Simultaneous Mapping of Molecular Proximity and Comobility Reveals Agonist-Enhanced Dimerization and DNA Binding of Nuclear Receptors

Abstract

Single Plane Illumination Microscopy (SPIM) revolutionized time lapse imaging of live cells and organisms due to its high speed and reduced photodamage. Quantitative mapping of molecular (co)­mobility by fluorescence (cross-)correlation spectroscopy (F­(C)­CS) in a SPIM has been introduced to reveal molecular diffusion and binding. A complementary aspect of interactions is proximity, which can be studied by Förster resonance energy transfer (FRET). Here, we extend SPIM-FCCS by alternating laser excitation, which reduces false positive cross-correlation and facilitates comapping of FRET. Thus, different aspects of interacting systems can be studied simultaneously, and molecular subpopulations can be discriminated by multiparameter analysis. After demonstrating the benefits of the method on the AP-1 transcription factor, the dimerization and DNA binding behavior of retinoic acid receptor (RAR) and retinoid X receptor (RXR) is revealed, and an extension of the molecular switch model of the nuclear receptor action is proposed. Our data imply that RAR agonist enhances RAR–RXR heterodimerization, and chromatin binding/dimerization are positively correlated. We also propose a ligand induced conformational change bringing the N-termini of RAR and RXR closer together. The RXR agonist increased homodimerization of RXR suggesting that RXR may act as an autonomous transcription factor.

Agonist binding directs dynamic competition among nuclear receptors for heterodimerization with retinoid X receptor

“…Calcitriol treatment had a triple effect on VDR: it enhanced its nuclear localization along with an augmented heterodimeri-zation with RXRa, and it increased its binding to its response elements, stronger to VDREs binding the heterodimer and to a lesser extent to NR half-sites. This is in line with our finding that heterodimerization of RARa and RXRa was also positively correlated with chromatin binding as measured by comobility (27).…”

“…Previously we have shown by fluorescence correlation spectroscopy that specific agonists increase chromatin binding of RARa and RXRa in a coactivatordependent manner (25,26). We have shown by light-sheet microscopy-based FRET and fluorescence cross-correlation measurements (SPIM-FRET-FCCS) that dimerization of RARa and RXRa and chromatin binding of the dimer are enhanced upon agonist treatment (27).…”

Of numbers and movement – understanding transcription factor pathogenesis by advanced microscopy

“…In addition, covariance in fluorescence intensity fluctuations from the two different fluorophores is analysed to assess the codiffusion of the investigated molecules (Bacia and Schwille, 2007;Krieger et al, 2014;Langowski, 2017;Rika and Binkert, 1989;Schwille et al, 1997). This method can be used to study the efficiency and stoichiometry of TF complex formation, such as hetero-/homo-dimers, as well as the chromatin-binding behaviour of these complexes (Bacia and Schwille, 2007;Krieger et al, 2014;Langowski, 2017;Papadopoulos et al, 2015;Reho et al, 2020).…”

“…This reduces out-of-focus light and improves signal-to-noise ratio, and enables imaging deeper into the sample. TF imaging and localisation studies can be combined with additional methods to study TF mobility and chromatinbinding dynamics, using SPIM (Huisken et al, 2004;Langowski, 2017;Pitrone et al, 2013;Reho et al, 2020), LLSM (Cai et al, 2019;Chen et al, 2014a;Liu et al, 2014;Mir et al, 2017;Strom et al, 2017), RLSM (Gebhardt et al, 2013) and HILO (Ball et al, 2016;Hansen et al, 2017;Hipp et al, 2019;Mehta et al, 2018;Morisaki et al, 2014;Tokunaga et al, 2008;Zhan et al, 2014). (Axelrod, 1981;Douglass and Vale, 2008).…”

Doxorubicin impacts chromatin binding of HMGB1, Histone H1 and retinoic acid receptor

“…Its diffusion in the nucleus can be described by a model assuming a fast, freely diffusing component corresponding to monomers or small complexes, and a slow, chromatin-bound component. Agonist treatment increased the slow fraction in a coactivator-dependent manner and enhanced dimerization with RXR 17 – 20 . In view of our ChIP-seq data, ligand treatment can enhance the binding of NRs to their response elements on DNA 18 , 21 .…”