Genetic mapping with multiple levels of phenotypic information reveals new determinants of lymphocyte glucocorticoid sensitivity
ABSTRACT: Clinical response to glucocorticoids (GCs), steroid hormones widely used as pharmaceuticals, varies extensively, with many patients (~30%) showing a weak response to treatment. Although little is known about the molecular basis of this variation, regulatory polymorphisms are likely to play a key role as GCs act largely through activation of a transcription factor, the GC receptor. In an effort to characterize the molecular basis of variation in GC sensitivity, we measured in vitro lymphocyte GC sensitivity (LGS) and transcriptome-wide response to GCs in peripheral blood mononuclear cells (PBMCs) from African-American healthy donors. We found that variation in LGS was correlated with transcriptional response at 27 genes (FDR<0.1). Furthermore, a genome-wide association scan revealed a quantitative trait locus (QTL) for LGS (rs11129354, P=4x10-8) that was also associated with transcriptional response at multiple genes, including many (14 of 27) where transcriptional response was correlated with LGS. Using allelic imbalance assays, we show that this QTL is a GC-dependent cis-regulatory polymorphism for RBMS3, which encodes an RNA-binding protein known as a tumor suppressor. We found that siRNA-mediated knockdown of RBMS3 expression increases cellular proliferation in PBMCs, consistent with the role of the gene as a negative regulator of proliferation. We propose that differences in LGS reflect variation in transcriptional response, which are influenced by a GC-dependent regulatory polymorphism that acts in cis relative to RBMS3 and in trans to affect the transcriptional response of multiple distant genes. Total RNA was obtained from paired aliquots of peripheral blood mononuclear cells treated with dexamethasone and phytohemagglutinin, vehicle (EtOH) and phytohemagglutinin, or blank (no treatment) for 6 hours.
Project description:Glucocorticoids (GCs) are steroid hormones widely used as pharmaceutical interventions, which act mainly by regulating gene expression levels. A large fraction of patients (~30%), especially those of African descent, show a weak response to treatment. To interrogate the contribution of variable transcriptional response to inter-ethnic differences, we measured in vitro lymphocyte GC sensitivity (LGS) and transcriptome-wide response to GCs in peripheral blood mononuclear cells (PBMCs) from African-American and European-American healthy donors. We found that transcriptional response after 8hrs treatment was significantly correlated with variation in LGS within and between populations. We found that NFKB1, a gene previously found to predict LGS within populations, was more strongly downregulated in European-Americans on average. NFKB1 could not completely explain population differences, however, and we found an additional 177 genes with population differences in the average log2 fold change (FDR<0.05), most of which also showed a weaker transcriptional response in AfricanAmericans. These results suggest that inter-ethnic differences in GC sensitivity reflect variation in transcriptional response at many genes, including regulators with large effects (e.g. NFKB1) and numerous other genes with smaller effects. Total RNA was obtained from paired aliquots of peripheral blood mononuclear cells treated with dexamethasone or vehicle (EtOH) for 8 and 24 hours.
Project description:Glucocorticoids (GCs) are key mediators of stress response and are widely used as pharmacological agents to treat immune diseases, such as asthma and inflammatory bowel disease, and certain types of cancer. GCs act mainly by activating the GC receptor (GR), which interacts with other transcription factors to regulate gene expression. Here, we combined different functional genomics approaches to gain molecular insights into the mechanisms of action of GC. By profiling the transcriptional response to GC over time in 4 Yoruba (YRI) and 4 Tuscans (TSI) lymphoblastoid cell lines (LCLs), we suggest that the transcriptional response to GC is variable not only in time, but also in direction (positive or negative) depending on the presence of specific interacting TFs. Accordingly, when we performed ChIP-seq for GR and NF-kB in two YRI LCLs treated with GC or with vehicle control, we observed that features of GR binding sites differ for up- and down-regulated genes. Finally, we show that eQTLs that affect expression patterns only in the presence of GC are 1.9-fold more likely to occur in GR binding sites, compared to eQTLs that affect expression only in its absence. Our results indicate that genetic variation at GR and interacting transcription factors binding sites influences variability in gene expression, and attest to the power of combining different functional genomic approaches. GR and NFkB ChIP-seq in lymphoblastoid cell lines treated with either dexamethasone or EtOH (vehicle for dexamethasone) for 1 hour.
Project description:Glucocorticoids (GCs) are key mediators of stress response and are widely used as pharmacological agents to treat immune diseases, such as asthma and inflammatory bowel disease, and certain types of cancer. GCs act mainly by activating the GC receptor (GR), which interacts with other transcription factors to regulate gene expression. Here, we combined different functional genomics approaches to gain molecular insights into the mechanisms of action of GC. By profiling the transcriptional response to GC over time in 4 Yoruba (YRI) and 4 Tuscans (TSI) lymphoblastoid cell lines (LCLs), we suggest that the transcriptional response to GC is variable not only in time, but also in direction (positive or negative) depending on the presence of specific interacting TFs. Accordingly, when we performed ChIP-seq for GR and NF-kB in two YRI LCLs treated with GC or with vehicle control, we observed that features of GR binding sites differ for up- and down-regulated genes. Finally, we show that eQTLs that affect expression patterns only in the presence of GC are 1.9-fold more likely to occur in GR binding sites, compared to eQTLs that affect expression only in its absence. Our results indicate that genetic variation at GR and interacting transcription factors binding sites influences variability in gene expression, and attest to the power of combining different functional genomic approaches. Total RNA obtained from lymphoblastoid cell lines treated with either dexamethasone or EtOH (vehicle for dexamethasone) for 2, 4, 8, 12, 16, or 24 hours.
Project description:Glucocorticoids (GCs) are commonly used to treat patients suffering from lymphoid malignancies i.e. leukemia and multiple myeloma. Although GCs are known to be strong inducers of apoptosis in lymphoid cells, the molecular determinants of GC therapy resistance are poorly understood. Although GC treatment triggers important changes in gene expression, few studies have addressed the regulatory role of small regulatory microRNAs (miRNAs) in GC therapy response. Only recently, aberrant microRNA expression has been linked to the development of haematological malignancies and microRNAs have become master regulators of drug resistance. We identified GC inducible mRNA and microRNA transcription profiles in GC sensitive MM1S as compared to GC resistant MM1R cells. Transcriptome analysis revealed that GCs regulate multiple genes involved in cell cycle control, cell organization and cell death in MM1S, which remain unaffected in MM1R cells. Correspondingly, GCs selectively trigger cell death in MM1S but not in MM1R. Out of 32 microRNAs responsive to GC in MM1S cells but not in MM1R cells, mir-150 was identified as the most persistent GC responsive microRNA. Furthermore, Ingenuity Pathways Analysis (IPA) revealed that ectopic transfection of a synthetic mir-150 mimics GC therapy response in MM1S cells, associated with selective changes in mRNA levels of typical GR transactivated and transrepressed target genes. Although mir-150 largely mirrors GC responsive changes in gene expression of the transcription factor Myb, GR chaperone FKBP5, cell cycle modulator proteins (IL23A, SKP2, CDKN1A), chemokine signaling proteins (CXCR4, CX3CR1, CCL3) and mTOR/UPR stress related proteins (DDIT4, TXNIP), we also observed mir-150 selective effects on transcription factors (NR3C2 (MR), Myb, Fos, Jun, C/EBP-beta, IRF4, NFE2L1, ATF3, ATF4,), chaperone molecules HSPA8, HSP90AB1), the sodium channel SCNN1G and UPR stress proteins (TRIB3, DDIT3). Remarkably, mir-150 overexpression was not able to overcome GC therapy resistance, since we could not detect GC like effects of mir-150 in GR (NR3C1) deficient MM1R cells. Altogether GC-inducible mir-150 adds a novel complex layer of regulation for fine tuning GC specific therapeutic responses in multiple myeloma. Overall design: 2 myeloma cellines, resistant (MM1R) and sensitive (MM1S), were treated treated with dexamethasone for 72hrs and compared to their untreated counterparts or transfected with synthetic mir-150-5p for 72hrs and compared to their mock transfected counterparts.
Project description:The use of glucocorticoids (GCs), which bind and activate the glucocorticoid receptor (GR), in systemic inflammatory response syndromes (SIRS) is disputed. Mice with a poor transcriptional response of dimer-dependent GR target genes were studied in a model of TNF-induced SIRS. These GR dim/dim mice display a significant increase in TNF sensitivity and a lack of protection by the GC dexamethasone (DEX). Unchallenged GR dim/dim mice have a strong interferon-stimulated gene (ISG) signature at the transcriptional level and this ISG signature is gut specific. Here, we used shotgun proteomics to study the regulation of ISG proteins in the ileum of GR dim/dim mice. Our data showed that unchallenged GR dim/dim mice have a strong interferon-stimulated gene (ISG) signature, along with STAT1 upregulation. Taken together, we show that GR dim/dim mice poorly control ISG expression resulting in excessive necroptosis induction by TNF. Our findings support a critical interplay between gut microbiota, interferons, necroptosis and GR in both the basal response to acute inflammatory challenges and in the pharmacological intervention by GCs.
Project description:Glucocorticoids (GC) and 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3) are steroid hormones with anti-inflammatory properties with enhanced effects when combined. We previously showed that transcriptional response to GCs was correlated with inter-individual and inter-ethnic cellular response. Here, we profiled cellular and transcriptional responses to 1,25(OH)2 D3 from the same donors. We studied cellular response to combined treatment with GCs and 1,25(OH)2 D3 in a subset of individuals least responsive to GCs. We found that combination treatment had significantly greater inhibition of proliferation than with either steroid hormone alone. Overlapping differentially expressed (DE) genes between the two hormones were enriched for adaptive and innate immune processes. Non-overlapping differentially expressed genes with 1,25(OH)2 D3 treatment were enriched for pathways involving the electron transport chain, while with GC treatment, non-overlapping genes were enriched for RNA-related processes. These results suggest that 1,25(OH)2 D3 enhances GC anti-inflammatory properties through a number of shared and non-shared transcriptionally-mediated pathways. Total RNA was obtained from aliquots of peripheral blood mononuclear cells treated with 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3) for 8 and 24 hours. These data were analyzed together with previously published data from expression analysis of PBMC aliquots collected in parallel to these and treated with dexamethasone or vehicle (EtOH).
Project description:Glucocorticoids (GCs) are steroid hormones produced by the human body in response to environmental stressors. Despite their key role as physiological regulators and widely administered pharmaceuticals, little is known about the genetic basis of inter-individual and inter-ethnic variation in GC response. As GC action is mediated by the regulation of gene expression, we profiled transcript abundance and protein secretion in EBV-transformed B lymphocytes from a panel of 114 individuals, including those of both African and European ancestry. Combining these molecular traits with genome-wide genetic data, we found that genotype-treatment interactions at polymorphisms near genes affected GC-regulation of expression for 26 genes and of secretion for IL6. A novel statistical approach revealed that these interactions could be distinguished into distinct types, with some showing genotypic effects only in GC-treated samples and others showing genotypic effects only in control-treated samples, with differing phenotypic and molecular interpretations. The insights into the genetic basis of variation in GC response and the statistical tools for identifying gene-treatment interactions that we provide will aid future efforts to identify genetic predictors of response to this and other treatments. Total RNA was obtained from paired aliquots of lymphoblastoid cell lines (collected as a part of the HapMap project) treated with dexamethasone or vehicle (EtOH) for 8 hours.
Project description:Glucocorticoids (GCs) are a central component of combination chemotherapy for childhood B-cell precursor acute lymphoblastic leukemia (B-ALL). GCs work by activating the glucocorticoid receptor (GR), a ligand induced transcription factor, which in turn regulates genes that induce leukemic cell death. Which GR-regulated genes are required for GC cytotoxicity, the pathways that affect their regulation, and how resistance arises are not well understood. Here we systematically integrate the transcriptional response of B-ALL to GCs with a next-generation shRNA screen to identify GC-regulated “effector” genes that contribute to cell death as well as genes that affect the sensitivity of B-ALL cells to dex. This analysis reveals a pervasive role for GCs in suppression of B-cell development genes that is linked to therapeutic response. Inhibition of PI3Kδ, a lynchpin in the pre-B-cell receptor and IL7R signaling pathways critical to B-cell development, with CAL-101 (idelalisib), interrupts a double-negative feedback loop, enhancing GC-regulated transcription to synergistically kill even highly resistant B-ALL with diverse genetic backgrounds. This work not only identifies numerous opportunities for enhanced lymphoid-specific combination chemotherapy that have the potential to overcome treatment resistance, but is also a valuable resource for understanding GC biology and the mechanistic details of GR-regulated transcription. Please note that the cell lines and primary samples were processed and normalized separately. Overall design: The data submitted include the gene expression profiles for 11 cell lines and three primary patient samples. The cells were all treated in the same way: grown in RPMI plus 10% FBS @ 37˚C with 5% CO2; treated for 4 hours with either 1µM dexamethasone, or 0.1% ethanol as a control; RNA was harvested then run on Illumina HT12 v 4 arrays. There are at least three reapeats for each treatment and control. Control repeats have the suffix U* whereas the treated have either I* or D* as a suffix.
Project description:Background and Aims: Gastric adenocarcinoma (gastric cancer, GC) is a major cause of global cancer mortality. Identifying molecular programs contributing to GC patient survival may improve our understanding of GC pathogenesis, highlight new prognostic factors, and reveal novel therapeutic targets. We aimed to produce a comprehensive inventory of gene expression programs expressed in primary GCs, and to identify those expression programs significantly associated with patient survival. Methods: Using a network modeling approach, we performed a large scale meta-analysis of GC transcriptome data integrating 940 gastric transcriptomes from multiple independent patient cohorts. We analyzed a training set of 428 GCs and 163 non neoplastic gastric mucosa (‘non-malignants’), and a validation set of 288 GCs and 61 non-malignants. Results: We identified 178 gene expression programs (modules) expressed in primary GCs, which were associated with distinct biological processes, chromosomal location patterns, cis-regulatory motifs, and clinicopathological parameters. Amongst these modules, expression of a TGF-B-signaling associated “super-module” of stroma-related genes consistently predicted patient survival in multiple GC validation cohorts. Analysis of histopathological tissue sections from gastrectomy specimens revealed that expression of this stromal module was associated with the proportion of intra-tumoral stroma (ITS). Further supporting the stromal module expression/ITS association, we found that the ITS proportion, measured directly from tissue sections, also predicted GC patient survival. Conclusion: Stromal gene expression predicts GC patient survival in multiple independent cohorts and may be closely related to the ITS proportion, a specific morphological GC phenotype. These findings suggest that therapeutic approaches targeting pathways associated with the GC stroma may merit evaluation. Overall design: Profiling of 86 gastric samples (83 tumors and 3 non-malignants) on Affymetrix GeneChip U133A Array. All samples were collected with approvals from National Cancer Centre of Singapore. Profiling of 34 gastric tumors on Custom cDNA Microarray(30K). All samples were collected with approvals from VU University Medical Centre, Amsterdam, The Netherlands. Profiling of 65 gastric samples (29 tumors and 36 non-malignants) on Affymetrix GeneChip Human Genome U133 set Array. All samples were collected with approvals from Leeds Institute for Molecular Medicine, St James’s University Hospital, Leeds, United Kingdom.
Project description:We used microarrays to expression profile peripheral blood mononuclear cells (PBMCs) from LGL leukemia patients and control subjects to identify survival pathways that render leukemic LGL resistant to activation induced cell death. Experiment Overall Design: Leukemic PBMC RNA from 30 patients was extracted for target preparation and hybridization onto Affymetrix microarrays. We also isolated PBMCs and PBMCs subjected to enrichment for CD8+ cells from control patients. RNA from these cells (naïve or activated with phytohemagglutinin) was extracted for target preparation and hybridization onto Affymetrix microarrays.