SynSTARR-seq with Glucocorticoid Receptor synthetic binding sites in GR18 cell line
ABSTRACT: We performed STARR-seq with synthetic libraries (synSTARR-seq) in GR18 cell line (derived from U2OS ATTC:HTB-96, stably transfected with an expression construct for GR), upon glucocorticoid (dexamethasone) or vehicle (ethanol) treatment. The synthetic libraries are variants of the glucocorticoid receptor binding sites (GBS). The "GBS half site" library contains 8 consecutive randomized nucleotides within the core binding sites, while the "Cgt/Sgk library" contains 5 consecutive randomized nucleotides on the flank of the GBS.
Project description:To identify the genes regulated by the Glucocorticoid Receptor (GR), we performed RNA-seq in GR18 cell line (derived from U2OS ATTC:HTB-96, stably transfected with an expression construct for GR), upon glucocorticoid (dexamethasone) or vehicle (ethanol) treatment.
Project description:To identify the genes regulated by the Glucocorticoid Receptor (GR), we performed RNA-seq in GR18 cell line (derived from U2OS ATTC:HTB-96, stably transfected with an expression construct for GR), upon glucocorticoid (dexamethasone) or vehicle (ethanol) treatment for 24 hours.
Project description:We performed ChIP-seq in the GR18 cell line (derived from U2OS ATTC:HTB-96, stably transfected with an expression construct for GR), upon glucocorticoid (dexamethasone)treatment for 90 minutes. Cells were cross-linked with 1% formaldehyde for 3 minutes.
Project description:We performed ATAC-seq in the GR18 cell line (derived from U2OS ATTC:HTB-96, stably transfected with an expression construct for GR), upon glucocorticoid (dexamethasone) or vehicle (ethanol) treatment for 90 minutes.
Project description:In the present study, genomic binding sites of glucocorticoid receptors (GR) were identified in vivo in the rat hippocampus applying chromatin immunoprecipitation followed by next-generation sequencing. We identified 2470 significant GR-binding sites (GBS) and were able to confirm GR binding to a random selection of these GBS covering a wide range of P values. Analysis of the genomic distribution of the significant GBS revealed a high prevalence of intragenic GBS. Gene ontology clusters involved in neuronal plasticity and other essential neuronal processes were overrepresented among the genes harboring a GBS or located in the vicinity of a GBS. Male adrenalectomized rats were challenged with increasing doses of the GR agonist corticosterone (CORT) ranging from 3 to 3000 μg/kg, resulting in clear differences in the GR-binding profile to individual GBS. Two groups of GBS could be distinguished: a low-CORT group that displayed GR binding across the full range of CORT concentrations, and a second high-CORT group that displayed significant GR binding only after administering the highest concentration of CORT. All validated GBS, in both the low-CORT and high-CORT groups, displayed mineralocorticoid receptor binding, which remained relatively constant from 30 μg/kg CORT upward. Motif analysis revealed that almost all GBS contained a glucocorticoid response element resembling the consensus motif in literature. In addition, motifs corresponding with new potential GR-interacting proteins were identified, such as zinc finger and BTB domain containing 3 (Zbtb3) and CUP (CG11181 gene product from transcript CG11181-RB), which may be involved in GR-dependent transactivation and transrepression, respectively. In conclusion, our results highlight the existence of 2 populations of GBS in the rat hippocampal genome. - See more at: http://press.endocrine.org/doi/10.1210/en.2012-2187?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dpubmed#sthash.LqK088DP.dpuf Overall design: Hippocampal Glucocorticoid receptor ChIP-seq using Santa Cruz H-300 Ab, for 300ug.kg and 3mg/kg corticosterone treatment and associated IgG controls Raw data are unavailable for this series because the raw data files have been lost. Available data include read alignments in bed format and genome coverage data in wiggle format.
Project description:The glucocorticoid receptor (GR), is a hormone-activated transcription factor which binds to GR binding sequences (GBS) encoded in the genome. Interestingly, of the many genomic sequences matching the GR consensus sequence only a small subset is actually bound. This indicates that the presence of a GBS alone is insufficient to specify where in the genome GR binds. We identified sequences that can locally prevent GR binding and mass-spec analysis was used to identify proteins that bind to these sequences.
Project description:Glucocorticoid drugs are widely used to treat immune-related diseases, but their use is limited by side effects and by resistance, which especially occurs in macrophage-dominated diseases. In order to improve glucocorticoid therapies, more research is required into the mechanisms of glucocorticoid action. In the present study, we have used a zebrafish model for inflammation to study glucocorticoid effects on the innate immune response. In zebrafish larvae, the migration of neutrophils towards a site of injury is inhibited by the synthetic glucocorticoid beclomethasone, while migration of macrophages is glucocorticoid resistant. RNA sequencing was done on on Fluorescence-Activated Cell Sorting (FACS)-sorted macrophages.The results show that the vast majority of the wounding-induced transcriptional changes in these cells are inhibited by beclomethasone, whereas a small subset is glucocorticoid-insensitive. As a result, beclomethasone decreases the number of macrophages that differentiate towards a pro-inflammatory (M1) phenotype, which we demonstrated using a tnfa:eGFP-F reporter line and analysis of macrophage morphology. We conclude that the glucocorticoid resistance of the wounding-induced macrophage migration is due to the insensitivity of the induction of macrophage-specific chemoattractants to glucocorticoid inhibition, which may explain the relative resistance of macrophage-dominated diseases to glucocorticoid therapy. However, the induction of pro-inflammatory genes in macrophages is strongly attenuated, which inhibits their differentiation to an M1 phenotype. Overall design: After anesthesia with 0.02% aminobenzoic acid ethyl ester (tricaine, Sigma Aldrich), the tails of 3 days post fertilization (dpf) embryos were partially amputated with a 1mm sapphire blade (World Precision Instruments) on 2% agarose-coated Petri dishes under a Leica M165C stereomicroscope (Chatzopoulou et al., 2016). Amputated and non-amputated (control) embryos were pretreated for 2 hours with 25 μM beclomethasone (Sigma Aldrich) or vehicle (0.05% dimethyl sulfoxide (DMSO)) in egg water prior to amputation and received the same treatment after the amputation. Macrophages were sorted from Tg(mpeg1.4:mCherry-F) embryos as previously described (Rougeot et al., 2014; Zakrzewska et al., 2010) at 4 hours post amputation (hpa). The sorted cells were collected in QIAzol lysis reagent (Qiagen) for RNA isolation. Extracted total RNA was amplified using the SMART-seq V4 kit (Clontech) for sequencing. The RNA seq libraries generated with the SMART-seq V4 kit were sequenced using an Illumina HiSeq 2500 instrument according to the manufacturer’s instructions with a read length of 50 nucleotides.
Project description:Protein translation is at the heart of cellular metabolism and its in-depth characterization is key for many lines of research. Recently, ribosome profiling became the state-of-the-art method to quantitatively characterize translation dynamics at a transcriptome-wide level. However, the strategy of library generation affects its outcomes. Here, we present a modified ribosomeprofiling protocol starting from yeast, human cells and vertebrate brain tissue. We use a DNA linker carrying four randomized positions at its 5’ and a reverse-transcription (RT) primer with three randomized positions to reduce artifacts during library preparation. The use of seven randomized nucleotides allows to efficiently detect library-generation artifacts. We find that the effect of polymerase chain reaction (PCR) artifacts is relatively small for global analyses when sufficient input material is used. However, when input material is limiting, our strategy improves the sensitivity of gene-specific analyses. Furthermore, randomized nucleotides alleviate the skewed frequency of specific sequences at the 3’ end of ribosome-protected fragments (RPFs) likely resulting from ligase specificity. Finally, strategies that rely on dual ligation show a high degree of gene-coverage variation. Taken together, our approach helps to remedy two of the main problems associated with ribosome-profiling data. This will facilitate the analysis of translational dynamics and increase our understanding of the influence of RNA modifications on translation. Ribosome profiling and mRNA-seq libraries from wt yeast comparing different library preparation approaches using different combinations of randomized and non-randomized linkers and RT primers.
Project description:The circadian transcriptional repressors cryptochromes 1 (Cry1) and 2 (Cry2) interact with the C-terminus of the glucocorticoid receptor (GR) and are required for transrepression in response to the synthetic GR ligand dexamethasone (Dex) in mouse embryonic fibroblasts. Dex induction of many genes was increased in Cry-deficient fibroblasts suggesting that cryptochromes oppose transactivation in addition to contributing to transrepression. In mice, genetic loss of Cry1 and/or Cry2 resulted in glucose intolerance and constitutively high levels of circulating corticosterone, suggesting reduced glucocorticoid suppression of the hypothalamic-pituitary-adrenal axis coupled with increased sensitivity to the hyperglycemic effects of glucocorticoid-mediated transactivation in the liver. Cry1 and Cry2 association with a GRE in the Pck1 promoter was stimulated by Dex, and Dex-induced transcription of pck1 was strikingly increased in Cry-deficient livers. Finally, cry1-/-;cry2-/- mice subjected to 8 weeks of chronic Dex treatment exhibited incomplete suppression of circulating corticosterone and greater glucose intolerance compared with wildtype littermates subjected to the same chronic treatment, consistent with enhanced transcriptional response to the synthetic glucocorticoid ligand. Overall design: Total RNA was obtained from WT and Cry1/2 KO MEFs treated with Dexamethasone (1uM) or control EtOH for 16 hours.