Ligand binding shifts highly mobile retinoid X receptor to the chromatin-bound state in a coactivator-dependent manner, as revealed by single-cell imaging.
Retinoid X receptor (RXR) is a promiscuous nuclear receptor forming heterodimers with several other receptors, which activate different sets of genes. Upon agonist treatment, the occupancy of its genomic binding regions increased, but only a modest change in the number of sites was revealed by chromatin immunoprecipitation followed by sequencing, suggesting a rather static behavior. However, such genome-wide and biochemical approaches do not take into account the dynamic behavior of a transcription factor. Therefore, we characterized the nuclear dynamics of RXR during activation in single cells on the subsecond scale using live-cell imaging. By applying fluorescence recovery after photobleaching and fluorescence correlation spectroscopy (FCS), techniques with different temporal and spatial resolutions, a highly dynamic behavior could be uncovered which is best described by a two-state model (slow and fast) of receptor mobility. In the unliganded state, most RXRs belonged to the fast population, leaving ∼ 15% for the slow, chromatin-bound fraction. Upon agonist treatment, this ratio increased to ∼ 43% as a result of an immediate and reversible redistribution. Coactivator binding appears to be indispensable for redistribution and has a major contribution to chromatin association. A nuclear mobility map recorded by light sheet microscopy-FCS shows that the ligand-induced transition from the fast to the slow population occurs throughout the nucleus. Our results support a model in which RXR has a distinct, highly dynamic nuclear behavior and follows hit-and-run kinetics upon activation.
Department of Biochemistry and Molecular Biology, Research Center for Molecular Medicine, University of Debrecen, Faculty of Medicine, Debrecen, Hungary.
Peter
P
Brazda
Jan
J
Krieger
Bence
B
Daniel
David
D
Jonas
Tibor
T
Szekeres
Jörg
J
Langowski
Katalin
K
Tóth
Laszlo
L
Nagy
György
G
Vámosi
D001665:Binding Sites / Q000235:genetics; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008954:Models, Biological; D009930:Organic Chemicals / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D047488:Retinoid X Receptors / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D059010:Single-Cell Analysis; D013050:Spectrometry, Fluorescence
D002843:Chromatin; C423914:LG 268; D008024:Ligands; D009930:Organic Chemicals; D011994:Recombinant Proteins; D047488:Retinoid X Receptors