Comment[ArrayExpressAccession] E-GEOD-43036 MAGE-TAB Version 1.1 Public Release Date 2013-09-20 Investigation Title Synergistic Activation of Inflammatory Cytokine Genes by Priming of Regulatory DNA Elements for Increased Transcription in Response to TLR Signaling Comment[Submitted Name] Synergistic Activation of Inflammatory Cytokine Genes by Priming of Regulatory DNA Elements for Increased Transcription in Response to TLR Signaling Experiment Description Synergistic activation of inflammatory cytokine genes by IFN-gamma and TLR signaling is important for innate immunity and inflammatory disease pathogenesis, but underlying mechanisms are not known. By obtaining over three billion bases of sequence from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of human primary monocytes under IFN-gamma-priming and TLR stimulation. We found that IFN-gamma induced genome-wide sustained occupancy of STAT1, IRF-1 and associated histone acetylation at TSS-proximal and distal regulatory elements and provided a synergy mechanism whereby IFN-gamma creates a primed chromatin environment to augment TLR-induced gene transcription, which suggest therapeutic approaches that selectively target priming mechanisms. Examination and comparison of the changes in TF binding and histone modification in human primary monocytes under different conditions. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Ivashkiv Qiao Li Giannopoulou Person First Name Lionel Yu Yushan Eugenia Person Email geo@ncbi.nlm.nih.gov Person Affiliation Hospital for Special Surgery Person Address Hospital for Special Surgery, 535 East 70th Street, New York, NY, USA Person Roles submitter Protocol Name P-GSE43036-4 P-GSE43036-1 P-GSE43036-3 P-GSE43036-2 Protocol Description Basecalls performed using CASAVA version 1.7 ChIP-seq reads were aligned to the hg19 genome assembly using BOWTIE with default configurations. peaks were called using ChIPSeeqer with the following setting: STAT1 -t_15 -t_fold 2; H3K27Ac -t_20 -t_fold 3 ; IRF1 -t_15 -t_fold 2; Genome_build: hg19 Supplementary_files_format_and_content: wig files were generated using ChIPSeeqer The headers for peak *txt files are as follows (from the left): Chromosome : chromosome name Start_Position : the first (genomic) coordinate of the peak End_Position : the second (genomic) coordinate of the peak Avg_p-value : average log p-value of the nucleotides in the normalized peak region Score : score estimated as the average ChIP reads/length of peak (minus the average INPUT reads/length of peak - if INPUT is available) Posmaxpeakheight : the position of the maximum height of the peak Maxpeakheight : the maximum peak height (in reads) RelPosMaxPeakHeight(%) : the relative position of the maximum height of the peak, e.g., 50% means the highest point is at the middle of the peak Peak_Size : the size of the peak (in bp) Mid_point : the middle position of the peak Summit_dist_from_mid : the distance of the maximum height from the middle of the peak Cells were treated with or without IFN-g (100U/ml) for 24 hours, and then stimulated with LPS (50ng/ml) for 3 or 6 hours as indicated. Sonicated cell lysates of nuclei and histone-DNA complexes were isolated with antibody. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, Klenow polymerase and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3’ to 5’ exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was purified using MinElute PCR Purification kit and PCR amplified with Illumina primers for 15 cycles. Library fragments of 200-300 bp were band isolated from an 1.5% agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on an Illumina HiSeq 2000 Sequencer following the manufacturer's protocols. Peripheral blood mononuclear cells were obtained from the blood of healthy donors by density gradient centrifugation using Ficoll (Invitrogen) and a protocol approved by the Hospital for Special Surgery Institutional Review Board. CD14+ human monocytes were purified from PBMCs by positive selection using anti-CD14 magnetic beads as recommended by the manufacturer (Miltenyi Biotec). Monocytes were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen) supplemented with 10% heat-inactivated defined FBS (HyClone), penicillin/streptomycin (Invitrogen), L-glutamine (Invitrogen), and 10 ng/mL human macrophage colony-stimulating factor (M-CSF; Peprotech). Protocol Type normalization data transformation protocol sample treatment protocol nucleic acid library construction protocol growth protocol Experimental Factor Name EXPERIMENTAL CONDITION CHIP ANTIBODY Experimental Factor Type experimental condition chip antibody PubMed ID 24012417 Comment[SecondaryAccession] GSE43036 Comment[GEOReleaseDate] 2013-09-20 Comment[ArrayExpressSubmissionDate] 2012-12-19 Comment[GEOLastUpdateDate] 2013-09-20 Comment[AEExperimentType] ChIP-seq Comment[SecondaryAccession] SRP017687 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR640563-SRR640565 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR640567-SRR640580 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR640582-SRR640583 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR642088-SRR642089 SDRF File E-GEOD-43036.sdrf.txt