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Nr3c1 null mutant zebrafish are viable and reveal DNA-binding-independent activities of the glucocorticoid receptor.

ABSTRACT: Glucocorticoids (GCs) play important roles in developmental and physiological processes through the transcriptional activity of their cognate receptor (Gr). Using CRISPR/Cas9 technology, we established a zebrafish null Gr mutant line and compared its phenotypes with wild type and a zebrafish line with partially silenced gr (gr ^s357/s357 ). Homozygous gr ^-/- larvae are morphologically inconspicuous and, in contrast to GR ^-/- knockout mice, viable through adulthood, although with reduced fitness and early life survival. Mutants gr ^-/- are fertile, but their reproductive capabilities fall at around 10 months of age, when, together with cardiac and intestinal abnormalities already visible at earlier stages, increased fat deposits are also observed. Mutants show higher levels of whole-body cortisol associated with overstimulated basal levels of crh and pomca transcripts along the HPI axis, which is unresponsive to a mechanical stressor. Transcriptional activity linked to immune response is also hampered in the gr ^-/- line: after intestinal damage by dextran sodium sulphate exposure, there are neither inflammatory nor anti-inflammatory cytokine gene responses, substantiating the hypothesis of a dual-action of the GC-GR complex on the immune system. Hence, the zebrafish gr mutant line appears as a useful tool to investigate Gr functions in an integrated in vivo model.

SUBMITTER: Facchinello N

PROVIDER: S-EPMC5491532 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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nr3c1 null mutant zebrafish are viable and reveal DNA-binding-independent activities of the glucocorticoid receptor.

Facchinello N N Skobo T T Meneghetti G G Colletti E E Dinarello A A Tiso N N Costa R R Gioacchini G G Carnevali O O Argenton F F Colombo L L Dalla Valle L L

Scientific reports 20170629 1

Glucocorticoids (GCs) play important roles in developmental and physiological processes through the transcriptional activity of their cognate receptor (Gr). Using CRISPR/Cas9 technology, we established a zebrafish null Gr mutant line and compared its phenotypes with wild type and a zebrafish line with partially silenced gr (gr s357/s357 ). Homozygous gr -/- larvae are morphologically inconspicuous and, in contrast to GR -/- knockout mice, viable through adulthood ...[more]

PMID: 28663543

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Project description:Glucorticoids (GCs) are steroid hormones with a wide range of physiological actions in different cell types and tissues. Their ability to induce apoptosis in malignant cells of the lymphoid lineage is exploited since decades in the treatment of childhood acute lymphoblastic leukemia (ALL), the molecular mechanism of this cell death induction being however still unknown. Using an integrative approach we delineated the transcriptional response of a preB- and a T-ALL model system employing Exon microarrays and identified in vivo interactions of the transcription factor GC receptor (GR) with genes' promoters in the same samples using the ChIP-on-chip technology. We did find differences in GR-DNA interaction in the genes' promoters between the two cell systems and also in extent and kinetics of GC mediated gene regulation. The overall response was stronger in CEM-C7H2 T-ALL cells where it was also accompanied by a stronger autoup-regulation of the GR gene. Considering the differing kinetics we furthermore defined a set of common response genes. These response genes were enriched in apoptosis related gene ontology processes, and about half of them yielded also GR enrichment in the respective promoter regions. Globally, the presence of GR at gene's promoters was higher for GC induced compared to repressed genes, indicating that GR mediates gene repression by interaction with distant enhancer regions or by cross-talk with other transcription factors. In order to identify the genes' GC regulated transcript variants we analyzed the data set also at the level of individual exons and predicted first exons of GC induced isoforms. We could thus define GC regulated variants of response genes, did however also identify first exons not corresponding to any known transcript. We reconfirmed these results for the genes ATP4B, GPR98, TBCD and ZBTB16 and identified for the latter also a novel exon employing 5' nested amplification of cDNA ends. Expression and regulation of this and all downstream exons of ZBTB16 was further verified by real time RT-PCR in 4 different preB-ALL cell lines. Thus, besides potential other novel gene isoforms, GCs induce a novel short transcript variant of ZBTB16 in preB-ALL cells. Finally, the implemented bioinformatic methods may be useful also for other high density microarray data sets to identify alternative splice events and transcript variant expression.

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Nr3c1 null mutant zebrafish are viable and reveal DNA-binding-independent activities of the glucocorticoid receptor.

Publications

nr3c1 null mutant zebrafish are viable and reveal DNA-binding-independent activities of the glucocorticoid receptor.

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