Computational protocol: BRD4 is a Histone Acetyltransferase that Evicts Nucleosomes from Chromatin

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Protocol publication

[…] Human histone H3.3 was subjected to HAT assays with p300, BRD4 or no enzyme (control). The acetylated histone bands were excised from a coommassie blue stained SDS-PAGE gel and subjected to in-gel tryptic digestion at 37°C overnight. Digested peptide samples were desalted by C18 ZipTip (Millipore), lyophilized and re-suspended in 16 μl of 0.1% formic acid for LC-MS analysis. Sample (6 μl) were loaded on an Easy LC 1000 nano-capillary HPLC system (Thermo Scientific) with a 15 cm Acclaim® PepMap™ RSLC C18 column (Thermo Scientific) connected to an electrospray ionization (ESI) emitter, coupled online with a dual-pressure linear ion trap (LTQ Velos Pro) mass spectrometer (Thermo Scientific) for μRPLC-MS/MS analysis. Peptides were eluted using a linear gradient of 2% mobile phase B (acetonitrile with 0.1% formiac acid) to 42% mobile phase B within 45 min at a constant flow rate of 200 nL/min. The fifteen most intense molecular ions in the MS scan were sequentially selected for collision-induced dissociation (CID) using a normalized collision energy of 35%. The mass spectra were acquired at the mass range of m/z 380–2000. The Easy Nano Spray ion source (Thermo Scientific) capillary voltage and temperature were set at 1.7 kV and 275 °C, respectively. The dynamic exclusion function on the mass spectrometer was enabled during the MS2 data acquisition. The MS/MS data were searched against the human histone H3.3 protein sequence using Proteome Discoverer 1.4 interfaced with SEQUEST HT (Thermo Scientific). Lysine acetylation (42.01 Da) and methionine oxidation (15.99 Da), were set as dynamic modifications. Up to two missed tryptic cleavage sites was allowed during the database search. The cut-off for legitimate identifications were: charge state dependent cross correlation (Xcorr) ≥ 2.0 for [M+H]1+, ≥ 2.5 for [M+2H]2+ and ≥ 3.0 for [M+3H]3+. The results were normalized by deducting the acetylated lysines detected in the no-enzyme control sample from those detected in the BRD4 and p300 acetylated samples. [...] All ChIP-seq datasets used in this study were downloaded from public repositories. BRD4, AcH3K122 ChIP-seq data sets for MCF-7 cells were from the NCBI Gene Expression Omnibus database as raw fastq files (accession numbers GSE35954 and GSE55923; ,. p300/CBP ChIP-seq data for MCF-7 cells were from the ArrayExpress ChIP-seq database (accession number E-MTAB-785). CTCF, MYC and Gata3 ChIP-seq data sets for MCF-7 cells were from the ENCODE project (The ENCODE Project Consortium 2012) at the UCSC Genome Browser. BRD4 and H3K27ac ChIP-seq datasets for diffuse large B cell lymphoma cell line (LY1) in the presence of JQ1 or DMSO were from NCBI GEO (accession number: GSE46663,). BRD4, H3ac and H4ac ChIP-seq datasets for 1 h LPS stimulated mouse bone marrow-derived macrophages in the presence of iBET BRD4 inhibitor or DMSO were from NCBI GEO (accession number: GSE21910). All ChIP-seq datasets were aligned using Bowtie2 to the reference genome (hg19 for human datasets and mm10 for mouse datasets). Only reads mapped uniquely to the genome were used for the downstream analysis. Peaks for ChIP-seq data were identified using HOMER (http://homer.salk.edu/homer/). AcH3K122 enrichment was calculated based on the center of each peak of BRD4 and the other transcription factors. The average profile plots at TSSs were generated using ngsplot (https://github.com/shenlab-sinai/ngsplot). […]

Pipeline specifications

Software tools Bowtie2, HOMER
Databases ArrayExpress GEO UCSC Genome Browser
Application ChIP-seq analysis
Organisms Mus musculus, Homo sapiens
Diseases Autoimmune Diseases, Neoplasms