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.
Highlights
In the past decade, imaging studies of fluorescently tagged proteins inside living cells have enormously increased our understanding of transcription factor dynamics [1,2,3,4,5,6,7,8,9]
We first investigated the nuclear dynamics of the glucocorticoid receptor (GR) by singlemolecule microscopy (SMM)
GR mobility was analyzed by assessing molecule displacements over image sequences with short time lags (6.25 and 12.5 ms; Figure 1C), using the Particle Image Correlation Spectroscopy (PICS) analysis method [33]
Summary
In the past decade, imaging studies of fluorescently tagged proteins inside living cells have enormously increased our understanding of transcription factor dynamics [1,2,3,4,5,6,7,8,9]. These studies have shown that transcription factors display a remarkably high mobility in the nucleus. Using GR mutants with reduced DNA-binding capacity or antagonist-bound GR, a correlation was shown between GR immobilization time and the capacity to initiate transcription [3,8,17]
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