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Formaldehyde-assisted isolation of regulatory elements with next generation sequencing (FAIRE-seq) in primary human macrophages and foam cells

ABSTRACT: We report the open chromatin landscape in primary human macrophages and foam cells using FAIRE-seq CD14+ monocytes were isolated from the blood of 3 healthy volunteers. Monocytes were differentiated into macrophages by culture for 7 days with 50ng/ml macrophage colony stimulating factor and then treated for 48 hours with either oxidized low density lipoprotein (oxLDL) to induce foam cell formation or with a control buffer that lacked oxLDL. The resulting six samples were then subjected to FAIRE-seq using an established protocol (Simon JM, Giresi PG, Davis IJ, Lieb JD. Using formaldehyde-assisted isolation of regulatory elements (FAIRE) to isolate active regulatory DNA. Nature protocols 2012;7:256-67).

ORGANISM(S): Homo sapiens

SUBMITTER: Christopher O'Callaghan

PROVIDER: E-GEOD-54971 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Lipid-induced epigenomic changes in human macrophages identify a coronary artery disease-associated variant that regulates PPAP2B Expression through Altered C/EBP-beta binding.

Reschen Michael E ME Gaulton Kyle J KJ Lin Da D Soilleux Elizabeth J EJ Morris Andrew J AJ Smyth Susan S SS O'Callaghan Christopher A CA

PLoS genetics 20150402 4

Genome-wide association studies (GWAS) have identified over 40 loci that affect risk of coronary artery disease (CAD) and the causal mechanisms at the majority of loci are unknown. Recent studies have suggested that many causal GWAS variants influence disease through altered transcriptional regulation in disease-relevant cell types. We explored changes in transcriptional regulation during a key pathophysiological event in CAD, the environmental lipid-induced transformation of macrophages to lipi ...[more]

PMID: 25835000

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Chromatin immunoprecipitation with next generation sequencing (ChIP-seq) of H3K27ac in primary human macrophages and foam cells

Project description:We report the enhancer landscape in primary human macrophages and foam cells using ChIP-seq for the H3K27ac histone mark CD14+ monocytes were isolated from the blood of 2 healthy male volunteers. Monocytes were differentiated into macrophages by culture for 7 days with 50ng/ml macrophage colony stimulating factor and then treated for 48 hours with either oxidized low density lipoprotein (oxLDL) to induce foam cell formation or with a control buffer that lacked oxLDL. The resulting 4 samples were then subjected to ChIP-seq for H3K27ac.

2015-04-08 | E-GEOD-54972 | biostudies-arrayexpress

A study of the effect of OxLDL on C/EBP beta binding in human macrophages using chromatin immunoprecipitation for C/EBP beta with high throughput sequencing (ChIP-seq)

Project description:We report the C/EBP beta transcription factor binding landscape in human macrophages and oxLDL-induced foam cells ChIP-seq for C/EBP beta binding was performed in human macrophages and oxLDL induced-foam cells with quadruple biological replicates

2015-04-08 | E-GEOD-66192 | biostudies-arrayexpress

Proteomic analysis of S-nitrosylation in HUVECs

Project description:HUVECs stimulated with HG+oxLDL or Mannitol+nLDL were subjected to the biotin-switch assay to enrich the S-nitrosylated proteins, the S-nitrosylated proteins were resolved by SDS-PAGE and gels were silver stained. Mass spectrometry was conducted to determine proteins with S-nitrosylation.

2021-06-03 | PXD025295 | Pride

Role of Tet1 and 5-hydroxymethylcytosine in cocaine action (5hmC-Seq and ChIP-Seq)

Project description:Here we show that Tet1 is down-regulated in mouse nucleus accumbens (NAc), a key brain reward structure, by repeated cocaine administration which enhances behavioral responses to cocaine. Through genome-wide 5hmC profiling, we identified 5hmC changes selectively clustered in both enhancer and coding regions of genes with several annotated neural functions. By coupling with mRNA sequencing, we found cocaine-induced alterations in 5hmC correlate positively with alternative splicing. We also demonstrated that 5hmC alteration at certain genes lasts up to a month after cocaine exposure. DNA Nac samples were collected at various time points after 7 daily cocaoine ip administration for 5hmC and transcriptome analysis

2015-02-23 | E-GEOD-63747 | biostudies-arrayexpress

Human endothelial gene expression under estradiol and oxLDL treatments

Project description:DNA microarrays were used to investigate global gene expression patterns in cultured human umbilical artery endothelial cells (HUAECs) exposed to 1 nmol/L estradiol and/or 100 µg/ml oxidized low density lipoprotein (oxLDL) for 24 hours compared to control cells. HUAECs from 15 separate cultures were exposed to control (0.1% ethanol), 1nmol/L estradiol, 100 µg/ml oxLDL, or 1nmol/L estradiol + 100 µg/ml oxLDL treatments for 24 h. Total cellular RNA was extracted. Equal amounts of RNA extracted from 3 control cells or 3 estradiol-treated cells obtained from three different cultures were pooled, achieving five biological replicates of the control, five replicates that were treated with estradiol, five replicates that were treated with oxLDL and five replicates that were treated with estradiol+oxLDL . Therefore, a total number of 20 microarrays were developed.

2011-06-10 | E-GEOD-29881 | biostudies-arrayexpress

Gene expression in primary human macrophages and foam cells

Project description:The aim of the experiment was to determine the effects of 48 hours of treatment with oxidized low density lipoprotein (oxLDL) on gene expression in primary human monocyte-derived macrophages. 6 independent biological replicates were processed. From each replicate half of the cells were treated with oxLDL and the other half were treated with the control buffer. For foam cell formation (after 7 days) macrophages were treated with oxLDL (50mcg/ml, enodtoxin free, Copper oxidized). Control macrophages were treated with a matching buffer without oxLDL. Treatment duration - 48 hours.

2015-04-08 | E-GEOD-54666 | biostudies-arrayexpress

ATRX tolerates activity-dependent histone H3 M-bM-^@M-^\methyl/phos switchingM-bM-^@M-^] to maintain repetitive element silencing in neurons

Project description:ATRX is a member of the SWI2/SNF2 family of chromatin remodeling proteins and primarily functions at heterochromatic loci via its recognition of M-bM-^@M-^XrepressiveM-bM-^@M-^Y histone modifications (e.g., H3K9me3). Despite significant roles for ATRX during normal neural development, as well as its relationship to human disease, ATRX function in the central nervous system is not well understood. Here, we describe ATRXM-bM-^@M-^Ys ability to recognize an activity-dependent combinatorial histone modification, H3K9me3S10ph, in post-mitotic neurons. In neurons, this M-bM-^@M-^\methyl/phosM-bM-^@M-^] switch occurs exclusively following periods of stimulation and is highly enriched at heterochromatic repeats associated with centromeres. Using a multifaceted approach, we reveal that H3K9me3S10ph bound Atrx represses non-coding transcription of centromeric minor satellite sequences during instances of heightened activity. Our results indicate an essential interaction between ATRX and a previously uncharacterized histone modification in the central nervous system and suggest a potential role for abnormal repetitive element transcription in pathological states manifested by ATRX dysfunction. For Atrx ChIP-seq, IPs were performed on three control vs. three KCl stimulated (all representing biological, and not technical replicates) primary cultured mouse cortical neurons at DIV 8. All samples were normalized to background input levels. For H3K9me3S10phos ChIP-seq, biological singlecates (control vs. forskolin) were analyzed against respective inputs.

2015-01-02 | E-GEOD-64062 | biostudies-arrayexpress

TE-associated chromatin variation in mice livers

Project description:Chromatin accessibility is a hallmark of active regulatory function in the genome and variation of chromatin accessibility across individuals has been shown to contribute to complex traits and disease susceptibility. However, the mechanisms responsible for chromatin variation among different individuals and how this variation contributes to phenotypic diversity remain poorly understood. We examined chromatin accessibility variation in liver tissue from seven strains of adult mice that have phenotypic diversity in response to a high-fat/high-sucrose diet. Remarkably, nearly 40% of the loci with the greatest degree of chromatin variability across the strains are associated with transposable elements (TEs), with evolutionarily younger TEs being particularly enriched for regions of chromatin variation. We found that evolutionary younger and older TEs have differential chromatin accessibility profiles and are enriched for binding sites of different transcription factors, indicating the role of TEs in the evolution of regulatory networks in the liver. We also demonstrate that TE polymorphisms and epigenetic regulation of TEs contribute to regulatory variation across different strains through providing binding sites for liver transcription factors. Intriguingly, variable chromatin loci that are associated with liver metabolism are primarily TE-associated. We demonstrate that TEs contribute to regulatory variation in liver and have downstream effects on metabolism. Our data reveal TEs as a novel and important contributor to regulatory and phenotypic variation in the liver and suggest that regulatory variation at TEs is a major contributor to phenotypic variation in populations. Examination of chromatin accessibility with FAIRE-seq in livers of male mice (A/J, AKR/J, BALB/cJ, C57BL/6J, C3H/HeJ, CBA/J, DBA/2J, BXH2/TyJ, and BXH19/TyJ) fed a high-fat, high-sucrose diet.

2016-07-14 | E-GEOD-75770 | biostudies-arrayexpress

Monocyte to macrophage differentiation and loading/deloading with lipoproteins (eLDL, oxLDL, HDL3)

Project description:Elutriated monocytes from 9 healthy donors with ApoE3/E3 genotype were differentiated for up to six days in vitro to macrophages using rhMCSF under serum-free conditions. Samples were taken on days 1, 4, 5 and 6 and analyzed for their transcriptomic profiles. After four days of differentiation cells were loaded for 24 hours with enzymatically modified LDL (eLDL, 40 µg/ml) or mildly oxidized LDL (oxLDL, 80 µg/ml). Finally on day five, lipid deloading was performed in serum free medium containing M-CSF and cells were incubated another 24 h with HDL3 (100 µg/mL).

2014-03-02 | E-MTAB-2298 | biostudies-arrayexpress

Transcriptional study of oxLDL-induced foam cell formation in WT and Atf3-/- murine bone marrow macrophages

Project description:Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipid-loaded macrophages in the arterial wall. Intimal macrophages internalize modified lipoproteins such as oxidized LDL (oxLDL) through scavenger receptors, leading to storage of excess cholesteryl esters in lipid bodies and a "foam cell" phenotype. In addition, stimulation of macrophage Toll-like receptors (TLRs) has been shown to promote lipid body proliferation. We investigated the possibility that there are transcriptional regulators that are common to both pathways for stimulating foam cell formation (modified lipoproteins and TLR stimulation), and identified the transcription factor ATF3 as a candidate regulator. In this specific microarray experiment, we studied the effect of genetic knockout of ATF3 on the transcriptional response of macrophages to oxLDL. Murine bone marrow-derived macrophages from two different mouse strains (Atf3-/- and WT) were incubated in the presence or absence of oxidized low-density lipoprotein (oxLDL), and then transcriptionally profiled using the Affymetrix Mouse Exon Array 1.0 ST. The goal was to study the pattern of differential expression between WT and Atf3-/- strains, in oxLDL-induced foam cells and in non-foamy control cells, to identify cellular pathways that may be dysregulated under loss of the transcription factor ATF3 in macrophage foam cells. Twelve female mice (six Atf3-/-, and six WT control mice) were sacrificed at 8-12 weeks of age, and macrophages were derived from the femoral bone marrow using rhM-CSF. oxLDL was introduced into the medium for six samples (3 WT and 3 Atf3-/-) at 25 ug/mL on day seven, and after 24 h of incubation, RNA was isolated using Trizol. Labeled cRNA derived from the RNA samples was hybridized to Affymetrix Mouse Exon Array 1.0 ST GeneChips. Microarray data were processed using transcript-level probesets, and are in log2 scale.

2012-03-23 | E-GEOD-32358 | biostudies-arrayexpress

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