Nascent transcriptional profiling of CEM-C7 ALL cells treated with dexamethasone
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE219041 | GEO | 2022/12/04
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:A mutation in the dimerization domain of the mouse glucocorticoid receptor (GR), dim1, has recently been shown to bind DNA and regulate gene expression. To expand these studies we created a stable osteosarcoma (U-2 OS) cell line expressing four mutations in the dimerization domain of the human GR, dim4 (N454D, A458T, R460D, D462C), and used whole human genome microarray analysis to compare differences in gene regulation between vehicle treated (CON) and those treated with the glucocorticoid receptor agonist dexamethasone (DEX) at 100nM concentration for 6 hours. Gene expression in U-2 OS hGRdim4 cells was measured after a 6 hour treatment with 100nM dexamethasone or vehicle (control) and the dexamethsone (dex) treated cells were compared to vehicle treated cells. The experiment was performed in triplicate.
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Project description:The beneficial effects of glucocorticoids (GCs) in acute lymphoblastic leukemia (ALL) are based on their ability to induce apoptosis. Omics technologies such as DNA microarray analysis are widely used to study the changes in gene expression and have been successfully implemented in biomarker identification. In addition, time series studies of gene expression enable the identification of correlations between kinetic profiles of glucocorticoid receptor (GR) target genes and diverse modes of transcriptional regulation. This study presents a genome-wide microarray analysis of both our and published Affymetrix HG-U133 Plus 2.0 data in GCs-sensitive and -resistant ALL. GCs-sensitive CCRF-CEM-C7-14 cells were treated with dexamethasone at three time points (0 h, 2 h and 10 h). The treated samples were then compared to the control (0 h). Dexamethasone-treated CCRF-CEM-C7-14 samples were divided into 3 groups based on time points: the untreated control (0 h), 2 h and 10 h.
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Project description:Gene expression array analysis component. Ligand-dependent transcription by the nuclear receptor glucocorticoid receptor (GR) is mediated by interactions with co-regulators. The role of these interactions in determining selective binding of GR to regulatory elements remains unclear. Recent findings indicate a large fraction of genomic GR binding coincides with chromatin that is accessible prior to hormone treatment, suggesting that receptor binding is dictated by proteins that maintain chromatin in an open state. Combining nucleolytic cleavage and chromatin immunoprecipitation with high-throughput sequencing, we identify the activator protein 1 (AP1) as a major partner for productive GR-chromatin interactions. AP1 is critical for GR-regulated transcription and recruitment to co-occupied regulatory elements, illustrating an extensive AP1-GR interaction network. Importantly, the maintenance of baseline chromatin accessibility facilitates GR recruitment and is dependent on AP1 binding. We propose a model where the basal occupancy of transcription factors act to prime chromatin and direct inducible transcription factors to select regions in the genome. Dexamethasone treatment of cells at zero hour untreated control and 4 hour treatment times, in the absence and presence of acidic-fos (A-fos), an AP1 dominant negative. A-fos expression is under tetracycline control (Tet-Off). This is part of a larger study: Transcription Factor AP1 Potentiates Chromatin Accessibility and Glucocorticoid Receptor Binding. There are 2 replicates per condition per time point.
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Project description:ARGLU1 is a Transcriptional Coactivator and Splicing Regulator Important for Stress Hormone Signaling and Development Stress hormones bind and activate the glucocorticoid receptor (GR) in many tissues including the brain. We identified arginine and glutamate rich 1 (ARGLU1) in a screen for new modulators of glucocorticoid signaling in the CNS. Biochemical studies show that the glutamate rich C-terminus of ARGLU1 coactivates multiple nuclear receptors including the glucocorticoid receptor (GR) and the arginine rich N-terminus interacts with splicing factors and binds to RNA. RNA-seq of neuronal cells depleted of ARGLU1 revealed significant changes in the expression and alternative splicing of distinct genes involved in neurogenesis. Loss of ARGLU1 is embryonic lethal in mice, and knockdown in zebrafish causes neurodevelopmental and heart defects. Treatment with dexamethasone, a GR activator, also induces changes in the pattern of alternatively spliced genes, many of which were lost when ARGLU1 was absent. Importantly, the genes found to be alternatively spliced in response to glucocorticoid treatment were distinct from those under transcriptional control by GR, suggesting an additional mechanism of glucocorticoid action is present in neuronal cells. Our results thus show that ARGLU1 is a novel factor for embryonic development that modulates basal transcription and alternative splicing in neuronal cells with consequences for glucocorticoid signaling.
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