H3K27ac ChIP-seq in primary inflammatory (TPP) macrophages
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ABSTRACT:
PROVIDER: EGAS00001007562 | EGA |
REPOSITORIES: EGA
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Project description:We investigated an intergenic haplotype on chr21q22, linked to five different inflammatory diseases. We used a functional approach (massively-parallel reporter assay; MPRA) to first identify active enhancers at the locus in primary human macrophages, and then determine if candidate variants within these regulatory regions might alter enhancer activity. In doing so, we discovered a mechanism that orchestrates macrophage responses during chronic inflammation and delineated how the risk haplotype increases expression of the causal gene, ETS2.
2024-05-01 | GSE229472 | GEO
Project description:We investigated an intergenic haplotype on chr21q22, linked to five different inflammatory diseases, and discovered a mechanism that orchestrates macrophage responses during chronic inflammation. We delineated how the risk haplotype increases expression of the causal gene, ETS2, and demonstrated that ETS2 is necessary for inflammatory macrophage effector functions. To establish whether ETS2 is sufficient to drive inflammatory responses, we overexpressed ETS2 in a dose-dependent manner and performed RNA-sequencing to characterise the transcriptional effects.
| EGAS00001007554 | EGA
Project description:By investigating an intergenic haplotype on chr21q22, independently linked to inflammatory bowel disease (IBD), ankylosing spondylitis, primary sclerosing cholangitis and Takayasu’s arteritis, we discover that the causal gene, ETS2, is a master regulator of inflammatory responses in human macrophages and delineate how the risk haplotype increases ETS2 expression. Using a database of cellular signatures, we identified drugs that could modulate this pathway. To validate the anti-inflammatory activity of one class of small molecules in vitro, we treated TPP macrophages with vehicle control or a selective, non-ATP competitive MEK inhibitor (PD-0325901) at 2 doses (100nM and 500nM) and performed RNA sequencing.
| EGAS00001007552 | EGA
Project description:By investigating an intergenic haplotype on chr21q22, independently linked to inflammatory bowel disease (IBD), ankylosing spondylitis, primary sclerosing cholangitis and Takayasu’s arteritis, we discover that the causal gene, ETS2, is a master regulator of inflammatory responses in human macrophages and delineate how the risk haplotype increases ETS2 expression. Using a database of cellular signatures, we identified drugs that could modulate this pathway. To validate the anti-inflammatory activity of one class of small molecules ex vivo, we obtained biopsies from patients with active IBD and cultured these biopsies with a selective, non-ATP competitive MEK inhibitor (PD-0325901), infliximab (positive control), or DMSO (vehicle control) for 18 hours, then performed RNA sequencing.
| EGAS00001007534 | EGA
Project description:We investigated an intergenic haplotype on chr21q22 that is associated with five different inflammatory diseases and discovered a mechanism that orchestrates macrophage responses during chronic inflammation. We delineated how the risk haplotype increases expression of the causal gene, ETS2, and showed that the pathway regulated by ETS2 is both necessary and sufficient for human macrophage responses during chronic inflammation. To better characterise ETS2 binding across the genome - and thereby identify ETS2 target genes - we performed Cleavage-Under-Targets-and-Release-Using-Nuclease (CUT&RUN) in inflammatory (TPP) macrophages. Anti-ETS2 (ThermoFisher) or IgG control (Cell Signaling) antibodies were used for targeted digestion of chromatin. Library preparation was performed according to a protocols.IO protocol (dx.doi.org/10.17504/protocols.io.bagaibse) using the NEBNext Ultra II DNA Library Prep Kit. Size selection was performed using AMPure XP beads (Beckman Coulter) and fragment sizes were determined using an Agilent 2100 Bioanalyzer (High Sensitivity DNA kit). Equimolar pools of indexed libraries were sequenced. Raw data are provided as 100 bp paired-end Illumina reads from n = 2 donors.
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Project description:A major goal of genetics research is to elucidate mechanisms explaining how genetic variation contributes to phenotypic variation. The genetic variants identified in genome-wide association studies (GWASs) generally explain only a small proportion of heritability of phenotypic traits, the so-called missing heritability problem. Recent evidence suggests that additional common variants beyond lead GWAS variants contribute to phenotypic variation; however, their mechanistic underpinnings generally remain unexplored. Herein, we undertake a study of haplotype-specific mechanisms of gene regulation at 8p23.1 in the human genome, a region associated with a number of complex diseases. The FAM167A-BLK locus in this region has been consistently found in the genome-wide association studies (GWASs) of systemic lupus erythematosus (SLE) in all major ancestries. Our haplotype-specific chromatin interaction (Hi-C) experiments, allele-specific enhancer activity measurements, genetic analyses, and epigenome editing experiments revealed that: (1) haplotype-specific long-range chromatin interactions are prevalent in 8p23.1; (2) BLK promoter and cis-regulatory elements cooperatively interact with haplotype-specificity; (3) genetic variants at distal regulatory elements are allele-specific modifiers of the promoter variants at FAM167A-BLK; (4) the BLK promoter interacts with and, as an enhancer-like promoter, regulates FAM167A expression and (5) local allele-specific enhancer activities are influenced by global haplotype structure due to chromatin looping. Although SLE causal variants at the FAM167A-BLK locus are thought to reside in the BLK promoter region, our results reveal that genetic variants at distal regulatory elements modulate promoter activity, changing BLK and FAM167A gene expression and disease risk. Our results suggest that global haplotype-specific 3-dimensional chromatin looping architecture has a strong influence on local allelic BLK and FAM167A gene expression, providing mechanistic details for how regional variants controlling the BLK promoter may influence disease risk.
2022-09-07 | GSE211246 | GEO
Project description:A haplotype block at the sclerostin (SOST) gene correlates with bone mineral density (BMD) and increased periodontitis risk in smokers. Investigating the putative causal variants within this block, our study aimed to elucidate the impact of linked enhancer elements on gene expression and to evaluate their role on transcription factor (TF) binding. Using CRISPR/dCas9 activation (CRISPRa) screening in SaOS-2 cells, we quantified disease-related enhancer activities regulating SOST expression. Additionally, in SaOs2-cells, we investigated the influence of the candidate TFs CCAAT/enhancer-binding protein beta (CEBPB) on gene expression by antisense (GapmeR) knockdown, followed by RNA sequencing. The periodontitis-linked SNP rs9783823 displayed a significant cis-activating effect (25-fold change in SOST expression), with the C-allele containing a CEBPB binding motif (position weight matrix (PWM) = 0.98, Pcorrected = 7.7 x 10-7). CEBPB knockdown induced genome-wide upregulation but decreased epithelial-mesenchymal transition genes (P= 0.71, AUC = 2.2 x 10-11). This study identifies a robust SOST cis-activating element linked to BMD and periodontitis, carrying CEBPB binding sites and highlights CEBPB's impact on epithelial-mesenchymal transition.
2024-06-27 | GSE269019 | GEO
Project description:GWAS studies in five different inflammatory diseases have identified a strong genetic association at a gene desert at the chr21q22 locus. We have shown that this locus contains a monocyte/macrophage-specific enhancer that regulates ETS2 - a gene whose role in primary human monocytes/macrophages is incompletely understood. We therefore used a CRISPR-Cas9-based approach to delete the enhancer region or to disrupt ETS2, and performed RNA-sequencing to examine the transcriptional consequences. One effect of ETS2 disruption was upregulation of genes involved in aerobic respiration and oxidative phosphorylation. We therefore treated ETS2-edited inflammatory macrophages with roxadustat, a HIF1α stabiliser that can promote glycolysis via HIF1α-mediated metabolic reprogramming, and performed RNA-sequencing to determine whether this drug might rescue the transcriptional effects of ETS2 disruption.
| EGAS00001007553 | EGA
Project description:Genome-wide association studies (GWASs) identified the MEIS1 locus for Restless Legs Syndrome (RLS), but causal single nucleotide polymorphisms (SNPs) and the functional relevance have remained to be elucidated. The MEIS1 locus contains an exceptionally large number of highly conserved non-coding regions (HCNRs), which potentially function as cis-regulatory modules. We analyzed the HCNRs in the RLS-associated linkage disequilibrium (LD) block for allele-dependent enhancer activity in both zebrafish and mouse, comparing the protective and risk alleles of RLS-associated common variants. We found one enhancer, harboring the lead SNP rs12469063, which showed an allele-dependent reduction of reporter gene expression exclusively in the embryonic ganglionic eminences at developmental stage E12.5. Notably, the reporter activity overlapped with the endogenous telencephalic Meis1 expression domain, which co-localized with transcripts of all four validated RLS loci. Thus, the developing telencephalon represents the first neuroanatomic region implicated for RLS based on GWAS findings. Total RNA obtained from 2-3 male and female mice E12.5 (wildtype, heterzygote, homozygote) and 3-4 male heterozygote and wildtype mice (adult)
2014-01-29 | E-GEOD-44592 | biostudies-arrayexpress
Project description:Biomechanical cues dynamically control major cellular processes but whether genetic variants actively participate in the mechano-sensing mechanisms remain unexplored. Vascular homeostasis is tightly regulated by hemodynamic forces. Exposure to disturbed blood flow at arterial sites of branching and bifurcation causes constant activation of vascular endothelium contributing to the development of atherosclerosis, the major cause of coronary artery disease (CAD). Conversely, unidirectional flow promotes the anti-inflammatory and anti-permeable endothelial phenotype resistant to atherogenesis. Genome-wide association studies (GWAS) have identified chromosome 1p32.2 as one of the loci most strongly associated with CAD susceptibility; however, the causal mechanism related to this CAD locus remain unknown. PhosphoLipid PhosPhatase 3 (PLPP3) is located at 1p32.2 and encodes a phosphatase that suppresses endothelial inflammation and promotes monolayer integrity by hydrolyzing lysophosphatidic acid. Our previous studies demonstrated that PLPP3 is significantly reduced in vascular endothelium exposed to disturbed flow while unidirectional flow significantly increases endothelial PLPP3 expression in vitro and in vivo. In addition, CAD protective allele at 1p32.2 locus is associated with increased PLPP3 in an endothelium-specific manner, shown by expression quantitative trait locus (eQTL). Using Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq), H3K27ac ChIP-Seq, H3K4me2 ChIP-Seq, and luciferase assays, here we identified a mechano-sensitive endothelial enhancer in PLPP3 intron 5 that is dynamically activated by unidirectional flow. Deletion of this enhancer by CRISPR/Cas9-based genome editing causatively reduces endothelial PLPP3 expression and promotes endothelial activation, and moreover, impairs endothelial PLPP3 induction by unidirectional flow. Chromatin accessibility quantitative trait locus (caQTL) mapping, allelic imbalance assay, and luciferase assays further demonstrated that CAD protective allele at rs17114036 in the PLPP3 intron 5 confers an increased enhancer activity. ChIP-PCR and luciferase assays showed that CAD protective allele C at rs17114036 creates a binding site (CACC) for mechano-sensitive transcription factor KLF2, leading to increased enhancer activity under unidirectional flow. These results elucidate the contributory role of CAD genetic predisposition in critical endothelial mechano-transduction mechanisms and suggest that human genetic variants provide a previously unappreciated layer of regulatory control in cellular mechano-sensing mechanisms.
2018-11-19 | GSE112336 | GEO