Project description:We performed ChIP-seq targeting the glucocorticoid receptor (GR) in the U2OS-GR cell line. The cell line is derived from U2OS ATTC:HTB-96 and stably transfected with an expression construct for rat GR. The cells were treated with 100 nM dexamethasone for 4 hours, washed 2x with PBS and cultured in hormone-free medium for 24 hours before harvest.
Project description:We performed ATAC-seq in the U2OS-GR cell line. The cell line is derived from U2OS ATTC:HTB-96 and stably transfected with an expression construct for rat GR. Cells were treated with dexamethasone (100 nM) or vehicle (ethanol) for 4 hours and either harvested immediately or washed 2x with PBS and subsequently cultured in hormone-free medium for 24 hours before harvest.
Project description:To identify genome-wide CCCTC binding factor (CTCF)-binding in A549 (ATCC CCL-185) and U2OS cells stably expressing rat GR (Rogatsky et al. , Mol Cell Biol, 1997. 17(6): p. 3181-93.) we performed ChIP-seq experiments upon hormone treatment (1.5 h, 1 M dexamethasone).
Project description:To investigate the role of HES1 during glucocorticoid signaling in bone sarcoma cells (U2OS-GR cells) , we employed whole-genome microarray expressions in mRNA extracted from control U2OS-GR and Hes1-overexpressed U2OS-GR cells. RNA isolated from control U2OS-GR and Hes1-overexpressed U2OS-GR cells treated with VEH or Dexamethasone.
Project description:We performed ChIP-seq targeting the glucocorticoid receptor (GR) in the U2OS-GR cell line and the androgen receptor (AR) in the U2OS-AR cell line. The cell lines are derived from U2OS ATTC:HTB-96 and stably transfected with an expression construct for either rat GR or human AR, respectively. The U2OS-GR cells were treated with dexamethasone (1 µM) for 90 minutes. The U2OS-AR cells were treated with R1881 (5 nM) for 4 hours.
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.
Project description:To investigate the interaction landscape at the GILZ locus we performed 4C-seq in A549 (ATCC CCL-185) and U2OS cells stably expressing rat GR (Rogatsky et al. , Mol Cell Biol, 1997. 17(6): p. 3181-93.) upon hormone treatment (1.5 h, 1 M dexamethasone). Experiments were performed in two biological replicates using hg19 chrX:106,960,488-106,960,865 as a viewpoint.
Project description:In response to environmental stressors and a variety of inflammatory cytokines, p38 MAPKs become directly activated. Here we report the human glucocorticoid receptor (GR) Serine 134 as a novel target for p38 MAPK. Unlike most other phosphorylation events that occur on the GR, phosphorylation of Ser134 was found to be hormone-independent in several human and rat cell types. Instead we found phosphorylation of Ser134 was induced by a variety of stress-activating stimuli, including: glucose starvation, ultraviolet irradiation, osmotic shock, and oxidative stress. Pharmacological inhibitors and shRNA-mediated knockdown experiments correlate this phosphorylation with the activation of p38 MAPK. Compared to wild-type GR, cells expressing a mutant receptor incapable of phosphorylation at Ser134 (S134A GR) had a significantly altered hormone-dependent genome-wide transcriptional response to glucocorticoids. Moreover, we show that although WT GR regulated roughly half as many genes as S134A GR, WT receptor selectively activated significantly more genes associated with endocrine and inflammatory disease than the mutant receptor, suggesting that the phosphorylation status of Ser134 is critical for modulating GR function. Phosphorylation of Ser134 did not alter either nuclear translocation or the stability of the GR protein in the absence or presence of ligand. However, phosphorylation of Ser134 significantly increased the association of the GR with the zeta isoform the 14-3-3 class of signaling proteins, resulting in a blunted hormone-dependent transcriptional response of LAD1 and IGFBP1 but not GILZ. Together these data suggest that the phosphorylation of Ser134 acts as a molecular sensor on the GR, monitoring the level of cellular stress to allow for altered 14-3-3zeta cofactor association, ultimately modifying glucocorticoid signaling in a gene-dependent manner. Our results reveal one mechanism that may allow cellular stress to dictate the transcriptional response of cells to hormone. U2OS cells, a human osteosarcoma cell line, were transfected with either WT GR or S134A GR and put under antibiotic selection to produce a stable mixed population of cells expressing comparable levels of GR. 10^6 cells were treated with 100nM Dexamethasone (DEX) or vehicle control for 6 hours. Three biological and one hybridization replicate are included for each sample.
Project description:We performed ChIP-seq targeting the H3K27ac, H3K4me1, H3K27me3 and H3K9me3 in the U2OS-GR and U2OS-AR cell lines. The cell lines are derived from U2OS ATTC:HTB-96 and stably transfected with an expression construct for either rat GR or human AR, respectively. The U2OS-GR cells were treated with dexamethasone (1 µM) or vehicle (ethanol) for 90 minutes. The U2OS-AR cells were treated with R1881 (5 nM) or vehicle (DMSO) for 4 hours.
