Comment[ArrayExpressAccession] E-GEOD-45023 MAGE-TAB Version 1.1 Public Release Date 2013-04-18 Investigation Title Genome-wide maps of chromatin state for H3.3 tagged cells Comment[Submitted Name] Genome-wide maps of chromatin state for H3.3 tagged cells Experiment Description The HIRA chaperone complex, comprised of HIRA, UBN1 and CABIN1, collaborates with histone-binding protein ASF1a to incorporate histone variant H3.3 into chromatin in a DNA replication-independent manner. To better understand its function and mechanism, we integrated HIRA, UBN1, ASF1a and histone H3.3 ChIP-seq and gene expression analyses. Most HIRA-binding sites co-localize with UBN1, ASF1a and H3.3 at active promoters and active and weak/poised enhancers. At promoters, binding of HIRA/UBN1/ASF1a correlates with the level of gene expression. HIRA is required for deposition of histone H3.3 at its binding sites. There are marked differences in nucleosome and co-regulator composition at different classes of HIRA-bound regulatory site. Underscoring this, we report novel physical interactions between the HIRA complex and transcription factors, a chromatin insulator and an ATP-dependent chromatin-remodelling complex. Our results map the distribution of the HIRA chaperone across the chromatin landscape and point to different interacting partners at functionally distinct regulatory sites. Examination of H3.3 histone modification in HeLA cells with accompanying FAIRE data 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 McBryan Pchelintsev McBryan Adams Person First Name Tony Nikolay Tony Peter Person Mid Initials A D Person Email tony@mcbryan.co.uk Person Affiliation University of Glasgow, Beatson Institute for Cancer Research Person Address University of Glasgow, Beatson Institute for Cancer Research, Switchback Rd, Bearsden, Glasgow, United Kingdom Person Roles submitter Protocol Name P-GSE45023-4 P-GSE45023-5 P-GSE45023-1 P-GSE45023-3 P-GSE45023-2 Protocol Description Basecalls performed using CASAVA 1.8 Sequenced reads were mapped to hg18 whole genome using bowtie v0.12.7 with parameter -p 8 -m 1 --best and remaining default settings Aligned reads normalised by library size, extended to 150bp and converted to bigWig format USeq 8.1.6 was used to perform peak finding with the following steps. BED files converted to Useq Point Format (Tag2Point). Satellite Repeat regions were removed from the dataset (FilterPointData). ScanSeqs used to identify enriched windows (-w 200 -p 150 -f). Enriched Regions Extracted (EnrichedRegionMaker) with 5% FDR and 2 fold change (-i 2,4 -s 13,1) Genome_build: hg18 Supplementary_files_format_and_content: bed files represent aligned reads (one line per read) in UCSC bed format. bigWig files represent pileups of the reads at each base. bigWig files were normalised by library size and represent the number of reads at a given genomic location per million mapped reads and generated using UCSC wigToBigWig. peak files were generated with USeq and thresholded at 5% FDR and 2 fold change cutoffs Library strategy: FAIRE-Seq Basecalls performed using CASAVA 1.8 Sequenced reads were mapped to hg18 whole genome using bowtie v0.12.7 with parameter -p 8 -m 1 --best and remaining default settings Aligned reads normalised by library size, extended to 150bp and converted to bigWig format USeq 8.1.6 was used to perform peak finding with the following steps. BED files converted to Useq Point Format (Tag2Point). Satellite Repeat regions were removed from the dataset (FilterPointData). ScanSeqs used to identify enriched windows (-w 200 -p 150 -f). Enriched Regions Extracted (EnrichedRegionMaker) with 5% FDR and 2 fold change (-i 2,4 -s 13,1) Genome_build: hg18 Supplementary_files_format_and_content: bed files represent aligned reads (one line per read) in UCSC bed format. bigWig files represent pileups of the reads at each base. bigWig files were normalised by library size and represent the number of reads at a given genomic location per million mapped reads and generated using UCSC wigToBigWig. peak files were generated with USeq and thresholded at 5% FDR and 2 fold change cutoffs Cells were harvested by trypsinization and precipitated by centrifugation. Cells pellets were resuspended in 1% formaldehyde in PBS and incubated for 15 more minutes. Cross-linking was quenched by adding 1/10 of volume of 1.25 M glycine (125mM final) and incubating for 5 min at room temperature. Media was aspirated and cells were rinsed twice with PBS, scraped into ice-cold PBS supplemented with protease inhibitors (Sigma, P8340) and collected by centrifugation (5 min at 200g, 4M-0C). Cell pellets were snap frozen in dry ice with ethanol and kept at -70M-0C before use. Cross-linked cells were solubilized by ultrasound treatment (Bioruptor Diagenode) to generate DNA fragmentation in 150-300 bp range. Protein-DNA complexes were isolated using corresponding antibody pre-immobilized on Dynabeads (Invitrogen). Libraries were prepared according to NEB's instructions accompanying the NEBNextM-. ChIP-Seq Library Prep Master Mix Set for IlluminaM-.(E6240S). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 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 3M-bM-^@M-^Y end. After adapter ligation library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were isolated using Agencourt AMPure XP beads (Beckman Coulter) and PCR-amplified using Illumina primers. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols. HeLa cells were cultured in DMEM supplemented with 10% FBS, 2 mM L-Glutamine, 50 U Pen-Strep and incubated at 37M-0C in a humidified 5% CO2 atmosphere. Every 3-4 days the cells were passaged to maintain 40-90% confluence level Protocol Type normalization data transformation protocol normalization data transformation protocol sample treatment protocol nucleic acid library construction protocol growth protocol Experimental Factor Name CHIP ANTIBODY Experimental Factor Type chip antibody Comment[SecondaryAccession] GSE45023 Comment[GEOReleaseDate] 2013-04-18 Comment[ArrayExpressSubmissionDate] 2013-03-11 Comment[GEOLastUpdateDate] 2013-04-19 Comment[AEExperimentType] ChIP-seq Comment[AEExperimentType] other Comment[SecondaryAccession] SRP019254 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR771824-SRR771826 SDRF File E-GEOD-45023.sdrf.txt