Glucocorticoid Receptor-DNA Dissociation Kinetics Measured in Vitro Reveal Exchange on the Second Time Scale.
The glucocorticoid receptor (GR) is a member of the steroid receptor family of ligand-activated transcription factors. Recent live cell imaging studies have revealed that interactions of GR with chromatin are highly dynamic, with average receptor residence times of only seconds. These findings were surprising because early kinetic studies found that GR-DNA interactions in vitro were much slower, having calculated residence times of minutes to hours. However, these latter analyses were conducted at a time when it was possible to work with only either partially purified holoreceptor or its purified but isolated DNA binding domain. Noting these limitations, we reexamined GR-DNA dissociation kinetics using a highly purified holoreceptor shown to be amenable to rigorous study. We first observe that GR-DNA interactions in vitro are not slow as previously thought but converge with in vivo behavior, having residence times of only seconds to tens of seconds. This rapid exchange is seen at six individual response elements and the multisite MMTV promoter used in live cell imaging. Second, GR dissociation rates are identical for all response elements. Thus, previously observed differences in receptor affinity toward these sequences are not due to differences in off rate but in on rate. Finally, dissociation kinetics are biphasic in character. A minimal kinetic model consistent with the data is that in which DNA-bound GR interconverts between states on a second time scale, with dissociation occurring via a multistep process. We speculate that receptor interconversion in this time frame can be recognized by the coregulatory proteins that interact with GR, leading to unique transcriptional responses.
Department of Pharmaceutical Sciences and ‡Department of Pathology, University of Colorado Anschutz Medical Campus , Aurora, Colorado 80045, United States.
Rolando W
RW
De Angelis
Department of Pharmaceutical Sciences and ‡Department of Pathology, University of Colorado Anschutz Medical Campus , Aurora, Colorado 80045, United States.
Nasib K
NK
Maluf
Department of Pharmaceutical Sciences and ‡Department of Pathology, University of Colorado Anschutz Medical Campus , Aurora, Colorado 80045, United States.
Qin
Q
Yang
Department of Pharmaceutical Sciences and ‡Department of Pathology, University of Colorado Anschutz Medical Campus , Aurora, Colorado 80045, United States.
James R
JR
Lambert
Department of Pharmaceutical Sciences and ‡Department of Pathology, University of Colorado Anschutz Medical Campus , Aurora, Colorado 80045, United States.
David L
DL
Bain
D000818:Animals; D055592:Biophysical Phenomena; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018983:DNA Footprinting; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D011401:Promoter Regions, Genetic; D011960:Receptors, Estrogen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020218:Response Elements
D011960:Receptors, Estrogen; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004247:DNA