Essential functions of the Glucocorticoid Receptor require direct DNA binding [ChIP-seq]
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE126654 | GEO | 2019/12/31
REPOSITORIES: GEO
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Project description:We performed RNA sequencing in Mouse Embryonic Fibroblasts (MEFs) of GR wild-type and GR Zn. Cells were treated with: vehicle (EtOH), Dexamethasone (Dex), Lipopolysaccharide (LPS) or Dex+LPS. We show that GR Zn does not regulate any GR target gene
2019-12-31 | GSE124919 | GEO
Project description:Purpose: To compare the binding profiles of mineralocorticoid and glucocorticoid receptors after corticosterone treatment in neuroblastoma cells. Methods: Tagged glucocorticoid receptors (eGFP-rat GR) and mineralocorticoid receptors (mCherry-rat MR) were transiently transfected into Neuro2A mouse neuroblastoma cells and treated with (i) vehicle, (ii) corticosterone 100 nM for 20 mins and (iii) corticosterone 100 nM for 20 mins followed by media repacement twice and 40 mins further incubation in charcoal-stripped serum media. ChIP-nexus samples were prepared in triplicate for deep sequencing using Illumina NextSeq, as well as control samples taken from cell lysates before immunoprecipitation. Sequence data was analyzed and processed for the identification of enriched binding sites, using MACS2, and for the characteristic staggered ChIP-nexus borders, using the MACE pipeline (Wang et al., 2014). Results: Corticosterone-sensitive glucocorticoid receptor (GR) binding sites and mineralocorticoid receptor (MR) binding sites showed considerable overlap, by MACS2 analysis. GR and MR binding sites were highly similar showing recruitment to the same DNA sites, by MACE analysis (Wang et al., 2014). Further analyses showed GR could tether MR to the DNA by GR in a functional manner such that gene expression could be altered by the presence of tethered MR. Conclusions: MR can be recruited to DNA-binding sites by GR, even in the absence of MR DNA-binding. MR can interact in multiple modes at genomic DNA binding sites.
2019-03-15 | GSE115417 | GEO
Project description:The RNA binding ability of the glucocorticoid receptor (GR) remains an understudied area of GR regulation. Through in vitro binding assays, we identified a separation-of-function mutation (SoF) in the DNA-binding domain that greatly reduces RNA affinity with a slight reduction in DNA affinity. To study the effects of RNA binding on GR function, we first generated stable U2OS cells expressing wild-type GR-HaloTag, the SoF mutant, or a control (Ctrl) mutant that mimics the SoF's reduction in DNA affinity without impairing RNA affinity. We then treated the cells with dexamethsone every hour over three hours with 4-thiouridine labeling, isolated the labeled RNAs, and used deep sequencing to measure changes in the dexamethasone-driven transcriptome.
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Project description:Glucocorticoid receptor (GR) is a ligand-inducible transcription factor with an intricate role in cancer biology. Using an in silico designed GR activity signature we show that GR is a tumor suppressor across diverse primary cancers. In breast cancer, GR activity status determines luminal identity, and importantly, relates to patients’ outcomes. We illustrate that GR suppresses tumor growth, mediated through its engagement with the estrogen receptor-α (ER). This steroid hormone receptor cross-talk leads to redistribution of ERα on chromatin, ultimately leading to expression of ZBTB16 gene. We define ZBTB16 as a transcriptional repressor and a tumor suppressor in ER-positive breast cancer. Importantly, highly aggressive ER-positive breast cancer cells displaying absence of GR activity can be eradicated if GR-induced gene repression is mimicked by inhibitors of the epigenetic pathway. In line with this, epigenetic regulators are highly expressed upon GR activity loss, leading to vulnerability of aggressive breast cancer cells to clinically available epigenetic inhibitors. Our findings indicate that GR functions as a tumor suppressor by repositioning ER to specific sites on chromatin, modulating targetable pathways, which has important implications for patients’ prognosis and therapeutic interventions.
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Project description:Glucocorticoids are primary stress hormones and their synthetic derivatives are widely used clinically. The therapeutic efficacy of these steroids is limited by severe side effects and glucocorticoid resistance. Multiple glucocorticoid receptor (GR) isoforms are produced by alternative translation initiation; however, the role individual isoforms play in controlling tissue-specific responses to glucocorticoids in vivo is unknown. We have generated knockin mice that exclusively express the most active receptor isoform, GR-C3. The GR-C3 knockin mice die at birth due to respiratory distress but can be completely rescued by antenatal glucocorticoid administration. To evaluate the GR-C3 transcriptome, we prepared mouse embryonic fibroblasts (MEFs) from E12.5 wild-type (WT) and GR-C3 knockin embryos and treated the cells with vehicle or the synthetic glucocorticoid Dexamethasone (Dex) for 6 hours. The GR-C3 isoform was found to have a markedly different gene-regulatory profile than GR in WT MEFs.
2018-04-23 | GSE103675 | GEO
Project description:We performed ChIP-Sequencing for E2A and GR in mouse livers and demonstrate that E47 co-occupies metabolic promoters and enhancers together with GR. Together with the corresponding RNA-Seq data, our results illustrate how GR and E47 synergize during the transcriptional activation of hepatic metabolism.
2018-11-30 | GSE111526 | GEO
Project description:The hormone stimulated Glucocorticoid Receptor (GR) modulates transcription by interacting with thousands of enhancers and GR binding sites (GBSs) throughout the genome. Here, we examined the effects of GR binding on enhancer dynamics and interrogated the contributions of individual GBSs to the hormone response. Hormone treatment resulted in genome-wide reorganization of the enhancer landscape in breast cancer cells. Upstream of the DDIT4 oncogene, GR bound to four sites constituting a hormone-dependent super enhancer. Three GBSs were required as hormone-dependent enhancers that differentially promoted histone acetylation and transcription frequency and burst size. Conversely, the fourth site suppressed transcription and hormone treatment alleviated this suppression. GR binding within the super enhancer promoted a loop-switching mechanism that allowed interaction of the DDIT4 TSS with the active GBSs. The unique functions of each GR binding site contributed to hormone-induced transcriptional heterogeneity and demonstrate the potential for targeted modulation of oncogene expression.
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Project description:The Glucocorticoid Receptor (GR) is a transcriptional regulator which co-ordinates behavioural, metabolic and immune responses to stressors. Previous studies have shown that wild-type and grs357 mutant zebrafish exhibit a range of behavioural differences, implying possible roles for GR function in the brain. As part of a study to elucidate the mechanisms underlying the behaviours that are regulated by GR, we compared the brain transcriptomes of wild-type and grs357 mutant adults. RNA-seq analysis identified 19 Biological Process and 4 Molecular Function Gene Ontology (GO) terms that were particularly sensitive to loss of GR function. Foremost among the Biological Process terms linked to GR function are Chaperone-mediated Protein Folding, Regulation of Circadian Rhythm and Regulation of Primary Metabolic Process, each of which is accompanied by several other closely related GO terms. In addition, the Biological Process GO term Behavior was significantly associated with 32 genes exhibiting GR-regulated gene transcription. The Molecular Function terms most closely linked to GR function are Heat Shock Protein Binding, Protein Serine/Threonine Kinase Activity, Transcription Regulator Activity and Unfolded Protein Binding. Taken together, our results identify key biological processes and novel molecular mechanisms through which the GR likely regulates responses to stress in the zebrafish adult brain.
2023-09-06 | GSE213505 | GEO
Project description:The glucocorticoid (GC) receptor (GR) is a ligand-induced transcription factor regulating metabolism and inflammation. While homologous downregulation of ligand-bound GR limits the response, it also reduces hormone responsiveness resulting in glucocorticoid resistance. Regulatory mechanisms that refine this equilibrium are less understood. Here, we have identified seven lysine acetylation sites in the amino terminal domain of GR, lysine 154 within the regulatory AF-1 region being the dominant acetyl-acceptor. GR-Lys154 acetylation is mediated by p300/CBP in an exclusively agonist-induced manner and correlates with nuclear translocation and transcriptional activity. GR-Lys154 acetylation is removed by the NAD+ dependent deacetylase, SIRT1, indicating dynamic regulation of this mark. Notably, agonist-binding to both the wild type and acetylation-deficient mutant GR elicits similar short-term target gene expression. In contrast, upon extended treatment, the acetylation-deficient mutant shows impaired ubiquitination and higher protein stability with a concomitant increase in chromatin association and transactivation. Taken together, p300/CBP and SIRT1 fine-tune glucocorticoid response by siphoning acetylated GR for proteasomal degradation while GR deacetylation sustains the transcriptional cascade for prolonged periods.
2024-01-16 | GSE236739 | GEO
Project description:The RNA binding ability of the glucocorticoid receptor (GR) remains an understudied area of GR regulation. Through in vitro binding assays, we identified hairpin RNAs as GR's preferred binding motif. To study how GR-bound RNAs change with dexamethasone treatment, we first generated stable U2OS cells expressing wild-type GR-HaloTag. We then treated the cells with dexamethsone every hour over three hours, UV crosslinked, harvested, and pulled down GR using a specific and covalently-bound HaloTag ligand. GR-bound RNAs were then isolated, library prepped, and sequenced.
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