyte Nuclear Factor 3-alpha / Q000378:metabolism; D008745:Methylation; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D011944:Receptors, Androgen / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D020218:Response Elements; D014157:Transcription Factors / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D051541:Hepatocyte Nuclear Factor 3-alpha; D011944:Receptors, Androgen; D011965:Receptors, Glucocorticoid; D014157:Transcription Factors; D004247:DNA