Glucocorticoid Receptor-DNA Dissociation Kinetics Measured in vitro Reveal Exchange on the Second Timescale

Abstract

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 GR interactions with chromatin are highly dynamic, with receptor residence times on the order of seconds. These findings were surprising since early in vitro studies suggested that GR-DNA interactions were more static, with receptor-DNA exchange occurring on the order of minutes to hours. However, these latter analyses were carried out without the modern-day advantages of recombinant receptor expression, established purification protocols or rigorous characterization methods. Here we examine the DNA dissociation kinetics of full-length human GR, using highly purified and homogenous receptor preparations. We find that in vitro dissociation kinetics are not slow as previously reported, but are quite fast, with calculated receptor-DNA residence times of seconds to tens of seconds. These findings are observed at both isolated response elements and the multisite mouse mammary tumor virus (MMTV) promoter used in live cell imaging. However, we also find that dissociation kinetics are identical for all response elements and are biphasic in character. These results indicate that differences in GR affinity toward these sequences are not due to differences in off-rate but on-rate, and further suggest that GR exists in multiple and interconverting states when bound to DNA. Since we previously established that GR-DNA binding affinity is a primary determinant of transcriptional activity at these sequences, the current findings implicate DNA sequence in controlling the extent of receptor interconversion and hence extent of transcriptional activation.