Ligand binding shifts highly mobile retinoid X receptor to the chromatin-bound state in a coactivator-dependent manner, as revealed by single-cell imaging

Mol Cell Biol. 2014 Apr;34(7):1234-45. doi: 10.1128/MCB.01097-13. Epub 2014 Jan 21.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites / genetics
  • Cell Nucleus / metabolism
  • Chromatin / genetics
  • Chromatin / metabolism
  • Fluorescence Recovery After Photobleaching
  • HeLa Cells
  • Humans
  • Kinetics
  • Ligands
  • Models, Biological
  • Organic Chemicals / pharmacology
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Retinoid X Receptors / agonists
  • Retinoid X Receptors / genetics
  • Retinoid X Receptors / metabolism*
  • Single-Cell Analysis
  • Spectrometry, Fluorescence

Substances

  • Chromatin
  • LG 268
  • Ligands
  • Organic Chemicals
  • Recombinant Proteins
  • Retinoid X Receptors