Comment[ArrayExpressAccession] E-GEOD-48085 MAGE-TAB Version 1.1 Public Release Date 2014-04-24 Investigation Title Genome-scale analysis in blood progenitor and mast cells reveals how lineage-affiliated transcription factors control distinct regulatory programs [ChIP-Seq/RNA-Seq Experiments] Comment[Submitted Name] Genome-scale analysis in blood progenitor and mast cells reveals how lineage-affiliated transcription factors control distinct regulatory programs [ChIP-Seq/RNA-Seq Experiments] Experiment Description Despite major advances in the generation of genome-wide binding maps, the mechanisms by which transcription factors (TFs) regulate cell type identity have remained largely obscure. Through comparative analysis of 10 key haematopoietic TFs in both mast cells and blood progenitors, we demonstrate that the largely cell-type specific binding profiles are not opportunistic, but instead contribute to cell-type specific transcriptional control, because (1) mathematical modelling of differential binding of shared TFs can explain differential gene expression, (2) cell-type specific binding is largely mediated through consensus binding sites, and (3) knock-down of blood stem cell regulators Gata2 and Erg in mast cells reveals mast cell specific genes as direct targets. Finally we show that the known mast cell regulators Mitf and c-Fos likely contribute to the global reorganisation of TF binding profiles. Taken together therefore, our study elucidates how key regulatory TFs contribute to transcriptional programmes in several distinct mammalian cell types. Comparison of HPC7 cell line (haematopoietic multipotent progenitor) and primary mast cells. 10 key haematopoietic transcription factors and mRNA expression were profiled in both cell types. 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 Ng Calero-Nieto Ng Wilson Hannah Diamanti Moignard Leal-Cervantes Jimenez-Madrid Wernisch Gottgens Person First Name Felicia Fernando Felicia Nicola Rebecca Evangelia Victoria Ana Isabel Lorenz Berthold Person Mid Initials J S K I Person Email geo@ncbi.nlm.nih.gov Person Affiliation Cambridge Institute for Medical Research Person Address Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Addenbrooke's Hospital, Hills Road, Cambridge, United Kingdom Person Roles submitter Protocol Name P-GSE48085-6 P-GSE48085-4 P-GSE48085-3 P-GSE48085-5 P-GSE48085-7 P-GSE48085-2 P-GSE48085-1 Protocol Description RNA-seq reads aligned to mm9 transcriptome using Tophat and assembled using Cufflinks with default parameters. Genome_build: mm9 Supplementary_files_format_and_content: fpkm values - tab-delimited text files include RPKM values for each sample ChIP-seq reads aligned to mm9 using Bowtie with default parameters. bedGraphs were created with in-house script. Genome_build: mm9 Supplementary_files_format_and_content: bedGraph – read density profiles Supplementary_files_format_and_content: fpkm values - tab-delimited text files include RPKM values for each sample ChIP-seq reads aligned to mm9 using Bowtie with default parameters. bedGraphs were created with in-house script. Genome_build: mm9 Supplementary_files_format_and_content: bedGraph – read density profiles Total RNA was isolated using Tri-Reagent (Sigma) and subsequently digested with DNase I using TurboDNA (ambion), following manufacturer’s specifications. mRNA was enriched with oligoT, fragmented to an average size of 200 nt and retrotranscribed using random hexamers. cDNA was then amplified and sequenced. DNaseI-digested total RNA was processed and sequenced by BGI-Hong Kong. RNAseq samples were sequenced using the Illumina HiSeq 2000 instrument. Total RNA was isolated using Tri-Reagent (Sigma) and subsequently digested with DNase I using TurboDNA (ambion), following manufacturer’s specifications. mRNA was enriched with oligoT, fragmented to an average size of 200 nt and retrotranscribed using random hexamers. cDNA was then amplified and sequenced. DNaseI-digested total RNA was processed and sequenced by BGI-Hong Kong. ChIP assays were performed as previously described (Wilson NK et al., 2010 Cell Stem Cell) using polyclonal antibodies against CTCF (Upstate, 07-729), E2A (Santa Cruz, sc-763x), Erg (Santa Cruz, sc354x), FLI-1 (Abcam, ab15289-500), GATA2 (Santa Cruz, sc9008x), LMO2 (R&D, AF2726), Meis1 (Santa Cruz, sc-10599x), PU.1 (Santa Cruz, sc-352x), RUNX1 (Abcam, ab23980-100), TAL1 (Santa Cruz, sc12984x), Mitf (Cosmo Bio Co., BAM-73-107-EX), cFos (Santa Cruz, sc-253x). Each sample was amplified and sequenced using the Illumina Genome Analyzer II x, following manufacturer's instructions. ChIP assays were performed as previously described (Wilson NK et al., 2010 Cell Stem Cell) using polyclonal antibodies against CTCF (Upstate, 07-729), E2A (Santa Cruz, sc-763x), Erg (Santa Cruz, sc354x), FLI-1 (Abcam, ab15289-500), GATA2 (Santa Cruz, sc9008x), LMO2 (R&D, AF2726), Meis1 (Santa Cruz, sc-10599x), PU.1 (Santa Cruz, sc-352x), RUNX1 (Abcam, ab23980-100), TAL1 (Santa Cruz, sc12984x), Mitf (Cosmo Bio Co., BAM-73-107-EX), cFos (Santa Cruz, sc-253x). Each ChIPseq sample was amplified and sequenced using the Illumina Genome Analyzer IIx, following manufacturer's instructions. Mast cells were derived from bone marrow cells collected from tibias and femurs of 3- to 5- month old adult mice. Cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% fetal bovine serum (Sigma), 1% penicillin/streptomycin (Sigma), 150 micromolar MTG (Sigma), 10% stem cell factor conditional media from BHK/MKL cells and 10ng/ml of recombinant mIL-3 (Peprotech). Cells were frequently transferred to new flasks to remove adherent cells and experiments were performed after 3 weeks, when cultures were homogenous. Homogeneity of culture was confirmed by presence of FcERI by FACS and toluidine blue staining of cytospins. Protocol Type normalization data transformation protocol normalization data transformation protocol normalization data transformation protocol nucleic acid library construction protocol nucleic acid library construction protocol nucleic acid library construction protocol growth protocol Experimental Factor Name CELL TYPE Experimental Factor Type cell type Comment[SecondaryAccession] GSE48085 Comment[GEOReleaseDate] 2014-04-24 Comment[ArrayExpressSubmissionDate] 2013-06-19 Comment[GEOLastUpdateDate] 2014-05-19 Comment[AEExperimentType] RNA-seq of coding RNA Comment[AEExperimentType] ChIP-seq Comment[SecondaryAccession] SRP026165 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR1171520-SRR908271 SDRF File E-GEOD-48085.sdrf.txt