Single-molecule Analysis of Glucocorticoid Receptor and Cofactor Action in Living Cells

DIEGO M PRESMAN; VILLE PAAKINAHO; BALL, DAVID A.; THOMAS A. JOHNSON; R. LOUIS SCHILTZ; PETER LEVITT; DAVIDE MAZZA; TATSUYA MORISAKI; TATIANA S. KARPOVA; GORDON L. HAGER

Baltimore

Congreso; 1st Chesapeake Bay Area Single Molecule Biology Meeting; 2017

John Hopkings/University of Maryland/NIH

For decades, transcription factors (TFs) have been thought to operate by binding their genomic targets stably to form well-defined macromolecular complexes that remain intact and biologically functional for minutes or hours at a time. However, this view was challenged when fluorescent tagged proteins enabled the visualization of TFs dynamics in live cells. Steroid receptors are a class of ligand-inducible TFs that respond to environmental stimuli and mediate the expression of genes involved in metabolic, developmental, and inflammatory pathways. Their hormone-dependent nature makes these proteins ideal for studying transcription factor dynamics. In this work, we compared the dynamic behavior of the glucocorticoid receptor (GR) by single-molecule tracking (SMT) under highly inclined and laminated optical sheet (HILO) illumination, labelling HaloTag fused GR with the Janelia Fluor 549 (JF549). Our results show that only a small percentage of GR molecules are specifically bound to chromatin at any given time. We also show that, given the multi-exponential nature of the distribution of residence time, the measured kinetic parameters for TF binding can depend on the image acquisition parameters. Therefore, the dwell time for a given factor must be evaluated over a range of acquisition conditions to develop a rigorous understanding of its dynamic behavior. Overall, our data indicate transient rather than stable TF-cofactors chromatin interactions at the single-molecule level.