Abstract
Estrogen-related receptor (ERR), a member of the nuclear receptor superfamily, consists of three subtypes (α, β, γ) and has strong homology with estrogen receptor. No endogenous ligands have been identified for ERRs, but they play key roles in metabolic, hormonal, and developmental processes as transcription factors without ligand binding. Although subnuclear dynamics are essential for nuclear events including nuclear receptor-mediated transcriptional regulation, the dynamics of ERRs are poorly understood. Here, we report that ERRs show subcellular kinetic changes in response to diethylstilbestrol (DES), a synthetic estrogen that represses the transactivity of all three ERR subtypes, using live-cell imaging with fluorescent protein labeling. Upon DES treatment, all ERR subtypes formed discrete clusters in the nucleus, with ERRγ also displaying nuclear export. Fluorescence recovery after photobleaching analyses revealed significant reductions in the intranuclear mobility of DES-bound ERRα and ERRβ, and a slight reduction in the intranuclear mobility of DES-bound ERRγ. After DES treatment, colocalization of all ERR subtypes with scaffold attachment factor B1 (SAFB1), a nuclear matrix-associated protein, was observed in dot-like subnuclear clusters, suggesting interactions of the ERRs with the nuclear matrix. Consistently, co-immunoprecipitation analyses confirmed enhanced interactions between ERRs and SAFB1 in the presence of DES. SAFB1 was clarified to repress the transactivity of all ERR subtypes through the ERR-response element. These results demonstrate ligand-dependent cluster formation of ERRs in the nucleus that is closely associated with SAFB1-mediated transrepression. Taken together, the present findings provide a new understanding of the pathophysiology regulated by ERR/SAFB1 signaling pathways and their subcellular dynamics.
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Acknowledgements
The authors sincerely thank Prof. Steffi Oesterreich (University of Pittsburgh) for kindly providing the pEGFP-SAFB1 expression vector and the colleagues in our laboratory including Dr. Shunji Yamada, Dr. Katsutoshi Taguchi, Yuki Takeda, Emiri Takemaru, and Yu Sakaue for helpful discussions and technical support. Kayo Tanida (Sakai, Osaka, Japan) helped with the FRAP analyses. The authors also thank Alison Sherwin, Ph.D., from Edanz Group (https://en-author-services.edanz.com/ac), for editing a draft of this manuscript. This work was supported by Grants-in-Aid for Young Scientists (B) (24791116 and 26860852 to T.T.) and Grants-in-Aid for Scientific Research (B) (24300128 to M.K.) and (C) (17K10188 and 20K08186 to T.T.) from The Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT).
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The Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) / Japan Society of Promotion of Sciences (JSPS): Grants-in-Aid for Young Scientists (B) (24791116 and 26860852 to T.T.) Grant-in-Aid for Scientific Research (B) (24300128 to M.K.) Grants-in-Aid for Scientific Research (C) (17K10188 and 20K08186 to T.T.)
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Study concept and design: TT; plasmid construction: TT and TH with support from KIM; time-lapse live-cell imaging: TT and TU.; FRAP analyses: TU, TY, and TT with support from TH; immunofluorescence cytochemistry: TT and KIM; co-immunoprecipitation, western blotting, and transcription assays: TT; statistical analyses: TT; interpretation of data: TT, KIM, TU, TY, TH, MK, and MT; writing and editing the manuscript: TT with input from KIM and MT.
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Tanida, T., Matsuda, K.I., Uemura, T. et al. Subcellular dynamics of estrogen-related receptors involved in transrepression through interactions with scaffold attachment factor B1. Histochem Cell Biol 156, 239–251 (2021). https://doi.org/10.1007/s00418-021-01998-7
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DOI: https://doi.org/10.1007/s00418-021-01998-7