Effect of reduced glucocorticoid receptor-RNA affinity on dexamethasone treatment
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE216337 | GEO | 2022/12/17
REPOSITORIES: GEO
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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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Project description:Glucocorticoids, including dexamethasone and prednisone, are key components of therapy for B-lymphoblastic leukemia (B-ALL) that work through the glucocorticoid receptor (GR). However, glucocorticoids are not effective for all patients, leading to poor outcomes, and the high doses used in chemotherapy cause many toxicities. We have shown that inhibition of the leukocyte restricted PIK3δ is a promising way to specifically enhance glucocorticoids in B-ALL, with the potential to improve outcomes without increasing toxicities. Here, we show that the PI3Kδ inhibitor idelalisib potentiates both prednisolone and dexamethasone in both B-ALL cell lines and primary patient specimens, particularly at sub-saturating doses. Potentiation is partially explained by a widespread enhancement of glucocorticoid gene regulation, including effector genes that drive B-ALL cell death. Idelalisib causes a reduction in direct phosphorylation of GR at S203 and S226. Ablation of these phospho-acceptor sites enhances glucocorticoid potency. Phosphorylation of S226 inhibits GR DNA binding, indicating that PI3Kδ mediated phosphorylation directly inhibits GR function in part though reduction of DNA binding affinity. Thus, PI3Kδ inhibition improves glucocorticoid efficacy in B-ALL in part by decreasing GR phosphorylation and enhancing DNA binding and downstream gene regulation. Identification of this and other contributing mechanisms will help identify patients who can most benefit clinically from this combination therapy.
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Project description:The glucocorticoid receptor (GR) binds to DNA in at least three stoichiometric ratios to regulate gene expression. The dimeric binding of GR to a 15 base pair core sequence has been well studied, and is associated with activation of gene expression. Monomeric GR has been associated with repression. GR has also been shown to form tetramers on DNA in live cells. The role of DNA sequence in directing DNA-binding stoichiometry and subsequent gene regulation is not clear. We took an unbiased approach using SELEX-seq to calculate comprehensive monomeric- and dimeric-binding profiles for GR. The monomeric site is composed of a canonical half-site, but is distinguished from the dimer site by a downstream TTTg motif and the absence of a second half-site. To probe the mechanism of how this motif favors monomeric binding, we performed Spec-/Coop-seq. The TTTg increases monomeric affinity while decreasing cooperative dimeric binding. Crystal structures indicated that TTTg results in a narrowed minor groove bringing residues Y455 and K471 of GR into the proximity of DNA, increasing affinity. Interestingly, although monomeric binding is more often associated with repression of gene expression than dimeric binding, the trend is not significant. Consistent with live-cell imaging, a meta-analysis of GR:DNA structures reveals that, regardless of binding sequence, GR binds to DNA as a dimer of dimers using highly similar interfaces. This suggests that the functional stoichiometry of GR in the cell is tetrameric, and that sequence may modulate GR activity.
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Project description:RNA metabolism is controlled by an expanding yet incomplete catalog of RNA binding proteins (RBPs), many of which lack characterized RNA binding domains. Approaches to expand the RBP repertoire to discover non-canonical RBPs are currently needed. Here, HaloTag fusion pull-down of twelve nuclear and cytoplasmic RBPs followed by quantitative mass-spectrometry (MS) demonstrates that proteins interacting with multiple RBPs in an RNA-dependent manner are enriched for RBPs. This motivated SONAR, a computational approach that predicts RNA binding activity by analyzing large-scale affinity precipitation-MS protein-protein interactomes. Without relying on sequence or structure information, SONAR identifies 1923 human, 489 fly and 745 yeast RBPs, including over 100 human candidate RBPs that contain zinc finger domains. Enhanced CLIP confirms RNA binding activity and identifies transcriptome-wide RNA binding sites for SONAR-predicted RBPs, revealing unexpected RNA binding activity for disease-relevant proteins and DNA binding proteins.
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Project description:The Glucocorticoid Receptor (GR) is both one of the most widely used clinical drug targets and a very potent metabolic regulator. GR belongs to the nuclear hormone receptor family of ligand-gated transcription factors that govern mammalian physiology. Upon ligand binding, GR enters the nucleus to regulate gene expression both positively and negatively. It is known to bind to consensus DNA sequences termed glucocorticoid response elements (GREs), but the mechanisms determining transcriptional activation versus repression remain an unresolved molecular paradox. Prevailing models suggest that tethering of GR to AP-1 or NF-κB via protein-protein interactions, rather than direct DNA binding, specifies negative regulation. However, here we show that the repression of inflammatory genes as well as all other glucocorticoid responses, require direct DNA binding of GR. Generating GR point mutant mice that retain the ability to tether via protein-protein interactions while unable to recognize DNA sequences, we demonstrate that response element recognition via the Zinc finger is absolutely required for both transcriptional activation and repression. We have used ChIP-Seq and RNA-Seq in inflammatory and metabolic cells and tissues together with proteomics to reveal that DNA binding of GR is necessary for the assembly of a functional SWI/SNF coregulator complex. Generally, the desired anti-inflammatory actions of GR are attributed to the silencing of inflammatory genes, while its adverse effects are believed to result from the transcriptional upregulation of metabolic targets. Our findings not only challenge classical models and dogmas of GR mediated gene regulation, but will provide an important basis for the development of novel immunosuppressants with reduced side effect profiles.
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Project description:We performed ChIP-Sequencing for GR in GR wild-type, GR Zn mutant and GR Knock-Out Mouse Embryonic Fibroblasts (MEFs). We show that GR Zn mutant chromatin binding is mostly lost except for some tethered binding sites. Together with the corresponding RNA-Seq data, our results illustrate how GR requires direct DNA binding for activation and repression of target genes.
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Project description:The glucocorticoid (GC) receptor (GR) is essential in development and inflammation. Healthy GRdim/dim mice with reduced dimerization propensity due to a point mutation (A465T) at the dimer interface of the GR DNA binding domain (DBD) (here GRD/D) have helped to define GR monomer and dimer functions of GR. Since GRD/D retains residual dimerization capacity, we generated the dimer-nullifying double mutant GRD+L/D+L mice, featuring an additional mutation (I634A) in the ligand binding domain (LBD) of GR. These mice are perinatally lethal, as are GRL/L mice, displaying improper lung and skin formation. We used embryonic fibroblasts, high and low doses of dexamethasone (Dex), nuclear translocation, RNAseq, dimerization assays and ligand binding assays (and Kd values), we suggest that the lethal phenotype is due to insufficient ligand binding. The data suggest cross talk between GR dimerization potential and ligand affinity. We conclude that a mutation as subtle as this I634A, at a position not directly involved in ligand interactions senso stricto, can thus still influence ligand binding and have a lethal outcome.
2021-12-03 | GSE189620 | GEO
Project description:The glucocorticoid (GC) receptor (GR) is essential in development and inflammation. Healthy GRdim/dim mice with reduced dimerization propensity due to a point mutation (A465T) at the dimer interface of the GR DNA binding domain (DBD) (here GRD/D) have helped to define GR monomer and dimer functions of GR. Since GRD/D retains residual dimerization capacity, we generated the dimer-nullifying double mutant GRD+L/D+L mice, featuring an additional mutation (I634A) in the ligand binding domain (LBD) of GR. These mice are perinatally lethal, as are GRL/L mice, displaying improper lung and skin formation. We used embryonic fibroblasts, high and low doses of dexamethasone (Dex), nuclear translocation, RNAseq, dimerization assays and ligand binding assays (and Kd values), we suggest that the lethal phenotype is due to insufficient ligand binding. The data suggest cross talk between GR dimerization potential and ligand affinity. We conclude that a mutation as subtle as this I634A, at a position not directly involved in ligand interactions senso stricto, can thus still influence ligand binding and have a lethal outcome.
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Project description:DNA methylation (mC) on CpG sites is the most common epigenetic modification. Recently methylation on non-CpG context was found to widely occur on genomic DNA. However, how non-CpG methylation influences DNA/protein interaction and regulates transcription is unknown. Using single-molecule assays, we studied the interactions of transcription factors (TFs) GR, ER and BMAL1-CLOCK with methylated or unmethylated cognate DNA binding sequences. The results demonstrated that these TFs interact with methylated DNA discriminatively. We then performed BisChIP-seq with GR and BMAL1-CLOCK in HEK-293T cells and showed that they can recognize and bind to the methylated sites within chromatin. The effects of non-CpG methylation on transcriptional regulation were validated by in vitro transcription assays with correlated RNA level, and further studied mechanistically by crystallographic analyses and molecular dynamics simulations. We conclude that the non-CpG methylation of DNA provides another general mechanism for regulating gene expression through affecting TFs binding affinity.
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