biostudies-arrayexpressCarsten CarlbergHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-GEOD-28319Gene 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)biostudies-arrayexpressNucleic Acid Extraction - PMA-differentiated THP-1 cells (per condition nine 175 cm2-flasks, each 2x107 cells, density of 1x106 cells/ml) were treated for 60 min with 1 M-5M T09 or vehicle (DMSO) and nuclear proteins were cross-linked to DNA by adding formaldehyde directly to the medium to a final concentration of 1% following incubation for 10 min at room temperature on a rocking platform. Cross-linking was stopped by adding glycine to a final concentration of 0.15 M and incubating at room temperature for 10 min on a rocking platform. The cells were collected by centrifugation and washed twice with ice cold PBS (140 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8.1 mM Na2HPO4.2H2O). Nuclear extraction was performed by adding 500 M-5l PIPES buffer (5 mM Pipes pH 8.0, 0.85 mM KCl, 0.5% Nonidet P-40, protease inhibitors), incubating for 10 min on ice and removing cytoplasmatic components by centrifugation. Nuclear pellets were dissolved in 500 M-5l SDS lysis buffer (1% SDS, 10 mM EDTA, protease inhibitors, 50 mM Tris- HCl, pH 8.1) and incubated 10 min on ice. Lysates were sonicated at high power with 22x30 s pulses in a Bioruptor (Diagenode, LiM-CM-(ge, Belgium) to result in DNA fragments of 100 to 600 bp. Cellular debris were removed by centrifugation. Aliquots of 100 M-5l of the lysate were diluted 1:10 in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl, protease inhibitors, 16.7 mM Tris-HCl, pH 8.1) and 2 M-5g of anti-LXR antibody (Jakobsson et al. 2009) or non-specific anti-IgG rabbit (sc-2027, Santa Cruz Biotechnologies, Santa Cruz, CA, USA) were added and the samples were incubated for overnight at 4 M-0C on a rotating platform. The immunocomplexes were collected using 60 M-5l of BSA-coated protein A agarose beads slurry (Millipore) for 3 h at 4 M-0C with rotation. The beads were washed sequentially for 4 min in rotating platform with 1 ml of the following buffers: low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH 8.1), high sa lt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, 20 mM Tris-HCl, pH 8.1) and LiCl wash buffer (0.25 M LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1). Finally, the beads were washed twice with 1 ml TE buffer (1 mM EDTA, 10 mM Tris-HCl, pH 8.0) and the immune complexes were eluted twice using 250 M-5l elution buffer (1% SDS, 100 mM NaHCO3) for 15 min at room temperature with rotation. The supernatants were combined and the immune complexes were reverse cross-linked overnight at 65 M-0C in the presence of proteinase K (Fermentas) in a final concentration of 40 M-5g/ml. DNA was extracted with the ChIP DNA Clean & Concentrator Kit (Zymo Research Cooperation, HiSS Diagnostics, Freiburg, Germany) according to manufacturer's instructions and eluted in 40 M-5l nuclease-free H2O. The ChIP templates were sequenced using a Solexa Gene Analyzer II platform (Illumina) at 36 bp read length using standard manufacturer protocols at the Genomics Co re Facility in Heidelberg, Germany. The anti-LXR antibody used was established in: Jakobsson T, Venteclef N, Toresson G, Damdimopoulos AE, Ehrlund A, Lou X, Sanyal S, Steffensen KR, Gustafsson JA, Treuter E. 2009. GPS2 is required for cholesterol efflux by triggering histone demethylation, LXR recruitment, and coregulator assembly at the ABCG1 locus. Mol Cell 34: 510-518.Growth Protocol - THP-1 human monocytic leukemia cells were grown in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine, 0.1 mg/ml streptomycin and 100 U/ml penicillin and the cells were kept at 37 M-0C in a humidified 95% air/ 5% CO2 incubator.Nucleic Acid Extraction - Total RNA was extracted using the Invisorb Spin Cell RNA Mini Kit (Invitek GmbH, Berlin, Germany) and checked for RNA integrity using a Biorad Experion automated electrophoresis system (Biorad, Nazareth, Belgium).Labeling - Biotinylated cRNA were prepared with the Ambion MessageAmp kit for Illumina arraysSample Processing - For differentiation into macrophages the cells were incubated for 3 days with 20 nM PMA. Prior to stimulation with 1 M-5M of the synthetic LXR ligand T09 (Sigma-Aldrich) or vehicle (DMSO), final concentration 0.1%) the medium was replaced by RPMI 1640 supplemented with 5% lipid-depleted fetal calf serum, 2 mM L-glutamine, 0.1 mg/ml streptomycin and 100 U/ml penicillin and 20 nM PMA.Sample Processing - For differentiation into macrophages the cells were incubated for 3 days with 20 nM PMA. Prior to stimulation with 1 M-CM-^BM-5M of the synthetic LXR ligand T09 (Sigma-Aldrich) or vehicle (DMSO), final concentration 0.1%) the medium was replaced by RPMI 1640 supplemented with 5% lipid-depleted fetal calf serum, 2 mM L-glutamine, 0.1 mg/ml streptomycin and 100 U/ml penicillin and 20 nM PMA.Hybridization - Standard Illumina hybridization protocolGrowth Protocol - THP-1 human monocytic leukemia cells were grown in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine, 0.1 mg/ml streptomycin and 100 U/ml penicillin and the cells were kept at 37 M-CM-^BM-0C in a humidified 95% air/ 5% CO2 incubator.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesFeature Extraction - The data were normalized using VST transformation and RSN normalization used as standard approach for Illumina arrays with the R/lumi packageImage Adquisition - Standard Illumina scanning protocolAssay Data Transformation - ID_REF = <br>VALUE = RSN normalized signal intensity<br>Detection Pval =Feature Extraction - Alignment: Sequence reads were obtained and mapped to the human genome (hg19) using Bowtie. Peaks: Peak detection was performed with the Model-based Analysis of ChIP-Seq (MACS) algorithm (http://liulab.dfci.harvard.edu/MACS/)MetabolomicsUnknownTranscriptomicsGenomicsProteomics<h4>Background</h4>The liver X receptors (LXRs) are oxysterol sensing nuclear receptors with multiple effects on metabolism and immune cells. However, the complete genome-wide cistrome of LXR in cells of human origin has not yet been provided.<h4>Results</h4>We performed ChIP-seq in phorbol myristate acetate-differentiated THP-1 cells (macrophage-type) after stimulation with the potent synthetic LXR ligand T0901317 (T09). Microarray gene expression analysis was performed in the same cellular model. We identified 1357 genome-wide LXR locations (FDR < 1%), of which 526 were observed after T09 treatment. De novo analysis of LXR binding sequences identified a DR4-type element as the major motif. On mRNA level T09 up-regulated 1258 genes and repressed 455 genes. Our results show that LXR actions are focused on 112 genomic regions that contain up to 11 T09 target genes per region under the control of highly stringent LXR binding sites with individual constellations for each region. We could confirm that LXR controls lipid metabolism and transport and observed a strong association with apoptosis-related functions.<h4>Conclusions</h4>This first report on genome-wide binding of LXR in a human cell line provides new insights into the transcriptional network of LXR and its target genes with their link to physiological processes, such as apoptosis.The gene expression microarray and sequence data have been submitted collectively to the NCBI Gene Expression Omnibus http://www.ncbi.nlm.nih.gov/geo under accession number GSE28319.ChIP-seqHomo sapiensGenome-wide landscape of liver X receptor chromatin binding and gene regulation in human macrophages.Pehkonen P, Welter-Stahl L, Diwo J, RyynM-CM-$nen J, Wienecke-Baldacchino A, Heikkinen S, Treuter E, Steffensen KR, Carlberg CEckardt TreuterLynn Welter-StahlSami HeikkinenJanine DiwoCarsten CarlbergAnke Wiencke-BaldacchinoKnut SteffensenPetri PehkonenfalseGenome-wide landscape of liver X receptor chromatin binding and gene regulationGene 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)2012-02-02T00:00:00Z2023-08-16T23:05:31.369Z2022-03-03T15:26:03.832ZE-GEOD-28319GSE2831922292898SRP006202EFO_000269210.1186/1471-2164-13-50