Comment[ArrayExpressAccession] E-GEOD-46044 MAGE-TAB Version 1.1 Public Release Date 2013-09-09 Investigation Title A compendium of hematopoietic regulators, chromatin modifiers and basal transcription factors occupy CBF-MYH11/RUNX1 target genes Comment[Submitted Name] A compendium of hematopoietic regulators, chromatin modifiers and basal transcription factors occupy CBF-MYH11/RUNX1 target genes Experiment Description Different mechanisms for CBF-MYH11 function in acute myeloid Leukemia (AML) with inv(16) have been proposed such as tethering of RUNX1 outside the nucleus, interference with transcription factor complex assembly and recruitment of histone deacetylases, all resulting in transcriptional repression of RUNX1 target genes. Here, through genome-wide CBF-MYH11 binding site analysis and quantitative interaction proteomics we found that CBF-MYH11 localizes to RUNX1 occupied promoters where it interacts with TAL1, FLI1 and TBP associated factors (TAFs) in the context of the hematopoietic transcription factors ERG, GATA2 and PU.1/SPI1 and the co regulators EP300 and HDAC1. Transcriptional analysis revealed that upon fusion protein knock down a subset of the CBF-MYH11 target genes show increased expression, confirming a role in transcriptional repression. However, the majority of CBF-MYH11 target genes, including genes implicated in hematopoietic stem cell (HSC) self-renewal such as ID1, LMO1 and JAG1, are actively transcribed and upon fusion protein knock down repressed. Together these results suggest an essential role for CBF-MYH11 in regulating expression of genes involved in maintaining a stem cell phenotype. 17 ChIP-seq samples using antibodies recognizing the indicated proteins and one RNA-seq file from ME-1 cells were analyzed. In addition 2 ChIP-seq profiles were generated using patient AML cells. A CBFβ-MYH11 inducible U937 system (U937CM) was used to examine binding patterns before (1 profile) and after (2 profiles) induction of CBFb-MYH11. In addition, expression was measured through RNA-seq analysis of the two states. The U937CM cells were maintained in the presence of tetracyclin (Tet, 1 uM) and grown in the absence of tetracycline for 3 days to induce expression of CBFβ-MYH11. Finally, a CBFb-MYH11 knock down system was developed in ME-1 cells. Two ME-1 cell lines were created, one with a stably integrated shRNA construct that targets CBFb-MYH11 (ME-1_knockdown) and one with a scrambled shRNA construct (ME-1_SCR). Expression of the shRNA constructs was induced using doxycyclin (dox; 1 mM) treatment for 3 days. 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 Martens Mandoli Singh Jansen Wierenga Riahi Franci Prange Saeed Vellenga Vermeulen Stunnenberg Martens Person First Name Joost A A P B H G K S E M H J Person Mid Initials A W G H Person Email geo@ncbi.nlm.nih.gov Person Affiliation Radboud University Person Address Molecular Biology, Radboud University, Geert Grooteplein 26, Nijmegen, Netherlands Person Roles submitter Protocol Name P-GSE46044-6 P-GSE46044-4 P-GSE46044-1 P-GSE46044-3 P-GSE46044-5 P-GSE46044-2 Protocol Description Basecalls performed using CASAVA Reads were aligned to the hg18 or hg19 genome assembly (see characteristics) using ELAND/BWA Peaks were called using MACS 1.3.3 on settings:mfold =8, tsize=32, p value = e-6 Genome_build: hg19 Supplementary_files_format_and_content: wig or bedgraph files were generated (extended to 300bps) with regions having more than 1 read in a window of 10 bps Basecalls performed using CASAVA Reads were aligned to the hg18 or hg19 genome assembly (see characteristics) using ELAND/BWA Peaks were called using MACS 1.3.3 on settings:mfold =8, tsize=32, p value = e-6 Genome_build: hg18 Supplementary_files_format_and_content: wig or bedgraph files were generated (extended to 300bps) with regions having more than 1 read in a window of 10 bps U937 CBFβ-MYH11 cells were maintained in the presence of tetracyclin and grown in the absence of tetracycline for induction of CBFβ-MYH11. shRNA expression was induced in ME-1 using dox. Chromatin was sonicated and used for ChIP seq using specific antibodies (avialable on suplementry table). Libraries were prepared according to Illumina's standard protocol. RNA was extracted using qiagen RNeasy kit according to manufacturer instructions and total RNA was treated using Ribo-zero to remove rRNA according to manufacturer instructions. Libraries were prepared according to Illumina's standard protocol. ME-1 cells were cultured in normal cell culture containing 10% serum. U937 cells were cultured in tet free culture media for maintenance. Protocol Type normalization data transformation protocol normalization data transformation protocol sample treatment protocol nucleic acid library construction protocol nucleic acid library construction protocol growth protocol Experimental Factor Name CELL LINE TREATMENT ANTIBODY CATALOGUE NUMBER ANTIBODY COMPANY CELL TYPE ANTIBODY TARGET Experimental Factor Type cell line treatment antibody catalogue number antibody company cell type antibody target Publication Title CBFB-MYH11/RUNX1 together with a compendium of hematopoietic regulators, chromatin modifiers and basal transcription factors occupies self-renewal genes in inv(16) acute myeloid leukemia. Publication Author List Mandoli A, Singh AA, Jansen PW, Wierenga AT, Riahi H, Franci G, Prange K, Saeed S, Vellenga E, Vermeulen M, Stunnenberg HG, Martens JH PubMed ID 24002588 Publication DOI 10.1038/leu.2013.257 Comment[SecondaryAccession] GSE46044 Comment[GEOReleaseDate] 2013-09-09 Comment[ArrayExpressSubmissionDate] 2013-04-15 Comment[GEOLastUpdateDate] 2013-09-11 Comment[AEExperimentType] ChIP-seq Comment[AEExperimentType] RNA-seq of coding RNA Comment[SecondaryAccession] SRP021072 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR826864-SRR826890 SDRF File E-GEOD-46044.sdrf.txt