Mapping the Dynamics of the Glucocorticoid Receptor within the Nuclear Landscape

Martin Stortz,Maria V Dansey,Valeria Levi,Enrico Gratton,Gerardo Burton,Adali Pecci,Luciana Bruno,Diego M Presman,Paolo Annibale

doi:10.1038/s41598-017-06676-0

Abstract

The distribution of the transcription machinery among different sub-nuclear domains raises the question on how the architecture of the nucleus modulates the transcriptional response. Here, we used fluorescence fluctuation analyses to quantitatively explore the organization of the glucocorticoid receptor (GR) in the interphase nucleus of living cells. We found that this ligand-activated transcription factor diffuses within the nucleus and dynamically interacts with bodies enriched in the coregulator NCoA-2, DNA-dependent foci and chromatin targets. The distribution of the receptor among the nuclear compartments depends on NCoA-2 and the conformation of the receptor as assessed with synthetic ligands and GR mutants with impaired transcriptional abilities. Our results suggest that the partition of the receptor in different nuclear reservoirs ultimately regulates the concentration of receptor available for the interaction with specific targets, and thus has an impact on transcription regulation.

Highlights

Many components of the transcription machinery ranging from transcription factors (TFs), coregulators, chromatin remodelers and RNA polymerases accumulate in distinct nuclear subdomains that dynamically exchange molecules with the nucleoplasm[3, 4]
We explore the dynamic nuclear organization of the glucocorticoid receptor (GR), a ligand-activated TF that plays a relevant role in physiology[6, 7]
We quantitatively characterized the organization of enhanced green fluorescent protein fused GR (GFP-GR) in BHK cells stimulated with dexamethasone (Dex), a selective and potent glucocorticoid receptor agonist or 21-hydroxy-6,19-epoxyprogesterone (21-OH), a dissociated GR agonist[6, 27]

Summary

Introduction

Many components of the transcription machinery ranging from transcription factors (TFs), coregulators, chromatin remodelers and RNA polymerases accumulate in distinct nuclear subdomains that dynamically exchange molecules with the nucleoplasm[3, 4]. The GR regulates gene expression through direct binding to specific DNA regions named Glucocorticoid Response Elements (GREs)[8], presumably as a tetramer[9]. It can modulate gene expression through interactions with other TFs10 either as a monomer or a dimer[11]. A complete understanding of GR response requires integrating the transcriptional events occurring upon GR recruitment to DNA with the spatial and temporal GR distribution imposed by nuclear compartmentalization We address this question using a combination of advanced fluorescence microscopy techniques that are able to quantitatively determine the dynamics and interactions of GR in different nuclear compartments

Methods

Results

Conclusion