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Rehó Bálint
Investigation of the interactions of nuclear receptors by modern fluorescence microscopic methods

Aug 29 - kedd

10:15 – 10:30

Molekuláris biofizika

E07

Investigation the interactions of nuclear receptors by modern fluorescence microscopic methods

Bálint Rehó¹, Péter Brazda², Lina Fadel¹, Katalin Tóth¹, László Nagy^3,4, and György Vámosi¹

¹ University of Debrecen, Faculty of Medicine, Doctoral School of Molecular Medicine, Department of Biophysics and Cell Biology

² Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary; Princess Maxima Centre for Pediatric Oncology, Utrecht, the Netherlands.

³ Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary; Department of Medicine and Biological Chemistry, Johns Hopkins University School of Medicine, Institute for Fundamental Biomedical Research, Johns Hopkins All Children's Hospital, Saint Petersburg, Florida, USA.

⁴ Department of Medicine and Biological Chemistry, Johns Hopkins University School of Medicine, Institute for Fundamental Biomedical Research, Johns Hopkins All Children's Hospital, Saint Petersburg, Florida, USA.

Nuclear receptors are transcription factors regulating gene expression in a ligand dependent manner. They play a central role in cell differentiation, growth, and death. We investigated interactions and dynamics of retinoic acid receptor (RAR), Vitamin D receptor (VDR) and retinoid X receptor (RXR) in live cells. Their operation is described by the relatively static nuclear receptor molecular switch model [1]. Nowadays, this model is being changed for more dynamic ones (for example the “Hit and run” model [2]) due to intense investigations in the field.

We found previously that nuclear receptors compete for heterodimerization with their common partner, RXR, in a ligand-dependent manner [3]. To investigate potential competition in their DNA binding, we monitored the mobility of RAR and VDR in live cells by fluorescence correlation spectroscopy (FCS) [4][5].

Our FCS studies showed that there are two distinct populations of nuclear receptors present in the nucleus: in the fast population they are bound to the chromatin with shorter, whereas in the slow population with much longer residence times. In the case of RAR, agonist treatment or RXR co-transfection increased the fraction of the slow population, which is due to an increased stability of chromatin-binding or increased residence time. In contrast to RAR, the slow population of the VDR only increased in the presence of both agonist and RXR, so the chromatin binding of the VDR is stable only in a liganded, RXR-bound form. By the triple co-transfection of RAR, VDR and RXR, we showed that the competition between RAR and VDR for the binding of RXR appeared on the level of chromatin-binding, which may at least be partly responsible for the side effects of nuclear receptor targeted therapies.

Our results can help in the designing and optimizing nuclear receptor-based therapies and raise attention to the importance of competition between nuclear receptors.

Acknowledgment

German Academic Exchange Service and the Tempus Public Foundation #73163,

GINOP-2.3.2-15-2016-00026 and OTKA NN 129371 by the National Research, Development, and Innovation Office, Hungary.

References

[1] Nagy, Laszlo, and John W R Schwabe. “Mechanism of the nuclear receptor molecular switch.” Trends in biochemical sciences vol. 29,6 (2004): 317-24. doi:10.1016/j.tibs.2004.04.006

[2] Gelman, Laurent et al. “Integrating nuclear receptor mobility in models of gene regulation.” Nuclear receptor signaling vol. 4 (2006): e010. doi:10.1621/nrs.04010

[3] Fadel, Lina et al. “Agonist binding directs dynamic competition among nuclear receptors for heterodimerization with retinoid X receptor.” The Journal of biological chemistry vol. 295,29 (2020): 10045-10061. doi:10.1074/jbc.RA119.011614

[4] Rehó, Bálint et al. “Simultaneous Mapping of Molecular Proximity and Comobility Reveals Agonist-Enhanced Dimerization and DNA Binding of Nuclear Receptors.” Analytical chemistry vol. 92,2 (2020): 2207-2215. doi:10.1021/acs.analchem.9b04902

[5] “Agonist-controlled competition of RAR and VDR nuclear receptors for heterodimerization with RXR is manifested in their DNA binding.” The Journal of biological chemistry vol. 299,2 (2023): 102896. doi:10.1016/j.jbc.2023.102896