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RNA sequencing in primary inflammatory (TPP) macrophages treated with a MEK1/2 inhibitor

ABSTRACT: By investigating an intergenic haplotype on chr21q22, independently linked to inflammatory bowel disease (IBD), ankylosing spondylitis, primary sclerosing cholangitis and TakayasuÃ¢Â€Â™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.

PROVIDER: EGAS00001007552 | EGA |

REPOSITORIES: EGA

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RNA sequencing on intestinal biopsies from inflammatory bowel disease patients treated with vehicle control, MEK inhibitor, or infliximab

Project description:By investigating an intergenic haplotype on chr21q22, independently linked to inflammatory bowel disease (IBD), ankylosing spondylitis, primary sclerosing cholangitis and TakayasuÃ¢Â€Â™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

MPRA in primary human macrophages

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

H3K27ac ChIP-seq in primary inflammatory (TPP) macrophages

Project description:We investigated an intergenic haplotype on chr21q22, linked to five different inflammatory diseases, and identified the molecular mechanism by which the risk haplotype increases expression of the causal gene, ETS2. This mechanism was predicted to alter enhancer activity at the disease-associated locus. We therefore performed H3K27ac ChIP-seq in inflammatory macrophages from minor and major allele homozygotes at the causal allele and investigated the effect on enhancer activity. In doing so, we mechanistically delineated how the risk haplotype increases expression of ETS2.

| EGAS00001007562 | EGA

Determining genome-wide binding of ETS2 by CUT&RUN in primary human macrophages

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.

| EGAS00001007560 | EGA

b9c4ae4a-8567-41c4-be0f-23f8f14b4c99 - samples

Project description:This dataset comprises raw RNA sequencing data (FASTQs) from primary inflammatory (TPP) macrophages that were unedited (non-targeting control, NTC), edited to delete the disease-associated chr21q22 enhancer region (n=5), or edited to disrupt ETS2 with 1 of 2 independent gRNAs (n=9). We also performed RNA-sequencing in NTC or ETS2-edited TPP macrophages that were treated for 12 hours with vehicle (DMSO) or roxadustat (30 uM, n=3). Macrophages were detached using Accutase and lysed. RNA was extracted from cell lysates using an AllPrep DNA/RNA Mini Kit (Qiagen). Sequencing libraries were prepared from 10ng RNA using the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara) following the manufacturerÃ¢Â€Â™s instructions. The quality and molarity of all libraries was assessed using a BioAnalyzer 2100 and the libraries were sequenced on a NextSeq500. Raw data are provided as 50 bp paired-end Illumina reads.

| EGAD00001011338 | EGA

RNA sequencing in primary inflammatory (TPP) macrophages following deletion of a disease-associated gene desert at chr21q22, disruption of ETS2, or treatment of ETS2-edited macrophages with a HIF1ÃŽÂ± stabiliser.

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 HIF1ÃŽÂ± stabiliser that can promote glycolysis via HIF1ÃŽÂ±-mediated metabolic reprogramming, and performed RNA-sequencing to determine whether this drug might rescue the transcriptional effects of ETS2 disruption.

| EGAS00001007553 | EGA

An Ets2-specific transcriptional program in tumor-associated macrophages promotes metastasis

Project description:Macrophages have been implicated in breast cancer progression and metastasis, but relatively little is known about the genes and pathways that are involved. Using a conditional allele of Ets2 in the mouse, we have identified Ets2 as a critical gene in tumor associated macrophages (TAMs) that specifically promotes mammary tumor metastasis. Loss of Ets2 in TAMs decreased the frequency and size of lung metastases without impacting primary tumor burden. Expression profiling of isolated tumor macrophages established that Ets2 deficiency resulted in the de-repression of a defined set of anti-angiogenic genes. Activation of this transcriptional program correlated with decreased angiogenesis in metastatic tumors and decreased metastatic growth. Comparison of this Ets2-specific TAM expression profile with human breast cancer profiles revealed a macrophage gene expression signature that could predict overall survival of estrogen receptor negative patients. In summary, we have identified a critical factor, Ets2, in TAMs that represses a transcriptional program to promote the growth of mammary tumor metastases in the lung. Breast TAMs were isolated from early-stage PyMT-induced mammary tumors expressing Ets2 and also from the tumors with Ets2-deficient TAMs. Since macrophages have also been implicated in normal mammary gland remodeling, normal remeodeling macrophages were also purified from females expressing Ets2 and the ones where Ets2 is deleted in the macrophages. One RNA sample was extracted from each genetic group for gene-expression profiling.

2010-01-05 | E-GEOD-12863 | biostudies-arrayexpress

f5cefdab-08bc-4954-bf36-bd12be05c89d - samples

Project description:This dataset consists of sequencing data from an ETS2 CUT&RUN experiment in primary inflammatory (TPP) macrophages (n = 2). Pre-cultured TPP macrophages were harvested and processed immediately using the CUT&RUN Assay kit (Cell Signaling) according to the manufacturerÃ¢Â€Â™s instructions with the following modifications (essentially, avoiding the use of ConA-coated beads). Anti-ETS2 (ThermoFisher) or IgG control (Cell Signaling) antibodies were used for targeted digestion of chromatin. For each donor, 5x10^5 cells were pelleted, washed in Wash Buffer, and resuspended in Antibody Binding buffer. Cells were incubated with antibodies (1:100 dilution for anti-ETS2) for 2h at 4Ã‚Â°C. After washing in Digitonin Buffer, cells were incubated with pA/G-MNase for 1h at 4Ã‚Â°C. Cells were washed twice in Digitonin Buffer, resuspended in the same buffer and cooled for 5 minutes on ice. Calcium chloride was added to activate pA/G-MNase digestion and cells were incubated for 30 minutes at 4Ã‚Â°C before Stop Buffer was added, and cells were incubated for 10 min at 37Ã‚Â°C to release cleaved chromatin fragments. Supernatants were collected by centrifugation and DNA extracted using DNA Purification Buffers and Spin Columns (Cell Signaling). 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 on an Illumina NovaSeq 6000 (100bp PE reads). Raw data are provided as 100 bp paired-end Illumina reads.

| EGAD00001011349 | EGA

An Ets2-specific transcriptional program in tumor-associated macrophages promotes metastasis

2010-01-05 | GSE12863 | GEO

RNA-seq of prostate cancer cell lines with acquired resistance to AKT inhibitors GDC-0068 (ATP competitive) or MK206 (allosteric)

Project description:Goal: Study resistance mechanisms to ATP-competitive vs. allosteric AKT inhibitors, in prostate cancer Methods: RNA-sequencing Results: Resistance to the ATP-competitive inhibitor GDC-0068 was driven by compensatory activity of parallel signaling pathways

2020-01-24 | GSE139178 | GEO

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