title: Quantitation of glucocorticoid receptor DNA-binding dynamics by single-molecule microscopy and FRAP.
abstract: Recent advances in live cell imaging have provided a wealth of data on the dynamics of transcription factors. However, a consistent quantitative description of these dynamics, explaining how transcription factors find their target sequences in the vast amount of DNA inside the nucleus, is still lacking. In the present study, we have combined two quantitative imaging methods, single-molecule microscopy and fluorescence recovery after photobleaching, to determine the mobility pattern of the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR), two ligand-activated transcription factors. For dexamethasone-activated GR, both techniques showed that approximately half of the population is freely diffusing, while the remaining population is bound to DNA. Of this DNA-bound population about half the GRs appeared to be bound for short periods of time (∼ 0.7 s) and the other half for longer time periods (∼ 2.3 s). A similar pattern of mobility was seen for the MR activated by aldosterone. Inactive receptors (mutant or antagonist-bound receptors) show a decreased DNA binding frequency and duration, but also a higher mobility for the diffusing population. Likely, very brief (≤ 1 ms) interactions with DNA induced by the agonists underlie this difference in diffusion behavior. Surprisingly, different agonists also induce different mobilities of both receptors, presumably due to differences in ligand-induced conformational changes and receptor complex formation. In summary, our data provide a consistent quantitative model of the dynamics of GR and MR, indicating three types of interactions with DNA, which fit into a model in which frequent low-affinity DNA binding facilitates the search for high-affinity target sequences.
authors.affiliation: Department of Medical Pharmacology, Leiden University/LUMC, Leiden, The Netherlands.
authors.forename: Femke L
authors.initials: FL
authors.lastname: Groeneweg
authors.affiliation: Department of Pathology, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Martin E
authors.initials: ME
authors.lastname: van Royen
authors.affiliation: Physics of Life Processes, Institute of Physics (LION), Leiden University, Leiden, The Netherlands; Cell & Developmental Biology, Biocenter, Würzburg University, Würzburg, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Fenz
authors.affiliation: Molecular Cell Biology, Institute of Biology, Leiden University, Leiden, The Netherlands.
authors.forename: Veer I P
authors.initials: VI
authors.lastname: Keizer
authors.affiliation: Department of Pathology, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Bart
authors.initials: B
authors.lastname: Geverts
authors.affiliation: Department of Medical Pharmacology, Leiden University/LUMC, Leiden, The Netherlands; Molecular Cell Biology, Institute of Biology, Leiden University, Leiden, The Netherlands.
authors.forename: Jurrien
authors.initials: J
authors.lastname: Prins
authors.affiliation: Department of Medical Pharmacology, Leiden University/LUMC, Leiden, The Netherlands.
authors.forename: E Ron
authors.initials: ER
authors.lastname: de Kloet
authors.affiliation: Department of Pathology, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Adriaan B
authors.initials: AB
authors.lastname: Houtsmuller
authors.affiliation: Physics of Life Processes, Institute of Physics (LION), Leiden University, Leiden, The Netherlands.
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Schmidt
authors.affiliation: Molecular Cell Biology, Institute of Biology, Leiden University, Leiden, The Netherlands.
authors.forename: Marcel J M
authors.initials: MJ
authors.lastname: Schaaf
mesh_terms: D000818:Animals; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D004247:DNA / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D006801:Humans; D008853:Microscopy / Q000379:methods*; D008962:Models, Theoretical; D011485:Protein Binding; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D018161:Receptors, Mineralocorticoid / Q000378:metabolism
chemical_list: D011965:Receptors, Glucocorticoid; D018161:Receptors, Mineralocorticoid; D004247:DNA