Zebrafish Mutant Lines Reveal the Interplay between nr3c1 and nr3c2 in the GC-Dependent Regulation of Gene Transcription.

Alberto Dinarello,Nicola Facchinello,Luisa Dalla Valle,Lorenzo Badenetti,Oliana Carnevali,Camilla Maria Fontana,Paolo Martini,Francesco Argenton,Davide Volpato,Chiara Romualdi,Annachiara Tesoriere,Francesca Terrin

doi:10.3390/ijms23052678

Abstract

Glucocorticoids mainly exert their biological functions through their cognate receptor, encoded by the nr3c1 gene. Here, we analysed the glucocorticoids mechanism of action taking advantage of the availability of different zebrafish mutant lines for their receptor. The differences in gene expression patterns between the zebrafish gr knock-out and the grs357 mutant line, in which a point mutation prevents binding of the receptor to the hormone-responsive elements, reveal an intricate network of GC-dependent transcription. Particularly, we show that Stat3 transcriptional activity mainly relies on glucocorticoid receptor GR tethering activity: several Stat3 target genes are induced upon glucocorticoid GC exposure both in wild type and in grs357/s357 larvae, but not in gr knock-out zebrafish. To understand the interplay between GC, their receptor, and the mineralocorticoid receptor, which is evolutionarily and structurally related to the GR, we generated an mr knock-out line and observed that several GC-target genes also need a functional mineralocorticoid receptor MR to be correctly transcribed. All in all, zebrafish mutants and transgenic models allow in vivo analysis of GR transcriptional activities and interactions with other transcription factors such as MR and Stat3 in an in-depth and rapid way.

Highlights

The glucocorticoid receptor (GR) is encoded by the NR3C1 gene and normally localized in the cytoplasm in a multimeric complex composed of heat shock protein (HSP) 70 and 90 and immunophilins
GR detaches from HSP90 [2] and regulates the transcription of target genes directly interacting with DNA at the level of hormone response elements (HRE)
Prior to the analysis of the differences between the two gr mutant lines at the transcriptional level, we confirmed the downregulation of nr3c1 mRNA expression only in gria30/ia30 compared to gr+/+ siblings (Figure 1A,B)

Summary

Introduction

The glucocorticoid receptor (GR) is encoded by the NR3C1 gene and normally localized in the cytoplasm in a multimeric complex composed of heat shock protein (HSP) 70 and 90 and immunophilins. GR detaches from HSP90 [2] and regulates the transcription of target genes directly interacting with DNA at the level of hormone response elements (HRE). Grdim/dim mice, characterized by the A458T mutation in the second zinc finger that abrogates GR dimerization and the subsequent HREdependent transactivation, are vital and reach adulthood, underlining alternative GRdependent mechanisms of transcription activation [6]. GR monomers expressed by Grdim/dim mice mutants have been proven to bind DNA efficiently [7,8], recent data obtained by Johnson and collaborators demonstrated that GR monomers poorly bind to chromatine and induce the transcription of a restricted number of GC-related genes [9]

Methods

Results

Conclusion