ER and GR crosstalk at the Genomic Level in mouse mammary cells
ABSTRACT: We report changes in ER and GR binding profiles genome-wide upon co-treatment with Dex and E2 when compared to Dex or E2 treatments alone. We examine ER and GR binding under four different treatments (unt, Dex, E2, and Dex + E2).
Project description:We report changes in DNaseI accessibility genome-wide upon co-treatment with Dex and E2 when compared to Dex or E2 treatments alone. We examine ER and GR binding under four different treatments (unt, Dex, E2, and Dex + E2).
Project description:We report changes in gene expression upon co-treatment with Dex and E2 when compared to Dex or E2 treatments alone. We examine expression under four different treatments (unt, Dex, E2, and Dex + E2).
Project description:We report changes in GR and Pol II binding profiles genome-wide upon treatment with corticosterone (Cort) for 20 minutes, treatment with Cort for 20 minutes followed by hormone withdrawal for 40 minutes, 60 minutes continuous stimulation with Cort, and 60 minutes continuous stimulation with Dexamethasone (Dex). We examine GR binding upon following treatments: 0' Cort, 20' Cort, 60' Cort Pulsed, 60' Cort Constant; Pol II binding upon following treatments: 0' Cort, 20' Cort, 60' Cort Pulsed, 60' Cort Constant, 60' Dex Constant; Pol II binding upon Mock treatments simulating 0' Cort, 20' Cort, 60' Cort Pulsed, 60' Cort Constant; CTCF binding profile of untreated cells.
Project description:DNase-seq and ChIP-seq determine that C/EBP maintains chromatin accessibility in liver and facilitates glucocorticoid receptor recruitment to steroid response elements DNase-seq and ChIP-seq (GR, C/EBPb and RNAPII) in intact liver from adrenalectomized mice injected with dex (1h)
Project description:A glucocorticoid-regulated BBM protein (35S:BBM-GR) was used in combination with microarray analysis to identify genes directly activated by BBM. We employed the system described by (Lloyd et al., 1994) in which dexamethasone (DEX) and cycloheximide (CHX) are applied together to respectively, induce nuclear localization of the BBM-GR protein and prevent translation of the primary targets mRNAs. In this way it is possible to identify direct targets of a transcriptional activator by comparing gene expression profiles between DEX+CHX-treated transgenic and wild-type tissues. The ability of the 35S:BBM GR construct to induce somatic embryogenesis in Arabidopsis seedlings was determined by phenotypic observation of 35S:BBM GR seeds germinated and grown in the presence of 10 µM dexamethasone (DEX). As in 35S:BBM seedlings, we observed somatic embryo formation on the cotyledons, first leaves and shoot meristem of DEX-treated 35S:BBM GR seedlings. We identified a set of 20 genes (including BBM itself) and our analysis indicates that BBM directly activates a signaling pathway comprising transcription factors and other signaling molecules, but which does not initially include genes known to induce somatic embryogenesis, such as LEC1, LEC2 or WUS. The functions of the BBM target genes are unknown, however a number of them have recently been identified in microarray screens for meristem-expressed genes. The identification of BBM-interacting partners and downstream targets provides new tools for unraveling pathways related to plant cell growth and organogenesis. Keywords: transcriptional activation To identify candidate BBM targets, we treated 4 day old 35S:BBM-GR seedlings for 8 hours with 10 µM DEX in the presence of 10 µM CHX and compared the mRNA population from these seedlings using Arabidopsis Operon microarrays (http://www.ag.arizona.edu/microarray/) to that of 4 day old wild-type seedlings treated in the same way. The seed batches used in this analysis showed 100% EFS when grown continuously on media with 10 µM DEX. Four day old seedlings were chosen as the experimental material because they are highly responsive to BBM GR activation and provide sufficient RNA for the microarray experiments. The biological reproducibility of the data was monitored using two independent single locus 35S:BBM GR lines. The technical reproducibility of the data was monitored using two independently DEX + CHX-treated samples from each transgenic line. Dye effects were monitored in a dye-swap experiment using one of the four RNA samples from the two biological replicates
Project description:Selective transcriptional activation and repression of genes throughout signaling cascades and development are poorly understood. Transcription factors (TF) orchestrate patterns and magnitude of transcriptional response, but TF action, or inaction, is highly dependent upon TF kinetics, distance from genes, chromatin architecture, and the local occupancy of other TFs. We integrated genomic transcription, chromosome looping, TF binding, and chromatin structure data to analyze the molecular cascade that results from estradiol-induced (E2) signaling in human MCF-7 breast cancer cells and addressed the context-specific nature of gene regulation. We analyzed kinetic ChIP-seq that profiled the master regulator of the E2-mediated response, estrogen receptor (ER), and found that transient ER binding sites are specifically associated with enhancers of repressed genes. We performed replicate ChIP-seq experiments prior to estrogen treatment and 2min, 5min, 10min, 40min, and 160min after E2 treatment.
Project description:Analysis of MCF-7 cells treated for 4h with Ethanol, Estradiol (E2), Dexamethasone (Dex), or Estradiol + Dexamethasone (E2 + Dex) In estrogen receptor (ER)-negative breast cancer (BC), high tumor glucocorticoid receptor (GR) expression has been associated with a relatively poor outcome. In contrast, using a meta-analysis of several genomic datasets, here we find that tumor GR mRNA expression is associated with improved ER+ relapse-free survival (RFS) (independently of progesterone receptor (PR) expression). To understand the mechanism by which GR expression is associated with a better ER+ BC outcome, the global effect of GR-mediated transcriptional activation in ER+ BC cells was studied. Analysis of GR chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) in ER+/GR+ MCF-7 cells revealed that upon co-activation of GR and ER, GR chromatin association became enriched at proximal promoter regions. Furthermore, following ER activation, increased association of GR was observed at ER, FOXO, and AP1 response elements. In addition, it was determined that ER associated with GR response elements, suggesting that ER and GR interact in a complex. Co-activation of GR and ER resulted in increased expression (relative to ER activation alone) of transcripts that encode proteins promoting cellular differentiation (e.g. KDM4B, VDR) and inhibiting Wnt-signaling (IGFBP4). Finally, expression of these individual pro-differentiation genes was associated with significantly improved RFS in ER+ BC patients. Together, these data demonstrate that the co-expression and subsequent activity of tumor cell GR and ER contribute to the less aggressive natural history of early-stage BC by coordinating the altered expression of genes favoring differentiation. Four treatment samples (Vehicle V, Dex D, E2, or Dex+E2). Three biological replicate experiments per sample. Vehicle sample is Ethanol control.
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:Plants typically contain two different types of cell walls: a primary wall that is being deposited around all growing cells, and a secondary wall that is produced in cells with specialized functions once they have ceased to grow. In Arabidopsis, VND7 is a transcription factor that is sufficient to activate secondary cell wall synthesis. To artificially turn on the secondary cell wall synthesis, VND7 was fused to the activation domain of the herpes virus VP16 protein and the glucocorticoid receptor (GR) domain. Thus, the transgenic plants harbouring the constructs can then be treated with dexamethasone (DEX), a glucocorticoid derivative, to induce the secondary cell wall formation. We used microarrays to investigate the global program of gene expression during the secondary cell wall development and identified up- and down-regulated genes associated with this process. Cultures were harvested at indicated time points after induction (1, 3, 6, 9, 12, 24, 30, 48 h) and at time point zero (0). Uninduced cultures (DMSO instead of DEX) served as controls for each time point, and thus, 48 microarray analyses for 8 time points (3 biological reps) for +/- induced VND7-VP16-GR cultures, and 3 arrays for time 0h. To ensure that the DEX did not influence the measurements, an empty vector construct (EV), i.e., without the VND7 gene, VP16-GR, was used at three time points, i.e., VP16-GR +/- DEX at 9h, 12h, and 24h after DEX induction.