Genomic determination of the glucocorticoid response reveals unexpected mechanisms of gene regulation.
ABSTRACT: The glucocorticoid steroid hormone cortisol is released by the adrenal glands in response to stress and serves as a messenger in circadian rhythms. Transcriptional responses to this hormonal signal are mediated by the glucocorticoid receptor (GR). We determined GR binding throughout the human genome by using chromatin immunoprecipitation followed by next-generation DNA sequencing, and measured related changes in gene expression with mRNA sequencing in response to the glucocorticoid dexamethasone (DEX). We identified 4392 genomic positions occupied by the GR and 234 genes with significant changes in expression in response to DEX. This genomic census revealed striking differences between gene activation and repression by the GR. While genes activated with DEX treatment have GR bound within a median distance of 11 kb from the transcriptional start site (TSS), the nearest GR binding for genes repressed with DEX treatment is a median of 146 kb from the TSS, suggesting that DEX-mediated repression occurs independently of promoter-proximal GR binding. In addition to the dramatic differences in proximity of GR binding, we found differences in the kinetics of gene expression response for induced and repressed genes, with repression occurring substantially after induction. We also found that the GR can respond to different levels of corticosteroids in a gene-specific manner. For example, low doses of DEX selectively induced PER1, a transcription factor involved in regulating circadian rhythms. Overall, the genome-wide determination and analysis of GR:DNA binding and transcriptional response to hormone reveals new insights into the complexities of gene regulatory activities managed by GR.
Project description:Glucocorticoids regulate gene expression by binding and activating the glucocorticoid receptor (GR). While ligand affinity determines the global sensitivity of the response, additional proteins act on the genome to tune sensitivity of some genes. However, the genomic extent and specificity of dose-specific glucocorticoid responses are unknown. We show that dose-specific glucocorticoid responses are extraordinarily specific at the genomic scale, able to distinctly express a single gene, the circadian rhythm gene for Period 1 (PER1), at concentrations consistent with the nighttime nadir of human cortisol. We mapped the PER1 response to a single GR binding site. The specific GR binding sequence did not impact sensitivity, and we instead attributed the response to a combination of additional transcription factors and chromatin accessibility acting in the same locus. The PER1 hypersensitive response element is conserved in the mouse, where we found similar upregulation of Per1 in pituitary cells. Targeted and transient overexpression of PER1 led to regulation of additional circadian rhythm genes hours later, suggesting that hypersensitive expression of PER1 impacts circadian gene expression. These findings show that hypersensitive GR binding occurs throughout the genome, drives targeted gene expression, and may be important to endocrine mediation of peripheral circadian rhythms.
Project description:Airway occlusion in obstructive airway diseases is caused in part by the overproduction of secretory mucin glycoproteins through the up-regulation of mucin (MUC) genes by inflammatory mediators. Some pharmacological agents, including the glucocorticoid dexamethasone (Dex), repress mucin concentrations in lung epithelial cancer cells. Here, we show that Dex reduces the expression of MUC5AC, a major airway mucin gene, in primary differentiated normal human bronchial epithelial (NHBE) cells in a dose-dependent and time-dependent manner, and that the Dex-induced repression is mediated by the glucocorticoid receptor (GR) and two glucocorticoid response elements (GREs) in the MUC5AC promoter. The pre-exposure of cells to RU486, a GR antagonist, and mutations in either the GRE3 or GRE5 cis-sites abolished the Dex-induced repression. Chromatin immunoprecipitation (ChIP) assays showed a rapid temporal recruitment of GR to the GRE3 and GRE5 cis-elements in the MUC5AC promoter in NHBE and in A549 cells. Immunofluorescence showed nuclear colocalization of GR and histone deacetylase-2 (HDAC2) in MUC5AC-expressing NHBE cells. ChIP also showed a rapid temporal recruitment of HDAC2 to the GRE3 and GRE5 cis-elements in the MUC5AC promoter in both cell types. The knockdown of HDAC2 by HDAC2-specific short interfering RNA prevented the Dex-induced repression of MUC5AC in NHBE and A549 cells. These data demonstrate that GR and HDAC2 are recruited to the GRE3 and GRE5 cis-sites in the MUC5AC promoter and mediate the Dex-induced cis repression of MUC5AC gene expression. A better understanding of the mechanisms whereby glucocorticoids repress MUC5AC gene expression may be useful in formulating therapeutic interventions in chronic lung diseases.
Project description:Alterations in circadian rhythms are closely linked to depression, and we have shown earlier that progressive alterations in circadian entrainment precede the onset of depression in mice exposed in utero to excess glucocorticoids. The aim of this study was to investigate whether treatment with the noradrenaline reuptake inhibitor desipramine (DMI) could restore the alterations in circadian entrainment and prevent the onset of depression-like behavior. C57Bl/6 mice were exposed to dexamethasone (DEX-synthetic glucocorticoid analog, 0.05 mg/kg/day) between gestational day 14 and delivery. Male offspring aged 6 months (mo) were treated with DMI (10 mg/kg/day in drinking water) for at least 21 days before behavioral testing. We recorded spontaneous activity using the TraffiCage™ system and found that DEX mice re-entrained faster than controls after an abrupt advance in light-dark cycle by 6 h, while DMI treatment significantly delayed re-entrainment. Next we assessed the synchronization of peripheral oscillators with the central clock (located in the suprachiasmatic nucleus-SCN), as well as the mechanisms required for entrainment. We found that photic entrainment of the SCN was apparently preserved in DEX mice, but the expression of clock genes in the hippocampus was not synchronized with the light-dark cycle. This was associated with downregulated mRNA expression for arginine vasopressin (AVP; the main molecular output entraining peripheral clocks) in the SCN, and for glucocorticoid receptor (GR; required for the negative feedback loop regulating glucocorticoid secretion) in the hippocampus. DMI treatment restored the mRNA expression of AVP in the SCN and enhanced GR-mediated signaling by upregulating GR expression and nuclear translocation in the hippocampus. Furthermore, DMI treatment at 6 mo prevented the onset of depression-like behavior and the associated alterations in neurogenesis in 12-mo-old DEX mice. Taken together, our data indicate that DMI treatment enhances GR-mediated signaling and restores the synchronization of peripheral clocks with the SCN and support the hypothesis that altered circadian entrainment is a modifiable risk factor for depression.
Project description:Small molecule inhibitors of lysine deacetylases (KDACs) are approved for clinical use in treatment of several diseases. Nuclear receptors, such as the glucocorticoid receptor (GR) use lysine acetyltransferases (KATs or HATs) and KDACs to regulate transcription through acetylation and deacetylation of protein targets such as histones. Previously we have shown that KDAC1 activity facilitates GR-activated transcription at about half of all cellular target genes. In the current study we examine the role of Class I KDACs in glucocorticoid-mediated repression of gene expression. Inhibition of KDACs through two structurally distinct Class I-selective inhibitors prevented dexamethasone (Dex)-mediated transcriptional repression in a gene-selective fashion. In addition, KDAC activity is also necessary to maintain repression. Steroid receptor coactivator 2 (SRC2), which is known to play a vital role in GR-mediated repression of pro-inflammatory genes, was found to be dispensable for repression of glucocorticoid target genes sensitive to KDAC inhibition. At the promoters of these genes, KDAC inhibition did not result in altered nucleosome occupancy or histone H3 acetylation. Surprisingly, KDAC inhibition rapidly induced a significant decrease in H3K4Me2 at promoter nucleosomes with no corresponding change in H3K4Me3, suggesting the activation of the lysine demethylase, LSD1/KDM1A. Depletion of LSD1 expression via siRNA restored Dex-mediated repression in the presence of KDAC inhibitors, suggesting that LSD1 activation at these gene promoters is incompatible with transcriptional repression. Treatment with KDAC inhibitors does not alter cellular levels of LSD1 or its association with Dex-repressed gene promoters. Therefore, we conclude that Class I KDACs facilitate Dex-induced transcriptional repression by suppressing LSD1 complex activity at selected target gene promoters. Rather than facilitating repression of transcription, LSD1 opposes it in these gene contexts.
Project description:Dexamethasone (DEX) an anti-inflamatory 9-fluoro-glucocorticoid, activates the cytosolic glucocorticoid receptor (GR) binding to its Ligand Binding Domain (LBD). The GR-ligand complex then translocates to the nucleus and binds to the Glucocorticoid Response Element (GRE) resulting up-regulation of target gene expression of anti-inflamatory proteins. DEX is one of the most effective ligand for GR activation but comply to side effects. Therefore, alternative for DEX - plant metabolites of Calotropis sp and Swertia chirata were screened using docking appraoch. These plants compounds were selected because; parts of these plants are widely used againsts inflamation, allergy, asthma etc. Three metabolites of Swertia chirata namely Gentianine (GENT), Xanthone (XANT) and Swerchirin (SWER) are found to be occupying the same binding pocket in the LBD of the GR (PDB ID 1M2Z). The binding affinity as reflected by binding energies of GENT-1M2Z, XANT-1M2Z and SWER-1M2Z are -5.6, -6.7 and -6.7, and all the output parameter of the respective compounds positively correlates with that of DEX-1M2Z with r = 0.9, 0.6 and 0.6 respectively indicating similar GR activation function. Visualization analysis of the models clearly indicates that GENT and SWER may be GR activators. Rest of the compounds mostly docked onto the surface of the receptor molecule.
Project description:Glucocorticoid signaling through the glucocorticoid receptor (GR) plays essential roles in the response to stress and in energy metabolism. This hormonal action is integrated to the transcriptional control of GR-target genes in a cell type-specific and condition-dependent manner. In the present study, we found that the GR regulates the angiopoietin-like 4 gene (ANGPTL4) in a CCCTC-binding factor (CTCF)-mediated chromatin context in the human hepatic HepG2 cells. There are at least four CTCF-enriched sites and two GR-binding sites within the ANGPTL4 locus. Among them, the major CTCF-enriched site is positioned near the ANGPTL4 enhancer that binds GR. We showed that CTCF is required for induction and subsequent silencing of ANGPTL4 expression in response to dexamethasone (Dex) and that transcription is diminished after long-term treatment with Dex. Although the ANGPTL4 locus maintains a stable higher-order chromatin conformation in the presence and absence of Dex, the Dex-bound GR activated transcription of ANGPTL4 but not that of the neighboring three genes through interactions among the ANGPTL4 enhancer, promoter, and CTCF sites. These results reveal that liganded GR spatiotemporally controls ANGPTL4 transcription in a chromosomal context.
Project description:Despite decades of clinical use, mechanisms of glucocorticoid resistance are poorly understood. We treated primary murine T lineage acute lymphoblastic leukemias (T-ALLs) with the glucocorticoid dexamethasone (DEX) alone and in combination with the pan-PI3 kinase inhibitor GDC-0941 and observed a robust response to DEX that was modestly enhanced by GDC-0941. Continuous in vivo treatment invariably resulted in outgrowth of drug-resistant clones, ~30% of which showed markedly reduced glucocorticoid receptor (GR) protein expression. A similar proportion of relapsed human T-ALLs also exhibited low GR protein levels. De novo or preexisting mutations in the gene encoding GR (Nr3c1) occurred in relapsed clones derived from multiple independent parental leukemias. CRISPR/Cas9 gene editing confirmed that loss of GR expression confers DEX resistance. Exposing drug-sensitive T-ALLs to DEX in vivo altered transcript levels of multiple genes, and this response was attenuated in relapsed T-ALLs. These data implicate reduced GR protein expression as a frequent cause of glucocorticoid resistance in T-ALL.
Project description:Compound A (CpdA), a dissociated glucocorticoid receptor modulator, decreases corticosteroid-binding globulin (CBG), adrenocorticotropic hormone (ACTH), and luteneinizing hormone levels in rats. Whether this is due to transcriptional regulation by CpdA is not known. Using promoter reporter assays we show that CpdA, like dexamethasone (Dex), directly transrepresses these genes. Results using a rat Cbg proximal-promoter reporter construct in BWTG3 and HepG2 cell lines support a glucocorticoid receptor (GR)-dependent transrepression mechanism for CpdA. However, CpdA, unlike Dex, does not result in transactivation via glucocorticoid-responsive elements within a promoter reporter construct even when GR is co-transfected. The inability of CpdA to result in transactivation via glucocorticoid-responsive elements is confirmed on the endogenous tyrosine aminotransferase gene, whereas transrepression ability is confirmed on the endogenous CBG gene. Consistent with a role for CpdA in modulating GR activity, whole cell binding assays revealed that CpdA binds reversibly to the GR, but with lower affinity than Dex, and influences association of [(3)H]Dex, but has no effect on dissociation. In addition, like Dex, CpdA causes nuclear translocation of the GR, albeit to a lesser degree. Several lines of evidence, including fluorescence resonance energy transfer, co-immunoprecipitation, and nuclear immunofluorescence studies of nuclear localization-deficient GR show that CpdA, unlike Dex, does not elicit ligand-induced GR dimerization. Comparison of the behavior of CpdA in the presence of wild type GR to that of Dex with a dimerization-deficient GR mutant (GR(dim)) strongly supports the conclusion that loss of dimerization is responsible for the dissociated behavior of CpdA.
Project description:Brain-derived neurotrophic factor (BDNF) is involved in many functions such as neuronal growth, survival, synaptic plasticity and memorization. Altered expression levels are associated with many pathological situations such as depression, epilepsy, Alzheimer's, Huntington's and Parkinson's diseases. Glucocorticoid receptor (GR) is also crucial for neuron functions, via binding of glucocorticoid hormones (GCs). GR actions largely overlap those of BDNF. It has been proposed that GR could be a regulator of BDNF expression, however the molecular mechanisms involved have not been clearly defined yet. Herein, we analyzed the effect of a GC agonist dexamethasone (DEX) on BDNF expression in mouse neuronal primary cultures and in the newly characterized, mouse hippocampal BZ cell line established by targeted oncogenesis. Mouse Bdnf gene exhibits a complex genomic structure with 8 untranslated exons (I to VIII) splicing onto one common and unique coding exon IX. We found that DEX significantly downregulated total BDNF mRNA expression by around 30%. Expression of the highly expressed exon IV and VI containing transcripts was also reduced by DEX. The GR antagonist RU486 abolished this effect, which is consistent with specific GR-mediated action. Transient transfection assays allowed us to define a short 275 bp region within exon IV promoter responsible for GR-mediated Bdnf repression. Chromatin immunoprecipitation experiments demonstrated GR recruitment onto this fragment, through unidentified transcription factor tethering. Altogether, GR downregulates Bdnf expression through direct binding to Bdnf regulatory sequences. These findings bring new insights into the crosstalk between GR and BDNF signaling pathways both playing a major role in physiology and pathology of the central nervous system.
Project description:Glucocorticoid (GC) actions are mediated through two closely related ligand-dependent transcription factors, the GC receptor (GR) and the mineralocorticoid receptor (MR). Given the wide and effective use of GCs to combat skin inflammatory diseases, it is important to understand the relative contribution of these receptors to the transcriptional response to topical GCs. We evaluated the gene expression profiles in the skin of mice with epidermal-specific loss of GR (GREKO), MR (MREKO), or both (double KO; DKO) in response to dexamethasone (Dex). The overall transcriptional response was abolished in GREKO and DKO skin suggesting dependence of the underlying dermis on the presence of epidermal GR. Indeed, the observed dermal GC resistance correlated with a constitutive decrease in GR activity and up-regulation of p38 activity in this skin compartment. Upon Dex treatment, more than 90% of differentially expressed genes (DEGs) in CO overlapped with MREKO. However, the number of DEGs was fourfold increased and the magnitude of response was higher in MREKO vs CO, affecting both gene induction and repression. Taken together our data reveal that, in the cutaneous transcriptional response to GCs mediated through endogenous receptors, epidermal GR is mandatory while epidermal MR acts as a chief modulator of gene expression.