Project description:We performed ChIP-seq in macrophage-type PMA-differentiated THP-1 cells after stimulation with the the natural VDR ligand 1,25dihydroxyvitamin D3 (1,25D). We identified in total 223 VDR binding locations on chromatin after 1,25D treatment. De novo analysis of VDR site sequences identified DR3-type binding sites as major motif.
Project description:Human monocyte THP-1 cells obtained from ATCC were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA) containing 10% FBS and supplemented with 10 mM Hepes (Gibco BRL). THP-1 was differentiated into macrophages by 24-h incubation with 160 nM phorbol 12-myristate 13-acetate (PMA; Sigma, St. Louis, MO) followed by 24-h incubation in RPMI medium. Macrophages were further polarized to M1 macrophages by incubation with 10 pg/ml of lipopolysaccharide (LPS; Sigma) and 20 ng/ml of interferon (IFN)-γ (R&D Systems, MN) and are referred to as M(LPS+IFN-γ) cells. M2 macrophages were obtained by incubation with 20 ng/ml of interleukin (IL)-4 (R&D Systems) and are referred to as M(IL4) cells. To test the represented polarization marker of PMA differentiated-THP-1 macrophages stimulated with 20 ng ml(-1) IFNγ + 10 pg ml(-1) LPS and 20 ng ml(-1) IL-4, which are known to influence macrophage polarization in vetro into the M1 and M2 state, respectively. We used microarrays to detail the gene expression pools to identify distinct M1 and M2 state during this process.
Project description:The genome-wide analysis of the binding sites of the transcription factor vitamin D receptor (VDR) is essential for a global appreciation the physiological impact of the nuclear hormone 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). Genome-wide analysis of lipopolysaccharide (LPS)-polarized THP-1 human monocytic leukemia cells via chromatin immunoprecipitation (ChIP) coupled with massive parallel sequencing (ChIP-seq) resulted in 1,318 high-confidence VDR binding sites, of which 789 and 364 occurred uniquely with and without 1,25(OH)2D3 stimulation, while only 165 were common. We re-analyzed five public VDR ChIP-seq datasets with identical peak calling settings (MACS, version 2) and found in total 23,409 non-overlapping VDR binding sites, 75% of which are unique within the six analyzed cellular models. LPS-differentiated THP-1 cells have 22% more genomic VDR locations than undifferentiated cells and both cell types display more overlap in their VDR locations than the other investigated cell types. In general, the intersection of VDR binding profiles of ligand-stimulated cells is higher than those of unstimulated cells. De novo binding site searches and DR3-type binding site screening using HOMER of the six VDR ChIP-seq datasets suggest that DR3 sites are strongly associated with the ligand-responsiveness of VDR occupation. Importantly, all VDR ChIP-seq datasets display the same relationship between the VDR occupancy and the percentage of DR3-type sequences below the peak summits. The comparative analysis of six VDR ChIP-seq datasets demonstrated that the mechanistic basis for the action of the VDR is independent of the cell type. Only the minority of genome-wide VDR binding sites contains a DR3-type sequence. Moreover, the total number of identified VDR binding sites in each ligand-stimulated cell line inversely correlates with the percentage of peak summits with DR3 sites.
Project description:The genome-wide analysis of the binding sites of the transcription factor vitamin D receptor (VDR) is essential for a global appreciation the physiological impact of the nuclear hormone 1M-NM-1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Genome-wide analysis of lipopolysaccharide (LPS)-polarized THP-1 human monocytic leukemia cells via chromatin immunoprecipitation (ChIP) coupled with massive parallel sequencing (ChIP-seq) resulted in 1,318 high-confidence VDR binding sites, of which 789 and 364 occurred uniquely with and without 1,25(OH)2D3 stimulation, while only 165 were common. We re-analyzed five public VDR ChIP-seq datasets with identical peak calling settings (MACS, version 2) and found in total 23,409 non-overlapping VDR binding sites, 75% of which are unique within the six analyzed cellular models. LPS-differentiated THP-1 cells have 22% more genomic VDR locations than undifferentiated cells and both cell types display more overlap in their VDR locations than the other investigated cell types. In general, the intersection of VDR binding profiles of ligand-stimulated cells is higher than those of unstimulated cells. De novo binding site searches and DR3-type binding site screening using HOMER of the six VDR ChIP-seq datasets suggest that DR3 sites are strongly associated with the ligand-responsiveness of VDR occupation. Importantly, all VDR ChIP-seq datasets display the same relationship between the VDR occupancy and the percentage of DR3-type sequences below the peak summits. The comparative analysis of six VDR ChIP-seq datasets demonstrated that the mechanistic basis for the action of the VDR is independent of the cell type. Only the minority of genome-wide VDR binding sites contains a DR3-type sequence. Moreover, the total number of identified VDR binding sites in each ligand-stimulated cell line inversely correlates with the percentage of peak summits with DR3 sites. Systematic reanalysis of 5 published VDR ChIP-seq datasets together with a new dataset from 24 h LPS-treated THP-1 cells at the unstimulated state and after 80 min ligand (10 nM 1M-NM-1,25(OH)2D3 (1,25D, calcitriol)) treatment. See GSM1280896 and GSM1280896 Sample records for data processing information. GSE53041_README.txt has additional details.
Project description:THP-1 cells were treated with phorbol 12-myristate 13-acetate (PMA) at 25μg/ml for 48h.Then remove PMA and rest for 24h to obtain M0 macropahges. Further applied LPS and IFNγ or IL-4 and IL-13 for 24h to obtain M1, M2 macrophages, respectively. During polarization, cells were treated with prostaglandin E2 or MEHP.Then M1,M2 cells were collected and applied to RNA-sequencing. We found that PGE2 and MEHP significantly impacted metabolic signature of macrophages.
Project description:THP-1 monocytes were differentiated, via PMA treatment, to macrophages. THP-1 macrophages were then treated with a mixed-oxysterol treatment (7-ketocholesterol and 7beta-hydroxycholesterol) for 24 hours. The mixed-oxysterol treatment was mixed to a ratio that represents the cytotoxic oxysterol content of human atherosclerotic plaque. Control and treated macrophages were analysed using both 2-DE with peptide mass-fingerprinting and tandem-mass spectrometry.
Project description:THP-1 cells were treated with phorbol 12-myristate 13-acetate (PMA) at 25μg/ml for 48h.Then remove PMA and rest for 24h to obtain M0 macropahges. Further applied LPS and IFNγ or IL-4 and IL-13 for 24h to obtain M1, M2 macrophages, respectively. During polarization, cells were growth within control medium(LN) or medium with extra lipid mixture(LH).M0,M1,M2 cells were collected and applied to RNA-sequencing. We found that differential activation of macrophage had distinct metabolic signature.Extra lipids supplement did not significantly alter transcriptomes of macrophages.
Project description:Gene expression: Identification of primary target genes of liver X receptor (LXR) in an immune-related cellular model (THP-1 cells) to study, in conjunction with LXR binding data from ChIP-seq, the genome-wide mechanisms of transcriptional regulation by LXR. ChIP-Seq: We performed ChIP-seq in macrophage-type PMA-differentiated THP-1 cells after stimulation with the potent synthetic LXR ligand T0901317 (T09). As a reference we performed microarray gene expression analysis in the same cellular model. We identified in total 1357 LXR binding locations on chromatin (FDR < 1%), of which 526 were observed after T09 treatment. De novo analysis of LXR site sequences identified DR4-type binding sites as major motif. gene expression: THP-1 cells were treated for 4 h with 1 M-BM-5M T09 or vehicle (DMSO) ChIP-Seq: PMA-differentiated THP-1 cells were treated for 60 min with 1 M-BM-5M T09 or vehicle (DMSO)