Relationship Between Homodimeric Glucocorticoid Receptor and Transcriptional Regulation Assessed via an In Vitro Fluorescence Correlation Spectroscopy-Microwell System

Scientific Reports
Sho OasaMasataka Kinjo

Abstract

Glucocorticoid receptor (GR) is a hormone-activated transcription regulatory protein involved in metabolism as well as adrenocortical responses to psychosocial stress. Ligand-activated GR localizes to the nucleus, where GR homodimers regulate gene transcription via direct binding to glucocorticoid response elements (GREs). The role of GR homodimers in transcriptional activation has not yet been elucidated. In this study, we determined the concentration of GR homodimer, and its dissociation constant (Kd), at the single-cell level, by using fluorescence correlation spectroscopy (FCS) combined with a microwell system. Results from dissociation constant analysis and diffusion analysis suggested that GR forms complexes with other proteins as well as homodimers. We determined the relationship between the concentration of GR homodimer and transcriptional activity using a triple-color FCS-microwell system-based fluorescent reporter assay. The binding affinity of GR to GREs was analyzed via fluorescence cross-correlation spectroscopy (FCCS). Our findings indicate that the GR homodimer is essential for activating target gene transcription.

References

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Citations

Jun 6, 2019·Optics Express·Johtaro Yamamoto, Masataka Kinjo
Apr 30, 2020·Biophysics Reviews·Tomoyasu AizawaMin Yao
Jan 30, 2021·The Biochemical Journal·Martin StortzValeria Levi

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Methods Mentioned

BETA
fluorescence
chips
chip
FCS
cross-correlation spectroscopy
electrophoretic mobility shift
electrophoretic mobility shift assay
nuclear translocation
electrophoretic mobility
PCR

Software Mentioned

ViaFect

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