Abstract
Population-based assays have been employed extensively to investigate the interactions of transcription factors (TFs) with chromatin and are often interpreted in terms of static and sequential binding. However, fluorescence microscopy techniques reveal a more dynamic binding behaviour of TFs in live cells. Here we analyse the strengths and limitations of in vivo single-molecule tracking and performed a comprehensive analysis on the intranuclear dwell times of four steroid receptors and a number of known cofactors. While the absolute residence times estimates can depend on imaging acquisition parameters due to sampling bias, our results indicate that only a small proportion of factors are specifically bound to chromatin at any given time. Interestingly, the glucocorticoid receptor and its cofactors affect each other's dwell times in an asymmetric manner. Overall, our data indicate transient rather than stable TF-cofactors chromatin interactions at response elements at the single-molecule level.
Publication types
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Research Support, N.I.H., Intramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Adaptor Proteins, Signal Transducing / metabolism
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Animals
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Chromatin / metabolism
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Corticosterone / pharmacology
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DNA Helicases / metabolism
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Fluorescent Dyes / chemistry
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Fluorescent Dyes / metabolism
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Green Fluorescent Proteins / genetics
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Mice
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Molecular Imaging / methods*
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Nerve Tissue Proteins / metabolism
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Nuclear Proteins / metabolism
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Protein Interaction Mapping
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Rats
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Receptors, Glucocorticoid / analysis
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Receptors, Glucocorticoid / genetics
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Receptors, Glucocorticoid / metabolism
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Receptors, Steroid / analysis
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Receptors, Steroid / genetics
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Receptors, Steroid / metabolism*
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Single-Cell Analysis / methods
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Time Factors
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Transcription Factor AP-1 / metabolism
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Transcription Factors / metabolism
Substances
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Adaptor Proteins, Signal Transducing
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Chromatin
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Fluorescent Dyes
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Grip1 protein, mouse
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Nerve Tissue Proteins
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Nuclear Proteins
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Receptors, Glucocorticoid
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Receptors, Steroid
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Recombinant Proteins
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Transcription Factor AP-1
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Transcription Factors
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Green Fluorescent Proteins
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Smarca4 protein, mouse
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DNA Helicases
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Corticosterone